Synthesis and characterization of chitosan–poly(acrylic acid) nanoparticles
Yong Hu
a,Xiqun Jiang
a,Yin Ding
a,Haixiong Ge
a,Yuyan Yuan
b,Changzheng Yang
a,*
aLab of Mesoscopic Materials, Department of Polymer Science and Engineering, College of Chemistry and Chemical Engineering, Nanjing University, 22 Hankou Road, Nanjing 210093, People’s Republic of China
bJiangsu Super-fine Center Powder Science and Technology, People’s Republic of China Received 14 August 2001; accepted 14 February 2002
Abstract
Chitosan (CS)–poly(acrylic acid) (PAA) complex nanoparticles,which are well dispersed and stable in aqueous solution,have been prepared by template polymerization of acrylic acid (AA) in chitosan solution. The physicochemical properties of nanoparticles were investigated by using size exclusion chromatography,FT-IR,dynamic light scattering,transmission electron microscope and zeta potential. It was found that the molecular weight of PAA in nanoparticles increased with the increase of molecular weight of CS,indicating that the polymerization of acrylic acid in the chitosan solution was a template polymerization. It was also found that the prepared nanoparticles carried a positive charge and showed the size in the range from 50 to 400 nm. The surface structure and zeta potential of nanoparticles can be controlled by different preparation processes. The experiment of in vitro silk peptide (SP) release showed that these nanoparticles provided a continuous release of the entrapped SP for 10 days,and the release behavior was influenced by the pH value of the medium.r2002 Elsevier Science Ltd. All rights reserved.
Keywords: Chitosan; Poly(acrylic acid); Nanoparticles; Drug delivery; Silk peptide
1. Introduction
Recently,polymer nanoparticles have been widely investigated as a carrier for drug delivery [1–3]. Among them,much attention has been paid to the nanoparticles made of synthetic biodegradable polymers such as poly- e-caprolactone (PCL) [4,5], polylactide (PLA) [6], and their copolymers [7–9] due to their good biocompat- ibility,biodegradability,and novel drug release beha- vior. However,these nanoparticles are not ideal carriers for hydrophilic drugs like peptides,protein and some anticancer drugs because of their strong hydrophobic property. To improve their hydrophilic property,many different hydrophilic nanoparticles have been developed as hydrophilic drug carriers. Among those hydrophilic systems,poly(ethylene glycol) (PEG) modified polyester nanoparticles are the promising carriers for the hydro- philic drugs due to the hydrophilic property and other outstanding physico-chemical and biological properties
of PEG [10–12]. But these hydrophobic–hydrophilic nanoparticles have a limitation in their preparation procedure,which requires the use of organic solvents and surfactants as well as sonication or homogenization.
Chitosan,a kind of nature polysaccharide,having structural characteristics similar to glycosaminoglycans, is non-toxic and biodegradable [13],which has rendered it widely,applicable in the pharmaceutical and biome- dical fields [14–16]. In the recent years,chitosan micro- spheres and beads have been investigated as drug delivery systems for anticancer drug or protein [17–19].
Many approaches have been developed to prepare the chitosan beads including water in oil method [20,21], emulsion-droplet coalescence technique [22] and spray drying process [23]. Usually,these preparation proce- dures are complex and need to use some organic solvents or surfactants. In addition,by these techniques, the beads are not appropriate for the routes of administration; For example,vein injection due to their large size (larger than 2mm) [20,21,23].
To overcome these drawbacks,many works have been done. Leong et al. [24] reported CS-DNA nanoparticles prepared by coacervation of CS and DNA in acidic
*Corresponding author. Tel.: +86-25-331-7807; fax: +86-25-331- 7761.
E-mail address:[email protected] (C. Yang).
0142-9612/02/$ - see front matterr2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 0 7 1 - 6
solution. The size of the CS-DNA nanoparticles was in the range of 100–250 nm,and such nanoparticles can protect the encapsulated plasmid DNA from nuclease degradation. Alonso et al. [25] developed a kind of hydrophilic chitosan–polyethylene oxide nanoparticles 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 features: (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-specific targeting in vivo [28,29]; (3) The nanoparticles have pH-depen- dent dissolution behavior.
2. Experimental 2.1. Materials
Chitosan (Nantong Shuanglin Biological Product Inc.) was refined twice by dissolving it in dilute acetic acid solution,filtered,precipitated with aqueous NaOH, and finally dried in vacuum at room temperature. The degree of deacetylation was about 90%,and the weight average molecular weights of chitosan were 40,80,100, 200 and 300 kDa,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 purification.
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 in 50 ml 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 at 3 mmol in all experiments.
Until the solution became clear,0.1 mmol of K2S2O8
was added to the solution with continued stirring. The pH value of the system was maintained at about 4.0.
Then,the polymerization was carried out at 701C under a nitrogen stream and magnetic stirring. When the opalescent suspension appeared,the reaction system was cooled,and the opalescent suspension was filtered with paper filter to remove any polymer aggregation. Finally, the residual monomers were removed by dialysis in a buffer solution of pH=4.5 for 24 h using a dialysis membrane bag with a molecular weight cut-off of 10 kDa.
