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Dendritic Polyglycerols for Biomedical Applications

By Marcelo Calderon, Mohiuddin Abdul Quadir, Sunil Kumar Sharma, and

Rainer Haag*

The application of nanotechnology in medicine and pharmaceuticals is a

rapidly advancing field that is quickly gaining acceptance and recognition as

an independent area of research called ‘‘nanomedicine’’. Urgent needs in this

field, however, are biocompatible and bioactive materials for antifouling

surfaces and nanoparticles for drug delivery. Therefore, extensive attention

has been given to the design and development of new macromolecular

structures. Among the various polymeric architectures, dendritic (‘‘treelike’’)

polymers have experienced an exponential development due to their highly

branched, multifunctional, and well-defined structures. This Review describes

the diverse syntheses and biomedical applications of dendritic polyglycerols

(PGs). These polymers exhibit good chemical stability and inertness under

biological conditions and are highly biocompatible. Oligoglycerols and their

fatty acid esters are FDA-approved and are already being used in a variety of

consumer applications, e.g., cosmetics and toiletries, food industries,

cleaning and softening agents, pharmaceuticals, polymers and polymer

additives, printing photographing materials, and electronics. Herein, we

present the current status of dendritic PGs as functional dendritic archi-

tectures with particular focus on their application in nanomedicine, in drug,

dye, and gene delivery, as well as in regenerative medicine in the form of

non-fouling surfaces and matrix materials.

1. Introduction

In recent years, research in the field of dendritic polymers hasexperienced an exponential development in both academic andtechnological areas.[1–15] Dendrimers represent a key stage inthe ongoing evolution of macromolecular chemistry, whichis a result of the wide range of applications that have beenforeseen for such materials in biomedical science. Applicationshave included solubility enhancement,[16–19] MRI contrastagents,[20–27] neutron capture therapy,[28–34] gene therapy,[35–41]

drug delivery,[42–44] nanocomposites,[45–48] and photodynamictherapy.[49–51] Such macromolecular structures have also been

[*] Prof. R. Haag, Dr. M. Calderon, M. A. QuadirOrganic and Macromolecular Chemistry, Department of Chemistryand BiochemistryFreie Universitat Berlin, Takustrasse 3, 14195 Berlin (Germany)E-mail: [email protected]

Dr. S. K. SharmaDepartment of Chemistry, University of DelhiDelhi – 110007 (India)

DOI: 10.1002/adma.200902144

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extensively explored in materials scienceand technical areas and therefore will not becovered in this Review.[5,52] Dendrimers arehighly branched, monodisperse macromo-lecules with a well-defined structure thathas great impact on their physical andchemical properties. Furthermore, theirsurface multifunctionality offers the oppor-tunity for multivalent interactions withbiological substrates.[53–55] The highlybranched, globular architecture of thesemolecules gives rise to a number ofinteresting properties when compared tolinear polymers of analogous molecularweight (MW).[56–60] For example, dendri-mers demonstrate significantly increasedsolubility[56,57] that can be readily tuned byderivatizing the periphery,[61] and theyexhibit very low intrinsic viscosities.[58,62]

Unlike linear polymers, properlydesigned high-generation dendrimers exhi-bit a distinct ‘‘interior’’ that is stericallyshielded within the dendrimer and enablestheir use as nanoscale transport systems.These ‘‘advanced materials’’ spurred theinterest of the scientific community toutilize them in biomedical applications.[63]

The synergy between their multivalency and size in nanoscalehave a range of options to impart chemical ‘‘smartness’’ alongtheir molecular scaffold to achieve environment-sensitivemodalities; these functional materials can be envisioned torevolutionize the existing therapeutic practice. Dendritic mole-cules such as polyamidoamine, polylysine, polyester, polyglycerol(PG), and triazine dendrimers have been introduced forbiomedical applications to molecularly amplify or multiplypathopharmacological effects.[64] In this report, we summarizethe recent developments of structural versatility and applicationsof dendritic PGs in biomedical applications. Our interest in suchmaterials is motivated by the potential application of poly-(ethylene oxide) (PEO) in biomedical and pharmaceutical areas.PEO, often referred to as PEG for poly(ethylene glycol), exhibitsspecific properties such as chemical stability under basic orneutral conditions, water solubility, non-toxicity, ion-transportingability, and presence of functional group(s) for the attachment ofbiologically active molecules.[65,66] In addition, the non-recognition of PEG by the immune system (PEG is ‘‘invisible’’to macrophages) allows its circulation in the human body for aprolonged time (‘‘stealth’’ effect).[67,68] The main limitation of allthese applications, however, lies in the fact that these linear PEG

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Rainer Haag obtained hisPh.D. with A. de Meijere at theUniversity of Gottingen in 1995.After postdoctoral work withS. V. Ley, University ofCambridge (UK), and G. M.Whitesides, Harvard University,Cambridge (USA), he com-pleted his habilitation at theUniversity of Freiburg in 2002.He then became AssociateProfessor at the University of

Dortmund and in 2004 was appointed Full Professor oforganic and macromolecular chemistry at the FreieUniversitat Berlin. In 2007, he started an intense collaborationwith Dr. Sunil Sharma, who is an Associate Professor inOrganic Chemistry at the University of Delhi and alsocoauthor of this Review. His research interests are in themimicry of biological systems by functional dentriticpolymers.

Marcelo Calderon received hisPh.D. in organic chemistry in2007 from the National Uni-versity of Cordoba, Argentina,under the supervision of Prof.Miriam Strumia. He is currentlya Postdoctoral Fellow in theResearch Group of Prof. RainerHaag at the Freie UniversitatBerlin. His research interest isthe development of nanotran-sporters based on dendritic

polyglycerol for intelligent delivery of drugs, gene, andimaging probes.

Mohiuddin Abdul Quadirreceived his Masters of Phar-macy in 2001 from the Faculty ofPharmacy, University of Dhaka,Bangladesh. Currently he ispursuing his Ph.D. in chemistryunder the supervision of Prof.Rainer Haag at the Freie Uni-versitat Berlin. His researchtopic focuses on the delivery ofdrugs and bioactive moleculesthrough the blood–brain barrierwith dendritic polymerarchitectures.

precursors possess a limited attachment capacity, with one or tworeactive sites depending on the chemical nature of the end groupson the polymer chain. On the other hand, aliphatic dendriticpolyethers, polyols with physical properties analogous to those ofPEG, present multiple attachment sites that allow an add-onsubstantial payload of biologically active molecules. Regularaliphatic dendritic polyethers with terminal hydroxy groupswere obtained by convergent synthesis[69–72] or by divergentapproaches as perfect dendrimers, pseudo-dendrimers andhyperbranched polymers.[73–79] Perfect glycerol dendrimers anddendrons are unique in their design based on the extremelybiocompatible glycerol building block. Hyperbranched PGrepresents the first hyperbranched polymer that can be preparedin a controlled synthesis via anionic ring-openingmultibranchingpolymerization (ROMBP). Both are characterized by thecombination of a stable, biocompatible polyether scaffold,high-end group functionality and a compact, well-defineddendrimer-like architecture. These characteristics can be usedto generate newmaterial properties for biomedical applications tocreate extremely high local concentrations of drugs, molecularlabels, or probemoieties or to modulate therapeutic efficacy of theactive molecules. Therefore, dendritic PGs present a multi-functional macromolecular scaffold and are expected to lead tonew strategies for ‘‘nanomedicine’’ as well as ‘‘regenerativemedicine.’’

This review describes the development of biocompatibledendritic PGs with a special emphasis on biomedical applica-tions. Besides synthesis and structural modifications of thedendritic structure, the current length scale (1–10 nm) has beenextended to 100 nm microgels. Furthermore, these structuresshow excellent protein resistant properties and can be easily builtinto novel core/shell architectures for drug and dye delivery.Dendritic PGs provide a new platform for life scienceapplications, especially in the treatment of cancer and inflamma-tion, because of their low toxicity, high transport capacity,multivalent charge, and ligand display for targeting biologicalcells and tissue.

2. Oligoglycerols and Linear Polyglycerols

Reduced toxicity of aliphatic polyethers in general and ofFDA-approved oligoglycerols in particular is an excellentmolecular platform for their use as newmacromolecular scaffoldsfor biomedical applications. The starting material of thesepolyether scaffolds is mainly glycerol and glycerol-basedcompounds, e.g., glycidol. Glycerol, the monomer used for thepreparation of oligoglycerols, is an increasingly abundant andextremely economic byproduct of biodiesel production (viatrans-esterification of vegetable oils).[80,81] This has led to a glutof crude glycerol on the market. The conversion of glycerol intovalue added products thus plays a crucial role from anenvironmental point of view. The simplest glycerol oligomer isdiglycerol, where two glycerol units can be coupled to form linear,branched, or cyclic oligomers. Oligomers up to the trimer arecommercially available in a medical grade (e.g., Solvay).Triglycerol is used as a moisturing agent in cosmetics and theirfatty acid esters are used as emulsifiers in a variety of health careapplications, toiletries, as well as in the food industry and forcleaning and softening agents, pharmaceuticals, polymers and

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polymer additives, printing and photographing materials, andelectronic applications. In order to generate higher oliogomers,Charlier and Raynard[82] as well as our group[83] have attemptedthe microwave assisted polycondensation of glycerol, however,only low molecular weight PG (up to 15 repeat units) could beobtained and in moderate yield. Recently, a microreactor-based

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Figure 1. Synthesis of glycerol–PEG copolymers and their modification todevelop amphiphilic polymers which spontaneously self-assemble to formstable micelles.

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synthesis of oligoglycerols utilizing the established ROMBPyielded well-defined hyperbranched compounds, but again withlimited molecular weights (up to 1000 gmol�1). Nevertheless, themicroreactor protocol illustrates the characteristics of themicrostructured reaction system and it is the first of its kindto engineer a continuous flow process for the preparation ofhyperbranched oligoglycerols.[84]

Attempts to polymerize glycidol date back to the earlierwork of Sandler and Berg[85] followed by several efforts topolymerize glycidol to linear products in the presence of variouscatalyst systems.[86] Linear PG has been obtained from thepolymerization of protected glycidols and the polymerization of3-hydroxyoxetane.[87–89] Dworak and coworkers[90] carried outcationic polymerization of glycidol under the action of Lewis acids(BF3OEt2, SnCl4) and protonic acids (CF3COOH, CF3SO3H)and polymers with number-average molecular weights varyingfrom 2500 to 6000 g mol�1 were obtained. Structural andmechanistic details of such polymerization process have beenreported and it was found that the structure of the polymers wasrelated to the contribution of activated monomer mechanism inchain growth.[91] Deffieux and coworkers[92] proposed a newinitiation system developed for monomer activated anionicpolymerization of propylene oxide (PO), i.e., onium salts/triisobutylaluminum, were applied to the polymerization ofglycidyl methyl ether. At low to moderate temperature theyyielded fast polymerization and allowed a controlled synthesis oflinear poly(glycidyl methyl ether) with high molar masses (up to100000 g mol�1). Linear PGs have also attracted considerableinterest due to their substantial hydrophilic character andbiocompatibility. Keul and coworkers[93] reported the synthesisof linear poly(glycidyl ether) by anionic ring-opening polymer-ization and copolymerization of allyl glycidyl ether, tert-butylglycidyl ether, and ethoxyethyl glycidyl ether using potassium3-phenyl-1-propanol as initiator. The encapsulation and phasetransfer efficiency of amphiphilic ‘‘core–fatty acid shell type’’linear and hyperbranched PGs toward congo red has beensystematically investigated and molecular encapsulation wasfound to be an exclusive peculiarity of hyperbranched architec-tures.[94,95] Linear methylated and hydroxylated oligo(glycidylethers) synthesized in our group showed reduced adsorption ofsingle proteins to surfaces modified with these oligomers.[96,97]

Rangelov and coworkers designed poly(glycidol)–poly(propylene oxide)–poly(glycidol) block copolymers analogousto PEO–poly(propylene oxide)–PEO block copolymers, so-calledpluronics. In aqueous solution, these polymers were found toself-associate to form stable micelles above a certain criticalconcentration value depending on PG content and tempera-ture.[98]

Our group has successfully carried out the polymerization ofglycerol and the dimethyl ester of a PEG bis(carboxymethyl)etherby adopting a biocatalytic approach using immobilized Candidaantarctica lipase (CAL-B, also known as Novozyme-435) asbiocatalyst. The polymerization was observed to occur in aregioselective manner via the two primary hydroxyl groups ofglycerol, leaving the secondary hydroxyl group intact. Thepolymerization was carried out in bulk, i.e., under solventlessconditions. The resulting polymers were then made amphiphilicby acylation of the secondary hydroxyl group of glycerol with acylchlorides of varying chain lengths[99] (Fig. 1).

