Conjugates of poly(DL-lactide-co-glycolide) on amino

Conjugates of poly(DL-lactide-co-glycolide)on amino cyclodextrins and their nanoparticles as protein delivery systemHui Gao,*Yi-Nong Wang,Yun-Ge Fan,Jian-Biao MaKey Laboratory of Functional Polymer Materials,Ministry of Education,Institute of Polymer Chemistry, Nankai University,Tianjin300071,People’s Republic of ChinaReceived18October2005;revised14March2006;accepted27March2006Published online7September2006in Wiley InterScience().DOI:10.1002/jbm.a.30861Abstract:Poly(DL-lactide-co-glycolide)(PLG)was chemi-cally conjugated on two amino cyclodextrins,mono(6-(2-aminoethyl)amino-6-deoxy)-b-cyclodextrin and ethylene-diamino bridged bis(b-cyclodextrin),to afford novel amphiphilic conjugates.Those conjugates were then char-acterized with infrared spectrometry(IR),proton nuclear magnetic resonance(1H NMR)and gel permeation chro-matography(GPC).A repeat-nanoprecipitation(RP-NP) method was also developed to fabricate the nanoparticles of the conjugates with a water-soluble model protein,bo-vine serum albumin(BSA).At the end of RP-NP process, the availability of BSA was over80%while the entrapment efficiency was40–50%for each nanoprecipitation.The nanoparticles were rigid and spherical with diameters of 110–180nm determined by transmission electron micro-scope(TEM),atomic force microscopy(AFM)and particle size analyzer.Nanoparticles possessed good steric stability during freeze-drying and resuspensions due to the exis-tence of cyclodextrins corona.Interactions between BSA and the conjugates in the nanoparticles were then eluci-dated with IR experiments.About25%BSA adsorbed on the surface of nanoparticles due to the interaction and was easy to release in thefirst day.The release of BSA from the nanoparticles was in three phases:a burst effect in the first day,a followed plateau in about a week,and a sus-tained release of the protein over14days.By changing the lactide/glycolide ratio,the degradation time of the conju-gates and the release rate of BSA could be controlled.The loss of CDs content was faster than that of overall Mw during degradation since CDs formed outer corona of the nanoparticles.Both the novel biomaterials and the nano-sphere fabrication technique contributed to the mainte-nance of protein structure.Ó2006Wiley Periodicals,Inc.J Biomed Mater Res80A:111–122,2007Key words:poly(DL-lactide-co-glycolide);amino b-cyclo-dextrin;biodegradable polymer;repeat-nanoprecipitation; protein deliveryINTRODUCTIONThere are many methods currently available for the preparation of polymeric nanoparticles,such as emulsification–evaporation method,1salting-out pro-cedure,2–4and nanoprecipitation method.However, several difficulties have been encountered for adopt-ing thefirst two methods.For example,the usage of chloric solvent may cause toxicity;the additives such as surfactants like poly(vinyl alcohol)and poloxamer may arouse toxicity and side-effects.1,5Salt perme-ates biological systems and is crucial for life.How-ever,salts also affect the stability of proteins.Neutral salts perturb various protein structures in ways that go well beyond simple,nonspecific charge effects, which has been reported for many years.6–8There-fore,the nanoprecipitation method becomes appeal-ing to us because it has been characterized to be ab-sence of oily–aqueous interfaces and avoid the usages of additives,toxic organic solvent,saturated electrolyte solution,and higher energy sources required for the formation of nanoparticles,all of which may damage the protein structure.The nano-precipitation technique wasfirst developed and patented by Fessi et al.9This method is based on the interfacial deposition of a polymer following dis-placement of a semipolar solvent miscible with water from a lipophilic solution,10which is mostly suitable for entrapment of hydrophobic drugs.11,12 Two modifications have been proposed to improve the versatility of the nanoprecipitation technique, particularly with respect to the entrapment of hydro-*Present address:Faculty of Pharmacy,University of Mon-treal,C.P.6128,Succ.Centre-ville,Montreal,Qc H3C3J7, CanadaCorrespondence to:J.