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Synthesis and characterization of PEG modified N-trimethylaminoethylmethacrylate chitosan nanoparticles
European Polymer Journal 43 (2007) 2244–2253
EUROPEAN POLYMER JOURNAL www.elsevier.com/locate/europolj
Macromolecular Nanotechnology
Synthesis and characterization of PEG modified N-trimethylaminoethylmethacrylate chitosan nanoparticles Siyu Zhu MACROMOLECULAR NANOTECHNOLOGY
a
a,b,1
, Feng Qian
a,b,1
, Yu Zhang
a,b
, Cui Tang a, Chunhua Yin
a,*
State Key Laboratory of Genetic Engineering, Department of Pharmaceutical Sciences, School of Life Sciences, Fudan University, Shanghai 200433, China b Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200433, China Received 7 November 2006; received in revised form 24 February 2007; accepted 26 March 2007 Available online 4 April 2007
Abstract Chitosan-N-trimethylaminoethylmethacrylate chloride–PEG (CS-TM–PEG) copolymers were synthesized in order to improve the solubility of chitosan in physiological environment, and enhance the biocompatibility of quaternized chitosan. The result of 1H NMR confirmed that PEG had been combined with amino groups of quaternized chitosan. The profile of hemolysis assay showed that Chitosan-N-trimethylaminoethylmethacrylate chloride (CS-TM) copolymer exhibited hemolytic activity from 10.31% to 13.58%, while CS-TM–PEG copolymer had hemolytic activity from 4.76% to 7.05% at copolymer concentrations from 250 to 2000 lg/ml. Through PEG modification, the hemolytic activity could be reduced to a half. CS-TM–PEG copolymer–insulin nanoparticles were prepared based on ionic gelation process of positively charged copolymers and negatively charged insulin. The nanoparticles were characterized in terms of particle size, TEM, association efficiency and in vitro release. These nanoparticles were 200–400 nm in size and insulin association efficiency of optimal formulations was found up to 90%. In vitro release showed that the nanoparticles provided an initial burst release followed by a sustained release with the sensitivity of ionic strength and pH values. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Quaternized chitosan; PEG modification; Insulin; Nanoparticles; Hemolysis
1. Introduction Chitosan (CS) is a (1,4) linked 2-amino-2-deoxyb-D-glucan and can be obtained from chitin, a kind of waste material from the ocean food industry, by
*
Corresponding author. Tel.: +86 21 6564 3797; fax: +86 21 5552 2771. E-mail address: [email protected] (C. Yin). 1 Both authors equally contributed to this work.
alkaline or enzymatic deacetylation. Due to its nontoxicity and high biocompatibility, CS has been formulated as films, beads, microspheres and nanoparticles in the pharmaceutical and biomedical fields [1,2]. CS could adhere to the mucosal surface and transiently open the tight junction between epithelial cells. It has been reported that CS can enhance the penetration of macromolecules across the intestinal and nasal barriers [3]. In recent years, CS has been investigated as drug delivery systems for genes and proteins because positively charged CS can be
0014-3057/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2007.03.042
easily complexed with negatively charged DNAs and proteins [4,5]. However, there are some limitations for CS to be applied in the pharmaceutical and biomedical fields. CS is only soluble in aqueous acidic solutions below pH 6.5, where primary amino groups of CS are protonated. In neutral and physiological environments, CS will lose charge and precipitate from solution, which indicates that it does not seem to be a suitable carrier for DNA and protein drugs. To improve the water solubility of CS, several derivatives of CS have been studied. For example, the modifications of CS by quaternization of the amino groups have been extensively reported. N-Trimethyl chitosan chloride (TMC) is a quaternized derivative of CS, and it has been studied and described by several research groups [6,7] for its absorption enhancing effect. It was concluded that the potential use of TMC, in neutral and physiological environments where unmodified CS is ineffective as absorption enhancer, could contribute significantly to the delivery of hydrophilic compounds such as protein and gene drugs. Although quaternized derivative of CS does possess outstanding properties for pharmaceutical and biomedical applications, those applications involve blood-contact problems such as hemolysis, thrombosis and embolization [8]. Many cationic polymers have been found to be toxic and it has been suggested that this toxicity comes from their effect to the plasma membranes [9]. Other possible toxic mechanisms are due to their interaction with negatively charged cell components and proteins [10]. Quaternized derivative of CS possess high positive charge, which can be easily contacted with negatively charged blood corpuscles, resulting in hemolysis and toxicity. To overcome these problems, poly(ethylene glycol) (PEG) was selected to modify CS derivatives because of its recognized biocompatibility and ability to reduce the interaction between cationic polymers and cell membranes [8]. PEG is a kind of hydrophilic flexible, non-ionic and biodegradable polymer. PEG-coated nanoparticles have been found to be of great potential in therapeutic application for controlled release of drugs and site-specific drug delivery [11–13]. PEG chains attached to the surface or formed the corona of a nanoparticle exhibit rapid motion in an aqueous medium and have a large excluded volume, and the steric repulsion result from a loss of configurational entropy of the bound PEG chains [14]. In addition, the hydrophilic
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PEG could form a hydrated outer shell, which can protect the nanoparticles from being quickly uptaken by the reticuloendothelial system (RES) [15], extend the half-lives of drugs, and change the tissue biodistribution of drugs. Mao et al. [16] synthesized trimethyl chitosan via two-step method, then prepared PEGylated trimethyl chitosan copolymer by grafting activated PEG onto primary amino groups of trimethyl chitosan, and the copolymer showed high solubility and low cytotoxicity. In the present study, a novel approach to prepare chitosan-N-trimethylaminoethylmethacrylate chloride–PEG (CS-TM–PEG) copolymer was described. CS-TM was synthesized by free radical polymerization of chitosan and N-trimethylaminoethylmethacrylate chloride, and CS-TM–PEG copolymer was prepared by coupling active ester of mPEG to –NH2 of CS-TM (Fig. 1). The goal of this study was to improve the solubility of chitosan as well as the biocompatibility of quaternized chitosan at physiological pH. The physicochemical properties of copolymers were analyzed by 1H NMR and hemolysis assay. Nanoparticles were prepared by electrostatic interaction between copolymers and insulin without any organic solvents or high-energy sources, and characterized for their particle size, morphology, association efficiency and in vitro release behavior. 2. Materials and methods 2.1. Materials Chitosan (deacetylation degree 85%) from crab shells was purchased from Sigma (St. Louis, MO, USA). Methoxypoly(ethylene glycol) (mPEG, MW 5000 Da) and N-trimethylaminoethylmethacrylate chloride (TMAEMC) were obtained from Fluka (Bushs, Switzerland). Ammonium persulfate (APS) was purchased from Sigma (St. Louis, MO, USA). Insulin (27.6 I.U./mg) was obtained from Xuzhou biochemical plant (Xuzhou, China). All the other reagents were analytical grade. 2.2. Preparation of chitosan-N-trimethylaminoethylmethacrylate chloride–PEG (CS-TM–PEG) copolymer 2.2.1. Preparation of CS-TM copolymer CS-TM copolymer was obtained by free radical polymerization of chitosan and TMAEMC according to Qian et al. [17]. Briefly, chitosan (1% w/v)
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Fig. 1. The reaction scheme of CS-TM–PEG copolymer.