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Major efforts, however, have been directed toward thesynthesis of dendritic PGs as well as toward post-syntheticincorporation of different functional groups onto PG scaffolds totailor functional architectures with specific physicochemical andtoxicological properties. The subtle changes in polymer structureaffecting the efficiency or mechanism of biological action arecrucial to the translation of PG architectures into clinical practice.In the following section, we focus on the syntheses and structuralaspects of dendritic PG structures, which are promisingcandidates for applications in drug delivery, gene transfection,blood-substitution, imaging and diagnostic, as well as non-fouling surface applications.

3. Synthetic Approaches to DendriticPolyglycerol-Based Nanostructures: FromOligomers to Megamers and Hydrogels

Multiple approaches to design different PG architectures havebeen reported that offer a great variety in the degree of branching,size, surface topology, and chemical properties in general. Alongwith the synthesis of hyperbranched PG, fabrication routes toperfect dendrimers, dendrons, microgels, and hydrogels havealso been reported over the last decade. A systematic library of PGarchitectures with varying properties was synthesized using acareful selection of starting materials through an economicsynthetic route that provided an option to perform post-modification on the PG scaffold. The synthetic progress ofPG-based architectures is presented in Figure 2 and will bediscussed throughout this section.

3.1. Bifuntional Glycerol Dendrons: Building Blocks for

Modular Synthesis

Novel approaches are consistently being tried with polyetherpolyols on oligomeric and dendritic levels to improve theapplication range, ease the scaling-up process, ensure syntheticreproducibility, and explore more structural diversity. Mono-

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Figure 2. Synthetic evolution of dendritic PGs: from dendrons to megamers.

amino PG dendrons have been synthesized by a completelydivergent approach using 2-aminopropane-1,3 diol orD,L-serine.[100,101] Allylation of alcohol functionalities underphase-transfer conditions and subsequent catalytic dihydroxyla-tion followed by catalytic hydrogenation yielded the desiredgenerations of monoamino glycerol dendrons (Fig. 3).

Commercially available triglycerol was utilized as startingmaterial for synthesizing dendrons of multiple generations. Inthis case, triglycerol was protected with acetone dimethylacetal onthe terminal diols, and the free secondary hydroxyl group wasconverted to the corresponding mesylate which was treated withsodium azide to give azido-[G.1] dendrons in good yield. After

Figure 3. Exemplary synthesis of [G.2] monoamino PG dendrons.Reagents and conditions: a) allyl bromide, 50% NaOH, TBAI, 40 8C,18 h; b) 1mol % K2OsO4, NMO, acetone/water/tert-butyl alcohol, 40 8C,18 h; c) 10% Pd/C, MeOH, 5 bar H2, 24 h.

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acetal deprotection, the iterative steps ofallylation and dihydroxylation were carriedout to generate monoazido [G.3] dendriticpolyol as the final product. Conversion of theazide functionality to primary amines wascarried out by catalytic hydrogenation. Theresulting polyols were consecutively attachedto the 4,4-biphenyldicarboxylic acid core bycarbodiimide mediated coupling to generatetriblock amphiphiles.

Zimmerman et al. presented the synthesisof azide-cored PG dendrons in an attemptto generate porphyrin cored dendrimersby clicking the dendrons withocta-alkynylporphyrin. The dendrons weresynthesized divergently starting fromTBDPS-protected allyl alcohol. Iterative cyclesof dihydroxylation-allyl etherification gavedendrons of different generations. Conversionof these dendrons into corresponding azide oralkyne variants and their subsequent clickingonto ‘‘polyalkyne’’ core generated porphyrincentered PG dendrons in satisfactory yield.[102]

The synthetic simplicity of copper(I)-catalyzed 1,3-dipolarcycloaddition, known as ‘‘click’’ chemistry, spurred the interest todesign clickable dendrons. Different generations of glyceroldendrons have been synthesized using 3-chloro-2-chloromethyl-1-propene (methallyl chloride or MDC) and acetal protectedtriglycerol as starting material. Using the Williamson ethersynthesis and an ozonolysis/reduction sequence as activation andgrowth promotion steps, PG dendrons up to the fourthgeneration have been synthesized. The focal point of the PGdendrons were then functionalized to azido groups by mesylationof the secondary hydroxyl group followed by subsequent reactionwith sodium azide.[103] For synthesizing modular core/shellarchitectures the azide terminated, acetal protected PG dendronscan be ‘‘clicked’’ onto diverse oligoacetylene cores. Upon removalof the acetal protection groups, amphiphilic compounds can beobtained in high yields ranging from 92% for [G.2] to 67% for[G.4] dendrons (Fig. 4). These ‘‘click’’ dendrons as well as theirconversion to more complex architectures are currently preparedon a 50 g scale in our laboratory. In contrast to previousapproaches there are no heavy metals involved.

3.2. Perfect Glycerol Dendrimers and Pseudodendrimers

The initial synthetic approach to perfect glycerol dendrimers andpseudodendritic PGs was reported by our group in early 2000.[104]

This divergent pathway was chosen to synthesize dendrimers withglycerol units as building blocks starting from trimethylolpropane(TMP) and to design a two-step approach to form analogous,perfectly branched structures, designated as ‘‘pseudo’’ dendrimers,bypost-modificationofwell-definedhyperbranchedPGs (Fig. 5). Indesigningageneration3 [G.3] glycerol dendrimer, a simple iterativetwo-step process based on allylation of TMP and catalyticdihydroxylation of the allylic double bond was developed. Theallylation of polyols under phase-transfer conditions was found tobe superior to the classical Williamson ether synthesis as far as the

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Figure 5. Synthesis of glycerol dendrimer and the pseudo dendritic analog:a) allyl bromide, NaOH, TBAB, water; b) NMO, OsO4 (cat), water, acetone,t-BuOH.

Figure 4. Synthetic scheme to ‘‘clickable’’ PG dendrons and subsequentreaction with an oligoacetylene core: a) NaH,[15] crown-5, KI, THF, reflux,24 h; b) i. O3, CH2Cl2/MeOH (1:1), ii. NaBH4; c) i. MsCl, EtN3; ii. NaN3; d)5–15mol % CuSO4, 10–30mol % sodium ascorbate, and 10–30mol %DIPEA, THF/H2O (1:1).

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quantitative completion of reaction is concerned. Catalyticdihydroxylation of the double bond with N-methyl morpholineoxide (NMO) as cooxidant leads to the formation of new glycerolunits on every available alcohol functionality. This perfect glyceroldendrimer contains only two types of structural units (terminal anddendritic) while the hyperbranched analogue contained terminal,linear, and dendritic units. Application of the same iterative steps,i.e., allylation and dihydroxylation, upon hyperbranched PGgenerates the pseudo dendritic variant of the polymer containingonly terminal and dendritic subunits. It is noteworthy that theproperties and analytical data (e.g., NMR) of both structures arealmost identical. The dihydroxylation protocol has recently beenoptimizedbyourgroup, therebyyieldingcolorless compoundswitha sub-ppmosmiumcontent.[105]An improvedsynthetic approach toglycerol dendrimers of multiple generations and their covalentmodification to contain perfluorinated shell showed interestingphysicochemicalproperties involvinghomogeneouscatalysis in thefield of fluorous phase chemistry. It also yielded useful propertiesfor surface coating for polymers, such as PMMA.[106,107]

3.3. Well-Defined Hyperbranched Polyglycerols

Hyperbranched PG is typically synthesized from the commerciallyavailable and highly reactive hydroxy epoxide, glycidol, which is alatent AB2 monomer by utilizing the ROMBP process.[73,108] Thisrouteavoidedtheproblemofhighpolydispersity associatedwith thesynthesis of hyperbranched polymers by polycondensation of ABm

monomers[109,110] or self-condensing vinyl polymerization(SCVP).[111,112] ROMBP is in turn a special polycondensation


variant of ABm monomers, where the complementary reactivegroups remain latent within the ring structure.

Controlled anionic ring-opening polymerization of glycidol isgenerally carried out utilizing partially deprotonated TMP as the


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Figure 6. Mechanism of anionic polymerization of glycidol to form well-defined hyperbranched PGs by slow monomer addition.

initiator. Slow addition of the monomer to establish a rapidcation-exchange equilibrium and to minimize polymerizationwithout initiator resulting in cyclization, favors the formation ofhyperbranchedpolyolswith awell-definedpolyether structure.Dueto controlled polymerization conditions, the monomer exclusivelyreacts with the growing multifunctional hyperbranched polymer,leading to a ‘‘living’’ type growth of themacromolecule (Fig. 6). Fastprotonexchangeequilibriummaintainsall hydroxyl groupspresentas potentially active propagation sites, thus leading to randombranching.[113] The step-wise mechanistic details are presented inFigure 6. Computer simulation carried out by Frey and cow-orkers[114] confirmedthatpolyfunctional initiatorssuchasTMPandslowmonomer addition provide a rigid control over the molecularweight and suppress the polydispersity of the resulting polymer.

The degree of branching for these polymers varies between 0.53and0.59,with a controllabledegreeofpolymerization ranging from15 to approx. 100 and a polydispersity index typically within 1.2–1.7which is exceptionally low for hyperbranched architectures.Detailed structural investigation revealed that such hyperbranchedPG contains linear 1,3- and 1,4-units, terminal and dendritic units,

Figure 7. Synthetic diversification of PG scaffolds: a modular toolbox.

and carries the initiator (TMP) incorporated ascore functionality in every PG molecule. Thescale up of this polymerization technology withtechnical details under economic aspects hasalso been documented.[115]

Recently, Brooks and coworkers[116]

reported the synthesis of very high molecularweight (up to 700 kDa) and narrowly dispersed(PDI¼ 1.1–1.4) hyperbranched PGby ROMBPof glycidol, using dioxane as an emulsifyingagent. The authors postulated that a fastercation exchange in the presence of dioxane ledto low polydispersities. These high molecularweight PGs showed low intrinsic viscosities,with very small hydrodynamic radii withdimensions similar to those of high generationdendrimers (5–10 nm). The study showed thatthe polymers were very compact with aspherical shape in water and did not showany indication of aggregate formation.

Also, the use of a macroinitiator can be anattractive way to control the above-mentioned


molecular weight limitations associated with the synthesis ofhyperbranched PG up to molecular weight of 24000 g mol�1. Afacile two-step strategy has been proposed by Frey and coworkersthat relies on the use of a lowmolecular weight PG (Mn¼ 500 and1000 g mol�1) as a macroinitator for the slow addition of glycidolthereby gaining more control over the molecular weight. Thereport was the first of its kind to describe a technique tosynthesize hyperbranched PG with molecular weights up to24 kDa. The polydispersities of these molecules were typicallywithin the range of 1.3–1.8 with a degree of branching from 0.60to 0.63, which approximate the theoretical limit of 0.66 for slowmonomer addition condition.[117]

3.4. Core Variation and Diversification of Polyglycerol Scaffold

Utilization of commercially available material, ease of scaling up,increment of the number of 1,2 diols (¼terminal units) render theROMBpolymerizationmediated synthesis of PG an attractive andcompetent approach. What makes PG evenmore attractive from asynthetic point of view is the diversity that can be incorporatedeither by a variation in the core, or by modification of theperipheral functional groups to fine tune the resulting functionalarchitecture for specific application areas (Fig. 7). For instance, toovercome the extremely high polarity of hyperbranched PG,which creates compatibility and solubility issues, the hydroxylgroups of the molecule can be hydrophobized by ‘‘capping’’ theend groups with a few units of PO to suppress hydrophilicity.[118]

The degree of propoxylation that can be carried out in the samepolymerization vessel subsequent to the glycidol polymerizationdoes not affect the inherently narrow polydispersity of thestructures, but decreases instead the glass-transition temperature(Tg) significantly. Extension of the concept led to the developmentof multiarm triblock all-ether-based copolymers formed byblock-copolymerization with ethylene oxide (EO) but left thelow polydispersity and high functionality of the PG coreunaffected.[119] In addition to EO and PO, a library of


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commercially available glycidol ethers can be copolymerized ineither block or random fashion to vary branching density or togenerate more fascinating architectures. An interesting exampleis the utilization of allyl glycidyl ether as the comonomer, therebyintroducing the allyl functionality into the hyperbranchedscaffold, which can be orthogonally modified along with thehydroxyl groups. Similarly, incorporation of phenyl glycidyl etherincreases the scaffold’s rigidity, resulting in higher Tg.

