Designer Biomaterials for Nanomedicine

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Designer Biomaterials for Nanomedicine

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By Nishit Doshi and Samir Mitragotri*
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Nanotechnology has had tremendous impact on medical science and has

resulted in phenomenal progress in the field of drug delivery and diagnostics.

A wide spectrum of novel nanomaterials including polymeric particles,

liposomes, quantum dots, and iron oxide particles have been developed for

applications in therapeutic delivery and diagnostics. This has resulted in

control over the rate and period of delivery and targeting of drugs to specific

organs in the human body. This feature article focuses on the delivery of drugs

using polymeric particles. The size, choice of polymer, surface chemistry,

shape, and mechanical properties of the particles are parameters that

critically affect particle function. Numerous biomaterials and fabrication

techniques have been developed in the last decade that focus on novel design

parameters, such as shape and mechanical properties and the interplay of

these parameters with the size and surface chemistry of particles. Recent

le shape are

are underscored.

1. Introduction

advances with particular focus on the importance of partic

highlighted, and the challenges that are yet to be fulfilled

Nanomedicine has emerged as a novel field which involves theapplication of nanotechnology to human health and is currently inits early stage of exponential growth.[1,2] Various therapeutic anddiagnostic modalities have been developed which can potentiallyrevolutionize disease diagnostics and treatment.[3–7] Examples ofnovel nanomaterials include polymeric nanoparticles, liposomes,micelles, composite nanoshells, carbon nanotubes, capsules,microbubbles, dendrimers, nucleic-acid-based nanoconstructs,engineered viral nanoparticles, magnetic nanoparticles, siliconoxide nanoparticles, and quantum dots to list a few.[1,8–10] Thesenanoconstructs offer significant advantages over traditionalmodalities since thephysical,mechanical, and electrical propertieschange significantly as the dimensions approach nanometer scale.Liposomes and polymeric particles have been widely studied asdrug delivery carriers[11,12] and imaging agents such as iron oxidenanoparticles and quantum dots have been explored for real time

[*] Prof. S. MitragotriDepartment of Chemical EngineeringUniversity of CaliforniaSanta Barbara, CA 93106 (USA)E-mail: [email protected]

N. DoshiDepartment of Chemical EngineeringUniversity of CaliforniaSanta Barbara, CA 93106 (USA)

DOI: 10.1002/adfm.200901538

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tracking of the particles in the body.[13]

Manyof thesedelivery and imagingsystemshave already been commercialized whereassome are in late-stage clinical trials.[14] Forexample, Doxil (doxorubicin liposomes),used for the treatment of AIDS-associatedKaposi’s Sarcoma was the first liposome-based delivery system to receive FDA(United States Food and Drug Administra-tion) approval[15] and Abraxane, a 130 nmpaclitaxel-decorated albumin, receivedFDAapproval for second-line treatment forbreast cancer patients.[16] Similarly, Com-bidex (ferumoxtran-10), an iron-oxide-par-ticle-based imaging contrast agent is beingused in conjunction with magnetic reso-nance imaging (MRI) for differentiatingcancerous from normal lymph nodes.Other nanoscale vehicles, such as Dauno-Xome (daunorubicin liposomes), Ambi-some (amphotericine B liposomes),

Genexol-PM (PEG-PLGA-Paclitaxel particle), and DE-310 (Dextran-Camptothecin), provide additional examples of nanocarriers.

In spite of these advances, current delivery and imaging systemssuffer from some major hindrances, e.g., rapid clearance by theimmune system, low targeting efficiency, and difficulty in crossingthe biological barriers.[17] Numerous solutions have been proposedwhere different physical, chemical, and functional properties of theparticles have beenmodified for improved efficacy.[18,19] Polymericparticles, in particular, hold enormous potential as delivery vehiclesdue to theeaseofmodificationof their designparameters.Themostimportant parameters that have been extensively studied includeparticle size and surface chemistry. Recent advances inmethods offabricating particles of different shapes have fueled interest inexploring the importance of shape in different biological interac-tions.[20] The importance of particle shape in various biologicalprocesses, such as internalization, transport through the bloodvessels, and targetingdiseasedsites, hasbeenrecently realized.[21,22]

Here, we review the literature demonstrating the growingimportance of particle shape in various particle functions. Shapehas added an additional dimension to the toolbox of particle designfor drug delivery. However, the interplay of different designparameters needs to be considered and optimized.

2. Particulate Medicine

Particulate medicine has received tremendous interest in recentyears since it has the potential to resolve the challenges currentlyfaced in the field of drug delivery. Application of nanotechnology tobiomedicine has already yielded numerous carriers for delivery of

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Nishit Doshi is a Ph.D. candi-date at the University of

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drugs and diagnostic imaging (Fig. 1). Some of these carriers arebriefly described below.

California, Santa Barbara inthe Chemical EngineeringDepartment and works withProf. Samir Mitragotri. Hereceived his Bachelor’s inChemical Engineering from theUniversity Institute of ChemicalTechnology, Mumbai, India. Hisresearch interests include thedesign of novel drug delivery

and diagnostic carriers and testing their biological function.

