DOI: 10.1002/cbic.200900383

Phosphate Functional Core-Shell Polymer Nanoparticles for the Release ofVascular Endothelial Growth Factor

Louisa Gilmore,[a] Sheila MacNeil,[b] and Stephen Rimmer*[a]

Heparin and heparin sulfate are negatively charged polysac-charides that regulate proteins of importance in numerous cel-lular interactions, which are vital aspects of human physiolo-gy.[1] The highly sulfated and anionic nature of the glycosami-noglycan, heparin, and the proteoglycan, heparin sulfate,mean that they are able to interact with proteins includinggrowth factors, extracellular matrix proteins, chemokines andenzymes. The activity of several important growth factors, forexample vascular endothelial growth factor (VEGF), is modulat-ed by binding to heparin.[2] Among other functions, VEGF[3]

controls the proliferation and migration of endothelial cells[4]

and recent work has provided evidence for its role in the stabi-lization of blood vessels.[5] Recently, the delivery of VEGF andother cytokine growth factors has become a key strategy in re-generative medicine as a means to control angiogenesis.[6]

However, given the need to arrest angiogenesis to prevent tu-morigenesis[7] it is clear that effective regenerative therapy willrequire control of both the temporal and geometric delivery ofgrowth factors. With these as-pects in mind delivery systemswith matrices derived from gela-tin or processed collagen havebeen used.[8] A collagen matrixhas also been modified by theaddition of heparin to bind VEGFand control its release.[9] Withthe aim of mimicking the releasefrom extracellular matrix, severalauthors have used polysaccha-ride-based matrixes including al-ginate[10] and hyaluronic acid[11]

systems. Encapsulation intopoly(lactide-co-glycolide) allowsfor the formation of particulatedelivery[12] systems or scaffoldsformed from fused particles[13]

However, as far as we are awareno attempts have been made tocombine the electrostatic bind-

ing of VEGF that is evident in the natural system with a partic-ulate delivery system. The binding of VEGF to heparin occursthrough interactions with highly basic peptide sequences, typi-cally containing multiple Arg and Lys units. We considered thatphosphate groups present on a synthetic polymer could alsobind to the highly basic VEGF heparin binding domains andthe presence of these features on polymer particles would pro-vide a convenient means of delivering VEGF. Therefore, withthis in mind, we set out to produce core shell polymer parti-cles capable of interacting with VEGF as a “heparin-mimic”,meaning it should interact with the protein through the hepa-rin-binding region and electrostatic interactions.

Core-shell particles were prepared with polystryrene coresand cross linked alkyl phosphate shells, as shown in Figure 1.Two core-shell submicron polymer particles (in which the shellwas molecularly imprinted with a peptide epitope) were pre-pared along with a nonimprinted material. Previously weshowed how polymerization of ethandiol dimethacrylate in the

presence of 1 and certain template molecules, including pep-tides, could be used to prepare molecularly imprinted parti-cles.[14–16] The inclusion of 1 probably occurs by transfer tomono ACHTUNGTRENNUNGmer rather than copolymerization. Core-shell particles im-printed with DKPRR bind to the sulfate groups on heparin. Wereasoned that phosphate groups could also interact with theheparin-binding region and we used imprinting methodologyto examine the effects of changing the spatial orientation ofthe phosphate groups on binding and release of VEGF. An un-charged peptide, GAA, was used to prepare alternative im-

Figure 1. Scheme showing polymerization of a surfactant-like binding (phosphate functional) monomer on thesurface of a polystyrene particle ; this produces a core-shell particle with a shell that can bind to species with anamino functionality.

[a] L. Gilmore, Dr. S. RimmerDepartment of Chemistry, University of SheffieldDainton Building, Brook Hill, Sheffield S3 7HF (UK)Fax: (+ 44) 114 222 9346E-mail : s.rimmer@shef.ac.uk

[b] Prof. S. MacNeilDepartment of Engineering MaterialsThe Kroto Research Institute, University of SheffieldSheffield, S1 3JD (UK)

Supporting information for this article is available on the WWW underhttp ://dx.doi.org/10.1002/cbic.200900383.

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printed particles and these particles were both compared tononimprinted core-shell particles.

