Caruso Caffo Raudino Tomasello Nanoparticles and Brain Tumor Treatment

8/10/2019 Caruso Caffo Raudino Tomasello Nanoparticles and Brain Tumor Treatment

1/111


2/111


3/111

2012, ASME, 3 Park Avenue, New York, NY 10016, USA (www.asme.org)

All rights reserved. Printed in the United States of America. Except as permitted under

the United States Copyright Act of 1976, no part of this publication may be reproducedor distributed in any form or by any means, or stored in a database or retrieval system,without the prior written permission of the publisher.

Co-published by Momentum Press, LLC, 222 E. 46th Street, #203, New York,NY 10017, USA (www.momentumpress.net)

INFORMAION CONAINED IN HIS WORK HAS BEEN OBAINEDBY HE AMERICAN SOCIEY OF MECHANICAL ENGINEERS FROM

SOURCES BELIEVED O BE RELIABLE. HOWEVER, NEIHER ASMENOR IS AUHORS OR EDIORS GUARANEE HE ACCURACYOR COMPLEENESS OF ANY INFORMAION PUBLISHED IN HISWORK. NEIHER ASME NOR IS AUHORS AND EDIORS SHALLBE RESPONSIBLE FOR ANY ERRORS, OMISSIONS, OR DAMAGESARISING OU OF HE USE OF HIS INFORMAION. HE WORK ISPUBLISHED WIH HE UNDERSANDING HA ASME AND ISAUHORS AND EDIORS ARE SUPPLYING INFORMAION BU ARENO AEMPING O RENDER ENGINEERING OR OHER PROFES-SIONAL SERVICES. IF SUCH ENGINEERING OR PROFESSIONALSERVICES ARE REQUIRED, HE ASSISANCE OF AN APPROPRIAEPROFESSIONAL SHOULD BE SOUGH.

ASME shall not be responsible for statements or opinions advanced in papers or . . .printed in its publications (B7.1.3). Statement from the Bylaws.

For authorization to photocopy material for internal or personal use under thosecircumstances not falling within the fair use provisions of the Copyright Act, contactthe Copyright Clearance Center (CCC), 222 Rosewood Drive, Danvers, MA 01923,

tel: 978-750-8400, www.copyright.com.

Requests for special permission or bulk reproduction should be addressed to the ASMEPublishing Department, or submitted online at: http://www.asme.org/kb/books/book-proposal-guidelines/permissions.

ASME Press books are available at special quantity discounts to use as premiums orfor use in corporate training programs. For more information, contact Special Sales [email protected].

A catalog record is available from the Library of Congress.

(Print) ISBN: 978-0-7918-6003-8ASME Order No.: 860038(Electronic) ISBN: 978-1-6065 -408-60


4/111

Series Editors Preface

Biomedical and Nanomedical Technologies (B&NT)Tis concise monograph series focuses on the implementation of variousengineering principles in the conception, design, development, analysis andoperation of biomedical, biotechnological and nanotechnology systems andapplications. Te primary objective of the series is to compile the latest re-search topics in biomedical and nanomedical technologies, specifically de-vices and materials.

Each volume comprises a collection of invited manuscripts, written inan accessible manner and of a concise and manageable length. Tese timelycollections will provide an invaluable resource for initial enquiries abouttechnologies, encapsulating the latest developments and applications withreference sources for further detailed information. Te content and formathave been specifically designed to stimulate further advances and applica-tions of these technologies by reaching out to the non-specialist across abroad audience.

Contributions to Biomedical and Nanomedical echnologies will inspireinterest in further research and development using these technologies andencourage other potential applications. Tis will foster the advancement ofbiomedical and nanomedical applications, ultimately improving healthcaredelivery.

Editor:Ahmed Al-Jumaily, PhD, Professor of Biomechanical Engineering &Director of the Institute of Biomedical echnologies, Auckland Universityof echnology.

Associate Editors:Waqar Ahmed, PhD, Chair, Nanotechnology and Advanced Manufac-turing, and Head, Institute of Nanotechnology and Bioengineering, Schoolof Computing, Engineering & Physical Sciences, University of CentralLancashire, UK.

Christopher H.M. Jenkins, PhD, PE, Professor and Head, Mechanical &

Industrial Engineering Department, Montana State University.


5/111


6/111

C

1. Introduction 12. Glioma biology 4 2.1 Invasion and angiogenesis 43. Blood-brain barrier 9 3.1 Blood-brain barrier physiology 9 3.2 Blood-brain barrier transport systems 114. Nanomedicine and nanotechnology 14 4.1 Nanoparticle drug delivery 19 4.1.1 Nanoparticle distribution 20

4.1.2 Nanoparticle functionalization 21 4.1.3 Nanoparticle targeting 23 4.2 Nanomedicine and cancer 25 4.3 Nanomedicine and toxicity 305. Nanoparticle technologies 33 5.1 Polymeric and polymer-drug conjugate nanoparticles 33 5.2 Micelle nanoparticles 35 5.3 Liposomes 37 5.4 Gold and silver nanoparticles 39

5.5 Metal oxide 41 5.6 Magnetic nanoparticles 42 5.7 Carbon nanotubes 43 5.8 Fullerenes 44 5.9 Peptides nanoparticles 45 5.10 Silica nanoparticles 46 5.11 Quantum dots 48 5.12 Dendrimers 496. Nanomedicine applications in brain tumors 51

6.1 Brain tumor drug targeting 55 6.1.1 Systemic approaches 55 6.1.2 Physiological approaches 56 6.1.2.1 Receptor-mediated transcytosis 57 6.1.2.2 Adsorptive-mediated transcytosis 58 6.1.2.3 Efflux pump inhibition 60 6.1.2.4 Cell-mediated drug transport 61 6.1.3 Direct CNS approaches 61 6.1.3.1 Intracerebral routes 65

6.1.4 Drug modifications and prodrugs 667. Experimental studies 698. Conclusions 77References 81


7/111


8/111

Abstract

Despite progresses in surgery, radiotherapy, and in chemotherapy, an effec-tive curative treatment of gliomas does not yet exist. Mortality is still closeto 100% and the average survival of patients with GBM is less than 1 year.Te efficacy of current anti-cancer strategies in brain tumors is limited bythe lack of specific therapies against malignant cells. Besides, the deliveryof the drugs to brain tumors is limited by the presence of the blood brainbarrier. Te oncogenesis of gliomas is characterized by several biologicalprocesses and genetic alterations, involved in the neoplastic transformation.Te modulation of gene expression to more levels, such as DNA, mRNA,

proteins and transduction signal pathways, may be the most effective mo-dality to down-regulate or silence some specific gene functions. Gliomas arecharacterized by extensive microvascular proliferation and a higher degreeof vasculature. In malignant gliomas targeted therapies efficacy is low. Inthis complex field, it seems to be very important to improve specific selectivedrugs delivery systems. Drugs, antisense oligonucleotides, small interferenceRNAs, engineered monoclonal antibodies and other therapeutic moleculesmay diffuse into CNS overcoming the BBB. Nanotechnology could be usedboth to improve the treatment efficacy and to reduce the adverse side effects.

Nanotechnology-based approaches to targeted delivery of drugs across theBBB may potentially be engineered to carry out specific functions as needed.Moreover, nanoparticles show tumor-specific targeting and long blood cir-culation time, with consequent low-short-term toxicity. Nanotechnologydeals with structures and devices that are emerging as a new field of re-search at the interface of science, engineering and medicine. Nanomedicine,the application of nanotechnology to healthcare, holds great promise forrevolutionizing medical treatments, imaging, faster diagnosis, drug deliveryand tissue regeneration. Tis technology has enabled the development of

nanoscale device that can be conjugated with several functional moleculesincluding tumor-specific ligands, antibodies, anticancer drugs, and imag-ing probes. Nanoparticle systems are, also emerging as potential vectors forbrain delivery, able to overcome the difficulties of the classical strategies. Byusing nanotechnology it is possible to deliver the drug to the targeted tis-sue across the BBB, release the drug at the controlled rate, and avoid fromdegradation processes. At the same time, it is also necessary to retain thedrug stability and ensure that early degradation of drugs from the nano-carriers does not take place. Large amounts of small molecules, such as

contrast agents or drugs, can be loaded into NPs via a variety of chemicalmethods including encapsulation, adsorption, and covalent linkage. Mosttargeting molecules can be added to the surface of NPs to improve targetingthrough a concept defined as surface-mediated multivalent affinity effects.


9/111

viii N B T T

Te future challenges may be the possibility to modify the cell genome andinduce it to a reversion to the wild-type conditions and the enhancing of im-mune system anti-tumor capacity. Recent advances in molecular, biologicaland genetic diagnostic techniques have begun to explore cerebral glioma-associated biomarkers and their implications for gliomas development andprogression. Realization of targeted therapies depends on expression of thetargeted molecules, which can also provide as specific biomarkers. Te de-velopment of multifunctional NPs may contribute to the achievement oftargeted therapy in glioma treatment.


10/111

1. Introduction

Gliomas are the most common primary brain tumors in adults, with aworldwide incidence of approximately 7 out of 100,000 individuals per year.Although brain tumors constitute only a small proportion of overall humanmalignancies, they carry high rates of morbidity and mortality. Mortalityis still close to 100% and the average survival of patients with glioblastomamultiforme (GBM) is less than 1 year when classical treatment is used.Recent progress in multimodal treatment of this disease has led to only aslight increase in average survival up to 1518 months. Te effectiveness ofthe actual chemotherapeutic approach and multimodal targeted therapiesremains modest in gliomas.

