Embed Size (px)
Citation preview
Research Collection
Doctoral Thesis
Preparation of drug nanocrystals stabilized by functionalizedpolymeric coatings
Author(s): Fuhrmann, Kathrin
Publication Date: 2014
Permanent Link: https://doi.org/10.3929/ethz-a-010175706
Rights / License: In Copyright - Non-Commercial Use Permitted
This page was generated automatically upon download from the ETH Zurich Research Collection. For moreinformation please consult the Terms of use.
ETH Library
DISS. ETH NO. 21903
PREPARATION OF DRUG NANOCRYSTALS
STABILIZED BY FUNCTIONALIZED
POLYMERIC COATINGS
Kathrin Fuhrmann
1
DISS. ETH NO. 21903
Preparation of Drug Nanocrystals Stabilized by
Functionalized Polymeric Coatings
A thesis submitted to attain the degree of
DOCTOR OF SCIENCES of ETH ZURICH
(Dr. sc. ETH Zurich)
presented by
KATHRIN FUHRMANN
Pharmacist, Freie Universität Berlin
born on 20.02.1981
citizen of Germany
accepted on the recommendation of
Prof. Jean-Christophe Leroux
Prof. Marc A. Gauthier
Prof. Bruno Alfred Gander
2014
2
3
Table of Contents
I. Summaries .............................................................................................................. 5I.1. Summary .......................................................................................................................... 7I.2. Zusammenfassung ........................................................................................................... 9I.3. List of Abbreviations ..................................................................................................... 11
II. Introduction ........................................................................................................ 13II.1. General Background on Drug Nanocrystals for Chemotherapy ................................... 15II.2. Targeting of Injectable Drug Nanocrystals .................................................................. 25
Introduction ......................................................................................................................... 25In Vitro ................................................................................................................................ 27In Vivo ................................................................................................................................. 32Stabilizer: To Shed or Not To Shed ..................................................................................... 36Altering Dissolution Profiles (by Means Other than Size) .................................................. 39Outlook ................................................................................................................................ 41
III. Results and Discussion ..................................................................................... 43III.1. PEG Nanocages as Non-Sheddable Stabilizers for Drug Nanocrystals ...................... 45
Introduction ......................................................................................................................... 45Experimental Section .......................................................................................................... 47Results and Discussion ........................................................................................................ 54Conclusion .......................................................................................................................... 69
III.2. Modular Design of Redox-Responsive Stabilizers for Nanocrystals .......................... 71Introduction ......................................................................................................................... 71Experimental Section .......................................................................................................... 73Results and Discussion ........................................................................................................ 78Conclusion .......................................................................................................................... 90
IV. Conclusion and Outlook .................................................................................. 91
V. Supplementary Information ............................................................................ 101
VI. Curriculum Vitae and Scientific Contributions .......................................... 117
VII. Acknowledgments ......................................................................................... 121
VIII. List of References ........................................................................................ 125
4
5
I. Summaries
6
7
I.1. Summary
The poor water-solubility of many newly discovered drugs causes problems for
several routes of administration. In the case of intravenous administration, the
dissolved drug should not precipitate lest it causes an embolism. One elegant
approach to overcome solubility problems is to formulate poorly water soluble drugs
as nanocrystals. Due to their small size, nanocrystals dissolve faster than the
corresponding bulk solid drug, can have a higher saturation solubility, and can be
administered intravenously. Nanocrystals are almost entirely composed of drug, with
only a small amount of stabilizing excipient. This is beneficial for parenteral use
because adverse side effects, such as pain and immune reactions, have been reported
for a variety of cosolvents and solubilizers. Unfortunately, the stabilizer cannot be
completely eliminated from the formulation as nanocrystals tend to aggregate due to
their high surface energy. Polymers and/or surfactants are added to provide steric
and/or electrostatic barriers to aggregation. Nevertheless, modification of the
stabilizer offers new opportunities for nanocrystal targeting and release in specific
tissues, which would be inaccessible to non-stabilized nanocrystals. This concept
could prove especially useful in the treatment of cancer, where most drugs are poorly
water-soluble.
Cancer is a disease in which the uncontrolled division and growth of cells results in
tumor formation and potential invasion of other locations throughout the body. The
prevalence for this disease is high, and lung and breast cancers are the worldwide
leading causes of cancer mortality. Cancer treatments have a high impact on the
patient’s well-being, because the pharmacological action of the toxic anticancer drugs
is often untargeted and therefore affects all dividing cells. Patients as well as
survivors suffer from effects of the disease, but also from complications caused by the
treatment, such as hair loss, nausea, and neurological pain. These adverse side effects
could be avoided if a drug formulation is made to only target and release the drug at
the tumor site.
In the work presented herein, stabilizers for nanocrystals of paclitaxel (poorly
soluble chemotherapeutic drug) were chemically modified to examine new
opportunities for improving stability, targeting, and stimuli-responsive release. The
stabilizer platform consisted of diblock copolymers of methoxy(polyethylene glycol)-
8
b-(-propargyl--valerolactone-co--caprolactone). These polymers stabilized
paclitaxel nanocrystals produced by wet milling. Moreover, they could be cross-
linked with different diazido compounds via the copper-catalyzed azide-alkyne
Huisgen cycloaddition reaction to form nanocages on the surface of nanocrystals. The
nanocages improved the size stability for the nanocrystals, and they were also less
shed from the surface of the nanocrystals.
In a second study, the stabilizers were modified with different alkane thiols by
radical thiol−yne addition. A library of polymers with different lipophilicities was
obtained and tested as stabilizers for paclitaxel nanocrystals. In addition, the acquired
thioether bonds in the polyester block were sensitive to oxidation, which reversed
their affinity for the hydrophobic nanocrystals’ surface. This could be beneficial
where drug release/uptake in response to a stimulus, e.g., presence of reactive oxygen
species in the tumor, is desired.
In summary, the proposed strategies for increased nanocrystal stabilization and
oxidation responsive release may be useful for improving chemotherapy and reducing
adverse side effects. Such methods can also be applicable for modifications of other
intravenously administered nanoformulations where a non-covalent attachment of the
coating and/or location-specific shedding of stabilizers is desired. Thus, in the long
term, this work can help improve the properties of drug nanocrystals for anticancer
therapy.
9
I.2. Zusammenfassung
Die schlechte Wasserlöslichkeit vieler neu entdeckten Arzneistoffe stellt ein
Problem für mehrere Applikationsarten dar. Im Falle einer intravenösen Gabe darf der
Wirkstoff nicht in der Blutbahn ausfallen, da er sonst eine Embolie verursacht. Eine
elegante Art diese Löslichkeitsprobleme zu überwinden, ist die Formulierung dieser
Klasse von Arzneistoffen als Nanokristalle. Wegen ihrer geringen Grösse lösen diese
sich schneller auf als der entsprechende grobe Arzneistoff, sie können eine höhere
Sättigungslöslichkeit haben und sie können auch intravenös gegeben werden.
Nanokristalle bestehen hauptsächlich aus reinem Arzneistoff und nur einem kleinen
Anteil an Hilfsstoff. Dies ist von Vorteil für den parenteralen Gebrauch, weil
unerwünschte Nebenwirkungen, wie Schmerzen und Immunreaktionen, für vielerlei
Lösungsmittel und Lösungsvermittler bekannt sind. Allerdings haben Nanokristalle
auf Grund ihrer hohen Oberflächenenergie die Tendenz zu aggregieren. Deshalb
werden Polymere und/oder Tenside hinzugefügt, welche als sterische und/oder
elektrostatische Barrieren fungieren. Durch Modifizieren dieser Polymere ergeben
sich neue Möglichkeiten für Nanokristall Targeting und Freigabe in spezifischen
Geweben. Dies könnte sich als besonders nützlich in der Behandlung von Krebs
erweisen, wo die meisten Arzneistoffe schlecht wasserlöslich sind.
Krebs ist eine Krankheit, bei welcher die unkontrollierte Zellteilung und -wachstum
zur Tumorbildung und möglicher Invasion in anderen Teilen des Körpers führen. Die
Prävalenz für diese Krankheit ist hoch, und Lungen- und Brustkrebs sind die weltweit
führenden Ursachen für Krebssterblichkeit. Krebsbehandlungen haben eine hohe
Auswirkung auf das Wohlbefinden eines Patienten, weil die pharmakologische
Wirkung des toxischen Chemotherapeutikums häufig ungerichtet ist und deshalb alle
sich teilenden Zellen betrifft. Patienten sowie Überlebende leiden unter den
Auswirkungen der Krankheit, aber auch an den Komplikationen die durch die
Behandlung entstehen, wie z.B. Haarausfall, Übelkeit und neurologische Schmerzen.
Diese unerwünschten Arzneimittelwirkungen könnten vermieden werden, wenn eine
Wirkstoffformulierung so gemacht ist, dass sie den Wirkstoff gezielt zum Tumor
bringt und dort freisetzt.
In dieser Arbeit wurden Polymere für Paclitaxel Nanokristalle (schlecht
wasserlöslicher chemotherapeutischer Arzneistoff) chemisch modifiziert, um neue
10
Mittel für verbesserte Größenstabilität, gewebespezifischer Pharmakotherapie und
bedarfsgesteuerter Freisetzung zu finden. Diese Polymere basierten auf einem
Diblock Kopolymer, Methoxy-polyethylenglycol-b-(-propargyl--valerolacton-
co-caprolacton), und konnten Paclitaxel Nanokristalle stabilisieren, welche durch
Nassvermahlung hergestellt wurden. Weiterhin konnten sie mit Hilfe verschiedener
Diazidverbindungen in einer Kupfer katalysierten Azid-Alkin Huisgen Zykloaddition
vernetzt werden, wodurch Nanokäfige auf der Oberfläche der Nanokristalle
entstanden. Die Nanokäfige verbesserten die Grössenstabilität der Nanokristalle und
neigten weniger dazu von deren Oberfläche abgestreift zu werden.
In einer zweiten Studie wurden diese Polymere mit verschiedenen Alkanthiolen
durch radikale Thiol-Alkin Reaktion modifiziert. Eine Sammlung neuer Polymere mit
unterschiedlichen Lipophilien wurde erhalten und als Stabilisierer für Paclitaxel
Nanokristalle getestet. Ausserdem waren die entstandenen Thioetherbindungen
empfindlich gegenüber Oxidation, welches ihre Affinität für die Oberfläche der
Nanokristalle umkehrte. Dies könnte dann von Vorteil sein wenn die
Wirkstofffreisetzung/-aufnahme in Abhängigkeit von einem Stimulus, z.B. Präsenz
von reaktiven Sauerstoff Spezies im Tumor, gewünscht ist.
Zusammenfassend kann gesagt werden, dass die vorgeschlagenen Strategien für
erhöhte Nanokristall Stabilisation und Oxidationsreaktionsfähigkeit durch
Polymermodifikation ein nützliches Mittel für die Verbesserung der Chemotherapie
mit weniger unerwünschten Arzneimittelwirkungen werden kann. Solche Methoden
könnten auch auf die Modifizierung anderer intravenös gegebener
Nanoformulierungen übertragen werden, da wo nicht-kovalentes Verknüpfen
und/oder ortsspezifisches Abstreifen des Polymers gewünscht ist. Daher kann diese
Arbeit in Zukunft dazu beitragen, die Eigenschaften von Nanokristallen für die
Chemotherapie zu verbessern.
11
I.3. List of Abbreviations
BSA Bovine serum albumin
CMC Critical micelle concentration
COSY Correlation spectroscopy (two-dimensional NMR)
DLS Dynamic light scattering
DMF Dimethylformamide
DMSO Dimethylsulfoxide
VL -Valerolactone
CL -Caprolactone
EPR Enhanced permeation and retention
FDA Food and drug administration
FTIR Fourier transform infrared (spectroscopy)
HMPA Hexamethylphosphoramide
HPLC High performance liquid chromatography
HPCD Hydroxypropyl--cyclodextrin
i.v. Intravenous
ICP-OES Inductively coupled plasma optic emission spectroscopy
LDA Lithium diisopropylamide
MALDI-TOF MS Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
mCPBA m-Chloroperoxybenzoic acid
mPEG Methoxy poly(ethylene glycol)
mPEG-b-[PVL-co-CL]
Methoxy poly(ethylene glycol)-b-[-propargyl--valerolactone-co--caprolactone]
mPEG-b-PSO Methoxy poly(ethylene glycol)-b-poly(styrene oxide)
MPS Mononuclear phagocyte system
MWCO Molecular weight cutoff
nab-Paclitaxel Nanoparticle albumin-bound paclitaxel
NC Nanocrystal
12
NMP 1-Methyl-2-pyrrolidone
NMR Nuclear magnetic resonance (spectroscopy)
P-gp P-glycoprotein
PCL Poly(caprolactone)
PEG Poly(ethylene glycol)
PTX Paclitaxel
PVL Propargyl--valerolactone
ROP Ring-opening polymerization
ROS Reactive oxygen species
SEC Size exclusion chromatography
TEM Transmission electron microscopy
THF Tetrahydrofuran
TPGS D-α-tocopheryl poly(ethylene glycol 1000) succinate
13
II. Introduction
14
15
II.1. General Background on Drug Nanocrystals for
Chemotherapy
What are drug nanocrystals?
Most drugs emerging from high throughput screenings are poorly water-soluble,1
which can be a disadvantage for their administration to a patient. For instance, a
drug’s oral bioavailability depends on its solubility (in addition to permeability), but
the same also applies to topical administration of a drug, because only dissolved drug
can be absorbed. Low or erratic bioavailabilty can have consequences for the safety
and efficacy of a drug. To overcome this problem crystalline drugs can, for example,
be converted to amorphous material.2 Alternatively drugs can be modified into
prodrugs,3 a process that involves considerable manufacturing efforts and risk of
lowered stability or change in efficacy. Furthermore, drugs can be formulated in a
liquid dosage form,4 which, however, can make it less appealing to the patient
because of the potentially required cold storage conditions, increased single dose
volume, and possibly unpleasant taste. In the case of the intravenous (i.v.)
administration route, an aqueous drug solution is the preferred type of pharmaceutical
formulation, however, cosolvents and solubilizers used to dissolve lipophilic
substances can pose problems. These can be systemic immune reactions
(hypersensitivity reactions), organ toxicity, and pain if the amount is high.5-8 When
poorly water soluble drugs are formulated as i.v. suspensions or emulsions one has to
bear in mind that particles larger than ca. 5 µm can potentially clog blood capillaries
and cause embolism. Therefore, an attractive approach for administering a drug in its
solid state is to engineer them as drug nanocrystals. These crystals are composed
almost entirely of drug, which differentiates them from other nanomedicines such as
liposomes,9, 10 micelles,11 or polymeric nanoparticles12 since only a small amount of
excipient is needed for stabilization. Typically the drug content ranges between 50
and 90%,13, 14 and the material is crystalline as the name states. In the 1990s, a wet
media milling process from the paint and photographic industry was adapted by
Liversidge and colleagues from Sterling Drug Inc./Eastman Kodak for reducing the
particle size of poorly soluble drugs in order to increase their oral bioavailability. This
process was patented as NanoCrystal® Technology, and currently there are five
16
products on the market (Emend®, Invega® Sustenna®, Megace® ES, Rapamune®, and
TriCor®)15 with another product (Panzem® NCD)16, 17 in the pipeline (Table 1). The
particularity of nanocrystals is that smaller particle size results in faster dissolution,18
according to the Noyes-Whitney equation (Equation 1):
dc
dt
A (CS CX )
h (1)
because the greater the specific surface area A of a solid, the faster is the dissolution
velocity dc/dt, (CS is the saturation solubility of the solute, CX is the solute
concentration in the medium, and h is the diffusion barrier). Furthermore, nanosizing
(reducing the radius r and increasing surface energy/interfacial tension ) can
increase the saturation solubility CS19 according to the Ostwald-Freundlich equation
(Equation 2):
logCS
C
2V
2.303RT r (2)
where C∞ is the solubility of very large particles, the interfacial tension between
the solid surface and the surrounding medium, V the molar volume of the compound,
R the gas constant, T the absolute temperature, and the density of the solid.
Particle size reduction generally improves the bioavailability of orally administered
drugs by increasing their solubility and dissolution rate. Furthermore, the fed/fasted
variability can also be attenuated. This benefit of nanocrystals becomes clear in the
example Emend® (aprepitant, anti-emetic). The conventional formulation showed
greater bioavailability in the presence of food but the fasted state is rather difficult to
obtain for a patient who suffers from nausea/vomiting.13, 20
Most nanocrystal formulations are transformed into tablets, which is a convenient
oral dosage form with high patient compliance. Nanocrystals are also amenable to
additional modifications, including the conjugation of targeting ligands or
environment-sensitive triggers to the stabilizer for increasing the bioavailability of the
drug specifically at the site of disease. Advantages and disadvantages of drug
nanocrystals as formulations for parenteral use specifically will be highlighted in
Chapter II.2.
17
Table 1. Products on the market produced by nanosizing technologies.13, 15, 21
Product Company Drug compound Nanosizing technology
Indication Administration route
Therapeutic benefit Year of FDA approval
Emend® Merck Aprepitant Wet milling Nausea, vomiting Oral Increased bioavailabi-lity, no food effects
2003
Invega® Sustenna®
Janssen Paliperidone palmitate
Wet milling Schizophrenia Intramuscular injection
Reformulated from tablets for sustained release
2009
Megace® ES Strativa/ PAR Pharmaceuticals
Megestrol acetate Wet milling Anorexia, chachexia
Oral Increased bioavailabi-lity, no food effects
2005
Rapamune® Pfizer/Wyeth Sirolimus Wet milling Prophylaxis of organ rejection (immunosuppress-ant)
Oral Reformulated from solution, better storage conditions and higher bioavailability
2000
TriCor® Abbot/Abbvie Fenofibrate Wet milling Hypercholesterol-emia
Oral Increased bioavailabi-lity, no food effects
2004
Triglide® Skye Pharma/ Shionogi
Fenofibrate High pressure homogeniza-tion
Hypercholesterol-emia
Oral Increased bioavailabi-lity, no food effects
2005
18
How are drug nanocrystals produced?
Nanocrystals can be produced by two major routes: (1) by decreasing size of the
starting material using various comminuting techniques (“top-down”) and (2) by
building new crystals from a solution via precipitation in an antisolvent (“bottom-
up”). In the “top-down” approach coarse drug powder is fractured by wet milling,22
sonication,23 high pressure homogenization,19 or even laser fragmentation24 in a wet
environment, and usually in presence of a surfactant to prevent aggregation of newly
formed nanocrystals.25, 26 Wet milling in particular is established for industrial scale
production and a number of products manufactured by this procedure are on the
market.13, 27 In this process, the coarse drug powder is suspended in aqueous media
and introduced into a milling chamber containing beads. These beads and often also
the milling chamber are of very hard material such as yttrium-stabilized zirconium
oxide to limit wear which could contaminate the product.22, 28 “Bottom-up”
approaches have been mostly used on laboratory scale, sometimes resulting in
amorphous material.29, 30 Many variations exist, but essentially they all have in
common a precipitation in an anti-solvent accompanied by sonication and/or stirring.
Due to a wider range of parameters that need to be controlled they have not yet
resulted in a commercialized pharmaceutical product. For further details on the
fabrication techniques the reader is directed to the thorough recent reviews by
Merisko-Liversidge and Liversidge 13 and by Chan and Kwok.31
How are nanocrystals stabilized?
The high surface energy of nanocrystals causes them to aggregate and form larger
particles. To prevent aggregation, a surfactant or polymer is added to the medium. It
physically adsorbs to the surface of the nanocrystals, thereby stabilizing them by
steric hindrance and/or electrostatic repulsion.22, 28 The stabilizer is in equilibrium
between its adsorbed and free state. A seemingly endless list of factors have been
studied for their influence on the preparation of stable nanosuspensions, including
intensity of grinding energy, drug content during milling, bead size, logP,
morphology, melting point, solubility, density, and mechanical properties of the drug
substance.22, 26, 28, 32, 33 Nevertheless, the choice of stabilizer remains often empirical,
although it seems that the affinity of the stabilizer to hydrophobic surfaces, which is
related to the hydrophobicity of the drug material itself, plays a crucial role.26
Commonly used stabilizers for nanocrystals include different types of poloxamers,
19
povidone, cellulose derivatives, and D-α-tocopheryl poly(ethylene glycol 1000)
succinate (TPGS).28, 34-36 The use of block copolymers containing hydrophilic flexible
polymers such as poly(ethylene glycol) (PEG) may also increase the circulation time
of i.v. injected nanoparticles by reducing opsonization and uptake by the mononuclear
phagocyte system (MPS).37, 38 Prominent examples of PEG-containing stabilizers are
poloxamers (a poly(propylene glycol) block flanked by two PEG chains) and TPGS.
Different attempts have been made to enhance adsorption and/or anchoring in order to
improve colloidal stability. These include, for example, the covalent cross-linking of
the surfactant network on the crystal surface to form nanocages (Chapter III.1),39 and
electrostatic cross-linking by layer-by-layer assembly of oppositely-charged polyions
in multiple layers around the crystal.23, 40, 41 Charged polymers can be of natural
source like chitosan (polycation) and alginic acid (polyanion), or synthetic such as
poly(allylamine hydrochloride) (polycation) and sodium poly(styrene sulfonate)
(polyanion).41
What is paclitaxel?
Paclitaxel is a poorly soluble drug with high potency in the treatment of cancer of
various types. Its long use in the clinic has shown that it is safe and effective,
however, there are issues related to the currently used formulations. Thus, it was
chosen as a model drug for the work in this doctoral thesis.
Paclitaxel was the first drug originating from the taxane family, a group of
microtubule-stabilizing agents that inhibit mitosis, and it is widely used in the
chemotherapy of cancer.42 Microtubules are essential during mitosis, a process during
which duplicated chromosomes of a cell are separated into two identical sets before it
divides into two daughter cells. Cancer cells constantly divide and thus the
importance of the microtubules in mitosis and cell division makes them a significant
target for anticancer drugs.42, 43 Paclitaxel is a natural component, which was
originally (about 40 years ago) isolated from the bark of the Pacific yew tree (Taxus
brevifolia). Due to its structural complexity (Figure 1) and the limited presence of
Pacific yew trees, it is now produced semisynthetically from 10-deacetylbaccatin III
from the European yew tree (Taxus baccata).44 Other representatives of the taxanes
include docetaxel and cabazitaxel.43 Paclitaxel is used in cancer chemotherapy to treat
ovarian cancer, breast cancer, small and non-small cell lung cancer, colon cancer, and
Kaposi’s sarcoma.42, 45 However, an important issue is its poor water solubility, which
20
requires the use of ethanol and Cremophor EL (macrogolglycerol ricinoleate).
Cremophor EL has been associated with hypersensitivity reactions.5
Figure 1. Chemical structure of paclitaxel
Adverse effects of taxanes generally include neurological and myeloid toxicities.
Neurological toxicity can be potentially severe and is a dose-limiting side effect that
manifests itself as a painful and debilitating peripheral axonal neuropathy for which
there are currently no effective symptomatic treatments.43 Symptoms tend to
disappear some months after the end of treatment, though in some cases patients
retain considerable sequelae several years after therapy.43 This toxicity is not yet
understood at a mechanistic level, however, it is apparently related to the relative
abundance of tubulin in neurons and the importance of an intact, functional
microtubule cytoskeleton for adequate nerve conduction.46 Myeloid toxicity is
frequently observed, with neutropenia as the most frequent and/or severe side effect
occurring in combination regimens, but is usually manageable.43
A novel surfactant-free taxane formulation which addresses the problems associated
with cosolvent induced hypersensitivity reactions is the nanoparticle albumin-bound
paclitaxel (nab-paclitaxel, Abraxane®), which is mainly amorphous.47, 48 Since its
approval in the US in 2005 (Europe in 2008) its use in the clinic allows the
administration of higher doses of drug without steroid or antihistamine prophylaxis
for hypersensitivity reactions. In a Phase III trial comparing Abraxane® and solvent
21
based paclitaxel formulation in the treatment of patients with metastatic breast cancer,
nab-paclitaxel was associated with a better outcome as well as with a lower rate of
severe neutropenia but a slightly higher rate of sensory neuropathy, which mostly
improved after treatment interruption.49 Abraxane® has also shown activity in other
contexts including melanoma, small cell lung cancer, and gynecological tumours.50, 51
Nevertheless, it still causes strong discomforting and dose limiting side effects such as
alopecia, fatigue, neutropenia, sensory neuropathy, and others.49, 52, 53 The importance
of these side effects will be discussed in the next section.
Why nanocrystals made of paclitaxel?
The issue with new oncology drugs is their high attrition rate during Phase II and III
trials.54, 55 Specifically 90% of all new oncology drugs do not obtain market
approval.56 Analyzing the cause for these drop outs, it has been stated that poor
pharmacokinetic properties or bioavailability were only of minor influence, whereas
lack of efficacy and low margins of safety were the major obstacles.54 Thus, paclitaxel
as well as microtubule stabilizing agents in general will likely continue to be
important drugs in the treatment of cancer, even as more selective approaches are
developed.42
“Old” drugs have the advantage that they have already been proven to be safe and
effective. Nevertheless, they can be reformulated to increase the therapeutic index and
improve the compliance of patients. Systemic drug administration, unless very
selective for the target tissue, commonly affects all cells. In particular, targeting of a
drug to the tumor in cancer therapy is of paramount importance because side effects
such as neutropenia,57 neuropathy,46 hair loss,58 and mucositis59 are caused by the vast
distribution of the drug to other tissues. Clearly, the quality of life and psychosocial
burden of cancer have an effect on therapeutic outcome because patients prefer
treatments with less harsh side effects that do not disrupt their everyday life.58, 60
A potential avenue for improving therapy with paclitaxel is its formulation as
nanocrystals. Owing to their small size, they can take advantage of the so-called
“passive targeting” via the enhanced permeation and retention (EPR) effect.61 This
term refers to the leaky vasculature combined with often dysfunctional lymphatic
clearance as a result of the tumor’s fast and chaotic growth.62 Nanoparticles which are
sufficiently small (less than 400 nm) can extravasate through the leaky endothelium
and accumulate in tumoral tissues.63 Nanocrystals are, in theory, the ideal formulation
22
approach for exploiting the EPR effect after i.v. injection because of their high drug
content paired with the poor solubility of the starting material. Furthermore, the
biodistribution could be favored to the tumor tissue if the nanocrystals carry a
targeting ligand and/or a trigger for selective drug release/uptake at the tumor site.
These features can potentially be included in the nanocrystal formulation by alteration
of the polymeric stabilizer. While an increase in tolerability of a formulation only
allows administration of higher doses, a targeted formulation would bring more drug
to the disease site, which effectively could reduce the overall dose to be given. This is
an opportunity to change the biodistribution of the drug, with more drug acting at the
site of disease and less causing adverse side effects because of systemic exposure.
What is the approach within this thesis?