The particle cores were formed from poly(styrene-co-divinylbenzene) with a diameter of 35 nm. Then the shell phase wasadded either in the presence of the DKPRR, GAA peptides orwithout added peptide. The resulting latexes were coagulatedwith 2-propanol and washed repeatedly with 0.1 m phosphoricacid (to remove the bound template), water and methanol toproduce particles containing shell-imprinted cavities ready forbinding experiments. Particle size analysis and transmissionelectron microscopy (TEM, Figure 2) imaging showed the core

particles to have average diameters of 35 nm while the nonim-printed, GAA-imprinted and DKPRR-imprinted had diameters of45–50 nm. The presence of the functional monomer in theouter shell was confirmed qualitatively by X-ray photoelectronspectroscopy and electron dispersive spectroscopy (EDS; seeESI) and quantitatively by inductively coupled plasma atomicemission spectroscopy (ICP-AES). ICP-AES revealed that nonim-printed particles contained 347 mg kg�1 of phosphorous, GAA-imprinted particles contained 345 mg kg�1 phosphorous andDKPRR-imprinted particles had a slightly higher phosphorouscontent at 390 mg kg�1. Table 1 shows that the zeta potentials(measured after removal of the template) were essentially un-changed on adding the shells regardless of whether the pep-tides were included in the polymerization (ANOVA, p>0.05).However, as shown in Figure 3, rebinding of DKPRR had a sig-

nificant effect on the z potential of each of the core-shellparticles.

Clearly, as expected, the addition of SSSSSSS (a hydrophilicuncharged peptide) had little effect on the z potential at 25 8C,whereas the addition of the highly charged peptide, DKPRR,produced a step change in z potential when added to thephosphate functional particles. There was no change whenDKPRR was added to the core particles. From these data thereappears to be no difference between the DKPRR-imprintedand the nonimprinted core-shell particles, but the magnitudeof the change appeared to be smaller in the GAA-imprintedmaterial than in either of the former particles. (At 15 mg thedifference between the GAA-imprinted and the blank or theDKPRR was significant: ANOVA post hoc (Tukey) p<0.01.)

Methyl red changes colour from yellow to red when the pHis lowered in an aqueous environment, and it is widely docu-mented that this colour change is the result of a structuralchange when the neutral species, the azo form, is convertedinto one of the protonated forms, either the ammonium orazonium species.[18, 19] It has previously been reported thatsteric hindrance of the methyl-red moiety can prevent proto-nation of the azo group and limits the colour change as thepH is altered. With the steric requirements of the colourchange in mind we hypothesised that methyl red attached totemplate bound in an imprint should produce a different spec-trum than the free molecule because the conformation of theformer would be restricted. A number of methyl-red taggedpeptides, methyl red–SSSDKPRR (mrSSSDKPRR), methyl red–SSSPRKRD (mrSSSPRKRD) and methyl red–SSSSSSSS(mrSSSSSSSS) were synthesised by solid phase peptide synthe-sis. These were then used to examine any qualitative differen-ces in the interactions of the peptides with the particles.

Figure 2. TEM images of: A) core particles, B) DKPRR-imprinted, C) nonim-printed, and D) GAA-imprinted.

Table 1. Physical data for core and core-shell particles.

Sample Particle size[nm]

z potential[mV]

Phosphorous contentACHTUNGTRENNUNG(ppm) [wt %]

core 34.9�0.2 �38.0�1.6 <10blank CS-MIP 44.4�0.8 �41.6�0.9 347.4DKPRR CS-MIP 48.2�0.1 �42.9�1.1 390GAA CS-MIP 45.4�0.3 �42.7�1.3 344.6

Figure 3. The z potential measurements at 25 8C obtained after addition of:A) SSSSSS, and B) DKPRR to the particles; &, core; *, nonimprinted; ~, GAA-imprinted; !, DKPRR-imprinted.