-B.Ma;email:jbma@'2006Wiley Periodicals,Inc.philic ender et al.have improved the incorporation of a water-soluble drug into the poly (DL -lactide-co -glycolide)nanoparticles by increasing the aqueous phase pH and thereby decreasing the solubility of the drug.13However,this method is not suitable for protein delivery since it often causes a protein deactivation when decreasing the solubility of the protein.Bilati et al.recently proposed that di-methyl sulfoxide (DMSO)could be one of the most useful organic solvents during nanoprecipitation,es-pecially for protein drugs.14However,they did not consider that some protein was not well soluble in DMSO and removing of DMSO is time-consuming.In our laboratory,a new technique was developed as the repeat-nanoprecipitation (RP-NP)method for protein delivery,which may solve the above-men-tioned problems.After the conventional precipitation of organic polymer solution in an aqueous protein solution,the protein solution in the supernatant re-covered from the centrifugation was not discarded but adjusted the concentration by adding protein for the next performance of nanoprecipitation.The pro-tein availability was enhanced after repeating nano-precipitation (RP-NP)several times,even though the entrapment efficiency of each time was relatively low.This technique keeps the advantages of the nanopre-cipitation method,but avoids loss of the drug during the fabrication of drug delivery system.It also allows us to prepare large amount of the protein nanopar-ticles since sonication and homogenization are not required in the process.When biodegradable polymers are used in protein delivery,the stabilization of proteins in delivery devices and the enhancement of entrapment effi-ciency into appropriate protein carriers are major issues.With this consideration in mind,we recently synthesized two kinds of biodegradable polymers composed of poly(lactic acid)(PLA)and cyclodextrin derivatives.15,16However,the degradation of PLA is time-consumed.17The copolymers of lactide and gly-colide,poly(lactide-co -glycolide)(PLG),as a matrix for drug delivery applications can preferably meet the requirements including suitable mechanical pro-perties,biodegradability,and tissue compatibility.18Furthermore,the degradation rate of a PLG copoly-mer can be controlled by the molar ratio of the lactic and glycolic acids in the polymer chain.19–23To cre-ate new biodegradable nanoparticles and evaluate their potential as protein carriers,two kinds of new conjugates of PLG with amino cyclodextrins,mono (6-(2-aminoethyl)amino-6-deoxy)-b -cyclodextrin and ethylenediamino bridged bis(b -cyclodextrin),were synthesized according to the synthesis route as illus-trated in the Scheme 1.The feasibility of the RP-NP method using these new materials was also eval-uated for bovine serum albumin (BSA)delivery.MATERIALS AND METHODSMaterialsPoly(DL -lactide-co -glycolide)copolymers (free acid end)with the molecular weight of 18KDa and monomer com-position of 50/50or 75/25(LA/GA)were obtained from Durect Company.b -Cyclodextrin (b -CD)was obtained from Aldrich Chemical Company.N ,N 0-Dicyclohexycarbo-diimide (DCC)was purchased from Shanghai Chemical Reagent Company,China.Bovine serum albumin (BSA)in electrophoresis purity was purchased from Institute of He-matology and Hospital of Blood Diseases,Chinese Acad-emy of Medical Sciences,Tianjin,China.The deuterated agents were obtained from Cambridge isotope laboratories Inc.N ,N 0-Dimethylformamide (DMF)was dried over cal-cium hydride for 2days and then distilled under a reduced pressure before use.Other chemicals were analyti-cal grade.Conjugating reaction of PLG with amino cyclodextrinsThe conjugating reactions were performed as reported pre-viously,15,16except that the reactant used was PLG instead of PLA.Briefly,PLG was respectively reacted with mono (6-(2-aminoethyl)amino-6-deoxy)-b -cyclodextrin (CDen)24and ethylenediamino bridged bis(b -cyclodextrin)(BCDen)25at a feeding ratio of 2:1(amino CD to PLG)using N ,N 0-Dicy-Scheme 1.Synthesis route of the CDenPLG and BCDenPLG copolymer.112GAO ET AL.Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.aclohexycarbodiimide(DCC)as conjugating agent in DMF. The former was reacted at room temperature while the latter was at608C.Both of them reacted for3days.After keeping at room temperature for another3days,solid dicyclohexylurea (DCU)wasfiltered out.Thefiltrate was diluted with DMF to 1%concentration of the conjugated product and fractioned with deionized water carefully.No complete phase separation took place after aging for about12h.After centrifuged at8000 rpm for0.5h,however,phase separation occurred.The sec-ond fraction in38–53%of total product weight was regarded as the principal component of the conjugated product. Characterization of the PLG-conjugated amino cyclodextrins1H NMR spectra of the products as well as PLG were measured in deuterated DMSO on the Varian UNITY-plus 400spectrometer at400MHz.Their infrared spectra were recorded on the NICOLET5DX FTIR apparatus using dif-fuse reflection technology.GPC was performed on a Waters510/410GPC apparatus with a polystyrene gel col-umn and a differential refractive index detector using lin-ear polystyrenes as the standards and DMF as thefluent.Nanoparticles fabricationA repetition nanoprecipitation(RP-NP)technique was developed.Typically,5mL of the polymer solution(10mg/ mL)in acetone was added dropwise to10mL of water with BSA(2mg/mL)at a rate of0.5mL/min using a syringe pump(74900series multichannel syringe pumps,Cole-Parmer Instrument Company,USA)under magnetic stirring. Acetone was eliminated by evaporation under reduced pres-sure using a rotation evaporator at308C for0.5h.The NPs were recovered by centrifugation at21,000rpm for20min. The supernatant concentration of BSA was measured accord-ing to the blue coomassie G250protein assay26and adjusted to2mg/mL through adding calculated weight of BSA.Then another batch of polymer acetone solution was added into the BSA supernatant with the adjusted concentration.These procedures were repeated for four times.Characterization of the nanoparticlesFTIR studies of the nanoparticlesThe NPs were made by RP-NP technique with the com-position of CDenPLG75.The IR spectra of blank NPs,BSA loaded NPs,and the physical mixture of BSA(10%)and CDenPLG75NPs(90%)were obtained by FTIR(NICOLET 5DX FTIR).Determination of entrapment efficiency andparticle sizeAfter centrifugation of NPs,the entrapment efficiency (EE)was determined by measuring the BSA concentration in the supernatant according to the blue coomassie G250protein assay.26NPs size was determined through BI-90 Plus Particle Size Analyzer(Brookhaven Instruments Cor-poration)at258C in distilled water.Electron spectroscopy for chemical analysis(ESCA) The presence of BSA at or near the surface of the sys-tems was investigated by electron spectroscopy for chemi-cal analysis(ESCA)(PerkinElmer Phi-1600ESCA System) using the K a ray of aluminum.The analysis depth was less than100A˚and it was possible to quantify the BSA at or near the surface,taking into account that an elemental composition in N(N(%))of14.4%corresponds to100%of BSA27at or near the surface.Thus,the percentage of BSA at or near the surface in the developed formulations(sur-face%BSA)can be calculated according to:Surface%BSA¼Nð%ÞÂ100=14:4:Morphology observation of the polymer nanoparticlesThe shape and surface morphology of the produced BSA-loaded nanoparticles(NPs)were investigated by transmission electron microscope(TEM,Tecnai,G220S-TWIN,Philips Co.,Netherlands)and atomic force micros-copy(AFM).A drop of NPs suspension(0.1mg/mL)was placed on copper grids with a carbonfilm and allowed to dry at room temperature before being loaded in the TEM microscope.Samples for AFM imaging were prepared by placing a drop of BSA-loaded NPs suspension(typical con-centration about0.01mg/mL)on a clean glass slide and allowing it to dry in the air.AFM imaging was performed using a Nanoscope IIIa system in a tapping mode at room temperature.Feasibility of RP-NP methodAt the end of the5th nanoprecipitation,the following experiments were performed to identify the feasibility of RP-NP method.The molecular weight of BSA in the super-natant was tested by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The parti-cle size was determined as described by particle size ana-lyzer.At last,the availability of BSA at the end of RP-NP performance was calculated as follows:Availability%¼100%ðW tÀW sÞ=W twhere W t and W s are the total weight of BSA inputted and that of BSA detected in the supernatant after nanoprecipi-tation forfive times.Results were represented as the aver-age value of triplicate experiments.Colloidal stability and lyophilized stabilityColloidal stability of NPs suspensions was studied through measuring their turbidity as a function of the elec-trolyte