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. Briefly,1 ml 0.02% CS solution (CS with a molecular weight being 80 kDa was dissolved in 1% (w/
v) acetic acid solution) was added dropwise into 5 ml 0.02% PAA (Mn=100 kDa) aqueous solution under magnetic stirring,the opalescent suspension was formed. The obtained suspension was then filtered by paper filter,the filtered suspension was incubated in a buffer solution of pH=4.5 for 24 h using a dialysis membrane bag for characterization. (b) Adding PAA solution into CS solution. 1 ml 0.02% PAA (Mn=100 kDa) solution was added dropwise into 5 ml 0.02% CS solution (CS with molecular weight 80 kDa was dissolved in 1% (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 dissolving 50 mg of SP in 50 ml CS–PAA nanoparticlate prepared by polymerization of AA in CS solution with the CS molecular weight 80 kDa and incubated for 48 h.
Then,these nanoparticles were separated from the aqueous phase by ultracentrifugation (Ultra ProTM80, Du Pont) with 50,000 rpm at 41C for 40 min. 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 66 V 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
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 ProTM80,Du Pont) with 50,000 rpm at 41C for 40 min. The weight of the sediment nanoparticles was defined as the weight of the resultant nanoparticles. The nanoparticle yields were calculated by the following equation:
Nanoparticle yieldð%Þ
¼ Weight of nanoparticles
Weight of chitosan and monomer fed initially100:
In order to investigate the molecular weight of PAA in nanoparticles,SEC measurement was carried out on Shimadzu LC-10AD HPLC using Ultrahydrogel 120 and 500 columns and a RID-10A detector. The deionized water was used as eluent and the flow rate was 0.5 ml min1. The SEC was calibrated with poly (ethylene oxide) standards. For 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 to 9,the solution was separated by ultracen- trifugation at 50,000 rpm for 40 min. 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; 3000 HS,Malvern,UK) in buffer solution with different pH values. All DLS measurements were done with a wavelength of 633.0 nm at 251C with an angle detection of 901. Each sample was repeatedly measured 3 times and the values reported are the mean diameter7SD for two replicate samples.
The zeta potential of the CS–PAA nanoparticles were measured on Zetasize 3000 HS. (Malvern,UK). The samples were diluted with 10 mmNaCl solution at a pH value of 4.5 (except when otherwise stated) in order to maintain a constant ionic strength. Each sample was
repeatedly measured 3 times and the values reported are the mean value7SD for two replicate samples.
2.9. SP encapsulation efficiency of the nanoparticles The SP-loaded CS–PAA nanoparticles were separated from the aqueous suspension medium by ultracentrifu- gation with 50,000 rpm at 41C for 40 min. The amount of free SP in the clear supertant was measured by fluorescence measurements on LS-50B,(Perkin Elmer) with excitation of 274 nm and emission of 302.4 nm.
SP encapsulation efficiency (AE) were calculated with the following equation:
AE¼Total amount SPFree 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 in 10 ml distilled water and placed in a dialysis membrane bag with a molecular cut-off of 10 kDa,tied and placed into 300 ml of water medium with various pH values on sink conditions.
The entire system was kept at 371C with continuous magnetic stirring. After a predetermined period,5 ml of the medium was removed and the amount of SP was analyzed by fluorescence measurement.
The released SP was determined by a calibration curve.
In order to maintain the original volume,each time, 5 ml 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 Scheme 1. First,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
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 Ferguson,[31] in template polymerization,the presence of template during the polymerization procedure has kinetic and structural effects,which influences 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 influenced 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 40,80,100,200 and 300 kDa. 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 1 shows the results of these experiments. From Table 1,it is interestingly found that the molecular weight (Mn) 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 NH2
NH2
NH2
NH2
CH2CHCOOH
NH3
NH3
NH3
NH3 OOCCHCH2
OOCCHCH2 OOCCHCH2
OOCCHCH2 NH3
NH3 NH3
NH3OOC OOC OOC OOC Polymerization
NH3
NH3
OOC
OOC
OOC NH3 OOC
NH3
CS
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,Mw
Polymerization time (h)
Molecular weight of PAA, Mn
Yield of PAA (%)
Yield of nanoparticles (%)
1 40,000 2 367 85 70.0
2 80,000 2 1087 83 68.6
3 100,000 2 2138 72 62.3
4 200,000 2 4526 78 63.1
5 300,000 2 8026 71 60.0
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,FT-IR studies were conducted.
Fig. 1 shows the FT-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 at 1662 cm1 and amide band II at 1586 cm1,which can be observed clearly in pure chitosan,decrease dramatically,and two new absorption bands at 1731 and 1628 cm1, which can be assigned to the absorption peaks of the carboxyl groups of PAA (the absorption peak of carboxyl groups in pure PAA appears at 1740 cm1), and the NH3+ absorption of CS,respectively,are observed. The broad peaks appeared at 2500 and 1900 cm1 also confirmed the presence of NH3+
in CS–PAA nanoparticles. Furthermore,the absorption peaks at 1532 and 1414 cm1 could be assigned to asymmetric and symmetric stretching vibrations of COO anion groups. These results indicate that the carboxylic groups of PAA are dissociated into COO groups which complex with protonated amino groups of CS through electrostatic interaction to form the polyelectrolyte complex during the polymerization procedure.
