Polymer Degradation and Stability 96 (2011) 107e113

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Polymer Degradation and Stability

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A dual microsphere based on PLGA and chitosan for delivering the oligopeptide derived from BMP-2
Mingbo Wang a, Qingling Feng a, *, Xiaodong Guo b, Zhending She c, Rongwei Tan a
a

State Key Laboratory of New Ceramics and Fine Processing, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084, China Union Hospital, Tongji Medical College, Department of Orthopaedics, Huazhong University of Science and Technology, Wuhan 430022, China c Center For Advanced Materials & Biotechnology, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057, China
b

a r t i c l e i n f o
Article history: Received 10 August 2010 Received in revised form 4 October 2010 Accepted 23 October 2010 Available online 2 November 2010 Keywords: Poly (lactide-co-glycolide) Chitosan Dual microsphere Osteoinductive Oligopeptide Release kinetic Stability

a b s t r a c t
In this paper, we document the process and ndings of preparing dual poly (lactide-co-glycolide)/chitosan microspheres (PLGA/CS MSs) for osteoinductive oligopeptide derived from BMP-2 (abbreviated as Peptide-24). Through adjusting the amount of Peptide-24, three kinds of PLGA/CS MSs were successfully constructed in twice encapsulations. We studied the morphology, size distribution and loading efciency of the PLGA/CS MSs. We also focused on the pH change of the environment and the molecular weight of the matrix during the degradation process of PLGA/CS MSs. More specically, the release of Peptide-24 from three kinds of PLGA/CS MSs was monitored in PBS at 37  C and pH 7.4. The structural stability of the released Peptide-24 was detected by Far-UV circular dichroism and MALDI-TOF-MS analysis. The mean sizes of the three kinds of PLGA/CS MSs are 47.5, 63.0 and 89.1 mm; and their drug-loading rates are 2.61, 3.21 and 2.21%, respectively. Comparing with Chitosan microspheres (abbreviated as CS MSs), the PLGA/ CS MSs have excellent release curves with zero-order kinetics and controllable model. The incubation solution of PLGA/CS MSs avoided producing acid environment as poly (lactide-co-glycolide) microspheres (PLGA MSs) did, which was explained by analyzing the molecular weight of the matrix. The released oligopeptide kept its original structure and relative molecular weight throughout the procedures of encapsulation, storage and release. This indicates its structure stability. Thus, we conclude that dual PLGA/CS MSs is a promising vehicle that is suitable for the delivery of bioactive factors. 2010 Elsevier Ltd. All rights reserved.

1. Introduction Bone morphogenetic protein-2 (BMP-2) is one of most important BMPs showing strong ability of bone formation. It has been demonstrated to elicit new bone formation at both orthotopic and ectopic sites in experimental animal models [1, 2]. However, BMPs are labile and expensive proteins which prevent its extensive application in bone repair. In another study, a novel synthetic oligopeptide (S[PO4]KIPKASSVPTELSAISTLYLDDD, Peptide-24) was synthesized by using FMOC/tBu solid phase peptide synthesis based on the antigenic determinants of BMP-2 [3]. The oligopeptide can precisely regulate biological behaviors of cells in vitro, such as adhesion and differentiation, and it also owns excellent osteoinductivity and ectopic bone formation property in vivo [4]. The delivery of bioactive proteins and peptides is becoming increasingly more important in modern medical applications. Two topics in particular that have been attracting a lot of research

* Corresponding author. Tel.: 86 10 62782770; fax: 86 10 62771160. E-mail address: biomater@mail.tsinghua.edu.cn (Q. Feng). 0141-3910/$ e see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymdegradstab.2010.10.010

attention are extending release and bioactivity retention. Microencapsulation is an effective formulation for bioactive factor delivery because it can protect proteins/peptides from degenerative environment, allowing the sustained delivery and enhance their therapeutic efciency. Biodegradable materials such as PLGA [5e7] and chitosan (CS) [8] have been extensively studied as the matrices, and as representatives of synthetic and natural polymers, respectively. PLGA was extensively investigated as a controlled delivery system of proteins due to its desirable biocompatibility and adjustable biodegradability [5,9,10]. However, some problems associated with local acidity within PLGA microspheres constrained its applications. For instance, PLGA degrades through hydrolysis and produces carboxylic acids [11]. The acidic condition further promotes the degradation of PLGA according to the acid catalyzed hydrolysis mechanism [12]. This kind of high acidic microenvironment may induce the aggregation and denaturation of proteins [13, 14]. Chitosan, a natural linear biopolyaminosaccharide, possesses the properties such as biodegradability, low toxicity and desirable biocompatibility, which make it suitable for the matrix [15]. The