In this doctoral work, paclitaxel nanocrystals were prepared by wet milling (Figures
2A and 2B) and stabilized with newly designed polymeric surfactants to prevent
aggregation (Figure 2C). In addition, it was the scope of this thesis to exploit
strategies for the improved targeting/accumulation and release of drug at the tumor
site by means of stabilizer modification. Novel biodegradable block copolymers were
developed for this task, which generally consisted of PEG (steric stabilization and
stealth properties) and a polyester (hydrophobic interaction with crystal surface,
possibility for postpolymerization modification). The polymers were modified with
cross-linking agents or reactive groups in order to improve their anchoring on the
particles’ surfaces and/or make their affinity responsive to stimuli such as oxidation.
In this context reactive oxygen species (ROS) are particularly interesting triggers as
they are associated with chronic inflammation and cancer.64, 65 Responsiveness to
ROS could eventually be exploited to promote deposition of the nanocrystals in the
tumor. Furthermore these polymeric stabilizers offer the possibility of more stably
attaching targeting moieties by use of heterotelechelic PEG derivatives, which may
favor accumulation or uptake of nanocrystals at specified locations. Nonetheless, it
remains critical that the surfactant−targeting agent construct is not shed rapidly after
administration due to changes in the equilibrium of adsorbed and desorbed stabilizer
upon dilution in the bloodstream.
23
Figure 2. Wet milling of paclitaxel nanocrystals. (A) Zirconium oxide beads are
agitated in a vessel containing coarse drug powder and polymeric stabilizer solution.
(B) Attrition between beads breaks powder particles into drug nanocrystals. (C)
Diblock copolymer adsorbs to nanocrystal surface (yellow: mPEG, red: polyester).
In chapter III.1, different stabilizers are cross-linked around paclitaxel nanocrystals
by click chemistry to form polymeric “nanocages”. These nanocages function as
sterically stabilizing barriers to particle−particle interactions and prevent aggregation
as observed by size measurements over time by dynamic light scattering (DLS) and
transmission electron microscopy (TEM). Dialysis experiments combined with TEM
revealed the presence of nanocages. Centrifugation experiments showed that these
were less shed from the nanocrystal surface than comparable non-cross-linked
stabilizers. The development of these nanocages contributes an important general
approach for the preparation of poorly sheddable stabilizing coatings for nanocrystals,
and potentially also for other classes of nanoparticles for which covalent attachment
of the stabilizer is inappropriate (e.g., a drug) or impossible (e.g., chemically inert
material).
Chapter III.2 presents a modular and systematic strategy for preparing ROS
sensitive polymeric stabilizers with different affinity for drug nanocrystals both
before and after oxidation. Using the thiol−yne addition reaction, a library of ten
redox-responsive polymer stabilizers was prepared from two parent block
copolymers. The stabilizing potential for paclitaxel nanocrystals as well as the
influence of oxidation on size after exposure to ROS was studied by DLS. TEM as
well as dissolution tests before and after treatment with ROS were carried out. The
versatility of the presented postpolymerization modification approach makes it a
24
potential platform for preparing triggered-sheddable stabilizing coatings for
nanoparticles.
25
II.2. Targeting of Injectable Drug Nanocrystals1
Kathrin Fuhrmann, Marc A. Gauthier, and Jean-Christophe Leroux
Introduction
“Nano” drug delivery carriers are in many respects established technologies for
improving the therapeutic index of chemotherapeutic drugs and overcoming critical
formulation challenges of poorly water-soluble compounds. The latter are, for
instance, difficult to administer intravenously (i.v.) because of their potential
aggregation in the bloodstream, which can lead to embolism and accumulation in the
lungs. A variety of anticancer drug nanocarriers based on, for example, liposomes,66
micelles,67 polymeric nanoparticles,68 and so forth, are in clinical trials, and some
have reached the market. For example, liposomal doxorubicin (Doxil® or Myocet®)
has now been used for more than 15 years in the treatment of myeloma, breast cancer,
ovarian cancer, and AIDS-related Kaposi's sarcoma (see refs in 69). In comparison to
conventional drug formulations, nanocarriers can be advantageous because of the
lesser use of solubilizing agents or cosolvents (excipients), which are often a source
of side-effects such as hypersensitivity reactions (e.g., Cremophor EL® in the
paclitaxel formulation Taxol®).70 Furthermore, nanocarriers can reduce side-effects of
the drug by targeting them specifically to sites of disease rather than to healthy tissues
(e.g., cardiotoxicity of free doxorubicin).71 Two important remaining technological
challenges of nanocarriers, however, are the need to formulate drugs on a case-by-
case basis (i.e., due to the specific chemistry of each drug) and the difficulty
associated with transporting large amounts of drug to the site of the tumor (i.e., in part
because of moderate to poor drug loadings).
One of the most valuable “nano” opportunities in this field is to address these
challenges by creating nanocarriers composed of the drug itself.72-76 This strategy
reduces the use of “non-drug” material within the formulation compared to the other
nanocarriers above, and, if successfully targeted to the site of disease, should deposit
a significant amount of drug at this location. To this end, several drugs have been
processed into colloidal dispersions known as "nanocrystals" (NCs),25 and have been
1 Published in Mol. Pharm. 2014, 11, 1762–1771
26
examined in cells, animal models, and in humans. In an excellent recent contribution,
Gao et al.21 describe the state-of-the-art of the in vivo performance of drug NCs. The
NC platform is particularly attractive because production can be achieved by a variety
of bottom–up and top–down approaches more-or-less irrespectively of the physical-
chemical properties of the drug.13, 31 Many techniques can lead to products with
reduced particle size, including sophisticated ones such as laser fragmentation24, 77 or
supercritical fluids with enhanced mass transfer,78, 79 but the most commonly used
techniques in industry are high pressure homogenization, and wet milling.80, 81 For
example, wet milling can produce unimodal NCs with mean diameters in the ca. ~200
nm range with little batch-to-batch variability.82 This process is suitable for many
different classes of compounds and there currently exist a variety of oral NC
formulations produced by wet milling on the market, including Rapamune®
(sirolimus), Emend® (aprepitant), TriCor® (fenofibrate), and Megace® ES (megestrol
acetate).73, 83
Opportunities That “Nano” Brings to Targeted NC Delivery. Because of their
adjustable sub- ~400 nm size, intravenously injected NCs can, in principle,
extravasate from the blood through the leaky endothelium and accumulate in tumoral
tissue via the enhanced permeation and retention effect.63 In addition to this passive
targeting phenomenon, the stabilizers used to mask the high-energy drug surfaces
created during the size-reduction process can be functionalized with
targeting/internalizing ligands to promote active tumor accumulation or uptake,
respectively. These stabilizers, for example polymers or surfactants, typically stabilize
NCs by adsorbing to the surface of the NCs and providing steric (e.g., poloxamers,
cellulose derivatives)28 or electrostatic (e.g., sodium dodecyl sulfate)84 barriers to
aggregation.25, 26, 82, 85 Steric stabilization is most efficient in a good solvent (for the
stabilizer) and a minimum layer thickness is required,86 while the comparable
guidelines for electrostatic repulsion are that the absolute value of zeta potential
should be at least 30 mV.26 As only a small amount of stabilizing agent is typically
required to mask NCs and prevent their aggregation, drug content of typically 50 to
90 wt% has been reported,40, 87 which is dramatically higher than for other nanocarrier
systems.
Challenges That “Nano” Brings to Targeted NC Delivery. NCs exhibit a
characteristic nonlinear increase of kinetic solubility upon miniaturization that is
described by the Ostwald–Freundlich equation.88 This phenomenon, which only
27
becomes evident when particles reach the submicrometer size range, dramatically
increases their rate of dissolution and is generally exploited for the “untargeted”
administration of insoluble drugs, with little need for solubilizing agents or
cosolvents.21 However, enhanced dissolution complicates targeted NC delivery due to
off-target drug delivery, insufficient circulation time for passive targeting, and
potential shedding of the stabilizing agent/targeting agents used for active targeting.
In addition, passive targeting via the enhanced permeation and retention effect is
increasingly becoming a subject of debate. 89
Overall, despite the fact that drug NCs have been studied and used clinically for
nearly three decades, enhancing NC uptake through specific interactions in vitro, and
targeting in vivo remain elusive objectives. This noncomprehensive review highlights
the opportunities offered by NCs as well as the important challenges that remain for
achieving targeted delivery. For this purpose, selected studies on drug NC
performance, irrespective of disease treated, in cell culture models and in vivo after
parenteral administration are presented, and current and future avenues of research for
enhancing their therapeutic potential are discussed. It should be noted that another
advanced nanoparticulate system, namely Abraxane®, is also presented in this review.
Abraxane® is an injectable paclitaxel formulation produced by high-pressure
homogenization in the presence of human serum albumin and used for the treatment
of metastatic breast cancer.90 Although not a (nano)crystalline material per se,48 the
clinical use of this product and its multiple physical–chemical similarities to other
NCs supports its inclusion in this discussion, for comparative purposes.
In Vitro
Cell-based assays are often used prior to in vivo experimentation to validate the
performance of drug NCs in comparison to other formulations. In the case of
anticancer drugs, the cytotoxicity of NCs compared to that of the free drug in solution
or within other nanocarriers is used as a parameter for establishing activity. For drugs
with limited cytotoxicity, uptake is either measured directly or via specific assays
associated with their mechanism of action. Unfortunately, due to the specific
conditions used in these assays, many studies involving drug NCs have yielded
disparate results, which complicate generalizations and extrapolation of in vitro
findings on NCs to the in vivo setting.
28
One fundamental, but sometimes forgotten, characteristic of cell culture assays is
that they are performed within a closed system, of finite volume. As a consequence,
the rate of drug dissolution from the NC, and its resulting consequence on therapeutic
efficacy, can cease to depend on the dose administered if it is above the saturation
solubility of the drug. In addition, the relationship between incubation time and
cytotoxicity will depend on the dissolution rate of the NC. For instance, when the
contact time with cells is short, rapidly dissolving NCs of cytotoxic drugs should have
a comparable effect on cell viability as the free drug in solution, and more slowly
dissolving NCs should be less cytotoxic.91 That is, when uptake is rapid compared to
NC dissolution, structural or chemical parameters associated with the NC may play a
role in performance. In support of this, Shegokar et al.92 have shown that the in vitro
uptake after 2 h of ca. 450-nm NCs formed of the antiretroviral drug nevirapine by
macrophages was sensitive to the nature of the stabilizer, indicating that the NCs are
still intact and have not shed their stabilizing coating within this time frame. The
authors notably observed that the NCs coated with poly(ethylene glycol) were less
taken up by cells in comparison to NCs coated with dextran or albumin, in accordance
with the stealth-like behavior previously reported for this polymer. NC endocytosis
appears to be clathrin and caveolae mediated, as observed for 240-nm NCs of
anticancer drug camptothecin with needle-like morphology.93
The situation changes when longer incubation times with NCs are used, given that
dissolution may occur early in the overall incubation process. For example, 240-nm
NCs of camptothecin showed similar cytotoxicity to that of the solution after 72 h of
incubation.93 Indeed, rapid dissolution has been observed in a number of reports. For
instance, 125-nm tamoxifen NCs coated with three bilayers of
poly(dimethyldiallylamide ammonium chloride) and poly(styrenesulfonate) were
~50% solubilized within 2 h under sink conditions.40 In vitro, Ben Zirar et al.94 have
evaluated the viability of both K562 and U937 cells after 48–72 h incubation with
melarsoprol either as a free drug solution or as 300–600 nm poloxamer-stabilized
NCs (Figure 1A). Under these incubation conditions, differences between the NC and
the free drug, when statistically significant, were generally small. Vergara et al.95
have assessed the cytotoxicity of ~150-nm paclitaxel NCs stabilized by electrostatic
layer-by-layer (LbL) assembly of alginic acid and chitosan. Interestingly, the authors
observed that cell viability ceased to decrease when the dose of paclitaxel was
increased beyond ca. 5–10 ng·mL–1. At first glance, this result appeared to indicate
29
that saturation (and thus prevention of NC dissolution) was occurring in the culture
medium, despite the fact that these concentrations were well below the saturation
solubility of paclitaxel in water (300 ng·mL–1).96 However, only marginal differences
were observed between the NC and freely soluble drug (Figure 1B), suggesting that
dissolution had occurred well within this timeframe in cell culture medium. The
authors attributed this phenomenon to the poor effectiveness of paclitaxel in OVCAR-
3 cells due to the expression of the multidrug resistance transporter MDR1. This study
points to the necessity of performing adequate control experiments with freely soluble
drug. More recently, the uptake and intracellular trafficking of larger (ca. 300–900
nm) NCs of the antiretroviral drug ritonavir has been examined in macrophages.97
Testing NCs of drugs with limited cytotoxicity allowed the authors to evaluate uptake
at higher concentrations (100 µM). The tested NCs were stabilized with a mixture of
poloxamer 188, 1,2-distearoyl-phosphatidyl ethanolamine-methyl-poly(ethylene
glycol) (2 kDa), and 1,2-dioleoyl-3-trimethylammonium propane. The authors
demonstrated by electron microscopy that these NCs loaded into macrophages and
remained mostly intact (68%) 24 h postuptake (Figure 2A). In addition, the NCs
appeared to aggregate with time within the cells and possessed rougher edges.
Sustained release of drug from the macrophages, which serve as drug NC reservoirs
in this example, was observed for a variety of antiretroviral drug NC loaded
macrophages for a prolonged period of time extending over a period of two weeks
and longer.98 In an extension of this work, the uptake of 21 different NCs (ca. 200–
400 nm) of four antiretroviral drugs has been evaluated in macrophages under
comparable conditions (100 µM).99 The authors observed that drug type, surfactant
coating, and NC shape had substantive effects on NC uptake, release, and
antiretroviral response. NCs with rounded and irregular edges showed diminished cell
uptake, while rod-like NCs with smooth and regular edges were taken up more
rapidly, and the loaded macrophages slowly released the drug in a period of days.
30
Figure 3. Drug solution-like behavior of NCs in vitro. (A) Viability of both K562
and U937 cells after 48–72 h incubation with melarsoprol either as a free drug
solution, as 300–600 nm poloxamer-stabilized NCs, or as a drug–hydroxypropyl--
cyclodextrin (HPCD) complex (*: p < 0.01 versus free melarsoprol). Redrawn from
Ben Zirar et al.,94 with permission from Elsevier (B) Cell viability of OCVAR-3 cells
decreases as a function of paclitaxel concentration for both free paclitaxel, and
paclitaxel NCs up to ca. 5–10 ng·mL–1 (24 h incubation time), after which it is
unaffected. This phenomenon was attributed to the expression of the multidrug
resistance transporter MDR1, rather than saturation of the medium with paclitaxel.
Little differences are observed in comparison to the free drug. Redrawn from Vergara
et al.,95 with permission from Elsevier.
31
Figure 4. Particle-like behavior and enhanced NC uptake in vitro. (A) Electron
micrographs of ritonavir NCs prior to macrophage uptake, within macrophages, and
after release from macrophages into the surrounding medium (24 h after uptake).
Adapted from Kadiu et al.,97 with permission from Future Medicine. (B) Paclitaxel
NCs targeting the folate receptor are more cytotoxic than untargeted ones in a human
folate-receptor-positive oral carcinoma cell line. The difference between the targeted
and untargeted NCs disappeared when excess free folic acid was added to compete for
the cell-surface receptor (*: p < 0.01 versus in presence of excess free folic acid).
Redrawn from Liu et al.,91 with permission from John Wiley and Sons.
Despite the apparent ability to maintain, in certain cases, the integrity of NCs in the
presence of cells over a certain period of time, to the extent of our knowledge, few
attempts have been made to modify the surface of the NCs with targeting ligands to
improve cellular uptake in vitro. Liu et al.91 have shown that ca. 150-nm paclitaxel
NCs coated with poloxamer 407 bearing 10% folic acid as targeting ligand were
significantly more cytotoxic than the comparable nontargeted NCs at short incubation
times (2 h; Figure 2B). This effect was abolished in a competition assay with free
folic acid potentially indicating that drug uptake was associated with folate-mediated
receptor-mediated endocytosis. The cytotoxicity of the untargeted NCs was not
affected by addition of folic acid. One caveat, however, is that cytotoxicity is an
indirect measurement of drug uptake that cannot distinguish the folate-mediated
uptake of free versus that of NC-associated paclitaxel. More recently, Bui et al.100
have prepared fluorescent and biotinylated squalene–gemcitabine (prodrug) NCs and
have observed increased cell uptake and improved anticancer efficiency in three
cancer cell lines.
32
Overall, several cell culture studies support that the drug NCs can remain intact for
a certain time and indeed behave like nanoparticles rather than freely dissolved drug.
The dominant factors in vitro for maintaining NC integrity are size and concentration
in the medium. Depending on the in vivo application foreseen, in vitro experiments
might sometimes benefit from being performed under more dilute conditions. This
would avoid saturating the medium with drug, which prevents NC dissolution.
Dissolution profiles in the absence of cells and under sink conditions are indeed not
always performed in the literature. Imaging of NCs within cells may also provide
more insight into how the performance of NCs can be rationally altered.
In Vivo
Quite often, NCs display pharmacokinetic profiles that are very similar to the drug
solution when administered i.v.30, 101-103 This is generally a consequence of their rapid
dissolution under in vivo sink conditions. In Mouton et al.’s101 report on an early
clinical study in humans, the authors compare 200–300 nm itraconazole NCs to an
itraconazole–hydroxypropyl--cyclodextrin complex. The NCs exhibited a higher
mean maximum plasma concentration at the end of infusion than those receiving the
cyclodextrin formulation (Figure 3). The authors speculated that this difference may
be explained by assuming that NCs were not yet dissolved, and were consequently
confined in the circulatory system, and unavailable for diffusion and distribution to
the peripheral tissues. However, after this time point, the differences between the two
formulations with regards to the other pharmacokinetic parameters was less
pronounced or was not significant at all. In addition, the authors also mentioned that
other (unpublished) data with several animal species showed that the drug NCs were
specifically trapped in Kupffer cells in the liver and in the macrophages of the spleen
and that pharmacokinetic changes were related to the size of the NCs (most
pronounced for NCs 340 nm). The similar plasma concentration profiles and high
drug concentrations early in the liver obtained for 200-nm NCs of an antitumor p-
terphenyl derivative versus the drug solution could also be explained by fast
dissolution and NC instability.102 In a previous study this NC formulation exhibited
complete dissolution within 2 h compared to less than 10% for the bulk drug.104
Sharma et al.103 have observed that the sub-150-nm NCs of the investigational
anticancer compound SN 30191, stabilized with poloxamer 407 and poly(ethylene
33
glycol)-15-hydroxystearate, were rapidly cleared from the blood of mice and
accumulated in the kidney, liver, and heart. The authors postulated that drug
accumulation in these tissues could be due to rapid dissolution of the NCs in the
blood, which facilitated distribution in highly perfused tissues. Unfortunately, as the
free soluble formulation of SN 30191 was four times less tolerated than the NCs,
comparison between the two was not possible. Sigfridsson et al.30 have compared
100–150 nm (amorphous drug) nanoparticles, 300–400 nm NCs, and the solution of
the investigational antipsychotic drug AZ68. Both nanoparticles and NCs were
stabilized with poly(vinylpyrrolidone) and a combination of small-molecule
surfactants. When administered i.v. to rats, no significant difference among the three
formulations was observed in terms of their plasma profiles.
Figure 5. Drug solution-like behavior of NCs in vivo. Multiple-dose study
comparing itraconazole NCs to an itraconazole–hydroxypropyl--cyclodextrin
(HPCD) complex administered i.v. to humans. Two hundred milligramm doses were
given every 24 h except on days 1 and 2, when the dose was given every 12 h. Note
that samples were collected just before and 1 h after each infusion for the first 5 days.
Redrawn from Mouton et al.,101 with permission from American Society for
Microbiology.
Differences between the NC and other formulations begin to manifest themselves
when the particle size is large. Ganta et al.105 have prepared 130−700 nm poloxamer
188-stabilized NCs of asulacrine and, when administered i.v. to mice, observed
preferential accumulation in the liver compared to the free drug in solution. The
authors rationalized this result to stem from uptake of the NCs by the mononuclear
34
phagocyte system, which removed them from the systemic circulation. From the
phagocytes, the drug was released over a period of a couple of hours, a timeframe that
was consistent with the in vitro dissolution profiles of the NCs in 1% polysorbate 80
solution. This is in line with the observation of enhanced liver accumulation and
retention of radioactive 450-nm nevirapine NCs in the liver compared to the drug in
solution (Figure 4A).92 Gao et al.106 have investigated the effects of particle size on
the pharmacokinetics and tissue distribution of two oridonin NCs with markedly
different size (ca. 100 and 900 nm) following i.v. injection in rabbits. In vitro,
complete dissolution occurred within 10 min and 2 h, for the smaller and larger NCs,
respectively. In vivo, the smaller NCs behaved similarly to the drug in solution,
whereas the larger NCs accumulated to a greater extent in the liver, spleen, and lungs.
Based on these findings, the authors suggested that the larger NCs were subjected to
mononuclear phagocyte system uptake. Indeed, Rabinow et al.107 have demonstrated,
seven days postinjection, that 600-nm itraconazole NCs stabilized with poloxamer
188 were taken up intact by the spleen in rats by histological analysis (Figure 4B).
Unfortunately, organ toxicity resulting from NC accumulation in the liver and spleen
was so far not assessed. Nevertheless, such toxicity would be drug dependent and
would have to be evaluated for each composition. In addition, continuous
accumulation and organ toxicity, as observed for biopersistent nanoparticles, such as
asbestos and carbon nanotubes, is unlikely since drug NCs dissolve with time and are
eliminated metabolically or by renal excretion.108, 109
35
Figure 6. Particle-like behavior of NCs in vivo. (A) Gamma scintigrams depicting
biodistribution of bare radiolabeled nevirapine 450-nm NCs and NCs surface-coated
with albumin and dextran at 1 h and 24 h in rat compared to the drug in solution.
Reproduced from Shegokar et al.,92 with permission from Elsevier. (B) Histological
analysis of rat spleen by transmission electron microscopy shows the presence of
itraconazole NCs within macrophages. Adapted from Rabinow et al.,107 with
permission from Elsevier.
In contrast to the aforementioned i.v. injection, however, 200-nm rilpivirine NCs
administered as a single intramuscular or subcutaneous injection achieve stable
sustained plasma concentration profiles detectable up to three months in dogs.36 With
respect to NC size, the 200-nm NCs displayed improved early release (higher Cmax) in
dogs, when compared with 400 or 800 nm particles. For instance, 40 × 150-nm
paclitaxel NCs stabilized with D--tocopheryl poly(ethylene glycol) 1000 succinate
(TPGS) exhibited greater antitumor efficacy than Taxol® at equivalent dose in a drug
resistant NCI/ADR-RES xenograft mouse model.34 Although it was hypothesized that
the improved activity of the NCs could be attributed to the inhibition of efflux pump
(permeability glycoprotein 1; P-gp) function by TPGS, as reported elsewhere,110 the
slow dissolution of the NCs observed in vitro (<20% in 24 h) might have contributed
to a better drug distribution to the tumor via the EPR effect. Unfortunately, in this
study the pharmacokinetics and biodistribution of the drug were not assessed.34 Zhang
et al.93 observed a greater antitumor efficacy for 240-nm uncoated NCs of
camptothecin in an MCF-7 tumor xenograft mouse model compared to the salt
solution of camptothecin in a mixture of propylene glycol and saline. They attributed
36
this to the EPR effect and the higher resistance of NCs against hydrolysis. This was
further supported by biodistribution data showing higher camptothecin deposition in
the tumor and reduced drug hydrolysis. However, the pharmacokinetic profiles were
difficult to interpret as the NCs displayed in rats a lower area under the plasma
concentration versus time curve than the drug solution and a comparable mean
residence time.111 Based on previous data,93 this may be indicative of retention of the
uncoated needle shape NCs in the lungs but could also result from their aggregation
and embolization in the lung capillaries. Hollis et al. developed 200-nm hybrid NCs
consisting of partially radiolabeled paclitaxel and the fluorescent dye FPI-749.89 Both
NCs and paclitaxel solution, injected i.v. into HT-29 tumor xenograft bearing mice,
accumulated less than 1% at the site of the tumor as determined by scintillation
counting. Additionally, repeated injections of both formulations gave no significant
difference in treatment efficacy at the defined end point.
While not a NC per se, Karmali et al.112 have modified Abraxane®, an amorphous
paclitaxel nanoformulation stabilized with albumin (130 nm),113 with tumor-targeting
peptides and observed a change in the biodistribution 3 h postadministration versus
the untargeted formulation. Indeed, at this time point, the targeted NC colocalized
with its target while untargeted form did not. Unfortunately, targeted Abraxane® only
showed a small effect in inhibiting MDA-MB-435 tumor growth in comparison to its
unmodified form. This may attest to some dissolution prior to efficient targeting of
the NC, and should be further investigated.
Stabilizer: To Shed or Not To Shed
One important limitation in the design of therapeutic drug NCs is the desire not to
chemically modify the NC itself with the stabilizing agent. As such, stabilizers cannot
be covalently anchored onto the NC surface, and non-specific interactions must thus
be exploited for adsorption. Independently of the dissolution of the NC that leads to
desorption of the stabilizer (vide infra), high dilution conditions encountered either in
vitro or in vivo will inevitably lead to loss of the stabilizing agent as well as any
appended targeting/internalizing agents. For instance, Deng et al.114 have shown
(through NC size increase) that poloxamer 407 desorbs from paclitaxel NCs upon
dilution. Another observation was that increasing the stabilizer-to-drug ratio resulted
in poorer NC stability. Supported by evidence that higher concentrations of stabilizer
37
led to the formation of micelles in addition to stabilizing NC coatings, the authors
hypothesized that stabilizers deposited as unimers (i.e., below the CMC) may have
higher affinity to NCs than stabilizers deposited as multimers (i.e., above the CMC),
whose deposition process was in competition with micellization. This result could
potentially attest to a different organization of the stabilizer on the surface of the NC.
Indeed, owing to the complexity of systematically altering the structure of
macromolecular stabilizing agents, few studies have attempted to rationally modulate
interactions between the stabilizer and the NC for preventing desorption. Our group
has recently presented a modular and systematic strategy for optimizing the affinity of
polymeric stabilizers for NCs based on the postpolymerization modification of
polymer precursors (containing -propargyl-δ-valerolactone)115 by thiol–yne click
chemistry.87 In this approach, two parent block copolymers of methoxy poly(ethylene
glycol)-b-(-propargyl-δ-valerolactone-co-ε-caprolactone) were used to create a
library of 10 different stabilizers in which the hydrophobic polyester block was
modified with alkanes of different length and structure. All stabilizers had equivalent
numbers of monomeric units and polydispersity indices to the parent stabilizers.
Under the production conditions used, all stabilizers produced dense polymer brushes
on the surface of the NC, and size-stability assays were found to strongly depend on
the structure of the hydrophobic block.