[75]

Another interesting outcome of utilizing multifarious ABm

monomers to manipulate scaffold design is that chiralhyperbranched PG and dendron analogues have been synthe-sized by polymerization of commercially available enantiomers ofglycidol.[74] Purported applications of chiral PG in general werefound in chiral catalysis, separation of enantiomers, orbiochemical applications. The synthetic principle involves thebase-catalyzed ROMB polymerization of two enantiomericglycidols using TMP as well as bis(2,3-dihydroxypropyl)-10-undecanylamine as initiator (Fig. 8). Depending on themonomer/initiator ratio, chiral hyperbranched PG analogueswith a molecular weight ranging from 1.3 to 4.8 kDa and a degreeof polymerization of 20–60 glycidol units per polymer weresynthesized. The degree of branching was in the range of0.53–0.57 with a narrow polydispersity index (Mw/Mn below 1.5).These chiral hyperbranched structures were characterized fortheir optical rotation power in methanol solution. Interestingly,all the samples exhibited the same specific optical rotation [a] asthe monomer used for polymerization which confirms the factthat in anionic epoxide polymerization, nucleophilic attack occursat the least substituted end of the epoxide ring.

Recently, we reported an efficient approach to hyperbranchedmonoamino dendrons (X¼NH2) by an efficient polymerization/deprotection process to generate more complex core/multishell(CMS) architectures (see below).[120]

A systematic investigation of utilizing different functionalcores within hyperbranched PGs has also been carried out by Freyand coworkers.[121] They reported the preparation of a novel seriesof hyperbranched polyether polyols with various n-alkyl aminecores (mono- and bifunctional) and photoactive cores (benzyla-mine and 1-naphthylmethylamine). Polymerization of glycidolwas carried out either directly from the primary amine initiatorsor from bisglycidolized amine initators. Molecular weights of thehyperbranched PGs prepared with these initiator cores were inthe range of 1.6–8.4 kDa with polydispersities ranging from 1.5 to

Figure 8. Synthesis of chiral hyperbranched PGs and bifunctional PGdendrons.


2.5. Bisglycidolized amine-cores offered better control ofmolecular weight as revealed by NMR spectroscopy and sizeexclusion chromatography (SEC). Direct amine-initiated poly-mers always showed the presence of non-functionalized PGhomopolymer, but if bisglycidolized amine-initiators were used,only functionally initiated polymers were observed.

An efficient synthesis of hydrophobically modified as well asPEG-grafted hyperbranched PGs was recently reported by Brooksand coworkers[122,123] using 1,2-epoxyoctadecane and a-epoxy,v-methoxy PEG 350 (MPEG-epoxide) as the monomer, respec-tively. Initially hyperbranched PG of molecular weight 7 kDa wasprepared by anionic ROMBP of glycidol. An equivalent of 2–5% ofthe OH groups were derivatized with C18 alkyl chains withhydrophobic modification and 20–40% of the OH groups wasmodified with MPEG 350 chains by sequential addition of thecorresponding epoxides (Fig. 9).

3.5. Block Copolymers of Polyglycerol

Along with the synthesis of homopolymers, block copolymers ofglycerol and PG have also been investigated. Double hydrophiliclinear hyperbranched block copolymers based on PEO and PGhave been reported by Frey and coworkers.[124] These polymers,which essentially contain an aliphatic polyether structure, wereprepared from linear PEO-b-(l-PG) precursor block copolymers,obtained by anionic polymerization of EO and subsequently ofethoxyethyl glycidyl ether. For generating initiator functionalitiesfor glycidol, the protected hydroxyl groups of the ethoxyethylglycidyl ether block were recovered by hydrolysis withHCl. Partialdeprotonation of the linear PG block with cesium hydroxidepermitted hypergrafting of glycidol onto the alkoxide initiatingsites using the slow monomer addition technique (Fig. 10).

Theresulting linear-hyperbranchedPEO-b-PGblockcopolymersexhibited low polydispersities ranging from 1.09 to 1.25, based onthe molecular weight of the hyperbranched block. Depending onthe linear PEO segment and hyperbranched segment, it waspossible to attain the MW as high as 15.7 kDa. Polydispersity wasfound to depend on the base used. For example, with cesiumcounterions, narrow polydispersities were obtained and the use ofpotassium resulted in larger polydispersities.

Figure 9. Synthetic scheme for the hydrophobically derivatized PG. Repro-duced with permission from [122]. Copyright 2008 Elsevier.


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Figure 10. a) Synthesis of poly(glyceryl glycerol)s. b) Synthesis of linear-hyperbranched PEO–PG blockcopolymers as well as dendronized PG(adapted from [121] and [122]).

Figure 11. Synthetic scheme for ‘‘thermal click’’ reaction to formPG-megamers.

Frey and coworkers[125] also reported the synthesis andcharacterization of poly(glyceryl glycerol) block copolymers pre-pared from PEO and poly(glyceryl glycerol) that can be viewed as aperfect first-generation dendronized polymer based on PG. Thissynthesis can be realized either by anionic polymerization ofD,L-1,2-isopropylidene glyceryl glycidyl ether and conversion intowell-defined block copolymers, or in another approach, byconsecutive polymerization of EO and allyl glycidyl ether, followedby a subsequent dihydroxylation step using osmium tetroxide(Fig. 10). Thewide and diversified range of comonomer that can beused along with glycidol polymerization has been reported.[126–128]

Copolymerization of e-caprolactone with hyperbranched PG asan initiator, in the presence of a tin catalyst, has been carried outto yield multiarm star polymers with biodegradable poly(e-caprolactone) arms.[129] In another approach, hyperbranched PGhas also been used as a macroinitiator for atom transfer radicalpolymerization (ATRP) by conjugating an initiator moiety to thehydroxyl end groups.[130]

3.6. Post-polymerization Modification: Tailoring the Properties

of Polyglycerol

The linear monohydroxy and terminal dihydroxy functionalitiesof PG scaffold can be modified/functionalized following classicalhydroxyl group chemistry to render a broad spectrum of products.High loading capacity, water-solubility, and ease of purification of


the product make PG an attractive architecture for carrying outpost-polymerization modifications. A substantial amount ofresearch has been directed to design different architectures bymodification of PG hydroxyl group into different functionalities(Fig. 7).[131]

A highly regio-selective synthesis of amino functionalizeddendritic PGs has been reported by a facile one-pot hydro-formylation/reductive amination sequence.[132] Selective rho-dium-catalysts were used for sequential hydroformylation/reductive amination of dendritic perallylated PGs with variousamines and resulted in dendritic polyamines in high yields. Sucharchitectures carrying neutral core structures with amine groupsin the shell proved to be candidates for supports in homogeneouscatalysis as well as in non-viral delivery of DNA. A comprehensiveapproach for the transformation of PG hydroxyl groups tomultifarious reactive functionalities has been reported.[131]

3.7. Synthesis of Giant Polyglycerols: Megamers, Microgels,

and Hydrogels

In order to close the gap of soluble PG nanoparticles between>20 nm, our group recently reported the synthesis of larger


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Figure 12. Synthetic pathways toward pure PG-m-gel and surface functionalized PG-m-gelparticles: i) cyclohexane/DMSO/block copolymer surfactant, sonic tip miniemulsification4� 1min; ii) p-TSA (cat.), 115 8C, 16 h; iii) p-TSA (cat.), 115 8C, varied time; iv) NaN3, DMF,60 8C, 24 h; v) propargyl derivative, CuSO4 � 5H2O, sodium ascorbate, H2O, 24 h.

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hyperbranched PG analogues to target an optimumdiameter ranging from 20 to 100 nm, the so-called‘‘PG megamer’’ particles.[133] The concept was tocross-link existing hyperbranched PG (2–3 nmdiameter, MW 5kDa) to higher homologues usingthe miniemulsion polymerization technique,whereby the surfactant-stabilized dispersed dro-plets act as ‘‘nanoreactors.’’ The hyperbranched PGmonomers were converted to their high-molecular-weight variant using the nanoreactortemplate, whereas cross-linking was achieved by aneasy ‘‘click’’-type Huisgen alkyne/azide cycloaddi-tion. It is noteworthy that due to the confinement ofspace, no copper was needed for this thermal [2þ 3]cycloaddition at only 80 8C. Both hydrophilic andhydrophobic nanoparticles could therefore beprepared by direct and inverse miniemulsionprocess (Fig. 11). In hydrophobic nanosystems,particles with an average diameter between 25 and85 nm were obtained with low polydispersity whilefor hydrophilic ones, nanoparticle size could bevaried from 45 to 90 nm depending on the amountof surfactant used, as evident from TEM andGPC.[134]

More recently, a new concept was developed byour group[135] to target this privileged size rangebetween 20 and 100 nm for biomedical applica-tions. Functional PG microgels which are homo-geneously dispersible nanoparticles were synthe-sized by an acid catalyzed polyaddition of glycerol totrisglycidyl glycerol ether utilizing the inverseminiemulsion technique where the polar reactantswere dispersed in non-polar cyclohexane (Fig. 12).A poly(ethylene-co-butylene)-block-PEO surfactantwas used as a stabilizer and a small amount ofDMSO was used to prevent Ostwald ripening.

Bulk PG hydrogels have been synthesized by taking advantageof the low viscosity of hyperbranched PG in water.[136] PG withMW 2kDa was derivatized with glycidyl methacrylate in dimethylsulfoxide using 4-(N,N-dimethylamino)pyridine as a catalyst toobtain methacrylated PG derivatives (HyPG-MA). Hydrogelswere obtained by cross-linking methacrylated PG in aqueoussolution using potassium peroxodisulfate as an initiatorand tetramethylethylenediamine as catalyst. The obtainedhydrogels showed a dimensionally stable network with limitedswelling capacity. Photopolymerization of PG methacrylate wasalso possible using Irgacure 2959 (2- hydroxy-1-[4-(2- hydroxyethox-y)phenyl]-2-methyl-1-propanone) as initiator, while the networksformed via this route showed high shear storage modulus andlimited swelling (Fig. 13).

Hennink et al. also reported the preparation and characteriza-tion of structured hydrogel microparticles based on cross-linkedhyperbranched PG by micromolding and photolithographytechniques. Soft lithography process was found to be suitablefor accurately tailoring the size and shape of HyPG-MAmicrogels in the size range of 200–1400mm. The differenttechniques for obtaining structured hydrogels are illustrated inFigure 14. In a micromolding technique, a non-flexible SU-8 gridwas filled with aqueous HyPG-MA solution. Exposure to UV light


and sonication in water released the microparticles. The softmicromolding technique, on the other hand, involves a flexiblePDMS grid which is compressed onto a drop of aqueousHyPG-MA solution deposited on a glass plate. Followingexposure to UV light, the grid can be peeled off and the particlescan be obtained by rinsing the plate with water. Photolithographywas performed by applying an aqueous HyPG-MA solutionbetween two glass plates which are separated by spacers andpolymerization was carried out through the patterned mask. Onremoving the upper plate, the microparticles are collected byrinsing the plate with water. Such dimensionally stable particlesare expected to possess extensive potential in tissue engineeringand drug delivery applications.[137]

4. Biocompatibility Profile of DendriticPolyglycerols

Several studies have been conducted to investigate the biocom-patibility of dendritic PGs to evaluate their applicability in thefield of nanomedicine. The benchmark material, PEG, one of themost studied biocompatible polymers, was commonly used forcomparison with PG structures.


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Figure 13. Schematic diagram for the preparation of PG hydrogels. Reproduced with permissionfrom [136]. Copyright 2006, Elsevier.