Samir Mitragotri is a professorof Chemical Engineering at theUniversity of California, SantaBarbara. He received his Ph.D.in Chemical Engineering from

2.1. Nanoparticles from Biodegradable Polymers

Nanoparticles have been fabricated using biodegradable syntheticpolymers, such as polylactide–polyglycolide copolymers (PLGA),polyacrylates, and polycaprolactones, or natural polymers, such asalbumin, gelatin, alginate, collagen, and chitosan.[23] Various fabric-ation methods, such as solvent evaporation, spontaneous emulsi-fication, solvent diffusion, salting out/emulsification-diffusion, useof super critical CO2, and various polymerization methods, havebeen used to synthesize nanoparticles.[24] A variety of therapeuticagents including low-molecular-weight drugs, proteins, and DNAhave been encapsulated in these nanoparticles.[25] Imaging contrastagents have also been successfully encapsulated in PLGAparticles.[26] The advances in the design of polymeric particles forimproved particle performance are discussed in detail later.

the Massachusetts Institute ofTechnology. His research inter-ests include the development ofnovel methods of drug delivery.His group is also workingon understanding transportprocesses in biological systems

through experimental and theoretical investigations.

2.2. Micelles of Diblock Copolymers

Micelles of amphiphilic diblock copolymers in aqueous solutionscan serve as excellent delivery vehicles, for example, PLA-PEGmicelles (where PLA is polylactic acid and PEG is polyethyleneglycol).[27] The hydrophobic core of micelles can entrap hydro-phobic drugs, and the hydrophilic shell, which usually consists ofeither PEO (polyethylene oxide) or PEG, offers excellent stealthproperties. Recently, efforts have focused on chemicalmodificationof the block copolymer building units such as the use ofcrosslinkable groups to increase the stability of the micellar drugcarriers or specific ligands that enable targeted drug delivery.[27]

2.3. Liposomes

Liposomes, lipid vesicles derived from self-assembled enclosedlipid bilayers, are one of the most widely researched drug deliverycarriers, and some liposomal delivery systems have alreadyentered clinical practice.[28] Enhanced safety and heightenedefficacy have been achieved for a wide range of drugs. Surfacemodificationof liposomeswithhydrophilic polymers, suchasPEGand targeting moieties, has been achieved.[29] Some of thelimitations of liposomes include relatively quick release of drugsand poor storage stability.[30]

2.4. Lipid Nanoparticles

Solid lipid nanoparticles (SLN) and nanostructured lipid carriers(NLC) have found a great deal of applications in cosmetics anddermatology.[31] Excipients ofSLN include lipidswith relatively lowmelting points, some of which are present in common foods.Advantages of SLN andNLC include a high surface area, favorablezeta potential, prolonged release profile of encapsulated drugs,rapid uptake by cells, and relatively simple productionmethods.[32]

Moreover, administration of SLNs by various routes, includingparenteral, topical, and oral, has been tested.

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2.5. Polysaccharide-Based Nanoparticles

Nanoparticles fabricated from polysaccharides offer distinctadvantages compared to other drug delivery systems since theyare highly stable, safe, nontoxic, hydrophilic, and biodegrad-able.[33] Moreover, polysaccharides are abundantly available, forexample, of algal origin (alginate), plant origin (pectin, guar gum),microbial origin (xanthan gum), and animal origin (chitosan,chondroitin). Polysaccharides have a large number of reactivegroups, a wide range of molecular weight, and varying chemicalcomposition, which contribute to their diversity in structure andproperties and which facilitate the ease of surfacemodification forefficient drug delivery. Hence, many crosslinked polysaccharide-based nanoparticulate delivery systems have been recently studiedfor therapeutic applications.[34]

2.6. Protein-Based Nanoparticles

Hydrophobic therapeutics, such as taxanes, have proved instru-mental in chemotherapy for cancer treatment. However, solvent-based delivery vehicles used for such therapeutics are associatedwith toxicity and hypersensitivity-related issues. Protein nano-particle-based delivery vehicles hold great potential to reduce thetoxic effects of solvents.[10,16] Abraxane, a 130 nm, albumin-boundpaclitaxel, is the first FDA approved commercial product based onprotein nanoparticles for the treatment of breast cancer in patientswho fail combination chemotherapy for metastatic disease orrelapse within 6 months of adjuvant chemotherapy.[35] The phaseIII trial conducted in women with metastatic breast cancer

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Figure 1. Carriers for drug delivery and diagnostics: a) scanning electron microscopy (SEM)

image of polymer microspheres; b) block copolymers used for synthesis of micelles;

c) liposomes; d) solid lipid nanoparticles; e) abraxane (albumin bound paclitaxel)-protein-

based nanoparticles; f) polyelectrolyte-based layer-by-layer capsules [37]; g) dendrimers [38];

h) microbubbles; i) carbon nanotubes and fullerenes [4]; j) PEG-coated polymeric particle;

k) quantum dots [103]; l) iron oxide nanoparticles encapsulated within polymeric carriers

coated with targeting moiety-composite nanoparticles. Previously published figures are repro-

duced with permission. Copyright 2006 Elsevier (f), 2005 Nature Publishing Group (g,k), 2005

Elsevier (i).

illustrated increased response rates with nab (nanometer albuminbound)-abraxane compared to Cremophor EL-paclitaxel (33%versus 19%, respectively) and prolonged time to tumor progres-sion. The potential use of nab-paclitaxel and nab-docetaxel is beingstudied in a variety of solid tumors.[16]

2.7. Layer by Layer (LbL) Capsules

LbL capsules have attracted particular interest due to the ability toreadily tailor their properties, such as size, composition, porosity,surface functionality, and colloidal stability.[36] Their fabricationprocedure involves coating a template colloidal particle withcomplementary/interacting layers of polyelectrolytes/proteinsand subsequently dissolving the sacrificial template. The stepwiseformation allows introduction of multiple functionalities, whichmakes them appealing for drug delivery applications. Variousencapsulation, targeting, and drug release strategies have beendeveloped for LbL capsules.[37]