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M. Wang et al. / Polymer Degradation and Stability 96 (2011) 107e113 Table 1 Theproperties of PLGA/CS MSs and CS MSs. Peptide-24 mass (mg) M1 8 8 4 e
a

cross-linked CS microspheres are loaded with varied drugs such as ampicillin [16], tetanus toxoid [17] and bovine serum albumin [18] have been proven to be preferable for the drug-delivery system. However, the release period of the drugs mentioned above (from several hours to 8 days) is usually too short to meet clinical requests. By adjusting the pH value to 5 and adding polyethylene glycol (PEG) to the protein solution, Celik [19] has not only increased the encapsulation efciency of superoxide dismutase loaded CS microspheres, but also obtained the long period release up to 15 days. However, the release period is still insufcient for long-effect applications [15]. In one of our previous studies, a spheres-in-sphere vehicle was fabricated for controlling burst release of drugs [20]. In this paper, the vehicle was improved on drug-delivery modus in order to release Peptide-24 while not disturbing its original structure. The results proved that PLGA/CS MSs are an effective vehicle for extended and controlled release model. 2. Materials and methods We described the methods of preparation and methods for detecting released peptide and environmental pH value in in vitro incubation experiments of PLGA/CS MSs. The structural stability of the released Peptide-24 was tested by Far-UV circular dichroism and MALDI-TOF-MS. 2.1. Materials PLGA (LA/GA 75/25, 5.01 104) used in our experiences was purchased from the Medical Equipment Research Institute in Shandong province, P.R. China. Chitosan (2.50 105, degree of deacetylation 90%) was purchased from Beijing Chemical Reagents Company, P.R. China. Dichloromethane (DCM), sodium dodecyl sulphate (SDS), sodium tripolyphosphate (TPP), Tween 80 and Span 80 were all reagent grades. 2.2. Preparation of PLGA/CS MSs We rstly prepared Peptide-24 loaded Poly (lactide-co-glycolide) microspheres (abbreviated as PLGA MSs) with the w/o/w method. 50 mg/ml PBS Peptide-24 solution was obtained by dissolving 4 mg (M1) Peptide-24 in 0.08 ml PBS. A 0.08 ml PBS Peptide24 solution was used to prepare Peptide-24 loaded PLGA MSs according to a previous publication [20]. This sequential product was abbreviated as P4. Another PLGA MSs (abbreviated as P8) was also prepared by the same procedure except that we set 8 mg of M1. Poly (lactide-co-glycolide)/chitosan microspheres (abbreviated as PLGA/CS MSs) were prepared in six steps. First of all, the 2% aqueous acetic acid solution was prepared and used as the solvent of 3% (w/v) aqueous CS solution. 2 ml PLGA MSs suspension was added into the 10 ml 3% aqueous CS solution to form a uniform PLGA/CS mixture. Secondly, 6 mg (M2) Peptide-24 was dissolved in 0.12 ml PBS, and then added into the PLGA/CS mixture while stirring mildly. After that, we poured the PLGA/CS/Peptide-24 mixture into the mixture of 60 ml liquid parafn and 1.8 ml Span 80 to form a w/o emulsion, which is then emulsied for 30 min at room temperature. As the forth step, we dropped a 5% (w/v) aqueous TPP solution slowly into the w/o emulsion while stirring and then stirred mildly for additional 3 hours. Settled overnight, the acquired microsphere suspension was repeatedly washed with excess amounts of petroleum ether and isopropyl alcohol. Finally, lyophilization was carried out at 45  C and 80 Pa for 48 h to obtain the dried microspheres (i.e. PLGA/CS MSs). The three kinds of PLGA/CS MSs listed in Table 1 were prepared through modulating the Peptide-24 mass (M1 and M2) with same mass matrix. CS microspheres (CS MSs) were prepared as

M2 6 15 6 6

Total 14 23 10 6

Mass ratio of Drug-loading Abbreviated Mean name diameter internal to external rates (%) (mm) P8/C6 P8/C15 P4/C6 C6 89.1 63.0 47.5 53.8 1/7.15 1/5.21 1/8.09 e 2.61 3.21 2.21 1.92

a M1 and M2 represent added Peptide-24 mass during the process of preparation PLGA MSs and CS crust, respectively.