In addition to altering the chemical structure of the stabilizer to promote
interactions with the NCs, another approach is to cross-link the stabilizer around the
NC and thus reduce shedding via physical entrapment. For instance, Kim and Lee116
have electrostatically cross-linked chitosan on the surface of paclitaxel with
tripolyphosphate, but have not evaluated the size stability of the NCs, nor the decrease
of shedding achieved after cross-linking. Other electrostatically cross-linked
stabilizers produced via the LbL deposition of polyelectrolytes are discussed in the
following section. Our group has more recently designed block copolymer stabilizers
that could be cross-linked directly on the surface of paclitaxel NCs by copper-
catalyzed 1,3-dipolar cycloaddition to form nanocage–NC constructs.39 Size-stability
analysis showed that nanocages acted as sterically stabilizing barriers to NC–NC
interactions and aggregation, which in turn imparted better size-stability to the NCs in
comparison to the non-cross-linked coating. By dosing the amount of polymer
released from nanocage–NC constructs, it was shown that the nanocages were 3–4-
38
fold less shed from the NCs than comparable non-cross-linked stabilizers. In addition,
transmission electron microscopy of the nanocages after complete dissolution of the
drug NC revealed the intactness of the nanocage, demonstrating a successful cross-
linking reaction (Figure 5A).
It should be noted, however, that shedding of the stabilizer may in fact be beneficial
under certain circumstances. For instance, Liu et al.34 have sought to exploit the
shedding phenomenon by stabilizing 40 × 150 nm paclitaxel NC rods with TPGS. The
rationale of this study was that the tocopheryl-functionalized stabilizer may inhibit P-
gp upon shedding, which may permit an enhanced treatment of multi-drug resistant
cells. Indeed, the authors observed that in NCI/ADR-RES cells, which overexpress P-
gp and are resistant to paclitaxel, NCs stabilized with TPGS exhibited a significantly
enhanced antiproliferative effect than free paclitaxel or paclitaxel NCs stabilized with
poloxamer 407 (Figure 5B). The authors also observed that as the amount of TPGS
increased compared to drug, the antiproliferative effect increased for both TPGS-
stabilized NCs and the physical mixture, indicating that TPGS modulated drug
resistance transporters. Interestingly, however, at low TPGS concentrations, TPGS-
stabilized NCs were more cytotoxic than the mixture, whereas at high surfactant
concentrations they were comparable. These observations indicate that additional
mechanistic investigations are warranted. In another example, our group has created
amphiphilic block copolymer stabilizers that are spontaneously shed in response to a
stimulus.87 More specifically, the hydrophobic block of the stabilizer, responsible for
physisorption on the investigated paclitaxel NCs, contained thioether groups that
became substantially more hydrophilic in the presence of reactive oxygen species,
thus driving the stabilizer from the NC and provoking its destabilization. Stabilizer
shedding in areas of oxidative stress in the body, which are associated with a variety
of diseases,117, 118 may provide a means of provoking selective aggregation or promote
uptake at these target locations, but should be tested in vivo.
39
Figure 7. Controlling or exploiting stabilizer shedding. (A) Preventing NC
stabilizers from shedding by cross-linking. Transmission electron microscopy images
of paclitaxel NCs before (I) and after cross-linking (II). Following dissolution of the
NCs from (I) and (II), an aggregate structure was observed for the non-cross-linked
NCs (III), whereas intact polymeric coatings were observed for the correspondingly
cross-linked NCs, in the form of discrete spheroids (IV). Reproduced from Fuhrmann
et al.,39 with permission from American Chemical Society. (B) Stabilizer shedding
promotes activity of NCs in vitro. Effects of paclitaxel/TPGS NCs (10 µM) with
different amounts of TPGS, in comparison to a physical mixture of paclitaxel and
TPGS demonstrating that the “shed” stabilizer potentiates the activity of paclitaxel.
Redrawn from Liu et al.,34 with permission from American Chemical Society.
Altering Dissolution Profiles (by Means Other than Size)
It is clear from the examples above that size plays a key role in the dissolution
characteristics of drug NCs, which alters their performance. Based on in vivo
evidence, NCs with sizes above ca. 300–400 nm (depending on the specific drug in
question) persist for a sufficiently long time that they could, in principle, accumulate
passively within tumors via the EPR effect. However, particles of this size may be
subject to greater uptake by the mononuclear phagocyte system and would poorly
diffuse in the extracellular tumoral matrix. Smaller NCs may have greater abilities to
penetrate tumors, but their targeting is more challenging because dissolution must be
delayed. Several approaches have been examined for this purpose.
40
Drug NCs can be reprocessed following miniaturization to alter dissolution kinetics.
For instance, paclitaxel NCs could be renanozised by an incubation–sonication
technique.114 In this technique, the authors incubated NCs at 37 ºC for a certain period
during which time NC size increased via ripening processes. This was then followed
by sonication to break the growing NCs into smaller ones. The authors observed that
the renanosized NCs displayed significantly greater size stability, which they
attributed to the disruption of the preferred growth pattern of the NCs. These
interesting findings should be pursued with analysis of dissolution kinetics under sink
conditions and a more in-depth characterization of this phenomenon. Lu et al. also
produced very stable 300-nm NCs of paclitaxel by adsorption of transferrin. The NCs
did not exhibit a size change during the 3 months study period. The increased stability
compared to the bare NCs was also reflected in a slightly slower drug release during
dissolution experiments.119
One of the most investigated systematic approaches for altering microparticle120, 121
and, more recently, NC dissolution kinetics via stabilizing coatings produced by LbL
assembly of polyeletrolytes. In this technique, the hydrophobic drug NC is first
covered by an anchoring layer typically composed of a small molecule amphiphile
and a polymer, and is followed by the sequential deposition of multiple layers of
charged polyelectrolytes. Model experiments have shown these coatings are
semipermeable (i.e., permeable to small molecules smaller than specific cut-offs),122
which points to the importance of well characterizing the coating. Despite this
semipermeability, an effect on dissolution rate of NCs, albeit a small one, has been
observed. For instance, the rate of drug release from 300-nm paclitaxel NCs was
independent of the thickness of the stabilizing coating, when this coating was thinner
than 3.5 bilayers of poly-L-lysine and sodium heparin (Figure 6).123 However, when
the number of bilayers increased from 4 to 12, a slight decrease in the drug release
rate was observed. Agarwal et al.40 have observed a marginal difference in the rate of
dissolution of 125-nm tamoxifen NCs stabilized with either 0.5 or 3 bilayers of
poly(dimethyldiallylamide ammonium chloride) /poly(styrenesulfonate). One
particularly interesting feature of the LbL approach is the large parameter space
available for constructing these stabilizing coatings including: polyelectrotyte
type/architecture/molecular weight, addition of salts and other additives, anchoring
layer chemistry. Future work should focus on assessing how these parameters inflence
41
the semipermeability of LbL-assembled coatings, which may provide the means to
rationally alter NC dissolution.
Figure 8. Altered NC dissolution via the stabilizing coating. Paclitaxel release from
300 nm NCs coated with (poly-L-lysine/heparin)n shells. Redrawn from Shutava et
al.,123 with permission from Royal Society of Chemistry.
Outlook
The studies presented above suggest that drug NCs may in the future play an
important role in targeting significant amounts of drug to sites of disease. NC size can
for all intents and purposes be selected using (universal) preparation approaches – NC
persistence in vitro/in vivo strongly correlates with size – and cellular uptake and
tumor accumulation can be promoted by modifying the surface chemistry of the
coating. While these aspects have been individually demonstrated in the examples
discussed herein, future research should question how to overcome the challenges
associated with assembling these individual properties into efficient and optimized
therapeutics. For instance, as small (sub-100 nm) nanocarriers are known to penetrate
even poorly permeable tumors,124, 125 could such an effect also be achieved in practice
with NCs? Can the rate of drug release of these smaller, dissolution prone NCs be
controlled by their stabilizing coating? Can stimuli-responsive stabilizers provide a
means for selective NC destabilization and accumulation at sites of disease? In
addition, other forms of targeting strategies in vivo could be foreseen. For instance, it
has been recently reported that composite nanoparticles of a gemcitabine prodrug and
magnetite yielded enhanced tumor accumulation and therapeutic activity via magnet-
assisted targeting.126 Finally, other opportunities for enhancing the targeting potential
42
of NCs should be explored and which are not part of the NC construct itself. Sugahara
et al.127 have indeed recently demonstrated that tumor-penetrating peptides,
coadministered with Abraxane®, increased vascular and tissue permeability leading to
a 12-fold increase of the tumor accumulation versus in the absence of peptide. This
area is ripe for discovery and answering these questions will require creative new
hypotheses to be tested.
43
III. Results and Discussion
44
45
III.1. PEG Nanocages as Non-Sheddable Stabilizers for Drug
Nanocrystals2
Kathrin Fuhrmann, Jessica D. Schulz, Marc A. Gauthier, and
Jean-Christophe Leroux
Introduction
Many modern and potent anticancer drugs are poorly water soluble, which can
create formulation problems when the drug is intended to be injected by the
intravenous (i.v.) route.128 Drug solubility may be altered on a number of levels
including modification of the drug’s structure (e.g., change of crystalline form)1 and
by formulation approaches.129 For example, paclitaxel (PTX), a potent antitumor
agent, is typically formulated in a vehicle composed of polyethoxylated castor oil and
dehydrated ethanol (Cremophor EL; Taxol).130 However, owing to the required dose
of Cremophor EL necessary to deliver a sufficient amount of PTX, a number of
undesirable side effects have been observed.5 In addition to the desire of eliminating
large amounts of excipient, modern formulation strategies are also designed to
increase the drug’s therapeutic index by exploiting passive131, 132 and more recently
active targeting. For instance, PTX has been formulated as a water-soluble prodrug,133
as a polymer nanoconjugate,134 conjugated to the natural lipid squalene,135 in
liposomes,136 with carbon nanotubes,137 as cyclodextrin complexes,138 and in
micelles.139 Formulations that have been successfully applied or tested in the clinic
are Abraxane, an albumin-bound nanoparticle system,113 and Opaxio, a poly(L-
glutamic acid)-based PTX conjugate (clinical phase III underway).140 Nanosized
systems have the potential to accumulate at cancer sites through the enhanced
permeation and retention (EPR) effect, and can possess targeting agents to promote
active uptake by target cells or deposition at specific tumoral sites.
Current limitations include insufficient solubilization capacity,141 potential
excipient-related toxicity,5, 142 instability upon dilution in the bloodstream,143, 144
manufacturing difficulties, and drug stability issues during processing.145 One
2 Published in ACS Nano 2012, 6, 1667-1676. J.D. Schulz synthesized a starting compound.
46
validated and robust approach for addressing these issues is to process the drug
powder into colloidal dispersions known as “nanocrystals” (NCs).25, 77, 80, 81 Wet
milling can produce uniformly sized drug NCs with mean diameters of less than 200
nm, and little batch-to-batch variability.82 This top-down process is suitable for many
different classes of compounds and there currently exist several oral formulations
produced by wet milling on the market.83 One key feature of NCs is the minimal use
of excipients compared to other formulation approaches, which implies both high
drug content and diminished excipient-related toxicity. For the i.v. route, NCs are
promising drug carriers because their very high loading can lead to higher deposition
of drug in cancer cells upon delivery.91 Indeed, there is increasing evidence that drug
NCs can accumulate in tumors via the EPR effect.34
NCs are generally stabilized with surfactants (or “stabilizers”), whose functional
role is to prevent aggregation between the high-energy crystal surfaces produced
during the size-reduction process. Consequently, much research is now directed
towards optimizing the coating.72 For example, Agarwal et al. have produced stable
NCs of hydrophobic drugs by sonication and layer-by-layer assembly of oppositely-
charged polyelectrolytes leading to electrostatic repulsion between NCs.40 Similarly,
Abraxane is an injectable NC formulation of PTX produced by high-pressure
homogenization in the presence of human serum albumin, which acts as a steric
stabilizer for the NCs.146 In addition, the stabilizer provides a means of anchoring
targeting moieties to the NCs. For example, Liu et al. have modified poloxamer 407
with folic acid and have shown enhanced cellular uptake of targeted PTX NCs, in
comparison to untargeted ones.91 A recent study by Sugahara et al. has investigated
the efficacy of PTX NCs when coadministered with a tumor-penetrating peptide and
of Abraxane decorated with the latter.127 The authors have shown that these
approaches could slightly enhance the antitumoral effect.112 However, a critical
challenge in achieving active targeting is the possibility that the targeting agents are
shed along with the stabilizer upon high dilution. For instance, Deng et al. have
indirectly, shown that poloxamer 407 can desorb from PTX NCs upon mild heating or
dilution.114 The authors also found that the conditions under which the NCs are
prepared can lead to different affinities between the polymer and the drug.
The objective of this study was to prepare non- or poorly-sheddable biodegradable
stabilizers for NCs that are maintained in place independently of specific stabilizer–
drug interactions. It was hypothesized that this could be accomplished by chemical
47
cross-linking of the stabilizer around the NC, thereby maintaining the drug in place by
a combination of physical entrapment and physisorption. Conceptually, the cross-
linked polymer coating forms a polymeric “nanocage” in which the NC is trapped.
Earlier work on nanocages has been reported by Turner et al.147 These were prepared
in a multistep process involving shell cross-linking of micellar structures, core
digestion and functionalization with lipids to improve subsequent drug loading. In
contrast to their method, the specific challenge addressed herein lies in performing
cross-linking reactions near the surface of PTX NCs, due to the presence of high-
reactive crystal surfaces and to reactions taking place in heterogeneous phase. As
illustrated in Figure 9, herein is reported the preparation of PTX NCs by wet milling
with amphiphilic biodegradable and cross-linkable copolymers, and conditions for
successfully cross-linking them to form a nanocage–NC construct. Evidence for the
lower sheddability of the nanocage in comparison to non-cross-linked stabilizers is
given. This approach is advantageous because it can easily be adapted to the grafting
of targeting ligands. In addition, as the stabilizing coating is maintained in place
more-or-less independently of specific drug–polymer interactions, it should be
amenable to other hydrophobic drugs.
Figure 9. Schematic representation of diblock-copolymer self-assembly on the
surface of drug NCs and nanocage formation following cross-linking.
Experimental Section
Materials
PTX was obtained from Bioxel Pharma Inc. (Sainte-Foy, QC, Canada) and
docetaxel from ScinoPharm Taiwan, Ltd. (Tainan County, Taiwan). Sepharose CL-
4B, Sephadex G10, lithium diisopropylamide (LDA), -valerolactone, propargyl
bromide, hexamethylphosphoramide (HMPA), -caprolactone, 2,6-bis(4-
48
azidobenzylidene)-4-methylcyclohexanone (97%, containing 35 – 45% water),
sodium azide, copper(II) sulfate, albumin from bovine serum (BSA; ≥ 96%), and
methoxy poly(ethylene glycol) (mPEG; 2000 g/mol) were purchased from Sigma-
Aldrich (Buchs, Switzerland) and used as received. Hydrogen chloride (1 N solution
in diethyl ether), m-chloroperoxybenzoic acid (mCPBA, 70 – 75%), and 1-methyl-2-
pyrrolidone were purchased from Chemie Brunschwig AG (Basel, Switzerland). 1,11-
Diazido-3,6,9-trioxaundecane was obtained from Santa Cruz Biotechnology (Santa
Cruz, CA), sodium L-(+)-ascorbate from Axon Lab AG (Baden-Dättwil, Switzerland),
and deuterium oxide from ReseaChem GmbH (Burgdorf, Switzerland). Ultra pure
water was prepared by a Barnstead Nanopure system (Thermo Fisher Scientific,
Reinach, Switzerland). Dry solvents were taken from a solvent purification system
(LC Technology Solutions Inc., Seabrook, NH). -valerolactone and -caprolactone
were distilled over calcium hydride under inert atmosphere before use. All other
solvents were of highest purity and bought from Sigma-Aldrich (Buchs, Switzerland).
Synthesis of -propargyl-δ-valerolactone (1)
The synthesis of 1 was adapted from Parrish et al.115 LDA (6.49 mL, 11.69 mmol,
1.1 eq.) was dissolved in THF and cooled to –78 °C. A solution of δ-valerolactone
(0.96 mL, 10.63 mmol, 1 eq.) in THF was added dropwise over 1 h and then stirred
for another 30 min. Propargyl bromide (1.14 mL, 12.75 mmol, 1.2 eq.) and HMPA
(2.22 mL, 12.75 mmol, 1.2 eq.) were added dropwise over 10 min. The brown
reaction mixture was warmed to approximately –42 °C and the temperature was
maintained while stirring for 3 h. After this period, 2.5 mL of a saturated solution of
ammonium chloride was added and the reaction was allowed to warm to room
temperature. Volatiles were removed by rotary evaporation and the residual yellow oil
was dissolved in diethyl ether (40 mL), which was washed three times with brine (40
mL). The organic phase was dried over MgSO4 and concentrated by rotary
evaporation. After column chromatography (30% ethyl acetate in hexane, Rf 0.35) and
distillation under reduced pressure (160 °C, 10 mbar) a colorless viscous liquid was
obtained (0.675 g, 4.9 mmol, 46% yield). The density of 1 was measured by weighing
pipetted volumes (n = 10) and determined to be 1.10 g/mL. 1H NMR (400 MHz,
CDCl3, δ) 4.37 – 4.27 (m, 2H, CH2O), 2.74 – 2.61 (m, 2H, COCHCH2C≡C), 2.53 –
2.46 (m, 1H, COCHCH2), 2.33 – 2.24 (m, 1H, CHCH2CH2), 2.01 (t, J = 2.6 Hz, 1H,
49
C≡CH), 1.98 – 1.87 (m, 2H, OCH2CH2CH2), 1.77 – 1.67 (m, 1H, COCHCH2CH2)
(for an annotated spectrum see Supplementary Figure S 1).
Synthesis of -azido--caprolactone (2)
The first step of the synthesis of 2 was adapted from Lenoir et al.148 2-
chlorocyclohexanone (1.98 g, 15 mmol, 1 eq.) was dissolved in 20 mL
dichloromethane to which mCPBA (4.1 g, 16.5 mmol, 1.1 eq.) was added. The
reaction was stirred for 96 h at room temperature then stopped by cooling to –20 °C
for 1 h. The precipitated mCPBA was removed by filtration to yield a pale viscous
liquid, which was purified by column chromatography (35% ethyl acetate in hexane,
Rf 0.32) to yield -chloro--caprolactone (1.033 g, 7 mmol, 45% yield). 1H NMR
(400 MHz, CDCl3, δ) 4.80 (dd, J = 7.9, 2.7 Hz, 1H, COCHCl ), 4.63 (ddd, J = 12.8,
7.8, 1.8 Hz, 1H CH2CH2O), 4.23 (ddd, J = 12.7, 7.4, 1.8 Hz, 1H, CH2CH2O), 2.23 –
1.77 (m, 6H, ClCHCH2CH2CH2) (1H NMR spectrum in Supplementary Figure S 2).
In a second step, -chloro--caprolactone (500 mg, 3.4 mmol, 1 eq.) was added to a
solution of sodium azide (1.1 g, 16.9 mmol, 5 eq.) in DMSO (30 mL) and stirred at 50
°C for 48 h. The turbid yellow mixture was diluted with water (150 mL) and extracted
three times against 50% ethyl acetate in hexane (40 mL). The organic phases were
combined, dried over sodium sulfate, and concentrated by rotary evaporation under
reduced pressure to yield 2 as a viscous pale liquid (0.299 g, 1.9 mmol, 60% yield). 1H NMR (400 MHz, CDCl3, δ) 4.45 – 4.40 (m, 1H, COCHN3), 4.18 – 4.12 (m, 2H,
CH2CH2O), 2.09 – 1.67 (m, 6H, N3CHCH2CH2CH2) (Supplementary Figure S 3). The
density of 2 (1.154 g/mL) was measured by weighing pipetted volumes (n = 6).
General polymerization procedure and synthesis of mPEG-b-(CL-co-1) (3)
The polymerization procedure was adapted from Kim et al.149 In a typical
experiment, mPEG (800 mg, 0.4 mmol, 1 eq.) was dried by azeotropic distillation
with dry toluene (50 mL) under a flow of nitrogen. The flask was sealed and the
mPEG was dissolved in dry dichloromethane (2 mL). Dry -caprolactone (132.8 μL,
1.2 mmol, 3 eq.) and 1 (150.5 μL, 1.2 mmol, 3 eq.) were then added using gastight
syringes. The polymerization was initiated by the addition of HCl in ether (1.2 mL,
1.2 mmol, 3 eq.) and stirred at room temperature for 24 h. The reaction mixture was
then precipitated twice in cold ether, the solvent evaporated in vacuo, the residue
50
taken up in water, and the polymer recovered by lyophilization (0.867 g, 80% yield).
The fully annotated NMR spectrum of 3 can be found in Supplementary Figure S 4.
This polymer contained on average two units of 1 per polymer chain. SEC (DMF): Mn
= 2,200 g/mol, Mw/Mn = 1.11.
Synthesis of mPEG-b-(CL-co-1) (4)
According to the general procedure described above, mPEG (1.50 g, 0.75 mmol, 1
eq.) was dried by azeotropic distillation with toluene and dissolved in dry
dichloromethane. Then, dry -caprolactone (166 µL, 1.5 mmol, 2 eq.) and 1 (376.4
µL, 3 mmol, 4 eq.) were introduced and the polymerization was started by the
addition of HCl in ether (1.5 mL, 1.5 mmol, 2 eq.). After 24 h, the polymer was
precipitated with diethyl ether, affording 4 as a white solid. The fully annotated NMR
spectrum can be found in Supplementary Figure S 5. This polymer contains on
average four units of 1 per polymer chain. SEC (DMF): Mn = 2,700 g/mol, Mw/Mn =
1.11.
Synthesis mPEG-b-(CL-co-2) (5)
According to the general procedure described above, mPEG (500 mg, 0.25 mmol, 1
eq.) was dried and dissolved in dichloromethane. Dry -caprolactone (83 µL, 0.75
mmol, 3 eq.) and 2 (134.4 µL, 1 mmol, 4 eq.) were introduced and the polymerization
started by the addition of HCl in ether (0.75 mL, 0.75 mmol, 3 eq.). After 24 h, the
polymer was precipitated twice in cold n-hexane to yield 5 as a white solid. The fully
annotated NMR spectrum can be found in Supplementary Figure S 6. This polymer
contains on average four units of 2 per polymer chain. SEC (DMF): Mn = 2,000
g/mol, Mw/Mn = 1.18.
NC preparation
PTX NCs were produced by wet milling. In a typical experiment, 2 mL of a 5%
(w/w) polymer solution in ultrapure water was filtered (0.2 μm pore size) into a 20
mL cylindrical glass vessel containing 10 mg of PTX and 4 mL of zirconium oxide
beads (~ 14.7 g, 0.3 mm in diameter, Union Process, Akron, OH). The vessel was
sealed with a plastic cap and then placed horizontally on a Ratek BTR5 blood tube
51
roller (with custom modified motor, Labortechnik Fröbel GmbH, Lindau, Germany)
and rolled at 220 rpm for 48 h in a cold room (6 °C). After milling, the beads were
separated from the suspension by filtration through polyamide sieve fabric (30 μm
pores, VWR, Dietikon, Switzerland), and the residue washed four times with 2 mL of
ultra pure water. The suspension was centrifuged at 12000 × g for 6 min to remove
larger aggregates. The supernatant was then subjected to SEC with Sepharose CL-4B
to remove excess polymer, typically yielding a suspension containing 0.55 mg/mL or
0.74 mg/mL PTX for polymers 3 or 4, respectively.
Cross-linking in solution
In a proof of principle experiment, a 1.8 mM solution of 3 was reacted with 7 at two
different ratios (1/1 and 1/10 azide/alkyne) in a solution containing 1.8 mM copper
sulfate and 9 mM sodium ascorbate for 20 min. The reaction mixture was then frozen
in liquid nitrogen, lyophilized and dissolved in CDCl3 for 1H NMR analysis and
subsequent FTIR analysis.
Cross-linking on NCs
For cross-linking in the presence of NCs, the procedure above was slightly
modified. As a general example, 7 (0.159 mg, 0.65 μmol, 2.5/1 molar equivalent
azide/alkyne) in ethanol was transferred to a round bottom flask and the solvent
removed under a stream of nitrogen. Then, 3 mL of NC suspension (~ 0.55 mg/mL
PTX, ~ 0.262 μmol alkyne) was added followed by 10 μL of a solution containing
100 mM sodium ascorbate and 20 mM copper sulfate, final concentrations 333 µM
and 67 µM, respectively. Owing to the different solubility characteristics of 5 and 6,
they were dissolved in water and NMP, respectively, and added after the catalyst. The
flask was placed on a rotary shaker (~ 100 rpm) at room temperature for 24 h. After
this period, the cross-linked NCs were separated from catalyst and excess cross-linker
by SEC using Sephadex G10. The fraction with the highest concentration (~ 0.5
mg/mL PTX) was used for measuring particle size with time by DLS at both 6 ºC and
20 ºC. For 1H NMR and FTIR analyses, the fractions were pooled, frozen in liquid
nitrogen and lyophilized to concentrate the NCs. Then, deuterated methanol was
added to the lyophilized powder, which was then sonicated at 45 ºC to form a
homogenous suspension that was then analyzed. A summary of all cross-linking
conditions examined herein is given in Table 2.
52
Imaging of NCs and the polymer coating after PTX removal
For transmission electron microscopy (TEM), samples (4 µL) of the NC
suspension, prepared at a concentration of ~ 0.5 mg/mL PTX, were adsorbed to glow
discharged carbon-coated copper grids for 1 min. After two washings with water to
remove excess NC, they were negatively stained with 2% (w/v) uranyl acetate for 1
min and air-dried after blotting with filter paper. The specimens were examined in a
Philips CM12 (tungsten cathode) transmission electron microscope (FEI, Hillsboro,
OR) at 100 kV, and images were recorded with a Gatan CCD 794 camera (Gatan Inc.,
Pleasanton, CA).
To dissolve the drug within nanocages, the NCs (3 mL in water, ~ 0.5 mg/mL) were
added to a 5 mL Spectra/Por Float-A-Lyzer G2 (MWCO 100 kDa, Sigma-Aldrich,
Buchs, Switzerland), and, after addition of 870 µL ethanol, were dialyzed against 2.5
L 25% ethanol/water mixture. The drug concentration inside the dialysis bag was
measured before medium change every day by HPLC. After complete removal of
drug, the contents inside the dialysis bag were concentrated with Spectra/Gel
Absorbent (VWR, International AG, Dietikon, Switzerland) to about 0.5 mL (~ 0.4
mg/mL of polymer) and analyzed by TEM. The presence of mPEG was tested by
adding CoSCN to the concentrate.150 Samples were prepared according to the protocol
for NC suspension, but without the extra washing steps.
Particle volume and polymer coverage calculations
From TEM images, the average dimensions of the NCs were measured (n = 138).
Using equation 3, the number of polymer chains (nPolymer) per NC could be calculated
nPolymer NA V (wtPolymer /wtPTX ) / Mw Polymer (3)
where NA is Avogadro’s constant, V is the average volume of a NC (assumed to be
a cylinder), is the density (1.4035 g/mL for PTX dihydrate), (wtPolymer/wtPTX) is the
weight ratio of drug to polymer after purification determined by 1H NMR
spectroscopy, and Mw Polymer is the weight average molecular weight of the polymer.
