纳米微粒的载药的研究

*Corresponding author.Tel.:+86-25-331-7807;fax:+86-25-331-7761.

E-mail address:czyang@https://www.360docs.net/doc/1214673033.html, (C.Yang).

0142-9612/02/$-see front matter r 2002Elsevier Science Ltd.All rights reserved.PII:S 0142-9612(02)00071-6

为了克服蛋白和多肽水溶性的问题丆做了很多的修饰改进工作丆其中PEG 修饰效果好丆但是也有一些不足之处。

微球的制备方法及不足之处

prepared by the ionic interaction between positively charged CS and negatively charged polymer-tripolypho-sphate(TPP),and the nanoparticles showed a great protein loading capacity and sustained release ability. In the present work,we report a new approach to prepare hydrophilic nanoparticles based on polymeriz-ing acrylic acid into chitosan template in aqueous solution[26].We think that this system has some interesting(1)The nanoparticles are obtained spontaneously under very mild conditions without the need of high temperature,organic solvent,surfactant and some other special experimental technology;(2)the nanoparticles have small particle size and positive surface charges,which may improve their stability in the presence of biological cations[27],and is favorable for some drugs due to the interaction with negatively charged biological membranes and site-speci?c targeting in vivo[28,29];(3)The nanoparticles have pH-depen-dent dissolution

2.Experimental

2.1.Materials

Chitosan(Nantong Shuanglin Biological Product Inc.)was re?ned twice by dissolving it in dilute acetic acid solution,?ltered,precipitated with aqueous NaOH, and?nally dried in vacuum at room temperature.The degree of deacetylation was about90%,and the weight average molecular weights of chitosan were40,80,100, 200and300kDa,respectively,determined by visco-metric methods[30].Potassium persulfate(K2S2O8)was recrystallized from distilled water.Acrylic acid(AA) (Shanghai Guanghua Chemical Company)was distilled under reduced pressure in nitrogen atmosphere.Silk peptide powder(SP)was kindly supplied by Nanjing Golden Balei Limited Company(People’s Republic of China)as a model drug.All other reagents were of analytical grade and used without further puri?cation.

2.2.Preparation of CS–PAA nanoparticles by polymerization

The CS–PAA nanoparticles were obtained by poly-merization of AA in CS solution.Chitosan was dissolved in50ml acrylic acid solution in the ratio of 1:1([aminoglucoside units]:[AA],except when otherwise stated)under magnetic stirring.The amount of AA was maintained constantly at3mmol in all experiments. Until the solution became clear,0.1mmol of K2S2O8was added to the solution with continued stirring.The pH value of the system was maintained at about4.0. Then,the polymerization was carried out at701C under a nitrogen stream and magnetic stirring.When the opalescent suspension appeared,the reaction system was cooled,and the opalescent suspension was?ltered with paper?lter to remove any polymer aggregation.Finally, the residual monomers were removed by dialysis in a buffer solution of pH=4.5for24h using a dialysis membrane bag with a molecular weight cut-off of 10kDa.

2.3.Preparation of CS–PAA nanoparticles by dropping method

CS–PAA nanoparticles were also prepared by mixing positively charged CS and negatively charged PAA with dropping method.(a)Adding CS solution into PAA solution.Brie?y,1ml0.02%CS solution(CS with a molecular weight being80kDa was dissolved in1%(w/ v)acetic acid solution)was added dropwise into5ml 0.02%PAA(M n=100kDa)aqueous solution under magnetic stirring,the opalescent suspension was formed.The obtained suspension was then?ltered by paper?lter,the?ltered suspension was incubated in a buffer solution of pH=4.5for24h using a dialysis membrane bag for characterization.(b)Adding PAA solution into CS solution.1ml0.02%PAA (M n=100kDa)solution was added dropwise into5ml 0.02%CS solution(CS with molecular weight80kDa was dissolved in1%(w/v)acetic acid solution)under magnetic stirring.The following procedure was as described in method a.

2.4.Preparation of drug loaded CS nanoparticles

The drug-loaded nanoparticles were prepared by dissolving50mg of SP in50ml CS–PAA nanoparticlate prepared by polymerization of AA in CS solution with the CS molecular weight80kDa and incubated for48h. Then,these nanoparticles were separated from the aqueous phase by ultracentrifugation(Ultra Pro TM80, Du Pont)with50,000rpm at41C for40min.Next,the gained SP loaded CS–PAA nanoparticles were washed by acetone three times,frozen by liquid nitrogen and lyophilized by free dryer system to obtain dried SP loaded CS–PAA nanoparticles.

