12 csr wooley design of polym nanocarries

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This article was published as part of the

Nanomedicine themed issue

Guest editors Frank Caruso, Taeghwan Hyeon and Vincent Rotello

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ISSN 0306-0012

0306-0012(2012)41:7;1-Q

www.rsc.org/chemsocrev Volume 41 | Number 7 | 7 April 2012 | Pages 252130

Themed issue: Nanomedicine

Chemical Society Reviews

Guest editors: Frank Caruso, Taeghwan Hyeon and Vincent M. Rotello

TUTORIAL REVIEWMahmoud Elsabahy and Karen L. WooleyDesign of polymeric nanoparticles for biomedical delivery applications


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This journal is c The Royal Society of Chemistry 2012 Chem. Soc. Rev., 2012, 41 , 25452561 2545

Cite this: Chem. Soc. Rev ., 2012, 41 , 25452561

Design of polymeric nanoparticles for biomedical delivery applications w

Mahmoud Elsabahy* abc and Karen L. Wooley* abd

Received 1st December 2011

DOI: 10.1039/c2cs15327k

Polymeric nanoparticles-based therapeutics show great promise in the treatment of a wide rangeof diseases, due to the exibility in which their structures can be modied, with intricate denitionover their compositions, structures and properties. Advances in polymerization chemistries andthe application of reactive, efficient and orthogonal chemical modication reactions have enabledthe engineering of multifunctional polymeric nanoparticles with precise control over thearchitectures of the individual polymer components, to direct their assembly and subsequenttransformations into nanoparticles of selective overall shapes, sizes, internal morphologies,external surface charges and functionalizations. In addition, incorporation of certainfunctionalities can modulate the responsiveness of these nanostructures to specic stimuli throughthe use of remote activation. Furthermore, they can be equipped with smart components to allowtheir delivery beyond certain biological barriers, such as skin, mucus, blood, extracellular matrix,cellular and subcellular organelles. This tutorial review highlights the importance of well-denedchemistries, with detailed ties to specic biological hurdles and opportunities, in the design of nanostructures for various biomedical delivery applications.

Introduction

Nanomedicine can be dened as the design of diagnostics and/or therapeutics on the nanoscale, which provides advantagesdue to the high degree of coincident transport and delivery of the active species with mediation of their navigation within thebiological systems for the treatment, prevention and diagnosisof diseases. The biological transport processes, anatomicallyand down to the cellular and sub-cellular levels, are affected bythe physical attributes of the nanocarriers, including their size,shape, and exibility, as well as their chemical characteristics,including for instance the incorporation of active ligands forrecognition by and triggering of biological receptors. Therefore,it is of critical importance to utilize procedures that preparenanostructures with high degrees of uniformity, and with controlover their physical and chemical traits. Nanoparticles can beconstructed from various materials ( e.g. polymers, lipids, metals)and can host a wide range of active components, includingchemotherapeutics, contrast agents, proteins and nucleic acids,

for various biomedical applications. In particular, polymericnanoparticles have received great interest due to the versatilityin which their structures can be modied to package anddeliver their cargoes to the desired site of action or to respondto specic physiological or external stimuli (Fig. 1 and 2).Incorporation of certain functionalities can modulate theresponsiveness (assembly/disassembly) of the nanoparticlesin biological environments under different pH, enzymatic,oxidative, and reductive conditions, etc. , or in response toexternal stimuli, such as variation in temperature, irradiationwith near-IR or UV-vis light, activation with magnetic elds,and application of ultrasound vibrations, etc. The chemistry of polymeric nanoparticles and their payloads affects their stability,biodegradability, biocompatibility, biodistribution and cellular andsubcellular fate. The design of polymeric nanoparticles depends onthe therapeutic application, target site (organs, tissues, cellular orsubcellular organelles) and the route of administration. Althoughintravenous injection is the main route of administration forpolymeric nanoparticles, there are other ways to deliver them vialess invasive ways, such as dermal/transdermal, oral and mucosaldelivery. In all cases, these nanoparticles have to be equipped withsmart components to allow their delivery beyond the differentbiological barriers, such as skin, mucus, blood, extracellular matrix,in addition to the cellular and subcellular barriers.

Polymeric nanoparticles-based therapeutics are being developedto improve the diagnosis and treatment of a wide range of diseases,ranging from cancer, viral infections, cardiovascular diseases topulmonary and urinary tract infections. Advances in controlledpolymerization have enabled the engineering of advancedmultifunctional polymeric nanoparticles with precise control

a Department of Chemistry, Texas A&M University, P.O. Box 30012,3255 TAMU, College Station, Texas 77842-3012, USA.E-mail: [email protected],[email protected]

b Laboratory for Synthetic-Biologic Interactions, Texas A&M University, P.O. Box 30012, 3255 TAMU, College Station,Texas 77842-3012, USA

c Department of Pharmaceutics, Faculty of Pharmacy,Assiut University, Assiut, Egypt

d Department of Chemical Engineering, Texas A&M University,P.O. Box 30012, 3255 TAMU, College Station,Texas 77842-3012, USA

w Part of the nanomedicine themed issue.

Chem Soc Rev Dynamic Article Links

www.rsc.org/csr TUTORIAL REVIEW
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2546 Chem. Soc. Rev., 2012, 41 , 25452561 This journal is c The Royal Society of Chemistry 2012

over architecture, shape, size, surface charge and functionalization.The careful design and control over the targeting properties of these nanoparticles will secure their future development andversatility. The proper selection of the chemistry of the buildingblocks of these nanocarriers and their payload can drasticallyimpact their safety, pharmacokinetics and intracellular fate. Ourgroup has recently reviewed the application of orthogonalchemistry in the synthesis, preparation and functionalizationof polymeric nanostructures of different architectures 1 and theavailable technologies to precisely control the self-assembly andstability of these nanostructures. 2 This review highlights theimportance of well-dened chemistries in the design of nano-structures to surmount certain biological barriers for variousbiomedical applications. In particular, the underlying potential,current challenges, and future directions of polymeric micelles,crosslinked knedel-like nanoparticles and unimolecular/hyper-branched nanostructures will be discussed.

Biological barriers

For therapeutic benets, the drugs, either free or loaded intonanoparticles, have to reach their destinations ( i.e. sites of action),which are on the cellular or molecular levels. The barriers towardsthe delivery of these nanoparticles can be classied into externalbarriers (skin and mucosa), en route (blood and extracellularmatrix) and cellular barriers (the limited cellular uptake,endosomal/lysosomal degradation and the inefficient transloca-tion to the targeted subcellular organelles) (Fig. 3).

External barriers

The body surface is covered and protected with either skin ormucus. Both skin and mucus hinder polymeric nanoparticlesfrom reaching their target sites that are located either locally inthe underlying tissues or systemically in the blood. Althoughskin and mucus have different structures, both of them canprevent the uptake of the polymeric nanoparticles through

different mechanisms. In addition to hindering the uptake of polymeric nanoparticles, they may alter the surface characteristicsand stability of the nanoparticles before they are able to reach thesurface of the underlying tissues.

Skin consists of several layers of different thicknesses andstructures, stratum corneum (SC), epidermis, dermis and subcu-taneous tissues. 3 The outermost layer, SC, is a highly-organizedand hydrophobic structure consisting of several layers (1020 mm)of terminally-differentiated nonliving corneocytes embedded inintercellular lipid matrix-forming bilayers. Corneocytes containintracellular crosslinked macrobrillar bundles of keratin givingthe SC a rigid and hydrophobic structure. The SC is considered asthe major permeability barrier and the rate-limiting step for the

delivery through the skin. The viable epidermis (50100 mm) anddermis are immunologically-active sites consisting mainly of keratinocytes and broblasts, respectively, and rich in immunecells, such as Langerhans cells in the epidermis and dendritic cells inthe dermis. The dermis and subcutaneous tissues (12 mm) are richin blood vessels that can be used for the systemic ( i.e. transdermal)delivery. Disrupting the lipid bilayers of the SC is utilizedcommonly to enhance the permeation through the skin.

Mahmoud Elsabahy

Mahmoud Elsabahy isCo-Assistant Director of theLaboratory for Synthetic-

Biologic Interactions at theDepartment of Chemistry,Texas A&M University, and a Lecturer at the Departmentof Pharmaceutics, Faculty of Pharmacy, Assiut University,Egypt. He received a BS inPharmaceutical Sciences fromAssiut University, Egypt and then he nished his MSc and PhD degrees from the Facultyof Pharmacy, Universityof Montreal under supervision

of Professor Jean-Christophe Leroux (currently at the Institute

of Pharmaceutical Sciences, Drug Formulation & Delivery,ETH, Zu rich). He spent one year as a postdoctoral fellow and another year as a Research Associate at the School of Phar-macy, University of Waterloo (Ontario, Canada), studying theapplication of non-viral vectors for dermal and transdermal genetherapy. Research interests include the rational design of nano-medicines for various biomedical applications. He has obtained the Academic merit award (J. A. DeSe ve), and has beennominated as a meritorious candidate for both a Natural Sciences and Engineering Research Council of Canada(NSERC) Visiting Fellowship and a NSERC Industrial R&DFellowship in the cellular and molecular biology areas of expertise.