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Introduction of aqueous solutions mrSSSDKPRR,mrSSSPRKRD or mrSSSSSSSS to the core, DKPRR-imprinted,GAA-imprinted or the nonimprinted core shell particles result-ed in complete adsorption after removal of the supernatantand following centrifugation. Release of the peptides from theparticles was investigated by decreasing the polarity of themedium and then increasing the pH. The former procedureprovided a comparison of the contributions of hydrophobic in-teractions to the adsorption. The relative fraction of peptideadsorbed by hydrophobic interactions was determined bywashing the polymers with water containing increasing pro-portions of 2-propanol. The data are shown in Figure 4. In100 v/% 2-propanol, only 8 % of the mrSSSDKPRR, mrSSSPRKRDremained on the core particles but mrSSSSSSSS was complete-ly removed by a solvent mixture containing only 40 v/%propan-2-ol (IPA). DKPRR-, GAA-, and the nonimprinted core-shell particles retained 80–90 % of the mrSSSDKPRR andmrSSSPRKRD peptides, and there were no significant differen-ces in the amounts of peptide released. Contrary to these ob-servations, release of mrSSSSSSSS was essentially complete onapplication of a aqueous/solvent mixture containing 40 v/%IPA and there was no difference between the behaviour of thispeptide on the blank or any of the core-shell particles. Thedata clearly indicate that the phosphate core-shell particles dis-play strong electrostatic interactions with mrSSSDKPRR andmrSSSPRKRD but as expectedthe interactions between theserine residues and the particlesarise from only hydrophobic/hy-drophilic forces in water.

As the bound methyl-redpeptide was exposed to an in-creasingly acidic environmentfrom deionised water to0.04 mol dm�3 phosphoric acid, acolour change on the polymermaterial was easily observed bythe naked eye (Figure 5). Addi-tion of phosphoric acid tomrSSSPRKRD and mrSSSDKPRRpeptides bound to both thenonimprinted and the GAA-im-printed particles resulted in animmediate colour change fromorange to red/pink. This colourchange was not observed forthe peptides bound to theDKPRR-imprinted particles. Inthis case, addition of phosphoric acid to the boundmrSSSPRKRD peptide resulted in only a slight colour change,to a darker orange, and addition of phosphoric acid to thebound mrSSSDKPRR peptide particles produced no colourchange at all. The minimum colour change for the peptides at-tached to the DKPRR-imprinted particles demonstrated thatthe protonation of the methyl-red moiety was prevented.These observations appear to provide strong evidence for thelocation of the methyl-red peptide in a specific cavity in the

DKPRR-imprinted particles. Strong binding in such a cavity pre-vents the structural change necessary for protonation.

Cavities formed by imprinting of DKPRR are not present onthe nonimprinted and GAA-imprinted polymers. Therefore, anybound material would be present on the surface of the parti-cles, where it would be susceptible to protonation. Figure 4shows the effect of increasing concentration of phosphoricacid on release of the peptides. There is a clear difference be-tween the behaviour of the DKPRR-imprinted material and

Figure 4. Release of peptide, after rebinding, in increasing concentrations of:A) isopropanol, and B) phosphoric acid; *: mrSSSSSSSS, &: mrSSSDKPRR, ~:mrSSSPRKRD.

Figure 5. Images demonstrating the colour change upon protonation of mrSSSDKPRR. A) A graph showing shift inwavelength for mrSSSDKPRR at pH 6, pH 2 and in 1 mol dm�3 phosphoric acid, B) images of mrSSSDKPRR solutionsexposed to (left–right) 1 m phosphoric acid, pH 2 and pH 6, C) images of mrSSSDKPRR peptide bound to (left–right) DKPRR-imprinted particles, blank-imprinted particles, core-particles and GAA-imprinted particles, all at0.05 mol dm�3 phosphoric acid. These images demonstrate the lack of colour change for the mrSSSDKPRR peptidewhen bound to the DKPRR-imprinted particles.

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both the GAA-imprinted and the nonimprinted material. BothmrSSSPRKRD and mrSSSDKPRR were released in equal quanti-ties from the GAA-imprinted and the nonimprinted materialsbut the amounts of mrSSSPRKRD retained on the DKPRR-im-printed material were lower relative to the amounts ofmrSSSDKPRR. These data therefore confirm an effect of im-printing the particles with DKPRR but the data also clearlyshow that both mrSSSDKPRR and mrSSSPRKRD were adsorbedin a pH dependant manner on the phosphate functional parti-cles. It therefore seemed reasonable to examine the release ofVEGF from the GAA-imprinted, DKPRR-imprinted and the non-imprinted particles.