2.5.FT-IR spectrum analysis

FT-IR spectra were measured by a Bruke IFS 66V vacuum-type spectrometer to determine the chemical interaction between CS and PAA.The CS–PAA nanoparticles were frozen by liquid nitrogen and lyophilized by free dryer system to obtain dried CS–PAA nanoparticles.These gained CS–PAA 23(2002)3193–3201

很多人做了工作来改进亲水性丆但是我们发现一种更为新颖的。

他们的优点在于丗123

CDA,TPP等丆我们用的是氨基酸

nanoparticles were mixed with KBr and pressed to a plate for measurement.

2.6.The yields of the CS–PAA nanoparticles and size exclusion chromatography(SEC)characterization

The nanoparticles were separated from the aqueous phase by ultracentrifugation(Ultra Pro TM80,Du Pont) with50,000rpm at41C for40min.The weight of the sediment nanoparticles was de?ned as the weight of the resultant nanoparticles.The nanoparticle yields were calculated by the following equation:

Nanoparticle yielde%T

?

Weight of nanoparticles

Weight of chitosan and monomer fed initially

?100:

In order to investigate the molecular weight of PAA in nanoparticles,SEC measurement was carried out on Shimadzu LC-10AD HPLC using Ultrahydrogel120 and500columns and a RID-10A detector.The deionized water was used as eluent and the?ow rate was0.5ml minà1.The SEC was calibrated with poly (ethylene oxide)standards.F or SEC measurement,CS–PAA nanoparticles were dissolved in diluted HCl aqueous solution,and then added NaOH into this system.When the pH value of the above solution was adjusted to9,the solution was separated by ultracen-trifugation at50,000rpm for40min.The supernatant was used for SEC measurement.

2.7.Transmission electron microscopy

Transmission electron microscopy(TEM)(JEOL TEM-100,Japan)was used to observe the morphology of the CS–PAA nanoparticles.Samples were placed onto copper grill covered with nitrocellulose.They were dried at room temperature,and then were examined using a TEM without being negative stained.

2.8.Particle size and zeta potential of CS–PAA nanoparticles

The mean size and size distribution of the CS–PAA nanoparticles were measured by dynamic light scattering (DLS)(Zetasize;3000HS,Malvern,UK)in buffer solution with different pH values.All DLS measurements were done with a wavelength of 633.0nm at251C with an angle detection of901.Each sample was repeatedly measured3times and the values reported are the mean diameter7SD for two replicate samples.

The zeta potential of the CS–PAA nanoparticles were measured on Zetasize3000HS.(Malvern,UK).The samples were diluted with10m m NaCl solution at a pH value of4.5(except when otherwise stated)in order to maintain a constant ionic strength.Each sample was repeatedly measured3times and the values reported are the mean value7SD for two replicate samples.

2.9.SP encapsulation ef?ciency of the nanoparticles

The SP-loaded CS–PAA nanoparticles were separated from the aqueous suspension medium by ultracentrifu-gation with50,000rpm at41C for40min.The amount of free SP in the clear supertant was measured by ?uorescence measurements on LS-50B,(Perkin Elmer) with excitation of274nm and emission of302.4nm.

SP encapsulation ef?ciency(AE)were calculated with the following equation:

AE?

Total amount SPàFree amount SP

Total amount SP

?100:

2.10.In vitro drug release from the nanoparticles Hundred mg SP-loaded CS–PAA nanoparticles were re-dispersed in10ml distilled water and placed in a dialysis membrane bag with a molecular cut-off of10kDa,tied and placed into300ml of water medium with various pH values on sink conditions. The entire system was kept at371C with continuous magnetic stirring.After a predetermined period,5ml of the medium was removed and the amount of SP was analyzed by?uorescence measurement. The released SP was determined by a calibration curve. In order to maintain the original volume,each time, 5ml of the medium was replaced with fresh water. The SP release experiments were repeated three times.