Dissolution test
Release of PTX from NCs was tested under sink conditions in 5% BSA solution,
which is expected to be the main solubilizing component for PTX in vivo.151 In a
53
preliminary experiment, the saturation solubility of PTX powder in the test conditions
was found to be 0.01 mg/mL. A 1 mL Spectra/Por Float-A-Lyzer G2 (MWCO 100
kDa, Sigma-Aldrich, Buchs, Switzerland) was filled by mixing 20 mM phosphate
buffer (pH 7.4) containing 10% BSA with NCs in water to yield a final volume of 1
mL containing 0.1 mg/mL PTX in 10 mM phosphate buffer (pH 7.4) and 5% BSA.
The dialysis device was placed in a 50 mL centrifugation tube containing 45 mL of 10
mM phosphate buffer (pH 7.4) with 5% BSA, on a rotary shaker ( 400 rpm) in an
incubator at 37 °C. Under these conditions, the maximum PTX concentration was 5
times below its saturation solubility. At selected time points, 30 μL aliquots were
taken from inside the dialysis device and prepared for HPLC analysis by addition of
60 L internal standard and 100 µL methanol. The samples were stored at –20 ºC
overnight to precipitate albumin. The precipitate was removed by centrifugation and
the supernatant was filtered through polyamide (0.2 µm pore size) and analyzed.
Polymer shedding test
Dispersions of NCs with either non-cross-linked or cross-linked coatings (4 cross-
linked with 7) were diluted to a PTX concentration of 0.4 mg/mL in water. The
dispersions were then repeatedly centrifuged at 20,000 × g for 45 min at 14 °C until
the supernatant contained a stable concentration of PTX (measured by UV-Vis
spectroscopy at 230 nm). This procedure lead to removal of ~95% of the initially
present PTX. The supernatant was lyophilized and residual mPEG content was
analyzed by 1H NMR spectroscopy in D2O using DMSO as internal standard for
quantitative analysis.
Statistical analysis
Differences in groups were calculated by one-way ANOVA (normal distribution
was assumed) followed by a Tukey post hoc test. A value of p < 0.05 was considered
significant.
Equipment 1H NMR spectra were recorded on a Bruker Av400 spectrometer (Bruker BioSpin,
Fällanden, Switzerland) operating at 400 MHz for protons. Analytical size-exclusion
chromatography (SEC) measurements were performed in 0.01 M LiBr in DMF using
54
a Viscotek TDAmax system (Viscotek, Houston, TX) equipped with a differential
refractive index detector. Molecular weights are given relative to narrow PEG
standards (PSS Polymer Standards Service GmbH, Mainz, Germany). Separation was
achieved using two Viscotek columns (CLM 3047) in series at a flow rate of 1.0
mL/min at 45 °C. Particle hydrodynamic diameter was determined by dynamic light
scattering (DLS) using a DelsaNano C Particle Analyzer (Beckman Coulter, Brea,
CA). The cumulants result calculated by the software was used to report the
hydrodynamic diameter of the NCs. PTX concentration was determined by HPLC
analysis, using an autosampler and pump system (CTC, Thermo Fisher) equipped
with a reversed phase column (Hypersil Gold column, 1 × 100 mm, Thermo Fisher)
heated to 30 °C (HotDog column oven) and an Accela PDA detector (Thermo Fisher
Scientific, Reinach, Switzerland). Gradient elution was performed starting with a mix
of 55% methanol in water (both containing 0.1% formic acid) rising to 85% methanol
within 15 min. PTX was detected at 227 nm. A solution of docetaxel in methanol was
added as internal standard. FTIR spectra were obtained using ATR geometry on a
Spectrum 65 infrared spectrophotometer (Perkin Elmer, Schwerzenbach,
Switzerland). After centrifugation of NC dispersions (180 min, at 19,000 × g), the
copper concentration in the supernatant was measured by ICP-OES relative to
aqueous CuSO4 standards (ULTIMA 2 ICP-OES, HORIBA Jobin Yvon Gmbh,
Unterhaching, Germany).
Results and Discussion
Polymer design and synthesis
A number of differing polymeric agents have been examined as steric stabilizers for
drug NCs. The currently used systems are maintained on the surface of NCs through
physisorption,25, 114 an equilibrium process which can lead to the reversible desorption
of the stabilizer upon dilution or heating.114 To examine the hypothesis that a non-
sheddable stabilizing nanocage can be made, a cross-linkable amphiphilic polymer
was prepared. The structure of the stabilizers and cross-linking agents examined in
this study are illustrated in Scheme 1. The stabilizing copolymer was designed to
contain mPEG as hydrophilic polymer segment to act as a steric stabilizing agent for
the NCs and thereby prevent NC agglomeration by masking their high-energy
surfaces. In addition, this polymer should decrease opsonization and convey “stealth-
like” properties to the NCs.37, 128, 152 Also, a variety of heterotelechelic variants of this
55
polymer are commercially available, which opens the opportunity for future
functionalization of the hydrophilic corona of stabilized NCs. Poly(epsilon
caprolactone) was selected in favor of other polyesters because of its hydrophobicity
and slow degradability. 153 It was rationalized that these properties would minimize
polymer degradation during the milling process and favor the hydrophobic
interactions with the drug NC. In addition, there are medical devices fabricated with
poly(epsilon caprolactone) which have FDA approval (suture materials like Coated
Monoderm or Monocryl (poliglecaprone 25)). The hydrophobic block was prepared
by statistical cationic ring-opening copolymerization (ROP) of ε-caprolactone with
functional lactone monomers.149 A short hydrophobic segment was selected (ca. 5 – 7
units) so that the overall strength of the interaction between the polymer and the drug
was moderate to low. As a consequence, findings obtained using this polymer may be
relatively independent of specifically strong (or weak) polymer–drug interactions.
Several recent reports have investigated other ROP approaches for preparing
functional polyesters.115, 154 Cationic ROP is advantageous for producing polymers for
pharmaceutical applications owing to the absence of commonly used tin containing
catalysts.115 ε-Caprolactone was copolymerized with variable ratios of 1 to produce 3
and 4. These polymers had monomodal and narrow molecular weight distributions
(Figure 10) and contained on average two and four alkynyl groups per polymer chain
for 3 and 4, respectively, as determined by 1H NMR spectroscopy (Supplementary
Figures S 4 and S 5). An azido analog of 3 and 4 was prepared in the same fashion as
above by replacing 1 with 2 in the polymerization reaction. The resulting polymer, 5,
also had a narrow and monomodal molecular weight distribution and contained on
average four azido units per polymer (Figure 10 and Supplementary Figure S 6).
Precipitation with ether or hexane was sufficient for removing unreacted monomer
and hydrophobic oligomers not connected to mPEG (i.e., initiated by trace water in
the reaction vessel) as evidenced by analytical SEC (Figure 10).
56
Scheme 1. (a) Synthesis of polymers 3, 4, and 5 from functional monomers 1 or 2
and-caprolactone (CL) from an mPEG macroinitiator; (b) structure of small
molecule cross-linking agents 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone
(6) and 1,11-diazido-3,6,9-trioxaundecane (7).
Figure 10. SEC traces of 3 (Mn = 2191 g/mol, Mw/Mn = 1.112), 4 (Mn = 2723 g/mol,
Mw/Mn = 1.112), and 5 (Mn = 2020 g/mol, Mw/Mn = 1.182) in DMF.
Preparation of cross-linkable NCs
PTX was milled in the presence of 3 and the size reduction monitored with time
(Figure 11). A stable and reproducible particle size of about 200 nm was achievable
by milling for 48 h, which was the smallest size achievable with the current
experimental setup. This time was substantially shorter than the seven days reported
for poloxamer 407,155 with similar milling equipment. The size and shape of particles
obtained was consistent with values reported for paclitaxel and other drug NCs
57
produced by wet milling.85, 155, 156 The characteristic needle-like structure of paclitaxel
NCs was always observed using this process (Figures 12A and 12B). Owing to their
similarity, comparable particle sizes were obtained using 3 and 4. Briefly centrifuging
the crude suspension of NCs to remove larger aggregates had only a small effect on
mean particle size and polydispersity (Figure 13) confirming a narrow distribution of
particle sizes. The suspensions were purified from excess polymer by SEC, yielding a
suspension with a typical concentration of 0.55 or 0.74 mg/mL PTX, when using 3 or
4, respectively. The recovered yields depended on the initial particle size after milling
and the centrifugation step, which removed less drug in case of the smaller particles.
Owing to the biodegradable nature of the hydrophobic block, the stability of the
polymer during the milling process was assessed by analytical SEC. In a drug-free
run, a slight decrease of polymer molecular weight was observed after 96 h of milling,
which is twice as long as the time used for NC preparation (Figure 14).
Figure 11. Optimization of wet milling of PTX with 3. A milling duration between
42−54 h was found to be ideal for size-reduction and narrow particle size distribution.
58
Figure 12. TEM images of NCs stabilized with 3 before (A) and after cross-linking
(B) with 7 (2.5/1 azide/alkyne). Following dissolution of the NCs from A and B in
25% ethanol, an aggregate structure was observed for the non-cross-linked NCs (C)
while intact polymeric coatings were observed for the correspondingly cross-linked
NCs, in the form of discrete spheroids (D).
Figure 13. Purification of NCs from large aggregates by centrifugation after milling
with 3. A reduction of both average NC size and polydispersity was observed
following centrifugation for 6 min at 12,000 g (n = 3).
59
Figure 14. SEC traces of 3 before and after milling for 96 h at 6 °C under the same
conditions as for NC preparation (without PTX). Polymer length was slightly reduced
due to hydrolysis.
To quantify the amount of polymer per drug NC, 1H NMR spectra of the NCs
stabilized with 3 and 4 were recorded in CDCl3, a good solvent for both polymer and
drug (Figure 15). By comparing the signals from the aromatic protons of PTX (8.12
ppm) and the signal of PEG (3.64 ppm), a molar polymer/drug ratio of 0.134:1
(approximately 1:2 w/w) was obtained. Thus the drug content of the PTX NCs was
approximately 67 wt %, a value which is comparable or higher to that reported for
other drug/stabilizer systems.114, 155 Drug content was comparable when either 3 or 4
were used as stabilizers. TEM analysis of the stabilized NCs showed the characteristic
needle-like shape of PTX and lack of aggregation (Figures 12A and 12B). From these
and additional images, the average length and diameter of the NCs were 206 × 26 nm
(n = 138), respectively. Considering the weight ratio of polymer to drug, and
assuming the drug NCs to be cylinders, one can estimate that approximately 1.35 ×
104 polymer chains are adsorbed per NC. This equates to 1.34 nm2/polymer chain or
an ethylene glycol monomer density of 34/nm2, which both point to the polymer
adopting a brush-like conformation.157, 158 This type of conformation is advantageous
for preventing clearance by the mononuclear phagocyte system.
60
Figure 15. Polymer/drug ratios were calculated by 1H NMR spectroscopy in CDCl3
using the characteristic peaks for PTX (8.12 ppm) and mPEG (3.64 ppm). NCs
prepared with 3 (A) and 4 (B) gave similar polymer/drug ratios.
Cross-linking and purification of cross-linked NCs
a) General considerations
In a proof of principle experiment, 3 was cross-linked with 7 in solution and in the
absence of NCs (Table 2). The reaction was monitored by 1H NMR and FTIR
spectroscopy to verify the consumption of alkynyl and azido peaks and the
appearance of the triazole product (Figures 16 and 17). At a stoichiometric azide to
alkyne ratio, both reacting groups were completely consumed within 20 min,
indicating that the alkynyl groups on 3 were readily available for reaction in the
absence of NCs.
In general, for cross-linking the NCs, a lower concentration of copper sulfate and
sodium ascorbate was used compared to the reaction in solution above because
insoluble copper(I) salts caused rapid destabilization and aggregation of the NCs.
Cross-linking performed at 6 °C also resulted in the formation of large aggregates
indicating that the reaction should be carried out at room temperature. An optimal
cross-linking time of 24 h was found for the NCs (no further evolution of their 1H
NMR spectra after this reaction duration). After the reaction, the salts were removed
61
from the cross-linked NCs by SEC. For this purpose, Sephadex G10 had to be
employed because the interactions between cross-linked NCs and Sepharose CL-4B
lead to column blockage or very low recovery of the NCs. Inductively coupled plasma
optical emission spectroscopy (ICP-OES) analysis revealed less than 1 ppm residual
copper after purification. This result could be improved by increasing the column
length or by performing a second purification by SEC.
Table 2. Conditions of cross-linking experiments
Polymer (mM)
Cross-linker Azide/alkyne molar ratio
CuSO4 (mM)
Ascorbate (mM)
Time (h)
In solution
3 (1.8) 7 1/1 1.8 9 0.3
3 (1.8) 7 1/10
On NCs
3 (0.087) 7 2.5/1
0.067 0.333 24
3 (0.087) 7 10/1
3 (0.087) 7 15/1
4 (0.114) 5 2/1
4 (0.114) 6 2/1
4 (0.114) 7 15/1
62
Figure 16. 1H NMR spectra in CDCl3 of 3 cross-linked with 7 at azide/alkyne ratios
of 1:10 (A) and 1:1 (B). The signal for the acetylene proton at 2.01 ppm disappeared
while at 8.12 ppm a signal of the resulting triazole appeared.
Figure 17. FTIR spectra of 3 cross-linked with 7. The starting compounds exhibit the
characteristic bands for the alkynyl group (≡C–H stretch at 3270 cm-1) and azido
group (N3 at 2100 cm-1), which are consumed after the reaction. The broad new peak
between 3700−3200 cm-1 is due to residual sodium ascorbate, which was not removed
from the reaction mixture.
63
b) Analysis of the extent of cross-linking
Following purification, analysis of the extent of cross-linking was complicated by
reduced possibilities for dissolving the nanocage. For instance, after reaction with
excess 7 (10/1 azide/alkyne), purification, and lyophilization, the NCs were no longer
fully soluble in chloroform, indicating successful interpolymer chain cross-linking.
Sonication of the cross-linked polymer coating while heating in deuterated methanol
lead to a homogeneous suspension which could be analyzed by 1H NMR spectroscopy
(Figure 18). From this spectrum, a decrease of the intensity of the alkynyl protons and
the appearance of a peak for the triazole groups could be observed, though both were
slightly shifted in comparison to those observed for the polymers in solution, which
were analyzed in deuterated chloroform. Quantitative disappearance of the alkynyl
group (2.59 ppm) was observed for 3 when cross-linked with 10 eq. or more of 7.
FTIR spectroscopy of the NCs before and after cross-linking also qualitatively
revealed the decrease of the band associated with the alkynyl group (3270 cm–1)
indicating reaction (Figure 19A). The absence of the band for the azido group (2100
cm–1) indicated both successful removal of excess diazido reagent during the
purification step and argues in favour of bivalent reaction of 7 with the polymer (i.e.,
reaction of both rather than a single azide of 7 with the alkynyl groups). In contrast,
NCs prepared with 4 had lower conversion of the alkynyl group (85%, 65%, and 75%
for 5, 6, and 7, respectively) and showed residual azide by FTIR spectroscopy (Figure
19B). As evidenced by TEM (Figure 12B), inter-NC cross-linking was not observed.
Figure 18. 1H NMR spectra of NCs stabilized with 3 before (A) and after (B) cross-
linking with 7 (azide/alkyne ratio 10/1). Signals belonging to the acetylene proton at
2.59 ppm were reduced and a peak for the resulting triazole at 7.78 ppm appeared. A
break on the x-axis between 7.6 ppm and 2.7 ppm was added for clarity.
64
Figure 19. FTIR spectra of NCs stabilized with 3 (A) or 4 (B) before cross-linking, in
a physical mix with 7, and following cross-linking. After cross-linking, signals from
the alkyne decreased or disappeared, while the azide band completely disappeared,
providing a strong indication of cross-linking. Only for the NCs cross-linked with 5
was residual azide observed, possibly due to lower reactivity of the polymeric tetra-
azide.
To verify that the polymer coating was indeed cross-linked, NCs were dialyzed
against an ethanol/water mixture to remove the drug from the polymer network. The
dialysis membrane was selected so that the polymer and dissolved drug molecules
could freely pass through the pores, while the cross-linked polymer, because of its
higher molecular weight, remained trapped inside. Following reaction with CoSCN,
residual mPEG was detected in the dialysis bag containing the cross-linked NCs,
while none was detected for the non-cross-linked analogs (Figure 20). TEM imaging
revealed the presence of intact polymeric spheroids for the cross-linked samples
(Figure 12D), proof for intermolecular cross-linking, but showed dense aggregates,
probably residual polyester, for the non-cross-linked analogs (Figure 12C). These
experiments demonstrated that the nanocage remained structurally intact even
following complete removal of the drug. Although different, this system is
reminiscent of hollow cage-like structures, as prepared by Turner and Wooley, which
were obtained after core degradation of shell cross-linked particles.159
65
Figure 20. Addition of cobalt thiocyanate to the concentrate of the polymer after
dialysis of NCs revealed the presence of mPEG in the case of the cross-linked coating
(D) compared to the non-cross-linked coating (C). (A) and (B) are negative and
positive controls for the absence and presence of mPEG, respectively.
Sheddability of the polymer nanocages
Owing to its adequate water solubility, the influence of cross-linking excess on
particle-size stability was examined using 7 and NCs stabilized with 3. Azide/alkyne
ratios of 2.5/1, 10/1, and 15/1 were examined, as listed in Table 2. NCs stored at 6 °C
showed very little or no change of size over a period of three weeks (Figure 21),
which was comparable to findings from the literature.85 At room temperature, an
increase of particle size with time was observed for all samples. As seen in Figure 22,
cross-linking led to enhanced size-stability in comparison to NCs with non-cross-
linked stabilizers. Owing to the relatively short hydrophobic anchoring moieties on 3
and 4, strong interaction between these and the PTX NCs is unlikely. Consequently,
while the cross-linked coating is likely to prevent NC growth by aggregation, it may
not efficiently prevent crystal growth by Ostwald ripening.160, 161 This is consistent
with the lack of crystal growth at 6 ºC (Figure 21), where the solubility of PTX is
lower than at 20 ºC, thereby slowing ripening. Future experiments involving more
polymer layers, longer hydrophobic segments and/or hydrophobic segments capable
of tightly packing on the surface of the NC may provide insight as to whether the
nanocages can fully prevent Ostwald ripening. For now, storage at 6 ºC was found to
be sufficient for preventing this process.
66
Figure 21. Results from size measurements over time of non-cross-linked and cross-
linked NCs coated with 3 (A) (n = 2−6) or 4 (B) (n = 6−13), which were stored at 6
°C.
Figure 22. Evolution of NC size over time. (A) Non-cross-linked and cross-linked
NCs stabilized with 3 and cross-linked with 7. On day six the size of non-cross-linked
NCs was significantly different from those cross-linked with 10 and 15 eq. of 7 (p <
0.05; n = 6). (B) Non-cross-linked and cross-linked NCs stabilized with 4 and cross-
linked with 5−7. On day 3, 6, and 20 the size of non-cross-linked NC was
significantly different from those which were cross-linked with 5 and 7, on day 13
only non-cross-linked and cross-linked with 7 were different (p < 0.05; n = 6−13).
Values represent means – SD.
It was observed that the size stability of the cross-linked NCs improved with
increased feed azide/alkyne ratios up to 10/1, at which point differences were no
longer observed. This indicated that maximal conversion of the alkynyl group was
achieved at this ratio. Overall, these results indicated that cross-linking is an
67
important parameter in slowing crystal growth, and argued in favor of nanocage
desorption being reduced in comparison to the non-cross-linked control. Increasing
the number of alkynyl groups on the polymer (i.e., NCs stabilized with 4, bearing four
cross-linkable groups per chain) did not influence the results significantly in
comparison to those obtained with 3 (Figure 22B).
The relative sheddability of the polymer coatings before and after cross-linking was
analyzed by quantifying by 1H NMR the amount of dissolved mPEG present in the
supernatant of freshly centrifuged NC dispersions (Figure 23). In comparison to non-
cross-linked NCs, the cross-linked analogs showed between 3–4-fold less mPEG in
the supernatant, as determined for two different batches of NCs. This experiment
demonstrated that cross-linking the surfactant to form a nanocage favors retention of
the latter on the surface of the NC.
Figure 23. 1H NMR spectra in D2O of the supernatant of centrifuged NCs with non-
cross-linked (A) and cross-linked polymer coating (B).
NC–nanocage interactions
Two additional cross-linking agents were selected based on their different physico-
chemical characteristics, which could potentially influence the nature of the
interactions between the polymer nanocages and the NCs. 5 is a polymeric cross-
linking agent with a structure analogous to 3 and 4, but with four azido groups in its
hydrophobic segment. These could promote greater inter-chain cross-linking.
However, owing to its amphiphilic nature, it was only added in small amounts as
competition with 4 for the surface of the NC prior to cross-linking was anticipated. 5
displayed a similar stabilizing effect to 7, though with less cross-linker added. In
68
addition, the FTIR spectrum of this NC revealed the presence of residual azido
groups, indicating incomplete conversion of this group on the NC. 6 is a hydrophobic
cross-linking agent that should preferentially partition to the surface of NCs, thereby
not only favoring cross-linking but also interaction between the hydrophobic
segments of the nanocage and the NC. However, only low concentrations could be
used, since the compound, owing to its poor water solubility, precipitated from its
organic solution upon mixing with water. When the nature of the cross-linking agent
was altered to more hydrophobic 6, no statistical difference in size was observed in
comparison to the non-cross-linked NCs (Figure 22B). These results indicate that
specific drug–nanocage interactions do not play a large role on size stability of the
NCs at least within the range of modifications examined (i.e., two hydrophobic
anchoring groups and three cross-linking agents). Due to its unknown toxicity and
small molecular weight, this compound was uniquely used for gaining a better
understanding of the nanocage system and was not intended for in vivo use.
Drug dissolution under sink conditions
NCs of PTX have previously been shown to possess sufficiently long circulation
times to benefit from passive accumulation in tumours.91, 162 The dissolution kinetics
of the NCs were evaluated herein under sink conditions to assess whether the lack of
desorption of the polymer nanocage was an influencial factor on the latter. The
dissolution characteristics of all NCs, cross-linked and non-cross-linked, were overall
statistically the same (Figure 24). Under the present experimental conditions, the
polymer nanocage was expected to remain intact over the entire time frame of the
experiment and maintain residual drug particle within its hydrophobic core due to a
combination of physical entrapment and physisorption. From these results it was
found that about 50% of the NCs dissolved in more than 3 h. This time frame would
be sufficient for high deposition of drug in the tumor tissue if a targeting ligand were
attached to the nanocage.127 In addition to the high surface coverage with mPEG, the
high aspect ratio of the NCs (Figures 12A and 12B) might increase circulation time,
as has been observed in other studies.163
69
Figure 24. Drug dissolution from NCs. Values represent means SD (n = 4 – 5). It
was noteworthy that free drug (control) released very rapidly from the dialysis device,
indicating that diffusion of the drug was not limited by the molecular weight cutoff of
the membrane
Conclusion
This work demonstrated successful cross-linking of polymeric stabilizers around
PTX NCs to form polymeric nanocages. These retained the particulate drug through a
combination of physical entrapment and physisorption. The nanocages were found to
act as sterically stabilizing barriers to particle–particle interactions and aggregation.
These were also shown to be shed to a lesser extent than non-cross-linked coatings,
thereby providing a means for enhanced retention of targeting agents on NCs. These
findings provide crucial tools for preparing non-sheddable stabilizing coatings for
NCs and potentially other classes of nanoparticles under circumstances where
covalent attachment between the coating and the particle is not possible or desired. In
addition, the proposed strategy should be applicable to other polyesters to thereby
modulate the rate of degradation of the nanocage. In theory, it should be possible to
attach targeting ligands to the nanocage by use of heterotelechelic PEG derivatives
and thus access active targeting or internalization of NCs.
70
71
III.2. Modular Design of Redox-Responsive Stabilizers for
Nanocrystals3
Kathrin Fuhrmann, Anna Połomska, Carmen Aeberli, Bastien Castagner,
Marc A. Gauthier, and Jean-Christophe Leroux
Introduction
Many key features of nanoparticles, such as size stability, propensity to
aggregation, photonic properties, dissolution profiles, interactions with biomolecules,
and circulation lifetime intrinsically depend on the efficacy of stabilizing agents to
mask their surface.37, 157 Correspondingly, much work has been devoted to the
development of stabilizing agents for drug nanocrystals (NCs), quantum dots, metal
nanoparticles, etc.164-166 Interestingly, developing methods for spatio-temporally de-
stabilizing nanoparticles has received much less attention,167-170 despite the fact that
this property may have applications in imaging and drug delivery. Triggered de-
stabilization can, in principle, be achieved by developing functional stabilizers whose
properties change in response to a local endogenous stimulus. However, designing
such a system requires that the dynamic non-covalent interactions between the
stabilizer in both the original and “triggered” state be carefully balanced to the surface
chemistry of the nanoparticle, which can vary broadly. This poses several synthetic
and developmental challenges that have impeded the design of tailored responsive
stabilizers.
This study presents a simple strategy for systematically adjusting the affinity of
polymeric stabilizers for nanoparticles, and which simultaneously introduces chemical
groups that are sensitive to endogenous oxidants, such as reactive oxygen species
(ROS). ROS are strongly associated with chronic inflammation and cancer65 and,
owing to their short lifetime, their action is limited to these locations. ROS are
notably associated with tumor tissues where hypoxic stress causes ROS-mediated
signaling.171 This endogenous stimulus is increasingly being investigated as a means
of triggering the response of smart polymeric systems. Examples include
3 Published in ACS Nano 2013, 7, 8243-8250. A. Połomska contributed to compound analysis.
72
nanoparticles made of polymers bearing (aryl) boronic esters,172, 173 of poly(propylene
sulfide),174-176 or of cross-linked oligo(proline).177 In the current study, paclitaxel
(PTX) NCs were selected as model for demonstrating how ROS-sensitive polymeric
stabilizers can be systematically and rationally tuned to achieve the best compromise
between stability prior to oxidation, and subsequent responsiveness upon exposure to
ROS. PTX NCs have already been examined for their therapeutic potential in treating
cancer and thus represent a pharmaceutically relevant system. Notably, the size of
PTX NCs is expected to strongly depend on their stabilizing coating, and triggered
de-stabilization may be of interest for promoting cellular uptake.170, 178
A library of ten redox-responsive amphiphilic block copolymer stabilizers was
prepared from two parent block copolymers (methoxy polyethylene glycol-b-[-
propargyl--valerolactone-co--caprolactone] (mPEG-b-[PVL-co-CL]); Figure
25A) by postpolymerization modification using the thiol–yne reaction.
Postpolymerization modification is a powerful tool for systematically altering the
functionality of polymers without influencing chain length or chain length
distribution.179 The radical thiol–yne reaction is well established as a mild, efficient,
and functional group tolerant reaction which conforms to the criteria of “click”
chemistry.180 This reaction was used to systematically alter the polarity of the
polyester block by grafting different hydrophobic thiols, while the two thioether
linkages produced at each PVL repeat unit render this block sensitive to oxidation by
ROS (Figure 25). Oxidation of the thioether to a sulfoxide or sulfone significantly
alters the polarity of the hydrophobic block,181 which may in turn cause desorption
from the surface of the NC. The influence of the nature of the hydrophobic thiol agent
grafted to the polymer on its stabilizing potential for PTX NCs is reported herein as
well as the response of stabilized PTX NCs towards oxidizing conditions.