Karen L. Wooley

Karen L. Wooley holds theW. T. Doherty-Welch Chairin the Department of Chemis-

try at Texas A&M University,with a joint appointment in theDepartment of Chemical En- gineering. She received a BS inchemistry from Oregon StateUniversity in 1988 and thenstudied under the direction of Professor Jean M. J. Fre chetat Cornell University, obtain-ing a PhD in polymer/organicchemistry in 1993. Researchareas include the synthesisand characterization of de-

gradable polymers, unique macromolecular architectures and

complex polymer assemblies, and the design and developmentof well-dened nanostructured materials, for which she hasreceived several awards, including an Arthur C. Cope ScholarAward, a Herman F. Mark Scholar Award, and awards from theNational Science Foundation, the Office of Naval Research, and the Army Research Office. Karen serves as an Editor for theJournal of Polymer Science, Part A: Polymer Chemistry. Shedirects a National Heart Lung and Blood Institute-supported Program of Excellence in Nanotechnology, serves on theNational Institutes of Health NANO study section, and alsoserves on the Scientic Advisory Panel for the National Institutes of Health Nanomedicine Development Centers and on the International Scientic Advisory Board for the DutchBioMedical Materials Program.


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Mucus coat regions are not covered by the skin, includingthe gastrointestinal tract, eyes, lung airways, nasal, rectal andvaginal cavities. Mucus is a viscoelastic hydrogel secreted bythe mucosal glands to protect the cells below. The composition,vascularity, surface area, thickness, permeability, enzymaticactivity and pH of mucus vary from one region to another(e.g. respiratory vs. vaginal) and depend on the disease status(e.g. mucus becomes thick and sticky in patients with cysticbrosis). 4 The mucus gel is mainly comprised of a network of crosslinked mucin bers. These bers are composed of alter-nating glycosylated hydrophilic regions and relatively hydro-phobic regions. Mucin is negatively-charged due to thepresence of N -acetylneuramic acids (sialic acids) and sulfatedmonosaccharides in the sugar chains. Mucus gels are alsoloaded with cells, bacteria, lipids, salts, proteins, macromolecules,and cellular debris. The various components work together toform a nanoscopic network that hinders nanoparticle transport.Mucociliary clearance is a major reason for the clearance of nanoparticles. Other contributing factors include the adhesivenessand enzymatic activity of the mucus gel and steric hindrance by thenanoscopic layer. In addition, binding of mucus ingredients to the

surface of nanoparticles may result in their destabilization andaggregation or charge neutralization and displacement of theircargoes. Other factors can also complicate mucosal delivery,depending on the delivery route. For instance, oral delivery ischallenging due to the harsh conditions that nanoparticlesexperience as they transit from the stomach to the intestine,such as the pH gradient from 13.5 in the stomach, 57 in thesmall intestine to 67.5 in the large intestine and digestiveenzymes, which can destabilize or degrade a wide range of polymers and therapeutics.

En route barriers

Injection of polymeric nanoparticles into the systemic circula-tion circumvents the skin and mucosal barriers. However,injection is an invasive and unfavorable way of administrationbecause it raises the requirements of quality control, such assterility, increases the cost, lowers the patient complianceand places a burden on both the patients and caregivers. Inaddition, nanoparticles in the blood are subject to renal andhepatic clearance, destabilization, aggregation, opsonization and

Fig. 1 Building blocks of various types of polymeric nanoparticles with examples of some commonly used polymers and linkages. The mainbuilding blocks of polymeric nanoparticles are usually comprised of core-forming polymers; hydrophobic or charged (a), shell-forming polymer;neutral, hydrophilic and exible properties are important for stealth nanoparticles (b), targeting ligand for selective cellular uptake andaccumulation at target sites (c), and linkages between the shell and core and/or targeting moieties (d). Stimuli-responsiveness (pH, temperature,enzymatic, reductive or oxidative, etc. ) can be imparted into the core, shell and/or the linkages. Shell or core-crosslinking can be also utilized toenhance the stability of nanoparticles (e).


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scavengers to attack and engulf foreign particles, when theyare tagged by the appropriate opsonin. The opsonization of nanoparticles and subsequent clearance by MPS could alsoinitiate severe immunological reactions. In addition, the adsorp-tion of these proteins can potentially destabilize nanoparticles andlead to the premature release of their payloads. The released drugloses the favorable pharmacokinetics of the nanocarrier andbecomes then available to induce toxicity. Plasma proteins canalso bind to or displace the encapsulated drug. The abilities of proteins to destabilize nanostructures are often measured, forinstance a series of proteins ( e.g. albumin, a - and b-globulins,g-globulins) were tested with polymeric micelles ( e.g. poly(ethyleneglycol) (PEG)- b-poly( propyl methacrylate- co-methacrylic acid)/poly(amidoamine) and PEG- b-poly( D ,L-lactide)), 68 for whichit was observed that, although most proteins were found tocontribute to micelle destabilization, a more signicant effectwas observed for a - and b-globulins. However, destabilizationof nanoparticles may also result from interactions with othermolecules in blood ( e.g. blood cells) and the degradation of thepolymeric constituents of the nanoparticle. In tissues, bindingand interactions with the extracellular matrix and immune cellscan also hinder nanoparticles from reaching their sites of action.

The extent of opsonization, destabilization and clearancedepends mainly on the nanoparticle characteristics and willbe discussed in detail in the following sections.

Cellular barriers

The previously-mentioned barriers can alter the characteristicsand distribution of nanoparticles before they reach the surfaceof the target cells. However, these are not the only barrierstowards delivery. Cellular uptake is highly challenging, especiallyfor hydrophilic drugs and macromolecules. Interactions of nano-particles with components of the outer surface of cells andcellular membranes followed by internalization into vesicles(invagination of the plasma membrane surrounds the nano-particle) are the initial steps for the endocytosis process. Thesevesicles are then pinched off to form membrane-bound vesiclesof different sizes, compositions and internal environments,called endosomes, phagosomes or macropinosomes, dependingon the internalization pathway. The endocytic pathway dependson the size, morphology and surface chemistry of the nano-particles and varies from one cell line to another. 9 Currently, thereare ve recognized mechanisms for the uptake of nanoparticles,

Fig. 4 Possible destabilization and degradation pathways of polymeric nanoparticles during in vivo circulation (a) and the EPR effect andintracellular fate of nanoparticles (b). Drug-leakage, disassembly or degradation, detachment of surface-decorating moieties, opsonization andclearance of nanoparticles during circulation can all be detrimental to the efficiency of nanoparticles. Tumor tissues are characterized by the leakyvasculature that allows nanoparticles to accumulate in the tumor tissues. The endocytosis of the nanoparticles can then occur via differentmechanisms ( e.g. via multivalent binding and receptor-mediated endocytosis), ending into endocytic vesicles of different microenvironmentsdepending on the composition and characteristics of the nanoparticle. Entrapment of nanoparticles into the endocytic vesicles (dashed arrow)prevents them from reaching their target sites (cytoplasm, mitochondria, nucleus). The disassembly of polymeric nanoparticles and drug releasecan occur at various steps during the circulation and the intracellular trafficking pathway.


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phagocytosis, macropinocytosis, clathrin-mediated, caveolin-mediated, and clathrin/caveolin-independent endocytosis,depending on the proteins assisting in the endocytosis process. 10

Entrapment of drugs into the vesicles ( e.g. endosomes) oftenresults in their degradation due to the acidic pH and enzymesfound in the late endosomes (lysosomes). Escape of nanoparticlesor their payloads from these vesicles into the cytoplasm is essentialfor them to reach the targeted subcellular organelles (Fig. 4). Theviscosity and intracellular enzymes of the cytosol can also alterthe stability and movement of nanoparticles. Translocation tosubcellular organelles ( e.g. nucleus or mitochondria) is challengingdue to the physiological nature of these organelles and their boundmembranes. Recycling (exocytosis) of the vesicle contents is also apossible pathway for the excretion of nanoparticles from cells.Direct translocation of nanoparticles across the plasma membraneis another suggested endocytic pathway that does not depend onthe metabolic activity of the cells. This pathway is known asenergy independent, receptor-independent uptake ortransduction. Although still controversial, this pathway ismost well studied with a unique category of peptides, calledcell penetrating peptides (CPPs), 11 which have been applied forthe transport of CPP-functionalized nanoparticles, and in fact,this mechanism has been found recently to be involved in theuptake of nanoparticles in the absence of CPPs. 12 The interestingfeature of this pathway is that nanoparticles have a direct andquick access to the cytoplasm after crossing the membrane,which could simplify the design of nanoparticles by eliminatingthe need for additional features to be built into the nanoparticleframework for the purpose of disrupting the endocytic vesicles.