3.Results and discussion

3.1.Synthesis of CS–PAA nanoparticles

The CS–PAA nanoparticles were prepared by two methods in our study.One was polymerization of AA in CS solution.Another is mixture of positively charged CS and negatively charged PAA with dropping method. The polymerization of acrylic acid in the presence of chitosan is showed in Scheme1.F irst,CS was dissolved in AA solution,and then the polymerization of AA was initiated by K2S2O8.When the polymerization of AA reached a certain level,the inter-and intra-molecular linkages occurred between carboxyl groups from PAA and positively charged amino groups of CS.These linkages could make the macromolecular chains of CS rolling up,which was responsible for the formation of the gelation of the CS solution.In this system,at the early stage of the polymerization,there was no or little amount of PAA in the solution,thus the system showed the property as a clear solution.As the polymerization

Y.Hu et al./Biomaterials23(2002)3193–32013195

time extended,the amount of PAA in the solution increased,and the system changed initially from a clear solution to an opalescent emulsion,indicating the formation of CS–PAA nanoparticles.

Ahn et al.[26]had reported that acrylic acid could have undergone template polymerization in CS solution.In our case,we thought that the CS–PAA nanoparticles were also prepared by template polymerization of acrylic acid in chitosan solution using chitosan as the template.As reported by F erguson,[31]in template polymerization,the presence of template during the polymerization procedure has kinetic and structural effects,which in?uences the molecular weight of the growing polymer chain.That is to say that the propagation will continue for longer on a higher molecular weight template than on a low molecular weight before termination occurs.In this experiment,we thought that the molecular weight of CS template also in?uenced the molecular weight of formed PAA.To study it,a series of CS–PAA nanoparticles was synthesized by polymerization of acrylic acid in the solution of chitosan with different molecular weights of 40,80,100,200and 300kDa.All the CS samples have a similar degree of deacetylation of about 90%.The aminoglucoside units of chitosan were equal to the units of AA fed initially.Table 1shows the results of these experiments.F rom Table 1,it is interestingly found that the molecular weight (M n )of PAA in the CS–PAA nanoparticles increased with the increase of the mole-cular weight of CS,while the yield of CS–PAA nanoparticles was maintained in the range 60–70%.Like in template polymerization,when the PAA reaches a critical molecular weight,the propagation of PAA chains is restricted by the CS template.Thus,it is

NH 2

NH 2NH 2NH 2CH 2CHCOOH

3NH 3NH 3NH 3OOCCHCH 2OOCCHCH2OOCCHCH2OOCCHCH2

NH 3NH 3NH 3NH 3OOC OOC OOC OOC

Polymerization

NH 3NH 3OOC

OOC

OOC

OOC NH 3NH 3CS

CS-PAA nanoparticles

Scheme 1.Preparation mechanism of CS–PAA nanoparticles.

Table 1

The relationship of the molecular weight of CS and PAA Sample no.

Molecular weight of CS,M w Polymerization time (h)Molecular

weight of PAA,M n Yield of PAA (%)Yield of

nanoparticles (%)140,00023678570.0280,000210878368.63100,000221387262.34200,000245267863.15

300,000

2

8026

71

60.0

Y.Hu et al./Biomaterials 23(2002)3193–3201

3196

reasonable to conclude that the polymerization of AA in CS solution is a template polymerization.

3.2.FT-IR analysis

To investigate the complex formation between PAA and chitosan,F T-IR studies were conducted. ig.1shows the T-IR spectra of PAA,CS and CS–PAA nanoparticles prepared by the polymerization of AA in CS solution.For CS–PAA nanoparticles, the intensities of amide band I at1662cmà1and amide band II at1586cmà1,which can be observed clearly in pure chitosan,decrease dramatically,and two new absorption bands at1731and1628cmà1, which can be assigned to the absorption peaks of the carboxyl groups of PAA(the absorption peak of carboxyl groups in pure PAA appears at1740cmà1), and the NH3+absorption of CS,respectively,are observed.The broad peaks appeared at2500and 1900cmà1also con?rmed the presence ofàNH3+in CS–PAA nanoparticles.F urthermore,the absorption peaks at1532and1414cmà1could be assigned to asymmetric and symmetric stretching vibrations of COOàanion groups.These results indicate that the carboxylic groups of PAA are dissociated into COOàgroups which complex with protonated amino groups of CS through electrostatic interaction to form the polyelectrolyte complex during the polymerization procedure.3.3.In?uence of the ratio of CS/AA on the mean diameter of nanoparticles

The particle size distributions of CS–PAA nanopar-ticles,prepared by polymerization of AA in CS solution with various CS/AA ratios([aminoglucoside uni-ts]:[AA]),were characterized by DLS at pH4.5.The results are displayed in Fig.2and Table2.It is shown that the diameter of each sample is smaller than300nm. Moreover,the diameter distribution of the nanoparticles is smallest when CS:AA=1:1.This result suggested that the ratio of CS to AA have an in?uence on the mean particle size.It also can be seen that CS–PAA nanoparticles could be obtained at a different ratio of CS to AA,and the preparation condition of CS–PAA nanoparticles was not very critical on the ratio of CS to AA compared to CS-DNA and CS-TPP systems[24,25].