Gliomas are brain tumors with histological, immunohistochemical andultra structural features of glial differentiation. Approximately 50% of pri-mary brain tumors are gliomas, arising from astrocytes, oligodendrocytes,or their precursors and ependymal cells. Gliomas are classified from I toIV according to the World Health Association (WHO) malignancy scale.Grade I gliomas are benign with a slow proliferation rate and include py-locitic astrocytoma most common in pediatric age. Grade II gliomas arecharacterized by a high degree of cellular differentiation and grow diffuselyinto the normal brain parenchyma and are prone to malignant progression.Tey include astrocytoma, oligodendroglioma and oligoastrocytoma. GradeIII lesions include anaplastic astrocytoma, anaplastic oligoastrocytoma andanaplastic oligodendroglioma. Tese tumors show a higher cellular densityand a notable presence of atypia and mitotic cells. Grade IV tumors are themost malignant and also the most frequent gliomas and include glioblas-toma and gliosarcoma. Tese tumors presented microvascular proliferationsand pseudopalisading necrosis.

Conventional brain tumor treatments include surgery, radiation therapyand chemotherapy. Surgical treatment is invasive but represents the firstapproach for the vast majority of brain tumors due to difficulties arisingin early stage detection. However, after surgical resection, the residual poolof invasive cells rises to recurrent tumor which, in 96% of cases arise ad-

jacent to the resection margins [1]. Aggressive treatment modalities haveextended the median survival from 4 months to 1 year, but the survival isoften associated with significant impairment in the quality of life. Radiationtherapy and chemotherapy are non-invasive options often used as adjuvanttherapy, but may also be effective for curing early-stage tumors. In patientswith recurrent GBM, the 6-months progression-free survival is only 21%after treatment with temozolomide [2]. Adjuvant radiotherapy gives limitedbenefits and causes debilitation side effects which reduce its efficacy [3]. Teeffectiveness of systemic chemotherapy is limited by toxic effects on healthycells, generally resulting in morbidity or mortality of the patient. Moreover,the presence of the BBB limits the passage of a wide variety of anticancer


11/111

2 N B T T

agents. Te high incidence of recurrence and poor prognosis of malignantgliomas compel the development of more powerful anti-cancer treatments.Te compromise of the quality of remaining life as well as the limited suc-cess of current treatment options in shrinking tumors, raise increasingconcerns about the adverse effects of cancer treatment on brain function.Deterioration in neurological function is accompanied by significant deteri-oration in the global quality of life in patients affected by malignant gliomas.Te advent of molecular studies allows evaluation of the possibility of re-examination of the biology of gliomas with, a level of precision that prom-ises interesting advances toward the development of specific and effectivetherapies. It is now generally understood that tumor genesis occurs either,

by over-expression of oncogenes, or inactivation of tumor suppressor genes.Te modulation of gene expression at more levels, such as DNA, mRNA,proteins and transduction signal pathways, may be the most effective mo-dality to down-regulate or silence some specific gene functions.

Cerebral gliomas represent an important challenge in modern oncology,and only in the last years has the development of new multimodal thera-peutic strategies given the beginning to a new research field of neuroon-cology: nanotechnology and nanomedicine. With the advancement in BBBstructure and pathophysiology knowledge, brain delivery and targeting

skills, and brain tumor biology, these new interesting possibilities couldlead to new perspectives in brain tumor treatment. Nanotechnology is anemerging field that deals with interactions between molecules, cells andengineered substances such as molecular fragments, atoms and molecules.Te impact of nanotechnology in medicine can mainly be seen in diagnosticmethods, drug-release techniques and regenerative medicine. In the recentpast, nanotechnology has garnered much attention due to its potential ap-plication in cancer, and the National Cancer Institute has constituted anAlliance of Nanotechnology in Cancer with focus on the development of

novel nanoplatform-based diagnostics, therapeutics and preventive agents.Nanomedicine could lead to new possibilities to overcome important prob-lems in malignant brain tumors, such as the non specificity of cancer cellsdrug-delivering and targeting, as well as the non complete passage of drugsthrough the BBB and into cancer cells avoiding side effects in normal braintissue. Nanoparticles are colloidal particles typically synthesized in eitheraqueous or organic phases. Due to their small size, nanoparticles can easilyflow through blood capillaries and enter the target cancer cells. Reductionof toxicity to peripheral organs can also be achieved with these systems

[4]. Nanoparticle-based drug-delivery systems, an antisense approach tomodify gene expression in cancer cell genome, and molecular-based cancercell targeting all represent important possibilities in cerebral gliomas treat-ment. Nanosystems with different compositions and biological propertieshave been extensively investigated for drug and gene delivery applications[45]. Te type and the number of linkers within and on the surface of


12/111

I 3

nanoparticles and the size of the nanoparticle itself can be modulated tocontrol the loading/releasing of the encapsulated or covalently linked drugcomponents or to add surface coating. Moreover, they can improve the effi-cacy of existing imaging and treatment regimens. Te ability to deliver con-trast or therapeutic agents selectively to tumors at effective concentrationsis a key factor for the efficacy of cancer detection and therapy. Additionally,encapsulation of drugs within nanoplatforms can provide a significant ad-vantage when employing poorly soluble, poorly absorbed or labile agents byincorporating them in the matrix of the nanoparticle during the formula-tion/synthetic process.

Tis study presents a review of the recent studies of nanoparticle systems

in cerebral gliomas treatment with a particular emphasis on the develop-ment of nanocarrier drug delivery systems for brain cancer therapy appli-cations. Tese technologies include polymeric and polymer-drug conjugatenanoparticles, micelle nanoparticles, liposomes, metallic and magneticnanoparticles, metal oxide, carbon derivates, peptide nanoparticles, inor-ganic nanopaerticles, quantum dots, and dendrimers.


13/111

2. Glioma biology

Genomic DNA aberrations are key genetic events in gliomagenesis.Recurrent genomic regions of alteration, including net gains and losses,have been found in gliomas. Whereas some of these regions containknown oncogenes and tumor suppressor genes, the biologically relevantgenes within other regions remain to be identified. Te phenotypic andgenotypic heterogeneity indicate that no isolated genetic event accountsfor gliomagenesis, but rather the cumulative effects of a number of alter-ations that operate in a concerted manner. In this pathological processare included various biological events, such as activation of growth factorreceptor signaling pathways, down-regulation of many apoptotic mecha-nisms, and imbalance of pro- and anti-angiogenic factors. Several growthfactor receptors, such epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDRGF), C-Kit, vascular endothelialgrowth factor receptor (VEGFR) are over-expressed, amplified and/ormutated in gliomas (Figure 2-1). In able 2-1 are summarized the mostcommon glioma genetic alterations frequently found. In the light of thisnovel information, the modulation of gene expression at more levels, suchas DNA, mRNA, proteins and transduction signal pathways, may repre-sent the most effective modality to down-regulate or silence some specificgenic functions or introduce genes, down-regulated or deleted selectively,into neoplastic cells.

2.1 Invasion and angiogenesis

Glioma cell invasion consists of an active translocation of glioma cellsthrough host cellular and extracellular matrix barriers [67]. Cerebralgliomas show a unique pattern of invasion and with rare exceptions do notmetastasize outside of the brain. How invasive glioma cells survive in thesetting of invasion, evading immune detection, and deferring commitment

to proliferation, remains unknown. Invading glioma cells normally migrateto distinct anatomical structures. Tese structures include the basementmembrane (BM) of blood vessels, the subependymal space, the glial limi-tans externa, and parallel and intersecting nerve fibre tracts in the whitematter. Glioma cells adhesion to proteins of the surrounding extracellularmatrix (ECM), degradation of ECM components by proteases secretionby neoplastic cells and migration of glioma cells are fundamental phases inthis process. ECM is composed of proteoglycans, glycoproteins, and colla-gens and also contains fibronectin, laminin, tenascin, hyaluronic acid, and

vitronectin. Critical factors in glioma invasion include the synthesis anddeposition of ECM components by glioma and mesenchymal cells, the re-lease of ECM-degrading activities for remodeling interstitial spaces, thepresence of adhesion molecules and the effects of cell-matrix interactionson the behavior of glioma cells. ECM modification aids the loss of contact


14/111

G B 5

inhibition, allowing tumor cells to freely migrate and invade the surround-ing tissues. Te proteolytic degradation of the BM is mediated by pro-teases, such as the matrix metalloproteases (MMPs), secreted by tumorand stromal cells [8]. MMPs play an important role in human brain tumorinvasion, probably due to an imbalance between the production of MMPsand tissue inhibitor of metalloproteases-1 (IMP-1) by the tumor cells[8]. MMP-1 is able to initiate breakdown of the interstitial collagens and to

Figure 2-1 Growth factors signaling pathways in cerebralgliomas (K-kinase, EGF-epidermal growth factor, PDGF-plateled derived growth factor, mOR-mammalian target of

rapamycin, PEN-tumor suppressor phosphatise and tensinhomolog, PKC-protein kinase C, PI3K phosphatidylinositol-3-kinase, PLC-phospholipase, Akt-, MEK-1/2-mitogen-activatedprotein kinase and extracellular signal-regulated protein kinase-1/2, kinase, MAPK/ERK-1/2-mitogen-activated protein kinase/extracellular signal-regulated protein kinase-1/2).