concentrations.Na2SO4(2.0mL,0.05–1M)wereCONJUGATES OF PLG ON AMINO CYCLODEXTRINS113Journal of Biomedical Materials Research Part A DOI10.1002/jbm.aadded to the NPs suspensions(0.25mL,0.15%w/v)in test tubes placed on a rotary shaker(SHZ-88Constant Temper-ature Water-bath Shaker,China).The samples were allowed to stand for20min at378C and their absorbencies were measured at680nm(U-3010spectrophotometer, HITACHI,Japan).Using a plot of suspension turbidity versus electrolyte concentration,the critical coagulation concentration was determined.28Suspensions of NPs were lyophilized and rehydrated. The nanospheres before and after lyophilization and rehy-dration were compared by particle size analyzer. Degradation of the polymeric nanoparticlesand protein releasePolymer degradation was studied by monitoring the changes in molecular weight of the polymer NPs.About10 mg of blank NPs were incubated with2mL of PBS(pH7.4) at378C in a water bath rotary shaker at72rpm.Three repli-cate tubes were used for each time point.At predetermined time intervals,the samples were centrifuged at21,000rpm and the pellets from each sample were lyophilized and then dissolved in DMSO.The insoluble inorganic salts from PBS solutions werefiltered before GPC measurement.GPC con-ditions were the same as earlier.The release tests have been described in Ref15.After release for14days,the centrifuged pellet was also ana-lyzed for the shape of degraded NPs by TEM.The protein released from NPs was analyzed by SDS-PAGE and circu-lar diachronic spectra.15The change of cyclodextrin content in the nanoparticles during degradation was then evaluated.In DMSO-d6,the content of CDen/BCDen in copolymer using1H-NMR was determined by comparing integrals of signals of1H of cyclodextrin glucose units(d¼3.64ppm)and of methyl groups of LA unit(d¼5.20ppm).Three samples were used for detection and the results were recorded as the av-erage value.Remained CDen/BCDen%was calculated according:Remained CDsð%Þ¼100ÂCDs content atdetermined time/original CDscontent in NPs:RESULTS AND DISCUSSION Characterization of the copolymer conjugatesThe1H-NMR spectrum of the CDenPLG75is exhibited in Figure1.The spectrum is dominated by the signals of the PLG chains.Overlapping doublets are at d¼1.45ppm(\a")for the methyl groups and at d¼5.20ppm(\d")for CHÀÀgroups of the lactic acid(LA)repeating units,and the multiplets at d¼4.85ppm(\c")are assigned to the methylene groups of GA residues,which confirm the chemical struc-ture of PLG.The LA/GA ratios in the PLG can be calculated from the ratios of peak\d"and\c".29The small peak\b"at d3.64ppm is aroused from theprotons C2–C6of the CDen unit.The integration ra-tio of\c"and\b"indicates that the approximatemolar content of PLG to CDen was1:1.Infrared spectra are exhibited in Figure 2.Theabsorption peak at1675cmÀ1can be attributed to the stretching vibration of amido carbonyl C¼¼O from thecopolymer CDenPLG75,suggesting that the amino group of CDen reacted with the carboxylic group ofPLG in the conjugating reaction.Their contrast spec-trum clearly presents the multihydroxy characteristic peak(3435cmÀ1)and d vibrations(815cmÀ1)of ÀÀNHÀÀfrom CDen.All the samples have the main peaks from the functional groups of PLG moleculesuch asÀÀCH,ÀÀCH2,ÀÀCH3stretching(2850–3000 cmÀ1),ÀÀC¼¼O stretching vibrations(1700–1800cmÀ1), CÀÀO stretching(1050–1250cmÀ1),andÀÀOH stretch-ing vibrations(3200–3500cmÀ1).30Molecular weights of the copolymer conjugates are shown in Table I.The two copolymers,CDenPLG and BCDenPLG,possess the average molecular weights(M n)about1KDa and2KDa larger than the corresponding initial PLG.This indicated that only one PLG chain is linked on one molecule of CDen or BCDen due to precise molecular weights of CDen and BCDen is1177and2294Da,respectively.In addition,the molecular weight of thefirst fraction of the crude products is also tested based on GPC.There are two peaks in GPC(Figure not shown).One minor peak of M w is corresponding to the double of the coupling product and the other is the coupling prod-uct.Two PLG chains couple on CDen,which appears as the double molecular weight of main product. With two PLG chains linking on CDen,thefirst frac-tion is more hydrophobic and precipitate preferen-tially using water as the precipitator.FTIR study of the nanoparticlesFTIR is a useful tool to study the interactions