was dissolved in 50 ml of 250 mM acetic acid solution. APS (0.045% w/v) and TMAEMC were added and the mixture was stirred at 60 °C under nitrogen stream with a speed of 500 rpm. Mole ratio of –NH2 of chitosan and TMAEMC was 2:1. The reaction was terminated after 2 h and the copolymer solution was dialyzed in demineralized water for 48 h.
2.2.2. Activation of mPEG The succinimidyl active ester of mPEG (mPEGSCM) was prepared according to the work of Andreas with some modifications [18]. Briefly, the mixture of naphthalin and sodium was dissolved in anhydrous tetrahydrofuran, which was away from light and oxygen. mPEG and ethyl bromoacetate were added and the reaction mixture was stirred at
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1
2.2.3. Preparation and H NMR of CS-TM–PEG copolymer mPEG-SCM (5% w/v) was dissolved in 8 ml of dimethyl sulfoxide (DMSO), then 10 ml of CS-TM copolymer (10 mg/ml) was added and the mixture was stirred at the room temperature for 24 h. After that, the product was dialyzed in demineralized water for 72 h. Both mPEG-SCM and CS-TM–PEG were characterized by 1H NMR. The samples were measured in D2O, using a 400 MHz spectrometer (Bruker, Switzerland) at room temperature. The degree of quaternization was calculated using data obtained from the 1H NMR spectrum according to the previously described method [6,19] by Eq. (1): %DQ ¼ ½ðCH3 Þ3 =½H 1=9 100
ð1Þ
where %DQ is the degree of quaternization in percentage, [(CH3)3] is the integral of the chemical shift of the trimethylamino group (quaternary amino group) at 3.2 ppm, and [H] is the integral of the 1 H peaks at 4.75 and 4.80 ppm. The degree of PEG modification was calculated using data from the same 1H NMR spectrum by Eq. (2): %DP ¼ ½CH3 O=½H 1=3 100
ð2Þ
where %DP is the degree of PEGylation in percentage, [CH3O] is the integral of the chemical shift of CH3O group at 3.3 ppm, and [H] is the integral of the 1H peaks at 4.75 and 4.80 ppm. 2.3. Preparation of copolymer–insulin nanoparticles The CS-TM–PEG copolymer–insulin and CSTM copolymer–insulin nanoparticles were prepared via self-assembly based on the ionic gelation of copolymers and insulin. Appropriate amount of copolymer and insulin were separately dissolved in 0.1 M phosphate buffer solution (PBS, pH 7.4).
Nanoparticles were prepared by adding equal volume of insulin solution (1 mg/ml) into copolymer solution under gentle magnetic stirring, and incubating for 15 min at room temperature. Preparation of nanoparticles in 0.1 M Tris buffer solution (Tris, pH 7.4) was completed in the same way as described above. 2.4. Particle size The particle size of the nanoparticles was determined by photon correlation spectroscopy (PCS) with a BI 90 particle sizer (Brookhaven Instruments Corp, Holtsville, NY, USA). All samples were done triplicately and particle size was expressed by mean effective diameter. 2.5. Transmission electron microscopy Transmission electron microscopy (TEM) (H-600A, Hitachi, Tokyo, Japan) was used to observe the morphology of the CS-TM–PEG copolymer–insulin nanoparticles. Two types of nanoparticles, which were prepared in PBS and in Tris buffer solution, respectively, were observed. 2.6. Association efficiency and in vitro release Nanoparticle suspensions were ultracentrifugated at 15,000 rpm under 25 °C for 30 min, then the amount of free insulin in the clear supernatant was determined by Lowry method using UV spectrophotometry at 750 nm [20]. The insulin concentration used during the loading process was 0.5 mg/ml. The Association efficiency was calculated with the following Eq. (3): Association efficiency ¼ ðA BÞ=A 100%
ð3Þ
where A is the total amount of insulin, and B is the amount of free insulin in the supernatant. To determine the release of insulin, the nanoparticles were incubated in 1 ml of pH 3.6, pH 5.8 and pH 7.4 PBS and pH 7.4 Tris buffer solution at 37 °C, respectively. The concentrations of nanoparticles in the release medium were adjusted in order to achieve sink conditions for insulin release. At appropriate time intervals, the individual sample was centrifugated and the amount of insulin in the supernatant was measured by the Lowry method.
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room temperature for 8 h. The product was hydrolyzed in 1 M NaOH followed by extracting with dichloromethane. Then, N-hydroxysuccinimide (NHS) and Dicyclohexylcarbodiimide (DCCI) were added into the solution and the system was refluxed for 12 h. After that, the solution was filtrated and stored at 4 °C. Subsequently, the solution was dissolved in dichloromethane and precipitated in ether for three times to remove excess NHS. Finally, mPEG-SCM with activity degree over 90% was obtained.
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All release experiments were done triplicately and the mean values were reported.