73
Figure 25.(A) Synthesis of a library of ten oxidation-sensitive polymeric stabilizers
by thiol–yne postpolymerization modification; (B) Oxidation of the stabilizer by
endogenous oxidants (e.g., ROS) triggers desorption from the surface of NCs, leading
to de-stabilization.
Experimental Section
Materials
PTX was obtained from Bioxel Pharma Inc. (Sainte-Foy, QC, Canada) and
docetaxel from ScinoPharm Taiwan, Ltd. (Tainan County, Taiwan). Lithium
diisopropylamide, -valerolactone (VL), propargyl bromide,
hexamethylphosphoramide, -caprolactone, ethane-1-thiol, butane-1-thiol, octane-1-
thiol, benzyl mercaptane, thiocholesterol, 2,2-dimethoxy-2-phenylacetophenone
(99%), pyrene (99%), iron sulfate heptahydrate (> 99%, FeSO4), albumin from bovine
serum (BSA; ≥ 96 %), and methoxy poly(ethylene glycol) (mPEG; 2 kDa) were
purchased from Sigma-Aldrich (Buchs, Switzerland) and used as received. Hydrogen
chloride (1 N solution in diethyl ether), was purchased from Chemie Brunschwig AG
(Basel, Switzerland) and hydrogen peroxide (30% solution, Perhydrol) from Merck
(Altdorf, Switzerland). mPEG-b-CL and mPEG-b-PSO were bought from Advanced
Polymer Materials (Montreal, QC, Canada). Ultra pure water was obtained from a
Barnstead Nanopure system (Thermo Fisher Scientific, Reinach, Switzerland). Dry
74
solvents were taken from a solvent purification system (LC Technology Solutions
Inc., Seabrook, NH). VL and CL were distilled over calcium hydride under inert
atmosphere before use. All other solvents were of highest purity and bought from
Sigma-Aldrich (Buchs, Switzerland).
Equipment. 1H NMR spectra were recorded on a Bruker Av400 spectrometer (Bruker BioSpin,
Fällanden, Switzerland) operating at 400 MHz for protons. Analytical size-exclusion
chromatography (SEC) measurements were performed in THF using a Viscotek
TDAmax system (Viscotek, Houston, TX) equipped with a differential refractive
index detector. Molecular weights are given relative to narrow PEG standards (PSS
Polymer Standards Service GmbH, Mainz, Germany). Separation was achieved using
two Viscotek columns (GMHHRM) in series at a flow rate of 1.0 mL.min–1 at 45 °C.
Particle hydrodynamic diameter was determined by dynamic light scattering using a
DelsaNano C Particle Analyzer (Beckman Coulter, Brea, CA). The cumulants result
calculated by the software was used to report the hydrodynamic diameter of the NCs.
PTX concentration was determined by HPLC analysis, using an autosampler and
pump system (Ultimate 3000, Dionex, Thermo Fisher) equipped with a reversed
phase column (Accucore C18 column, 2.6 µm particle size, 100 × 2.1 mm, Thermo
Fisher) held in a column oven at 30 °C and a diode array detector (Thermo Fisher
Scientific, Reinach, Switzerland). Gradient elution was performed starting with a mix
of 40 % acetonitrile in water rising to 70% acetonitrile within 8 min. PTX was
detected at 230 nm. A solution of docetaxel in acetonitrile was added as internal
standard. FTIR spectra were obtained using ATR geometry on a Spectrum 65 infrared
spectrophotometer (Perkin Elmer, Schwerzenbach, Switzerland).
Synthesis of polymer precursor 3
PVL was synthesized as described elsewhere.39 In a typical experiment, mPEG
(800 mg, 0.4 mmol, 1 eq.) was dried by azeotropic distillation with dry toluene (50
mL) under a flow of nitrogen. The flask was sealed and the mPEG was dissolved in
dry dichloromethane (2 mL). Dry CL (132.8 μL, 1.2 mmol, 3 eq.) and PVL (150.5
μL, 1.2 mmol, 3 eq.) were then added using gastight syringes. The polymerization
was initiated by the addition of HCl in ether (1.2 mL, 1.2 mmol, 3 eq.) and stirred at
75
room temperature for 24 h. The reaction mixture was then precipitated twice in cold
ether, the solvent evaporated in vacuo, the residue taken up in water, and the polymer
recovered by lyophilization (0.867 g, 80% yield). The synthesis of 13 followed the
protocol above with the following quantities: mPEG (1.50 g, 0.75 mmol, 1 eq.), PVL
(564.6 µL, 4.5 mmol, 6 eq.), HCl in ether (1.5 mL, 1.5 mmol, 2 eq.). The fully
annotated NMR spectrum of 3 and 13 and the compositions of the polymers can be
found in Supplementary Figures S 4 and S 7 and Table 3, respectively.
Postpolymerization modification by radical thiol–yne addition.
In a general procedure, 3 (30 mg, 0.0115 mmol, 1 eq.) was dissolved in 100 μL of
stabilizer-free tetrahydrofuran. A fresh stock solution of 2,2-dimethoxy-2-
phenylacetophenone (0.89 mg, 0.0035 mmol, 5 mol% of thiol-compound) in
stabilizer-free tetrahydrofuran THF was added (8.9 μL) to the polymer solution in a
quartz cuvette. After addition of the thiol compound (6 or 12 eq. for modification of 3
and 13, respectively), the solution was subjected to UV light at 365 nm for 30 min.
The solution was then precipitated twice with about 15 volume parts of cold hexane,
recovered by centrifugation (unless otherwise stated, see Supporting Information),
and dried under a flow of nitrogen. The polymer was dissolved/dispersed in water and
lyophilized. Fully annotated 1H NMR spectra of all polymers and can be found in
Supplementary Figures S 8–S 17.
Preparation of PTX NCs.
PTX NCs were produced by wet milling. In a typical experiment, 2 mL of a 0.5%
(w/w) polymer solution in ultra pure water was filtered (0.2 μm pore size) into a 20
mL cylindrical glass vessel containing 10 mg of PTX and 4 mL of zirconium oxide
beads (~ 14.7 g, 0.3 mm in diameter, Union Process, Akron, OH). When the stabilizer
was not soluble in water, an aqueous solution was first prepared by micellization
following dialysis from an ethanol/water or tetrahydrofuran/water solution. The vessel
was closed with a plastic cap and then placed horizontally on a Ratek BTR5 blood
tube roller (with custom modified motor, Labortechnik Fröbel GmbH, Lindau,
Germany) and rolled at 220 rpm in a cold room (6 °C) for 18 – 48 h. After milling,
the beads were separated from the suspension by filtration through polyamide sieve
fabric (30 μm pores, VWR, Dietikon, Switzerland), and the residue washed four times
76
with 2 mL of ultra pure water. The suspension was centrifuged at 12,000 × g for 6
min to remove larger aggregates.
Imaging of stabilized PTX NCs.
NCs were imaged by TEM. 4 μL of a NC suspension was adsorbed to a glow
discharged carbon-coated copper grid for 1 min. After two washings with water, NCs
were negatively stained with 2% (w/v) uranyl acetate for 1 min and air-dried after
blotting with filter paper. The specimens were examined in a Philips CM12 (tungsten
cathode) transmission electron microscope (FEI, Hillsboro, OR) at 100 kV and
images were recorded with a Gatan CCD 794 camera (Gatan Inc., Pleasanton, CA).
Drug to stabilizer ratio.
NC dispersions were centrifuged at 20,000 × g for 90 min at 15 °C. Afterwards, the
supernatant was carefully and quantitatively removed and the pellet was dried by
lyophilization. The dried pellet was dissolved in deuterated chloroform for 1H NMR
spectroscopy. Integration of the aromatic proton signal of PTX at 8.12 ppm versus the
proton signal of PEG at 3.64 ppm provided an estimate of the drug to stabilizer ratio.
NC volume and density of the stabilizer coating.
Average dimensions of NCs were measured in TEM images. The number of
polymer chains (nPolymer) per NC can be calculated with equation (3) below.
nPolymer NA V (wtPolymer /wtPTX ) / Mw Polymer (3)
where NA is Avogadro’s constant, V is the average volume of a NC (assumed to be
a cylinder), is the density (1.4035 g/mL for PTX dihydrate), (wtPolymer/wtPTX) is the
weight ratio of drug to polymer after centrifugation determined by 1H NMR
spectroscopy, and Mw Polymer is the weight average molecular weight of the polymer as
calculated by 1H NMR spectroscopy.
Dissolution test
Release of PTX from NCs was tested under sink conditions in a 5% BSA solution,
which is expected to be the main solubilizing component for PTX in vivo.151 In a
preliminary experiment, the saturation solubility of PTX powder in the test conditions
was found to be 0.01 mg.mL–1. A 1 mL Spectra/Por Float-A-Lyzer G2 (MWCO 100
77
kDa, Sigma-Aldrich, Buchs, Switzerland) was filled by mixing 20 mM phosphate
buffer (pH 7.4) containing 10% BSA with NCs in water to yield a final volume of 1
mL containing 0.1 mg/mL PTX in 10 mM phosphate buffer (pH 7.4) and 5% BSA.
The dialysis device was placed in a 50 mL centrifugation tube containing 45 mL of 10
mM phosphate buffer (pH 7.4) with 5% BSA, on a rotary shaker ( 400 rpm) in an
incubator at 37 °C. Under these conditions, the maximum PTX concentration was 5
times below its saturation solubility. At selected time points, 30 μL aliquots were
taken from inside the dialysis device and prepared for HPLC analysis by addition of
30 L internal standard (docetaxel in acetonitrile) and vortexed. After addition of 200
L 0.1 M ZnSO4 solution and 500 L acetonitrile the samples were vortexed for 1
min. Following two centrifugation steps of 10 min at 15,000 × g 200 L of the
supernatant was mixed with 100 L water and filtered through polyamide (0.2 µm
pore size) and analyzed by HPLC as described under “Equipment”.
Sensitivity to ROS, dissolution and polymer shedding
NC suspensions were diluted with water to a PTX content of 0.3 mg/mL. In the
case of NCs formulated with 16 this step was neglected. The NC dilution (0.5 mL)
was mixed with Fenton’s reagent (H2O2/FeSO4 final concentrations of either 1
mM/0.1 mM or 0.1 mM/0.01 mM) directly in a UV cuvette and size was monitored at
25 °C for 12 h. To determine polymer shedding after oxidation, diluted NC
suspensions (0.2 and 0.3 mg/mL PTX for polymers 16 and 14/15 respectively) were
incubated with 1 mM H2O2/0.1 mM FeSO4 for 2 h at room temperature, then they
were treated and analyzed as described under “Drug to stabilizer ratio”. In a further
experiment, Fenton’s reagent (H2O2/FeSO4 final concentration in the mixture 1
mM/0.1 mM) was added to a NC suspension at the beginning of a dissolution test, and
then the experiment was conducted as described in the previous section. For the NMR
characterization of the shed polymer, a NC suspension stabilized with polymer 14 was
incubated with 1 mM H2O2 and 0.1 mM FeSO4 for 4 h. The particles were then
centrifuged at 20,000 × g for 90 min. The supernatant containing shed polymer was
collected, lyophylized, and later analyzed by 1H NMR spectroscopy.
78
Results and Discussion
Stabilizer design and synthesis
As illustrated in Figure 25A, a block copolymer structure was chosen as template
for the preparation of redox-responsive polymeric stabilizers. Two parent copolymers
bearing alkynyl groups in the hydrophobic block were prepared by cationic ring-
opening polymerization of mixtures of PVL and CL from a mPEG initiator (3 and
13 in Table 3). This polymerization approach is advantageous for producing polymers
for pharmaceutical applications owing to the absence of transition metal catalysts.149
mPEG was selected as hydrophilic polymer segment to prevent NC agglomeration by
masking their high energy surfaces and to convey “stealth-like” properties to the
NCs.38, 128, 152 The number of repeat units in the polyester block was targeted to be low
(ca., 4–5) so that affinity to the NC surface would not be too strong to prevent
desorption upon oxidation, and so that individual oxidation events within this short
block would stand a chance of significantly influencing the overall polarity of the
chain (i.e., for responsiveness). 3 and 13 possessed narrow molecular weight
distributions (Figure 26A), and analysis of their 1H NMR spectra (Figure 26B,
Supplementary Figures S 4 and S 7) allowed for the accurate determination of the
final composition of the short hydrophobic segment (Table 3).
79
Table 3.Characteristics of stabilizers examined in this study
Composition R Mn, SEC
(kDa)a Mw/Mn
Thiol–ynecoupling
(%)b 3 PVL2 CL3 2.6 1.1 –
8 PVL2 CL3 Et 2.8 1.3 100
9 PVL2 CL3 Bu 2.8 1.3 100
10 PVL2 CL3 Oct 3.3 1.5 100
11 PVL2 CL3 Bn 3.6 1.8 90
12 PVL2 CL3 Chol 3.9 1.9 100
13 PVL4 2.8 1.1 –
14 PVL4 Et 2.8 1.4 100
15 PVL4 Bu 3.1 1.4 100
16 PVL4 Oct 3.8 1.8 100 c
17 PVL4 Bn 3.6 2.4 95
18 PVL4 Chol 2.9 1.3 100
19 CL5 2.6 1.1 –
20 PSO9 2.7 1.1 –
a: in THF; b: by 1H NMR spectroscopy; c: by MALDI-TOF MS Abbreviations: ethyl
(Et), n-butyl (Bu), n-octyl (Oct), benzyl (Bn), and cholesteryl (Chol).
80
Figure 26. Polymer library synthesis by postpolymerization modification. (A) SEC
traces of parent polymers 3 and 13 in THF; (B) Representative 1H NMR spectra of 13
and 14 demonstrating quantitative functionalization by thiol–yne coupling.
Assignments based on the 1H–1H correlation spectrum of 14 (Supplementary
Figure S 18).
Postpolymerization modification of 3 and 13 by radical thiol–yne coupling using
various alkyl, aromatic, and multi-cyclic thiols led to the straightforward and efficient
preparation of the library of polymeric stabilizers 8-12, 14-18 (Table 3) with
systematically different number and nature of side chains (Figure 25A). Quantitative
grafting of all thiols except benzyl mercaptane was achieved in less than 30 min as
determined by 1H NMR spectroscopy (Figure 26B). For 11 and 17, small peaks at 5.4
81
and 5.9 ppm corresponding to the mono-substituted vinyl sulfide were observed
(Supplementary Figures S 11 and S 16). For 16, which bore eight octyl units,
integration of the 1H NMR spectrum did not match expectations, though peaks
associated with mono-substituted vinyl sulfide were not observed (Supplementary
Figure S 15). Consequently, matrix-assisted laser desorption ionization time-of-flight
mass spectrometry was used to verify complete functionalization of the polymer. The
latter showed a multiple peak distribution with mass differences of about 430.29,
which corresponds to one -valerolactone monomer conjugated with two octanethiols.
In addition, FTIR spectroscopy revealed complete disappearance of the characteristic
stretching vibration of the alkynyl group at 3270 cm–1 following thiol–yne
functionalization (Figure 27). Analytical size-exclusion chromatography in THF
showed in most cases a mono-modal molecular weight distribution, though as the
hydrophobicity (and bulk) of the polyester block increased, the chromatograms
became less well defined, likely due to potential enthalpic interaction with the
stationary phase of the column (Figure 28).
Figure 27. FTIR spectra of parent polymers 3 (A) and 13 (B) and their respective
thiol–yne adducts showing the disappearance of the alkynyl peak at 3270 cm–1.
Figure 28. SEC traces of polymers 8-12 (A) and 14-18 (B) after thiol–yne coupling
and control polymers 19, 20 (C).
82
NC production and stability
PTX NCs were produced by wet-milling in the presence of 8-12, 14-18 as well as
with mPEG-b-CL (19) or mPEG-b-poly(styrene oxide) (mPEG-b-PSO, 20) which
are un-branched aliphatic and aromatic controls, respectively. Overall, fourteen
polymers were investigated for their ability to sterically stabilize PTX NCs. With the
exception of 12, 17, and 18, wet-milling of PTX for 18 – 48 h led to the production of
NCs with mean diameters around 200 nm, as determined by dynamic light scattering,
which is in accordance with requirements for systemic administration. The size and
shape of particles obtained by wet-milling were consistent with values reported for
PTX NCs elsewhere.85, 156 NCs were subjected to a short centrifugation step to
remove larger aggregates, and then PTX concentration was measured by high-
performance liquid chromatography (Table 4). PTX recovery was lower with
increasing hydrophobicity of the polymer, which indicates inefficient milling in cases
where self aggregation is favored versus the interaction of the polymer with the NC
surface. While the polymers bearing the pendant cholesteryl and benzyl units were
poorly soluble and inefficient stabilizers for PTX NCs, the ability to quantitatively
introduce complex and bulky groups such as these by the thiol–yne reaction
demonstrates the versatility of the chemistry employed. Inefficient stabilization of
NCs observed using 12, 17, and 18 may be related the presence of branching within
the hydrophobic segment. Indeed polymer functionality can be broadly tuned using
the extensive list of commercially available thiol agents.
83
Table 4. Characteristics of stabilized PTX NCs examined in this study
Stab. R PTX conc. (mg/mL) a
Size (nm) a
PDI b PTX
content (wt%)
L/2Rg c
8 Et 0.82 180 0.234 77 0.40
9 Bu 0.60 285 0.255 79 0.43
10 Oct 0.29 189 0.211 77 0.43
11 Bn 0.42 298 0.219 78 0.45
14 Et 0.77 232 0.206 76 0.42
15 Bu 0.54 193 0.218 73 0.37
16 Oct 0.19 176 0.133 69 0.32
19 – 0.91 136 0.137 82 0.41
20 – 0.64 229 0.190 74 0.41
a: after centrifugation; b: polydispersity index; c: L is the average mean distance
between mPEG chains; Rg is the radius of gyration of “free” mPEG in aqueous solution; L/2Rg >1 mPEG is well-separated and in mushroom conformation, <1 mPEG in brush conformation, <0.5–0.7 enhanced protein resistance regime.158
The characteristic needle-like shape of PTX NCs was observed by transmission
electron microscopy (TEM) for all stabilizers (examples in Figure 29). Under the
simplification that the NCs are cylindrical, the average specific surface area of the
NCs was determined from these images. This, in combination with drug content
determined by 1H NMR spectroscopy, allowed for the calculation of the amount of
polymer per surface area of the NC which can be expressed as L, the average mean
distance between mPEG chains. According to the geometric model of Pasche et al.,158
the ratio L/2Rg (Rg being the radius of gyration for free mPEG) was calculated to
provide an indication of the packing structure of the stabilizer on the NC (Table 4).
An L/2Rg higher than 1 suggests that mPEG is well-separated and in mushroom
conformation, while an L/2Rg below 1 indicates brush conformation. mPEG modified
surfaces with L/2Rg below 0.5–0.7 have been reported to efficiently reduce protein
adsorption.158 Based on this model, all stabilizers on the surface of PTX NCs were
within a dense brush regime (L/2Rg ranges from 0.32–0.45, see Table 4), suggesting
84
intimate contact and interaction between neighboring polymer chains. While stabilizer
surface density for all polymers was roughly the same, a trend towards increased
stabilizer density was observed from 14 to 16 (increasing side-chain length).
Figure 29. Representative transmission electron micrographs of PTX NCs stabilized
with (A) 9, (B) 10, (C) 14, and (D) 15. Scale bars represent 200 nm.
Following purification, the stabilized NCs were stored at room temperature (Figure
30) and their size over time was monitored by dynamic light scattering. Compared to
the branched polymers, the un-branched aliphatic control 19 was the least efficient
stabilizer, pointing to the importance of multiple interactions and intimate contact
within the hydrophobic domain. Polymers 8-11 (i.e., with four branches) provided
improved size stability compared to 19 (Figure 30C). Increasing the number of
branches to eight (polymers 14-16, Figure 30B) led to significant stabilization of the
NCs over a period of over 21 days. At 6 °C, little or no evolution in size was observed
for any of the stabilized NCs (Figure 31), suggesting that the size increase observed at
room temperature was due to Ostwald ripening rather than aggregation. It should be
noted that the influence of mass transport, which is affected by initial NC size and
concentration, on NC stability cannot be discounted, the relative similarity of these
parameters between samples suggest that their effect is secondary to stabilizer
chemistry in Figure 30.
85
Figure 30. Size-stability of PTX NCs stabilized with (A) 8-11, (B) 14-16, (C) 19, and
20 at room temperature. Values represent mean ± SD (n = 3–6).
Figure 31. Size stability of NCs milled with different polymers and stored at 6 °C.
Values represent mean ± SD, n = 3–6.
Response of PTX NCs to reactive oxygen species
Stabilizers 8-12 and 14-18 contain on average four and eight thioether bonds per
polymer chain, respectively. Oxidation of these groups produces a sulfoxide and
ultimately a sulfone.181 This process is expected to impart a large change of polarity to
the hydrophobic block of the stabilizer. For instance, while the thioether side-chain of
methionine in proteins is one of the most hydrophobic, its sulfoxide/sulfone form is
more hydrophilic and bulky, which is used in practice for protein secondary structure
characterization by breaking -sheet assembly.182, 183 As PTX NCs stabilized with 14-
16 demonstrated excellent size stability (Figure 30B), their response to ROS was
investigated. These NC suspensions are characterized by a high drug content (69–
82%, see Table 4), which implies that the ROS-responsiveness of only a minor
component (i.e., the stabilizer) has the potential to trigger the destabilization of a
correspondingly large amount of drug. To examine the sensitivity of 14 and 15, a low
concentration of ROS was first examined (100 M H2O2, 10 M FeSO4). As shown
in Figure 32A and B this concentration did not influence NC stability. However,
exposure to an increased oxidizing environment (1 mM H2O2, 0.1 mM FeSO4) led to
the rapid de-stabilization of NCs prepared with 14 and 15. Oxidation of the thioether
86
was monitored by 1H NMR spectroscopy analysis of the fraction of 14 that was shed
from the NC. Examination of the 1H-1H correlation spectrum revealed the appearance
of a new signal consistent with an ethylsulfoxide group, at lower field than the
corresponding ethylthioether (Figure 33), as expected from prior observations from
the literature.184 Overlapping signals in the 1D 1H NMR spectrum does not allow for
precise quantification as only a low level of oxidation was observed. Semi-
quantitative analysis suggests that only a single, or very few oxidation events within
the very short hydrophobic block were able to drive 14 from the surface of the PTX
NCs. Thus a greater responsiveness to oxidation in comparison to other thioether-
based materials was observed. For the latter, multiple oxidation events are required to
significantly alter the polarity of the individual polymer chains. Responsiveness was
manifested by a rapid increase in size beginning well within the first hour (Figure 32),
which then leveled off thereafter. The size distribution of the NCs also increased to a
certain extent after exposure to ROS. TEM images of NCs before and after exposure
to ROS suggest that the increased size observed by light scattering could be the result
of aggregation, due to exposure of hydrophobic PTX areas (Figures 34 and 35).
Bearing in mind that this is a closed system, oxidation of 14 produced a shedding of
about 20% of the stabilizer from the NC (polymer to drug ratio (w/w) decreased from
0.311 ± 0.014 to 0.251 ± 0.006), suggesting that more shedding may be observed
within an open system such as the body. NCs stabilized with 16, bearing eight
pendant octyl chains in its hydrophobic block, showed a much slower and less
pronounced response to ROS, likely because the longer hydrophobic alkane chain
(octane) provided a stronger anchor to the surface of the NC than the shorter ones
(i.e., ethane or butane). These combined results suggest that the change of polarity of
the stabilizer’s hydrophobic block due to low level of oxidation event is sufficient to
induce desorption from the surface of the NC when the pendant groups are short and
when the concentration of oxidant is sufficiently high. Exposure to ROS did not
significantly influence the release kinetics of PTX from stabilized NCs, which points
to the absence of deleterious effects of the oxidation reaction on the NC itself (Figure
36).
87
Figure 32. Size-stability of PTX NCs stabilized with 14 (A), 15 (B), 16 (C) after
exposure to oxidizing agent (oxidant concentration: 1: 0.1 mM H2O2/0.01 mM
FeSO4 or 10: 1 mM H2O2/0.1 mM FeSO4). Data represent mean ± SD, n = 3–4.
Figure 33. Oxidation of 14. 500 MHz 1H-1H COSY NMR in CDCl3 of the polymer
shed from nanoparticles following exposure to 1mM H2O2 and 0.1 mM FeSO4 for 4 h.
The particles were centrifuged at 20,000 x g for 90 min and the supernatant
containing shed polymer was collected and lyophylized. The spectrum exhibits a new
peak at 1.31 ppm that correlates with a signal at 2.74 ppm, which is consistent with an
ethylsulfoxide moiety.185
88
Figure 34. Representative TEM overview and zoomed images of NCs stabilized with
14 before (A) and after (B) exposure to 1 mM H2O2/0.1 mM FeSO4 for 4 h,
demonstrating signs of aggregation. The specimens were examined in a FEI Morgagni
268 (tungsten cathode) transmission electron microscope (FEI Company,
Netherlands) at 100 kV and images were recorded with a Keen View camera (Soft
Imaging System, Germany).
Figure 35. Representative size distribution of NCs stabilized with 14 (A, B) and 15
(C) before and after exposure to 1 mM H2O2/0.1 mM FeSO4 for 4 h and 12 h,
demonstrating growth of the particles and increase in polydispersity index.
89
Figure 36. Dissolution profiles of NCs stabilized with 14 (A) and 15 (B) before and
after exposure to an oxidizing environment (1 mM H2O2/0.1 mM FeSO4), a solution
of PTX (A) served as control for the free diffusion of dissolved drug through the
dialysis device.
Sensitivity and responsiveness compared to other ROS-sensitive systems
In contrast to other ROS-sensitive drug delivery systems reported in the literature,
the stabilized NCs presented herein represent the first example of a core-shell system
in which the redox-sensitivity of the shell is designed to release the “insensitive” bulk.
As a consequence, ROS-sensitive stabilized NCs stand to respond faster and have the
potential to be more sensitive to oxidation than bulk-type systems, for which the
oxidation process is slow due to diffusion phenomena, swelling, erosion, etc. Indeed,
other bulk thioether-based systems have shown to respond over a period of a couple
days to very high concentrations of H2O2 (5 – 10 vol%).176 For instance, Mahmoud et
al. have reported the release of a dye from thioether-containing particles to occur over
1 day upon exposure to 100 mM H2O2.186 Indeed, it should be noted that Allen et al.
have recently shown rapid (<10 min) dye release from thioether-containing particles
in response to 25 – 2000 ppm sodium hypochlorite or ROS generated by enzymatic
processing of H2O2.175 This study provides an indication that thioether bonds can be
very sensitive to other endogenous and potentially more reactive ROS than H2O2, and
that existing oxidants can be potentiated with enzymes. Interestingly, Almutairi and
co-workers have recently reported that polymers bearing aryl boronic ester protecting
groups were substantially more sensitive than thioethers to oxidation. Indeed,
concentrations of H2O2 as low as 100 μM led to polymer backbone degradation and
release of about 50% of cargo within 24 h.172 The system reported in our work is
sensitive to oxidant concentrations that are higher than that in the aforementioned
90
studies. However, the reaction was relatively fast (in under 2 h) making the system
suitable to target regions with high/moderate ROS concentrations with lower chances
of off target effects (due to oxidation in low ROS concentration regions).