Rational design of polymeric nanoparticles

Signicant efforts continue to be exerted towards the designand development of polymeric nanocarriers with tailored

physical, chemical and biological properties. 13 The size, shapeand surface characteristics of nanoparticles dictate the bloodstability, biodistribution, endocytic pathway and intracellulardistribution and bioavailability of nanoparticles. In the followingsections, the rational design and characteristics of polymericnanoparticles, and their crucial constituents will be highlighted,with emphasis on polymer micelles, crosslinked knedel-likenanoparticles, and unimolecular/hyperbranched nanostructures(Table 1).

Stealth nanocarriers

One of the most successful strategies to avoid interaction of nanoparticles with components of biological uids is coatingwith a dense brush layer of neutral hydrophilic exible polymersto prevent adsorption of plasma proteins and avoid recognitionby the MPS and subsequent clearance (Fig. 5). PEG is abiocompatible, hydrophilic, biologically-inert polymer thathas been approved by the U.S. Food and Drug Administration(FDA) for internal use, for a number of applications. PEG isthe most commonly utilized polymer for coating nanoparticles.PEGylation and the length and number of PEG chains pernanoparticle have been shown to produce signicant differencesin blood circulation times vs. elimination in clearance organs forpolymeric nanoparticles. 1416 In addition, it partially preventsthe aggregation of nanoparticles and hinders the non-specicinteractions with cells by forming a neutral stabilizing interface,providing that the net surface charge of the nanoparticles isneutral. It could also provide steric hindrance and protectionfor cargoes that are sensitive towards a particular environment.For instance, incorporation of docetaxel (anticancer drugsusceptible to hydrolytic degradation) and nucleic acidsinto PEG-decorated polymeric micelles could protect themagainst hydrolytic and enzymatic degradations, respectively. 17,18

Importantly, the PEG chains may also provide chemically-active

Table 1 Summary of the important features of polymeric nanoparticles designed for biomedical delivery applications

Feature Description

Stealth A dense brush of neutral hydrophilic exible polymer to prevent adsorption of plasma proteins and avoid recognition by theMPS and prolong the blood circulation time, and thereby allowing the accumulation of the nanoparticles at sites withimpaired vasculature via the EPR effectPEG is the most commonly utilized polymer

Surface chemistry The surface chemistry of the nanomaterials greatly impact their toxicity, immunogenicity and biodistributionExcess positive charge results in rapid opsonization and clearance with possible blood vessels and capillary occlusion

Smart ingredients Cell-recognition moieties enhance the selective cellular uptakeTargeting subcellular compartments ( e.g. nuclear localization sequence for nuclear delivery)Skin, mucus (mucolytic) or cell-penetration enhancersEndosomolytic polymers or lipids to increase the intracellular delivery and avoid lysosomal degradationStimuli-responsiveness (temperature, pH, oxidation, reduction, light, magnetic eld, ultrasound, etc. ) allows the controlled-drug release at specic sites or under applied conditionsMultivalency, bio-mimicking or self-communication are recently-applied approaches to enhance the deliveryefficiencyAny added component to the composition of the nanoparticle may impact their biodistribution, clearance andimmunogenicity

Size Intermediate sizes (20200 nm) have the highest potential for in vivo applicationsShape/aspect ratio Various morphologies and/or aspect ratios can have different solubilization capacities, blood circulation time, biodis-

tribution, toxicity, cellular uptake and intracellular fateRoute of administration

The route of administration inuences the stability and delivery efficiency of the nanoparticlesThere are different barriers at the various entry routes of the body ( e.g. pH, enzymes, etc. )Auxiliary devices can enhance the delivery efficiency by localizing the nanoparticles at the target sites

Stability Drug leakage, dissociation of nanoparticles and detachment of the surface-decorating moieties are detrimental to the efficacyof the nanoparticlesStereocomplexation, non-covalent interactions and crosslinking are efficient techniques to enhance the kinetic stability of nanoparticles


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centers for further functionalization or modication of nano-particles. The usefulness of PEG shielding extends to other admin-istration routes and not only to systemic administration. Forexample, PEG can reduce interactions with the extracellular matrixand mucus components and decrease the mucosal clearance. 4

However, these shielded nanoparticles have limited cellular uptakeand ability to escape from endosomes. Therefore, research effortshave also included the decoration of nanoparticles with cellrecognition moieties to enhance their cellular uptake via receptor-mediated endocytosis. At the same time, these nanoparticles aredesigned to lose their PEG-shell at cell surfaces or in the acidifyingendosomesto facilitate their release into thecytoplasm. 13,19,20 Otherhydrophilic polymers have also been used to coat the surface of nanoparticles, such as poly(acrylic acid) (PAA), poly( N -vinylpyr-rolidone), poly( N -isopropylacrylamide) and poly(carboxybetaine).The latter is another promising class of shell-forming polymers.The zwitterionic characteristic of poly(carboxybetaine) impartsanti-biofouling properties to the coated nanoparticles, due to the

ability of the zwitterionic-based materials to electrostaticallybind water molecules more tightly than the hydrogen bondingin the case of other hydrophilic polymers ( e.g. PEG) and thusresulting in higher hydration of the corona. 21 Effective hydra-tion is critical in minimizing the protein adsorption and inimparting stealth properties to nanoparticles.

Smart ingredients

Incorporation of certain functionalities on the surface or into thecore/shell architecture of polymeric nanoparticles can facilitatetheir diffusion through various biological barriers and direct theirbiodistribution to specic tissues, cells or organelles. In addition,they can modulate and remotely control the responsiveness of these nanostructures to specic stimuli.

Skin and mucus permeation enhancers. There are manyphysical and chemical methods that can hydrolyze mucin ordestabilize SC, which then enhance the permeation of the

Fig. 5 Characteristics of polymeric nanoparticles: (a) stealth: imparts biocompatibility, steric stability and protection of the encapsulated drugand reduces the opsonization and clearance of nanoparticles, but may also reduce the cellular uptake and endosomal escape capabilities, (b)charge: cationic character enhances cellular uptake and endosomal escape, but subject to uncontrolled tissue distribution and often associated withtoxicity, (c) targeting: enhances cellular uptake and specicity, but sometimes can accelerate the clearance and/or immunogenicity, (d) stimuli-responsiveness: controls the dynamics of nanoparticles with possibility of releasing their cargoes at specic sites (selectivity). The stability andresponsiveness of these materials under physiological and pathological conditions may vary and may result in premature release of the drug.(e) size: B 100 nm particles is optimal for delivery, being large enough to avoid renal clearance and small enough to reduce clearance and toxicity,(f) morphology: expanded morphology results in higher drug-loading capacity, lower clearance and cellular uptake, (g) aspect ratio: the shell vs.core volume and length vs. diameter can greatly affect the cellular uptake, clearance, drug loading and release, and toxicity, (h) assembly vs.unimolecular structures: unimolecular structures are more stable (no dissociation) but can be cleared rapidly depending on the size and usuallyhave low drug-loading capacity, and (i) stability: intermediate stability to circumvent physiological barriers and at the same time be able to releasethe drug at the target sites is required and can be achieved with different methods, for instance, by crosslinking.


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delivery vehicles through the mucus and skin. However, wewill focus here on the design of the polymeric nanoparticlesthemselves. Decorating nanocarriers with mucolytic, mucusdissolving, agents can reduce the mucus viscosity by degradingthe biopolymers ( e.g. proteins) in the mucus mesh, which in turnreduces the interactions between the nanoparticles and mucusand enhances the mobility and permeation of nanoparticles.For instance, decoration of insulin-loaded chitosan nano-particles with the mucolytic agent acetylcysteine enhanced thenasal delivery of insulin in rats as compared to the plainnanoparticles. 22 Nanoparticles, depending on their compositionand delivery method, may penetrate skin via three main path-ways, intercellular (lipid bilayers), transcorneocyte and thetransappendageal (hair follicles) pathways. The intercellularpathway is the most characterized pathway for the delivery of nanostructured vehicles. Diffusion of nanoparticles throughthe skin is usually restricted and requires the incorporation of some chemical enhancers into the composition of nano-particles to disrupt the lipid bilayers and create pathways forthe penetration of nanoparticles. Lipid-based nanoparticleshave been used more extensively than polymeric ones, due totheir ability to destabilize the lipid channels of the SC andenhance the permeation through the skin. 3