of mo-lecular group.Hyvonen et al.reported that IR spectra of the PLA nanoparticles remained the same as matrix homopolymer PLA.31For CDenPLG75,as seen from Figure3(a),the same characteristic peaks aroused from the NPs present as those from the matrix.However, the relative sharp multihydroxyl peaks become broaden after the nanoparticle fabrication,which may be due to the formation of hydrogen bond.To our knowledge,the hydrogen bond formation after the NPs fabrication with the nanoprecipitation technique has not been put forward yet and this hydrogen bond may contribute to the NPs stabilization.Comparing IR spectrum of the BSA-loaded NPs to the blank NPs[Fig.3(b)],two additional peaks present in the BSA-loaded NPs spectrum at1653cmÀ1114GAO ET AL. Journal of Biomedical Materials Research Part A DOI10.1002/jbm.a(amides I)and 1518cm À1(amides II),which can be attribute to BSA.The lower wavenumber of amides I band indicates the existence of hydrogen bond because the free amides I band appears due to at higher wavenumber (1685–1690cm À1).Meanwhile,thereis no significant difference in the spectrum of the BSA-loaded NPs and their physical mixture.This sug-gests that some BSA may be absorbed on NPs during the RP-NP performance and the physicochemical in-tegrity of BSA retains within the NPs.Morphology of nanoparticlesTEM photographs in Figure 4show a spherical shape and a rough surface of CDenPLG75NPs with-out any aggregation or adhesion.However,TEM can only give morphology of NPs.Herein,AFM is used to observe the three-dimensional structures,which is capable of resolving surface details down to the atomic level and acquiring morphological images in high resolution.30AFM images reveal that the NPs of CDenPLG50are in fine spherical shape with an average diameter of 130nm and an average height of 119nm,indicating that the NPs surface is rigid and has sufficient mechanical resistance to collapse.Moreover,the particle size observed by microscope is very consistent with the result measured by parti-cle sizeanalyzer.Figure 2.IR spectra of CDenPLG75(a)and PLG75(b)and their contrast spectrum(c).Figure 1.1H-NMR spectrum of CDenPLG75.CONJUGATES OF PLG ON AMINO CYCLODEXTRINS 115Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.aEffect of copolymer composition on particle size and EEAs shown in Table II,the EE of the conjugate NPs has been improved compared with the PLG NPs,which can be explained by the interactions of the CD moieties with BSA.32Furthermore,the binding ability of BCDen with BSA is stronger than that of CDen due to the coopera-tive action of two CDs cavity,32which also contributes to the enhancement of the EE.The type of CD composi-tion has no effect on the mean diameter because the hydrophilicity of CDen is similar to that of BCDen.Onthe other hand,the particle size of NPs decreases after introduction of the CD units due to enhancing the hydrophilicity of the products.As for the composition of PLG,when the ratio of LA/GA decreases,the hydro-philicity of the polymer increases,which is the benefit for the NPs formation.Thereafter,the smaller NPs are obtained with the decreased ratio of LA/GA and the size distribution becomes narrower as well.Little effect of LA/GA ratio on EE is observed,whereas almost no difference of EE is either found between the nanopar-ticles of CDs-PLG with corresponding CDs-PLA nano-particles.This evidences that introduction of GA unit has little effect on the EE.Effect of fabrication method on particle size and EEThe effect of fabrication method on particle size and EE is displayed in Table III.Stirring provides the energy to disperse acetone in aqueous phase.As expected,a higher stirring speed yields smaller NPs because nanoprecipitation is broken up into smaller droplets at a higher input power.However,EE becomes lower with higher stirring rate because the exchange rate of acetone with water is too fast at a high stirring rate and less BSA molecules can be incorporated into the formed NPs,and as a result more BSA remain in aqueous phase.Therefore,the stirring speed needs to be optimized in order to obtain a sufficiently high EE with a desired particle size.All following experiments are performed at 500rpm.The increase in concentration of copolymer causes higher viscosity of organic phase,accordingly indu-ces a higher EE and a larger particle