control group. Note that we stored all the microspheres in desiccant at 4  C before they are used in the experiences. 2.3. Drug-loading rates and the matrix microstructure of PLGA/CS MSs We detected the amount of Peptide-24 in PLGA/CS MSs with high performance liquid chromatography system (HPLC, LC-10ATvp SCL-1, Japan Shimadzu) after Peptide-24 was extracted from the microspheres through the following steps. 10 mg (W1) PLGA/CS MSs was dispersed in 2 ml 2% aqueous acetic acid solution and the external CS was dissolved thoroughly. After centrifugation, the supernate (marked as S1) was segregated and the precipitate was dried and weighed (marked as W2). The precipitate was further dissolved after adding 0.7 ml acetonitrile and 1.3 ml 0.01 M aqueous HCl solution while stirred. After centrifugation at 5600 g for 2 min, the supernate (marked as S2) was obtained. S1 and S2 were used to detect the total amount of Peptide-24 within PLGA/CS MSs. The concentration detection of Peptide-24 in S1 and S2 was carried out with HPLC according to the known Peptide-24 concentration as follows: 20 mL of the obtained supernatant was injected in a chromatograph equipped with a UV detector and a reversed phase column. The mobile phase systems consisted of 0.1% triuoroacetic acid in acetonitrile and 0.1% triuoroacetic acid in H2O. The ow rate was 1.0 mL/min and the wavelength was set at 220 nm. The total amount of Peptide-24 encapsulated in PLGA/CS MSs was obtained and the drug-loading rates of the microspheres were calculated by the equation (1).

Drugloading rate %

Loaded Peptide24 weight 100% Total microspheres weight (1)

By following the above process, the mass ratio of the internal (PLGA BSA within PLGA) to the external (CS BSA within CS) of PLGA/CS MSs was calculated with the equation (2).

Mass ratio of internal to external

W2 W1 W2

(2)

2.4. Size analysis of PLGA/CS MSs The mean size and the size distribution of PLGA/CS MSs and CS MSs were evaluated by a laser particle size analyzer (Mastersizer 2000, UK). 2.5. Morphological analysis The surface shapes of Peptide-24 loaded PLGA MSs and PLGA/CS MSs were observed by SEM (LEO Gemini 1530 Field Emission Gun

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SEM, Germany). PLGA MSs and PLGA/CS MSs were dispersed in water and ethyl alcohol respectively; and then they were dropped on metal stubs and dried at room temperature for the observation. PLGA/CS MSs were also grinded after quenched with liquid nitrogen for the purpose of inspecting their cross-section. The dried samples were all sputter coated with gold under the 105 vacuum and examined at an accelerating voltage of 10 kV.

hsp hr

t 1 t0

(5)

t t0

(6)

2.9. The stability analysis of the oligopeptide 2.6. In vitro release kinetics For the release experiments, 10 mg of Peptide-24 loaded PLGA/ CS MSs and CS MSs were added into a 2 ml capped centrifuge tube containing 1 ml PBS (pH 7.4). Then, it was placed in a shaking bath (Model THZ-C, Taicang Laboratorial Equipment Factory in China) that was rotating at 60 rpm and 37  C. The microsphere suspensions were centrifuged at 500 g for 3 min and replaced with 0.5 ml fresh PBS at scheduled intervals. The aforesaid HPLC method was used to determine the Peptide-24 concentrations in the released supernate. The Peptide-24 release proles from PLGA/CS MSs and CS MSs were described in cumulative release percentage and absolute amount versus incubation time, respectively. The samples were tested in triplicate and data points are shown as mean standard deviation. 2.7. The detection of pH value in incubation experiment PLGA/CS MSs and CS MSs with the weights of 10 mg were incubated in a 1 ml PBS (pH 7.4) and placed in a shaking bath (Model THZ-C, Taicang Laboratorial Equipment Factory in China) that was rotated at 60 rpm and 37  C. The incubation suspensions were centrifuged at 500 g for 3 min to gain the supernate and the deposits of degradation residue for further use. Then the pH values in the supernate were tested with pH meter (Model PHS-3C, Shanghai Precision & Scientic Instrument Co. Ltd.) at 15, 30, 60, 90 and 120 days after the incubation. 2.9.1. Far-UV circular dichroism analysis Far-UV circular dichroism (J-715-150L) was used to monitor the secondary structure of the released oligopeptide after 30 days of incubation. 20 ml released oligopeptide solution were added into the quartz cuvette with 0.1 cm path length, and the spectra were scanned between 190 and 260 nm with 0.5 nm resolution and 100 nm/min scanning speed. 2.9.2. MALDI-TOF-MS analysis After 30 days of incubation, the molecular weights of the oligopeptide in the released supernate were detected by matrixassisted laser desorption/ionization time-of-ight Mass Spectrometer (MALDI-TOF-MS, AutoextI, Bruker Dalton). Firstly, 2 ml released supernate and 2 ml a-cyano-4-hydroxy-cinnamic acids were mixed uniformly. Secondly, 1 ml the consequential mixture was dried in the room temperature. After that, the sample was further dissociated by nitrogen gas laser under positive ion reector mode. Finally, the peak signals about the samples m/z were collected and analyzed. 3. Results and discussion 3.1. Drug-loading rates The drug-loading rates of PLGA/CS MSs and CS MSs are shown in Table 1. We can observe that the drug-loading rates of PLGA/CS MSs are in the range of 2e3% due to the low drug amount administrated. We can also observe that as more drugs were administrated, the higher drug-loading rate was obtained under the condition of same matrix mass. 3.2. The size analysis of the microspheres The size distribution of P8/C6, P8/C15, P4/C6 and C6 is depicted in Fig. 1. The size distributions of the three PLGA/CS MSs and C6