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2.7. Hemolysis assay The effect of the copolymers on the integrity of erythrocyte membranes was investigated by in vitro hemolysis assay. The release of hemoglobin from the erythrocytes was used as a measure of toxicity of these copolymers. Briefly, rat erythrocytes were separated from 10 ml blood by centrifugation at 1500 rpm for 20 min and washed three times with 10 ml of PBS. Two milliliters of the purified erythrocytes was resuspended in 38 ml of PBS to a final hematocrit of 5%. Then 0.4 ml of the resulting suspension was incubated with 1 ml of the CS-TM or CS-TM–PEG copolymers in PBS at 37 °C and shaked at 300 rpm for 30 min. The polymer concentrations were selected ranging from 250 to 2000 lg/ ml. The content of hemoglobin released from disrupted erythrocytes was determined photometrically at 541 nm in the supernatant after centrifugation at 12,000 rpm for 20 min. In controls, erythrocytes were incubated in pure PBS (negative control) and PBS containing 10 mg/ml Triton 100 (positive control), respectively. 3. Results and discussion 3.1. Preparation of copolymers and nanoparticles CS-TM has been prepared by free radical polymerization of chitosan and TMAEMC. Redox initiator is usually used in the graft copolymerization, and persulfate can form a redox system to initiate the graft copolymerization of vinyl monomers and chitosan [21,22]. In the present study, the initiator of APS and NH2 group of chitosan combined a redox system, which could open the vinyl of TMAEMC and then trigger the graft copolymerization of chitosan and TMAEMC. Through free radical polymerization, the quaternized chitosan was prepared conveniently via one-step method. The activation of mPEG was designed according to the second-generation PEGylation chemistry, which avoided the shortcomings of the first-generation PEGylation chemistry, such as unstable linkages, side reactions and so on. mPEG with a molecular weight of 5000 Da was chosen for its low toxicity [23]. mPEG was firstly converted to carboxylic acid derivative under the catalysis of the naphthalene and sodium. Then, NHS-terminated
mPEG (mPEG-SCM) was prepared by reacting the PEG–carboxylic acid with NHS. After activation, the electrophilic mPEG was coupled to the nucleophilic –NH2 of CS-TM, to form the CS-TM–PEG graft copolymer. The 1H NMR spectra of mPEG-SCM and CS-TM–PEG copolymers were shown in Fig. 2. Fig. 2a showed signals of mPEG-SCM at 2.7 (–NHS), 3.3 (CH3O–), and 3.6 (CH2–) ppm. The signal at 3.3 ppm corresponded with the CH3O groups of mPEG appeared in the spectrum of the CS-TM–PEG copolymer (Fig. 2b), indicating that mPEG had been combined with the backbone chain of polysaccharide. Additionally, Fig. 2b showed strong absorption at 3.2 (–N+(CH3)3) ppm, which was from the CS-TM. These results confirmed that the CS-TM–PEG copolymer had been synthesized. From 1H NMR analysis, the degree of quaternization of about 44% and the degree of PEGylation of about 34% were calculated. The nanoparticles were formed as a result of complex electrostatic interactions between the positively charged copolymers and negatively charged insulin [24]. The cationic copolymers and anionic insulin could spontaneously gelled and formed nanoparticles at pH 7.4. Nanoparticles could be prepared by the ionic gelation method under mild conditions avoiding the side effects of cross-linking agents. CS-TM and CS possess the ability to gel spontaneously with multivalent polyanions due to the formation of inter- and intramolecular crosslinkage mediated by these polyanions [7,25]. The insulin, which was negatively charged at pH 7.4, was used here to mediate the cross-linkage and prepare nanoparticles. The formation of nanoparticles is only possible for some specific mass ratio of copolymer and insulin: too high ratio (higher than 4:1) led to non-particulate solution while too low ratio (lower than 0.4:1) led to aggregation. 3.2. Particle size The particle sizes determined by PCS were shown in Table 1. All nanoparticles showed the size in the range of 200–400 nm. The nanoparticles prepared in PBS were larger than those prepared in Tris buffer solution. It was found that the particle size was not evidently affected by the copolymer/insulin mass ratio. When mass ratio of copolymer to insulin was 3:1, the nanoparticles obtained were most stable and no sedimentation or agglomeration occurred within 3 months, and they were chosen for further studies.
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Fig. 2. The 1H NMR spectra of mPEG-SCM (a) and CS-TM–PEG (b) in D2O.
Table 1 Particle size and association efficiency of different nanoparticles (n = 3) Nanoparticlesa
Solvent (pH 7.4)
Size (nm)
Association efficiency (%)
CS-TM–PEG, 0.4:1 CS-TM–PEG, 0.75:1 CS-TM–PEG, 1.5:1 CS-TM–PEG, 2:1 CS-TM–PEG, 3:1 CS-TM, 0.4:1 CS-TM, 0.75:1 CS-TM, 1.5:1 CS-TM–PEG, 3:1 CS-TM–PEG, 4:1
PBS PBS PBS PBS PBS PBS PBS PBS Tris Tris
315.3 ± 7.5 334.9 ± 1.7 362.1 ± 15.4 360.6 ± 0.0 375.6 ± 0.6 329.9 ± 1.2 295.7 ± 3.2 293.6 ± 5.5 267.8 ± 4.3 216.4 ± 6.3
10.8 22.0 40.8 51.1 84.0 28.4 33.1 32.4 94.6 67.1
a
Ratio represent copolymer/insulin mass ratio for preparation of nanoparticles.
Without specific depiction, the nanoparticles mentioned below were prepared at the copolymer/insulin mass ratio of 3:1. Transmission electron micrographs (TEM) of the nanoparticles prepared in Tris buffer solution and PBS were shown in Fig. 3. CS-TM–PEG copolymer–insulin nanoparticles prepared in Tris buffer solution exhibited an irregular shape surrounded by a diffuse and fuzzy coat (Fig. 3a). The nanopar-
ticles prepared in PBS were observed to be spherical and were generally separated from each other (Fig. 3b). It has been previously reported that CS derivatives and PEG could form intermolecular hydrogen bond between the electro-positive amino hydrogen and the electro negative oxygen atom, forming a loose structure [26]. In addition, the negative groups of insulin and oxygen atom of PEG may compete in their interaction with –N+(CH3)3 of copolymers, so the possibility of interaction between insulin and copolymers were reduced. Consequently, the structure of PEG modified nanoparticles became looser (Fig. 3a). While the phosphate ions of PBS could interact strongly with –N+(CH3)3 of copolymers [27], which resulted in strong interchain gelation and more compact formation (Fig. 3b). 3.3. Association efficiency and in vitro release Considering the isoelectric point of insulin is 5.3, copolymer–insulin nanoparticles were prepared at pH 7.4, which favored the interaction between insulin and the copolymers. As shown in Table 1, the association efficiency of nanoparticles prepared with CS-TM–PEG and insulin in PBS improved with the
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Fig. 3. TEM of nanoparticles prepared in Tris buffer solution (a) and PBS (b). Bar represents 100 nm.