Accumulation at a specic site. Nanoparticles presentingligands at their surfaces have been designed to enhance theirselective binding to specic receptors overexpressed on thetarget cells. This approach is benecial in terms of enhancingaccumulation at target sites and decreasing the exposure of normal cells to the drug. 23 The nanoparticles can be modiedwith these moieties either before or after assembly. However, itis a perquisite that they are available for receptor binding andinternalization, which is usually achieved by tethering them tolonger PEG chains than the ones forming the shell of thenanoparticle (Fig. 2). Nanoparticles can be decorated with avariety of targeting ligands, depending on the delivery location.For example, targeting transferrin and folate receptors isgenerally helpful in tumor therapy as they are overexpressedin many tumor cells. In particular, targeting asialoglycoproteinreceptors and prostate specic membrane antigen is benecialfor treatment of hepatocellular carcinoma and prostate cancer,respectively. 23 Targeted nanoparticles based on cyclodextrin-basedpolymers as the core matrix, PEG-adamantane (adamantaneforms inclusion complexes with cyclodextrin) as the stealth corona,and transferrin as the tumor-targeting moieties are currently inPhase I clinical trials for systemic delivery of small interferingRNA (siRNA). 24 Davis et al. have observed a dose-dependentaccumulation of these nanoparticles inside the tumor cells byexamining the tumor tissues (by transmission electron micro-scopy and confocal microscopy) after staining them with 5 nmPEGylated gold nanoparticles decorated with adamantane atthe surface. The role of chemistry here was not limited to thedesign and development of the nanoparticles, but was extendedto also study their fate. In another study, folate moieties weretethered to the distal end of COOH-functionalized PEG-shellcrosslinked knedel-like nanoparticles (SCKs). In vitro andin vivo studies demonstrated the enhanced and selective uptakeof the folate-targeted polymeric nanoparticles, as well as thepreferential accumulation in small-size tumors, as compared to

the non-targeted ones. However, no signicant accumulationin the folate-overexpressing large-tumors was observed. 25 It isalways controversial whether the use of targeting moietiesin vivo is benecial. The decoration of nanoparticles withtargeting ligands may not signicantly impact the tumoraccumulation of nanoparticles, if the rate-determining stepfor tumor uptake is based on the enhanced permeability andretention (EPR) effect and the receptors are within the tumors.However, it enhances the cellular uptake after accumulation,thereby enhancing the therapeutic efficacy. 24,26 In addition totargeting specic cellular types, vascular targeting is alsopossible by recognition of specic receptors overexpressedon the endothelial cell surface. 27 Targeted delivery was notonly limited to cells-overexpressing recognition receptors, butalso to subcellular organelles, such as endosomes/lysosomes,cytosol, nucleus and mitochondria. 28 Care should always betaken because the targeting ligand on the surface of nanoparticlesmay accelerate the clearance and/or increase the immunogenicityof the nanoparticles. 29,30

Remote control of the dynamics of polymeric nanoparticles.There are many different approaches that have been developedto interplay with the dynamics of nanoparticles, for example,by controlling the dissociation of nanoparticles and the releaseof the encapsulated drug at specic sites or time intervals.Many pathological sites have different environments ( e.g. pH,temperature, oxidative or reductive conditions) than the normalphysiological ones. Change in pH is probably the mostexploited stimulus to trigger drug release. Indeed, variationfrom physiological pH (7.4) occurs at different sites, such astumors, endosomes and in the gastrointestinal tract. An exampleof how to benet from these stimuli is the incorporation of pH-sensitive ingredients into the nanoparticles, to maintain theintegrity of the carrier at one pH value while destabilizing it at adifferent pH. 19 For instance, incorporation of endosomolyticpolymers, lipids or fusogenic peptides into the composition of nanoparticles is essential for endosomal destabilization andsubsequent cytoplasmic release. Most endosomolytic polymersare designed to be bi-functional, by incorporating primary and/or tertiary amines at different ratios. 3133 Some of these aminegroups are involved in electrostatic complexation with negatively-charged drugs while the unbound amine groups becomeprotonated at the acidic pH of the endosomes, which causesinux of protons together with chloride ions and inducesosmotic swelling and subsequent disruption of the endosomes,which is known as the proton sponge effect. 34 Anothersuggested mechanism is that these positively-charged polymersmight interact with and disrupt the endosomal membrane. 35

Anionic polymers have been also designed to change theirhydrophobicities and/or conformations at different pH valuesand, thus, utilized for either endosomal destabilization or fororal delivery to remain intact at one pH while dissociating andreleasing encapsulated drugs at another pH. 36,37 Alternatively,fusogenic peptides and lipids can induce endosomal destabiliza-tion via bilayer-to-micelle or lamellar-to-inverted hexagonal(H II ) transitions.

38 The presence of oxygen-reactive speciesreleased by activated macrophages in the inamed tissues andcertain tumors has been investigated as yet another stimulusto trigger the release of drugs from polymeric nanocarriers.


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Thioketal nanoparticles have been developed for oral delivery of siRNA to inammation sites in the intestine. 39 These nanoparticleswere built from poly(1,4-phenyleneacetone dimethylene thioketal)polymers, which contain thioketal linkages that are stable in acidic,basic and digestive environments, but degrade and release theircontents in tissues with high levels of reactive oxygen species ( i.e. atthe inammation sites). Another mechanism is to take theadvantage of the reductive conditions met in the cytosol, whichcan cleave disulde linkages used to link a drug or to stabilizenanoparticles. 40 Nanoparticles were also constructed with the shelland core connected via reducible or pH-sensitive bonds, which canbe degraded and release the uncoated core in the cytosolicreducing media or acidic pH of endosomes, respectively, and thusenhancing the intracellular bioavailability. 41,42 Alternatively,external stimuli can be utilized to trigger drug release fromnanoparticles after they accumulate at the desired sites. Examplesof external stimuli include temperature, visible or near-infraredlight, electric or magnetic elds, and ultrasound, among others. 27

Recently, Bhatia and co-workers have experimentally exploredin detail two important features for the rational design of smartnanoparticles. The rst feature is to decorate nanoparticles withclusters of targeting ligands to encourage the uptake via multi-binding mechanisms. 43 A dendritic polymer was rst functionalizedwith variable amounts of folate groups and then mixed with non-functionalized polymers at different proportions to form mixedmicelles that presented the same amount of folate moieties, butwith different spatial arrangements in variable sized clusters(i.e. different numbers of folate groups per cluster). It was foundthat the optimal number of ca. 3 folate groups per cluster enhancedthe cellular uptake due to the multivalent binding and the resultantlonger residence time on the cell surface. The second feature is thedesign of self-communicating nanoparticles. 44 In this case, nano-particles were used for two purposes. Firstly, tumor-targetedsignaling nanoparticles were used to create or amplify a target(coagulation cascade, a harmonized biological process) via differentmechanisms in the tumor tissues. Two mechanisms were used toinitiate the coagulation process, PEG-gold nanorods followed byphotothermal heating or by utilizing a tumor-targeted humanprotein tissue factor. Secondly, the receiving nanoparticles werethen loaded with doxorubicin and tagged with peptides thatrecognize brin (product of the coagulation process) and injectedin mice-bearing tumors. Hence, the receiving nanoparticles wererecruited from the circulation to the clotted tumor areas. Thiscommunicating system resulted in enhanced delivery to the tumortissues and was able to deliver more than 40 times higher amountsof doxorubicin than the non-communicating (without targetamplication) controls. Meijer and co-workers have conductedanother intricatestrategy for enhancing the selective cellular uptakeof dendritic nanoparticles by combining both multivalent bindingand natural system-mimicking. 45 The dendrimer-based nano-particles decorated with phage peptides (phage mimicking) in amultivalent platform (multivalency or cluster binding) had a higherreceptor-binding affinity than did the non-mimicking or mono-valent species.

Route of administration

Although injection is the most common way of deliveringnanoparticles, nanoparticles can be also delivered through the

skin, oral, nasal, pulmonary, vaginal, rectal, ocular and buccalroutes. Selecting the appropriate route of administration is asimportant as the nanoparticle design. Awareness of the differentbarriers and challenges in every route of administration isimperative for the proper design of nanocarriers. For instance,the pH of lung and nasal mucus is neutral, whereas it is slightlybasic and acidic in the eye mucus ( B 7.8) and vaginal secretions(3.54.5), respectively. 4 Many nanoparticles are designed todissociate at acidic pH, for example, to destabilize endosomes.The use of these polymeric nanoparticles for vaginal administra-tion can be detrimental due to the premature release of theirpayloads. The use of mucoadhesive nanoparticles can be alsouseful in the case of mucosal delivery. For instance, thiolatedcrosslinked chitosan nanocomplexes were used for intranasalDNA delivery and showed high transfection due to theirmucoadhesiveness, high stability and sustained release of thecomplexed DNA. 46 Generally, the use of semisolid matrices orscaffolds ( e.g. hydrogels or creams) is preferred for topical andmucosal delivery to allow prolonged contact with these tissuesand sustained release of the encapsulated drugs. Polymericnanoparticles can be modied, for instance, via crosslinking, toform semi-solid matrices. 47 Auxiliary devices can be also usefulto introduce the drug directly to the target organ ( e.g. catheter fordelivery to the bladder, aerosols for pulmonary delivery andmicroneedles for topical delivery). These devices localize thenanoparticles at the target organ, which together with targeteddelivery, maximize the therapeutic benets and minimize thetoxicity to healthy tissues.