2.8. Dendrimers

Dendrimers are branched synthetic polymers with a layeredarchitecture. The chemical composition and molecular weight of

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dendrimers can be precisely controlled unlikemost linear polymers, and hence, it is relativelyeasy to predict its biocompatibilty and pharma-cokinetics.[38] Dendrimers find applications inbiomedicine as targeted carrier of chemother-apeutics, tissue repair scaffolds, optical oxygensensors, and anti-viral drugs.[39]

2.9. Hydrogels for Drug Delivery

Hydrogels, with their 3D architecture, arecapable of imbibing high amounts of water orbiological fluids and exhibit unique physico-chemical and biological characteristics. This hasresulted in considerable attention being given tothese hydrophilic polymeric materials as excel-lent candidates for delivery systems of therapeu-tic agents.[40] Crosslinking provides stability inaqueous systems and results in swelling of thehydrogels. Hydrogels—prepared either fromnatural polymers, such as chitosan and alginate,or fromsynthetic ones, suchaspoly(vinyl alcohol),poly(ethylene oxide), poly(ethyleneimine), poly-(vinyl pyrrolidone), and poly-N-isopropylacryl-amide—have been extensively studied for con-trolled release of therapeutics, stimuli responsiverelease, andapplications inbiological implants.[41]

2.10. Microbubbles

Microbubbles have been explored as noveldiagnostic agents in conjunctionwith ultrasound

since they are near-perfect reflectors of acoustic energy.[42] Wheninjected intravenously into the bloodstream, microbubbles reflectultrasound waves within the capillaries without disrupting thelocal environment. Hence, microbubble ultrasound contrastagents are clinically useful in enhancing ultrasound imagesand improving the accuracy of diagnosis. More recently,ultrasound contrast agents have laid a new paradigm for diagnosisand treatment of atherosclerosis. Targeted delivery to specificdiseased sites followed by burst release of the drug usingmicrobubbles is being explored.[43]

2.11. Carbon-Based Nanoparticles

Carbon-based nanomaterials attract particular interest, since theyare chemically inert, but can be surface-functionalized for thegrafting of nucleic acids, peptides, and proteins. Hence, carbonnanotubes, fullerenes, and nanodiamonds[44] have been exten-sively studied for drugdelivery applications.[45] The size, geometry,and surface characteristics of single-wall nanotubes (SWNTs),multiwall nanotubes (MWNTs), and C60 fullerenes makes themappealing for drug carrier usage. SWNTs and C60 fullerenes havediameters on the order of 1 nm, about half the diameter of theaverage DNA helix. MWNTs have diameters ranging from severalnanometers to tens of nanometers depending on the number of

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walls in the structure. Fullerenes and carbon nanotubes aretypically fabricated using electric arc discharge, laser ablation,chemical vapor deposition, or combustion processes. Theapplication of these nanomaterials in the fields of vaccine delivery,gene delivery, smallmolecule transporters, and targeted delivery isbeing explored.

2.12. Silica-Based Nanoparticles

Silicon-based structures can be fabricated by photolithography,etching, and deposition techniques which are commonly used inthe manufacture of semiconductors and microelectromechanicalsystems (MEMS). Architectures, such as calcified nanopores,platinum-containing nanopores, porous nanoparticles, and nano-needles fabricated out of porous silicon or silica and silicondioxide, find applications in drug delivery.[46] Using sacrificialnanoscale templates such as calcium carbonate, porous hollowsilica nanoparticles can be fabricated. Some of the silicon-baseddelivery systems that havebeen investigated includeporous siliconembedded with platinum as an antitumor agent, calcified poroussilicondesigned as an artificial growth factor, siliconnanopores forantibody delivery, and porous silica nanoparticles containingantibiotics, enzymes, and DNA.[4]

2.13. Metal-Based Nanoparticles

When linked to or embedded within polymeric drug carriers,metal nanoparticles can be used as thermal release triggers whenirradiated with infrared light or excited by an alternatingmagneticfield.[3] Biomolecular conjugation methods of metals includebifunctional linkages, lipophilic interaction, silanization, electro-static attraction, and nanobead interactions.[4]

2.13.1. Gold Nanoparticles

Goldnanoparticles have received great attention in the synthesis ofmultifunctional drug delivery carriers due to the unique capabilityof attachment of thiol-derivatized molecules.[47] Not only can goldnanoparticles be decorated with a targeting ligand, hydrophilicpolymers such as PEG for imparting stealth properties andtherapeutics (drug or nucleic acids), but they can also be imagedusing contrast imaging techniques. Moreover, once the goldnanoparticles are targeted to the diseased site, such as tumor,hyperthermia treatment can be used for tumor destruction.

2.13.2. Magnetic Particles for Drug Delivery and Imaging

Magnetic particles such as iron oxide nanoparticles find greatapplication as contrast agents in MRI, magnetic cell sorting, andimmunoassays in pathology laboratories.[48] Efforts are underwayto developmagnetic particles for controlled and directed transportof therapeutics as well as hyperthermia treatment for canceroustumors.[49]

2.14. Composite Particles

It is clear that a varietyofparticleplatformshavebeendeveloped fora wide spectrum of applications, and they have unique advantages


and limitations. Combinations of materials have also beendeveloped to benefit from the advantages of various materialswhile addressing the limitations of these materials. For example,inorganic nanoparticles possess unique optical and magneticproperties, but lack favorable bulk mechanical properties andsurface processing characteristics. In contrast, polymeric particlesoffer flexibility with respect to manipulation of surface chemistry,bulk mechanical properties, and particle geometry. Hence, coreshell particles with an inorganic core (quantum dots, iron oxidenanoparticles) and polymeric shell have been developed, whichcan be used for therapeutic delivery and imaging.[50–52] Additionalfunctionalities, such as luminescence of the core, can be easilyintroduced. Similarly, CaCO3–polystyrene composites have beendeveloped where CaCO3 provides required strength and poly-styrene provides compatibility. In yet another example, silica,which possesses high surface area, large pore volume, uniformpore size, and low cytotoxicity, is functionalized with magnetic,luminescent, fluorescent, or imaging moieties.[53]