F rom the results of zeta potential listed in Table2,it is found that the surfaces of CS–PAA nanoparticles have positive charges of about20–30mV.The positive-charged surface of CS–PAA nanoparticles is common to other CS nanoparticles reported by other authors because of the cationic characteristic of CS.However, it is interesting to?nd that as the ratio of CS/AA increases,the zeta potential also increases.It is reason-able that CS is a cationic polysaccharide,when the content of CS(aminoglucoside units)excesses than AA, some of the excessive CS will be absorbed onto the surface of CS–PAA nanoparticles,which will increase 1001000

5

10

15

20

25

V

o

l

u

m

e

%

Particle Diameter (nm)

Fig.2.Size distribution of CS–PAA nanoparticles with various CS/PAA ratios(wt/wt)at pH=4.5.

Wavelength(cm-1)

F ig.1.F T-IR spectra of CS,PAA,and CS–PAA.

Table2

Mean particle size and zeta potential of CS–PAA complex nanoparticles

Sample AA:CS(wt:wt)Mean diameter a(nm)Polydispersity Zeta potential(mV) I1:22507200.32570.046+27.373.5

II1:12067220.16570.009+25.373.2

III2:12937250.35270.032+23.172.8

a Mean diameter was characterized at pH=4.5.

Y.Hu et al./Biomaterials23(2002)3193–32013197

the surface charges of CS–PAA nanoparticles and resulting in the increase of zeta potential.

3.4.In?uence of pH value on the mean diameter and morphology of CS–PAA nanoparticles

In order to investigate the effect of pH values on CS–PAA nanoparticles prepared by polymerizing AA in CS solution,a series of experiments were carried out.The obtained CS–PAA nanoparticles,with CS molecular weight80kDa,were incubated in buffer solution with different pH values(pH=1,2,3,4.5,5.8,7.4,9).Results of these experiments showed that these nanoparticles were stable in distilled water and acidic media in a range of pH values from4.0to7.4but dissolved in a few minutes in0.1n HCl and aggregated quickly at pH values larger than9.Table3shows the result of the mean diameter of the CS–PAA nanoparticles under different pH values.F rom Table3,it can be seen that, the diameter of the nanoparticles increases with the increase of pH value from4.0to7.4.

Fig.3shows the TEM photographs of CS–PAA nanoparticles prepared by template polymerization. All these nanoparticles were incubated in buffers for 48h.The nanoparticles,which were in acetic buffer solution at a pH value of4.5(F ig.3(a)),exhibit solid and consistent spherical shapes,indicating that the CS–PAA nanoparticles have a matrix structure.However these nanoparticles in PBS at a pH of7.4shown in Fig.3(b)exhibit a compact core surrounded by a diffuse and fuzzy coat.These facts can be explained by the following.These CS–PAA nanoparticles were formed by ionic interaction between positively charged chitosan and negatively charged PAA.CS is a kind of weak alkali and PAA is a kind of weak acid.The p K a values of PAA