Conclusion
This study demonstrates that postpolymerization modification via the thiol–yne
reaction is a powerful tool for rapidly, rationally, and systematically preparing ROS-
responsive biodegradable polymeric stabilizers for NCs. Achieving a fine balance
between stabilization of the NCs and observing a tangible and rapid response upon
oxidation was possible using this approach. The prepared NCs were sensitive to
oxidation and, in the case of PTX, this phenomenon may be self-amplifying due to the
known triggering of ROS production in PTX-treated cells.187, 188 This paves the way
for the design of ROS-sensitive systems based on nanoparticles and drug NCs, and
offers many opportunities in the biotechnological field for location-specific shedding
of stabilizers, which can be used for imaging 189-191 or for improving cellular uptake. 178, 192 While hydrophobic thiols were used herein for affinity with the hydrophobic
surface of PTX NCs, the extensive list of other commercially available (and
hydrophilic) thiols render this a universal approach for preparing libraries of
polymeric surfactants with precisely the same number of repeat units for the
reversible stabilization of drug NCs and potentially other classes of nanoparticles such
as quantum dots and gold nanoparticles, for which aggregation strongly influences
their photonic properties. 193
91
IV. Conclusion and Outlook
92
93
What was the reason for developing nanocrystals with functional stabilizers?
The successful therapy of cancer remains challenging because of the similarity of
cancer cells to those of the host, the difficulty associated with accessing them, and
evolving drug resistance mechanisms. In the US, the most underlying cause of death
in women, aged 40 to 79 years, and in men, aged 60 to 79 years, is cancer, whereas
about 15 years ago it was heart disease.194 In Europe, the cancer prevalence (the
number of patients diagnosed with cancer and survivors of cancer) was close to 13
million in 2010.195 This is related to the age distribution shifting toward the elderly
due to increased life expectancy and reduced mortality. Lung and breast cancers are
worldwide leading causes of cancer mortality.194 These figures will probably continue
to increase because the incidence of all epithelial cancer entities rises steeply with
age.195 By 2040, approximately one in four people in the United Kingdom aged 65
and over will be cancer survivors.196 Survivors of cancer suffer not only from effects
caused by the cancer and its treatment, but also from myriad treatment
complications.197 Some effects may quickly resolve (e.g., hair loss or nausea), while
others are long-term or permanent (e.g., infertility or neurological pain)43, 198
Understandably, adherence to life-saving cancer therapies remains a challenge.197
Should treatments be more selective to cancerous tissues, improved quality of life for
patients during therapy and beyond could ensue. Nanocrystals of poorly soluble
chemotherapeutic drugs, paclitaxel in particular, could be an avenue for improved
chemotherapy with less adverse effects. Different from conventional formulations,
nanocrystals correspond to small size drug systems almost entirely composed of drug,
and stabilized by a low amount of excipient.13, 14 Patients thus benefit from
formulations devoid of excessive amounts of cosolvents and excipients (e.g.,
surfactants), which can cause, for example, hypersensitivity reactions.5, 142
Furthermore, because of their small size, they are ideal for i.v. administration and
current ongoing research is attempting to promote their accumulation at the tumor site
by passive or active targeting. The more drug is selectively deposited at the site of
disease, the less adverse effects in healthy tissues (Figure 37A).
94
Figure 37. Rationale of the presented work. (A) Systemic exposure to a drug can
cause adverse effects which are, in chemotherapy, often severe. The tumor’s unique
physiology, however, offers possibilities for selective accumulation of drug carriers.
This includes leaky vasculature (paired with poor lymphatic drainage in the core of
the tumor) for passive targeting via the EPR effect and/or special ligands for active
targeting. (B) Nanocrystals are prone to aggregation due to their high surface energy.
Steric stabilization with a polymeric stabilizer can be enhanced by cross-linking and
formation of nanocages. Furthermore this non-sheddable nanocage can be modified
with a targeting ligand for tumor targeting. (C) The preferential release of the
nanocrystal can be achieved by tailoring the stabilizer to be reactive to the tumors
microenvironment, in this case the presence of ROS. Quick shedding can potentially
facilitate uptake by tumor cells and eventually accelerate drug dissolution.
There are two known mechanisms for concentrating a nanosized drug carrier in a
tumor: by passive targeting via the EPR effect and/or active targeting (e.g., as
antibodies or DARPins).199-201 In certain cases the selective targeting of nanocarriers
to specific cells (i.e., tumor cells expressing certain receptors) in the body is
achievable using targeting ligands. 202 However, passive targeting, which has
nonetheless been studied excessively in animal models, has met with varied
95
outcomes. For example, Yuan et al. concluded from injecting sterically stabilized
liposomes that the cutoff size (for exclusion from the tumor) lies somewhere above
~400 nm, because liposomes of 600 nm could not penetrate the tumor vessel wall.63
This cutoff size, however, depends on the type of tumor and its own physiology. For
example, the cutoff of polymeric micelles in hypervascular tumor tissue was 100 nm,
in contrast to a hypovascular tumor tissue that had a cutoff larger than 50 nm. The
coadministration of transforming growth factor signaling inhibitor enabled larger
micelles of 70 nm to accumulate in the tumor tissue, because this factor transiently
decreased the pericyte coverage of the endothelium in the neovasculature of
tumors.124 In humans, these less accessible tumors have been treated by using
angiotensin II to elevate blood pressure, which increased the diffusion of the colloid
into the tumor.203 Overall, the EPR effect in humans remains a highly heterogenous
phenomenon with variation from tumor model to tumor model and from patient to
patient.204, 205 For example, PEGylated liposomal doxorubicin failed to improve
overall survival for patients compared to conventional doxorubicin in metastatic
breast cancer.206 This is perhaps related to the fact that the tumor deposition of
liposomes in this type of tumors may not be sufficient. On the opposite, the good
response of Kaposi’s sarcoma and malignant pleural mesothelioma to this liposomal
formulation are attributed to accumulation via the EPR effect.207, 208 In the latter case,
tumor accumulation was emphasized by scintigraphic images showing in 80% of the
cases a significantly higher uptake of radio-labeled liposomal doxorubicin in the
tumor than in soft tissue.208 Based on these examples it appears that passive drug
targeting is possible, although many factors interplay and its success cannot be
predicted. Key parameters seem to include successful shielding from MPS uptake by
PEGylation for long circulation times and small size of the carrier. Therefore, within
this doctoral thesis, a method was sought out to stabilize nanocrystals with a PEG
copolymer to enable long circulation times and increased size stability for passive
accumulation via EPR effect. In addition, new approaches for a non-sheddable
coating to facilitate potential active targeting were developed as well as a coating for
fast release of the drug in response to a stimulus.
The potential change in biodistribution of a drug by using a nanocrystal formulation
can increase the therapeutic benefit for patients.
96
What was shown within this work?
An outline of the work achieved in this thesis is given in Figure 37. This work was
directed towards the study of whether a certain design of polymer coating can slow
down the dissolution of nanocrystals. Although this could not be achieved within this
work, findings showed that coatings can be designed to be either less sheddable from
the surface or reactive to cause nanocrystal destabilization in response to a stimulus.
As model system, nanocrystals of the drug paclitaxel were produced by wet milling.
Special emphasis was put on the design of a stabilizer to prevent aggregation. A non-
covalently attached coating on the nanocrystal surface can, in principle, permit active
targeting of the whole construct. This purpose can be achieved via ligand attachment
to the polymer without compromising the drug molecule’s integrity. Therefore,
functional monomers carrying alkyne moieties (-propargyl--valerolactone) were
synthesized and copolymerized with -caprolactone from a methoxy-PEG
macroinitiator to obtain custom polymeric stabilizers. These stabilizers were cross-
linked (Figure 37B) with different diazido compounds by a copper-catalyzed 1,3-
dipolar cycloaddition (Figure 18). In addition to increased size stability for the
nanocrystals, the formed nanocages resisted dissolution, and were less prone to be
shed from the surface of the nanocrystal. In a second study, the stabilizers were used
to prepare a library of new polymers by systematic grafting of different alkane thiols
to the polymer by radical thiol−yne addition. These additions produced polyester
blocks with different polarities, which were tested for optimal nanocrystal size
stabilization. Furthermore, oxidation of their thioether bonds changed the stabilizer’s
lipophilicity and affinity for the nanocrystal surface (Figure 37C), which in certain
cases destabilized the system as seen by an increase in nanocrystal size (Figures 32
and 34). Although not tested within this doctoral thesis, the fast shedding from the
crystal surface can, in principle, be useful for fast release of the drug and increased
uptake by tumoral cells. Non-sheddable nanocages and ROS responsive stabilizers
could contribute to increased drug uptake in the tumor by passive targeting via the
EPR effect.
How does this relate to other work?
Within this doctoral thesis, special emphasis was put on the design of a stabilizer,
which was amenable for modifications to improve distribution of the drug to the
97
tumor. Traditional stabilizers in use for parenteral nanocrystal formulations are
usually members of the poloxamer type.36, 103 However, these can desorb from the
surface upon dilution, as seen for poloxamer 407-stabilized nanocrystals.114 This can
cause nanocrystal destabilization with aggregation and increase in size, which is a
disadvantage in the use of nanosuspensions for anticancer therapy. Other groups have
evaluated stabilizer-free fluorescent paclitaxel nanocrystals but only observed
minimal accumulation via the EPR effect.89, 162 These “hybrid” (precipitate of
paclitaxel and dye) nanocrystals could be tracked via fluorescence imaging. When
injected into mice bearing MCF-7 tumor xenografts, the nanoparticle-incorporated
dye was retained within the tumor and surrounding tissue for several days compared
to the rather fast clearance (ca. one day) of the free dye.162 Fluorescence images
showed dye deposition in tumor and lung (possibly due to the accumulation of
nanocrystals) and kidney (excretion pathway for the hydrophilic free dye). However,
a further study using hybrid nanocrystals containing radiolabeled paclitaxel revealed
that less than 1% drug accumulated in the tumor (HT-29 xenograft in mice).89 Close
to 40% of injected drug was found in the liver. The lack of stabilizer might have
caused the nanocrystals to aggregate and/or to be recognized by MPS after injection.
Therefore, the high liver and the low tumor accumulation possibly resulted from low
size stability and uptake by MPS. Masking the surface of paclitaxel nanocrystals with
transferrin increased size stability over time but could not improve anticancer
efficiency compared to the conventional dissolved formulation.119 Indeed, it is a
challenging task to secure surfactants to the surface of nanocrystals, especially during
the administration and blood circulation stages.89 The approach presented in this
thesis can provide the means for addressing this issue.
Modifications to standard polymers are possible, but often limited in the number of
introducible functional groups. The commonly used poloxamers only offer
modification at the two terminal hydroxyl groups,209 which is a restriction to testing a
range of introduced modifications. The custom polymerization of functional
monomers has the advantage of including functional moieties of desired number.
The nanocrystals within this work were prepared with a coating susceptible to
cross-linking via click-chemistry to form a non-sheddable nanocage. This nanocage is
expected to not only increase colloidal stability by introducing steric hindrance, but
also to reduce opsonization and uptake by MPS. Furthermore, similar stabilizers were
modified by the postpolymerization approach with thiol bearing organic compounds
98
(of increasing length and hydrophobicity) and investigated in a systematic fashion for
their stabilizing capabilities. In some cases, relatively stable nanocrystals (about 20%
size increase over 3 weeks at room temperature) were obtained. The increased
colloidal stability is an important stepping stone for passive accumulation via the EPR
effect. On the other hand, the reactivity of the thioether containing polymers to
predominant ROS presence in the tumor tissue could trigger oxidation-induced
shedding of the stabilizer. After removal of the polymer coating the nanocrystals
could potentially be taken up to a higher extent by tumoral cells. Increased cell uptake
after PEG shedding from the surface has been shown for other nanoformulations.210
Both, the non-sheddable nanocage and the ROS-reactive stabilizer, are new
approaches of how to make the most of passive targeting.
What more could be done?
This doctoral thesis covered the thorough in vitro characterization involving size
stability, nanocrystal drug content and integrity after milling, polymer
characterization before and after cross-linking, sheddability, and oxidation. However,
in order to fulfill requirements for pharmaceutical application, these formulations
should be compared to benchmark formulations from the literature and more in vitro
characterization should be included, such as size stability measurements at elevated
temperatures, for longer durations, and at higher concentrations; interaction with cells
and cell uptake; and more investigation into the degradability of the polymeric
nanocage and the adsorption strength of the designed polymers in general. The in vivo
characterization of the pharmacokinetics and biodistribution of the formulated
nanocrystals is the next step to perform. The relevance of a non-sheddable coating for
tumor targeting with a conjugated ligand relies on a sufficiently long circulation time
of the nanocrystals. In theory, the PEG coating should protect the particles from rapid
clearance by the MPS. Adjustments in length and density of PEG on the crystal
surface can potentially help in achieving an optimal circulation time. Furthermore, the
dissolution velocity of paclitaxel nanocrystals enclosed by nanocages appeared to be
independent of the extent of cross-linking tested. Optimization in terms of coating
thickness can potentially prevent premature dissolution. Possible ways for increasing
coating thickness could include layer-by-layer assembly,23, 40 the adsorption and
cross-linking of more polymer layers, or the choice of a different stabilizer which
inherently covers the nanocrystal surface to a higher degree. Although the preparation
99
of a thicker coating on the nanocrystal might make the system more complex and less
elegant, it is nevertheless vital for a sufficient reduction in dissolution velocity to
enable a longer circulation time of the drug nanocrystal. Once a sufficiently long
circulation time for the nanocrystals has been achieved, further experiments with a
targeting ligand attached to the nanocage could be designed. New polymers amenable
for coupling to targeting ligands can be obtained by use of a heterotelechelic PEG as
macroinitiator for the polymerization of -propargyl--valerolactone and -
caprolactone. This polymer could be added at, for example, 5 to 10 wt. % of the
regular polymer amount during milling. After cross-linking and purification, a
targeting ligand could be attached through simple one step reactions such as the
reaction of a thiol group with a maleimide. This targeted nanocrystal formulation
could first be tested in vitro in cell culture for uptake and cytotoxic activity and then
in vivo for toxicity and efficacy in tumor-bearing mice.
Similar experiments should be designed for nanocrystals carrying the ROS sensitive
polymer. Here it would be important to assess the production of ROS in the tumor and
try to correlate it with the performance of the nanocrystal system. Additionally, the
selectivity of the ROS reactivity for the desired target tissue and the elimination of the
oxidized polymer should be studied. Further polymer modification may be necessary
as the conditions tested herein do not reflect the in vivo situation (high ROS
concentrations, closed system).
As discussed above, the diblock copolymers described in this work are intended to
be administered intravenously. PEG is known to be safe for i.v. administration and is
used in a variety of commercialized products.142, 211 However the coupled polyester
block has not been studied so far. Care needs to be taken that residual free alkyne
groups of -propargyl--valerolactone do not react in vivo with thiol-containing
molecules, which are vastly present as proteins such as enzymes or covering cell
surfaces. Indeed the successful cross-linking reaction with azides and
postpolymerization modification with thiols should in theory consume all available
alkyne groups. Otherwise a quenching reaction with a small alkane thiol (e.g.,
mercaptoethanol) could be included. The biodegradable nature of the polyester should
allow fast degradation into smaller fragments, which are expected to be readily
excretable through the kidneys. Studies need to be undertaken to characterize the
elimination and safety profile of these novel block copolymers.
100
The nanocrystal formulations presented herein have not been compared to other
benchmark formulations and a thorough evaluation is needed to see whether this
process is developable. To satisfy pharmaceutical quality aspects these formulations
need to show their potency, selectivity and safety. The milling process and subsequent
cross-linking would need to be done under sterile conditions for i.v. purposes. The
residual content, such as wear from milling or catalyst from polymer synthesis and
cross-linking, must be removed or reduced to below authorized thresholds. Many
parameters in the preparation would have to be optimized for a reliable production on
larger scale, such as milling apparatus, milling media composition, drug-to-polymer
ratio and concentration for input, separation of the nanocrystals from milling media
and from excess polymer, cross-linking conditions and purification, and so forth.
In conclusion, new routes for controlled nanocrystal size stabilization were
developed and studied by creating cross-linkable and ROS sensitive polymers. The
knowledge obtained from these studies could potentially help in the development of a
safe nanocrystal formulation for improved, because selective, chemotherapy of
cancer.
101
V. Supplementary Information
102
103
Figure S 1. 1H NMR spectra of -propargyl--valerolactone (1) in CDCl3 (A) and in
MeOD (B). The signals at 2.01 and 4.25 ppm confirm the presence of an acetylene
proton and an intact lactone ring, respectively. The acetylene peak was found to be
very solvent sensitive and moved to 2.33 ppm in MeOD.
104
Figure S 2. 1H NMR spectrum of -chloro--caprolactone. The proton signals at 4.63
and 4.23 ppm indicated oxidation of cyclohexanone to the lactone, while the signal at
4.80 ppm showed the incorporation of chlorine.
Figure S 3. 1H NMR spectrum of -azido--caprolactone (2). The proton signals at
4.40 and 4.12 ppm indicated incorporation of the azide and the lactone integrity,
respectively. The peak at 2.61 belongs to residual DMSO.
105
Figure S 4. 1H NMR spectrum of 3. The signal at 2.01 ppm showed successful
incorporation of -propargyl--valerolactone. Polymer composition was calculated
from signals at 2.01 ppm for -propargyl--valerolactone units and 1.31 ppm for -
caprolactone units using the signal from the methoxy group of mPEG at 3.38 ppm as
reference.
106
Figure S 5. 1H NMR spectrum 4. The proton signal at 2.01 ppm showed successful
incorporation of -propargyl--valerolactone units. Polymer composition was
calculated as for 3.
107
Figure S 6. 1H NMR spectrum of 5. The proton signal at 3.85 ppm indicated
successful incorporation of -azido--caprolactone units. Polymer composition was
calculated from signals at 3.85 ppm for -azido--caprolactone units (from which the 13C satellite from mPEG was removed) and 2.28 ppm for -caprolactone units using
the signal from the methoxy group of PEG at 3.38 ppm as reference.
108
Figure S 7. 1H NMR spectrum of polymer 13.
109
Figure S 8. 1H NMR spectrum of polymer 8.
Figure S 9. 1H NMR spectrum of polymer 9.
110
Figure S 10. 1H NMR spectrum of polymer 10.
Figure S 11. 1H NMR spectrum of polymer 11.
111
Figure S 12. 1H NMR spectrum of polymer 12. The polymer was purified by
extensive dialysis against 63% THF.
112
Figure S 13. 1H NMR spectrum of polymer 14.
Figure S 14. 1H NMR spectrum of polymer 15.
113
Figure S 15. 1H NMR spectrum of polymer 16. The polymer was purified by
extensive dialysis against 63% THF.
114
Figure S 16. 1H NMR spectrum of polymer 17.
115
Figure S 17. 1H NMR spectrum of polymer 18. The polymer was purified by
extensive dialysis against 63% THF.
116
Figure S 18. 1H–1H correlation spectrum of 14.
117
VI. Curriculum Vitae and Scientific Contributions
118
119
Kathrin FUHRMANN née Lüling born 20 Feburary 1981 in Hagen, Germany German citizen Doctoral Studies 09/2008−08/2013 Research project: Preparation of Drug Nanocrystals Stabilized by
Functionalized Polymeric Coatings, under supervision of Prof. Jean-Christophe Leroux, Institute of Pharmaceutical Sciences (IPW), ETH Zurich, Switzerland and co-supervised by Marc A. Gauthier, Énergie Matériaux Télécommunications Research Centre (INRS-EMT), Varennes, Canada
Undergraduate Studies and professional experience 07/2008
Licensure as pharmacist
12/2007−05/2008
Pharmacy internship at „Apotheke im Schweizer Viertel“ in Berlin, Germany
05/2007−10/2007 Research internship at the laboratory of Prof. Jean-Christophe Leroux, Faculty of Pharmacy, Université de Montréal, QC, Canada Research project: Preparation of Drug Nanocrystals by Femtosecond Laser Ablation
02/2006−03/2006 Research internship at the laboratory of Prof. Fritz Pragst, Institut f. Rechtsmedizin, Charité Berlin, Germany Research project: Untersuchung von Rohtabak auf seine enthaltenen Alkaloide
04/2003−04/2007 Undergraduate studies in pharmacy, Freie Universität Berlin, Germany
Original publications:
K Fuhrmann, A Połomska, C Aeberli, B Castagner, MA Gauthier, JC Leroux (2013) Modular Design of Redox-Responsive Stabilizers for Nanocrystals. ACS Nano, 7, 8243-8250. K Fuhrmann, JD Schulz, MA Gauthier, JC Leroux (2012) PEG Nanocages as Non-sheddable Stabilizers for Drug Nanocrystals. ACS Nano 6, 1667-1676. K Fuhrmann, MA Gauthier, JC Leroux (2010) Crosslinkable Polymers for Nanocrystal Stabilization. J. Control. Release 148, e12-e13. S Kenth, JP Sylvestre, K Fuhrmann, M Meunier, JC Leroux, (2011) Fabrication of Paclitaxel Nanocrystals by Femtosecond Laser Ablation and Fragmentation. J. Pharm. Sci. 100, 1022-1030.
120
Oral presentations K Fuhrmann, MA Gauthier, JC Leroux (2012), PEG Nanocages as Non-sheddable Stabilizers for Drug Nanocrystals, Swiss Galenic Meeting, ETH Zürich, Switzerland, K Fuhrmann, MA Gauthier, JC Leroux (2011), Stabilization of Drug Nanocrystals by Functionalized Polymeric Coatings, PharmSciFair, Prague, Czech Republic K Fuhrmann, MA Gauthier, JC Leroux (2011), Drug Nanocrystals Stabilized by Functionalized Polymeric Coatings, Doktorandentag (Doctoral Students Day), ETH Zürich, Switzerland K Fuhrmann, MA Gauthier, JC Leroux (2010), Functionalized Polymers for Nanocrystal Stabilization, Zürich-Geneva-Basle Meeting, University of Geneva, Switzerland K Lüling, JC Leroux (2009), Preparation of Drug Nanocrystals Stabilized by Polymeric Coatings, Zürich-Geneva Meeting, ETH Zürich, Switzerland Poster presentations K Fuhrmann, MA Gauthier, JC Leroux (2010), Cross-linkable Polymers for Nanocrystal Stabilization, Swiss Pharma Science Day, Bern, Switzerland K Fuhrmann, MA Gauthier, JC Leroux (2010), Cross-linking Polymers for Nanocrystal Stabilization, 3rd International NanoBio Conference 2010, Zurich, Switzerland K Fuhrmann, MA Gauthier, JC Leroux (2010), Cross-linkable Polymers for Nanocrystal Stabilization, 11th European Symposium on Controlled Drug Delivery, (ESCDD), Egmond aan Zee, Netherlands
121
VII. Acknowledgments
122
123
First of all, I would like to thank Prof. Jean-Christophe Leroux for the unique
opportunity to do doctoral studies in his research group. I am very thankful for his
great scientific input, constant availability, honesty, and fairness.
My sincerest thanks go to Prof. Marc A. Gauthier for his extensive scientific input,
great support in writing, and neverending motivation.
I thank Prof. Bruno Gander for accepting to be co-examiner of this doctoral thesis.
Furthermore, I am grateful for all his help with everyday matters and orientation in
the organization of ETH not only during the beginning but throughout my doctoral
studies.
I would like to thank the institution ETH for its innovativeness, management and
structure, and motivated employees. I enjoyed conducting research here.
Many thanks also go to Prof. Werner (Pharmaceutical analytics group, IPW) for the
extensive use of the FTIR instrument whenever needed.
I thank every former and present member of the Drug Formulation and Delivery
Group for the time spent inside and outside of the lab. Special thanks go to Dr. Paola
Luciani, Dr. Bastien Castagner, Dr. Nuria Bayo-Puxan, Dr. Marie-Hélène Dufresne,
Dr. Arnaud Felber, Dr. Vincent Forster, Dr. Gregor Fuhrmann, Dr. Davide Brambilla,
Dr. Soo Hyeon Lee, Jessica Schulz, Lorine Brülisauer, Athanasia Dasargyri, Estelle
Durantie, Yuhui Gong, Mattias Ivarsson, Monica Langfritz, Elena Moroz, Anna
Połomska, Maurizio Roveri, and Mi Liu for their advice and help.
Finally, I thank my friends and family for their support.
124
125
VIII. List of References
126
127
1. Lipinski, C. A. Drug-like Properties and the Causes of Poor Solubility and Poor Permeability. J. Pharmacol. Toxicol. Methods 2000, 44, 235-249.
2. Bhugra, C.; Pikal, M. J. Role of Thermodynamic, Molecular, and Kinetic Factors in Crystallization from the Amorphous State. J. Pharm. Sci. 2008, 97, 1329-1349.
3. Stella, V. J.; Nti-Addae, K. W. Prodrug Strategies to Overcome Poor Water Solubility. Adv. Drug Del. Rev. 2007, 59, 677-694.
4. Kawakami, K. Modification of Physicochemical Characteristics of Active Pharmaceutical Ingredients and Application of Supersaturatable Dosage Forms for Improving Bioavailability of Poorly Absorbed Drugs. Adv. Drug Del. Rev. 2012, 64, 480-495.
5. Gelderblom, H.; Verweij, J.; Nooter, K.; Sparreboom, A. Cremophor EL: The Drawbacks and Advantages of Vehicle Selection for Drug Formulation. Eur. J. Cancer 2001, 37, 1590-1598.
6. Weiszhár, Z.; Czúcz, J.; Révész, C.; Rosivall, L.; Szebeni, J.; Rozsnyay, Z. Complement Activation by Polyethoxylated Pharmaceutical Surfactants: Cremophor-EL, Tween-80 and Tween-20. Eur. J. Pharm. Sci. 2012, 45, 492-498.
7. Morshed, K. M.; Jain, S. K.; McMartin, K. E. Propylene Glycol-Mediated Cell Injury in a Primary Culture of Human Proximal Tubule Cells. Toxicol. Sci. 1998, 46, 410-417.
8. Petrou, E.; Iakovou, I.; Boutsikou, M.; Girasis, C.; Mavrogeni, S.; Pavlides, G. Acute Epigastric and Low Back Pain during Amiodarone Infusion; Is It the Drug or the Vehicle to Blame? Heart & Lung 2014, 43, 60-61.
9. Drummond, D. C.; Noble, C. O.; Hayes, M. E.; Park, J. W.; Kirpotin, D. B. Pharmacokinetics and In Vivo Drug Release Rates in Liposomal Nanocarrier Development. J. Pharm. Sci. 2008, 97, 4696-4740.
10. Yang, T.; Choi, M.-K.; Cui, F.-D.; Kim, J. S.; Chung, S.-J.; Shim, C.-K.; Kim, D.-D. Preparation and Evaluation of Paclitaxel-loaded PEGylated Immunoliposome. J. Control. Release 2007, 120, 169-177.