VEGF was added to the particles and then its concentrationwas determined in the supernatants using a sandwich ELISA.Figure 6 shows the release from particles loaded with 100 or

150 ng mg�1 of VEGF per dry mass of particle. With both load-ings, the growth factor was released at physiologically relevantconcentrations[20–22] from each of the phosphate functionalizedparticles. Most release occurred within the first 12 h but after12 h the release was relatively constant and the releasedgrowth factor maintained activity to anti-VEGF for at least 48 h.Because VEGF typically has a clearance half life of less than 1 hin vivo,[20, 21] the particles provide an important route for im-proving the continuous release of VEGF. There were no signifi-cant differences between the particles at either loading (two-way ANOVA p>0.05). Although particles imprinted with theterminal heparin binding sequence, DKPRR, displayed differentbehaviour to nonimprinted particles and particles imprintedwith GAA, when it came to release of VEGF there was no differ-ences in the release of this labile protein between any of theparticles.

All the phosphate-functional particles achieved release ofimmunoreactive VEGF for at least 48 h. We hypothesise that

physical binding of the VEGF by these nanoparticles stabilisesthis labile peptide; this offers a new approach to clinical appli-cations with this peptide, and these applications can now betested experimentally. Considering the short half-life of VEGFin vivo, the various regenerative therapies that have been pro-posed for this growth factor could be facilitated by thisapproach.

Experimental Section

Synthesis of oleyl phenyl hydrogen phosphate : Oleyl phenyl hy-drogen phosphate was synthesised by adding oleyl alcohol (65.5 g,0.237 mol) dropwise to stirring phenyl phosphoro-dichloridate(50.0 g, 0.237 mol) over 90 min at room temperature. The resultingsolution was then stirred for a further 60 min. The temperaturewas then increased to 50 8C and stirring was continued for a fur-ther 16 h. The resultant reaction mixture was then added dropwiseto rapidly stirring ice-cold water (300 mL) and after 60 min the or-ganics were extracted into diethyl ether (3 � 100 mL). Anhydroussodium sulfate was used to dry the combined organic extracts,which were then filtered and rotary evaporated to produce a vis-cous yellow oil. Purification was carried out by using column chro-matography on silica with DCM (100 %) to methanol (2–10 %)/DCM(98–90 %) to give a straw-coloured oil as the final product of 8.39 g(16.68 %), Rf = 0.26 on silica (37:2:1 CH2Cl2/MeOH/AcOH). 1H NMR(CDCl3, 250 MHz): d= 0.87 (t, 3 H; CH3), 1.26 (br m, 22 H; �CH2�),1.62 (br m, 2 H; �CH2CH2�OP), 2.00 (br m, 4 H; �CH2CH=), 4.05 (dt,2 H; �CH2OP, JPH = 6.71, JHH = 6.71), 5.38 (br m, 2 H; �CH=CH�),7.10–7.32 (m, 5 H; �C6H5�), 10.94 ppm (s, 1 H; OH); 31P NMR (CDCl3,162 MHz) d=�4.0228 ppm; elemental analysis calcd (%) forC24H41O4P: C 67.90, H 9.73; found: C 68.14, H 10.43.

Emulsion polymerizations : Synthesis of divinylbenzene-cross-linked polystyrene core particles was carried out by firstly produc-ing a solution of MES buffer (50 mmol dm�3) in deionised water.This solution was purged with nitrogen for 15 min, sodium dodecylsulfate surfactant (1 g, 3.47 � 10�3 mol) was added, and the solutionwas stirred until the SDS dissolved. The solution with surfactantwas sonicated for 10 min and adjusted to pH 6 through dropwiseaddition of NaOH (aq., 0.5 mol dm�3). Polymerisation was conduct-ed in a 100 mL jacketed glass flange reaction vessel with a fivenecked lid equipped with mechanical stirrer, nitrogen inlet, refluxcondenser, temperature probe and pressure equalised droppingfunnel. The buffer solution was introduced to the reaction vessel,which was heated and had been previously purged with nitrogen.The buffer solution was stirred at 400 rpm and brought to 70 8C.Styrene (4.5 g, 43.2 mmol dm�3) and divinylbenzene (DVB, 0.5 g,3.84 mmol dm�3) were combined, purged with N2 for 5 min andadded dropwise to the solution through an addition funnel over a30 min period. Potassium persulfate (0.2092 g, 0.774 mmol) and as-corbic acid (0.0682 g, 0.387 mmol) were dissolved in H2O (5 mL),and this initiator solution was then purged with nitrogen for 5 minbefore being added to the stirring monomer suspension by a sy-ringe in a one-shot initiation. Emulsion polymerisation was carriedout, overnight. EGDMA (0.226 g, 1.14 mmol) and oleyl phenyl hy-drogen phosphate (0.204 g, 0.482 mmol) were combined andadded dropwise to the stirring emulsion of core particles with a sy-ringe over 15 min. The monomer was allowed to equilibratearound the core particles for 1 h. The template compound(0.482 mmol, 1 equiv) was added in MES buffer (1 mL,50 mmol dm�3) at pH 6 and stirring was continued for 1 h. The ini-tiator solution was produced by dissolving potassium persulfate(0.32 g, 1.184 mmol) in deionised H2O (8 mL) and then purging the