and CS are 4.75and 6.5[26],respectively.Under stronger acidic conditions,such as pH o4.0,most carboxylic groups of PAA are in the form ofàCOOH. The interaction between NH3+and COOàin the CS–PAA nanoparticles could be disrupted by the acid of small molecules,which leads to chain stretch of CS and PAA.So the CS–PAA nanoparticles would be dissolved quickly.When the pH value is in the range from4.5to 5.8,CS and PAA are partly ionized.The partly ionized CS and PAA can form compact polyelectrolytes complex by ionic interaction,which results in a matrix structure with solid and consistent spherical shapes. When pH values increased from4.5to7.4,as listed in Table3,the ionized degree of PAA increased,and the charge density of the PAA molecules signi?cantly increased.Thus,the electrostatic repulsive forces of inter-and intra-PAA molecules increased,resulting in the increase of swelling degree of PAA and the increase of the mean size of these CS–PAA nanoparticles.When these CS–PAA nanoparticles were incubated in PBS, (pH=7.4),the morphology of nanoparticles was chan-ged because of the difference of the solubility of CS and PAA.At this pH value,PAA was highly swollen while CS was insoluble,which results in the phase separation of nanoparticles,that is,CS was just physically coated on this nanoparticles.Thus these CS–PAA nanoparti-cles formed the core-shell-like structure as shown in F ig.3(b).On contrary,under extremely basic condition, the COOH groups from PAA were neutralized by OHà, and almost all amine groups from CS were in the form of NH2.Thus,the CS–PAA nanoparticles would be destroyed,resulting in an aggregation of CS due to its

Table3

The mean diameter of CS–PAA nanoparticles under various pH values pH Value Mean diameter(nm) 4.0175732

4.5206722

5.8400746

7.46257

106Fig.3.Electron transmission microphotography of CS–PAA nano-particles at(a)pH=4.5and(b)at pH=7.4.

Y.Hu et al./Biomaterials23(2002)3193–3201 3198

insolubility in basic solution.

These processes can be shown as follows:When pH o 4.0,

NH t3àCOO

à

H t

-

NH t3tCOOH CS-PAA nanoparticles Clear solution

:e1T

When pH>9.0,

NH t3àCOO

à

OH à

-

NH 2tCOO àtH 2O

CS-PAA nanoparticles

Aggregation

e2T

F rom Eqs.(1)and (2),it is evident that,to obtain stable nanoparticles,the system should be in suitable pH value.

rom these results,it could be seen that these nanoparticles are pH-sensitive,which would be good as carriers to load ocular drug because there are different pH values in the alimentary canal.

3.5.In?uence of different preparation procedures on the morphology of CS–PAA nanoparticles

The CS–PAA nanoparticles were also synthesized by dropping method.The in?uence of different preparation procedures on the CS–PAA nanoparticles was investi-gated.Fig.4(a)shows the TEM photograph of CS–PAA nanoparticles prepared by dropping PAA solution into CS aqueous solution.Fig.4(b)shows the nanoparticles obtained by dropping CS aqueous solution into PAA solution.Because many factors in?uence TEM photo-graph,such as the sample structure,staining condition,in this experiment,all of those nanoparticles are not negative stained with phosphotungstic acid solution.The difference between the TEM photographs of samples might be mainly conduced by the sample structure.

CS–PAA nanoparticles shown in Fig.4(a)have a circular shape consisting of a dark shell and a light core.Fig.4(b)exhibits CS–PAA nanoparticles with a dark,solid and consistent structure.These results indicated that different preparation procedures have signi?cant in?uence on CS–PAA nanoparticles morphology.In the case of dropping PAA into CS solution,a PAA core was initially generated and a complex coacervate membrane is formed on the surface of PAA core.Thus,the nanoparticles with core-shell structure were formed.The formed CS–PAA complex membrane is so dense that it prevents the CS molecular solution from diffusing into the core to further complex with PAA.When these CS–PAA nanoparticles were dried to the TEM char-acterization,the water,which swelled the PAA cores,was removed off and formed some cavities in the cores.In this region,electron beams could easily pass through the CS–PAA nanoparticles,which resulted in a light region in the TEM photograph.For the CS–PAA complex membrane,which is very dense,it prevents

majority of the electron beams passing through it.As a result,the region of CS–PAA complex membrane is dark.Similar structures were also observed by TEM from polystyrene-block-poly(acrylic acid)dissolved in water [32].When dropping CS into PAA solution,CS–PAA nanoparticles with a CS core and a CS–PAA membrane are formed.Because CS does not swell in acidic condition,there are no cavities formed in CS–PAA nanoparticles when they are dried for TEM characterization,thus a dark,solid structure was observed.In addition,similar results were also observed in the chitosan-alginate beads [33].