Liposomes andpolymericnanoparticles are the twomostwidelyresearched drug delivery platforms. Attempts have been made tocombine the advantages of both systems. For example, polymericnanoparticles have been encapsulated within fusogenic liposomesto regulate the intracellular pharmacokinetics of gene-based drugsby protecting them from enzymatic and hydrolytic degradation.[54]

Sengupta et al. have reported the use of nanocells consisting ofnuclear PLGA nanoparticles within an extranuclear PEGylatedphospholipid envelope for temporal targeting of tumor cells andneovasculature.[55] Moreover, liposomes are routinely coated witha hydrophilic polymer, such as PEG or PEO, to improve thecirculation time in vivo, which is another example of a liposome–polymer composite.[29] Similarly, hydrogel–metal composites havebeen developed for burst release of the drug.[56] For example,composites of thermally sensitive hydrogels and optically activenanoparticles (gold nanoshells) can result in photothermallyactivated drug delivery.

Active research on composite nanoparticles forms the basis ofdevelopment of multifunctional carriers.[53,57]

3. Polymeric Particles

Polymeric particles are the most widely researched therapeuticcarriers due to the unique flexibility with respect to fabricationtechniques and ability to tailor properties. Important parametersthat determine particle functions include: i) polymer chemistry,ii) particle size, iii) surface chemistry, iv) mechanical properties,and v) shape (Fig. 2). In order to further improve the efficacy ofpolymeric particles, it is important to understand the effect of eachdesign parameter on biological interactions at each level ofanatomical hierarchy. Moreover, the interplay of various designparameters needs to be studied and optimized for specificapplications. Recent advances in the design of polymeric particlesespecially with respect to particle shape are reviewed here.

3.1. Polymer Chemistry

Biodegradable polyesters, such as poly(lactic acid), poly(glycolicacid), their co-polymer PLGA, and poly(e-caprolactone), have beenof particular interest for drug delivery applications.[24] Other


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Figure 2. Important parameters for the design of drug delivery carriers.

biodegradable polymers used for particle fabrication includepoly(orthoesters), poly(anhydrides), poly(amides), poly(phospha-zenes), and poly(phosphoesters).[23] The use of co-polymers tosynthesize particles with desired physical properties has recentlybecome popular. These include co-polymers of above-mentionedpolymers with hydrophilic polymers such as PEG, PEO, orpoly(propylene oxide) (PPO)[58] to impart stealth properties.Polymer properties such as molecular weight, charge, branching,and stability have agreat impact on theparticle functions includingthe mode of degradation, encapsulation efficiency, release rates,and cellular internalization.[59]

Polymers that respond to an external stimulus, such astemperature, pH,or IR irradiationfindawide rangeof applicationsin drug delivery.[8,60] For example, poly(4-styrene sulfonate)/poly-(allyl hydrochloride) capsules can reversibly be made permeabledepending on the pH.[61] Nanoparticles fabricated from a novelhydrophobic polymer poly(1,4-phenyleneacetone dimethyleneketal) undergo acid-catalyzed hydrolysis into low-molecular-weighthydrophilic components and can potentially release encapsulatedtherapeutics at an accelerated rate in acidic environments, suchas tumors or endosomes.[62] In addition, thermosensitive andphotosensitive polymers are also widely used.[56,63]

In order to facilitate multiple functionalization on a singleparticle, strategies have been developed to fabricate biphasic[64]

and triphasic particles.[65] These particles are synthesized bysimultaneous electrohydrodynamic jetting of parallel polymersolutions under the influence of an electrical field. Apart fromcompartmental surface functionalization, the different compart-ments can be loaded with different biomolecules for performingspecific applications.

3.2. Size

Polymeric particles ranging in size from a few nanometers tohundredsofmicrometershavebeen fabricatedby varyingdifferent

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fabrication parameters, such as polymer or surfactant concentra-tion, agitation method (vortexing, sonication, and stirring), speedof agitation, nozzle/capillary diameter, andmaterial flow rate.[24,66]

Size of the particle plays a key role in particle functions, such asdegradation, vascular dynamics, targeting, clearance, and uptakemechanism.[20] Particles have been shown to have differentvelocities, diffusion characteristics, and adhesion propertiesdepending on the size.[67] Particles less than 200 nm are generallyconsidered optimal for intravascular applications due to theircirculation half-life compared to larger particles.[68,69] Transportacross biological barriers, such as skin[70] and mucosa,[71,72]

localization in tissues, and intracellular transport of particles arealso size-dependent.