In preliminary cell culture experiments, hyperbranched PGwith a molecular weight of 5 kDa showed absolutely no toxicity atcellular level.[113] Brooks and coworkers[138–141] reported severalstudies including a comprehensive analysis of PGs in a broadMW distribution and with different compositions. Both linearand hyperbranched PGs were reported to have a similar or evenbetter biocompatibility profile than PEG with MW ranging from4.2 to 670 kDa. Different scaffolds were evaluated in vitro forblood compatibility, viscosity, complement activation, plateletactivation, plasma protein precipitation, and cytotoxicity. In allcases PG appeared to have very little effect on all testedparameters and outperformed PEG in some cases. As anexample, the effect of hyperbranched PG on red cell aggregationas revealed by microscopic examination is shown in Figure 15.The response of red blood cells (RBC) to PGwhen added to wholeblood in vitro in different concentrations was analyzed. PGsamples were found to have no effect on normal aggregationwhen polymer concentrations from 0.01 to 10mg mL�1 wereused. However, PEG (Mn 40000) at concentrations of 1 and 10mgmL�1 was found to enhance aggregation of the red cells whichcan be attributed to their large hydrodynamic radii.

In vivo studies conducted on mice revealed no sign of toxicityafter i.v. injection of a dose up to 1 g kg�1. For a period of 28 daysno sign of weight disturbances or untoward effects wereobserved. Although the biocompatibility of polymers in general

Figure 14. Fabrication process of HyPG-MA structured microparticles (200–1400mm) usingthree different techniques: a) The micromolding technique using a non-flexible SU-8 grid, b) softmicromolding is carried out with a flexible PDMS grid, c) photolithography. Reproduced withpermission from [137]. Copyright 2007, Elsevier.


is a function of molecular weight, no MWdependant toxicity was found up to 540 kDa fordendritic PG architectures. Pharmacokineticanalysis revealed exceptionally high plasmahalf-life for PG extending from 32 to 57 h forMW 106 and 540 kDa, respectively. The lowerMW variant of PG has a 2.5 times higherelimination rate constant from plasma than itshigh MW counterpart. Tissue accumulationwas found to decrease with time in the kidney,lung, and heart, but due to very limited urinaryexcretion and slow polymer degradation,accumulation in the liver and spleen wasobserved for at least 30 days after application.

Sharma and coworkers[142] recently reportedthe in vitro compatibility of hyperbranchedPGs with MW> 25 kDa, prepared by cationic

ring-opening polymerization. The structural differences of thePGs obtained by the cationic approach did not seem to have aneffect on the in vitro compatibility of the polymer. The resultsindicated a comparable toxicity of these structures with PEGagainst human peripheral blood mononuclear cells and tumorderived human B cell line.

Table 1 summarizes the biocompatibility profile ofdendritic PG architectures. Low toxicity, multiple function-ality, and a relatively higher thermal and oxidative stabilitycompared to PEG makes dendritic PG a promising materialin biomedical applications (see also biocompatible surfacefunctionalization).

5. Biomedical Applications

Due to their highly biocompatible nature, dendritic PGs have abroad range of potential applications in medicine and pharma-cology. Here, we present some recent trends in the use ofPG scaffolds for therapeutic purposes such as smart andstimuli-responsive delivery and release of bioactive molecules,enhanced solubilization of hydrophobic compounds, surface-modification and regenerative therapy, as well as transport ofactive agents across biological barriers (cell membranes, tumortissue, etc.).

5.1. Polymer Conjugates

The covalent attachment of bioactive mole-cules to dendritic scaffolds is a promisingroute for controlling the loading and release ofactive species. Chemical conjugation to adendritic scaffold allows covalent attachmentof different kinds of active molecules (imagingagents, drugs, targeting moieties, or biocom-patible molecules) in a controlled ratio. Theapproach is to some extent beneficial overphysical/non-specific encapsulation withinpolymeric networks since strict control overactive pay-load can be imparted. The load-ing as well as the release can be tuned by

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Figure 16. Conjugation of biologically active molecules to dendritic PG.Triggered release by pH-drop, enzyme activity, reductive environment, etc.

Figure 15. RBC aggregation effects of (a) PG versus (b) PEG 40 K after30min incubation at 37 8C in whole blood. Reproduced with permissionfrom [139]. Copyright 2007, Elsevier.

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incorporating cleavable bonds which can be degraded underspecific conditions present at the site of action (endogeneousstimuli, e.g., acidic pH, over-expression of specific enzymes, orreductive conditions as well as exogeneous stimuli, e.g., light, saltconcentration, electrochemical potential) as shown in Figure 16.

Kannan and coworkers[143] presented the first example of usinghyperbranched PG as a scaffold in the synthesis of polymer–drugconjugate containing high ibuprofen loading for enhancement ofcellular uptake. By a post-modification of the scaffold with anaverage MW of 6 kDa, a multiple functional system bearing 67%w/w of drug conjugated to PG by an ester linkage was obtained.The scaffold also contained fluorescein isothiocyanate (FITC) as afluorescent marker. The conjugate was able to rapidly deliver thedrug inside the cells after distribution into the cytosol. Theanti-inflammatory activity of the conjugate, as investigated bymonitoring prostaglandin inhibition, was considerably fasterthan the free drug.

The increasing development of maleimide-bearing prodrugsand diagnostic dyes[144,145] instigated us to synthesize thiolatedPG nanocarriers. Therefore, we developed a strategy to synthesizethiolated hyperbranched PGs that was used as a general, flexiblemethod to couple diagnostic or therapeutic agents underphysiological conditions (Fig. 17).[146,147]

In a recent communication,[148] we reported the use of thethiolated PG scaffold for conjugation to maleimide-bearingprodrugs of doxorubicin or methotrexate which incorporateeither a self-immolative para-aminobenzyloxycarbonyl (PABC)spacer coupled to dipeptide Phe–Lys or the tripeptideD-Ala–Phe–Lys as the protease substrate. Both prodrugs werecleaved by cathepsin B, an enzyme over-expressed by several solidtumors,[149] to release doxorubicin or a methotrexate lysinederivate. An effective cleavage of PG–Phe–Lys–DOXO andPG–D-Ala–Phe–Lys–Lys–MTX and release of doxorubicin

Table 1. Safety profile of dendritic PG in vitro and in vivo.

Parameters Result

Blood compatibility No effect

Blood viscosity No effect

Complement activation Negative

Platelet activation Negative

Plasma protein precipitation Negative

Cytotoxicity Negative

Acute toxicity in mice model Negative with dose of 1 g kg�1

Plasma half life 57 h for PG 540 kDa


and methotrexate–lysine in the presence of the enzyme wasobserved. Cytotoxicity of the conjugates against human tumor celllines showed that the activity of the drugs was primarily retained,which confirmed the macromolecular pro-drug concept.

In an ongoing investigation we prepared a series of conjugatesof hyperbranched PG, 10 kDa with (6-maleimidocaproyl)hydra-zone derivative of doxorubicin (DOXO–EMCH).[150] The con-jugates showed an acid-triggered release at pH 5.0 while only amarginal release was observed at pH 7.4. The antiproliferativeactivity assessed against two human tumor cell lines, i.e., AsPC1LN (pancreatic carcinoma) and MDA-MB-231 LN (mammacarcinoma), showed a 2- to 10-fold lower cytotoxicity thanthe corresponding free drugs. With respect to antitumor efficacy,the conjugates manifested excellent antitumor effects withcomplete tumor remission up to day 30 without significantchanges in body weight, even after administration of 3-fold themaximal tolerated dose for free doxorubicin.

To determine the optimal size of the conjugates for achievingenhanced permeability and retention (EPR) mediated targeting ofthe drug, the molecular weight of the PG scaffold was variedsystematically. Using the same synthetic strategy, indotricarbo-cyanin–maleimide was coupled to these scaffolds. Theseconjugates were injected i.v. in tumor bearing mice and thesystemic localization and tumor accumulation was followed byfluorescence imaging technique. The results from the in vivoimaging study showed an increased half-life of the conjugatescompared to the dye coupled to a small PG dendron, sincethe fluorescence signal was still measurable 24 h after theadministration.

Photodynamic therapy (PDT) is another stimuli-responsivesystem where the light of specific wavelengths is used to triggerthe therapeutic activity. In a preliminary study, the PDTefficacy oftetrapyrrols coupled to hyperbranched PG was evaluated.Conjugates of PG amine with porphyrins were tested for theirPDTeffect in human colon adenocarcinoma cell line HT29 whichshowed a strong phototoxic effect at concentration of 2–10mM.These results, in addition to the absence of dark toxicity, suggestthat the PG scaffolds are potential candidates for the developmentof new photosensitizable carrier systems.[151,152]

RGD, a short peptide sequence of Arg–Gly–Asp thatsuppresses platelet cross-linking during thrombosis, was con-jugated to high MW hyperbranched PG to make a model of a newclass of antithrombotics.[153] The RGD peptides are not suitablefor clinical applications due to their high inhibitory concentrationas indicated by IC50 and low in vivo residence times.Gyongyossy-Issa and coworkers[153] presented a method toimprove the RGD peptides activities by conjugation to hyper-branched PGs via a divinyl sulfone linker. The conjugates showedan increase in platelet inhibitory function of RGD by two to three


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Figure 17. Synthetic concept for conjugation of bioactive molecules to thiolated PG, derivedfrom in situ aminothiolane reaction with readily available PG–NH2.

orders of magnitude, thereby increasing its local concentration aswell as decreasing its dissociation kinetics. The effectiveness ofthe PG–RGD conjugate was dependent upon MW and thenumber of RGD peptides attached to each PG molecule. Thesemultivalent inhibitors of platelet aggregation decreased the IC50

of RGD in an inverse linearmanner based on the number of RGDpeptides per PG.

Queiroz et al.[154] presented the synthesis of boron-complexedPG–chitosan conjugates. Hyperbranched PG was esterified withO-carboxymethylated chitosan and further reaction with boricacid yielded viscous conjugates with 10–30% of PG content. Theglass transition temperature values of these products were �19and �26 8C, respectively, which favors their potential use as asurface coating for several polymers. Preliminary studies of theantimicrobial activity suggest that these compounds may beuseful to prevent the bacterial contamination and may acteffectively against Staphylococcus aureus and Pseudomonasaeruginosa. Being minimally cytotoxic, these compounds wereshaped into membranes and placed in wound contact to study thein situ degradation process of the films in rat model. No evidenceof necrosis was observed for the PG–chitosan membranes. Afteran initial inflammatory response, the post-operative evolutionwas found to be uniform, displaying a complete wound healingwith a normal disruption of the collagen fibers ordering in thedermis. The best response was found for membranes with 30%PG load, which exhibited higher synthesis of collagen fibers.

Figure 18. Unimolecular dendritic nanocarriers for encapsulation of bio-logically active compounds. Controlled release after triggered shell clea-vage (e.g., pH-controlled).

5.2. Physical Encapsulation

Physical entrapment of drugs or other bioactive molecules withinpolymeric networks by non-specific, non-covalent interactionbetween complementary functional modalities of the involvedspecies is another attractive approach to design nanotransportsystems for drug delivery. One of the major problems faced by thepharmaceutical industries is the poor water solubility of manyexisting and novel bioactive species (e.g., drugs and imagingprobes). Many potent candidates fail in preclinical studiesbecause of limited solubility, stability, and toxicity, in closerelation to the hydrophobic character of the concerned species.Thus, a number of nanocarriers has been designed and developedto overcome the solubility issue. This includes physicalaggregates of amphiphilic molecules such as polymeric micellesas well as stable unimolecular micelles, which have beenconsidered as powerful nanocarriers in the dawning era ofpolymer therapeutics.

Although physical aggregates such as liposomes and micellesare frequently used as drug-delivery systems, they can be unstableunder shear force and other environmental stresses such as high


dilution, temperature, and pressure required,for example, for sterilization. An alternativeapproach is the covalent modification ofdendritic macromolecules with an appropriateshell that results in stable micelle-type struc-tures suitable for non-covalent encapsulationof guest molecules (Fig. 18). Dendritic poly-mers with their regular and well-definedunimolecular architecture can be chemicallymodified either at the core (to increase

hydrophobicity) or at the shell (to increase hydrophilicity) therebytailoring the solubility profile of such nanotransport systems.

Among the various polymeric drug carriers knowntoday,[7,155,156] dendritic polymers based on PG, with theirdefined core/shell-type architectures, have shown good transportcapacities for several poorly water-soluble bioactive molecules.