Characteristics of polymeric nanoparticles

Size

Physiological barriers are the main limiting factors for theefficacy of nanoparticles (Fig. 3). The accepted size limit of molecules for passive delivery through the skin is below 500 Da.The mesh spacing between mucin bers of mucus ranges from100 to 1000 nm. The blood capillaries are B 540 mm indiameter. The mammalian vasculature has an average pore sizeof B 5 nm and below this size, nanoparticles can transverse theendothelium and equilibrate with the extracellular space. Plasmaconstituents have diameters o 4 nm, whereas most plasma proteinsare 4 7 nm ( e.g. the hydrodynamic diameter of human immuno-globulin is 11 nm). The fenestrations in the liver sinusoids andspleen are less than 500 nm in width. The renal molecular weightcutoff size is B 48 kDa (for some polymers such as PEG anddextran) and B 10 nm in diameter. In many tumors, the openingsof blood vessels are less than 200 nm in width. For brain tumor(e.g. malignant glioma), the pore size upper limit of the blood brainbarrier (BBB) is B 12 nm and smaller for healthy brain tissues. Onthe cellular level, the cell membrane blocks diffusion of complexeslarger than 1 kDa and the nuclear pore complexes, which regulatethe nuclear entry of materials, are around 1025 nm in diameter.The size of internalized vesicles (endosomes) ranges from 60 to120 nm, depending on the type of endocytosis and is usually muchlarger (micrometre range) for macropinocytosis and phagocytosis.It is important to note that these size limits are approximate anddepend on the physiological condition ( e.g. disease status, location,cell type), and the nanoparticle composition, size, morphology,


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geometry, charge and surface chemistry. For example, smalland hydrophobic molecules permeate through skin or plasmamembranes much easier and faster than do hydrophilicmacromolecules.

It is clear now how designing nanoparticles of denite sizecan greatly inuence the circulation time, clearance, selectivetissue distribution and intracellular fate. Large particles(4 1 mm) are usually opsonized and accumulate in the liverand spleen, with possibility of aggregation and capillaryocclusion. Small nanoparticles ( o 5 nm) are cleared rapidlyfrom the blood via extravasation or renal clearance. Rapidrenal clearance is expected for nanoparticles smaller than 10 nmin diameter or for their dissociated polymer chains, if they arebelow the renal molecular weight cutoff size. The renal ltrationis dependent also on the charge, with negatively-charged largeparticles being excluded from excretion. Sometimes, the smallersize can be useful to allow rapid renal clearance ( e.g. contrastagents) or to help crossing the BBB, although they may nothave enough circulation time for brain accumulation. Smallernanoparticles are transported more easily to lymph nodes vialymphatic drainage. Polymeric nanoparticles of intermediatesize (20100 nm) have the highest potential for in vivo applica-tions, due to their ability to circulate in the blood for longperiods of time, when they are designed appropriately. Thesenanoparticles are large enough to avoid renal and lymphaticclearance and small enough to avoid opsonization. In addition,nanoparticles within this size range are believed to be internalizedeasily by cells, in comparison to smaller or larger particles. 9

Furthermore, they may enter certain tissues with leaky vascula-ture or high vascular permeability such as tumors and sites of inammation, or in other tissues, such as the liver and spleen.Accumulation in tumor tissues is particularly useful for main-taining high concentrations of therapeutics and is augmentedby the impaired lymphatic drainage at these areas. This pheno-menon is known as the EPR effect. The EPR effect dependsalso on the charge and shape of nanoparticles and physiologicalconditions of the tumor ( e.g. tumor perfusion and permeability).

Morphology

There is currently great effort to study the effect of shape anddimensions of nanoparticles on their behavior both in vitroand in vivo (Fig. 5). The spherical morphology is the mostcommon shape of polymeric nanoparticles, although othermorphologies are attainable depending on the polymer structureand nanoparticle composition. Elongation of spherical entitiesinto cylindrical or vesicular architectures has the potential todisplay different characteristics, such as solubilization capacity,in vivo circulation time and cellular uptake. It is generally foundthat spherical nanoparticles enter cells to a greater extent than doelongated cylindrical ones. For instance, Discher and co-workershave prepared biodegradable PEG- b-poly( e-caprolactone) andnon-biodegradable PEG- b-polyethylethylene lamentous micelles(lomicelles) and compared them with spheres of similarchemistry. 48 In rodents, lomicelles, with a length of 18 mmand a diameter of B 2060 nm, were reported to persist in thecirculation about ten times longer than their spherical counter-parts, due to the reduced rate of phagocytosis and clearance by theMPS. The clearance of lomicelles occurred upon the persistent

decrease in length, which was more signicant for the biodegrad-able poly( e-caprolactone) than the non-degradable polyethyl-ethylene, due to the hydrolysis of poly( e-caprolactone) overtime. However, for cylindrical nanoparticles designed to prolongthe circulation (by reducing the MPS clearance), it is importantto ensure enough exibility/deformability of the nanoparticlesand to maintain at least one dimension less than 200 nm to avoidentrapment in the spleen or other tissues. The Wooley group hasalso developed expertise in preparing polymeric nanoparticles of well-dened morphologies from block copolymers. 49,50 Conjuga-tion of CPP onto both spherical (11 nm) and cylindrical (20 nmdiameter and 200 nm length) SCKs of the same composition wascarried out to study the effects of nanoparticle shapes on thecellular uptake. 51 Generally, higher cellular uptake was observedwith increasing levels of CPP-functionalization, and the effectsof shape and size were signicant, where the smaller sphericalSCKs were internalized by cells more rapidly than were longercylindrical nanoparticles. On the contrary, folate-functionalizedcylinders were taken up by cells to a greater extent than werefolate-functionalized spherical SCKs. 52 This discrepancy washypothesized to result from receptor clustering, where cylinderswere more capable of multivalent interactions (multiple receptorsbinding) due to their longer dimensions, and thereby triggeredmore efficient cellular uptake. The particle geometry was alsofound to be an important parameter that affects the cellularuptake of polymer nanoparticles produced via DeSimonesPRINT s (particle replication in non-wetting templates) tech-nology. Nanoparticles of the same shape ( i.e. cylindrical) butwith aspect ratios of 3 (450 nm height 150 nm diameter) and1 (200 nm height 200 nm diameter) had different cellularuptakes with the nanoparticles of the higher aspect ratio beingtaken up more rapidly. 53 This behavior is in agreement withobservations made with polymer microparticles of Mitragotriet al. 54,55 There are different techniques to predict, visualizeand control the morphology of nanoparticles. 13,56 However,care should be taken because sometimes these morphologiesare in equilibrium and can transform from one shape toanother. The transformation between different morphologiescan also be controlled by adjusting polymer composition or byaltering the ionic strength and pH of the solution or byaddition of organic solvents. 50,56

Surface chemistry

The chemical groups that coat the surface of nanoparticlescan be modied to modulate hydrophilicity, surface charge,immunogenicity, in vivo circulation, biodistribution and intra-cellular bioavailability (Fig. 5). The surface charge can greatlyaffect the fate of nanoparticles at different stages, namelyphysical stability, skin and mucus penetration, interactionswith extracellular matrix, blood stability, cellular uptake andendocytosis. In blood, excess positive charge results in opsoniza-tion, aggregation of particles and clearance with possible bloodvessels and capillary occlusion. The types, quantities andconformations of the opsonins adsorbed on the surface of nanoparticles are dictated by the nanoparticle chemistry.Interactions of positively- or negatively-charged nanoparticleswith components of the extracellular matrix hinder theirpenetration and may result in their clearance. At the cellular


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level, the surface charge is directly proportional to the cellularbinding and association, due to the negatively-charged proteo-glycans on the cell surface. The uptake of nanoparticles usuallyincreases with increasing the zeta potential values. However,excess positive charge can induce toxicity and initiate immuno-logical reactions. Nanoparticles of different surface chemistriesmay be taken up via various endocytic mechanisms, ending upin different intracellular trafficking pathways. For instance,decorating the surface of nanoparticles with folic acid, albuminor cholesterol favors the caveolin-mediated endocytosis, whichavoids the lysosomal degradation pathways. Alternatively,transferrin and CPP encourage the uptake via clathrin-mediatedendocytosis and macropinocytosis, respectively. Designing nano-particles for specic endocytic pathways remains challenging. 57

The endocytosis process depends on the composition, size,charge, shape and geometry of nanoparticles and cell densityand type. It is complicated to determine the main contributingfactor because changing one of these factors can affect othercharacteristics. For instance, increasing the +/ charge ratio of nanoparticles to impart higher positive surface charge can affectthe stability, size and morphology of the formed particles. Inaddition, each mechanism depends on the cell type and treatmentconditions. Some cells ( e.g. hepatocellular carcinoma cells(HepG2), neurons and leukocytes) do not endogenously expresscaveolin and, hence, do not allow the cellular uptake viacaveolae-mediated endocytosis. 10,58 Finally, these endocyticpathways can be interchangeable, which means blocking theuptake via a specic pathway may enhance the uptake throughother endocytic pathways.