There are some established rules of thumb with respect toparticle internalization into cells; particles>1mmare internalizedby phagocytosis, and those with diameters between 0.2 and 1mmare internalized by endocytosis. However, recent findings suggestthat particles as large as 5mm can be endocytosed throughreceptor-mediated endocytosis, whichmay open new applicationsin targeted delivery to the vasculature.[73,74]

3.3. Surface Chemistry

Surface chemistry plays a key role in targeted delivery andenhanced systemic circulation of particles. One of the majorbreakthroughs in this area was the finding that particles coatedwithhydrophilic polymermolecules, suchasPEGcan resist serumprotein adsorption and prolong the particle’s systemic circula-tion.[19] Since then, numerous variations of PEG and otherhydrophilicpolymershavebeen tested for improvedcirculation.[75]

Polaxamers and polaxamines, block co-polymers of PEO and PPOhave been extensively studied in this context.[58,76] Block co-polymers of PEG (hydrophilic surface) with PLGA (biodegradable)hold great potential as delivery vehicles.[58]

Targeted delivery is achieved by surface functionalization ofparticles[77] with biomolecules, such as peptides,[78] aptamers,[79]

and antibodies.[80] A number of tumor-specific antibodies arebeing investigated for therapeutic applications. Researchers havealso developed strategies to optimize surface concentrations ofPEG and targeting ligands to strike a balance between prolongedcirculation and effective tissue accumulation. These includeoptimization of randomly distributed PEG and aptamers onnanoparticle surface[81] and fabrication of compartmentalizedparticles.[64,65]

The charge on the particlemight also affect other functions, suchas internalization bymacrophages. Positively charged particles havebeen shown to exhibit higher internalization by macrophages anddendritic cells compared toneutral ornegatively chargedparticles.[82]

Excellent reviews on the effect of particle size and surface chemistryon particle function have been published elsewhere.[19,69,75]

3.4. Mechanical Properties

Recently, there have been several reports which emphasize theimportance of mechanical properties in biological functions.[83] It

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has been shown that macrophages engulf rigid particles to asignificantly higher extent compared to soft particles.[84] A stiff andordered cytoskeleton changes to an irregular biopolymer networkwhen a mature normal cell gets transformed into an immortal,replicating, and motile tumor cell, which can be used in cancerdiagnostics.[85] Discher et al. have shown that the tissue cells cansense the stiffness of the matrix, which can affect its cytoskeleton,differentiation, and development.[86] Thus, mechanical propertiesof biocompatible synthetic materials used in implants, assubstrates for tissue engineering and for fabrication of particulatedrug carriers are important.

3.5. Particle Shape

The effect of polymer chemistry, size, and surface chemistry onparticle functions has been extensively studied. However, recentstudies have shown that apart from size and surface chemistry ofthese particles, shape of the particle can have an intriguing effecton particle functions.[21,22,74] Hence, there has been remarkableinterest in investigating the role that particle shape plays ininternalization, circulation, distribution, and targeting.

4. Particles of Different Geometries: Motivation,Fabrication and Biomedical Applications

4.1. Motivation

The motivation of studying the effect of shape in biologicalinteractions emerges partly from the fact that amajority of entitiesthat we encounter in the biological world are nonspherical inshape.Fromorgans to intracellular organelles,moreoften thannotall biological entities exhibit a nonspherical shape. The peculiarshape of the different biological entities plays an important role intheir function. For example, spleen has a structure in the form ofslits with openings around 200 nm wide and allows filtration ofblood without trapping red blood cells, which have an averagediameter of 7mm owing to their discoidal shape and flexibility.[87]

Similarly, bacteria and viruses exhibit a variety of peculiar shapes.For example, rod-shaped Escherichia coli and Bacillus anthraci,spiral-shaped Campylobacter jejuni, rod-shaped Tobacco mosaicvirus, and bullet-shaped Rabies virus.[88] These peculiar shapesmight enable thebacteria and viruses toperformspecificbiologicalfunctions.

4.2. Fabrication of Particles of Different Geometry

Themajor bottleneck in performing experiments with particles ofdifferent shapes was the difficulty in their fabrication. However,with recent advances in materials science and technology, thislimitation is being addressed. Some of themethods for fabricationof anisotropic shapes include self-assembly,[89] photolithogra-phy,[90] nonwetting template molding,[74] and microfluidics.[90]

Some of the examples of anisotropic particles that have been


already fabricated include PEG-based trapezoids, bars, cubes,cones, discs, cylinders, and many other shapes fabricated by thetop-down particle replication in nonwetting templates (PRINT)technology.[74,91] Dendukuri et al. have developed a high-throughput continuous-flow lithographic technique that com-bines the advantages of microscope projection lithography andmicrofluidics to form morphologically complex polymericparticles of a variety of different shapes. Some of the shapesgenerated by this technique include rings, triangles, cylinders,cuboids, polygonal structures, and curved particles.[90] Wedge-shaped particles bearing segregated hydrophilic and hydrophobicsections have also been synthesized using the same technique.[92]

Velev et al. have demonstrated fabrication of complex particles bygrowing colloidal crystals in aqueous droplets suspended onfluorinated oil. Disks of varying flatness, dimpled particles,doughnut-shaped particles, aswell as anisotropic particles, such asdoughnut-shaped latex particles with a ring consisting of goldnanoparticles, have been fabricated by this technique.[93] A directreplica method reported by Sozzani et al. enabled them tosynthesize an array of plastic micro-objects of different shapes,such as cones, bicones, hollow cylinders, rings, test tubes, clubs,and vases (Fig. 3).[94] Dimethyl formamide-based colloidalnanoparticles with tunable size and shape have been fabricatedby wet chemical methods.[95] Palladium nanocrystals of controlledshape and size have been fabricated by polyol synthesis.[96]

Our laboratory has developed a simple, yet versatile method ofpreparing anarrayofnonspherical particles (Fig. 4).[97,98] Sphericalparticles were used as a startingmaterial for preparing particles ofcomplex shapes. These particles were suspended in an aqueoussolution of polyvinyl alcohol (PVA) and cast into films, which werethenmanipulated to engineer particle shape. In its simplestmode,polystyrene particles were liquefied using solvent or by heatingabove the glass transition temperature (Tg) of polystyrene and thenstretched in one or two dimensions. In another modality, PVAfilmswere stretchedfirst to create voids around theparticles.Thesevoids were then filled by liquefying the particles using solvent orheat. Re-solidifying the particles after manipulation, by solventextraction or cooling, set their new shape. The particles werecollected by dissolving the film and purified by multiple washes.Final particle shape is dictatedby thematerial properties of thefilm(Tg and thickness), the material properties of the particles (Tg andviscosity), interactions between particles and film (adhesionstrength), and the stretching parameters (extent and dimension-ality). Particle volume remains constant during stretching,governed entirely by the volume of the initial sphere. Thus, sizeand shape of particles can be independently controlled.