5.2.1. Solubilization of Hydrophobic Compounds

Park and coworkers[157–159] studied the effect of differentgenerations of perfect dendritic architectures on aqueoussolubilization and release of paclitaxel, a poorly water-solubledrug widely used for cancer chemotherapy. Comparison betweenperfect PG dendrimers (generation 3–5) with star-shaped PEGsshowed a higher ability of the dendritic structures to enhance thesolubility of the drug. The results clearly demonstrated thegeneration and concentration dependent enhancement ofpaclitaxel solubility: [G.3], [G.4], and [G.5] PG dendrimersincreased paclitaxel solubility by 270, 370, and 434-fold,respectively, for 10wt % solutions. PG dendrimers, at aconcentration level of 80wt % in water, increased the solubilityof paclitaxel by 10000-fold. NMR studies revealed the presence ofhydrophobic segments of paclitaxel within the dendritic scaffoldthereby exerting the hydrotropic effect. The results, however,suggested that the probable mechanism of solubilization is likelyto happen through aggregate formation.

The use of PG dendrons may allow a great diversity of core/shell architectures by the convergent synthetic approach as shownin Figure 19.

The preparation of a library of core/shell architectures, basedon a variety of aromatic cores attached to different generations ofPG dendrons was recently reported by our group.[100,103] Thesynthetic protocol included the linking of the dendrons throughamidation or click coupling. These architectures were evaluatedas solubilizing agents for the hydrophobic dye Nile red. For PGdendrons coupled to a biphenyl core via amide bonds, nocorrelation between encapsulation and dendron generation wasfound. However, because the complexes formed between Nilered and the dendritic PG derivatives were lower than 1:1

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Figure 19. Structural variety of core/shell architectures based on PGdendrons.

Figure 20. Structure of an idealized fragment of the pH-labile core/shellarchitecture with R¼O(CH2CH2O)nCH3 with n¼ 4, 7, 16, and 24.

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compositions in all cases, the formation of larger aggregates islikely to occur. On the other hand, in the core/shell architecturesobtained by the ‘‘click’’ approach, the UV–vis absorption spectrarevealed a strong red shift of the absorption band of Nile red with[G.1] dendron complex which suggests the presence of Nile red ina very polar environment, e.g., when it is surrounded by glycerolunits. In higher generations, where themaximum absorption wasshifted to lower wavelengths, Nile red was likely to be located inthe hydrophobic core. The transport capacity of the dye wassignificantly improved by expanding the core size which indicatesthat an extended aromatic core is necessary for efficientencapsulation and transport of hydrophobic compounds. Theencapsulation of Nile red was significantly improved by a factor of�200 by enlarging both core and dendrimer size.


5.2.2. Post-modification of Dendritic Outer Shells

Several investigations covered the influence of post-modificationof the PG scaffold in an attempt to increase transport capacitiesand/or efficiencies, decrease inherent toxicity, tune structuraltopology, and impart targeting modalities. Micelles as well asinverted micellar structures were constructed and their carrierproperties were investigated for several bioactive molecules. Onlywater-soluble systems are specifically focused in the followingdiscussion for their general acceptability in biology andmedicine.

Pegylation of hyperbranched polymers is a promising methodto render water-solubility, minimize immunogenicity, andincrease blood circulation half-life of the resulting nanocarriersmimicking the structure of so-called ‘‘stealth’’ liposomes.[160] PGpegylation resulted in efficient encapsulation, although therelease profiles need to be improved to overcome the stronghost–guest interaction. Paleos and coworkers[161] preparedpegylated hyperbranched PG derivates bearing folate targetingligands. They showed that PEG chains attached to the surface ofhyperbranched PG enhanced the encapsulation of fluorescentprobes, pyrene, and the anticancer drug tamoxifen. Dynamic lightscattering (DLS) revealed unimicellar encapsulation of the guestmolecules within the PG–PEG structure. A salt-triggered releasewas observed upon addition of sodium chloride.

A similar core/shell structure where a PEG shell was attachedto hyperbranched PG with a pH-labile linker has recently beenreported.[162] The dendritic nanocarriers were prepared byattaching tri-PEGylated benzaldehydes of varying lengths to thehyperbranched PG amine by using pH-labile imine bonds asshown in Figure 20.

The designed structures were able to encapsulate the antic-ancer agent doxorubicin and indotricarbocyanine, a near-infraredimaging dye. It was found that nanocarriers with the shortestPEG chain (n¼ 4) and a denser shell showed the bestencapsulation efficiency (up to 5molecules/pegylated PG). Inan early study in nudemice, the doxorubicin nanocarrier could bedosed up to 24mg kg�1 free doxorubicin equivalents as anintravenous administration which was a significant increase inthe maximum tolerated dose (MTD) compared to free doxor-ubicin MTD.[163] Although the pH-triggered cleavage of the PEGshell was confirmed by IR, the high IC50 values obtained for threecancer cell lines (3.3–31mM) may be due to an insufficient release


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Figure 21. Fluorescence image of a tumor-bearing mouse (F9 teratocar-cinoma) 6 h after injection of nanocarrier–NIR dye complex.

Figure 22. a) Synthesis of CMS architecture. b) Schematic representationof a typical liposome structure (top) and the dendritic multishell archi-tecture (bottom).

of doxorubicin from the complex (plateau level at 20% reachedafter 4 h).

The ability of the nanocarriers to localize in tumors in vivo wasdemonstrated by fluorescence imaging of tumor-bearing mice(Fig. 21) with the indotricarbocyanine–nanocarrier complex. Thenear-infrared fluorescence imaging allowed an easy visualizationof the tumor-accumulation of the dye–PG complexes sincenear-infrared and far-red light (650–900 nm) can avoid strongabsorption by RBC and water to allow light to pass through thebody of the mice in the depth of several centimeters.

For a more universal transport behavior our group developed anovel kind of CMS architecture that was inspired by themolecular mimicry of a liposome based on a hyperbranched PGcore surrounded by double-layered shells.[164,165] The synthesis ofsuch dendritic multishell architectures was performed bycoupling an alkyl chain to monomethylated PEGs which werein turn coupled to hyperbranched PG amine as shown inFigure 22. Above a critical concentration, single nanocarriers witha size range of 8–9 nm coexisted with their larger aggregates withdiameters of 20–50 nm. These supramolecular aggregates canencapsulate and transport a wide variety of compounds rangingfrom non-polar to ionic species in a broad matrix spectrumincluding non-polar and polar organic as well as aqueousenvironments. In contrast to already existing micelle-analoguesystems, this new architecturemimics the structure of a liposomeon a unimolecular basis.

As an example of the therapeutic potential of CMS structures,their skin penetration properties were evaluated and compared tothose of solid lipid nanoparticles and oil-in-water (o/w) cream.Nile red was used as a probe molecule and it was found that CMSsystems significantly enhanced the skin penetration of the dye.

The encapsulation process, shown in Figure 23, was time andconcentration dependant. With increasing Nile red concentra-tion, the surface is assumed to be totally clad with the dye


molecules. From this concentration on, corresponding to adiffusion time of 10–20min, Nile red diffuses into anagglomerate of the particles that first sit at the inter-particularspaces and then, with still increasing diffusion time, within thematrix of individual particles.

It was found that the concentration of Nile red, physicallyencapsulated into a stable dendritic CMS system, increased 8-foldin the stratum corneum and 13-fold in the epidermis as opposedto when loaded in o/w cream. Despite the degradation at thestratum corneum surface, solid lipid nanoparticles enhanced skinpenetration less efficiently (3.8- and 6.3-fold) than CMSnanoparticles. Viable human keratinocytes showed an inter-nalization of both nanocarriers within 30min of incubation.


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Figure 23. At low core multishell nanocarrier concentrations, Nile red is attached to the surfaceof the aggregates. At higher concentrations, Nile red molecules diffuse into the aggregates asexemplified by the shift of maximum wavelength from 548 to 505 nm as measured by UV–visspectroscopy. Reproduced with permission from [164]. Copyright 2009, Elsevier.

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In further studies,[166,167] the capability of the CMS system wastested as a drug delivery system for the topical treatment of skindiseases. A study of the influence of CMS in the wound healingprocess in combination with opioids showed the potential use ofsuch systems in formulations for topical pain reduction, becauseno acute toxic effects were observed. Their suitability for carryinghydrophilic drugs through the skin was confirmed by usingrhodamin B as model compound. The dye amount increasedmore significantly in viable epidermis and dermis than withconventional cream and solid lipid nanoparticles. As seen inFigure 24, the penetration of the loaded CMS was higher, whichshows the potential of such structures as topical drug deliverysystems.

A new CMS architecture was recently developed in our groupin an attempt to obtain core-double-shell architectures withdifferent densities and flexibility than those previouslydescribed.[120] New hyperbranched PG-based architectures(Fig. 25) with a highly branched outer shell were synthesizedby post-modification of hyperbranched PG with different lengthaliphatic chains which were further linked to PG dendrons. Asystematic study was carried out to analyze the effect of thevariation of the hydrophobic and hydrophilic domains of thepolymers on the guest encapsulation efficiency. The universalnanotransport character of the obtained CMS was confirmed byencapsulation of polar guest molecules as rose bengal and congored, and water insoluble compounds, as nimodipine, Nile red,and pyrene. Moreover, this new type of CMS system, in contrastto the previously reported one, acts as an unimolecular

Figure 24. Rhodamin B penetration into pig skin: staining of pig skin following the application of0.004% rhodamin B loaded cream (a), solid–lipid nanoparticle (b), and CMS architecture (c) for6 h. Reproduced with permission from [166]. Copyright 2009 Elsevier.

� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinh

nanocarrier system for non-polar guest mole-cules, with transport capacities of 1.5 guestmolecules per nanocarrier. However, for polarguest molecules, the formation of uniformaggregates with a diameter of 70 nm and withtransport capacities of up to 10 guests pernanocarrier was detected. This may be due tothe fact that the dye molecules are preferen-tially located on the outer shell of the polymerand therefore may be acting as non-covalentlinkers between molecules.

Modification of the hyperbranched PGsurface with electron rich moieties shouldallow the design of a PG-based scaffold forencapsulation of cationic bioactive species,including metal ions. Due to the criticalimportance of metal ions and their complexesin physiological homeostasis and their applica-tion in different pathological conditions, asignificant amount of research has been

directed to design PG-based scaffolds for metal encapsulationand transport.

Cisplatin [cis-diamminedichloroplatinum(II)], a potent anti-cancer compound, has been shown to form strongly boundcomplexes with succinic acid ester of hyperbranched PG with astable complex for over 5 days at 37 8C.[168] A sustained release ofcisplatin into physiological saline over 7 days was observed.

Toth and coworkers[169] attached two different Gd3þ chelators,tetraazatetracarboxylate DOTA-pBn4- and tetraazatricarboxylatemonoamide DO3A-MA3- to PG amines (Fig. 26) to designmagnetic resonance imaging (MRI) contrast agent. After in vitroand in vivo characterization on tumor-bearing mice, the PGcomplexes were found to be suitable for angiography and study ofvascular parameters like blood volume and tumor vesselpermeability. Attachment of PEG chains to the dendriticstructures does not influence relaxivity, which is a promisingresult since the toxicity as well as the circulation time of thecomplexes can be further improved by pegylation.

Efforts have been undertaken to combine pH- and tempera-ture-responsive modalities within one carrier molecule and todevelop smart delivery systems based on dendritic PG. Theresulting properties are promising for different fields inbiomedical applications, since the temperature and pH of thetarget sites, could be modulating factors for triggering activity ofthe bioactive molecules.

Kono and coworkers[170] reported the preparation of hyper-branched PG with NIPAM moieties that imparted thermosensi-tivity and pH-responsiveness to the PG scaffold in ranges around

normal physiological conditions. As shown inFigure 27, the synthetic pathway included acontrolled attachment of succinic acid moi-eties, as pH-sensitive units, followed by aconjugation of the temperature-sensitiveNIPAM unit.

The responsiveness of the NIPAM-Suc-PGstructures with a different NIPAM loading wasanalyzed by phase transition analysis. Acombination of pH, in the range of 5–7, andtemperature influence on thermosensitivity of

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Figure 25. Schematic representation of double-shell architecture withhyperbranched core and highly branched outer shell linked by an alkylchain of varying length.