Immunological properties of polymeric nanoparticles

Although nanoparticles can stimulate or suppress the immunesystem and potentially induce severe toxicity, there is limited dataavailable on the toxicological and immunological reactions inducedby nanoparticles. 5,59 Nanoparticles can be specically designedto stimulate ( i.e. vaccine) or suppress ( i.e. anti-inammatory)immunity. The immune system recognizes many of the compo-nents of nanoparticles as foreign materials and initiates animmune response through a complex process. The rst stepusually is the adsorption of the blood proteins on the surface of nanoparticles. The type and quantity of the adsorbed proteinsdetermine the fate of nanoparticles in terms of uptake byimmune cells and interactions with other molecules. Thecomposition of nanoparticle, size, charge, morphology, and,most importantly, surface chemistry dictate the toxicity andimmune response induced by nanoparticles. Binding of nano-particles to the cell surface can initiate signaling processes thatmay induce toxicity or immunogenicity. Activation of theimmune system can also release cytokines that act as mediatorsof local and systemic inammatory and hypersensitivity reactions.Signaling through the endosomal Toll-like receptors (TLRs) is alsoa well-characterized pathway for the induction of cytokines,especially with polymeric nanoparticles used for nucleic acidsdelivery. In this case, the immune response is usually due to thenucleic acid cargoes, which can stimulate different TLRs andinitiate serious immunological reactions.

The rst parameter to be considered in the design of nanoparticles is the shell thickness, density and type, and

accessibility of any molecule used for surface decoration(e.g. targeting ligands, contrast agents). Cationic nanoparticlesare believed to be more toxic, rapidly-cleared and inducehigher inammatory reactions than their anionic or neutralcounterparts. Encapsulation of therapeutics inside the nano-carrier is expected to reduce the immune response induced bythe drug. Considering the approximate size of plasma proteinsand constituents ( B 110 nm), it is desirable to keep thespacing between PEG chains as small as possible to minimizethe interactions between the plasma components and the corematerial. Crosslinking the corona by biodegradable cross-linkers is also important to retain the spacing and avoid thedissociation and segregation of the PEG chains. Shell decora-tion with different moieties is necessary for the preparation of multifunctional nanocarriers. Hence, it is a perquisite to keepthe functionalization ratio as low as possible. In addition, theuse of moieties of low immunogenicity ( e.g. galactose insteadof antibody for targeting) is recommended. Comprehensivestudies to identify the critical parameters ( e.g. PEG length anddensity) 1416 that inuence the in vivo clearance, toxicity andimmunogenicity of nanoparticles are required, although it isdifficult to conclude such data from one study. In vitro studiesto determine which blood components are involved in thedestabilization/opsonization of nanoparticles are equallyimportant for the rational design of nanoparticles. Polymerbiodegradability and biocompatibility are essential for patientsafety. Biodegradability can be attained, for example, byendowing polymers with ester or disulde linkages, providingthat they will remain stable until delivering their cargoes. Carefuldesign of the drug itself can modulate the immunotoxicity of nanoparticles. For instance, while the injection of plasmid DNAusually induces strong immune responses, designing plasmidstructures that do not contain unmethylated CpG motifs(immunostimulatory sequences in the plasmid structure) didnot induce immunological reactions. 60 Similarly, chemicalmodications of other small nucleic acids ( e.g. siRNA) canmodify/amend their immunogenicity. 61

Polymeric nanoparticles as versatile nanomedicineplatforms

There has been extensive research for designing polymericnanoparticles of different types, sizes, morphologies, stabilitiesand biocompatibilities to surmount the various biologicalbarriers. In this section, block copolymer micelles (self-assemblies),crosslinked knedel-like nanoparticles (stabilized self-assemblies)and branched polymeric structures (single macromolecularentities) will be briey discussed with emphasis on theirrational design (Fig. 6).

Block copolymer micelles

Polymeric micelles are formed via the self-assembly of block- orgraft-copolymer chains in aqueous milieu. 56,62,63 They present acore/shell architecture wherein the hydrophobic core serves as amicroenvironment for the incorporation of drugs while thehydrophilic corona imparts colloidal stability against aggrega-tion. In water, hydrophobic interactions are generally the maindriving force behind the micellization process. However, the self-association of polymeric chains can involve additional forces.


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For example, electrostatic interactions were shown to inducethe complexation and neutralization of oppositely-chargedpolymers, thereby allowing the formation of polyion complexmicelles. 41,63 In addition, polymermetal complex micelles canalso be formed via substituting the ligands on a metal drug bythe charged groups of the copolymer through coordinationbonds. Most of these assemblies have fairly narrow sizedistributions with diameters ranging from 10 to 100 nm.Hydrophobic and hydrophilic blocks of different structures,charges and lengths have been utilized to prepare micelles of various size, shape and stability. PEG is probably the mostcommonly used hydrophilic block, although other hydrophilicsegments have been also utilized, such as poly( N -vinylpyrro-lidone), poly( N -isopropylacrylamide) and PAA. The core of these polymeric micelles plays the major role in dictating thenature of the molecules to be encapsulated. Hydrophobicpolymers provide good microenvironments for solubilizationof poorly water-soluble drugs. Cationic or anionic polymersare often used to complex nucleic acid materials or metallicdrugs by driving the formation of polyion complex micellesand polymermetal complex micelles, respectively. Biodegrad-able polymers, such as poly( e-caprolactone), poly( D ,L-lactide)and poly(amino acids), are expected to be more promising forin vivo administration than the non-degradable ones. The core-forming block can also be modied with hydrophobic moieties

or conjugated with the drug to enhance the hydrophobicityand affinity to the encapsulated drug. Many polymeric micelleformulations are in different phases of clinical trials (NK-105,NK-012, Genexol and NC-6004) for the delivery of severalpotent anticancer drugs. 64,65 Most of these formulationscould alter the pharmacokinetics and/or reduce the toxicityof drugs or the commercial surfactants used for their delivery(e.g. Cremophor s EL and Tween s 80). Even when theseformulations exhibit similar pharmacokinetics and antitumorefficacy to the commercial formulations, the lower toxicityassociated with polymeric micelles allows increase in themaximum tolerated dose of the encapsulated drug, therebyleading to better response among patients. Different blockcopolymers have been utilized for the preparation of thesepolymeric micelles, such as PEG- b-polyaspartate (NK-105),where the polyaspartate is esteried with 4-phenyl-1-butanolto increase the hydrophobicity 66 and PEG- b-poly( D ,L-lactide)(Genexol) for the delivery of paclitaxel. PEG- b-poly(glutamicacid) copolymer was also utilized to form coordination complexeswith cisplatin (NC-6004) or conjugated with 7-ethyl-10-hydroxy-irinotecan hydrochloride (NK-102) to form polymermetalcomplex micelles and polymeric micelles, respectively. The averagesizes of these micelles range from 20 to 85 nm, which is suitable forEPR targeting while minimizing the rapid clearance via the renalexcretion. For instance, NK105 exhibited B 86-fold higher area

Fig. 6 Types of polymeric nanoparticles and possible chemical modications: polymeric blocks (dendrimer, brush, hyperbranched, blockcopolymer) can be either chemically modied or supramolecularly-assembled into polymeric nanoparticles. Post-modication of thesenanoparticles via ligand modication, core- or shell-crosslinking or drug-loading is possible.


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under the plasma concentration time curve than the free drug atpaclitaxel-equivalent dose of 50 mg kg 1.66 In addition, tumorsdisappeared in mice treated with NK105 at a paclitaxel-equivalentdose of 100 mg kg 1, and all mice remained tumor-free thereafter.