increase of copolymer/insulin mass ratio, ranging from 10.8% to 84.0% while the mass ratio from 0.4:1 to 3:1. The more the CS-TM–PEG was fed, the more the positive charges existed in the solution. The increasing amount of positive charges would provide stronger electrostatic attraction and associate with more negatively charged insulin. Therefore, the association efficiency increased when more copolymer was fed. However, the formation of nanoparticles was only possible for some specific mass ratio of copolymer and insulin. It was found that too high mass ratio (P4:1) would form a non-particulate solution, which might be due to excessive positive charge existed in the solution. The non-particulate solution was difficult to separate by ultracentrifugation, resulting more insulin was dispersed in the supernatant which was used to determined and calculated for unassociated drug. Therefore the association efficiency of nanoparticles prepared in Tris decreased from 94.6% to 64.1% when the mass ratio changed from 3:1 to 4:1. For the same reason, CS-TM, which has strong positive charge, would form non-particulate solution even at a low mass ratio (<0.4:1). So, the association efficiencies of three kinds of nanoparticles prepared with CS-TM were below 35%. The association efficiency of the nanoparticles prepared in Tris buffer solution was higher than those prepared in PBS at the fixed copolymer/insulin mass ratio. The phosphate ions of PBS could bind to the positive copolymer, and then compete for binding sites with insulin, so the quantity of insulin associated to
copolymer would be reduced. On the other hand, the surface charge of nanoparticles in PBS was less than those in demineralized water due to charge shielding and the compression of diffuse layer by increased surrounding ions [28], resulting in decrease of electrostatic interaction and association efficiency. The in vitro release of insulin from nanoparticles indicated that they had a sustained release behavior. Fig. 4 showed the release of insulin from copolymer–insulin nanoparticles at various time intervals in PBS and Tris buffer solution at 37 °C. There was an obvious initial release of the associated 100
80
Insulin release (%)
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2250
PBS pH 7.4 Tris pH 7.4 PBS pH 3.6
60
40
20
0 0
4
8
12
16
Time (hours) Fig. 4. The release profiles of insulin from nanoparticles in pH 7.4 Tris buffer solution and PBS at 37 °C (mean ± SD, n = 3).
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3.4. Hemolysis assay The hemolysis assay would give additional information about the biocompatibility in the case of an in vivo application [30]. The detailed profiles of hemolysis assay of CS-TM and CS-TM–PEG copolymers were shown in Fig. 5. CS-TM copolymer exhibited hemolytic activity from 10.31% to 13.58% while CS-TM–PEG copolymer had hemolytic activity from 4.76% to 7.05% at copolymer concentra-
20
CS-TM CS-TM-PEG
15
% Hemolysis
10
5
0 500
1000
1500
2000
Concentration (µg /ml) Fig. 5. The hemolytic effects of CS-TM and CS-TM–PEG copolymers on rat erythrocytes in PBS at copolymer concentrations from 250 to 2000 lg/ml (mean ± SD, n = 3).
tions from 250 to 2000 lg/ml. When blood contacts with cationic macromolecules such as quaternized chitosan, there is the initial adsorption of plasma proteins, followed by adhesion and activation of platelets, which leads to hemolysis, thrombosis and embolization [8]. PEG modified CS-TM could obviously decrease the hemolytic activity. The effect of PEG could be explained by steric repulsion, which acts to shield a proportion of positive charge presented on quaternized chitosan, and prevents plasma protein adsorption and platelet adhesion. It is reported that the reduction of hemolysis is related to the MW of PEG, and PEG 5000 is preferable to small MW PEG as its comparatively long chain can shield the positive charge of quaternized chitosan more efficiently [16]. In addition, the copolymer–insulin nanoparticles reduced the hemolytic activity of copolymers after 30 min of incubation with erythrocytes (data not shown). The electrostatic interactions between copolymers and insulin, which decreased the interaction of positively charged copolymers with negatively charged erythrocytes, led to lower hemolysis. The hemolysis assay indicated that PEG modification could significantly reduce the cytotoxicity and improve the biocompatibility of quaternized chitosan. 4. Conclusions CS-TM–PEG copolymer was successfully synthesized. CS-TM–PEG copolymer–insulin nanoparticles were prepared based on ionic gelation process
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insulin, and about 55% of insulin was released within 1 h in pH 7.4 PBS. It could be found that a small initial burst release followed by a slowly sustained release of insulin occurred in Tris buffer solution, and this was quite different from conventional core–shell type nanoparticles. This result indicated that very little insulin located on the surface of nanoparticles prepared in Tris while most of insulin was firmly integrated with copolymer. The process of the release was disassociation of associated insulin from nanoparticles. Ionic strength in release medium may significantly influence the disassociation and release properties of nanoparticles [7]. Ionic strength of PBS may reduce the surface charge of nanoparticles, weaken the interactions between insulin and copolymers, and accelerate disassociation of insulin from nanoparticles due to its competition with insulin for binding sites. So it could be observed that the release of insulin from nanoparticles occurred very rapidly in PBS. On the other hand, the release rate was very fast and about 70% of the associated insulin was released from nanoparticles within 0.5 h in pH 3.6 PBS (Fig. 4), which indicated that the release of insulin related to the pH values of the release medium. Pan et al. [29] reported that the release of insulin nanoparticles at pH 5.8 had the slowest release rate, and at pH 4.0, the nanoparticles hardly show any sustained release property. The changed release rate could be a result of the changed solubility of insulin and different interactions between insulin and nanoparticles at the different pH values. The solubility of insulin at pH 5.8 (pI 5.3) was very limited, and the associated insulin could not be release easily. When the pH value was below 4.0, insulin would disassociate quickly due to its positive charge and higher solubility, which led to the very fast release effect. These results suggested the possibility to adjust the drug release rate of the CS-TM–PEG copolymer– insulin nanoparticles by changing the ionic strength and pH values.
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of copolymers and insulin. These nanoparticles have small size and high insulin association efficiency. The in vitro release of insulin from nanoparticles showed an initial burst release followed by a slowly sustained release with the sensitivity of ionic strength and pH values. The hemolysis assay indicated that PEG modification could significantly reduce the cytotoxicity of quaternized chitosan. In the further studies, the in vivo absorption and bioavailability of these nanoparticulate carriers will be investigated.