2.8. The detection of the matrix The deposits of degradation residue at 15, 30, 60, 90 and 120 days after incubation were used to detect the molecular weight changes of chitosan and PLGA inside PLGA/CS MSs and CS MSs. First, the deposits were dried and weighed before they were dissolved in water with 0.1 mol/L CH3COONa and 0.2 mol/L CH3COOH, and then centrifuged. The sequent supernate was tested for the molecular weight of chitosan. The sequent deposits and PLGA MSs (abovementioned P8) were dried and dissolved in CHCl3 for analyzing molecular weights of PLGA. Second, the molecular weights of chitosan and PLGA were calculated based on MarkeHouwink equation (3):

h KM a

(3)

where [h] and M were solutions intrinsic viscosity and solutes average molecular weight, respectively. K and a were solutions characteristic constant at determined temperature. [h] was tested with Ubbelohde viscometer (FUNGILAB CV003, Germany Julabo) through equation (4) in the paper [21]:

hsp 3lnhr
4C

(4)

where C was the concentration of tested solution. hsp and hr were the specic viscosity and relative viscosity of tested solution, respectively, and were obtained through equations (5) and (6):

Fig. 1. Size distribution of P8/C6, P8/C15, P4/C6 and C6 microspheres.

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clearly follow the Gaussian distribution, except C6 and P4/C6 show small aggregation peaks at near 600 mm of diameter. We believe that the aggregations are due to the smaller mean diameters of C6 and P4/C6 than P8/C6 and P8/C15. All the diameters of the microspheres distributed from 101 to 102 mm. 3.3. The morphology of PLGA MSs and PLGA/CS MSs Peptide-24 loaded PLGA MSs were used as the internal-spheres of PLGA/CS MSs. SEM micrographs of PLGA MSs are shown in Fig. 2a, b. The gures demonstrate that the surface of PLGA MSs is dense and smooth. In addition, PLGA/CS MSs are discrete and show a corrugated and compact surface (Fig. 2c, d), which is similar to CS MSs [18,22]. The cross-section of PLGA/CS MSs is demonstrated in Fig. 2eef. A number of PLGA MSs were encapsulated inside a continuous CS matrix and dispersed within the CS crust about 1e2 mm thick. 3.4. The analysis of microstructure of PLGA/CS MSs During the preparation of PLGA/CS MSs, the formation of CS crust is based on ionic gelation mechanism including deprotonation and cross-linking of CS through TPP [23,24]. To disclose the microstructure of PLGA/CS MSs, the CS crust was separated by dissolving it in a 2% aqueous acetic acid solution. We

also calculated the mass ratios of internal to external by the obtained data. The mass ratio of internal PLGA MSs to external CS ranged from 1/8.09 to 1/5.21 as shown in Table 1. The cross-section morphology of PLGA/CS MSs in Fig. 2 demonstrates that they are dual microspheres with two-grade global microstructures.