15/111

Table 2-1 Main genetic alterations in cerebral gliomas.

Gene Chromosome

Molecular

alteration

Molecular alteration

effectsP53 Cr17p13.1 Mutation Cell cycle control loss,

proliferationAs(Wsec

PDGFR-aPDGF-A

Cr4q11-q12 Amplification/over-expression

Proliferation/invasion As(W

Unknown tumorsuppressor genes

1p, 19q, 4q, 9pand 11p loss

Loss ofheterozygosity

Proliferation, invasiveness,angiogenesis

As(W

Unknown tumor

suppressor genes

Cr22q Deletion Proliferation As

(WRb 1 Cr13q14.2 Mutations/

deletionCell cycle control loss,proliferation

As(W

P16 Cr9p CDKN2/p16deletion

Cell cycle control loss,proliferation

As(W

PEN Cr10q23 LOH Regulation Akt/PKB signal-ing pathway loss; prolif-eration and tumor growth;invasiveness, angiogenesis

As(W

BAX Cr19q24 LOH Pro-apoptotic action loss,

proliferation

As

(WEGFR (c-erb-2) Cr7p11-p12 Amplification/

over-expressionCell transformation andproliferation

De

MDM2 Cr12q14.3-q15 Over-expression Cell cycle control loss andproliferation

De


16/111

G B 7

activate the other MMPs which allow glioma cell infiltration. Cell adhesionis the binding of the cells to each other and to the ECM through cell adhe-sion molecules such as integrins, selectins, cadherins, the immunoglobulinsuperfamily and lymphocyte homing receptors. Te extracellular ligandsthat anchor these adhesions include laminin, fibronectin, vitronectin, andvarious collagens. Integrins are heterodimers of a- and b-subunits that reg-ulate many aspects of the cell behavior including survival, proliferation, mi-gration and differentiation. Integrins are expressed on different cell types,including neurons, glial cells, meningeal and endothelial cells. b2 integrinsare specifically expressed by leukocytes and they are found on microgliaand on infiltrating leukocytes within the CNS. Down-regulated b1 inte-

grin protein levels in vivo probably affect interactions of glioma cells withECM components, leading to reduced migration along vascular basementmembranes [9]. Tese data can be interpreted as contributing to the locallyinvasive behavior of astrocytic tumors, favoring the regulation of proteasesactivation.

Cerebral gliomas are characterized by extensive microvascular prolifera-tion and a higher degree of vasculature. Angiogenesis, the formation of newblood vessels from existing microvessels, is a histological indicator of thedegree of malignancy and prognosis. Angiogenesis also includes vessel pen-

etration into avascular regions of the tissue, and is critically dependent onthe correct interactions among endothelial cells, pericytes and surroundingcells and their association with the ECM and the vascular BM. Caffo et al.[10] demonstrated that, the presence of endothelial glomeruloid-like pro-liferation in neoplastic vessels, was predictive of active tumor invasiveness(Figure 2-2). Endothelial cells are guided into avascular areas via macro-molecules such as VEGF-A, a pro-angiogenic factor and endothelial cellmitogen. VEGF-A activation causes endothelial cell differentiation and aVEGF-A gradient induces stalk cell proliferation along an opening in the

BM in the formation of a new vessel sprout. VEGF also induces expression

Figure 2-2 Presence of marked endothelial glomeruloid-likeproliferations in neoplastic vessels. Tis feature is indicative ofactive tumor progression and invasiveness, and of neoplasticcellular migration.


17/111

8 N B T T

of the delta-like ligand, DLL-4, in tip cells that bind to its receptors, as wellas Notch 1 and Notch 4, on adjacent stalk endothelial cells. DLL-4-Notchsignaling functions act as a dampening mechanism in preventing excess an-giogenesis and promoting orderly development of new vessels. Membranetype 1-matrix metalloproteinase M1-MMP on the endothelial cell sur-face, are also required for the subsequent step in the angiogenesis cascadeof tube formation, by playing a role in endothelial intracellular vacuole andlumen formation. Te BM is built up of scaffolding laminins and essentialcomponents such as collagen IV and collagen XVIII [11]. Part of the finalstage of angiogenesis is the recruitment of pericytes as their association withendothelial and vascular smooth muscle cells, is essential for the maturation

of endothelial tubes into blood vessels.


18/111

3. Blood-brain barrier

Te brain is a unique organ highly protected by two major barriers, the BBBwhich displays the largest surface area and the bloodcerebrospinal fluidbarrier (BCSFB). BBB is responsible for several functions, such as main-tenance of neuronal microenvironment, tissue homeostasis, vasotonousregulation, fibrinolysis and coagulation, blood cell activation and migrationduring physiological and pathological processes. Tere are several gatewayswhich offer entry to brain parenchyma, the most important are blood circu-lation and cerebrospinal fluid (CSF) circulation. In the human brain, thereare about 100 billion capillaries in total, providing a combined length ofbrain capillary endothelium of approximately 650 km and a total surfacearea of approximately 20 m2[12]. Despite the rapid development in under-standing of the molecular structure of components of the BBB, knowledge ofreceptor expression at the BBB, advances in medical technology, and break-throughs in nanotechnology-based approaches, many of the CNS associ-ated diseases remain under-treated by effective therapies. Since the majorityof drugs and large molecular weight particulate agents such as recombinantproteins, peptides, monoclonal antibodies, small-interfering RNA (siRNA)and gene therapeutics do not readily permeate into the brain parenchyma,one of the most significant challenges facing CNS drug development, is theavailability of effective brain drug targeting technology.

3.1 Blood-brain barrier physiology

Physiologically BBB is made up of three layers such as the inner endothelialcell layer which forms the wall of the capillary and contains tight junctions,followed by the presence of a basement membrane upon which pericytesand astrocytic feet processes lie [13]. Te BBB endothelial cells differ fromendothelial cells in the rest of the body by the absence of fenestrations, moreextensive tight junctions (Js), and sparse pinocytic vesicular transport.

Endothelial cells Js limit the paracellular flux of hydrophilic moleculesacross the BBB. In addition to brain capillary endothelial cells, extracellularbase membrane, pericytes, astrocytes, and microglia are all integral parts ofthe BBB supporting system. Te capillary endothelial cell line the microves-sels, which are coupled by much more J (zonulae occludentes) than foundin peripheral vessels. Te endothelial cells secrete and are surrounded bya basal lamina (BL), with the end-feet of astrocytic glial cells close on itsopposite side. Astrocytes are the most abundant non-neuron cells and playmany essential roles in the healthy CNS, including biochemical support of

endothelial cells which form the BBB, regulation of blood flow, provisionof nutrients to the nervous tissue, maintenance of extracellular ion bal-ance, and in the repair and scarring process of the brain and spinal cordfollowing traumatic injuries. Pericytes are embedded in the BL between en-dothelial cells and astrocyte cells, making particularly close contact with the


19/111

10 N B T T

endothelial cells. Pericytes provide microvasculature structural support andvasodynamic capacity.

BCSFB function, together with the BBB and the meninges, is the con-trol of the brain internal environment. It is sited at the choroid plexus epi-thelium, secreting CSF, which circulates through the ventricles and aroundthe outside of the brain and spinal cord [14]. Te choroidal epithelium isa complex organ with many additional functions including neuroendocrinesignaling, neuroimmune and neuroinflammatory responses, drug and toxinmetabolism, and transport. On the external surface of the brain the ependy-mal cells fold over upon themselves to form a double layered structure. Tisvirtual space is known as subarachnoid space and acts in CSF drainage.

Te passage of substances from the blood through the arachnoid membraneis prevented by tight junctions. Te capillary endothelium in the choroidplexus is fenestrated, allowing the passage of small molecules. Te arach-noid membrane is generally impermeable to hydrophilic substances and itsrole in the formation of the blood-CSF barrier is largely passive.

Js are located on the apical region of endothelial cells and are structur-ally formed by a complex network made of a series of parallel, intercon-nected, transmembrane and cytoplasmatic strands of proteins [15]. Jsconsist of three integral membrane proteins, namely, claudin, occludin, and

junction adhesion molecules, and a number of cytoplasmic accessory pro-teins including ZO-1, ZO-2, ZO-3, cingulin. Te high level of integrity ofJs is reflected by the high electrical resistance of the BBB (15002000 cm2), which depends on a proper extracellular Ca2+ ion concentration. Tetightness of the BBB is due to the physical complexity of its junctional struc-ture and the molecular substructure, in particular, the presence of trans-membrane proteins claudins 1 and 5 which help to seal the intercellular cleft.Cytoplasmic proteins link membrane proteins to actin, which is the primarycytoskeleton protein for the maintenance of structural and functional integ-

rity of the endothelium. In a recent study, treatment of claudin-5 by cyclicAMP (cAMP) led to enhancement of claudin-5 activity along cell borders,rapid reduction in transendothelial electrical resistance (ER), and loosen-ing of the claudin-5-based endothelial barrier against mannitol [16]. Tesesuggest that manipulation of claudin-5, or potentially other J proteins maypermit drug transport by altering the function at the BBB but without itstotal disruption. Occludin is a phosphoprotein with four transmembranedomains. Occludin appears to be a regulatory protein that can alter para-cellular permeability. Occludins and claudins assemble into heteropolymers

and form intramembranous strands.Adherens junctions (AJs) are located below the Js in the basal region ofthe lateral plasma membrane. Tey are composed of trans-membrane gly-coproteins (cadherins) linked to the cytoskeleton by cytoplasmatic proteins,thus providing an additional tightening structure between the adjacent en-dothelial cells at the BBB. Te cytoplasmic domains of cadherins bind to the


20/111

B-B B 11

submembranal plaque proteins h- or g-catenin, which are linked to the actincytoskeleton via a-catenin. In addition to supporting the barrier function,AJs mediate the adhesion of brain endothelial cells to each other, the initia-tion of cell polarity and the regulation of paracellular permeability [17].