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Synthesis and characterization of PEG modified N-trimethylaminoethylmethacrylate chitosan nanoparticles Siyu Zhu MACROMOLECULAR NANOTECHNOLOGY
a
a,b,1
, Feng Qian
a,b,1
, Yu Zhang
a,b
, Cui Tang a, Chunhua Yin
a,*
State Key Laboratory of Genetic Engineering, Department of Pharmaceutical Sciences, School of Life Sciences, Fudan University, Shanghai 200433, China b Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200433, China Received 7 November 2006; received in revised form 24 February 2007; accepted 26 March 2007 Available online 4 April 2007
Abstract Chitosan-N-trimethylaminoethylmethacrylate chloride–PEG (CS-TM–PEG) copolymers were synthesized in order to improve the solubility of chitosan in physiological environment, and enhance the biocompatibility of quaternized chitosan. The result of 1H NMR confirmed that PEG had been combined with amino groups of quaternized chitosan. The profile of hemolysis assay showed that Chitosan-N-trimethylaminoethylmethacrylate chloride (CS-TM) copolymer exhibited hemolytic activity from 10.31% to 13.58%, while CS-TM–PEG copolymer had hemolytic activity from 4.76% to 7.05% at copolymer concentrations from 250 to 2000 lg/ml. Through PEG modification, the hemolytic activity could be reduced to a half. CS-TM–PEG copolymer–insulin nanoparticles were prepared based on ionic gelation process of positively charged copolymers and negatively charged insulin. The nanoparticles were characterized in terms of particle size, TEM, association efficiency and in vitro release. These nanoparticles were 200–400 nm in size and insulin association efficiency of optimal formulations was found up to 90%. In vitro release showed that the nanoparticles provided an initial burst release followed by a sustained release with the sensitivity of ionic strength and pH values. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Quaternized chitosan; PEG modification; Insulin; Nanoparticles; Hemolysis
1. Introduction Chitosan (CS) is a (1,4) linked 2-amino-2-deoxyb-D-glucan and can be obtained from chitin, a kind of waste material from the ocean food industry, by
*
Corresponding author. Tel.: +86 21 6564 3797; fax: +86 21 5552 2771. E-mail address: [email protected] (C. Yin). 1 Both authors equally contributed to this work.
alkaline or enzymatic deacetylation. Due to its nontoxicity and high biocompatibility, CS has been formulated as films, beads, microspheres and nanoparticles in the pharmaceutical and biomedical fields [1,2]. CS could adhere to the mucosal surface and transiently open the tight junction between epithelial cells. It has been reported that CS can enhance the penetration of macromolecules across the intestinal and nasal barriers [3]. In recent years, CS has been investigated as drug delivery systems for genes and proteins because positively charged CS can be
0014-3057/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2007.03.042
easily complexed with negatively charged DNAs and proteins [4,5]. However, there are some limitations for CS to be applied in the pharmaceutical and biomedical fields. CS is only soluble in aqueous acidic solutions below pH 6.5, where primary amino groups of CS are protonated. In neutral and physiological environments, CS will lose charge and precipitate from solution, which indicates that it does not seem to be a suitable carrier for DNA and protein drugs. To improve the water solubility of CS, several derivatives of CS have been studied. For example, the modifications of CS by quaternization of the amino groups have been extensively reported. N-Trimethyl chitosan chloride (TMC) is a quaternized derivative of CS, and it has been studied and described by several research groups [6,7] for its absorption enhancing effect. It was concluded that the potential use of TMC, in neutral and physiological environments where unmodified CS is ineffective as absorption enhancer, could contribute significantly to the delivery of hydrophilic compounds such as protein and gene drugs. Although quaternized derivative of CS does possess outstanding properties for pharmaceutical and biomedical applications, those applications involve blood-contact problems such as hemolysis, thrombosis and embolization [8]. Many cationic polymers have been found to be toxic and it has been suggested that this toxicity comes from their effect to the plasma membranes [9]. Other possible toxic mechanisms are due to their interaction with negatively charged cell components and proteins [10]. Quaternized derivative of CS possess high positive charge, which can be easily contacted with negatively charged blood corpuscles, resulting in hemolysis and toxicity. To overcome these problems, poly(ethylene glycol) (PEG) was selected to modify CS derivatives because of its recognized biocompatibility and ability to reduce the interaction between cationic polymers and cell membranes [8]. PEG is a kind of hydrophilic flexible, non-ionic and biodegradable polymer. PEG-coated nanoparticles have been found to be of great potential in therapeutic application for controlled release of drugs and site-specific drug delivery [11–13]. PEG chains attached to the surface or formed the corona of a nanoparticle exhibit rapid motion in an aqueous medium and have a large excluded volume, and the steric repulsion result from a loss of configurational entropy of the bound PEG chains [14]. In addition, the hydrophilic
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PEG could form a hydrated outer shell, which can protect the nanoparticles from being quickly uptaken by the reticuloendothelial system (RES) [15], extend the half-lives of drugs, and change the tissue biodistribution of drugs. Mao et al. [16] synthesized trimethyl chitosan via two-step method, then prepared PEGylated trimethyl chitosan copolymer by grafting activated PEG onto primary amino groups of trimethyl chitosan, and the copolymer showed high solubility and low cytotoxicity. In the present study, a novel approach to prepare chitosan-N-trimethylaminoethylmethacrylate chloride–PEG (CS-TM–PEG) copolymer was described. CS-TM was synthesized by free radical polymerization of chitosan and N-trimethylaminoethylmethacrylate chloride, and CS-TM–PEG copolymer was prepared by coupling active ester of mPEG to –NH2 of CS-TM (Fig. 1). The goal of this study was to improve the solubility of chitosan as well as the biocompatibility of quaternized chitosan at physiological pH. The physicochemical properties of copolymers were analyzed by 1H NMR and hemolysis assay. Nanoparticles were prepared by electrostatic interaction between copolymers and insulin without any organic solvents or high-energy sources, and characterized for their particle size, morphology, association efficiency and in vitro release behavior. 2. Materials and methods 2.1. Materials Chitosan (deacetylation degree 85%) from crab shells was purchased from Sigma (St. Louis, MO, USA). Methoxypoly(ethylene glycol) (mPEG, MW 5000 Da) and N-trimethylaminoethylmethacrylate chloride (TMAEMC) were obtained from Fluka (Bushs, Switzerland). Ammonium persulfate (APS) was purchased from Sigma (St. Louis, MO, USA). Insulin (27.6 I.U./mg) was obtained from Xuzhou biochemical plant (Xuzhou, China). All the other reagents were analytical grade. 2.2. Preparation of chitosan-N-trimethylaminoethylmethacrylate chloride–PEG (CS-TM–PEG) copolymer 2.2.1. Preparation of CS-TM copolymer CS-TM copolymer was obtained by free radical polymerization of chitosan and TMAEMC according to Qian et al. [17]. Briefly, chitosan (1% w/v)
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Fig. 1. The reaction scheme of CS-TM–PEG copolymer.