3.5. Release curve It is considered that the polymer microspheres deliver drugs in a diffusion-controlled or erosion-controlled fashion, or a combination of both [24]. It is shown in Fig. 3a that the release patterns of Peptide-24 from the three PLGA/CS MSs are different from CS MSs. CS MSs (C6) presents an obvious burst release (48.9%) of Peptide-24 in the rst three days and a short release period about 15 days. It has been reported that CS microspheres release proteins or peptides only for a short period [17,19]. The release percentage of Peptide-24 from PLGA/CS MSs in the rst three days is kept at 19.5e35%, which is much lower than CS MSs. After 84 days of incubation, P8/C6, P8/C15 and P4/C6 released 70.4%, 84.2% and 84.7% Peptide-24 respectively, indicating a long release period. The release curves of PLGA/CS MSs (including P8/C6, P8/C15 and P4/C6) made great improvements on both burst release and release period. PLGA/CS MSs achieved nearly zero-order release throughout the release process. On the other hand, there are some differences between the release curves of P8/C6, P8/C15 and P4/C6, which is

Fig. 2. SEM micrographs of PLGA MSs (a, b: surface) and PLGA/CS MSs P8/C15 (c, d: surface; e, f: cross-section).

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Fig. 3. Cumulative release percentage (a) and release mass (b) of Peptide-24 from P8/C6, P8/C15, P4/C6 and C6 microspheres.

attributed to the different amount of Peptide-24 in twice encapsulations. It can be seen from Fig. 3b that P8/C15 released more Peptide-24 than P8/C6 in the rst 12 days, which was due to the more Peptide-24 loaded in the CS crust of P8/C15 than P8/C6. P4/C6 presented a bigger burst and a bigger release mass of Peptide-24 than P8/C6 with the same matrix. This phenomenon of P4/C6 was unexpected and mainly due to its smaller diameter than P8/C6 (Table 1). The release curves of drug loaded microspheres are inuenced by the drug/polymer ratio, polymer molecular weight and the particle size according to corresponding publications [25e28]. For the smaller microspheres, most of the proteins are distributed near surface so that the proteins are released easier and faster. These PLGA/CS MSs were cross-linked with equal TPP to avoid the inuence of cross-linking degree on the release of the drugs. P4/C6 and C6, with the same CS matrix and the same crosslinking degree, have obvious different release model due to the presence of PLGA MSs. It may be considered that Peptide-24 distributed mainly at the interface of PLGA MSs and CS matrix, but not on the surface of CS like CS MSs, it needs to be proved. The release phase relevant to chitosan degradation is affected by the properties of the polymeric device such as release mode, polymer molecular weight and cross-linking degree [29]. The improved release curves of Peptide-24 from PLGA/CS MSs are different from CS MSs because they went through different degradation processes [20]. Especially, after 30 days incubation of PLGA/CS MSs, the degradation-controlled Peptide-24 release from external CS cooperated with the diffusion-controlled Peptide-24 release from PLGA MSs, which results in a zero-order kinetics. It is very attractive for the dual PLGA/CS MSs in that the release curves can be regulated through adjusting Peptide-24 amount in the twice encapsulations. 3.6. pH value analysis of the incubation solutions Fig. 4 shows the pH changes of the incubation solutions containing PLGA MSs (P4, P8), CS MSs (C6) and PLGA/CS MSs (P8/C6, P8/C15 and P4/C6) as a function of incubation time. Obvious differences are observed between PLGA MSs and PLGA/CS MSs. Te pH values in the incubation solution of P4, P8 have slight uctuations within 30 days. It was followed by a drop down to 2.76 and 2.78 after 90 days incubation respectively. This was caused by the degradation of PLGA MSs. The pH values of incubation solution of PLGA/CS MSs (P8/C6, P8/C15 and P4/C6) retained 7e8 over the whole incubation period. It is not a surprise that the pH value of incubation solution of Peptide-24 loaded PLGA/CS MSs did not drop because of CS in the matrix. The eNH2 groups of the external CS mainly contributed the retention of pH value [30,31]. Considering the acid problem of PLGA MSs, PLGA/CS with CS crust is a more