11. Gaucher, G.; Dufresne, M.-H.; Sant, V. P.; Kang, N.; Maysinger, D.; Leroux, J.-C. Block Copolymer Micelles: Preparation, Characterization and Application in Drug Delivery. J. Control. Release 2005, 109, 169-188.
12. Musumeci, T.; Ventura, C. A.; Giannone, I.; Ruozi, B.; Montenegro, L.; Pignatello, R.; Puglisi, G. PLA/PLGA Nanoparticles for Sustained Release of Docetaxel. Int. J. Pharm. 2006, 325, 172-179.
13. Merisko-Liversidge, E.; Liversidge, G. G. Nanosizing for Oral and Parenteral Drug Delivery: A Perspective on Formulating Poorly-water Soluble Compounds Using Wet Media Milling Technology. Adv. Drug Del. Rev. 2011, 63, 427-440.
14. Sun, B.; Yeo, Y. Nanocrystals for the Parenteral Delivery of Poorly Water-soluble Drugs. Curr. Opin. Solid State Mater. Sci. 2012, 16, 295-301.
128
15. Möschwitzer, J. P. Drug Nanocrystals in the Commercial Pharmaceutical Development Process. Int. J. Pharm. 2013, 453, 142-156.
16. Harrison, M.; Hahn, N.; Pili, R.; Oh, W.; Hammers, H.; Sweeney, C.; Kim, K.; Perlman, S.; Arnott, J.; Sidor, C., et al. A Phase II Study of 2-Methoxyestradiol (2ME2) NanoCrystal® Dispersion (NCD) in Patients with Taxane-refractory, Metastatic Castrate-resistant Prostate Cancer (CRPC). Invest. New Drugs 2011, 29, 1465-1474.
17. Tevaarwerk, A. J.; Holen, K. D.; Alberti, D. B.; Sidor, C.; Arnott, J.; Quon, C.; Wilding, G.; Liu, G. Phase I Trial of 2-Methoxyestradiol NanoCrystal Dispersion in Advanced Solid Malignancies. Clin. Cancer. Res. 2009, 15, 1460-1465.
18. Butcher, J.; Garg, S.; Kim, D.; Sharma, P. A Modified Approach to Predict Dissolution and Absorption of Polydisperse Powders. Pharm. Res. 2008, 25, 2309-2311.
19. Müller, R. H.; Peters, K. Nanosuspensions for the Formulation of Poorly Soluble Drugs: I. Preparation by a Size-reduction Technique. Int. J. Pharm. 1998, 160, 229-237.
20. Wu, Y.; Loper, A.; Landis, E.; Hettrick, L.; Novak, L.; Lynn, K.; Chen, C.; Thompson, K.; Higgins, R.; Batra, U., et al. The Role of Biopharmaceutics in the Development of a Clinical Nanoparticle Formulation of MK-0869: A Beagle Dog Model Predicts Improved Bioavailability and Diminished Food Effect on Absorption in Human. Int. J. Pharm. 2004, 285, 135-146.
21. Gao, L.; Liu, G.; Ma, J.; Wang, X.; Zhou, L.; Li, X. Drug Nanocrystals: In Vivo Performances. J. Control. Release 2012, 160, 418-430.
22. Cerdeira, A. M.; Gander, B.; Mazzotti, M. Role of Milling Parameters and Particle Stabilization on Nanogrinding of Drug Substances of Similar Mechanical Properties. Chem. Eng. Technol. 2011, 34, 1427-1438.
23. Lvov, Y. M.; Pattekari, P.; Zhang, X.; Torchilin, V. Converting Poorly Soluble Materials into Stable Aqueous Nanocolloids. Langmuir 2011, 27, 1212-1217.
24. Kenth, S.; Sylvestre, J.-P.; Fuhrmann, K.; Meunier, M.; Leroux, J.-C. Fabrication of Paclitaxel Nanocrystals by Femtosecond Laser Ablation and Fragmentation. J. Pharm. Sci. 2011, 100, 1022-1030.
25. Rabinow, B. E. Nanosuspensions in Drug Delivery. Nat. Rev. Drug Discov. 2004, 3, 785-796.
26. Peltonen, L.; Hirvonen, J. Pharmaceutical Nanocrystals by Nanomilling: Critical Process Parameters, Particle Fracturing and Stabilization Methods. J. Pharm. Pharmacol. 2010, 62, 1569-1579.
27. Gao, L.; Liu, G.; Ma, J.; Wang, X.; Zhou, L.; Li, X.; Wang, F. Application of Drug Nanocrystal Technologies on Oral Drug Delivery of Poorly Soluble Drugs. Pharm. Res. 2013, 30, 307-324.
129
28. Van Eerdenbrugh, B.; Vermant, J.; Martens, J. A.; Froyen, L.; Van Humbeeck, J.; Augustijns, P.; Van den Mooter, G. A Screening Study of Surface Stabilization During the Production of Drug Nanocrystals. J. Pharm. Sci. 2009, 98, 2091-2103.
29. Dhumal, R. S.; Biradar, S. V.; Yamamura, S.; Paradkar, A. R.; York, P. Preparation of Amorphous Cefuroxime Axetil Nanoparticles by Sonoprecipitation for Enhancement of Bioavailability. Eur. J. Pharm. Biopharm. 2008, 70, 109-115.
30. Sigfridsson, K.; Forssén, S.; Holländer, P.; Skantze, U.; de Verdier, J. A Formulation Comparison, Using a Solution and Different Nanosuspensions of a Poorly Soluble Compound. Eur. J. Pharm. Biopharm. 2007, 67, 540-547.
31. Chan, H.-K.; Kwok, P. C. L. Production Methods for Nanodrug Particles Using the Bottom-up Approach. Adv. Drug Del. Rev. 2011, 63, 406-416.
32. Ghosh, I.; Schenck, D.; Bose, S.; Liu, F.; Motto, M. Identification of Critical Process Parameters and Its Interplay with Nanosuspension Formulation Prepared by Top Down Media Milling Technology – A QbD Perspective. Pharm. Dev. Technol. 2013, 18, 719-729.
33. George, M.; Ghosh, I. Identifying the Correlation between Drug/Stabilizer Properties and Critical Quality Attributes (CQAs) of Nanosuspension Formulation Prepared by Wet Media Milling Technology. Eur. J. Pharm. Sci. 2013, 48, 142-152.
34. Liu, Y.; Huang, L.; Liu, F. Paclitaxel Nanocrystals for Overcoming Multidrug Resistance in Cancer. Mol. Pharm. 2010, 7, 863-869.
35. Smet, L.; Colin, P.; Ceelen, W.; Bracke, M.; Bocxlaer, J.; Remon, J.; Vervaet, C. Development of a Nanocrystalline Paclitaxel Formulation for Hipec Treatment. Pharm. Res. 2012, 29, 2398-2406.
36. Baert, L.; van ‘t Klooster, G.; Dries, W.; François, M.; Wouters, A.; Basstanie, E.; Iterbeke, K.; Stappers, F.; Stevens, P.; Schueller, L., et al. Development of a Long-acting Injectable Formulation with Nanoparticles of Rilpivirine (TMC278) for HIV Treatment. Eur. J. Pharm. Biopharm. 2009, 72, 502-508.
37. Alexis, F.; Pridgen, E.; Molnar, L. K.; Farokhzad, O. C. Factors Affecting the Clearance and Biodistribution of Polymeric Nanoparticles. Mol. Pharm. 2008, 5, 505-515.
38. Bertrand, N.; Leroux, J.-C. The Journey of a Drug-carrier in the Body: An Anatomo-physiological Perspective. J. Control. Release 2012, 161, 152-163.
39. Fuhrmann, K.; Schulz, J. D.; Gauthier, M. A.; Leroux, J.-C. PEG Nanocages as Non-sheddable Stabilizers for Drug Nanocrystals. ACS Nano 2012, 6, 1667-1676.
40. Agarwal, A.; Lvov, Y.; Sawant, R.; Torchilin, V. Stable Nanocolloids of Poorly Soluble Drugs with High Drug Content Prepared Using the Combination of Sonication and Layer-by-layer Technology. J. Control. Release 2008, 128, 255-260.
41. Becker, A. L.; Johnston, A. P. R.; Caruso, F. Layer-By-Layer-Assembled Capsules and Films for Therapeutic Delivery. Small 2010, 6, 1836-1852.
130
42. Jordan, M. A.; Wilson, L. Microtubules as a Target for Anticancer Drugs. Nat. Rev. Cancer 2004, 4, 253-265.
43. Dumontet, C.; Jordan, M. A. Microtubule-binding Agents: A Dynamic Field of Cancer Therapeutics. Nat. Rev. Drug Discov. 2010, 9, 790-803.
44. Kingston, D. G. I. The Shape of Things to Come: Structural and Synthetic Studies of Taxol and Related Compounds. Phytochemistry 2007, 68, 1844-1854.
45. Zhang, Z.; Mei, L.; Feng, S.-S. Paclitaxel Drug Delivery Systems. Expert Opin. Drug Deliv. 2013, 10, 325-340.
46. Windebank, A. J.; Grisold, W. Chemotherapy-induced Neuropathy. J. Peripher. Nerv. Syst. 2008, 13, 27-46.
47. Green, M. R.; Manikhas, G. M.; Orlov, S.; Afanasyev, B.; Makhson, A. M.; Bhar, P.; Hawkins, M. J. Abraxane®, a Novel Cremophor®-free, Albumin-bound Particle Form of Paclitaxel for the Treatment of Advanced Non-small-cell Lung Cancer. Ann. Oncol. 2006, 17, 1263-1268.
48. Desai, N. P.; Tao, C.; Yang, A.; Louie, L.; Soon-shiong, P., Formulations of Pharmacological Agents, Methods for the Preparation Thereof and Methods for the Use Thereof, United States Patent 8137684 B2, 2012
49. Gradishar, W. J.; Tjulandin, S.; Davidson, N.; Shaw, H.; Desai, N.; Bhar, P.; Hawkins, M.; O'Shaughnessy, J. Phase III Trial of Nanoparticle Albumin-Bound Paclitaxel Compared With Polyethylated Castor Oil-Based Paclitaxel in Women With Breast Cancer. J. Clin. Oncol. 2005, 23, 7794-7803.
50. Teneriello, M. G.; Tseng, P. C.; Crozier, M.; Encarnacion, C.; Hancock, K.; Messing, M. J.; Boehm, K. A.; Williams, A.; Asmar, L. Phase II Evaluation of Nanoparticle Albumin–Bound Paclitaxel in Platinum-Sensitive Patients With Recurrent Ovarian, Peritoneal, or Fallopian Tube Cancer. J. Clin. Oncol. 2009, 27, 1426-1431.
51. Stinchcombe, T.; Socinski, M.; Walko, C.; O’Neil, B.; Collichio, F.; Ivanova, A.; Mu, H.; Hawkins, M.; Goldberg, R.; Lindley, C., et al. Phase I and Pharmacokinetic Trial of Carboplatin and Albumin-bound Paclitaxel, ABI-007 (Abraxane®) on Three Treatment Schedules in Patients with Solid Tumors. Cancer Chemother. Pharmacol. 2007, 60, 759-766.
52. Ibrahim, N. K.; Samuels, B.; Page, R.; Doval, D.; Patel, K. M.; Rao, S. C.; Nair, M. K.; Bhar, P.; Desai, N.; Hortobagyi, G. N. Multicenter Phase II Trial of ABI-007, an Albumin-Bound Paclitaxel, in Women With Metastatic Breast Cancer. J. Clin. Oncol. 2005, 23, 6019-6026.
53. Hersh, E. M.; O'Day, S. J.; Ribas, A.; Samlowski, W. E.; Gordon, M. S.; Shechter, D. E.; Clawson, A. A.; Gonzalez, R. A Phase 2 Clinical Trial of nab-Paclitaxel in Previously Treated and Chemotherapy-naive Patients with Metastatic Melanoma. Cancer 2010, 116, 155-163.
131
54. Kola, I.; Landis, J. Can the Pharmaceutical Industry Reduce Attrition Rates? Nat. Rev. Drug Discov. 2004, 3, 711-716.
55. Paul, S. M.; Mytelka, D. S.; Dunwiddie, C. T.; Persinger, C. C.; Munos, B. H.; Lindborg, S. R.; Schacht, A. L. How to Improve R&D Productivity: The Pharmaceutical Industry's Grand Challenge. Nat. Rev. Drug Discov. 2010, 9, 203-214.
56. Kelloff, G. J.; Sigman, C. C. Cancer Biomarkers: Selecting the Right Drug for the Right Patient. Nat. Rev. Drug Discov. 2012, 11, 201-214.
57. Crawford, J.; Dale, D. C.; Lyman, G. H. Chemotherapy-induced Neutropenia. Cancer 2004, 100, 228-237.
58. Paus, R.; Haslam, I. S.; Sharov, A. A.; Botchkarev, V. A. Pathobiology of Chemotherapy-induced Hair Loss. Lancet Oncol. 2013, 14, e50-e59.
59. Rubenstein, E. B.; Peterson, D. E.; Schubert, M.; Keefe, D.; McGuire, D.; Epstein, J.; Elting, L. S.; Fox, P. C.; Cooksley, C.; Sonis, S. T. Clinical Practice Guidelines for the Prevention and Treatment of Cancer Therapy–induced Oral and Gastrointestinal Mucositis. Cancer 2004, 100, 2026-2046.
60. Fallowfield, L.; McGurk, R.; Dixon, M. Same Gain, Less Pain: Potential Patient Preferences for Adjuvant Treatment in Premenopausal Women with Early Breast Cancer. Eur. J. Cancer 2004, 40, 2403-2410.
61. Matsumura, Y.; Maeda, H. A New Concept for Macromolecular Therapeutics in Cancer Chemotherapy: Mechanism of Tumoritropic Accumulation of Proteins and the Antitumor Agent Smancs. Cancer Res. 1986, 46, 6387-6392.
62. Brown, J. M.; Giaccia, A. J. The Unique Physiology of Solid Tumors: Opportunities (and Problems) for Cancer Therapy. Cancer Res. 1998, 58, 1408-1416.
63. Yuan, F.; Dellian, M.; Fukumura, D.; Leunig, M.; Berk, D. A.; Torchilin, V. P.; Jain, R. K. Vascular Permeability in a Human Tumor Xenograft: Molecular Size Dependence and Cutoff Size. Cancer Res. 1995, 55, 3752-3756.
64. Pelicano, H.; Carney, D.; Huang, P. ROS Stress in Cancer Cells and Therapeutic Implications. Drug Resist. Update 2004, 7, 97-110.
65. Fang, J.; Seki, T.; Maeda, H. Therapeutic Strategies by Modulating Oxygen Stress in Cancer and Inflammation. Adv. Drug Del. Rev. 2009, 61, 290-302.
66. Papahadjopoulos, D.; Allen, T. M.; Gabizon, A.; Mayhew, E.; Matthay, K.; Huang, S. K.; Lee, K. D.; Woodle, M. C.; Lasic, D. D.; Redemann, C. Sterically Stabilized Liposomes: Improvements in Pharmacokinetics and Antitumor Therapeutic Efficacy. P. Natl. Acad. Sci. USA 1991, 88, 11460-11464.
67. Kang, N.; Perron, M.-È.; Prud'homme, R. E.; Zhang, Y.; Gaucher, G.; Leroux, J.-C. Stereocomplex Block Copolymer Micelles: Core−Shell Nanostructures with Enhanced Stability. Nano Lett. 2005, 5, 315-319.
132
68. Soppimath, K. S.; Aminabhavi, T. M.; Kulkarni, A. R.; Rudzinski, W. E. Biodegradable Polymeric Nanoparticles as Drug Delivery Devices. J. Control. Release 2001, 70, 1-20.
69. Barenholz, Y. Doxil® — The First FDA-approved Nano-drug: Lessons Learned. J. Control. Release 2012, 160, 117-134.
70. Singla, A. K.; Garg, A.; Aggarwal, D. Paclitaxel and Its Formulations. Int. J. Pharm. 2002, 235, 179-192.
71. Olson, R. D.; Mushlin, P. S. Doxorubicin Cardiotoxicity: Analysis of Prevailing Hypotheses. The FASEB Journal 1990, 4, 3076-3086.
72. Wu, L.; Zhang, J.; Watanabe, W. Physical and Chemical Stability of Drug Nanoparticles. Adv. Drug Del. Rev. 2011, 63, 456-469.
73. Junghanns, J.-U. A. H.; Müller, R. H. Nanocrystal Technology, Drug Delivery and Clinical Applications Int. J. Nanomed. 2008, 3, 295-309.
74. Muller, R. H.; Keck, C. M. Challenges and Solutions for the Delivery of Biotech Drugs – a Review of Drug Nanocrystal Technology and Lipid Nanoparticles. J. Biotechnol. 2004, 113, 151-170.
75. Choi, J.-Y.; Yoo, J. Y.; Kwak, H.-S.; Uk Nam, B.; Lee, J. Role of Polymeric Stabilizers for Drug Nanocrystal Dispersions. Curr. Appl. Phys. 2005, 5, 472-474.
76. Keck, C. M.; Muller, R. H. Drug Nanocrystals of Poorly Soluble Drugs Produced by High Pressure Homogenisation. Eur. J. Pharm. Biopharm. 2006, 62, 3-16.
77. Sylvestre, J.-P.; Tang, M.-C.; Furtos, A.; Leclair, G.; Meunier, M.; Leroux, J.-C. Nanonization of Megestrol Acetate by Laser Fragmentation in Aqueous Milieu. J. Control. Release 2011, 149, 273-280.
78. Chattopadhyay, P.; Gupta, R. B. Production of Antibiotic Nanoparticles Using Supercritical CO2 as Antisolvent with Enhanced Mass Transfer. Ind. Eng. Chem. Res. 2001, 40, 3530-3539.
79. Chattopadhyay, P.; Gupta, R. B. Production of Griseofulvin Nanoparticles Using Supercritical CO2 Antisolvent with Enhanced Mass Transfer. Int. J. Pharm. 2001, 228, 19-31.
80. Date, A. A.; Patravale, V. B. Current Strategies for Engineering Drug Nanoparticles. Curr. Opin. Colloid Interface Sci. 2004, 9, 222-235.
81. Merisko-Liversidge, E. M.; Liversidge, G. G. Drug Nanoparticles: Formulating Poorly Water-Soluble Compounds. Toxicol. Pathol. 2008, 36, 43-48.
82. Merisko-Liversidge, E.; Liversidge, G. G.; Cooper, E. R. Nanosizing: A Formulation Approach for Poorly-water-soluble Compounds. Eur. J. Pharm. Sci. 2003, 18, 113-120.
133
83. Van Eerdenbrugh, B.; Van den Mooter, G.; Augustijns, P. Top-down Production of Drug Nanocrystals: Nanosuspension Stabilization, Miniaturization and Transformation Into Solid Products. Int. J. Pharm. 2008, 364, 64-75.
84. Cerdeira, A. M.; Werner, I. A.; Mazzotti, M.; Gander, B. Simultaneous quantification of polymeric and surface active stabilizers of nanosuspensions by using near-infrared spectroscopy. Drug Dev. Ind. Pharm. 2012, 38, 1360-1370.
85. Cerdeira, A. M.; Mazzotti, M.; Gander, B. Miconazole Nanosuspensions: Influence of Formulation Variables on Particle Size Reduction and Physical Stability. Int. J. Pharm. 2010, 396, 210-218.
86. Ploehn, H. J.; Russel, W. B., Interactions Between Colloidal Particles and Soluble Polymers. In Advances in Chemical Engineering; James, W., Eds.; Academic Press: San Diego, 1990, pp 137-228.
87. Fuhrmann, K.; Połomska, A.; Aeberli, C.; Castagner, B.; Gauthier, M. A.; Leroux, J.-C. Modular Design of Redox-Responsive Stabilizers for Nanocrystals. ACS Nano 2013, 7, 8243-8250.
88. Shchekin, A. K.; Rusanov, A. I. Generalization of the Gibbs–Kelvin–Köhler and Ostwald–Freundlich Equations for a Liquid Film on a Soluble Nanoparticle. J. Chem. Phys. 2008, 129, 154116.
89. Hollis, C. P.; Weiss, H. L.; Leggas, M.; Evers, B. M.; Gemeinhart, R. A.; Li, T. Biodistribution and Bioimaging Studies of Hybrid Paclitaxel Nanocrystals: Lessons Learned of the EPR Effect and Image-guided Drug Delivery. J. Control. Release 2013, 172, 12-21.
90. Moreno-Aspitia, A.; Perez, E. A. North Central Cancer Treatment Group N0531: Phase II Trial of Weekly Albumin-Bound Paclitaxel (ABI-007; Abraxane®) in Combination with Gemcitabine in Patients with Metastatic Breast Cancer. Clin. Breast Cancer 2005, 6, 361-364.
91. Liu, F.; Park, J.-Y.; Zhang, Y.; Conwell, C.; Liu, Y.; Bathula, S. R.; Huang, L. Targeted Cancer Therapy with Novel High Drug-loading Nanocrystals. J. Pharm. Sci. 2010, 99, 3542-3551.
92. Shegokar, R.; Singh, K. K. Surface Modified Nevirapine Nanosuspensions for Viral Reservoir Targeting: In Vitro and In Vivo Evaluation. Int. J. Pharm. 2011, 421, 341-352.
93. Zhang, H.; Hollis, C. P.; Zhang, Q.; Li, T. Preparation and Antitumor Study of Camptothecin Nanocrystals. Int. J. Pharm. 2011, 415, 293-300.
94. Ben Zirar, S.; Astier, A.; Muchow, M.; Gibaud, S. Comparison of Nanosuspensions and Hydroxypropyl-β-cyclodextrin Complex of Melarsoprol: Pharmacokinetics and Tissue Distribution in Mice. Eur. J. Pharm. Biopharm. 2008, 70, 649-656.
95. Vergara, D.; Bellomo, C.; Zhang, X.; Vergaro, V.; Tinelli, A.; Lorusso, V.; Rinaldi, R.; Lvov, Y. M.; Leporatti, S.; Maffia, M. Lapatinib/Paclitaxel
134
Polyelectrolyte Nanocapsules for Overcoming Multidrug Resistance in Ovarian Cancer. Nanomedicine: NBM 2012, 8, 891-899.
96. Cho, Y. W.; Lee, J.; Lee, S. C.; Huh, K. M.; Park, K. Hydrotropic Agents for Study of In Vitro Paclitaxel Release from Polymeric Micelles. J. Control. Release 2004, 97, 249-257.
97. Kadiu, I.; Nowacek, A.; McMillan, J.; Gendelman, H. E. Macrophage Endocytic Trafficking of Antiretroviral Nanoparticles. Nanomedicine 2011, 6, 975-994.
98. Nowacek, A.; McMillan, J.; Miller, R.; Anderson, A.; Rabinow, B.; Gendelman, H. Nanoformulated Antiretroviral Drug Combinations Extend Drug Release and Antiretroviral Responses in HIV-1-Infected Macrophages: Implications for NeuroAIDS Therapeutics. J Neuroimmune Pharmacol 2010, 5, 592-601.
99. Nowacek, A. S.; Balkundi, S.; McMillan, J.; Roy, U.; Martinez-Skinner, A.; Mosley, R. L.; Kanmogne, G.; Kabanov, A. V.; Bronich, T.; Gendelman, H. E. Analyses of Nanoformulated Antiretroviral Drug Charge, Size, Shape and Content for Uptake, Drug Release and Antiviral Activities in Human Monocyte-derived Macrophages. J. Control. Release 2011, 150, 204-211.
100. Bui, D. T.; Nicolas, J.; Maksimenko, A.; Desmaele, D.; Couvreur, P. Multifunctional Squalene-based Prodrug Nanoparticles for Targeted Cancer Therapy. Chem. Commun. 2014, 50, 5336-5338.
101. Mouton, J. W.; van Peer, A.; de Beule, K.; Van Vliet, A.; Donnelly, J. P.; Soons, P. A. Pharmacokinetics of Itraconazole and Hydroxyitraconazole in Healthy Subjects After Single and Multiple Doses of a Novel Formulation. Antimicrob. Agents Chemother. 2006, 50, 4096-4102.
102. Tian, X.; Li, H.; Zhang, D.; Shen, J.; Jia, L.; Zheng, D.; Liu, G.; Hao, L.; Shen, Y.; Zhang, Q. Parenteral nanosuspension of a novel synthesized antitumor candidate: Investigation of tissue biodistributions and plasma pharmacokinetics. Colloids Surf. Physicochem. Eng. Aspects 2013, 436, 868-872.
103. Sharma, P.; Zujovic, Z. D.; Bowmaker, G. A.; Denny, W. A.; Garg, S. Evaluation of a Crystalline Nanosuspension: Polymorphism, Process Induced Transformation and In Vivo Studies. Int. J. Pharm. 2011, 408, 138-151.
104. Tian, X.; Li, H.; Zhang, D.; Liu, G.; Jia, L.; Zheng, D.; Shen, J.; Shen, Y.; Zhang, Q. Nanosuspension for parenteral delivery of a p-terphenyl derivative: Preparation, characteristics and pharmacokinetic studies. Colloids Surf. B. Biointerfaces 2013, 108, 29-33.
105. Ganta, S.; Paxton, J. W.; Baguley, B. C.; Garg, S. Formulation and Pharmacokinetic Evaluation of an Asulacrine Nanocrystalline Suspension for Intravenous Delivery. Int. J. Pharm. 2009, 367, 179-186.
106. Gao, L.; Zhang, D.; Chen, M.; Duan, C.; Dai, W.; Jia, L.; Zhao, W. Studies on Pharmacokinetics and Tissue Distribution of Oridonin Nanosuspensions. Int. J. Pharm. 2008, 355, 321-327.
135
107. Rabinow, B.; Kipp, J.; Papadopoulos, P.; Wong, J.; Glosson, J.; Gass, J.; Sun, C.-S.; Wielgos, T.; White, R.; Cook, C., et al. Itraconazole IV Nanosuspension Enhances Efficacy through Altered Pharmacokinetics in the Rat. Int. J. Pharm. 2007, 339, 251-260.
108. Shegokar, R.; Müller, R. H. Nanocrystals: Industrially feasible multifunctional formulation technology for poorly soluble actives. Int. J. Pharm. 2010, 399, 129-139.
109. Sanchez, V. C.; Pietruska, J. R.; Miselis, N. R.; Hurt, R. H.; Kane, A. B. Biopersistence and potential adverse health impacts of fibrous nanomaterials: what have we learned from asbestos? WIREs Nanomed Nanobiotechnol 2009, 1, 511-529.
110. Dintaman, J. M.; Silverman, J. A. Inhibition of P-Glycoprotein by D-α-Tocopheryl Polyethylene Glycol 1000 Succinate (TPGS). Pharm. Res. 1999, 16, 1550-1556.
111. Zhang, H.; Wang, X.; Dai, W.; Gemeinhart, R. A.; Zhang, Q.; Li, T. Pharmacokinetics and Treatment Efficacy of Camptothecin Nanocrystals on Lung Metastasis. Mol. Pharm. 2013, 11, 226-233.
112. Karmali, P. P.; Kotamraju, V. R.; Kastantin, M.; Black, M.; Missirlis, D.; Tirrell, M.; Ruoslahti, E. Targeting of Albumin-embedded Paclitaxel Nanoparticles to Tumors. Nanomedicine: NBM 2009, 5, 73-82.