Figure 6. Release of VEGF; instantaneous concentrations determined byELISA. Initial loading of VEGF: A) 100 ng mL�1, B) 150 ng mL�1; &: DKPRR-im-printed; *: GAA-imprinted; ~: nonimprinted.

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solution for 5 min with nitrogen. The second stage of polymeri-sation was then initiated by the addition of this solution to the stir-ring latex in one shot with a syringe. The emulsion was allowed toreact at 70 8C, overnight. Nonimprinted particles were also pre-pared by repeating the polymerisation procedure in the absenceof a template molecule.

Zeta potential measurements : An aliquot of the core or CS-MIPemulsion (2 mL) was injected into a Slide-A-Lyzer� cassette. Theemulsions were then dialysed against phosphoric acid(1 mol dm�3), with the addition of sodium dodecyl sulfate (2.5 %) toprevent coagulation, over three days with twice daily solventchanges. Finally, the emulsions were dialysed against sodium dode-cyl sulfate (2.5 %)/distilled water for three days with solventchanged twice daily. Emulsion (100 mL) was added to KCl (2 mL,1 mmol) and was agitated to produce a homogeneous latex. The z

potentials were analysed at 25 8C in five analysis cycles, each con-sisting of ten repeat runs. peptide solution (10 mL, 0.5 mg mL�1)was added to each emulsion and agitated before measuring thepH of the solution and the z potential. In total, seven aliquots ofeach peptide were added and analysed for pH and zeta potential.The zeta potentials were measured using a Brookhaven ZetaPALSinstrument.

Electron microscopy : In preparation for TEM and EDS studies analiquot (1 mL) of the CS-MIP emulsion or the core emulsion wasadded to a preswollen slide-a-lyzer dialysis cassette. The emulsionswere dialysed against phosphoric acid (1 m, 1 mol dm�3) with SDS(2.5 %, 200 mL) over three days. The dialysis solution was replacedtwice a day. The emulsions were then dialysed against SDS (2.5 %)/distilled water for three days, again the solution was changedtwice daily. Materials were prepared for TEM and EDS studies byevaporating the colloidal suspensions on to copper grids (600mesh, 3 mm diameter) coated with a carbon film. The particleswere visualised for TEM by negative staining with uranyl acetate.

ICP-AES : To prepare solid resins for inductively coupled plasmaatomic emission spectroscopy (ICP-AES) a surface-templated emul-sion (2.0 mL) was added to a centrifugation cartridge (20 mL,MWCO 100 000) followed by the dropwise addition of IPA (1.1 mL)to induce coagulation. The centrifuge cartridge was agitatedgently until the solids had precipitated out. The sample was al-lowed to stand for 10 min to allow complete precipitation andthen it was centrifuged at 4500 rpm at room temperature using abenchtop centrifuge with swing-bucket rotor for 30 min. Thedamp solids remaining were washed with IPA/H2O (7:3, 5.0 mL)and the suspension was then agitated and centrifuged at4500 rpm for 30 min. This procedure was carried out six times. Thesolids were then washed with deionised water (5.0 mL), gently agi-tated to produce a suspension and centrifuged at 4500 rpm for5 min. This washing step was also repeated six times. The finalwashing step was washing the solids with methanol (5.0 mL), fol-lowed by agitation and centrifuging at 4500 rpm for 5 min. Thisstep was also repeated six times. The polymer was then dried to aconstant mass in a vacuum oven at 60 8C for 48 h. The polymerwas then refluxed in concentrated nitric acid (5 mL) at 160 8C for4 h followed by perchloric acid at 200 8C for 30 min and the result-ing solution was then analysed for phosphorous content by ICP-AES.