preferred vehicle for bioactive peptides because bioactive peptides are easy to aggregation and degradation in acid environment [5,32]. 3.7. The analysis of the matrixs molecular weight It is shown in Fig. 5 that the molecular weights of chitosan inside PLGA/CS MSs changes with the increase of the incubation time. In the rst 15 days, less change took place, and the chitosans molecular weights kept at (2.43e2.48) 105. So, the PLGA/CS MSs retained ne spheres at 15 days post-degradation [20]. From 15 to 30 days, the chitosan degraded violently and molecular weights dropped to (5.99e6.88) 104 while releasing lots of PLGA MSs showed in the morphology of post-incubation [20]. From 30 to 120 days, the cross-linked chitosan eroded slowly. On the other hand, the produced chitosamine through the chitosan degradation resulted in the less change of pH value in incubation suspensions, while PLGA was eroded inside PLGA/CS MSs. The molecular weight changes of PLGA extracted from PLGA/CS MSs and PLGA MSs is depicted in Fig. 6. The PLGA degraded slowly in rst 30 days, and molecular weights changed from 5.01 104 to (4.18e4.70) 104. From 30 to 60 then to 90 days, the PLGA degraded intensively and the molecular weights decreased from (4.18e4.70) 104 to (1.30e1.90) 104 then to (1.07e1.99) 103. The change of the molecular weights of PLGA in P8 is similar to the pH change of P8 as shown in Fig. 4. The more acidic oligomers were produced when the more PLGA molecules degrade intensively, the lower the pH value dropped. As to PLGA/CS MSs, the degradation of

Fig. 4. PH values of the incubation solutions of P4, P8, P8/C6, P8/C15, P4/C6 and C6 microspheres.

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Fig. 5. The molecular weight changes of chitosan extracted from PLGA/CS MSs (P8/C6, P8/C15, P4/C6) and CS MSs (C6).

chitosan stopped the decrease of pH value. From Fig. 6, we can see that the molecular weight of PLGA from P8 decreased swiftly than that from PLGA/CS MSs, which is due to extensive exposure of PLGA to the environmental uid. Herein, the analysis of the matrixs molecular weights partly explains the morphology changes and pH changes of the microspheres during the degradation process in vitro. 3.8. The stability analysis of Peptide-24 3.8.1. The Far-UV circular dichroism spectra analysis The Far-UV circular dichroism spectrum is an effective method for detecting the second structure of protein/peptide [33,34]. It is shown in Fig. 7 that the Far-UV circular dichroism spectra of Peptide-24 released from P8/C6, P8/C15, P4/C6 and C6 are similar to the spectrum of original Peptide-24. In these curves, there are two minus peaks at 195.5 and 201.0 nm attributing to a-helix of Peptide-24, and an intense positive peak at 198.0 nm attributing to b-sheet of Peptide-24. These protein characteristic peaks partly indicate the integrity of secondary structure of Peptide-24. The results here show that the encapsulation, storage and release processes did not induce any change in secondary structure of Peptide-24.

Fig. 7. Far-UV circular dichroism spectra of Peptide-24 released from PLGA/CS MSs and CS MSs (The positive peak head upward, and the minus peak head downward).

3.8.2. The MALDI-TOF-MS analysis The relative molecular weight of bioactive protein/peptide is one of most important identications for its integrity, and can be tested accurately with MALDI-TOF-MS, especially for the trace level peptide detection [35e37]. The MALDI-TOF-MS herein was used to obtain the m/z spectra of Peptide-24 released from PLGA/CS MSs and CS MSs as shown in Fig. 8. The relative molecular weights of Peptide-24 were obtained from the peaks of the spectra. In comparison to original Peptide-24, the relative molecular weights of released Peptide-24 retained at 2550 and 2630 throughout the procedures of encapsulation, storage and release. The retention of the relative molecular weights indicates that no degradation and aggregation happened to Peptide-24, which is a key of expressing their bioactive functions [38,39]. Incorporating with the results of Far-UV circular dichroism spectra analysis, it can be concluded that the encapsulated Peptide-24 kept its integrity. Other studies are currently investigating the function of dual PLGA/CS MSs in animal tests.

Fig. 6. The molecular weight changes of PLGA extracted from PLGA/CS MSs (P8/C6, P8/ C15, P4/C6) and PLGA MSs (P8).

Fig. 8. MALDI-TOF-MS spectra of the original Peptide-24 and the Peptide-24 released from PLGA/CS MSs and CS MSs.