3.2 Blood-brain barrier transport systems

Tere are different mechanisms by which solutes move across membranesas they enter and leave the brain. Te transport may occur due to diffu-sion, either simple diffusion or facilitated transport across aqueous chan-nels (Figure 3-1). Passive diffusion is a concentration gradient dependentprocess that allows molecules to move across cellular membranes betweencells (paracellular way) or across cells (transcellular way) down their electro-

chemical gradient without the requirement of metabolic energy. Smallwater-soluble molecules simply diffuse through the Js but not to any greatextent. Small lipid soluble substances like alcohol and steroid hormonespenetrate transcellularly by dissolving in their lipid plasma membrane. Inaddition to concentration differences, other factors can affect the diffusionof a drug across the BBB such as lipophilicity and molecular weight. Onlylipid soluble small molecules with a molecular weight of 400 Daltons cancross the BBB. However, the majority of small molecule drugs have a highermolecular weight or current water solubility which prevents their simple

diffusion across the barrier. In addition, even though some small moleculessuch as HIV protease inhibitors exhibit a high degree of lipophilicity, theirCSF and brain concentrations are often undetectable [18]. Tis effect is

Figure 3-1 Molecular transport across the blood-brain barrier.


21/111

12 N B T T

believed to be attributed to the functional expression of several ABC mem-brane associated drug transporters, which can actively export these agentsout of the brain [18]. For almost all other substances, including essentialmaterials such as glucose and amino acids, transport proteins (carriers), spe-cific receptor-mediated or vesicular mechanisms (adsorptive transcytosis)are required to pass the BBB.

Different substances are transported through free diffusion mecha-nism either paracellularly or transcellularly. Paracellular diffusion is a non-saturable and noncompetitive movement of compounds between cells. Itoccurs to a limited extent at the BBB, due to the Js. ranscellular diffusion(transcytosis) is a non-saturable and noncompetitive movement across cells

of lipophilic substances. Facilitated diffusion is a form of carrier-mediatedendocytosis in which solute molecules bind to specific membrane proteincarriers that trigger a conformational change in the protein. Tis results in acarrying through of the substance to the other side of the membrane, fromhigh to low concentration (passive diffusion). Tis mechanism contributesto the transport of various substances including amino acids, nucleoside,small peptide, monocarboxylates, and glutathione.

Carrier mediated transport (CM) or carrier mediated influx processesinvolve putative proteins that facilitate the movement of poorly permeable

solutes across cellular membranes. Te CM system is expressed on boththe luminal and abluminal membranes of the brain capillary endotheliumand operates in both directions. CM systems can be exploited for braindrug-delivery after reformulating the drug in such a way that the drug as-sumes a molecular structure mimicking that of the endogenous ligand. Ifcompounds need to be moved against a concentration gradient, AP mayprovide the energy to facilitate the process. Gabapentin (a g-amino acid) suc-cessfully crosses the BBB because the structure does mimic that of aa-aminoacid and is recognized by large neutral amino acid transporter [19]. Several

other drugs which have been successfully transported into the brain includemelphalan for brain cancer, laevodopa (L-Dopa) for Parkinsons disease anda-methyl-DOPA for treatment of high blood pressure. Te uptake of nu-trients from blood into the brain is facilitated by the solute carrier (SLC)transporter families. Tese influx carriers are involved in the transport of abroad range of substrates including glucose, amino acids, nucleosides, fattyacids, minerals and vitamins in various human tissues, including the brain.SLCO/SLC21, the organic anion transporting superfamily (OAPs), andSLC22, the organic cation/anion/zwitterions transporter family, are heavily

involved in the uptake of many diverse substrates [20].Te active efflux transport is responsible for extruding drugs from thebrain and this mechanism is a major obstacle for the accumulation of a widerange of biologically active molecules in the brain. Te AP binding cas-sette (ABC) transporter P-glycoprotein and multidrug resistant protein(MRP) represent the principle efflux mechanism of these agents [21]. Te


22/111

B-B B 13

most abundantly present component of this system is efflux P-glycoprotein,which is a product of the ABCB1gene. Inhibition of P-glycoprotein in pre-clinical studies has enhanced the penetration of paclitaxel into the brain,indicating the feasibility of achieving improved drug delivery to the brain bysuppression of P-glycoprotein [22].

Endocytosis and transcytosis allow the internalization, sorting and traf-ficking of many plasma macromolecules. Endocytosis is a process wheremolecules from the circulation are internalized in vesicles and are directedto endosomes or lysosomes within the cell. Endocytosis can be isolated intobulk-phase (fluid phase or pinocytosis) endocytosis and mediated endo-cytosis (receptor and absorptive mediated). Bulk-phase endocytosis is the

noncompetitive, non-saturable, temperature and energy dependent non-specific uptake of extracellular fluids. ranscytosis refers to the transcellularmovement of molecules.

Receptor mediated endocytosis or clathrin-dependent endocytosis pro-vides for a highly specific and energy mediated transport enabling eukary-otic cells to selective uptake macromolecules as specific cargo. Cells havedifferent receptors for the uptake of many different types of ligands, includ-ing hormones, growth factors, enzymes, and plasma proteins. Tis processoccurs at the brain for macromolecular substances, such as transferrin, in-

sulin, leptin, and IGF-I & IGF-II, and is a highly specific type of energydependent transport [23].Adsorptive endocytosis/transcytosis facilitates the transport of large

peptides such as IgG, histone, albumin, native ferritin, horse radish per-oxidase and dextran. Adsorptive-mediated endocytosis is characterized byan electrostatic interaction between a positively charged substance and thenegatively charged sites on the brain endothelial cell surface (e.g. glycopro-tein) [24]. Adsorptive processes largely depend upon electrostatic interac-tions that allow the positively charged moiety of the substrate to bind to the

negatively charged cell membrane. Receptor mediated transport is mainlyemployed in the transport of macromolecules like peptides and proteinsacross the BBB, by conjugating the substance with ligands such as lacto-ferrin, transferrin and insulin. It is an important transport mechanism ofpredominant interest in drug delivery.

Cell-mediated transcytosis is a recently identified route of drug transportacross the BBB [25]. Tis transport route relies on immune cells such asmonocytes or macrophages to cross the intact BBB. Unlike the aforemen-tioned transport pathways which normally permit only solute molecules

with specific properties, cell-mediated transcytosis is unique in that it canbe used for virtually any type of molecule or material as well as particulatecarrier systems.


23/111

4. Nanomedicine and nanotechnology

Nanotechnology is a collective definition referring to every technology andscience which operates on a nanoscale and refers to the scientific principlesand new properties that can be found and mastered when operating inthis range. When we bring materials down to the nanoscale, the propertieschange and nanoparticles have other optical, magnetic or electrical prop-erties than larger particles. Tese properties are and will be utilized in awide spectre of areas as in medical applications, information technologies,energy production and storage, materials, manufacturing, instrumentation,environmental applications and security. Nanotechnology in biomedical re-search has emerged as an interdisciplinary science that has quickly foundits own niche in clinical methodologies including imaging, diagnostic andtherapeutic. Te nano-based technology is expected to expand multi-directionally to provide unmet needs in medicine and has potential to gen-erate innovations that will bring breakthrough treatments to various humandiseases, including cancer. Nanotechnology is characterized by the manipu-lation of atoms and molecules leading to the construction of structures inthe nanometer scale size range [2627]. Te National Institute of Healthdefines nanomedicine as the application of nanotechnology to diseases treat-ment, diagnosis, monitoring, and to the control of biological systems. Tefield of nanomedicine aims to use the properties and physical characteristicsof nanomaterials, which have been extensively investigated as novel intra-vascular or cellular probes, for both diagnostic and therapeutic purposes.Te sub-micron size of nanoparticle systems confers considerable advan-tages as compared to large sized systems including targeted delivery, higherand deeper tissue penetrability, greater cellular uptake and greater ability tocross the BBB [28]. NPs consist of molecules with dimensions in the orderof 109nm, of different kind and compositions capable of containing drugsand DNA-RNA fragments and able to regulate their transport and intakeinto target tissues and cells. NPs show some peculiar features, such as theirsurface to mass ratio, which is higher than that of other particles, their quan-tum properties, and their capacity to transport other compounds [2930].Nanomedicine is applied in many fields of biology and medicine, such asfluorescent biological labels, drug and gene delivery, detection of pathogens,detection of proteins, probing of DNA structure, tissue engineering, tumordestruction via heating, separation and purification of biological moleculesand cells, MRI contrast enhancement, and phagokinetic studies [31]. NPdrug delivery vehicles have shown the ability to encapsulate a variety of ther-apeutic agents such as small molecules (hydrophilic and/or hydrophobic),peptides, protein-based drugs, and nucleic acids (Figure 4-1). By encapsu-lating these molecules inside a nanocarrier, the solubility and stability ofthe drugs can be improved, providing an opportunity to reevaluate potentialdrugs previously ignored because of poor pharmacokinetics. Encapsulated


24/111

N N 15

molecules can be released from nanocarriers in a controlled manner overtime to maintain a drug concentration within a therapeutic window or therelease can be triggered by some stimulus unique to the delivery site [32].