Peptide rebinding and release studies : mrSSSDKPRR,mrSSSPRKRD and mrSSSSSSSS binding and release data was deter-mined by adding of an aqueous peptide solution (0.5 mL,0.29 mg mL�1) to pre-coagulated and pre-washed particles. Theparticles were prepared as follows. An aliquot of emulsion (0.2 mL)

was pipetted into a 30 000 MWCO Vivaspin centrifugal concentra-tor, followed by isopropyl alcohol (0.5 mL) to coagulate the latex.The sample was agitated and then left on an orbital shaker plateat 150 rpm for 10 min before being centrifuged at 10 000 rpm for10 min. The coagulated polymer was then washed with an IPA/H2O(7:3) solution, agitated, left for 10 min on an orbital shaker plate at150 rpm and then removed by centrifugation for 10 min at10 000 rpm. This wash step was carried out six times with IPA/H2O(7:3), six times with H3PO4 (1 m, solvent removed by centrifugingfor 5 min at 5000 rpm), six times with H2O, six times with methanoland finally six times with H2O. The polymer slurry was then keptdamp before use.

Each sample was agitated and then allowed to equilibrate on ashaker plate at 200 rpm for 30 min. The samples were then centri-fuged at 6000 rpm for 10 min and the supernatant removed forUV/Vis analysis to determine MRSSSDKPRR concentration. Anumber of washes with mixtures of water and isopropyl alcoholfrom deionised water (100 %) to IPA (100 %) were carried out byadding an aliquot (0.5 mL) of the wash solution, agitating thesample and allowing it to reach equilibrium on a shaker plate at200 rpm for 30 min. The sample was then centrifuged at 6000 rpmfor 10 min and the supernatant was removed for analysis. Methyl-red tagged peptide remaining on the polymer after a IPA (100 %)wash for the mrSSSDKPRR and the mrSSSPRKRD studies was thenremoved by sequential phosphoric acid washes/IPA washes withincreasing phosphoric acid concentrations (from 50 %0.005 mol dm�3 phosphoric acid (50 v/% IPA/water) to 1 mol dm�3

phosphoric acid (50 v/% IPA/water)). For each of these steps acid/IPA solution (0.5 mL) was added to the polymer slurry, the suspen-sion was agitated and then left on an orbital shaker plate at200 rpm for 30 min. The liquid was removed by centrifugation for20 min at 10 000 rpm.

VEGF release studies : Three polymer types were analysed in dupli-cate; DKPRR-imprinted particles, GAA-imprinted particles and non-imprinted particles. The polymer emulsions (0.1 mL) were coagulat-ed with IPA (0.5 mL) in a polypropylene tube and were thenwashed as described in the section on Peptide rebinding and re-lease studies above. Solutions of human VEGF (either 100 ng or150 ng in 0.5 mL PBS) were then added to the polymer. The solu-tions were agitated and then allowed to interact for 24 h at 4 8C.The suspensions were then centrifuged at 13 000 rpm for 20 minand the supernatant was removed and stored at �20 8C prior toanalysis of the VEGF concentration by using an ELISA kit (Quanti-kine, R&D Systems, Minneapolis, MN, USA) according to the manu-facture’s instructions.

Phosphate-buffered saline (PBS) solution containing fresh BSA(1 wt %, 0.5 mL) was added to the polymer samples, which werethen agitated and incubated at 37 8C for the release study. At de-fined time points the protein solution was removed and replacedwith fresh PBS solution containing BSA (1 wt %, 0.5 mL). The pro-tein solutions were stored at �20 8C in polypropylene tubes untilanalysis. The BSA was utilized to stabilize the VEGF during the re-lease study and storage period.

Acknowledgements

The authors thank the BBSRC for a PhD studentship for LouisaGilmore.

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Keywords: controlled release · drug delivery · growth factors ·nanoparticles · VEGF

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Received: June 20, 2009Published online on July 30, 2009

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