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4. Conclusions The dual PLGA/CS MSs were fabricated while encapsulating Peptide-24 in PLGA internal-spheres and CS external-crust, respectively. The mean sizes of the three kinds of PLGA/CS MSs are 47.5, 63.0 and 89.1 mm, and their drug-loading rates are 2.61, 3.21 and 2.21% respectively. Based on our experiences, we conclude that PLGA/CS MSs have excellent release curves with a less burst release and a zero-order release that can be extended up to nearly 3 months, when compare with CS MSs. The release curves of PLGA/CS MSs can be regulated through adjusting the Peptide-24 amount in twice encapsulations in order to satisfy clinical requests. PLGA/CS is an excellent vehicle to prevent acidity, proven by the pH value of the incubation solution. The pH value changes were explained through analyzing the molecular weights of the vehicle matrix. Furthermore, the structure integrity of Peptide-24 is not disturbed during the encapsulation, storage and release procedure, proven by Far-UV circular dichroism spectra and MALDI-TOF-MS. Thus, we conclude that dual PLGA/CS MSs is an effective vehicle for sustained release of bioactive peptides. Acknowledgements The authors are grateful for the nancial support from National Natural Science Foundation of China (50772052) and Doctor Subject Foundation of the Ministry of Education of China under Grant (20070003004). References
[1] Gautschi OP, Frey SP, Zellweger R. Bone morphogenetic proteins in clinical applications. ANZ J Surg 2007;77:626e31. [2] Niu X, Feng Q, Wang M, Guo X, Zheng Q. Porous nano-HA/collagen/PLLA scaffold containing chitosan microspheres for controlled delivery of synthetic peptide derived from BMP-2. J Controlled Release 2009;134:111e7. [3] Duan Zhixia, Zheng Qixin, Guo Xiaodon, Yuan Quan, Chen Shunguang. Experimental research on ectopic osteogenesis of BMP2-derived peptide P24 combined with PLGA copolymers. Journal of Huazhong University of Science and Technology-Medical Sciences 2007;27:179e82. [4] Yuan Quan, Guo Xiaodong, Zheng Qixin, Zhao Ming, Pan Zhengqi, Chen Shunguang, et al. Bioinspired growth of hydroxyapatite nanocrystals on PLGA-(PEG-ASP)n scaffolds modied with oligopeptide derived from BMP-2. Advances in Composite Materials and Structures 2007;334e335(1e2): 1261e4. [5] Giteau A, Venier-Julienne MC, Aubert-Pouessel A, Benoit JP. How to achieve sustained and complete protein release from PLGA-based microparticles? Int J Pharm 2008;350:14e26. [6] Jain RA. The manufacturing techniques of various drug loaded biodegradable poly (lactide-co-glycolide) (PLGA) devices. Biomaterials 2000;21:2475e90. [7] Thomasin C, Ho NT, Merkle HP, Gander B. Drug microencapsulation by PLA/ PLGA coacervation in the light of thermodynamics. 1. Overview and theoretical considerations. J Pharm Sci. 1998;87:259e68. [8] Hong Yun-Jia, Li Tan-Yao, Chen Bo, Yao Shou-Zhuo. Immobilization of angiotensin converting-enzyme on chitosan microspheres. Chem J Chin Univ Chinese 2009;30:328e31. [9] Gomes AJ, Lunardi CN, Lunardi LO, Pitol DL, Machado AEH. Identication of psoralen loaded PLGA microspheres in rat skin by light microscopy. Micron 2008;39:40e4. [10] Wischke C, Schwendeman SP. Principles of encapsulating hydrophobic drugs in PLA. Int J Pharm 2008;364:298e327. [11] Kim JH, Taluja A, Knutson K, Bae YH. Stability of bovine serum albumin complexed with PEG-poly(L-histidine) diblock copolymer in PLGA microspheres. J Controlled Release 2005;109:86e100. [12] Bezemer JM, Radersma R, Grijpma DW, Dijkstra PJ, van Blitterswijk CA, Feijen J. Microspheres for protein delivery prepared from amphiphilic multiblock copolymers 2. Modulation of release rate. J Controlled Release 2000; 67:249e60. [13] Bilati U, Allemann E, Doelker E. Strategic approaches for overcoming peptide and protein instability within biodegradable nano- and microparticles. Eur J Pharm Biopharm 2005;59:375e88.