Te surface of the nanocarrier can be engineered to increase the blood circu-lation half-life and influence the bio-distribution, while attachment of tar-geting ligands to the surface can result in enhanced uptake by target tissues.Te net result of these properties is to lower the systemic toxicity of thetherapeutic agent, while increasing the concentration of the agent in the areaof interest, resulting in a higher therapeutic index for the therapeutic agent.In addition to therapeutic drugs, imaging agents can also incorporated intonanocarriers to improve tumor detection and imaging [33]. Finally, nano-particles can be engineered to be multifunctional with the ability to target

diseased tissue, carry imaging agents for detection, and deliver multipletherapeutic agents for combination therapy [34]. Te NPs penetrate easilyin the neoangiogenic vessels interstitium, Fig. 4-2 remaining entrapped inthe tumor, with evident higher retention times of drug into tumor. NPs maybe delivered to specific sites by size-dependent passive targeting or by activetargeting. Passive targeting is directly linked to intrinsic cancer cellular andmicro-environmental features. Active targeting involves the use of peripher-ally conjugated targeting moieties for enhanced delivery of NP systems. Tismethod has been performed to obtain a high degree of selectivity to specific

tissues and to enhance the uptake of NPs into cancer cells and angiogenicmicrocapillaries. With these strategies, NPs drug delivering systems mini-mize the uptake and the toxic side effects of the anticancer agent by nor-mal cells and enhance the entry and accumulation of the drug into tumorcells. NPs behavior within the biological microenvironment, stability, andextracellular and cellular distribution varies with their chemical makeup,

Figure 4-1 Drug encapsulation in a nanocarrier.


25/111

16 N B T T

morphology, and size. When injected intravenously, particles are cleared

rapidly from the circulation, predominantly by the liver and the spleen mac-rophages [35]. Opsonization, which is surface deposition of blood opsonicfactors such as fibronectin, immunoglobulins, and complement proteins,often aid particle recognition by these macrophages. Size and surface char-acteristics of nanoparticles both play an important role in blood opsoniza-tion processes and clearance kinetics. Larger particles (200 nm and above)are more efficient at activating the human complement system and henceare cleared faster from the blood by Kupffer cells. Te binding of bloodproteins and opsonins to NPs differ considerably in amount and in pattern

depending on surface properties, such as the presence and type of functionalgroups and surface charge density [3536]. Indeed, precision surface engi-neering with synthetic polymers can resolve aggregation and afford controlover nanoparticle interaction and their fate with biological systems. Tisstrategy suppresses macrophage recognition by an array of complex mecha-nisms, which collectively achieve reduced protein adsorption and surface

Figure 4-2 Schematic structure of different nanocarriers fordrug delivery in brain tumors.


26/111

N N 17

opsonization. Here, the efficiency of the process is dependent on the poly-mer type, their surface stability, reactivity, and physics (surface density andconformation) [35]. Suppression of opsonization favors enhanced passiveretention of NPs at sites and compartments.

Prolonged circulation properties are ideal for slow or controlled releaseof therapeutic agents into the blood to treat vascular disorders. Long cir-culating particles may have application in vascular imaging, or even act asartificial nanoscale red blood cells. Recent advances in synthetic polymerchemistry afford precise control over the architecture and polydispersity ofpolymers, polymer-conjugates, and block copolymers. Some of these novelmaterials can form sterically stabilized nanoscale self-assembling structures

with macrophage-evading properties. Molecular signatures related to par-ticular vascular and lymphatic beds and types of endothelial cells have beenidentified, providing landmarks for circulating cells and molecules [37]. Tisrequires assembly of the appropriate targeting ligands on nanocarriers andlong circulating nanosystems. However, the ultimate characteristics suchas ligand density, spacing and conformation are dependent on ligand andparticle properties (curvature and surface reactivity). Tese modificationsdetermine the extent of particle stability and aggregation in vivo, as well asthe efficiency of receptor binding and follow up events, such as the mode of

particle internalization and associated signaling processes.Te macrophages represent a valid pharmaceutical target and there arenumerous opportunities for a focused macrophage-targeted approach [38].Many pathogenic organisms have developed means of resisting macrophagedestruction following phagocytosis. Passive targeting of nanoparticulate ve-hicles with encapsulated antimicrobial agents to infected macrophages canrepresent a natural strategy for effective microbial killing [39]. Degradationof the carrier by lysosomal enzymes releases the drug into the phagosome-lysosome vesicle itself, or into the cytoplasm, either by diffusion or by specific

transporters depending on the physicochemical nature of the drug molecule.Intravenous injection of tuftsin-bearing liposomes to infected animals havenot only resulted in delivery of liposome-encapsulated drugs to the mac-rophage phagolysosomes, but also in the nonspecific stimulation of liver andspleen macrophage functions against parasitic, fungal and bacterial infec-tions [40]. Recently nanocarrier-mediated macrophage suicide (delivery ofmacrophage toxins) has proved to be a powerful approach in removing un-wanted macrophages in gene therapy and other clinically relevant situations.Numerous polymeric and ceramic nanospheres, nanoemulsions, liposomes,

protein cage architectures, and viral-derived nanoparticles act as powerfuladjuvants, if they are physically or covalently associated with protein antigens[41]. After endocytic uptake of nanoparticles, macrophages partially degradethe entrapped antigens and channel peptides into the MHC molecules (classI or II), for processing and presentation. Tus, there is considerable poten-tial for nanoparticulate adjuvants for the development of new-generation


27/111

18 N B T T

vaccines made either recombinant or from synthetic peptide antigens thatare less or no immunogenic in their own right. Recent advances in cell bi-ology have provided new information regarding the structure, recognitionproperties, and signaling functions of a variety of macrophage/dendritic cellsreceptors, particularly those that affect immunogenicity. Harnessing these re-ceptors as therapeutic targets may prove a better strategy for antigen deliveryand targeting with particulate nanocarriers. Dendritic cell receptors such asDEC-205 and DECSIGN have been implicated in antigen internalizationand presentation to cells [42].

A unique attribute of nanoplatform-based delivery systems is their mul-tifunctionality, characterized by multiple components, which include, imag-

ing agents, therapeutic agents, targeting ligands, and cloaking agents thatavoid interference with the immune system. Nanotheranostic platforms arepowerful tools for imaging and treatment of cancer. Multifunctionality ofthese nanovehicles offers a number of advantages over conventional agents.Tese include targeting to a diseased site thereby minimizing systemic toxic-ity, the ability to solubilize hydrophobic or labile drugs leading to improvedpharmacokinetics and their potential to image, treat and predict therapeuticresponse. argeted nanoparticle-based treatment technologies with diag-nostic capabilities are referred to as theranostic agents as they form a class of

agents which can serve diagnostic and therapeutic functions simultaneously.In the current state of technology, tumor detection and therapy are mostlyperformed separately. A more efficient and effective method can be achievedwith theranostic nanoparticles, which would integrate the efforts for detec-tion, treatment and follow-up monitoring of tumor response, and assistin the decision-making process for the need for further treatment (Figure4-3). Recently, Bhojani et al. [43] has developed a modular theranostic

Figure 4-3 Schematic structure of a theranostic nanoparticle(therapeutic agent-yellow; imaging contrast agent-white).


28/111

N N 19

nanoplatform, based on a polyacrylamide (PAA) nanoparticle core, withencapsulated components for synergistic cancer detection, diagnosis andtreatment. Tis platform combined MRI contrast enhancement, photody-namic therapy and specific targeting to tumor sites using F3 peptide [44].F3 peptide, a 31-amino acid fragment of a high mobility group protein, wasshown to home to the vasculature of a number of tumor types by interactingdirectly with endothelial cells [4546]. In some human cancers F3 peptidecan interact directly with tumor cells, where it is specifically taken up at thecell surface, then internalized into the cell and transported to the nucleus[4546]. Te authors have shown that significant therapeutic benefit withphotodynamic therapy was obtained when an F3-targeted polymeric nano-

particle formulation consisting of encapsulated imaging agent (iron oxide)and photosensitizer (Photofrin) was administered to glioma bearing rats.Using these multifunctional nanoparticles the authors demonstrated thatnanoparticles could be targeted to intracerebral rat 9L gliomas and detectedusing MRI [47]. F3-targeted nanoparticles provided a significantly increasedsurvival time over that of nontargeted Photofrin encapsulated nanoparticlesor Photofrin alone [47].

issue engineering brings together principles and innovations from en-gineering and the life sciences for the improvement, repair or replacement

of tissue/organ function. Since its inception, this multidisciplinary field hasbeen governed by the generic concept of combining cell, scaffold (artificialextracellular matrix) and bioreactor technologies, in the design and fabri-cation of neo-tissues/organs. Microenvironment of organs and tissues iscomposed of parenchymal cells and mesenchymal cells (support cells) im-mersed in the extracellular matrix. Te objective is to enable the body (cel-lular components) to heal itself by introducing a tissue engineered scaffoldthat the body recognizes as part of itself and uses this process to regenerateneo-native functional tissues [48]. Furthermore the construction of organs

by regenerative therapy has been presented as a promising option to addressthis deficit. Nanotechnology has the potential to provide instruments thatcan accelerate progress in the engineering of organs. Achievement of themore ambitious goals of regenerative medicine requires control over the un-derlying nanostructures of the cell and extracellular matrix. Cells, typicallymicrons in diameter, are composed of numerous nanosized componentsthat all work together, to create a highly organized, self-regulating machine.Cell-based therapies, especially those based on stem cells, have generatedconsiderable excitement in the media and scientific communities, and are

among the most promising and active areas of research in regenerativemedicine [49].