The liquefied particle is stretched due to its strong associationwith the film arising from hydrogen bonding. However, the finalshape depends strongly on the details of key parameters.Increasing the liquefaction temperature decreases the particleviscosity and results in sharp-ended, worm-like particles withnearly circular cross-sections. The 2D stretching of heat-liquefiedparticles leads to oblate ellipsoids with aspect ratios dictated by theextent of stretching. Replacing heat with toluene, as a mode ofliquefying particles, leads to entirely different shapes due todecreased polystyrene viscosity. Specifically, 1D stretching of filmsafter toluene-liquefaction formed very thin elliptical disks withcurved ends under conditions where heat-liquefied particlesformed rectangular disks with blunt ends. Moderate stretching of


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Figure 3. Particles with peculiar shapes fabricated from different strategies: a, b, c, and e are PEG particles fabricated by PRINT technology with

trapezoidal, bar, cube, and conical shape, respectively [74, 91] (scale bar of c: 20mm). d) Fluorescence image of worm-shaped fillomicelle fabricated from

di-block copolymers [22] (scale bar 2mm). f) A ring of gold nanoparticles formed on a doughnut-shaped latex particle by growing colloidal crystals in

aqueous droplets suspended on fluorinated oil [93] (scale bar: 500 nm). g,h,i,j) Particles fabricated in microfluidic channels by continuous-flow lithography

with triangular, key-shaped, elongated particles with triangular cross-section and wedge-shaped particles bearing segregated hydrophilic and hydrophobic

sections, respectively [90, 92] (scale bars: 30 (g) and 10 (h,i) mm). k) Vase-shaped particle made by direct replication technology [94] (scale bar: 1mm).

Images are reproduced with permission. Copyright 2005 American Chemical Society (a,b,e), 2008 National Academy of Sciences (c), 2007 Nature

Publishing Group (d), 2000 American Association for the Advancement of Science (f), 2006 Nature Publishing Group (g,h,i), 2005 Nature Publishing

Group (k), and 2007 American Chemical Society (j).

toluene-liquefied particles led to UFO-shaped particles due topreferential stretching of the particle around the equator. Manyadditional shapes also resulted from other combinations ofparameters (Fig. 5).

Film-stretching method thus leads to the generation of a widerange of geometries, including predominantly 1D (e.g., worms),2D (e.g., elliptical and circular disks), and 3D shapes (e.g., UFOsand barrels). It has also been adopted to prepare particles frompolymers other than polystyrene, for example biodegradablePLGA.[20] The method can be used to independently control sizeandshapeofparticles. Suchparticleswill be crucial inmethodicallyidentifying the role of geometric parameters in particle function indrug delivery. The method was further modified to controladditional design features, such as surface texture and porositywhile keeping the size and shape constant (Fig. 6). By stretchingthe film in air and allowing it to relax before liquefaction, particleswith a rough surface were obtained. Rapid removal of solvent afterstretching resulted in porous particles. In addition, particles thatdisplay regions of varying concavity, curvature, aspect ratio, andsurface texture were synthesized. In another strategy, particles ofdifferent shapes were fabricated by utilizing more complexstarting materials such as hollow microspheres. In addition tomaking hollow particles of different shapes, we also preparedparticle micelles from hollow microspheres using the self-assembly technique.Diversity of particles can be further enhanced


by incorporating additional features into particles of differentshapes (Fig. 6). Since magnetic particles have received immenseattention in drug delivery and imaging, we synthesized elongatedmagnetic particles using the film-stretching method, therebycombining the importance of particle shapewith the advantages ofmagnetic particles. Moreover, novel parameters such as particleorientation in a magnetic field can be explored with elongatedmagnetic particles, which is not possible with spherical particles.

Such particles with diverse physical features will open up newavenues in engineering carriers for drug delivery and imaging.These particles can also be used asmodels to study the importanceof shape in biological functions of organisms, such as bacteria andviruses, since the film stretching method can efficiently mimicmany peculiar shapes exhibited by biological entities; for example,rod-shaped particles mimic clostridium difficile bacteria.

4.3. Biological Processes Impacted by Shape

Particle shape is likely to influence many biological interactionsinvolved in drug delivery including transport through the bloodvessels, through organs such as the liver and spleen, targeting adiseased site, internalization into cells and intracellular transport.Below, we summarize these processes briefly.


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Figure 4. Film stretching technique for fabrication of particles of different shapes. a) Schematic

representation of 1D and 2D stretching method using custom-made stretchers. b) Modified

stretching procedure for fabrication of more complex particles. Image (b) is reproduced with

permission from Ref. [97]. Copyright 2007 National Academy of Sciences.