3.3. Influence 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 pH 4.5. The results are displayed in Fig. 2 and Table 2. It is shown that the diameter of each sample is smaller than 300 nm.
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 influence 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].
From the results of zeta potential listed in Table 2,it is found that the surfaces of CS–PAA nanoparticles have positive charges of about 20–30 mV. 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 find 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
100 1000
0 5 10 15 20 25
c b
a
5000 a: CS:AA=2:1 b: CS:AA=1:1 c: CS:AA=1:2
Volume%
Particle Diameter (nm)
Fig. 2. Size distribution of CS–PAA nanoparticles with various CS/PAA ratios (wt/wt) at pH=4.5.
4000 3500 3000 2500 2000 1500 1000
CS-PAA PAA
CS
1410
1532 1414
1628 1586 1662
1731 1740
Wavelength(cm-1)
Fig. 1. FT-IR spectra of CS,PAA,and CS–PAA.
Table 2
Mean particle size and zeta potential of CS–PAA complex nanoparticles
Sample AA:CS (wt:wt) Mean diametera(nm) Polydispersity Zeta potential (mV)
I 1:2 250720 0.32570.046 +27.373.5
II 1:1 206722 0.16570.009 +25.373.2
III 2:1 293725 0.35270.032 +23.172.8
aMean diameter was characterized at pH=4.5.
the surface charges of CS–PAA nanoparticles and resulting in the increase of zeta potential.
3.4. Influence 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 weight 80 kDa,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 from 4.0 to 7.4 but dissolved in a few minutes in 0.1n HCl and aggregated quickly at pH values larger than 9. Table 3 shows the result of the mean diameter of the CS–PAA nanoparticles under different pH values. From Table 3,it can be seen that, the diameter of the nanoparticles increases with the increase of pH value from 4.0 to 7.4.
Fig. 3 shows the TEM photographs of CS–PAA nanoparticles prepared by template polymerization.
All these nanoparticles were incubated in buffers for 48 h. The nanoparticles,which were in acetic buffer solution at a pH value of 4.5 (Fig. 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 of 7.4 shown 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 pKavalues of PAA and CS are 4.75 and 6.5 [26],respectively. Under stronger acidic conditions,such as pHo4.0,most carboxylic groups of PAA are in the form ofCOOH.
The interaction between NH3+
and COO in 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 from 4.5 to 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 from 4.5 to 7.4,as listed in Table 3,the ionized degree of PAA increased,and the charge density of the PAA molecules significantly 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 Fig. 3(b). On contrary,under extremely basic condition, the COOH groups from PAA were neutralized by OH, 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
Table 3
The mean diameter of CS–PAA nanoparticles under various pH values
pH Value Mean diameter (nm)
4.0 175732
4.5 206722
5.8 400746
7.4 6257106
Fig. 3. Electron transmission microphotography of CS–PAA nano- particles at (a) pH=4.5 and (b) at pH=7.4.
insolubility in basic solution. These processes can be shown as follows:
When pHo4.0,
NHþ3 COO H-þ NHþ3 þCOOH
CS-PAA nanoparticles Clear solution : ð1Þ When pH>9.0,
NHþ3 COO OH- NH2þCOOþH2O CS-PAA nanoparticles Aggregation : ð2Þ From Eqs. (1) and (2),it is evident that,to obtain stable nanoparticles,the system should be in suitable pH value.
From 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. Influence of different preparation procedures on the morphology of CS–PAA nanoparticles
The CS–PAA nanoparticles were also synthesized by dropping method. The influence 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 influence 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 significant influence 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 4 lists 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.
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 2 and 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. 5 shows the release profiles of SP from CS–PAA nanoparticles with an encapsulation efficiency 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 4.5 and 7.4.
Moreover,these nanoparticles provided a continuous release of the entrapped peptide for up to 10 days. On the other hand,at pH values of 2.0 and 3.0,the SP release rate was very fast and about 90% of the loaded SP was released from CS–PAA nanoparticles within 25 h. It is obvious from the results that the release of the SP depends on pH values of the release medium. The release profile at a pH of 4.5 has 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 o4.0, 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 4 to 8,and aggregate at pH>9.
Furthermore,different preparation procedures have a great influence 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. Furthermore, 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 financial support of this study.
0 50 100 150 200 250 300 350
0 20 40 60 80 100
pH=7.4 pH=4.5 pH=3.0 pH=2.06
SP released(%)
Time (hours)
Fig. 5. Release profiles of SP from CS–PAA nanoparticles at various pH values at 371C (n¼3).
Table 4
Zeta potential of CS–PAA PEC nanoparticles obtained by different procedure
Sample Mean sizea(nm) Zeta potential (mv)
1 436778 22.273.6
2 358746 47.272.8
3 206722 25.373.2
CS dropping into PAA solution.
PAA dropping into CS solution.
Free radical polymerization.
aMean size was characterized at pH=4.5.
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