Although a great success has been accomplished to enhancethe stability of polymeric micelles, these self-assemblies remainprone to dissociation and are in equilibrium with non-micellizedfree polymer chains. Dilution of polymeric micelles upon in vivoadministration can lead to premature dissociation and drug release.Micelle stabilization can be achieved via different strategies,such as stereocomplexation, non-covalent interactions andcrosslinking. 6769 Mixed polymeric micelles of PEG- b-poly-(acid carbonate)/PEG- b-urea-functionalized polycarbonatehave been prepared and loaded with doxorubicin. The mixedmicelles have demonstrated higher kinetic stability than thePEG- b-poly(acid carbonate) micelles, due to the hydrogenbonding interactions between the carboxylate groups of thepolycarbonate and the urea-derivatized polycarbonate. 69 Inthis particular case, hydrogen bonding can occur betweenureaurea, carboxylatecarboxylate, ureacarboxylate orbetween any of the two groups and the drug ( i.e. doxorubicin).Micelle stabilization can be also achieved by crosslinkingeither the core or the corona of the pre-formed micelles orby designing unimolecular/hyperbranched structures that areintrinsically-stable against dissociation (Fig. 6).

Crosslinked knedel-like nanoparticles

SCKs are a member of a large family of crosslinked blockcopolymer micelles that have shown great potential andversatility for biotechnology and medicine, due to the easeby which the shape, composition, functionality, and stabilitycan be tailored for a particular application (Fig. 6). 70 Thechemical structure of the forming copolymers, type of crosslinkerand surface chemistry can affect stability, size, morphology andthe type of the drug that can be encapsulated. Wooley andco-workers have focused on developing and screening SCKs asversatile carriers for a variety of biomedical applications,ranging from the delivery of large payloads of chemothera-peutics, nucleic acids, antimicrobials and diagnostic agents tothe in vivo targeting of such entities to tumors, lung or bladder viathe external multivalent presentation of tissue-specic ligandsand/or by passive targeting. The size of SCKs is usually in thesame range as polymeric micelles although crosslinking isoften associated with a slight decrease in the diameter. Thedynamics of these nanostructures could be remotely controlledby (1) synthesizing polymers that can respond to environmentalor physiological triggers ( e.g. pH, temperature, cytosolicreduction), (2) integrating additional functionalities that canstabilize these entities ( e.g. crosslinking) and (3) triggering theircellular uptake by specic receptors that are overexpressed at thetarget sites. To prepare these nanostructures, a combination of controlled radical and ring-opening polymerizations, chemicaltransformations and supramolecular assembly is utilized. 1,2,68,71,72

In one study, a linear triblock terpolymer of ethylene oxide,N -acryloxysuccinimide acrylate, and styrene, PEO 45 -b-PNAS 105 -b-PS 45 was synthesized and used to prepare photonic multi-compartmental nanostructures. 73 While the PEO enhancedthe water solubility and biocompatibility, the PNAS and PS

segments were required for reactivity (pyrazine crosslinking)and supramolecular self-assembly, respectively. The relativemolecular weight of each block was also important to form themulticompartmental micelles ( i.e. longer PNAS and shorter PS).For instance, PEO 45 -b-PNAS 95 -b-PS 60 formed only discretespherical micelles. These nanocarriers exhibited different uores-cence emission intensities at different pH values. Interestingly,they could be designed to intrinsically-uoresce or to switch theiruorescence ( e.g. ON/OFF) at different physiological environmentsand may be also used for in vivo imaging for signicantcontrast enhancement. PAA- b-PS SCKs consisting of a hydro-phobic core and a highly-functionalizable PAA shell have beenused to encapsulate, protect and deliver silver-based antimicrobialagents for the treatment of pulmonary and urinary tractinfections. 74 Sustained release of the encapsulated drug fromthe SCKs was observed over several days and was associatedwith high antimicrobial activities against Gram-negativebacteria. The same copolymer has been also utilized to solubilizedoxorubicin, a widely used anticancer drug, in SCK cores. Then,the effect of crosslinking and relative block length of PAA- b-PScopolymer on the characteristics of SCKs was studied. 75 SCKs of different core volumes were formed depending on the ratio of acrylic acid to styrene block lengths. The nanoparticles withlarger core volume (lower relative proportion of PAA to PS)were associated with higher loading capacity and higher rateand extent of drug release. Crosslinking could reduce therelease rate of doxorubicin. The release rate was higher atpH 5 vs. pH 7.4 for both crosslinked and non-crosslinkednanoparticles ( i.e. pH-responsive SCKs). The carboxylic acidgroups of PAA could be chemically converted into primaryand/or tertiary amines to invert the surface charge of SCKsfrom negative to positive. The micelle was then stabilized viacovalent crosslinking of the shell by amide formation betweenchains with an activated diester to form cationic SCKs. Thesenanoparticles were shown to condense nucleic acid materials of different structures and chemistry, protect them against enzymaticdigestion and to afford high transfection efficiency. 3133,40

Multifunctional SCKs have been also used for theranosticapplications. Theranostic nanoparticles nanotheranosticsinvolve the use of multifunctional nanoparticles loaded withtherapeutic drugs and imaging probes for the combinedtherapy and diagnosis. Advantages of combinational therapyare the ability to measure pharmacokinetics, visualize biodistribu-tion and quantify drug release and accumulation at the target sitesnon-invasively in real time, which aid in predicting drug responseand in the diagnosis and treatment of diseases. 76 Folate-targetedSCKs were loaded with uorescein thiosemicarbazide (for in vitrotracking), functionalized with 1,4,8,11-tetraazacyclotetradecane-N ,N 0,N 0 0,N 0 0 0-tetraacetic acid (TETA) and used for delivering64 Cu to tumors-overexpressing folate receptors (for in vivotracking). TETA is a macrocyclic ligand used to form stablecomplex with 64 Cu, which is commonly used for both positronemission tomography and radiotherapy. 25 The specicity of cellular uptake was conrmed both in vitro and in vivo byblocking the uptake of SCKs after the saturation of folatereceptors. In mice-bearing tumors, the targeted nanoparticleswere accumulated in the small-size solid tumors to a greaterextent as compared to the non-targeted formulations, while nopreferential accumulation was observed between both formulations


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in large tumors. One concern associated with such chemicalstabilization of micelles is that it may impair the end elimina-tion of the system, especially in the case of non-biodegradablematerials. Crosslinked micelles are often large entities that canno longer be eliminated by glomerular ltration. Likewise,crosslinking may impair the biodegradability of some polymers.To overcome these potential problems, hydrolysable-crosslinkershave recently been used. 77,78 Moreover, although the early workinvolving the development of SCKs for biomedical applicationsinvolved the use of non-degradable polymer components, toensure the study of the robust nanoparticle systems, current effortsfocus on transformation to degradable nanoparticle structures.

Unimolecular structures

Small entities that present covalently-bound polymeric chains(single macromolecules) have been synthesized as an alternativeapproach to provide intrinsic stability. Examples of thesestructures include dendrimers, star polymers and polymerbrushes (Fig. 6). This review is not detailing these structures,but highlighting their rational design. These structures weredesigned such that their formation and dissociation areintrinsically independent of polymer concentration and donot require a supramolecular assembly. In addition, their sizesand morphologies can be controlled precisely. Importantly,they can be decorated with different numbers and types of functional groups per molecule. Hence, different tasks can beachieved simultaneously and co-delivery of multiple drugs/probesat the desired ratio and at the same site can be ascertained.

Dendrimers are probably the most commonly utilized andcharacterized unimolecular structures. 79,80 Dendrimers arewell-dened tree-like structures that their size is usuallyo 510 nm, although decoration with different polymers orligands on their surfaces can increase their size and alter theirbehavior. 81 They consist of a central core, radially-branchedbackbone and surface functional groups. The core andbackbone can be degradable or non-degradable and can havedifferent chemical structures. Amine, carboxylic and hydroxyl-functionalized surfaces are examples of cationic, anionic andneutral dendrimers that have been studied for various biomedicalapplications. The surface can be also modied with polymers(e.g. PEG) or with targeting ligands for active targeting. 82

Depending on the specic structure, they can solubilize orconjugate a wide range of therapeutics. The low polydispersityof dendrimers (close to 1.0) gives high control over theircharacteristics both in vitro and in vivo and results in lessvariability from batch to batch. The multivalency could bealso useful to complex and condense nucleic acid materials(e.g. poly(amidoamine) dendrimers). However, the multivalencyof dendrimers and their high binding affinity could adverselyresult in complications upon in vivo administration. The toxicityof these structures greatly depends on the surface functionalitythat dictates their interactions with cells and biomolecules,opsonization, immunogenicity, clearance and tissue distribution. 81

Imparting biodegradability and attachment of PEG on the surfaceof these structures were found to signicantly improve theirbiocompatibility and reduce their toxicity. 83 The use of PEGwas not only limited to the surface modication but also to theconstruction of the core of dendrimers. For instance, dendrimers

with PEG polyester core have shown longer circulation timesas compared to the same dendrimers with trisphenol core. 84

The chemistry of these dendrimers could be also controlled tocomply with other routes of administration. For instance,thiopyridyl functionalized poly(amidoamine) dendrimers havebeen crosslinked via disulde linkages through 8-arm PEGchains bearing thiol terminations to form biodegradable hydrogelsfor intravaginal administration and controlled release of amoxicillin, a commonly used antibiotic. 47 The gel lasted forup to 3 days in the cervicovaginal area before biodegradationoccurred after intravaginal administration in guinea pigs. In thisstudy, the hydrolysable crosslinkers were designed to degradeunder reductive conditions and not at acidic pH, otherwise, theywould have degraded rapidly in the acidic vaginal environment.