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4.3.1. Phagocytosis

In order to explore the effect of particles shape, somerepresentative shapes (spheres and elliptical disks) were selectedto study the importance of particle shape on internalization byphagocytosis. It was observed that the local geometry of the particleat the particle–cell interface plays a crucial role in internaliza-tion.[21] Hence, when an elliptical disk-shaped particle approachedthe macrophage with its major axis perpendicular to the cellmembrane, the particlewas internalizedwithin 6minutes, similarto a spherical particle.[21] However, when the elliptical disk-shapedparticle approached the macrophage with its minor axisperpendicular to the cell membrane, the macrophage was unableto internalize the particle for hours (Fig. 7a). Internalization ofparticles of different shapes from the library was then tested, and apeculiar trend was observed. A parameter omega (V) was definedto quantify the local geometry of the particle–cell interface, whichindicates themean angle made by themembrane with the normalas it travels around the particle during phagocytosis (Fig. 7b). Forparticles with V> 45, the cells were able to spread around theparticles but could not internalize the particles, whereas forV� 45, the cells internalized the particles easily which is quitestriking (Fig. 7b). Phagocytosis is mediated by formation andprogressionof anactin ringor cup,whichwasnot seen forparticleswithV> 45.Thus, the local geometryplays a very important role indetermining phagocytosis of particles of different shapes. More-

� 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

over, internalization of both opsonized andnonopsonized particles exhibited a strongdependence on local particle shape from theperspective of the phagocyte.

The importance of local geometry duringinternalization can be explored to engineerparticle shapes that either promote or inhibitphagocytosis. For therapeutic delivery to targetsother than macrophages, internalization viaphagocytosis needs to be avoided. With thisaim, worm-shaped particles were fabricated,which have a large aspect ratio (>20) to avoidphagocytosis.[99] In the case of worm-shapedparticles, the V< 45 exists only at two discreteendpoints whereas the rest of the structure isrelatively flat (V¼ 87.5), and hence the prob-ability of the particles being internalized isextremely low which was verified experimen-tally. The worm-shaped particles fabricatedfrom 3 and 1mm spheres exhibited a six- and20-fold lower internalization with respect to 3and 1mm spheres (Fig. 7c).

4.3.2. Endocytosis

Although shape plays an important role invarious biological processes, it is important tounderstand the interdependence of shape withother particle design parameters. Gratton et al.reported the interdependence of shape, size,and surface chemistry on cellular internaliza-tion.[74] A top-down particle fabrication techni-que, PRINT was utilized to fabricate uniform

populationsof cationic, crosslinkedpoly(ethyleneglycol) hydrogel-based micro- and nanoparticles with control over size, shape, andsurface chemistry. Cellular internalization studies of differentaspect ratios of cubic and cylindrical particleswere performed, andit was found to have a strong dependence on the particle size andshape (Fig. 8a). Moreover, cationic particles were internalized to asignificantly higher extent compared to anionic particles which isconsistent with previous reports.

In another study, Muro et al. showed that particle size andshape plays an intriguing role in endothelial targeting (Fig. 8b),receptor-mediated endocytic internalization and intracellulartrafficking. Spheres (0.1–10mm) and elliptical disks(0.1mm� 1mm� 3mm) were coated with anti-ICAM1 to targetICAM1 expressed on the surface of endothelial cells. Gratton et al.and Muro et al. showed that particles up to severalmicrometers in size could be endocytosed. Disks showedhigher circulation half-life and significantly better targetingefficiency in comparison with spheres of all sizes studied.[73]

Thus, carrier geometry plays an important role in therapeuticdelivery to the endothelium.

4.3.3. Circulation

Discher and co-workers synthesized highly stable polymermicelleassemblies known as fillomicelles which exhibit a prolonged

Adv. Funct. Mater. 2009, 19, 3843–3854

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Figure 5. Library of particles of different shapes fabricated by the film stretching technique [97]: a) spheres, b) rectangular disks, c) rods, d) worm-shaped,

e) oblate ellipsoids, f) elliptical disks, g) UFO-shaped particles, h) circular disks, i) ribbons with curled ends, j) bicones, k) diamond disks, l) emarginate

disks, m) flat pill-shaped, n) elongated hexagonal disks, o) ravioli-shaped, p) taco-shaped, q) lemon-shaped particles, r) circular disks with a hump,

s) truncated bicones, and t) pulley-shaped particles (scale bar: 2mm). Images (a–p) and (t) reproduced with permission. Copyright 2007 National Academy

of Sciences.

circulation life and delivery of anti-cancer drugs for tumorannihilation.[22] Cylindrical micelles were self-assembled in waterfrom block co-polymers with lipid-like amphiphilicity (PEG withpolyethylethylene (PEE) and poly(e-caprolactum)). In vitro experi-ments showed that the fillomicelles enter cells under staticconditions, but avoid internalization under flow conditions. Invivo, these fillomicelles could circulate in rodents for around aweek, which is about ten times more than circulation time ofspherical counterparts and significantly higher than that of anyknown synthetic nanoparticle. Most nano-vehicles are clearedfrom the body within hours of administration. The length of thefillomicelles decreases over a period of time since fragmentsof fillomicelles break and are cleared from the body. Thesefillomicelles have been demonstrated to be excellent drug carriersfor cancer therapy. In particular, the delivery of anti-cancer drug


paclitaxel to tumor-bearing mice led to significant reduction intumor volume.