113. Gardner, E. R.; Dahut, W. L.; Scripture, C. D.; Jones, J.; Aragon-Ching, J. B.; Desai, N.; Hawkins, M. J.; Sparreboom, A.; Figg, W. D. Randomized Crossover Pharmacokinetic Study of Solvent-Based Paclitaxel and nab-Paclitaxel. Clin. Cancer. Res. 2008, 14, 4200-4205.
114. Deng, J.; Huang, L.; Liu, F. Understanding the Structure and Stability of Paclitaxel Nanocrystals. Int. J. Pharm. 2010, 390, 242-249.
115. Parrish, B.; Breitenkamp, R. B.; Emrick, T. PEG- and Peptide-Grafted Aliphatic Polyesters by Click Chemistry. J. Am. Chem. Soc. 2005, 127, 7404-7410.
116. Kim, S.; Lee, J. Folate-targeted Drug-delivery Systems Prepared by Nano-comminution. Drug Dev. Ind. Pharm. 2011, 37, 131-138.
117. Trachootham, D.; Alexandre, J.; Huang, P. Targeting Cancer Cells by ROS-mediated Mechanisms: a Radical Therapeutic Approach? Nat. Rev. Drug Discov. 2009, 8, 579-591.
118. Lin, M. T.; Beal, M. F. Mitochondrial Dysfunction and Oxidative Stress in Neurodegenerative Diseases. Nature 2006, 443, 787-795.
119. Lu, Y.; Wang, Z.-h.; Li, T.; McNally, H.; Park, K.; Sturek, M. Development and Evaluation of Transferrin-stabilized Paclitaxel Nanocrystal Formulation. J. Control. Release 2014, 176, 76-85.
120. Ai, H.; Jones, S. A.; de Villiers, M. M.; Lvov, Y. M. Nano-encapsulation of Furosemide Microcrystals for Controlled Drug Release. J. Control. Release 2003, 86, 59-68.
136
121. Qiu, X.; Leporatti, S.; Donath, E.; Möhwald, H. Studies on the Drug Release Properties of Polysaccharide Multilayers Encapsulated Ibuprofen Microparticles. Langmuir 2001, 17, 5375-5380.
122. Sukhorukov, G. B.; Brumen, M.; Donath, E.; Möhwald, H. Hollow Polyelectrolyte Shells: Exclusion of Polymers and Donnan Equilibrium. J. Phys. Chem. B 1999, 103, 6434-6440.
123. Shutava, T. G.; Pattekari, P. P.; Arapov, K. A.; Torchilin, V. P.; Lvov, Y. M. Architectural Layer-by-layer Assembly of Drug Nanocapsules with PEGylated Polyelectrolytes. Soft Matter 2012, 8, 9418-9427.
124. Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.; Terada, Y.; Kano, M. R.; Miyazono, K.; Uesaka, M., et al. Accumulation of Sub-100 nm Polymeric Micelles in Poorly Permeable Tumours Depends on Size. Nat. Nanotechnol. 2011, 6, 815-823.
125. Wong, C.; Stylianopoulos, T.; Cui, J.; Martin, J.; Chauhan, V. P.; Jiang, W.; Popović, Z.; Jain, R. K.; Bawendi, M. G.; Fukumura, D. Multistage Nanoparticle Delivery System for Deep Penetration into Tumor Tissue. P. Natl. Acad. Sci. USA 2011, 108, 2426-2431.
126. Arias, J. L.; Reddy, L. H.; Couvreur, P. Magnetoresponsive Squalenoyl Gemcitabine Composite Nanoparticles for Cancer Active Targeting. Langmuir 2008, 24, 7512-7519.
127. Sugahara, K. N.; Teesalu, T.; Karmali, P. P.; Kotamraju, V. R.; Agemy, L.; Greenwald, D. R.; Ruoslahti, E. Coadministration of a Tumor-Penetrating Peptide Enhances the Efficacy of Cancer Drugs. Science 2010, 328, 1031-1035.
128. Aggarwal, P.; Hall, J. B.; McLeland, C. B.; Dobrovolskaia, M. A.; McNeil, S. E. Nanoparticle Interaction with Plasma Proteins as it Relates to Particle Biodistribution, Biocompatibility and Therapeutic Efficacy. Adv. Drug Del. Rev. 2009, 61, 428-437.
129. Lindfors, L.; Forssen, S.; Skantze, P.; Skantze, U.; Zackrisson, A.; Olsson, U. Amorphous Drug Nanosuspensions. 2. Experimental Determination of Bulk Monomer Concentrations. Langmuir 2005, 22, 911-916.
130. Meerum Terwogt, J. M.; Nuijen, B.; Ten Bokkel Huinink, W. W.; Beijnen, J. H. Alternative Formulations of Paclitaxel. Cancer Treat. Rev. 1997, 23, 87-95.
131. Fahr, A.; Liu, X. Drug Delivery Strategies for Poorly Water-soluble Drugs. Expert Opin. Drug Deliv. 2007, 4, 403-416.
132. Chan, J. M.; Zhang, L.; Tong, R.; Ghosh, D.; Gao, W.; Liao, G.; Yuet, K. P.; Gray, D.; Rhee, J.-W.; Cheng, J., et al. Spatiotemporal Controlled Delivery of Nanoparticles to Injured Vasculature. P. Natl. Acad. Sci. USA 2010, 107, 2213-2218.
133. Hayashi, Y.; Skwarczynski, M.; Hamada, Y.; Sohma, Y.; Kimura, T.; Kiso, Y. A Novel Approach of Water-Soluble Paclitaxel Prodrug with No Auxiliary and No Byproduct: Design and Synthesis of Isotaxel. J. Med. Chem. 2003, 46, 3782-3784.
137
134. Van, S.; Das, S. K.; Wang, X.; Feng, Z.; Jin, Y.; Hou, Z.; Chen, F.; Pham, A.; Jiang, N.; Howell, S. B., et al. Synthesis, Characterization, and Biological Evaluation of Poly(L-γ-glutamyl-glutamine)-paclitaxel Nanoconjugate. Int. J. Nanomed. 2010, 5, 825-837.
135. Dosio, F.; Reddy, L. H.; Ferrero, A.; Stella, B.; Cattel, L.; Couvreur, P. Novel Nanoassemblies Composed of Squalenoyl−Paclitaxel Derivatives: Synthesis, Characterization, and Biological Evaluation. Bioconjugate Chem. 2010, 21, 1349-1361.
136. Zhang, J. A.; Anyarambhatla, G.; Ma, L.; Ugwu, S.; Xuan, T.; Sardone, T.; Ahmad, I. Development and Characterization of a Novel Cremophor® EL Free Liposome-based Paclitaxel (LEP-ETU) Formulation. Eur. J. Pharm. Biopharm. 2005, 59, 177-187.
137. Liu, Z.; Chen, K.; Davis, C.; Sherlock, S.; Cao, Q.; Chen, X.; Dai, H. Drug Delivery with Carbon Nanotubes for In Vivo Cancer Treatment. Cancer Res. 2008, 68, 6652-6660.
138. Brewster, M. E.; Loftsson, T. Cyclodextrins as Pharmaceutical Solubilizers. Adv. Drug Del. Rev. 2007, 59, 645-666.
139. Le Garrec, D.; Gori, S.; Luo, L.; Lessard, D.; Smith, D. C.; Yessine, M.-A.; Ranger, M.; Leroux, J.-C. Poly(N-vinylpyrrolidone)-block-poly(D,L-lactide) as a New Polymeric Solubilizer for Hydrophobic Anticancer Drugs: In Vitro and In Vivo Evaluation. J. Control. Release 2004, 99, 83-101.
140. Li, C.; Wallace, S. Polymer-drug Conjugates: Recent Development in Clinical Oncology. Adv. Drug Del. Rev. 2008, 60, 886-898.
141. Kogan, A.; Garti, N. Microemulsions as Transdermal Drug Delivery Vehicles. Adv. Colloid Interface Sci. 2006, 123-126, 369-385.
142. Strickley, R. G. Solubilizing Excipients in Oral and Injectable Formulations. Pharm. Res. 2004, 21, 201-230.
143. Chiappetta, D. A.; Sosnik, A. Poly(ethylene oxide)-poly(propylene oxide) Block Copolymer Micelles as Drug Delivery Agents: Improved Hydrosolubility, Stability and Bioavailability of Drugs. Eur. J. Pharm. Biopharm. 2007, 66, 303-317.
144. Soussan, E.; Cassel, S.; Blanzat, M.; Rico-Lattes, I. Drug Delivery by Soft Matter: Matrix and Vesicular Carriers. Angew. Chem. Int. Ed. 2009, 48, 274-288.
145. Serajuddin, A. T. M. Solid Dispersion of Poorly Water-soluble Drugs: Early Promises, Subsequent Problems, and Recent Breakthroughs. J. Pharm. Sci. 1999, 88, 1058-1066.
146. Ibrahim, N. K.; Desai, N.; Legha, S.; Soon-Shiong, P.; Theriault, R. L.; Rivera, E.; Esmaeli, B.; Ring, S. E.; Bedikian, A.; Hortobagyi, G. N., et al. Phase I and Pharmacokinetic Study of ABI-007, a Cremophor-free, Protein-stabilized, Nanoparticle Formulation of Paclitaxel. Clin. Cancer. Res. 2002, 8, 1038-1044.
138
147. Turner, J. L.; Chen, Z.; Wooley, K. L. Regiochemical Functionalization of a Nanoscale Cage-like Structure: Robust Core-shell Nanostructures Crafted as Vessels for Selective Uptake and Release of Small and Large Guests. J. Control. Release 2005, 109, 189-202.
148. Lenoir, S.; Riva, R.; Lou, X.; Detrembleur, C.; Jérôme, R.; Lecomte, P. Ring-Opening Polymerization of α-Chloro-ε-caprolactone and Chemical Modification of Poly(α-chloro-ε-caprolactone) by Atom Transfer Radical Processes. Macromolecules 2004, 37, 4055-4061.
149. Kim, M. S.; Seo, K. S.; Khang, G.; Lee, H. B. Ring-Opening Polymerization of ε-Caprolactone by Poly(ethylene glycol) by an Activated Monomer Mechanism. Macromol. Rapid Commun. 2005, 26, 643-648.
150. Ghebeh, H.; Handa-Corrigan, A.; Butler, M. Development of an Assay for the Measurement of the Surfactant Pluronic F-68 in Mammalian Cell Culture Medium. Anal. Biochem. 1998, 262, 39-44.
151. Letchford, K.; Liggins, R.; Wasan, K. M.; Burt, H. In Vitro Human Plasma Distribution of Nanoparticulate Paclitaxel is Dependent on the Physicochemical Properties of Poly(ethylene glycol)-block-poly(caprolactone) Nanoparticles. Eur. J. Pharm. Biopharm. 2009, 71, 196-206.
152. Torchilin, V. P. Multifunctional Nanocarriers. Adv. Drug Del. Rev. 2006, 58, 1532-1555.
153. Ahmed, F.; Discher, D. E. Self-porating Polymersomes of PEG-PLA and PEG-PCL: Hydrolysis-triggered Controlled Release Vesicles. J. Control. Release 2004, 96, 37-53.
154. Riva, R.; Schmeits, S.; Stoffelbach, F.; Jérôme, C.; Jérôme, R.; Lecomte, P. Combination of Ring-opening Polymerization and "Click" Chemistry towards Functionalization of Aliphatic Polyesters. Chem. Commun. 2005, 42, 5334-5336.
155. Merisko-Liversidge, E.; Sarpotdar, P.; Bruno, J.; Hajj, S.; Wei, L.; Peltier, N.; Rake, J.; Shaw, J. M.; Pugh, S.; Polin, L., et al. Formulation and Antitumor Activity Evaluation of Nanocrystalline Suspensions of Poorly Soluble Anticancer Drugs. Pharm. Res. 1996, 13, 272-278.
156. Van Eerdenbrugh, B.; Vermant, J.; Martens, J. A.; Froyen, L.; Humbeeck, J. V.; Van den Mooter, G.; Augustijns, P. Solubility Increases Associated with Crystalline Drug Nanoparticles: Methodologies and Significance. Mol. Pharm. 2010, 7, 1858-1870.
157. Budijono, S. J.; Russ, B.; Saad, W.; Adamson, D. H.; Prud’homme, R. K. Block Copolymer Surface Coverage on Nanoparticles. Colloids Surf. Physicochem. Eng. Aspects 2010, 360, 105-110.
158. Pasche, S.; De Paul, S. M.; Vörös, J.; Spencer, N. D.; Textor, M. Poly(L-lysine)-graft-poly(ethylene glycol) Assembled Monolayers on Niobium Oxide Surfaces: A Quantitative Study of the Influence of Polymer Interfacial Architecture on Resistance
139
to Protein Adsorption by ToF-SIMS and in Situ OWLS. Langmuir 2003, 19, 9216-9225.
159. Turner, J. L.; Wooley, K. L. Nanoscale Cage-like Structures Derived from Polyisoprene-Containing Shell Cross-linked Nanoparticle Templates. Nano Lett. 2004, 4, 683-688.
160. Horn, D.; Rieger, J. Organic Nanoparticles in the Aqueous Phase—Theory, Experiment, and Use. Angew. Chem. Int. Ed. 2001, 40, 4330-4361.
161. Lindfors, L.; Skantze, P.; Skantze, U.; Rasmusson, M.; Zackrisson, A.; Olsson, U. Amorphous Drug Nanosuspensions. 1. Inhibition of Ostwald Ripening. Langmuir 2005, 22, 906-910.
162. Zhao, R.; Hollis, C. P.; Zhang, H.; Sun, L.; Gemeinhart, R. A.; Li, T. Hybrid Nanocrystals: Achieving Concurrent Therapeutic and Bioimaging Functionalities Toward Solid Tumors. Mol. Pharm. 2011, 8, 1985–1991.
163. Venkataraman, S.; Hedrick, J. L.; Ong, Z. Y.; Yang, C.; Ee, P. L. R.; Hammond, P. T.; Yang, Y. Y. The Effects of Polymeric Nanostructure Shape on Drug Delivery. Adv. Drug Del. Rev. 2011, 63, 1228-1246.
164. Gaucher, G.; Asahina, K.; Wang, J.; Leroux, J.-C. Effect of Poly(N-vinyl-pyrrolidone)-block-poly(D,L-lactide) as Coating Agent on the Opsonization, Phagocytosis, and Pharmacokinetics of Biodegradable Nanoparticles. Biomacromolecules 2009, 10, 408-416.
165. Cormode, D. P.; Skajaa, G. O.; Delshad, A.; Parker, N.; Jarzyna, P. A.; Calcagno, C.; Galper, M. W.; Skajaa, T.; Briley-Saebo, K. C.; Bell, H. M., et al. A Versatile and Tunable Coating Strategy Allows Control of Nanocrystal Delivery to Cell Types in the Liver. Bioconjugate Chem. 2011, 22, 353-361.
166. Schweiger, C.; Pietzonka, C.; Heverhagen, J.; Kissel, T. Novel Magnetic Iron Oxide Nanoparticles Coated with Poly(ethylene imine)-g-poly(ethylene glycol) for Potential Biomedical Application: Synthesis, Stability, Cytotoxicity and MR Imaging. Int. J. Pharm. 2011, 408, 130-137.
167. Romberg, B.; Hennink, W.; Storm, G. Sheddable Coatings for Long-Circulating Nanoparticles. Pharm. Res. 2008, 25, 55-71.
168. Sankaranarayanan, J.; Mahmoud, E. A.; Kim, G.; Morachis, J. M.; Almutairi, A. Multiresponse Strategies To Modulate Burst Degradation and Release from Nanoparticles. ACS Nano 2010, 4, 5930-5936.
169. Knipe, J. M.; Peters, J. T.; Peppas, N. A. Theranostic Agents for Intracellular Gene Delivery with Spatiotemporal Imaging. Nano Today 2013, 8, 21-38.
170. Motion, J. P. M.; Nguyen, J.; Szoka, F. C. Phosphatase-Triggered Fusogenic Liposomes for Cytoplasmic Delivery of Cell-Impermeable Compounds. Angew. Chem. Int. Ed. 2012, 51, 9047-9051.
140
171. Preston, T. J.; Abadi, A.; Wilson, L.; Singh, G. Mitochondrial Contributions to Cancer Cell Physiology: Potential for Drug Development. Adv. Drug Del. Rev. 2001, 49, 45-61.
172. de Gracia Lux, C.; Joshi-Barr, S.; Nguyen, T.; Mahmoud, E.; Schopf, E.; Fomina, N.; Almutairi, A. Biocompatible Polymeric Nanoparticles Degrade and Release Cargo in Response to Biologically Relevant Levels of Hydrogen Peroxide. J. Am. Chem. Soc. 2012, 134, 15758-15764.
173. Broaders, K. E.; Grandhe, S.; Fréchet, J. M. J. A Biocompatible Oxidation-Triggered Carrier Polymer with Potential in Therapeutics. J. Am. Chem. Soc. 2010, 133, 756-758.
174. Khutoryanskiy, V. V.; Tirelli, N. Oxidation-responsiveness of Nanomaterials for Targeting Inflammatory Reactions. Pure Appl. Chem. 2008, 80, 1703-1718.
175. Allen, B. L.; Johnson, J. D.; Walker, J. P. Encapsulation and Enzyme-Mediated Release of Molecular Cargo in Polysulfide Nanoparticles. ACS Nano 2011, 5, 5263-5272.
176. Rehor, A.; Hubbell, J. A.; Tirelli, N. Oxidation-Sensitive Polymeric Nanoparticles. Langmuir 2004, 21, 411-417.
177. Yu, S. S.; Koblin, R. L.; Zachman, A. L.; Perrien, D. S.; Hofmeister, L. H.; Giorgio, T. D.; Sung, H.-J. Physiologically Relevant Oxidative Degradation of Oligo(proline) Cross-Linked Polymeric Scaffolds. Biomacromolecules 2011, 12, 4357-4366.
178. Gao, W.; Langer, R.; Farokhzad, O. C. Poly(ethylene glycol) with Observable Shedding. Angew. Chem. Int. Ed. 2010, 49, 6567-6571.
179. Gauthier, M. A.; Gibson, M. I.; Klok, H.-A. Synthesis of Functional Polymers by Post-Polymerization Modification. Angew. Chem. Int. Ed. 2009, 48, 48-58.
180. Hoogenboom, R. Thiol–Yne Chemistry: A Powerful Tool for Creating Highly Functional Materials. Angew. Chem. Int. Ed. 2010, 49, 3415-3417.
181. Napoli, A.; Valentini, M.; Tirelli, N.; Muller, M.; Hubbell, J. A. Oxidation-responsive Polymeric Vesicles. Nat. Mater. 2004, 3, 183-189.
182. Szela, S.; Avtges, P.; Valluzzi, R.; Winkler, S.; Wilson, D.; Kirschner, D.; Kaplan, D. L. Reduction−Oxidation Control of β-Sheet Assembly in Genetically Engineered Silk. Biomacromolecules 2000, 1, 534-542.
183. Dado, G. P.; Gellman, S. H. Redox Control of Secondary Structure in a Designed Peptide. J. Am. Chem. Soc. 1993, 115, 12609-12610.
184. Carampin, P.; Lallana, E.; Laliturai, J.; Carroccio, S. C.; Puglisi, C.; Tirelli, N. Oxidant-Dependent REDOX Responsiveness of Polysulfides. Macromol. Chem. Phys. 2012, 213, 2052-2061.
141
185. Abraham, R. J.; Byrne, J. J.; Griffiths, L. 1H Chemical Shifts in NMR. Part 27: Proton Chemical Shifts in Sulfoxides and Sulfones and the Magnetic Anisotropy, Electric Field and Steric Effects of the SO Bond. Magn. Reson. Chem. 2008, 46, 667-675.
186. Mahmoud, E. A.; Sankaranarayanan, J.; Morachis, J. M.; Kim, G.; Almutairi, A. Inflammation Responsive Logic Gate Nanoparticles for the Delivery of Proteins. Bioconjugate Chem. 2011, 22, 1416-1421.
187. Alexandre, J.; Batteux, F.; Nicco, C.; Chéreau, C.; Laurent, A.; Guillevin, L.; Weill, B.; Goldwasser, F. Accumulation of Hydrogen Peroxide is an Early and Crucial Step for Paclitaxel-induced Cancer Cell Death Both In Vitro and In Vivo. Int. J. Cancer 2006, 119, 41-48.
188. Alexandre, J.; Hu, Y.; Lu, W.; Pelicano, H.; Huang, P. Novel Action of Paclitaxel against Cancer Cells: Bystander Effect Mediated by Reactive Oxygen Species. Cancer Res. 2007, 67, 3512-3517.
189. Duncan, R.; Gaspar, R. Nanomedicine(s) under the Microscope. Mol. Pharm. 2011, 8, 2101-2141.
190. Basiruddin, S. K.; Saha, A.; Pradhan, N.; Jana, N. R. Advances in Coating Chemistry in Deriving Soluble Functional Nanoparticle. J. Phys. Chem. C 2010, 114, 11009-11017.
191. Yu, S.; Scherer, R.; Ortega, R.; Bell, C.; O'Neil, C.; Hubbell, J.; Giorgio, T. Enzymatic- and Temperature-sensitive Controlled Release of Ultrasmall Superparamagnetic Iron Oxides (USPIOs). J. Nanobiotechnol. 2011, 9, 7.
192. Mailänder, V.; Landfester, K. Interaction of Nanoparticles with Cells. Biomacromolecules 2009, 10, 2379-2400.
193. Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. In Vivo Imaging of Quantum Dots Encapsulated in Phospholipid Micelles. Science 2002, 298, 1759-1762.
194. Schottenfeld, D.; Beebe-Dimmer, J. L.; Buffler, P. A.; Omenn, G. S. Current Perspective on the Global and United States Cancer Burden Attributable to Lifestyle and Environmental Risk Factors. Annu. Rev. Public Health 2013, 34, 97-117.
195. Gatta, G.; Mallone, S.; van der Zwan, J. M.; Trama, A.; Siesling, S.; Capocaccia, R.; Group, t. E. W. Cancer Prevalence Estimates in Europe at the Beginning of 2000. Ann. Oncol. 2013, 24, 1660-1666.
196. Maddams, J.; Utley, M.; Moller, H. Projections of Cancer Prevalence in the United Kingdom, 2010-2040. Br. J. Cancer 2012, 107, 1195-1202.
197. Mao, J. J.; Bowman, M. A.; Stricker, C. T.; DeMichele, A.; Jacobs, L.; Chan, D.; Armstrong, K. Delivery of Survivorship Care by Primary Care Physicians: The Perspective of Breast Cancer Patients. J. Clin. Oncol. 2009, 27, 933-938.
142
198. Jefford, M.; Rowland, J.; Grunfeld, E.; Richards, M.; Maher, J.; Glaser, A. Implementing Improved Post-treatment Care for Cancer Survivors in England, with Reflections from Australia, Canada and the USA. Br. J. Cancer 2013, 108, 14-20.
199. Danhier, F.; Feron, O.; Préat, V. To Exploit the Tumor Microenvironment: Passive and Active Tumor Targeting of Nanocarriers for Anti-cancer Drug Delivery. J. Control. Release 2010, 148, 135-146.
200. Hrkach, J.; Von Hoff, D.; Ali, M. M.; Andrianova, E.; Auer, J.; Campbell, T.; De Witt, D.; Figa, M.; Figueiredo, M.; Horhota, A., et al. Preclinical Development and Clinical Translation of a PSMA-Targeted Docetaxel Nanoparticle with a Differentiated Pharmacological Profile. Sci. Transl. Med. 2012, 4, 128ra139.
201. Chen, T.; Öçsoy, I.; Yuan, Q.; Wang, R.; You, M.; Zhao, Z.; Song, E.; Zhang, X.; Tan, W. One-Step Facile Surface Engineering of Hydrophobic Nanocrystals with Designer Molecular Recognition. J. Am. Chem. Soc. 2012, 134, 13164-13167.
202. Ferrari, M. Cancer Nanotechnology: Opportunities and Challenges. Nat. Rev. Cancer 2005, 5, 161-171.
203. Nagamitsu, A.; Greish, K.; Maeda, H. Elevating Blood Pressure as a Strategy to Increase Tumor-targeted Delivery of Macromolecular Drug SMANCS: Cases of Advanced Solid Tumors. Jap. J. Clin. Oncol. 2009, 39, 756-766.
204. Lammers, T.; Kiessling, F.; Hennink, W. E.; Storm, G. Drug Targeting to Tumors: Principles, Pitfalls and (Pre-) Clinical Progress. J. Control. Release 2012, 161, 175-187.
205. Jain, R. K.; Stylianopoulos, T. Delivering Nanomedicine to Solid Tumors. Nat. Rev. Clin. Oncol. 2010, 7, 653-664.
206. O’Brien, M. E. R.; Wigler, N.; Inbar, M.; Rosso, R.; Grischke, E.; Santoro, A.; Catane, R.; Kieback, D. G.; Tomczak, P.; Ackland, S. P., et al. Reduced Cardiotoxicity and Comparable Efficacy in a Phase III Trial of Pegylated Liposomal Doxorubicin HCl (CAELYX™/Doxil®) versus Conventional Doxorubicin for First-line Treatment of Metastatic Breast Cancer. Ann. Oncol. 2004, 15, 440-449.
207. Northfelt, D. W.; Dezube, B. J.; Thommes, J. A.; Miller, B. J.; Fischl, M. A.; Friedman-Kien, A.; Kaplan, L. D.; Du Mond, C.; Mamelok, R. D.; Henry, D. H. Pegylated-liposomal Doxorubicin versus Doxorubicin, Bleomycin, and Vincristine in the Treatment of AIDS-related Kaposi's Sarcoma: Results of a Randomized Phase III Clinical Trial. J. Clin. Oncol. 1998, 16, 2445-2451.
208. Arrieta, O.; Medina, L. A.; Estrada-Lobato, E.; Hernandez-Pedro, N.; Villanueva-Rodriguez, G.; Martinez-Barrera, L.; Macedo, E. O.; Lopez-Rodriguez, V.; Motola-Kuba, D.; Corona-Cruz, J. F. First-line Chemotherapy with Liposomal Doxorubicin plus Cisplatin for Patients with Advanced Malignant Pleural Mesothelioma: Phase II Trial. Br. J. Cancer 2012, 106, 1027-1032.
209. Bae, K. H.; Choi, S. H.; Park, S. Y.; Lee, Y.; Park, T. G. Thermosensitive Pluronic Micelles Stabilized by Shell Cross-Linking with Gold Nanoparticles. Langmuir 2006, 22, 6380-6384.
143
210. Suresh, A. K.; Weng, Y.; Li, Z.; Zerda, R.; Van Haute, D.; Williams, J. C.; Berlin, J. M. Matrix metalloproteinase-triggered denuding of engineered gold nanoparticles for selective cell uptake. Journal of Materials Chemistry B 2013, 1, 2341-2349.
211. Alconcel, S. N. S.; Baas, A. S.; Maynard, H. D. FDA-approved Poly(ethylene glycol)-protein Conjugate Drugs. Polym. Chem. 2011, 2, 1442-1448.