Polymer brushes are another promising class of unimolecularstructures, consisting of a polymeric backbone with densely-grafted polymeric side chains. Polymer brushes of differentstructures and compositions have been synthesized and usedfor delivery of drugs, diagnostic agents and nucleic acidmaterials. The concept of stimuli-responsiveness has been alsoapplied to molecular brushes and the use of pH-, temperature-,magnetic- and light-responsive polymer brushes has beeninvestigated. 85 Recently, unimolecular structures ( e.g. polymerbrush and unimolecular micelles) could be loaded with drugs, suchas paclitaxel, doxorubicin and camptothecin, which were attachedto the polymers via photo-cleavable 86,87 or pH-sensitive 88 linkers.The release of the drugs could be then controlled by exposureto light, or by changing the pH. For instance, doxorubicin andcamptothecin-loaded polymer brushes induced Z 10-foldhigher toxicity to human cancer cells after photo-initiateddrug release. 87 Limitations of unimolecular structures includethe low loading capacity, as the solubilization of drugs intothese structures is usually limited. Conjugation of drugs tothe polymeric backbone or onto the surface often providesimproved loading and retention of drugs, however, it alsoplaces a burden on the synthesis and preparation of thedelivery vehicles. Unless the unimolecular structure is of signicant size ( i.e. , nanoscopic dimensions) to be larger thanthe drug being contained within, they will provide incompleteprotection of the payloads, unlike multi-polymeric nanoparticleassemblies, where the drugs are shielded in the core. Further-more, small sizes can be rapidly cleared from the body, whereasthe biodegradability and clearance of covalently-linked largerones is questionable. Worth mentioning is that unimolecularstructures can be also utilized as building blocks for polymericnanoparticles ( e.g. the use of dendrimers as core forming blockof micelles 19,43 and polymer brushes to construct cylindricalnanostructures) 50 (Fig. 6).

Limitations

Although signicant progress has been made in the eld of nanotechnology and in controlling the physical, chemical andbiological properties of nanomaterials, expectations for theirstability in the blood, preferential distribution to the targetsites, and their capability of curing diseases are still far beyondbeing met. The development of in vivo and live-cell imaginginstrumentations and the availability of various chemicals thatcan inhibit specic biological processes have aided in understanding


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the cellular and in vivo fate of the different components of

nanoparticles. However, there are still many questions to beanswered. Many discrepancies are found in the literature,which could be due to biological, technical and experimentalcomplexities. Limitations are generally related to nanoparticles,instruments, biological and physiological variations (Table 2).Assembly/disassembly of nanoparticles, cellular processes andin vivo pharmacokinetics are all dynamic processes that dependon many factors, some of which are difficult to control. Thesize, polydispersity and charge of nanoparticles depend on theconditions, measurement techniques and hydration state, andcan vary during storage, blood circulation, tissue distributionand cellular uptake. For instance, adsorption of plasmaproteins and cellular binding can modify the size, geometry

and surface charge of nanoparticles. Cells also behave differentlydepending on the cell type, cycle, density and passage number.For instance, great efforts have been focused recently to designnanoparticles for specic intracellular pathways. However, this ischallenging because the endocytic pathways are interchangeableand one inhibitor for a specic pathway can inhibit otherpathways. Many biological processes, either in cells (endocytosis,cytoplasmic release, nuclear uptake) or in vivo (pharmacokinetics,biodistribution), are analyzed based on tracking uorescentprobes that are either linked or encapsulated into polymericnanoparticles. The stability and quenching of uorophores, theleakage or dissociation from nanoparticles and the effect of theuorophore on the stability and pharmacokinetics of nano-particles are always questionable and need further investigations.The use of inappropriate controls, impure uorophores, improperuse of imaging instrumentations ( e.g. the use of high laser power,detector saturation, spectral overlap) can all result in misunder-standing and misinterpretation of cellular and in vivo fate of nanoparticles. Besides the complexities of biological systems, theinherent heterogeneity and distribution of populations innanoparticle samples, and difficulties with exact reproductionof comparable materials, even within a particular laboratory,also pose signicant challenges to making direct comparisonswithin and across nanosystems under development. Furthermore,degradability of nanoparticles is considered to be of primary

importance, however, release of the degradation products

and the resulting compositional, structural, size, etc. changesthat the nanoparticles undergo during the degradation processlead to variations in the nature of the materials and thebiological responses as a function of time.

Conclusions

Polymeric nanoparticles hold great promise as versatile nano-carriers for a wide range of therapeutics for various biomedicalapplications. The ease with which the size, shape, geometry,charge, structure and composition can be controlled will securetheir future development and success. Challenges towards thedevelopment of these nanostructures include, but are not limitedto, the complexity of both the nanoparticles and availabletechnologies for physical, chemical and biological evaluations,the possible toxicity and immunological reactions, lack of scalability, batch-to-batch variation and the need for case-by-case evaluation. For toxicity and immunogenicity considerationswith intravenous administration, it is attractive to have nano-particles of sizes that can be cleared by the kidney or dissociateinto individual polymer chains that have molecular weightsbelow the renal molecular weight cutoff size, or to have thenanoparticles undergo hepatobiliary clearance. However, otherroutes of administration and clearance are also possible.Alternatively, biodegradable nanoparticles would be safer touse. In all cases, biocompatibility is essential for the safety of patients and should be tested both in vitro and in vivo. Theserestrictions are rm with systemic administration of nano-particles, whereas they are less signicant for nanoparticlesdesigned for topical applications. The control of polymer chemistryand supramolecular assembly could be managed to design poly-meric nanoparticles for imaging, pH-sensing, cancer therapy, aswell as, for other applications, such as treatment of pulmonary andurinary tract infections. The chemistry of these copolymers couldbe also controlled to design stimuli-responsive and self-communicating nanoparticles of different structures andmorphologies, such as spherical and cylindrical nanoparticles.Robust nanoparticles ( e.g. crosslinked nanoparticles) could

Table 2 Limitations towards the development of polymeric nanoparticles

Nanoparticles 1. The heterogeneous populations of nanoparticles, within any particular sample2. The size, polydispersity and charge of nanoparticles depend on the measurement techniques and hydration state3. Assembly and disassembly of nanoparticles are dynamic processes that change during storage, blood circulation and tissueand cellular distribution4. Changing one of the nanoparticle characteristic ( e.g. size) affects others5. Premature release of the linked uorophore: the stability of the link used to label the nanoparticles in the blood and tissues andthe effect of uorophores on the stability and pharmacokinetics of nanoparticles are important considerations6. Impure uorophore results in tracking two populations (free and conjugated dyes)

Cellular 1. The cellular processes are dynamic and interchangeable and inhibition of specic endocytic pathways can inhibit otherpathways2. Nanoparticle efficacy varies in different cell lines, cell cycles, growth media, and passage number

In vivo 1. Pharmacokinetics depend on the animal model and disease status; different tumors and different sizes of the same tumorrespond differently to therapy and may accumulate different amounts of the injected nanocarriers2. Physiological factors: age, sex, weight, etc.

Instrumentations 1. The use of two uorophores: unresolved emission spectra lead to misinterpretation2. Confocal microscopya. Three-dimensional imaging is essentialb. Apparent co-localization may be obtained from structures in close proximity without real co-localization in the sameorganellec. Difficult to differentiate between cellular binding and uptaked. Background, detector saturation and spectral overlap should be avoided


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enhance the in vitro and in vivo stability, prolong the circulationtime and enhance the accumulation at the target sites. However,elimination of these entities and hindrance of the release of theircargoes should be considered. Control over the polymer chemistryand supramolecular assembly together with the availability of newtargets and cell-recognition moieties will improve the stabilityand efficacy of polymeric nanoparticles.

Acknowledgements

We gratefully acknowledge nancial support from the NationalHeart Lung and Blood Institute of the National Institutes of Health as a Program of Excellence in Nanotechnology(HHSN268201000046C), the National Institute of Diabetes andDigestive and Kidney Diseases of the National Institutes of Health(R01-DK082546) and Covidien Pharmaceuticals, Inc. The WelchFoundation is gratefully acknowledged for support through theW. T. Doherty-Welch Chair in Chemistry, Grant No. A-0001.

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