Table 4lists the results of mean sizes and zeta potentials of CS–PAA nanoparticles obtained by different preparation procedures described above.As shown in Table 4,the preparation procedures have an effect on the CS–PAA nanoparticles size.CS–PAA nanoparticles obtained by template polymerization has the smallest mean diameter.In the case of dropping PAA into CS solution,PAA solution cannot be dispersed homogeneously,and polyelectrolyte complex can be formed instantly,which makes the nanoparticles to have a large size and a broad size distribution.The same happens when CS drops into PAA solution.In the

Fig.4.Morphology of CS–PAA nanoparticles prepared by different procedure at pH 4.5:(a)CS dropping into PAA solution;(b)PAA dropping into CS solution.

Y.Hu et al./Biomaterials 23(2002)3193–32013199

case of template polymerization,since the CS and PAA are homogeneously dispersed in the solution,the smallest and uniform size nanoparticles can be obtained.Interestingly,when CS was dropped into PAA solution,the CS–PAA nanoparticles have negative zeta potential,which is quite different from samples 2and 3.This might be due to the fact that there is excessive PAA in the solution,and that the PAA is negatively charged.Some of the negatively charged PAA molecules were adsorbed onto the surface of CS–PAA nanoparticles,which resulted in the negative zeta potential.

These results indicate that the surface structure and the surface charge of these nanoparticles can be adjusted by different preparation processes.3.6.SP release

In order to investigate the feasibility of using CS–PAA nanoparticles as hydrophilic drug carriers SP as a model peptide was loaded by CS–PAA nanoparticles prepared by template polymerization.Fig.5shows the release pro?les of SP from CS–PAA nanoparticles with an encapsulation ef?ciency of 82%(7.96%,Wt/Wt)for various time intervals in various pH values release media at 371C.An initial burst release followed by a slow release of SP occurred in pH values of 4.5and 7.4.Moreover,these nanoparticles provided a continuous release of the entrapped peptide for up to 10days.On the other hand,at pH values of 2.0and 3.0,the SP release rate was very fast and about 90%of the loaded SP was released from CS–PAA nanoparticles within 25h.It is obvious from the results that the release of the SP depends on pH values of the release medium.The release pro?le at a pH of 4.5has the slowest release rate,and at a pH of 2.0,the CS–PAA nanoparticles almost do not have any sustained release property.This can be explained by the fact that the release of the SP depends greatly on the swelling of the nanoparticles.At a pH of 4.5,there is very limited swelling,and the SP entrapped in the nanoparticles cannot be released easily.However,at a pH of 7.4,the nanoparticles are swollen to a great extent,resulting in a fairly fast release of SP compared

with the nanoparticles at pH of 4.5.This result is also in good agreement with the effect of the pH values on nanoparticles morphology as mentioned above.At strong acidic condition,for example,pH value o 4.0,the nanoparticles will dissolve quickly,which leads to the very fast release effect.These results suggest the possibility to adjust the drug release rate of the CS–PAA nanoparticles by changing the pH values.

4.Conclusion

The CS–PAA nanoparticles can be prepared by polymerizing acrylic acid into chitosan template.The remarkable advantage of this system is that it is solely made of hydrophilic polymers:chitosan and poly(acrylic acid),which are non-toxic,and biodegradable.All these CS–PAA nanoparticles are obtained under mild condi-tions without any organic solvents and surfactants.These nanoparticles are stable under acidic and neutral conditions ranging from 4to 8,and aggregate at pH>9.F urthermore,different preparation procedures have a great in?uence on these CS–PAA nanoparticles.The preliminary results of model drug (silk peptide)loading and release experiments indicate that this system seems to be a very promising vehicle for the administration of hydrophilic drugs,proteins and peptides.F urthermore,

due to their pH-sensitive behavior,these CS–PAA nanoparticles are appropriate carriers for the delivery of drugs in the gastric cavity.

Acknowledgements

The authors are thankful to Natural Science Founda-tion of Jiangsu Province,China for the partial ?nancial support of this study.

50

100

150

200

250

300

350

20406080100S P r e l e a s e d (%)

Time (hours)

Fig.5.Release pro?les of SP from CS–PAA nanoparticles at various pH values at 371C (n ?3).

Table 4

Zeta potential of CS–PAA PEC nanoparticles obtained by different procedure

Sample Mean size a (nm)Zeta potential (mv)1436778à22.273.6235874647.272.83

206722

25.373.2

CS dropping into PAA solution.PAA dropping into CS solution.Free radical polymerization.a

Mean size was characterized at pH=4.5.

Y.Hu et al./Biomaterials 23(2002)3193–3201

3200ph 敏感性的应用，自己文章以后用的着

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