size.Neverthe-less,too much polymer in acetone prevents the pre-cipitation,which is probably due to the high viscos-ity of the polymeric solution that hampers an appropriate diffusion of the solvent toward the non-solvent.14Therefore,10mg/mL concentration is cho-sen to make biodegradable NPs.Increased concentration of BSA in aqueous phase causes a decrease of particle size and a slight decrease of EE.BSA may serve as a surfactant,high concentration of which results in the decrease in par-ticle size,while the decrease in particle size causesaFigure 3.IR spectra of CDenPLG75(A)matrix and blank NPs;(B)blank NPs,BSA-loaded NPs,mixture of blank NPs (90%)and BSA (10%).[Color figure can be viewed in the online issue,which is available at .]TABLE IConjugating Reaction of PLG with Amino CDs and the Molecular Weights of the ConjugatesFeeding PLA Feeding CD Product Samples M n (KDa)M w (KDa)M w /M n Yield a (%)PLG50CDen CDenPLG501924 1.2653.6PLG50BCDen BCDenPLG502125 1.2038.4PLG75CDen CDenPLG752028 1.4351.2PLG75BCDenBCDenPLG7521301.4443.3aThe yield was calculated from the ratio of principal component to the crude product.116GAO ET AL.Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.aCONJUGATES OF PLG ON AMINO CYCLODEXTRINS117Figure4.TEM images(a and b with deferent magnified ratio)of BSA-loaded CDenPLG75NPs,AFM images(c and d represents two or three dimensionsfigure,respectively)of BSA-loaded CDenPLG50NPs,e:TEM image of BSA-loaded CDenPLG75NPs after released for14days.[Colorfigure can be viewed in the online issue,which is available at www. .]Journal of Biomedical Materials Research Part A DOI10.1002/jbm.adecrease amount of BSA encapsulated in NPs and thus a decreased EE.On the basis of the abovementioned results,fine-tuning of various formulation parameters,including the copolymer composition,the stirring speed,con-centration of polymer solution and BSA solution,will be necessary to optimize the polymer EE and particle size.Electron spectroscopy for chemical analysis ESCA clearly reveals the presence of N,hence of BSA,in the surface of the nanoparticles.BSA remains on the surface of copolymer with a single wash of NPs due to the binding interactions between CDs and BSA.CDenPLG50and CDenPLG75were used to test for ESCA.Since their elemental composition in N is less than BCDenPLG copolymers,the effect on analysis of N on the surface is less correspondingly.The blank nanoparticles made by CDenPLG50and CDenPLG 75were text as a control and N%is 0.0960.04and 0.160.02,repectively (calculation value is 0.07),while for BSA-loaded nanoparticles,the calculated \Surface %BSA "is 2363%and 2864%,correspondingly.The fabrication method determines the amount of protein existing near the surface of NPs.When the excipients,i.e.polymers,are not soluble in water,drug moleculesdissolved in water may be very close to the outer NPs surface,forming a layer of molecules,susceptible to be easily and rapidly released.33Feasibility of the RP-NP methodAt the end of RP-NP,the supernatant is analyzed by the SDS-PAGE and a single binding correspond-ing to the Mw of BSA is presented (Figure not shown),indicating the maintenance of structure in-tegrity of BSA.On the other hand,the particle size of nanoprecipitation for the last time remains the same as the first time.The availability of BSA in the NPs is listed in Table II.Even though the EE is rela-tively low using RP-NP method,the availability of all formulations can exceed 80%after five-time pre-cipitation.Therefore,the RP-NP is a feasible tech-nique to increase the protein availability without any change of both protein integrity and particle size.Colloidal stability of NPsThe colloidal stability of NPs was examined at various Na 2SO 4concentrations.The difference in floc-culation provides a simple method to detect modifi-cation in the surface characteristics of the NPs.Above 0.05M Na 2SO 4,suspensions of unmodifiedTABLE IIIInfluence of Processing Factors on EE and Particle Size Using RP-NP Technique (CDenPLG50was Used)Polymer Concentration (mg/mL)BSAConcentration (mg/mL)Stirring Rate (rpm)Entrapment Efficiency (%)Mean Diameter (nm)PolydispersityIndex10230048.8162.40.14210250046.4137.80.11210270040.9125.50.0395250040.3112.20.05815250047.5175.10.12310150047.9175.30.13310350042.3112.50.042TABLE IIEffect of Copolymer Composition on EE and Particle SizeCopolymer CompositionEntrapment Efficient (%)Mean HydrodynamicDiameterPolydispersityIndexAvailabilityPLG7531.5235.20.19369.7PLG5028.1203.80.16762.6CDenPLA17a 48.3140.90.10482.9CDenPLG5046.4137.80.11281.2CDenPLG7547.8178.90.14882.1BCDenPLA17b 49.3166.50.04483.0BCDenPLG5049.6139.10.06383.1BCDenPLG7550.3174.30.08583.3Typical condition:5mL of the polymer solution (10mg/mL)in acetone was added dropwise to 10mL of water with BSA (2mg/mL)at a rate of 0.5mL/min,and the stirring rate was 500rpm.aFrom Ref.15.bFrom Ref.16.118GAO ET AL.Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.aPLG NPs are no longer stable,due to the screening of the surface charge by salt ions.Therefore,floccula-tion as the result of Van der Waals attraction occurs when salt concentration increases.34As for the CDenPLG50and BCDenPLG50NPs,the critical coagulation concentration is observed at Na 2SO 4con-centrations up to 0.6M and 0.8M ,respectively.The observed colloid stability of the conjugate NPs under high ionic strength strongly suggests the presence of a nonelectrostatic contribution to nanosphere stabili-zation,such as steric component to the stabiliza-tion.28The BCDenPLG NPs show even more stability in the presence of electrolyte.The two nearly situ-ated CDs cavity can offer more steric stability.Stability of freeze-dried samplesThe industrial development of polymeric nanopar-ticle suspensions,as drug delivery systems,is lim-ited due to the problems in maintaining stability of suspensions.According to the literature,freeze-dry-ing was a good method to dry nanospheres in order to increase the stability of these colloidal systems.35SiO 235or glucose/threalose was added to permit adequate reconstitution of the freeze-dried product.36In our systems,the resulting fluffy powder is easily resuspendable in water after lyophilization of super-natant without any additives.Moreover,there is no change in the size of NPs.The particle suspension exhibits good steric stability.Similar stability is observed with methoxy poly(ethylene glycol)-poly (lactide)(MPEG-PLA)nanoparticles due to the pres-ence of PEG on the particles surface to prevent the coagulation cascade.37CDenPLG/BCDenPLG nanoparticles are of a core-corona structure 15:the hydrophobic PLG comprises the core while thehydrophilic CDen/BCDen extends to the outer aqueous environment to form a corona.This struc-ture is believed to possess self-stabilization function.NPs degradation and protein releaseFigure 5illustrates the in vitro release profiles.For all NPs,BSA release occurs as following:(1)an initial burst release (the 1st day)of a certain amount of pro-tein which releases from the NPs surface,primarily due to the adsorption of the protein to the NPs surface,(2)a plateau followed for about a week,from an equi-librium between the amount of BSA released in the su-pernatant and the amount of BSA adsorbed on the NPs,(3)a sustained release of the drug over 14days resulting from the erosion of the polymers.To our knowledge,the burst release is often con-sidered to avoid when the NPs are used as drug delivery systems.However,the burst effect can mimic the effect of booster doses required for cur-rent vaccination programs.The release characteristics are also considered advantageous because the dose of antigen necessary to stimulate the secondary response is much lower than that required for evok-ing a primary response.3830–37%of BSA released for the first day.As discussed above,about 25%of BSA appeared on the surface of the nanoparticles,which led to the bust release of BSA.It has been generally accepted that the third phase of the sustained release profiles correspond to the erosion of the polymers 39To investigate the release mechanism of protein from the NPs,we evaluated the polymer Mw by GPC.The Mw of NPs immedi-ately after the fabrication remains the same as that of before device preparation.Results in Figure6Figure 5.In vitro release profiles of BSA from the conju-gate NPs.[Color figure can be viewed in the online issue,which is available at.]Figure 6.Weight molecular weight changes (solid line)and remained CDen/BCDen%(dash line)of the degrading NPs as a function of incubation time.[Color figure can be viewed in the online issue,which is available at .]CONJUGATES OF PLG ON AMINO CYCLODEXTRINS 119Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a。