Figure 26. PG derivatives as Gd3þ chelator for MRI.

the PG scaffold confirmed the stimuli sensitivity imparted bysuccinic acid and NIPAM units. The thermosensitive compoundshowed that it could potentially act as a nanocapsule for carrying/releasing bioactive molecules. The temperature-dependentencapsulation of rose bengal was demonstrated by a UV–visanalysis, where aqueous solutions with and without NIPAM-Suc-PG were prepared and heated to 40 8C. It was found that theabsorption spectrum of rose bengal in the presence ofNIPAM-Suc-HPG almost disappeared after heating while inthe absence of NIPAM-Suc-HPG, the spectrum remainsunchanged. The spectrum at 40 8C in the presence of PG, which

Figure 27. Synthetic scheme for the synthesis of pH and temperature re(modified from ref. [167).


is not thermosensitive, was the same as that for 4 8C. Thus thethermosensitive hyperbranched polymer could be a promisingcandidate as a temperature-dependent carrier of bioactivemolecules. Similar methodology was used for the preparationof stimuli-responsive gold nanoparticle assemblies by encapsula-tion of the nanoparticles within NIPAM-HPG polyelectrolytes,leading to a sharp phase transition upon change of temperatureor pH value.[171]

Another approach to obtain such kind of stimu-li-responsiveness was developed by Huang and coworkers.[172]

They prepared nanoparticles by postmodification of hyper-branched PG with N,N-dimethylaminoethyl acrylate (DMA)followed by polymerization and intramolecular cross-linking.The hydroxyl groups of PG were converted to trithiocarbonates,and the later were used to mediate the surface graft polymeriza-tion of DMA. The poly(DMA) shell was cross-linked by1,6-dibromohexane and then parted from the core by thecleavage of trithiocarbonates with sodium borohydride to yieldparticles with thiol groups located on the interface between PGcore and poly(DMA) shell. The thermal and pH-sensitiveproperties of poly(DMA) that were utilized can be potentiallyused in biomedical applications. However, these thermorespon-sive PG-derivatives have not yet been tested for actual drug releaseand require further investigation.

5.2.3. Core Modification of Hyperbranched Structures

As mentioned above, hyperbranched PG possesses linearhydroxyl groups in proximity to the core as well as terminalhydroxyl groups in the periphery of the macromolecule (Fig. 28).Unlike dendrimers, hyperbranched polymers show no distin-guishable interior or periphery. Instead they possess two types ofhydroxyl functionalities arising from linear and terminal glycerolunits. An effective and highly reproducible strategy has beenreported, where these two types of hydroxyl groups can bechemically differentiated to generate a core/shell type architec-ture within the hyperbranched PG scaffold.[173] To this end, the1,2-diols of the terminal glycerol units were selectivelytransformed to the corresponding ketals in order to distinguishbetween the interior (close to the focal unit) and periphery (distantfrom the focal unit) of the macromolecule, which was possiblebecause the remaining linear glycerol units remained unaffectedby this transformation. A subsequent reaction of the linear unitswith alkyl halides, such as allyl or benzyl chloride, under phasetransfer condition yielded the corresponding polyether polyketals.

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Selective deprotection of 1,2-ketals wasachieved to give core-functionalized PGs.

The procedure allowed selective tailoring ofthe PG scaffold to contain hydrophobicsubstituents in the interior or in the periphery,thereby modulating the distribution coeffi-cients of the generated structure betweenorganic and aqueous phase. Such selectiveketal-functionalized hyperbranched PGs werefound to be effective for the encapsulation andtransport of polar guests (e.g., dyes or drugs)and the creation of special microenvironmentas demonstrated by the shaded area inFigure 28.

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Figure 28. Selective functionalization of hyperbranched PG scaffold. a) Hydrophilic outer shell.b) Hyperbranched PG. c) Hydrophobic outer shell.

Figure 29. PG core/shell architecture after post-modification of the cor-e-hydroxyl groups with biphenylmethyl ether groups and its specific inter-action with the drug nimodipine as exemplified by a batochromic shift inthe UV–vis spectrum.

Figure 30. Aggregation of core modified PG as revealed by AFM.

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Our group also investigated the effect of modified linearhydroxyl groups on encapsulation and transport capacities(Fig. 29).[17] A library of core-modified nanocarriers bearinghydrophobic biphenyl groups was synthesized in an effectivethree- or four-step procedure by employing Suzuki-couplingreactions. These structures were then used to solubilize pyreneand nimodipine, a calcium-channel blocker widely used for thetreatment of heart diseases and neurological disorders. A majoradvantage of these amphiphilic PGs was that a hydrophobic corecould be designed with them for specific polymer–druginteractions. Consequently, it was possible to design dendriticarchitectures with a specific interaction profile for functional drugmolecules. As example, the core/shell PG derivative with30,40-dimethoxybiphenyl-4-methyl ether groups in the coreshowed specific p–p interactions as detected by UV spectroscopyin the case of nimodipine complexation, in addition tonon-specific hydrophobic host–guest interactions.

The modified dendritic polymers were found to self-assemblearound the drug molecules in a controlled manner, as revealed byAFM analysis and DLS (Fig. 30). In addition to the transport ofdifferent drug molecules, these structures exhibited substantialrelease of nimodipine in SEC column filtration where highdilution conditions could be mimicked.


5.2.4. Core and Surface Modification of

Hyperbranched Structures

As mentioned in Section 3.5, an effective andeasy method for the synthesis of unimolecularmicelles was developed by Brooks and cow-orkers.[141] The obtained structures carriedalkyl chains at the core and PEG moietiesgrafted on the shell (see Fig. 9). The unim-olecular micellar nature of the molecules withdifferent alkyl chains/PEG composition wasprobed by multiangle laser light scattering(MALLS). Due to low intrinsic viscosity, thesescaffolds were an extremely promising candi-date for human serum albumin (HSA) sub-stitutes.[122] The encapsulation efficiency wasevaluated using paclitaxel and pyrene as modelcompounds. Fluorescence studies revealedthat the hydrophobic molecules are most likely

to be located in the hydrophobic pockets of the unimolecularstructures. The solubility of paclitaxel in water was increasedfrom 0.3–1mg mL�1 up to 2mg mL�1 after encapsulation in thenanocarriers, without any considerable effect on the size of theunimolecular micelles. The release profile was characterized by arapid-release phase followed by a slower sustained-release phasereaching 80% of paclitaxel released.

Since these structures presented mucoadhesive properties,their complex with paclitaxel was evaluated as an intravesicalagent against non-muscle-invasive bladder cancer.[175] Thoughthe encapsulated paclitaxel was slightly less potent than the freedrug in vitro, the in vivo studies showed that the mucoadhesiveformulation of paclitaxel was significantly more effective inreducing orthotopic bladder tumor growth than the standardCremophor-EL formulation of paclitaxel (Fig. 31). Relative tumorgrowth with paclitaxel complex was reduced to 15% of the controlcompared to 66% for the free paclitaxel group. The complex waswell tolerated in mice and the resulting stabilized level ofhematuria and body weight with zero mortality indicated no signof systemic toxicity.

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Figure 31. In vivo evaluation of hydrophobic PG–paclitaxel complexagainst orthotopic bladder tumor (Adapted from [172]).

Figure 32. One-pot selective condensation reactions for the generation ofamphiphilic unimolecular micelles (see also Fig. 28 and 33).

Figure 33. Transport and functional group selectivity relationship of PGcore/shell architectures. Reactive OH groups are labeled in red for terminalunits and blue for inner (linear) units: Unselective alkylester formation(top) results in moderate transport capacities. Selective ketal formation onterminal OH groups results in high transport due to larger polar coreenvironment (bottom).

5.2.5. Non-aqueous Encapsulation Systems

A different attempt for the preparation of delivery systemsincluded the preparation of water insoluble architectures. Thesimple post-modification of the terminal hydroxyl groups fromthe PG scaffolds (see Fig. 28) allows the preparation of core/shellstructures with reverse micellar character.

In non-aqueous solvents the self-association of amphiphilicpolymers should yield nanostructures with a polar coresurrounded by a hydrophobic shell. In some instances,amphiphilic hyperbranched architectures may self-assemble intosupramolecular assemblies, primarily when the shell density wasinsufficient to prevent interactions between adjacent polymersthrough hydroxyl–hydroxyl interactions.

The inverted micellar systems have been extensively investi-gated in encapsulation of oligonucleotides, metal complexes, andseveral water-soluble dyes and drugs.[4] The potential use of suchsystems in pharmaceutical applications is highly promising sincethey can be used in oleaginous formulations thereby providingbetter stability against enzymatic degradation and facilitatingabsorption through biological barriers such as the intestinalmembrane. In addition, these systems could allow the controlleddelivery of active compounds by subcutaneous administration.The hydrophobic character of the shell also makes thesearchitectures attractive candidates for use in hydrophobic deviceslike Teflon-based bone replacement materials and transdermalpatches.

Our first attempt to synthesize such scaffolds from dendriticPG embraces a simple concept based on the selective andreversible shell functionalization for the generation ofpH-responsive nanocarriers.[176,177] The synthetic route involveda selective condensation reaction of the 1,2-diol units by a one-potpathway, based on two consecutive transketalization reactions(Fig. 32). The selective core/shell architectures obtained werehighly amphiphilic with a hydrophobic shell and hydrophilic


interior due to the remaining, unreacted hydroxyl groups. Such‘‘molecular nanocarriers’’ do not exhibit aggregation in dilutesolution, as demonstrated by DLS, and thus represent inverseunimolecular micelles.

We observed that the transport capacity of these systems isdirectly governed by core-molecular weight, alkyl-chain length,and degree of functionalization. A limiting core size of 3 kDa anda highly branched architecture are required for successfulencapsulation of the guest molecules while for optimum guestentrapment, the degree of alkylation should be around 50% andthe alkyl chains should have a minimum length of 10 carbonatoms.

Transport capacity of the PG-core/shell architecture toward agiven guest species critically depends on the packing density ofthe shell. It was found that, upon reaction with an activated longchain fatty acid, both linear and terminal units of PG arenon-selectively esterified. Though significant degree of alkylation(55%) was achievable, absence of selectivity results in a statisticaldistribution of the non-polar moiety over entire core/shellstructure, thereby giving rise to non-continuous shell andmoderate transport capacity in the order of 1.6 dye molecules.On the other hand, ketalization converts almost half of theterminal diols, thereby forming a densely packed core/shellstructure that shows substantial transport capacity (13 dyemolecules). The relationship amongst functional group selectiv-ity, functionalization level and transport capacity is presented inFigure 33.

In addition, a controlled release of encapsulated congo redfrom such a system was achieved in chloroform by loweringthe pH of the aqueous phase, which promoted the cleavage of the


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Figure 34. Encapsulation of polar guest molecules into organic phase. Cleavage of the shell leadsto the release of the encapsulated guest back to the aqueous phase.

Figure 35. Schematic representation of allylated (1), RCM cross-linked (2),dihydroxylated (3), and RCM cross-linked and dihydroxylated (4) hyper-branched PGs (HPG).

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hydrophobic shell and the further release of the dye to theaqueous phase (Fig. 34).

The self-assembly behavior of a series of palmitoyl chloridegrafted hyperbranched PGs was systematically investigated byYan and coworkers.[178] The group showed that, depending on thesolvent, it is possible to control the unimolecular character ofthe amphiphilic PG architectures by varying the content of thehydrophobic shell. Core/shell architectures with a relatively highalkyl grafting ratio can form unimolecular micelles in THF andassemble into giant vesicles around 1–10mm in THF/watermixed solvents. However, the architectures with a low alkylgrafting ratio of 15.6% can directly assemble into vesicles in THFand form micelles in water.

Adeli et al.[179] reported the preparation of core/shellarchitectures using hyperbranched PG as macroinitiator forring-opening polymerization of lactid and L-lactide monomers.Different molar ratios of monomer to end functional groups ofPG were used to prepare the core/shell architectures withdifferent shell thickness. These core/shell architectures were ableto encapsulate and transport small guestmolecules. The transportcapacity and release of guest molecules, e.g., rose bengal, fromchloroform to water depended on the type and thickness of theshells. The transport capacity of the architecture containingpoly(L-lactide) shell was higher than for their analogues contain-ing poly(lactid) shell.

In order to generate closed shell systems, polyallyl glyceroldendrimers of generation 3.5 were reported by Zimmerman andour group, where cross-linking was performed using ring-closingmetathesis with Grubbs I catalyst. These cross-linked dendrimersact as selective ionophores in organic solvents, and can readily bedihydroxylated to make fully water-soluble nanoparticles.[180]

Furthermore, a series of PG scaffolds with a cross-linked outershell was recently reported. Different smart systems withcleavable or tunable moieties at the core or at the shell wereinvestigated. The ring-closing metathesis of the allyl groupslocated on the PG surface generated the cross-linked dendriticarchitectures with a covalently closed shell system (Fig. 35).[180]


The cross-linking process provided a uniquearchitecture with a dense shell which, uponcomplete hydrogenation of the alkene groups,displayed a crown ether-type binding of picrateions in organic phases, with ion affinity andselectivity comparable to several crown ethers.Investigations to assess the capacity of thesearchitectures to effectively host organic dyeslike rose bengal, thymol blue, and congo reddemonstrated that larger loop sizes in the shellexhibited better complexation properties whilesmaller cavities within the PG scaffold assureda higher stability of the host–guest complexes.