4.3.4. Targeting

In principle, particle shape should affect the targeting efficiencysince particles of different shapes possess different surface areaper unit volume that can potentially interact with the targetreceptors (Fig. 8d). Park et al. have demonstrated that dextran-coated iron oxide nanoworms show enhanced magnetic relaxivityin MRI, which make them better contrast agents. Moreover, theiron oxide nanoworms showed a higher tumor targeting efficiencycompared to spherical counterparts which can be attributed to thehigher contact surface area of the nanoworms (Fig. 8c).[100]

Similarly, the fillomicelles used by Geng et al. exhibited highercirculation and better tumor targeting compared to spherical


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Figure 6. Library of particles of different shapes with additional features and complexity [97]:

a,b) wrinkled prolate and oblate ellipsoids, c) porous bicones, d) fluorescent image of worm-

shaped particles with non-convex geometry (inset shows SEM image), e)magnetic elliptical disks,

f) particle micelles using self-assembly. Scale bars: 2mm (a,c,d); 400 nm (b). Images

(a–c) reproduced with permission. Copyright 2007 National Academy of Sciences.

Figure 7. Effect of shape of particles fabricated by film-stretching technique on various biological

processes. a) Phagocytosis of elliptical disks depends on the local geometry of particles in contact

with the macrophage. In the end-on orientation, the disk is easily phagocytosed, whereas in the

side-on orientation, the macrophage struggles to phagocytose the particle for a long time. On

the other hand, spheres are always easily phagocytosed [21]. b) Effect of local particle geometry

defined in terms of V on internalization by macrophages [21]. c) SEM image of a macrophage

internalizing IgG-adsorbed worm-shaped particle and the effect of internalization on the shape of

the cell. The rate of internalization of worms is significantly less than the spherical counterparts

[99]. d) Effect of size of IgG opsonized (open circles) and non-opsonized (closed circles) particles

on phagocytosis (attachment and internalization) by macrophages [104]. Images are reproduced

with permission. Copyright 2006National Academy of Sciences (a,b), 2009 Springer (c), and 2008

Springer (d).

3852 � 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

micelles.[22] Muro et al. have also demonstratedenhanced accumulation of targeted ellipticaldisks in lungs.[73]

4.3.5. Transport Through the Vasculature

Using theoretical models to supplementexperimental data, Decuzzi et al. have investi-gated theeffect of shape on the transport of cellsand carriers through the blood vessels.[101]

Margination, firm adhesion, and internaliza-tion are important features of transport ofcarriers through capillaries. Particle interactionwith the vasculature includes receptor–ligandinteractions and nonspecific interactions, suchas van der Waal, electrostatic, and stericinteractions. A neutrally buoyant sphericalparticle moving in proximity to a wall can driftlaterally only in thepresenceof anexternal forcewhereas nonspherical particles exhibit morecomplex motions with tumbling and rollingwhich can be exploited to control theirmargination dynamics without any need forlateral external forces. For nonspherical parti-cles, it has been shown that the lateral driftingvelocity is directly related to their aspect ratio,with a maximum between the two extremes:sphere, with aspect ratio unity, and disk, withaspect ratio infinity. Discoidal particles havebeen shown to marginate more than quasi-hemispherical which in turn marginated morethan spherical particles in a gravitationalfield.[102] Such models provide profoundfundamental understanding and predictivecapabilities for carrier behavior in flow andwill prove to be instrumental in efficient designof carriers.

5. Summary and FutureDirections

It is clear that the shape of colloidal carriersplays a crucial role in many biological interac-tions. Shape of the particle adds a newdimension in the toolbox of designing carriersfor drug delivery and imaging. While the effectof particle shape on various biological interac-tions is being realized, it is worthwhile to notethat different design parameters are highlyinterlinked, and the optimization of all thedesign parameters for a particular applicationwill be the key to success. The ultimate aim isto synthesize a vehicle which is optimized toperform a wide spectrum of desired tasks in adefined sequence, in other words, a multi-functional delivery carrier (Fig. 9). With theadvances in the field of particulate medicine,the futuristic multifunctional delivery carriersseem close to reality.

Adv. Funct. Mater. 2009, 19, 3843–3854

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Figure 8. Effect of shape of particles fabricated by different techniques on various biological

processes. a) Effect of particle geometry on internalization in HeLa cells [74]; AR¼ aspect ratio.

b) Immunospecificity index (ISI, ability of particles coated with targeting moiety to accumulate in

a particular organ in comparison with particles coated with nonspecific moiety) of particles with

different geometries in the liver and lung (target site) [73]. c) Biodistribution of iron oxide

nanoworms (NW) in comparison with nanospheres (NS) for targeting tumors in mouse. FRI

refers to fluorescence index. Arrows point towards tumor and liver [100]. d) Schematic

representation of higher contact surface area presented by elongated particles in comparison

with spherical particles, thereby having a higher potential of targeted delivery [100]. e) Circulation

profile of fillomicelles fabricated by Geng et al. In comparison with long circulating stealth vesicles

and phages, the fillomicelles remain in circulation for a significantly longer time [22]. Previously

published images are reproduced with permission. Copyright 2008National Academy of Sciences

(a), 2008 The American Society of Gene Therapy (b), and 2007 Nature Publishing Group (e).

Figure 9. Schematic of a multifunctional delivery carrier for drug delivery.

a) A flexible elliptical disk-shaped carrier decorated with PEG chains that

possess a targeting moiety at the end. Iron oxide nanoparticles and desired

therapeutic entity can be encapsulated within the carrier. b) The multi-

functional delivery carrier flowing through the vasculature.

Adv. Funct. Mater. 2009, 19, 3843–3854 � 2009 WILEY-VCH Verlag GmbH & Co. KGaA,

Acknowledgements

This work was supported by National Institute ofHealth Program of Excellence in Nanotechnology(1U01HL080718). Authors acknowledge Peter Allenfor help with Figure 9 and Professor Julie Championfor electron microscopy images of the particles.

Received: August 14, 2009

Published online: November 20, 2009

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