was dissolved in 50 ml of 250 mM acetic acid solution. APS (0.045% w/v) and TMAEMC were added and the mixture was stirred at 60 °C under nitrogen stream with a speed of 500 rpm. Mole ratio of –NH2 of chitosan and TMAEMC was 2:1. The reaction was terminated after 2 h and the copolymer solution was dialyzed in demineralized water for 48 h.
2.2.2. Activation of mPEG The succinimidyl active ester of mPEG (mPEGSCM) was prepared according to the work of Andreas with some modifications [18]. Briefly, the mixture of naphthalin and sodium was dissolved in anhydrous tetrahydrofuran, which was away from light and oxygen. mPEG and ethyl bromoacetate were added and the reaction mixture was stirred at
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1
2.2.3. Preparation and H NMR of CS-TM–PEG copolymer mPEG-SCM (5% w/v) was dissolved in 8 ml of dimethyl sulfoxide (DMSO), then 10 ml of CS-TM copolymer (10 mg/ml) was added and the mixture was stirred at the room temperature for 24 h. After that, the product was dialyzed in demineralized water for 72 h. Both mPEG-SCM and CS-TM–PEG were characterized by 1H NMR. The samples were measured in D2O, using a 400 MHz spectrometer (Bruker, Switzerland) at room temperature. The degree of quaternization was calculated using data obtained from the 1H NMR spectrum according to the previously described method [6,19] by Eq. (1): %DQ ¼ ½ðCH3 Þ3 =½H 1=9 100
ð1Þ
where %DQ is the degree of quaternization in percentage, [(CH3)3] is the integral of the chemical shift of the trimethylamino group (quaternary amino group) at 3.2 ppm, and [H] is the integral of the 1 H peaks at 4.75 and 4.80 ppm. The degree of PEG modification was calculated using data from the same 1H NMR spectrum by Eq. (2): %DP ¼ ½CH3 O=½H 1=3 100
ð2Þ
where %DP is the degree of PEGylation in percentage, [CH3O] is the integral of the chemical shift of CH3O group at 3.3 ppm, and [H] is the integral of the 1H peaks at 4.75 and 4.80 ppm. 2.3. Preparation of copolymer–insulin nanoparticles The CS-TM–PEG copolymer–insulin and CSTM copolymer–insulin nanoparticles were prepared via self-assembly based on the ionic gelation of copolymers and insulin. Appropriate amount of copolymer and insulin were separately dissolved in 0.1 M phosphate buffer solution (PBS, pH 7.4).
Nanoparticles were prepared by adding equal volume of insulin solution (1 mg/ml) into copolymer solution under gentle magnetic stirring, and incubating for 15 min at room temperature. Preparation of nanoparticles in 0.1 M Tris buffer solution (Tris, pH 7.4) was completed in the same way as described above. 2.4. Particle size The particle size of the nanoparticles was determined by photon correlation spectroscopy (PCS) with a BI 90 particle sizer (Brookhaven Instruments Corp, Holtsville, NY, USA). All samples were done triplicately and particle size was expressed by mean effective diameter. 2.5. Transmission electron microscopy Transmission electron microscopy (TEM) (H-600A, Hitachi, Tokyo, Japan) was used to observe the morphology of the CS-TM–PEG copolymer–insulin nanoparticles. Two types of nanoparticles, which were prepared in PBS and in Tris buffer solution, respectively, were observed. 2.6. Association efficiency and in vitro release Nanoparticle suspensions were ultracentrifugated at 15,000 rpm under 25 °C for 30 min, then the amount of free insulin in the clear supernatant was determined by Lowry method using UV spectrophotometry at 750 nm [20]. The insulin concentration used during the loading process was 0.5 mg/ml. The Association efficiency was calculated with the following Eq. (3): Association efficiency ¼ ðA BÞ=A 100%
ð3Þ
where A is the total amount of insulin, and B is the amount of free insulin in the supernatant. To determine the release of insulin, the nanoparticles were incubated in 1 ml of pH 3.6, pH 5.8 and pH 7.4 PBS and pH 7.4 Tris buffer solution at 37 °C, respectively. The concentrations of nanoparticles in the release medium were adjusted in order to achieve sink conditions for insulin release. At appropriate time intervals, the individual sample was centrifugated and the amount of insulin in the supernatant was measured by the Lowry method.
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room temperature for 8 h. The product was hydrolyzed in 1 M NaOH followed by extracting with dichloromethane. Then, N-hydroxysuccinimide (NHS) and Dicyclohexylcarbodiimide (DCCI) were added into the solution and the system was refluxed for 12 h. After that, the solution was filtrated and stored at 4 °C. Subsequently, the solution was dissolved in dichloromethane and precipitated in ether for three times to remove excess NHS. Finally, mPEG-SCM with activity degree over 90% was obtained.
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All release experiments were done triplicately and the mean values were reported.