4.1 Nanoparticle drug delivery

Within past few years, rapid developments have been made to use nano-materials in a wide variety of applications in various fields of medicine such


29/111

20 N B T T

as oncology, cardiovascular and orthopedics. Nanomaterials have been usedin specific applications such as tissue engineered scaffolds and devices, sitespecific drug delivery systems, cancer therapy and clinical bioanalytical di-agnostics and therapeutics. An area of research where nanotechnology andnanomedicine applications have been particularly prolific pertains to the de-livery of diagnostic and therapeutic agents.

Drug delivery can be defined as the process of releasing a bioactiveagent at a specific rate and at a specific site. As current advances in bio-technology and related areas are aiding the discovery and rational designof many new classes of drugs, it is crucial to improve specific drug-deliverymethods, to turn these new advances into clinical effectiveness. Several

drugs are limited by their poor solubility, high toxicity, and high dosage,aggregation due to poor solubility, nonspecific delivery, in vivo degrada-tion and short circulating half-lives. argeted drug-delivery systems canincrease patient compliance, extend the product life cycle, provide prod-uct differentiation and reduce healthcare costs. Nanotechnology can becorrectly envisioned as the future of drug-delivery technology as it hasthe potential to provide useful therapeutic and diagnostic tools in thenear future. NPs offer a suitable means to deliver small molecular weightdrugs as well as macromolecules such as proteins, peptides or genes in the

body using various routes of administration. Te ability of the engineeredNPs to interact with cells and tissues at a molecular level provides themwith a distinct advantage over other polymeric or macromolecular sub-stances. Drug delivery carriers are macromolecular assemblies that canincorporate imaging and therapeutic compounds of distinct nature, suchas small chemicals, fluorophores and biosensors, peptides and proteins,oligonucleotides and genes. Tey can be designed to improve the solubil-ity of these cargo molecules and their bioavailability, and also to controltheir circulation, biodistribution in the body, and release rate, together

enhancing their efficacy [5051]. Surface property modifications conferadvantageous properties to the particle, such as increased solubility andbiocompatibility which are useful in the crossing of biophysical barriers.Te use of biodegradable materials in the NPs formulation permits drugrelease for prolonged periods. For their small size, NPs can extravasatethrough the endothelium in inflammatory sites, epithelium, tumors, orpenetrate microcapillaries.

4.1.1 Nanoparticle distribution

Te natural clearance and excretion mechanisms of the human body pro-

vide a framework for the rational design of effective nanoparticles for usein medical therapies. Once a pharmaceutical agent is introduced into thecirculatory system, it is distributed systemically via the vascular and lym-phatic systems. Te distribution of a drug in a tissue is correlated with therelative amount of cardiac output passing through that tissue. Accordingly,


30/111

N N 21

tissues and organs with high blood flow (brain, liver, heart, intestines, lungs,kidneys, and spleen) may be exposed to higher concentrations of a drug,provided that the drug is able to penetrate into the tissues from the vascu-lature. Particle size and size distribution determine the in vivo distribution,biological fate, toxicity, and targeting ability of these delivery systems. Inaddition, they can influence drug loading, drug release, and the stability ofnanoparticles. Generally, nanoparticles have relatively high cell uptake andare available to a wider range of cellular and intracellular targets due to theirsmall size and mobility. Very small nanomaterials, on the order of 120 nm,have long circulatory residence times and slower extravasation from the vas-culature into interstitial spaces. Tis may cause an altered volume of dis-

tribution when administered intravenously. Smaller particles have a largersurface area-to-volume ratio and thus most of the drug associated withsmall particles would be at or near the particle surface, leading to faster drugrelease. Smaller particles also have a greater risk of aggregation during stor-age, transport and dispersion.

Surface manipulation can control the extent of localization at intersti-tial sites and limit clearance. As nanomaterials are stealthed via hydrophilicPEGylation, their circulatory residence times increase. Te zeta potential ofa nanoparticle is commonly used to characterize the surface charge property

of nanoparticles [52]. It reflects the electrical potential of the particles andis influenced by the composition of the particle and the medium in whichit is dispersed. NPs with a zeta potential above 30 mV have been shownto be stable in suspension, as the surface charge prevents aggregation of theparticles. Endothelial damage or alteration may modify the distributionparameters of nanoparticles. Inflammation, solid tumors, and deliberatedisruption of endothelial contribute to an increased leakiness that providesvascular contents greater access to extravascular targets. Te presence ofdisturbed, porous vascular beds at the tumor allows for selective targeting

by this passive mechanism. Generally speaking solubility, diffusion, and bio-degradation of the particle matrix influence the drug release process. It isevident that the method of incorporation has an effect on the release profile.If the drug is loaded by the incorporation method, then the system has arelatively small burst effect and sustained release characteristics. If the nano-particle is coated by polymer, the release is then controlled by diffusion ofthe drug from the polymeric membrane. Membrane coating acts as a drugrelease barrier and thus drug solubility and diffusion in or across the poly-mer membrane becomes a determining factor in drug release. Furthermore,

the release rate also can be affected by ionic interactions between the drugand auxiliary ingredients.

4.1.2 Nanoparticle functionalization

NP functionalization represents the first step towards NP drug deliverysystems. Drug delivery carriers can be functionalized to improve control


31/111

22 N B T T

of their circulation and biodistribution in the body at the tissue, cellular,and sub-cellular level. Tis can be achieved by incorporating immune-evading moieties and/or affinity molecules, that favor adhesion to eithergeneral or specific biological markers, depending on the degree of selectiv-ity required. In addition, when carriers are targeted to cellular receptorsinvolved in endocytic transport or coupled to cell penetrating peptides, orif they are designed to modify the permeability of cellular barriers, theyalso provide delivery to a variety of intracellular compartments, such as thelysosome, cytosol, and nuclei [53]. When administered in vivo, therapeuticagents are recognized as foreign substances and rapidly cleared from thebody. Clearance of foreign compounds in the body occurs mainly by the

reticuloendothelial system (RES), and other elements of the immune sys-tem, as well as by renal filtration. For most applications, rapid clearanceis detrimental as it minimizes the chances of the delivered agent to reachits targets in the body and accumulate there, at amounts amenable to ren-der significant efficacy. Tis can be achieved by coating nanoparticles withhydrophilic polymers/surfactants or formulating nanoparticles with bio-degradable copolymers with hydrophilic characteristics, e.g., polyethyleneglycol (PEG), polyethylene oxide, polyoxamer, poloxamine, and polysorbate80. PEG helps form a hydrophilic brush around NP cargoes and/or their

carriers, minimizing interactions with plasma opsonins, the complement,professional phagocytes, and lymphocytes which provide specific immunity.As a consequence, certain physiochemical properties of the cargo are al-tered, allowing the platform to gain solubility and to remain elusive fromimmune detection. Tis prolongs the circulation in the bloodstream froma few hours to days, which favors lengthened medicinal effects and less fre-quent administrations [54]. Another strategy to minimize drug removaltakes advantage of the natural mechanism by which red blood cells in thebody avoid clearance by elements of the innate immune system. Tis is the

case for CD47, a transmembrane protein that acts like a marker of the self by binding to its cognate receptor expressed on leukocytes. CD47 inhibitsphagocytosis, in part via regulation of the cytoskeleton and inhibition of en-gulfing structures. Incorporation of CD47 on drug carrier surfaces reducesattachment to neutrophils and macrophages, therefore prolonging circula-tion and inhibiting inflammation [55]. In addition nanocarriers can alsoimprove control of the drug efficacy upon release in the case of therapeuticinterventions where the administration is local. Localized implantation ofbioactive agents embedded within porous matrices and/or hydrogels ca-

pable of responding to microenvironment properties can provide controlledrelease and effects [56]. Encapsulation within these formulations can alsoprovide sustained release over prolonged periods of time, as oppose to bulkdelivery of a naked therapeutic, which can apply to the release of encapsu-lated drugs and also bioactive substances produced by cells encapsulatedwithin these matrices [56].