To further investigate the host–guest stabi-lity, the polymer–dye solutions were extractedfor a few minutes with water. After phaseseparation, the UV absorption of the organicphase was measured again (Fig. 36) and a cleardifference between 1 and 2 was observed.Whereas the hydrophilic rose bengal andthymol blue sodium salt were extracted tothe water layer from its organic soluble

complex with 1, the dye complex with 2 was sufficiently stablethat the dye remained in the chloroform layer. Indeed, thecross-linked host 2 did not liberate the dye even after 12 h ofshaking.

In an attempt to achieve a controlled release of the guestmolecules, photoresponsive cross-linked systems were preparedby the introduction of o-nitrobenzyl groups within the shell,shown in Figure 37.[181] The photodegradable nanocapsulesretained the capacity and selectivity for encapsulating bioactive

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Figure 36. Photographs of encapsulated rose bengal sodium salt in poly-mers 1 and 2 before (left) and after 5min extraction with water (right).

molecules, while modification of the building blocks allowedsubstantial control over host–guest stability.

The light-triggered release of rose bengal from the dye complexin chloroform was confirmed by irradiation of the solution at350 nm monochromatic light. Figure 38 shows the controlledrelease of the dye as a function of UV irradiation time.

The versatility of the systemwas proven by hydroxylation of theshell which yielded fully water-soluble cross-linked analogues.These tend to form aggregates of about 100 nm diameter withnarrow size distribution in pure water which break down athigher ionic strength. A weak interaction of these systems wasfound with different water insoluble species, e.g., nimodipine,pyrene, and Nile red.

A PG dendrimer monomolecularly imprinted withD-(�)-fructose was reported byHashidzume and Zimmerman[182]

utilizing the ring-closing metathesis. The synthetic pathway,shown in Figure 39, includes the synthesis of allyl-terminateddendrons linked to fructose through a boronic linker, followed byring-closing metathesis as the final step. After treating the

Figure 38. Release of rose bengal from photoresponsive cross-linkedsystems by UV irradiation (l¼ 350 nm).

Figure 37. Formation of light-responsive nanotransporters with o-nitrobenzyl gshell obtained by intramolecular ring-closing metathesis.


cross-linked dendrimer with aqueous acidic THF, the imprintedscaffold was able to form adducts with several monosaccharides,i.e., fructose, D-(þ)-galactose, D-(þ)-glucose, D-(þ)-mannose, andmethyl-a-D-mannopyranoside (MMan). It was shown that thebinding affinity could be switched by addition of a chemical agentwhich could modify the spatial arrangement of the boronic acidgroups, e.g., N,N,N0,N0-tetramethyldiaminomethane.

A related synthetic approach presented by Leroux andcoworkers.[183–185] involves the formation of reversed micellesby ATRP. Four-, 5-, 6-, and 8-arm ATRP initiators were preparedand used to polymerize glycidylmethacrylate. After hydrolysis ofthe resulting polymers, a partial esterification of pendant hydroxylfunctions with acyl chlorides (12–18 carbons) was performed toform the inverted micellar structures. The alkylated PG scaffoldswere shown to self-assemble into reverse micelles (RMs) withdiameters in the range of 20–60 nm in organic solvents and/or oil.

These derivates were able to encapsulate different water-soluble anionic dyes (Congo red, brilliant blue R, methyl orange)and various proteins including vasopressin, myoglobin, andalbumin in organic solvents and ethyl oleate. It was demonstratedthat the ability to accommodate guest molecules and the size ofthe micelles increased with the degree of branching. Interest-ingly, a linear analogue synthesized with the same chemicalstrategy showed encapsulation to some extent but with a lowercapacity. Preliminary studies showed that the encapsulation ofvasopressin improved its pharmacological activity after s.c.injection and oral administration. Addition of RMs significantlyimproved the loading of model solutes in poly(ester) particles. Asa specific example, the RMs were embedded into poly(D,L-lactide)nanospheres to serve as hydrophilic reservoir for polar guestmolecules.[186] By enhancing the concentration of RMs within thenanoparticles, improved encapsulation efficiency of Nile red,

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congo red, methylene blue, methyl orange, andvasopressin was observed.

In another approach the stability of theself-assembled micelles was increased througha cross-linking reaction between the hydroxylgroups located within the micelles(Fig. 40).[187] The core cross-linked RMs wereproduced by an original method based on theencapsulation of divinyl sulfone into thehydrophilic core of the micelle and subsequentcross-linking via Michael addition. The core

cross-linking did not affect the micelles’ sphericity and aggrega-tion number, but it reduced their solvodynamic diameter.Compared to the non-cross-linked analog, the capacity to retainguest molecules was increased, although this occurred at theexpense of the loading capacity. The approachmay be of particularinterest for designing sustained drug delivery or diagnosissystems.

5.3. Multivalent Interactions

Multivalency describes the binding of two or more entities thatinvolves the simultaneous interaction between multiple andcomplementary functionalities. Multivalent interactions (Fig. 41)are considerably stronger than the individual bonding of acorresponding number of monovalent ligands to a multivalent

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Figure 40. Encapsulation stability of cross-linked micelles in dichloromethane (reproduced withkind permission of Prof. J. C. Leroux).

Figure 41. Comparison of monovalent and multivalent interactions.

Figure 39. Synthetic pathway for the preparation of monomolecularly imprinted PG (adaptedfrom [179]).

210 � 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinh

receptor and are the most predominantphenomena in biological systems, parti-cularly for recognition and attachment,signal transduction, and numerous cellularinteractions.[7,188]

The driving potential of multivalent systemsis the high entropic gain in the formation ofmultivalent complex. For example, the bindingconstants of bivalent interactions can be afactor of 1000-fold higher than the correspond-ing monovalent binding, and for tri- andpentavalent interaction, values up to 108 havebeen reported.

A challenging approach to the applicationof multivalent interactions is the mimicry offunctional biomacromolecules with therapeu-tic relevance. Several attempts have been madeto mimic specific proteins, e.g., histones orpolysaccharides like heparin. In these cases,mimicry is mostly based on the surface chargeof the polymer molecules (Fig. 42). In theparticular case of dendritic PG, the neutralspecies with hydroxyl end groups represents agood analogue of polysaccharides, polyanionicderivates present similar activities to nega-tively charged polysaccharides, e.g., heparin,polysialic acid, and the amine terminatedPGs can act in a similar fashion as histonesbinding and compacting DNA. Applicationsrange from protein resistant coatings (neutralspecies) to DNA-transfection agents (polyca-tionic systems), anticoagulating and anti-inflammatory drugs (polyanionic systems).

5.3.1. Neutral PGs as Mimicry of

Oligosaccharides for Surface Modification

The ability to resist non-specific proteinadsorption is a major indicator of a material’s

biological inertness or biocompatibility. Applications of proteinresistant materials include prostheses, sensors, substrates forenzyme-linked immunosorbent assays (ELISAs), materials foruse in contact lenses, and implanted devices.[189] More recentapplications include systems for patterned cell cultures,[190] tissueregeneration,[191] microfluidic systems,[192] drug delivery,[193] andsystems for high throughput screening of proteins[194,195] orcells.[196] With the development of new biomedical applications,there is a great need to develop new biomaterials with novelstructures and properties.

In recent years a variety of substances have been studied fortheir resistance to protein adsorption.[197–201] PEG is one of themost commonly used biomaterials because of its exceptionalbiocompatibility. It shows unsurpassed resistance to non-specificprotein binding. Polyethylene glycol also meets the fourmolecular-level characteristics defined by Whitesides et al. forresisting the adsorption of proteins because it is hydrophilic,includes hydrogen-bond acceptors, does not include hydrogenbond donors, and is neutral. However, PEGs can only befunctionalized at the chain ends and the polymer is not

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Figure 42. Mimicry of biologically active macromolecules.

biodegradable. Based on the analysis of the best protein resistantsurfaces known in literature it becomes apparent that branchedarchitectures, for example with highly flexible aliphatic polyethersand hydrophilic H-donor groups, are some of the criticalstructural features that enable protein resistance. This, inaddition to the higher thermal stability compared to PEG[202]

makes the dendritic PGs optimal candidates for surfacemodification with the scope of getting a biocompatible surface.[97]

In addition, PGs resemble the structure of polysaccharides toprevent unspecific adhesion of cells as major constituents of theextracellular glycomatrix.

Our group has synthesized several disulfide-functionalizedhyperbranched PGs by coupling of thioctic acid.[202] Thespontaneously formed self-assembled monolayers (SAMs) on agold surface prevented the adsorption of proteins to the samelevel as for PEG SAMs and significantly better than dextran(Fig. 43). A structure–activity relationship revealed that the moreglobular the structures, the more inert the assembly is towardsprotein adsorption, while the molecular weight changes did notappear to have a significant effect. A small improvement in theprotein resistance was observed by substitution of the acidichydrogen atoms by methyl groups, which was in agreement withthe hypothesis referring to the absence of hydrogen-bonddonors.[203]

Figure 43. Preparation of protein resistant surfaces by self-assembled dendritic PG monolayerson gold chips (adapted from [202]).


In a recent publication, a different synthesisfor a thiolated hyperbranched PG wasreported by ring-opening polymerizationof glycidol from partially deprotonated2,20-dihydroxyethane disulfide as the initiatorand the subsequent reduction of the disulfidegroup.[204] Two molecular weights (1.59 and4.26 kDa) were obtained by varying theinitiator to glycidol ratio. By a comprehensivestudy, it was shown that the branchedpolymers produced more uniform structureson gold surfaces with low surface roughness.Although the low molecular weight speciesgave a higher graft density, the high MWcoated surface was much more resistant toprotein adsorption than the PEG coated sur-face. In addition, the protein repulsion ofPG-coated surfaces was found to increase withincreasing graft density of chains on thesurface.

In order to study the mechanism of proteinresistance in more detail our group recently

prepared a large variety of PG derivatives to assess theirprotein-resistance behavior.[96,205] Two different approaches tomodify the gold surfaces namely the ‘‘anhydride’’ method anddirect chemisorption of alkanethiolates were used. In the firstcase, a library of mono-amino oligoglycerols (linear, dendrons,and hyperbranched) with terminal hydroxyl or methoxy function-ality were synthesized and subsequently coupled to gold surfacescoated with mercaptoundecanoic acid. Surface plasmon reso-nance (SPR) spectroscopy with parallel adsorptionmeasurementsof four model proteins (fibrinogen, albumin, lysozyme, andpepsin) revealed that the capability of PG dendrons to resistnon-specific protein adsorption strongly depends on the coatingdensity, surface functionality, size, and structural freedom of thePG structures. The coating density using the anhydride methodwas considerably lower for higher generation ([G.3] and [G.4])which results in a lower protein resistance. By comparison of theperfect [G.3] PG dendron and its hyperbranched analog weconcluded that the conformational freedom is critical forachieving a high protein resistance (reduction of the proteinadsorption from 47 to 17%). In the case of the methylatedhyperbranched PG the best protein resistant surface (0.5%fibrinogen adsorption) was obtained, although contact anglemeasurements indicated a quite hydrophobic surface.

In the direct chemisorption of alkanethiolates approach, there
was better coating efficiency. Self-assembledmonolayers with different terminal function-alities (OH, OCH3) were prepared by chemi-sorption of the different PG derivatives ontogold chips from ethanolic solution. Surpris-ingly, gold surfaces modified with [G.1]�OHthiolate already showed a dramatic decrease ofthe fibrinogen adsorption on the SAMs,indicating that already relatively small oligo-mers can be protein resistant.
A related application for the PG coatingwas presented by Wang et al.[206] wherehyperbranched structures were grown from

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Figure 44. Preparation of hyperbranched PG-modified magnetic nanopar-ticles (MNPs) via anionic ring-opening polymerization of glycidol (modi-fied from [202]).

Figure 45. Multivalent glycoarchitectures based on PG-galactose (R¼H)and PG-sulfated galactose (R¼ SO3Na).