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2.7. Hemolysis assay The effect of the copolymers on the integrity of erythrocyte membranes was investigated by in vitro hemolysis assay. The release of hemoglobin from the erythrocytes was used as a measure of toxicity of these copolymers. Briefly, rat erythrocytes were separated from 10 ml blood by centrifugation at 1500 rpm for 20 min and washed three times with 10 ml of PBS. Two milliliters of the purified erythrocytes was resuspended in 38 ml of PBS to a final hematocrit of 5%. Then 0.4 ml of the resulting suspension was incubated with 1 ml of the CS-TM or CS-TM–PEG copolymers in PBS at 37 °C and shaked at 300 rpm for 30 min. The polymer concentrations were selected ranging from 250 to 2000 lg/ ml. The content of hemoglobin released from disrupted erythrocytes was determined photometrically at 541 nm in the supernatant after centrifugation at 12,000 rpm for 20 min. In controls, erythrocytes were incubated in pure PBS (negative control) and PBS containing 10 mg/ml Triton 100 (positive control), respectively. 3. Results and discussion 3.1. Preparation of copolymers and nanoparticles CS-TM has been prepared by free radical polymerization of chitosan and TMAEMC. Redox initiator is usually used in the graft copolymerization, and persulfate can form a redox system to initiate the graft copolymerization of vinyl monomers and chitosan [21,22]. In the present study, the initiator of APS and NH2 group of chitosan combined a redox system, which could open the vinyl of TMAEMC and then trigger the graft copolymerization of chitosan and TMAEMC. Through free radical polymerization, the quaternized chitosan was prepared conveniently via one-step method. The activation of mPEG was designed according to the second-generation PEGylation chemistry, which avoided the shortcomings of the first-generation PEGylation chemistry, such as unstable linkages, side reactions and so on. mPEG with a molecular weight of 5000 Da was chosen for its low toxicity [23]. mPEG was firstly converted to carboxylic acid derivative under the catalysis of the naphthalene and sodium. Then, NHS-terminated
mPEG (mPEG-SCM) was prepared by reacting the PEG–carboxylic acid with NHS. After activation, the electrophilic mPEG was coupled to the nucleophilic –NH2 of CS-TM, to form the CS-TM–PEG graft copolymer. The 1H NMR spectra of mPEG-SCM and CS-TM–PEG copolymers were shown in Fig. 2. Fig. 2a showed signals of mPEG-SCM at 2.7 (–NHS), 3.3 (CH3O–), and 3.6 (CH2–) ppm. The signal at 3.3 ppm corresponded with the CH3O groups of mPEG appeared in the spectrum of the CS-TM–PEG copolymer (Fig. 2b), indicating that mPEG had been combined with the backbone chain of polysaccharide. Additionally, Fig. 2b showed strong absorption at 3.2 (–N+(CH3)3) ppm, which was from the CS-TM. These results confirmed that the CS-TM–PEG copolymer had been synthesized. From 1H NMR analysis, the degree of quaternization of about 44% and the degree of PEGylation of about 34% were calculated. The nanoparticles were formed as a result of complex electrostatic interactions between the positively charged copolymers and negatively charged insulin [24]. The cationic copolymers and anionic insulin could spontaneously gelled and formed nanoparticles at pH 7.4. Nanoparticles could be prepared by the ionic gelation method under mild conditions avoiding the side effects of cross-linking agents. CS-TM and CS possess the ability to gel spontaneously with multivalent polyanions due to the formation of inter- and intramolecular crosslinkage mediated by these polyanions [7,25]. The insulin, which was negatively charged at pH 7.4, was used here to mediate the cross-linkage and prepare nanoparticles. The formation of nanoparticles is only possible for some specific mass ratio of copolymer and insulin: too high ratio (higher than 4:1) led to non-particulate solution while too low ratio (lower than 0.4:1) led to aggregation. 3.2. Particle size The particle sizes determined by PCS were shown in Table 1. All nanoparticles showed the size in the range of 200–400 nm. The nanoparticles prepared in PBS were larger than those prepared in Tris buffer solution. It was found that the particle size was not evidently affected by the copolymer/insulin mass ratio. When mass ratio of copolymer to insulin was 3:1, the nanoparticles obtained were most stable and no sedimentation or agglomeration occurred within 3 months, and they were chosen for further studies.
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Fig. 2. The 1H NMR spectra of mPEG-SCM (a) and CS-TM–PEG (b) in D2O.
Table 1 Particle size and association efficiency of different nanoparticles (n = 3) Nanoparticlesa
Solvent (pH 7.4)
Size (nm)
Association efficiency (%)
CS-TM–PEG, 0.4:1 CS-TM–PEG, 0.75:1 CS-TM–PEG, 1.5:1 CS-TM–PEG, 2:1 CS-TM–PEG, 3:1 CS-TM, 0.4:1 CS-TM, 0.75:1 CS-TM, 1.5:1 CS-TM–PEG, 3:1 CS-TM–PEG, 4:1
PBS PBS PBS PBS PBS PBS PBS PBS Tris Tris
315.3 ± 7.5 334.9 ± 1.7 362.1 ± 15.4 360.6 ± 0.0 375.6 ± 0.6 329.9 ± 1.2 295.7 ± 3.2 293.6 ± 5.5 267.8 ± 4.3 216.4 ± 6.3
10.8 22.0 40.8 51.1 84.0 28.4 33.1 32.4 94.6 67.1
a
Ratio represent copolymer/insulin mass ratio for preparation of nanoparticles.
Without specific depiction, the nanoparticles mentioned below were prepared at the copolymer/insulin mass ratio of 3:1. Transmission electron micrographs (TEM) of the nanoparticles prepared in Tris buffer solution and PBS were shown in Fig. 3. CS-TM–PEG copolymer–insulin nanoparticles prepared in Tris buffer solution exhibited an irregular shape surrounded by a diffuse and fuzzy coat (Fig. 3a). The nanopar-
ticles prepared in PBS were observed to be spherical and were generally separated from each other (Fig. 3b). It has been previously reported that CS derivatives and PEG could form intermolecular hydrogen bond between the electro-positive amino hydrogen and the electro negative oxygen atom, forming a loose structure [26]. In addition, the negative groups of insulin and oxygen atom of PEG may compete in their interaction with –N+(CH3)3 of copolymers, so the possibility of interaction between insulin and copolymers were reduced. Consequently, the structure of PEG modified nanoparticles became looser (Fig. 3a). While the phosphate ions of PBS could interact strongly with –N+(CH3)3 of copolymers [27], which resulted in strong interchain gelation and more compact formation (Fig. 3b). 3.3. Association efficiency and in vitro release Considering the isoelectric point of insulin is 5.3, copolymer–insulin nanoparticles were prepared at pH 7.4, which favored the interaction between insulin and the copolymers. As shown in Table 1, the association efficiency of nanoparticles prepared with CS-TM–PEG and insulin in PBS improved with the
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Fig. 3. TEM of nanoparticles prepared in Tris buffer solution (a) and PBS (b). Bar represents 100 nm.