32/111

N N 23

4.1.3 Nanoparticle targeting

One of the major challenges in drug delivery is to carry the drug at the place

where it is needed and to avoid potential side effects on non diseased organs.After reaching the targeted tissue, drugs should have the ability to selec-tively kill diseased cells without affecting normal cells. Tese basic strategiesare also associated with improvements in patient survival and quality of lifeby increasing the intracellular concentration of drugs and reducing dose-limiting toxicities simultaneously. In some cases, general enhanced deliverythroughout the body, rather than specific delivery to particular organs, ispreferred. Tis is the case for genetic conditions that affect multiorgan sys-tems due to ubiquitous distribution of the molecular markers or functions

affected, such as in many monogenic disorders with both peripheral andcentral nervous system components. argeted drug delivery can be achievedby active targeting of the drugs, or through passive targeting to the site ofaction. Active targeting requires the therapeutic agent to be achieved by con-

jugating the therapeutic agent or carrier system to a tissue or cell-specificligand [57]. Te success of drug targeting depends on the selection of thetargeting moiety, which should be abundant, have high affinity and specific-ity of binding to cell surface receptors, and should be well suited to chemi-cal modification by conjugation. Te active targeting can be achieved by

molecular recognition of the diseased cells by various signature moleculesover-expressed at the diseased site, either via the ligand-receptor, antigen-antibody interactions or by targeting through aptamers. Te therapeuticagent can be actively targeted by conjugating the carrier with a cell or tissue-specific ligand, thereby allowing a preferential accumulation of the drug atthe diseased site. PEGylated gold NPs are decorated with various amountsof human f by Choi et al. [58] to enhance active targeting. Teir resultssuggest that targeted NPs can provide greater intracellular delivery of thera-peutic agents to the cancer cells within solid tumors than their non-targeted

analogs.Passive targeting exploits the anatomical differences between normal anddiseased tissues to deliver the drugs to the required site, because the physiol-ogy of diseased tissues may be altered in a variety of physiological conditionsthrough the enhanced permeability and retention (EPR) effect [59]. Tedifference between infection-induced EPR effect and that of cancer is theduration of the retention period. Te retention in normal tissue, where in-flammation occurs, is shorter than with cancer because the lymphatic drain-age system is still operative. Te EPR effect has been greatly exploited for

delivering various therapeutics at the site of action, and many studies po-tentially support this mechanism of passive targeting. Drugs encapsulatedin nanoparticles or drugs coupled to macromolecules can passively targettumors through the EPR effect. One of the examples is Doxil, a stericallystabilized PEGylated liposome that encapsulates doxorubicin. Doxil has


33/111

24 N B T T

shown good drug retention in the liposomal formulation. In experimentalstudies, such systems showed significant improvements in tumor size re-duction working through the EPR mechanism. Recently, Chytil et al. [60]have exploited the EPR effect for targeting HPMA copolymer-based drugcarriers with covalently bound hydrophobic substituents for targeting solidtumors. reatment of mice bearing EL-4 -cell lymphoma with the aboveconjugates resulted in significant tumor regression. Tese nanoconjugatesalso enhanced tumor accumulation, indicating an important role of the EPReffect in excellent anticancer activity of the conjugate. Since most therapeu-tics agents do not present intrinsic affinity to cells, coupling them to carri-ers with affinity properties provides advantages. Hydrophilic and slightly

positively-charged polymers provide affinity to the negatively-chargedplasma membrane of cells [51].Direct intratumor delivery of anticancer agents using NPs can be used

in the treatment of local cancers such as prostate, head and neck cancers.Recently, Sahoo et al. [61] have demonstrated that transferrin (f) conju-gated paclitaxel (x)-loaded biodegradable NPs are more effective in dem-onstrating the antiproliferative effect of the drug than its solution or withun-conjugated x-loaded NPs. NPs are emerging as a promising tool forthe intracellular delivery of practically insoluble drugs and sensitive drugs.

Intracellular targeting refers to the delivery of therapeutic agents to specificcompartments or organelles within the cell, and the delivered cargoes mustgain access to intracellular compartments where their molecular targets arelocated. Interventions related to RNA interference or delivery of antisenseoligonucleotides requires transport of these cargoes to the cytosol of thecell.

Gene therapy is a promising new approach for treating a variety of ge-netic and acquired diseases. Tese macromolecules are unstable and showa poor cellular uptake and are rapidly degraded by nucleases. o overcome

these limitations, various chemical modifications of oligonucleotides havebeen tried. Tese modifications have disadvantages such as decreasedmRNA hybridization, elevated cytotoxicity, and increased nonspecific tar-geting. In order to overcome the disadvantages of viral carriers (high cyto-toxicity, cost, small transgene size), nonviral carriers have been developed.Te advantages associated with nonviral carriers include facile large scalemanufacture, low immunogenic response, versatile modifications, and thecapacity to carry large inserts. Gene therapies require delivery to the cytosol,with subsequent transport to the cell nucleus. Te drug can be delivered into

target cells by simple diffusion, or it may involve complex cellular machinery.Te major route of intracellular therapeutic uptake is through endocytosis.Tis strategy is ideal in the case of delivery of therapeutic agents whose ac-tion is required at said sub-cellular compartments, such as in the case ofcarrier-assisted delivery of enzyme replacement for lysosomal storage dis-orders. Carriers themselves can also be designed to overcome endosomal


34/111

N N 25

membranes, such as in the case of pH-sensitive poly(acid) carriers andtemperature-responsive poly(electrolyte) hydrogels [6263]. Other strate-gies have been designed to directly overcome the plasmalemma. Tese in-clude electroporation and ultrasound, where a local electric or ultrasoundpulse is exerted in the immediate post-administration period causing tran-sient enhancement of the plasmalemma permeability [64], and biolisticparticle delivery systems, where penetration into cells is gained by meansof tungsten or gold particles that are propelled by a gene gun across theplasma membrane [65]. Amphiphilic and biodegradable cationic copoly-mers are efficient gene delivery systems, which can condense nucleic acidand form controlled nanosized complexes. Polyamidoamine (PAMAM) and

poly[2-(dimethylamino) ethyl methacrylate] (PDMAEMA) are low toxicpolymers which have shown great potential as carriers. Polycaprolactone(PCL) is another promising delivery system. PCL-g-PDMAEMA nano-particle/DNA complexes could escape from the endosome and release theirpayloads effectively in cytoplasm, which may be induced by the enhancedinteraction between the complexes and cell membrane, due to hydropho-bic modification [66]. Small interfering RNA (siRNA) has attracted muchattention because it enables sequence-specific manipulation of expressionfor multiple endogenous genes. Te intracellular release of siRNA from

pluronic/poly(ethylenimine) nanocapsules was achieved by changing thenanocapsules from a collapsed state to a swollen state using a brief coldshock treatment [67]. Weber et al. [68] reported an amino-terminated car-bosilane dendrimer-bound siRNA delivery system. Tese RNase-resistantcarbosilane/siRNA dendriplexes have a high and prolonged gene-silencingeffect, and can be safely used in serum and antibiotics containing medium,without affecting cell viability and metabolic activity at relatively high den-drimer concentrations. One of the most common methods used for thesystemic delivery of siRNA involves their electrostatic interaction with cat-

ionic liposomes. Self-assembled liposome-protamine-hyaluronic acid nano-particles, modified by DSPE-PEG with conjugated ligand have been usedto overcome innate immune responses of siRNA-based therapy. Te devel-oped nanoparticle formulation has a siRNA encapsulation efficiency of 90%and showed a reduced systemic immunotoxicity [69].

4.2 Nanomedicine and cancer

Cancer, a disease characterized by the uncontrolled growth and spread ofabnormal cells, is still the second most common cause of death in the U.S.According to the American Cancer Society, about 571,950 Americans are

expected to die in 2011 due to cancer, and that means more than 1,500deaths per day. Current treatments for various cancers include surgery, ra-diation, hormone therapy, and chemotherapy. Although these conventionaltherapies have improved patients survival, they have also shown several limi-tations. Te National Cancer Institute (NCI) has identified nanotechnology


35/111

26 N B T T

as having the potential to make paradigm-changing impacts on the detec-tion, treatment, and prevention of cancer. Te growing interest in nanotech-nology by both academic and industrial investigators has led to increaseddevelopment of novel nanotechnology platforms for medical applications,sharp increases in government funding, and venture capital investment. Inthe cancer context, nanotechnology will lead to a new generation of diag-nostic and therapeutic technologies, creating a range of new solutions fordiagnoses and treatment of neoplastic diseases [45, 26, 7073].