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magnetic nanoparticles via surface-initiated anionic ring-openingpolymerization of glycidol. The general mechanism presented inthe Figure 44, similar to the one earlier reported by Khan andHuck[207] for the coating of Si/SiO2, included the initial coating ofthe nanoparticles by 3-mercaptopropyl trimethoxysilane followedby the deprotonation of the thiols with sodiummethoxide to formthe polymerizable initiator. The product obtained after polymer-ization not only presented the expected excellent proteinresistance properties described before, but also improved thestability and dispersability of the magnetic nanoparticles whilethe supermagnetic behavior was retained.

Due to a higher thermal and oxidative stability of the bulk PGthan PEG and the easy accessibility of these materials, dendriticPGs are promising candidates for surface coatings in biomedicalapplications. In addition, the multiple free hydroxy groups ofdendritic PGs can be further functionalized with ligands forspecific protein interaction and avoidance of non-specificadhesion. The conjugation of PG to proteins may be an elegantsolution for systems where proteins to be immobilized, e.g., forbiosensors. For example, streptokinase, glucose oxidase, andphosphorylcholine have been conjugated to hyperbranched PG toconstruct a glucose biosensor.

In an earlier study, Queiroz and coworkers[208] conjugatedstreptokinase, a thrombolytic enzyme, to hyperbranched PG. In acareful study of the platelet adhesion and thrombus formation onpolystyrene surfaces, it was found that the conjugate can lyseblood clots. This indicates that the conjugation process does notaffect the fibrinolytic property of the enzyme. This concept wastransferred to design a biosensor,[209] where the PG–streptokinaseconjugate was linked to glucose oxidase and phosphorycholineand further entrapped in polyaniline nanotubes. After depositionin an aluminum electrode, the platelet adhesion, fibrinolytic


activity, protein adsorption, and amperometic response to glucosewere studied to examine the interaction of blood with thePG-based biosensor, and the electrochemical response. The invitro results on the blood compatibility and glucose sensingproperties suggest that the bioconjugated PG could be avery useful material for development of implantable glucosebiosensor.

5.3.2. Negatively Charged Derivates

Lectins are multivalent carbohydrate-binding proteins whichspecifically bind different sugar structures. They have receivedconsiderable attention recently due to their importance in cellsurface interaction and biological recognition. Multivalency inlectins has been discussed in detail and is considered to be a goodmodel for studying multivalency of dendritic polymer derivatives.

Our group recently explored the use of multivalent glycoarch-itectures based on PG (Fig. 45) for the inhibition of L- andP-selectins, a general class of receptors which displays a selectiveadhesion and includes a lectin-like domain.[210] Two structureswere compared, namely, free PG–galactose (R¼H in Fig. 44) andPG–sulfated galactose (R¼ SO3Na).

Selectin inhibition studies carried out with SPR measure-ments indicated a clear enhanced effect due to multivalency(Fig. 46). Using L-selectin, the nanomolar binding affinity ofPG–galactose was observed. Notably, sulfated dendritic galactoseshowed a further enhancement with an IC50 of 1 nM. Thisindicates the importance of negatively charged sulfate groups onthe surface of these polysaccharide analogues.

In an ongoing project by our group, a similar study wasperformed using hyperbranched PG sulfates (Fig. 47), primarilyreported as a heparin analogue. These structures were found toprolong the time of activated partial thromboplastin as well asthrombin and to inhibit both the classical and alternativecomplement activation more effectively than heparin


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Figure 46. SPR competitive study of L- and P-selectin affinity of various PG glycoconjugates.[211]

itself.[212,213] The biocompatible and well-tolerated PG sulfate actas multivalent selectin ligand mimetics and efficiently blocksleukocyte migration. L- and P-selectin binding to immobilizedligands was drastically reduced by the PG sulfates in vitro andgave IC50 values in the low nanomolar range. The inhibition wasstrongly dependent on core size and degree of sulfation fordifferent derivatives. In an in vivo model it was observed that theadministration of dendritic PG sulfates in a contact dermatitismodel dampened the inflammatory response, which is anoticeable phenomenon related to selectin binding.

5.3.3. Applications of Polycationic Derivates of Dendritic PGs

Another kind of interaction related to multivalency is genedelivery by polymeric carriers. The electrostatic interactionbetween the negative phosphates backbones of the DNA/RNAstrand with agents that carry multiple positively charged groups isthe principle which is used in non-viral gene delivery (Fig. 48).Cationic lipids and cationic polymers are the best studiedcompounds for this purpose.[214,215] The interaction between asingle protonated amine and the phosphate backbone of DNA isrelatively weak and competes with salt binding under biologicalconditions. Therefore tetra-amines such as spermine are used toachieve DNA binding.[216]

Several amine functionalized hyperbranched PGs have beenreported as potential gene vectors. In comparison to other

Figure 47. Schematic representation of highly anti-inflammatory dendriticPG sulfate. Figure 48. Dendritic


dendritic structures, these scaffolds have theadded advantage of being open and flexible,which is a prerequisite for gene interaction.Due to the polyether structure, the toxicityremains in considerably low range.

Kizhakkedathu and coworkers[217] pre-sented a complete study of blood compatibilityand DNA binding of PG decorated with PEGchains and tris(2-aminoethyl)amine. The scaf-folds with different degrees of amine quater-nization showed insignificant effects onhemolysis, erythrocyte aggregation, comple-ment activation, platelet activation, and coa-

gulation. By comparison with standard cationic polymers like PEIa much lower cytotoxicity of the PG derivatives was observed. InDNA binding, a high affinity in the nanomolar range was found atlow levels of quaternization, comparable to the binding affinitiesof cationic multivalent dendrimers with their much lowerbiocompatibility.[218] In a different approach, hyperbranchedPG was partially functionalized with quaternary and tertiaryammonium groups with different loading degrees.[41] All thepolymers showed interaction with DNA and formed neutralpolyplexes. These quarternized PG-derivatives showed a similartransfection efficiency as PEI but with significantly diminishedcytotoxicity in mammalian cells.

In a recent investigation, our group synthesized severalhyperbranched PG derivates that were post-modified withspermine, spermidine, and pentaethylenehexamine. Thesedendritic polyamine compounds were clearly multivalent ingene complexation (Fig. 49).[219,220] By an ethidium bromidedisplacement assay, it was possible to demonstrate the highaffinity of these amine functionalized PGs for DNA. The affinityof the amine moieties increased significantly after theirattachment onto the PG scaffold. Preliminary cytotoxicity studiesshowed that the dendritic nanocarriers exhibited a safe profile atthe required concentrations and have decreased bioluminescencein a U87-luciferase cell line by 50–80%. In a preliminary in vivoexperiment significant gene silencing of some of the structurestested was observed.

5.4. Functional Hydrogels and Biocompatible Matrix Materials

Hydrogels are cross-linked hydrophilic polymer structures whichcan imbibe large amounts of water or biological fluids. Althoughhydrogels have a number of non-biomedical applications, e.g., inagriculture, their use in the field of medicine and pharmacy is

nanocarriers for cell internalization of gene vectors.

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Figure 49. Representation of multivalent hyperbranched cationic PG forgene delivery.

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auspicious. The highly hydrophilic nature of PG, together withthe high multifunctionality of the dendritic structures, allows theuse of this polymer to design a new generation of hydrogels. Freyand coworkers[221] were pioneers in the synthesis of structuredPGhydrogels based on PEOmultiarm stars with a hyperbrancheddendritic core. The materials obtained showed excellent formstability with high compression moduli. Furthermore, theypossessed a large number of hydroxyl groups that could be furthermodified, e.g., with cell growth factors. Substantial suitability ofthese hydrogels as substrates for cell growth has beendemonstrated.

Recently, the application of dendritic PG was reported in thefield of tissue engineering. Alblas and coworkers[222] encapsu-lated bone marrow derived multipotent stromal cells (MSC) in aphotopolymerizable hydrogel synthesized as described in Figures13 and 14, for the development of printed bone grafts. Theydemonstrated the adverse effects of photopolymerization on theviability and cell cycle progression of exposed MSC monolayers,but their differentiation potential remained intact. The hydrogelwith incorporated MSC supported survival and osteogenicdifferentiation of the embedded cells to a variable degree.

Figure 50. Fluorescence microscopy shows clear evidence for cellular uptake


Queiroz et al.[223] prepared a novel bone scaffoldingmaterial byelectrospinning solutions of hyperbranched PG that containednanoparticles of hydroxyapatite (HA). The potential use of theelectrospun fibrous PG–HA scaffolds for bone regeneration wasevaluated in vitro with human osteoblasts (SaSO2) in terms ofalkaline phosphatase (ALP) activity of the cells and cytotoxicity.The biocompatibility evaluation of the PG–HA providedencouraging indications for long-term safety. The electrospunfibers exhibited highest ALP activity and appeared to promoteboth proliferation and differentiation of human osteoblasts.

Efficient routes to produce biocompatible microgels derivedfrom PG in the privileged nanosized range of 10–200 nm wererecently reported by our group.[135] Microgels are special highmolecular weight polymers with characteristics of both hyper-branched and macroscopically cross-linked materials. Microgelscan be dissolved somewhat analogously to linear polymers butdue to sporadic cross-linking the conformation is almost fixed.Based upon mounting insights into the mechanisms ofnanoparticle endocytosis, we hypothesized that PG microgels(PG-m-gels) of optimum size may be efficiently and non-disruptively uptaken into cells, making them attractive scaffoldsfor drug delivery agents and probing biological phenomena.Particles with diameters in the range of 25–50 nm are mostdesirable according to precedent studies. Of further significanceto the design of targeting delivery agents, particles in this sizerange are known to passively accumulate in disease distressedtissues by the so-termed ‘‘enhanced permeation and retention’’effect. Initial tests on cellular uptake show that these nanopar-ticles localize in the perinuclear region of human lung cancercells at 37 8C with significantly lower uptake when incubated at4 8C. In comparison, the low MW control did not lead to cellularfluorescence signals. These data, shown in Figure 50, may beevidence for a size dependent endocytotic mechanism of cellentry. In addition, such PG-m-gels architectures showed a safecytotoxicity profile in the mg/mL range.

6. Conclusions

Smart and biocompatible materials are critical for the futuredevelopment of the newly emerged field of nanomedicine andthe increased demand for better biomaterials in regenerativemedicine. Dendritic PGs, as characterized by tunable end groupfunctionalities, defined topological 3D architecture, and inertness

of fluorescently labeled PG-microgels via an endocytotic pathway.


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to unspecific interactions with biological systems, presents anovel platform for next generation biomaterials. The versatilesynthesis allows for a structural range of PG architectures fromperfect dendrons to well-defined hyperbranched polymers,megamers, microgels, and hydrogels.

Although these PG-based scaffolds demonstrate optimalbiocompatibility on the cellular and systemic levels, biodegrad-ability of the PG core itself remains an issue to be solved. Thetoxicity profile of PG runs parallel with PEG and classifies bothcompounds as highly non-toxic for in vivo applications. Thepolyol structure of PG, however, makes it a better candidate formultivalent interactions, as demonstrated by various examplesincluding targeting inflammation.

A continuous effort is necessary to modify and tailor PGarchitectures to fit the future demands of biomedical applications.The big challenge that still remains is the synthesis ofbiodegradable PG nanoparticles with sizes of 20–100 nm, whichshow a high targeting potential and can be cleared by the kidneys(cut-off �40 kDa). Furthermore, new intelligent release systemsare required to liberate an encapsulated or conjugated drug with adesigned release profile. There are still many open questions onhow to design sophisticated architectures based on PG as well ashow to control their biological availability and activity in vivo.Additionally, many more PG derivatives should be biologicallyevaluated to further improve their protein resistant properties,which are higher than for the current benchmark, PEG.

Finally, controlled synthetic methodologies that yield highlymonodisperse and well-defined dendritic PG derivatives areprimarily required to translate this state-of-the-art macromoleculefrom the laboratory to clinic.

Acknowledgements

We acknowledge financial support from German Science Foundation(DFG), Deutscher Akademischer Austausch Dienst (DAAD), Bundesmi-nisterium fur Bildung und Forschung (BMBF Nanonachwuchswettbe-werb), and the SFB 765. Furthermore, Dr. Pamela Winchester and Ms.Juliane Keilitz are thanked for careful proofreading and Ms. Christine Pachfor graphical support and help in the preparation of this manuscript.

Received: June 26, 2009

Published online: October 7, 2009

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