increase of copolymer/insulin mass ratio, ranging from 10.8% to 84.0% while the mass ratio from 0.4:1 to 3:1. The more the CS-TM–PEG was fed, the more the positive charges existed in the solution. The increasing amount of positive charges would provide stronger electrostatic attraction and associate with more negatively charged insulin. Therefore, the association efficiency increased when more copolymer was fed. However, the formation of nanoparticles was only possible for some specific mass ratio of copolymer and insulin. It was found that too high mass ratio (P4:1) would form a non-particulate solution, which might be due to excessive positive charge existed in the solution. The non-particulate solution was difficult to separate by ultracentrifugation, resulting more insulin was dispersed in the supernatant which was used to determined and calculated for unassociated drug. Therefore the association efficiency of nanoparticles prepared in Tris decreased from 94.6% to 64.1% when the mass ratio changed from 3:1 to 4:1. For the same reason, CS-TM, which has strong positive charge, would form non-particulate solution even at a low mass ratio (<0.4:1). So, the association efficiencies of three kinds of nanoparticles prepared with CS-TM were below 35%. The association efficiency of the nanoparticles prepared in Tris buffer solution was higher than those prepared in PBS at the fixed copolymer/insulin mass ratio. The phosphate ions of PBS could bind to the positive copolymer, and then compete for binding sites with insulin, so the quantity of insulin associated to
copolymer would be reduced. On the other hand, the surface charge of nanoparticles in PBS was less than those in demineralized water due to charge shielding and the compression of diffuse layer by increased surrounding ions [28], resulting in decrease of electrostatic interaction and association efficiency. The in vitro release of insulin from nanoparticles indicated that they had a sustained release behavior. Fig. 4 showed the release of insulin from copolymer–insulin nanoparticles at various time intervals in PBS and Tris buffer solution at 37 °C. There was an obvious initial release of the associated 100
80
Insulin release (%)
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2250
PBS pH 7.4 Tris pH 7.4 PBS pH 3.6
60
40
20
0 0
4
8
12
16
Time (hours) Fig. 4. The release profiles of insulin from nanoparticles in pH 7.4 Tris buffer solution and PBS at 37 °C (mean ± SD, n = 3).
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3.4. Hemolysis assay The hemolysis assay would give additional information about the biocompatibility in the case of an in vivo application [30]. The detailed profiles of hemolysis assay of CS-TM and CS-TM–PEG copolymers were shown in Fig. 5. CS-TM copolymer exhibited hemolytic activity from 10.31% to 13.58% while CS-TM–PEG copolymer had hemolytic activity from 4.76% to 7.05% at copolymer concentra-
20
CS-TM CS-TM-PEG
15
% Hemolysis
10
5
0 500
1000
1500
2000
Concentration (µg /ml) Fig. 5. The hemolytic effects of CS-TM and CS-TM–PEG copolymers on rat erythrocytes in PBS at copolymer concentrations from 250 to 2000 lg/ml (mean ± SD, n = 3).
tions from 250 to 2000 lg/ml. When blood contacts with cationic macromolecules such as quaternized chitosan, there is the initial adsorption of plasma proteins, followed by adhesion and activation of platelets, which leads to hemolysis, thrombosis and embolization [8]. PEG modified CS-TM could obviously decrease the hemolytic activity. The effect of PEG could be explained by steric repulsion, which acts to shield a proportion of positive charge presented on quaternized chitosan, and prevents plasma protein adsorption and platelet adhesion. It is reported that the reduction of hemolysis is related to the MW of PEG, and PEG 5000 is preferable to small MW PEG as its comparatively long chain can shield the positive charge of quaternized chitosan more efficiently [16]. In addition, the copolymer–insulin nanoparticles reduced the hemolytic activity of copolymers after 30 min of incubation with erythrocytes (data not shown). The electrostatic interactions between copolymers and insulin, which decreased the interaction of positively charged copolymers with negatively charged erythrocytes, led to lower hemolysis. The hemolysis assay indicated that PEG modification could significantly reduce the cytotoxicity and improve the biocompatibility of quaternized chitosan. 4. Conclusions CS-TM–PEG copolymer was successfully synthesized. CS-TM–PEG copolymer–insulin nanoparticles were prepared based on ionic gelation process
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insulin, and about 55% of insulin was released within 1 h in pH 7.4 PBS. It could be found that a small initial burst release followed by a slowly sustained release of insulin occurred in Tris buffer solution, and this was quite different from conventional core–shell type nanoparticles. This result indicated that very little insulin located on the surface of nanoparticles prepared in Tris while most of insulin was firmly integrated with copolymer. The process of the release was disassociation of associated insulin from nanoparticles. Ionic strength in release medium may significantly influence the disassociation and release properties of nanoparticles [7]. Ionic strength of PBS may reduce the surface charge of nanoparticles, weaken the interactions between insulin and copolymers, and accelerate disassociation of insulin from nanoparticles due to its competition with insulin for binding sites. So it could be observed that the release of insulin from nanoparticles occurred very rapidly in PBS. On the other hand, the release rate was very fast and about 70% of the associated insulin was released from nanoparticles within 0.5 h in pH 3.6 PBS (Fig. 4), which indicated that the release of insulin related to the pH values of the release medium. Pan et al. [29] reported that the release of insulin nanoparticles at pH 5.8 had the slowest release rate, and at pH 4.0, the nanoparticles hardly show any sustained release property. The changed release rate could be a result of the changed solubility of insulin and different interactions between insulin and nanoparticles at the different pH values. The solubility of insulin at pH 5.8 (pI 5.3) was very limited, and the associated insulin could not be release easily. When the pH value was below 4.0, insulin would disassociate quickly due to its positive charge and higher solubility, which led to the very fast release effect. These results suggested the possibility to adjust the drug release rate of the CS-TM–PEG copolymer– insulin nanoparticles by changing the ionic strength and pH values.
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of copolymers and insulin. These nanoparticles have small size and high insulin association efficiency. The in vitro release of insulin from nanoparticles showed an initial burst release followed by a slowly sustained release with the sensitivity of ionic strength and pH values. The hemolysis assay indicated that PEG modification could significantly reduce the cytotoxicity of quaternized chitosan. In the further studies, the in vivo absorption and bioavailability of these nanoparticulate carriers will be investigated.
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