Diagnostic methods are essential for the early detection of diseases toenable their prompt treatment, minimizing possible damage to the rest ofthe organism. Conventional imaging technologies represent static images

of tumors, rather than a continuous visualization of tumor proliferation.Nanodiagnostics, defined as the use of nanotechnology for clinical diagnos-tic purposes [74], was developed to meet the demand for increased sensi-tivity in clinical diagnoses and earlier disease detection. NP-based systemsimaging allows an early detection of tumor, as well as opportunities forreal-time monitoring, thereby increasing both the sensitivity and accuracyof anticancer therapies. Initial results in nanotechnology-enabled molecularimaging have been made in all imaging modalities, including optical, nu-clear, ultrasound, computed tomography, and magnetic resonance imaging

(MRI). MRI contrast agents have made a significant impact in the use ofMRI for various clinical indications. MRI contrast agents contain paramag-netic or superparamagnetic metal ions that affect the MRI signal propertiesof surrounding tissue. Tese contrast agents are used primarily to increasethe sensitivity of MRI for detecting various pathological processes and alsofor characterizing various pathologies. In addition, the contrast agents areused for depicting normal and abnormal vasculature, or flow-related abnor-malities and pathophysiologic processes like perfusion. A conglomerate ofnumerous nano-sized iron oxide crystals coated with dextran or carboxydex-

tran forms superparamagnetic iron oxide (SPIO) contrast agents [75]. woSPIO particle formulations are now clinically available, namely ferumoxidesand ferucarbotran. Both are approved specifically for MR imaging of theliver. After intravenous administration, clinical approved SPIO particles arecleared from the blood by phagocytosis accomplished by reticuloendothelialsystem so that uptake is observed in the normal liver, spleen, bone marrow,and lymph nodes. After the intracellular uptake, SPIOs are metabolized inthe lysosomes into a soluble, nonsuperparamagnetic form of iron that be-comes part of the normal iron pool [75]. Following intravenous injection,

SPIO is incorporated into macrophages via endocytosis. Te uptake ofSPIO by phagocytic monocytes and macrophages provides a valuable in-vivo tool by which MRI can be used to monitor involvement of macrophagesin inflammatory processes, such as multiple sclerosis, traumatic nerve injury,stroke, brain tumors, and vulnerable plaque in carotid artery. Neuwelt et al.[76] conducted clinical studies with MRI monitoring of macrophages in


36/111

N N 27

brain tumours. Te macrophage MRI detection with SPIO of tumor mor-phology might facilitate the surgical resection or biopsy of brain tumors.

Te main goal of nanotechnology in brain tumor imaging is an accu-rate and early diagnosis without side toxic effects and the evaluation of theefficacy of non-invasively treatments [5, 77]. Tese new cellular targetingbased imaging detection methods can reach the specific and selective mo-lecular recognition only for tumor cells, through the recognition of tumorspecific molecules into ligand-receptor, antibody-antigene interaction, orother interaction processes between nanoparticle drug-loaded systems andcancer cells, leading to a diffuse and complete delivering of drug into can-cer cells [78]. Te achievement of higher targeting efficiency per NP will

require the finding of more efficient bio-markers for cancer and correspond-ing targeting moieties. By detecting and analyzing tumor cells and tissueswith nanotechnologies, the internal biological features of cancer during itsoccurrence and development can be revealed. Generally speaking, the ap-plication of nanotechnology in medical diagnostics can be subdivided intoin vitro diagnostic devices and in vivo imaging. Te improvements in thetechnologies to characterize cells or cell compartments in vitro(optical andluminescence microscopy, scanning probe microscopy, electron microscopyand imaging mass-spectrometry) have been important for the development

of nanomedicine. Te miniaturization and integration of different functionsin a single device, based on nanotechnology-derived techniques, have led toa new generation of devices that are smaller and faster, and give accuratereadings. Tey require much smaller samples, implying less invasive andtraumatic sample extraction methods, and deliver more complete and moreaccurate biological data from a single measurement. Te use of these de-vices in research has become routine, and has improved the understandingof the molecular basis of disease, as well as helping to identify new therapeu-tic targets. In vitro diagnostic devices mainly include nanobiosensors and

microarrays. Te nanobiosensors are systems composed by biological andbiomimetic recognition elements. Interaction between the compound of in-terest and the recognition element produces a variation of physical-chemicalproperties (pH, electron transfer, heat, potential, mass, and optical proper-ties). Prototype sensors have been successfully used to detect nucleic acids,proteins and ions. Tey can operate in liquid or gas phase, opening up anenormous variety of downstream applications. Tese detection systems useinexpensive low-voltage measurement methods and detect binding eventsdirectly [49].

Microarray-based studies have enormous potential in the exploration ofdiseases such as cancer, and in the design and development of new drugs.Microarrays have been widely applied in the study of various pathologicalconditions, including inflammation, atherosclerosis, breast cancer, colon can-cer and pulmonary fibrosis [79]. As a result, functions have been assigned topreviously unannotated genes, and genes have been grouped into functional


37/111

28 N B T T

pathways. Several types of microarray have been developed for different tar-get materials, which can be DNA, cDNA, mRNA, protein, small molecules,tissues, or any other material that can be quantitatively analyzed. A DNAarray consists of a large number of DNA molecules in an orderly arrange-ment on a solid substrate to form a matrix of sequences in two dimensions.cDNA microarrays and oligonucleotide microarrays are used for microarrayexpression analysis, and to determine the level or volume of expression of agiven gene. Single nucleotide polymorphism microarrays detect mutationsor polymorphisms in a gene sequence [80]. Tis technology is used to testan individual for disease expression patterns, and to determine whether ornot individuals are susceptible to a disease.

Nanotechnology has produced advances in imaging diagnosis, develop-ing novel methods and increasing the resolution and sensitivity of existingtechniques. Tese systems include positron-emission tomography (PE),single-photon-emission C (SPEC), fluorescence reflectance imaging,fluorescence-mediated tomography (FM), fiber-optic microscopy, opticalfrequency-domain imaging, bioluminescence imaging, laser-scanning confo-cal microscopy and multiphoton microscopy [81]. Te main benefits of mo-lecular imaging for in vivo diagnosis lie in the early detection of disease andthe monitoring of disease stages, supporting the development of individu-

alized medicine and the real-time assessment of therapeutic and surgicalefficacy. MRI, C, PE and SPEC are the most widely used and studiedmodalities in cancer patients. Overall, nuclear imaging by PE or SPECoffers greater sensitivity, but is limited by the lack of anatomical context,whereas MRI provides accurate anatomical detail but no data on cell vi-ability and shows poor sensitivity [82]. Although none of these modalitiesis ideal, MRI is the preferred option for cellular tracking. Detecting protonrelaxations in the presence of a magnetic field yields tomographic imageswith excellent soft tissue contrast, and can locate the cells of interest in the

context of the surrounding milieu (oedema or inflammation) without theuse of harmful ionizing radiations. In addition, MRI offers a longer track-ing window in comparison to PE and SPEC, which are limited by thedecay of the short-lived radioactive isotopes. New contrast agents, used toincrease the sensitivity and contrast of imaging techniques are increasinglycomplex and formed by synthetic and biological NPs. NPs possess certainsize-dependent properties, particularly with respect to optical and magneticparameters, which can be manipulated to achieve a detectable signal. Te pri-mary event, in most nanoparticle-based assays is the binding of a nanopar-

ticle label or probe to the target biomolecule that will produce a measurablesignal characteristic of the target biomolecule. A probe that is to functionin a biological system must be water-soluble and stable and have mini-mal interaction with the surrounding environment. Although remarkableachievements have been made in nanodiagnostics during recent years, mostof these techniques are still under laboratory investigation. Nida et al. [83]


38/111

N N 29

used quantum dots that were attached to epithelial growth factor receptorand were conjugated with anti-growth antibody, to detect early biomarkersof cervical cancer. Cross et al. [84] installed a tiny probe on a spring usingnanotechnology, and used it to explore a cell surface and measure its soft-ness, which was used as a marker to determine whether carcinogenesis hadoccurred in the cells. Gao et al. [85] used quantum dots to locate and imagetumors in vivo. Tey coated quantum dots with a layer of polymer NPs andpolyethylene glycol, and attached them to a prostatic gland specific mono-clonal antibody. Fluoerescent image analysis revealed multi-color fluores-cent images that were sensitive to tumor cells in vivo, as well as informationregarding tumor volume and location. Nasongkla et al. [34] performed a

study on polymer micelle loaded with superparamagnetic iron oxide andfound it promising in the dual-targeting delivery and hypersensitive MR incancer cells.

Nanomedicine can improve the targeting ability of chemotherapeuticagents. Rapaport et al. [86] managed to deliver chemotherapeutic agentsaccurately into tumor cells using multifunctional NPs which improved thetargeting ability of chemotherapeutic agents and helped destroy cancercells effectively. In a recent study was reported that a polyethylene glycol-phospholipid nano micelle loaded with adriamycin could selectively accu-

mulate in tumor tissue, and penetrate thick layer of tumor tissue. Integratedquantum dots and glucose-binding protein antibodies selectively recognizecancer cells. Tese cells, when irradiated by ultra-violet ray showed greenfluorescence. Tis strategy allows the differentiation of normal cells andcancer cells. A prolonged ultra-violet irradiation can eliminate the cancercells [87]. Recently, Chakravarty et al. [88] coated a carbon nanotube witha monoclonal antibody against specific targets on lymphoma cells. Whenthese signed cells were exposed to near infrared light, the carbon nanotubestarted to kill these cells by heating them up. A large number of NPs can

serve as carriers of anti-cancer drugs. Drugs incorporated in the nanocarri-ers, either physically entrapped or chemically tethered, have the potential totarget physiological disorder zones sparing normal cells from collateral con-sequences. Te pharmacokinetic profile, especially the transportation capa-bilities, of the drug substances have been greatly modified by incorporationin a nanodrug delivery system. Tese include enhanced accommodation fortargeting moieties such as chaperones, and alteration in release rates com-prising of controlled release and site-specific delivery, by use of molecularengineering techniques. Additionally, encapsulation of the drug substances

in various polymeric and inorganic composites have also been evaluated fortheir rationalization of the drug delivery systems. Such encapsulations aregenerally made for protecting the biologically active protein and peptide-based drug compounds from the detrimental effects of biological fluids.In gene therapy, exogenous genes are introduced into cells by properl

Documents

Caruso Caffo Raudino Tomasello Nanoparticles and Brain Tumor Treatment