[14] Bilati U, Allemann E, Doelker E. Nanoprecipitation versus emulsion-based techniques for the encapsulation of proteins into biodegradable nanoparticles and process-related stability issues. Aaps Pharm; 2005:6. [15] Sinha VR, Trehan A. Biodegradable microspheres for protein delivery. J Controlled Release 2003;90:261e80. [16] Anal AK, Stevens WF, Remunan-Lopez C. Ionotropic cross-linked chitosan microspheres for controlled release of ampicillin. Int J Pharm 2006;312: 166e73. [17] Ahire VJ, Sawant KK, Doshi JB, Ravetkar SD. Chitosan microparticles as oral delivery system for tetanus toxoid. Drug Dev Ind Pharm 2007;33:1112e24. [18] Niu Xufeng, Feng Qingling, Wang Mingbo, Guo Xiaodong, Zheng Qixin. Preparation and characterization of chitosan microspheres for controlled release of synthetic oligopeptide derived from BMP-2. J Microencapsul 2008. doi:10.1080/02652040802319742. [19] Celik O, Akbuga J. Preparation of superoxide dismutase loaded chitosan microspheres: characterization and release studies. Eur J Pharm Biopharm 2007;66:42e7. [20] Mingbo Wang. A spheres-in-sphere structure for improving protein-loading poly (lactide-coglycolide) microspheres. Polym Degrad Stab 2010;95:1e13. [21] Fan Jinshi, Chen Guohua, Sun Mingkun, Hua Zhe. Rapidly determining the intrinsic viscosity of chitosan. J Ocean Univ Qingdao 2002;32:296e300. [22] Niu X, Feng Q, Wang M, Guo X, Zheng Q. In vitro degradation and release behavior of porous poly(lactic acid) scaffolds containing chitosan microspheres as a carrier for BMP-2-derived synthetic peptide. Polym Degrad Stab 2009;94:176e82. [23] Mi FL, Shyu SS, Kuan CY, Lee ST, Lu KT, Jang SF. Chitosanepolyelectrolyte complexation for the preparation of gel beads and controlled release of anticancer drug. I. Effect of phosphorous polyelectrolyte complex and enzymatic hydrolysis of polymer. J Appl Polym Sci. 1999;74:1868e79. [24] Mi FL, Shyu SS, Wong TB, Jang SF, Lee ST, Lu KT. Chitosanepolyelectrolyte complexation for the preparation of gel beads and controlled release of anticancer drug. II. Effect of pH-dependent ionic crosslinking or interpolymer complex using tripolyphosphate or polyphosphate as reagent. J Appl Polym Sci. 1999;74:1093e107. [25] Bittner B, Witt C, Mader K, Kissel T. Degradation and protein release properties of microspheres prepared from biodegradable poly (lactide-co-glycolide) and ABA triblock copolymers: inuence of buffer media on polymer erosion and bovine serum albumin release. J Controlled Release 1999;60:297e309. [26] Sinha VR, Singla AK, Wadhawan S, Kaushik R, Kumria R, Bansal K, et al. Chitosan microspheres as a potential carrier for drugs. Int J Pharm 2004;274:1e33. [27] Hora MS, Rana RK, Nunberg JH, Tice TR, Gilley RM, Hudson ME. Release of human serum-albumin from poly(lactide-co-glycolide) microspheres. Pharm Res 1990;7:1190e4. [28] Bodmer D, Kissel T, Traechslin E. Factors inuencing the release of peptides and proteins from biodegradable parenteral depot systems. J Controlled Release 1992;21:129e37. [29] Jiang T. A tissue engineering approach to bone regeneration using novel hydrid scaffolds based on natural chitosan and synthetic poly (lactide-coglycolide); 2008. [30] Ohtakara A. Microbial chitosanases e the degradation of chitosan and their application. Nippon Nogeikagaku Kaishi [J Jpn Soc Biosci Biotechnol Agrochem] 1988;62:1231e8. [31] Rinaudo M. Properties and degradation of selected polysaccharides: hyaluronan and chitosan. Corr Eng Sci Technol 2007;42:324e34. [32] van de Weert M, Hennink WE, Jiskoot W. Protein instability in poly(lactic-coglycolic acid) microparticles. Pharm Res 2000;17:1159e67. [33] Baginska K, Makowska J, Wiczk W, Kasprzykowski F, Chmurzynski L. Conformational studies of alanine-rich peptide using CD and FTIR spectroscopy. J Peptide Sci 2008;14:283e9. [34] Lee JY, Choo JE, Choi YS, Suh JS, Lee SJ, Chung CP, et al. Osteoblastic differentiation of human bone marrow stromal cells in self-assembled BMP-2 receptor-binding peptide-amphiphiles. Biomaterials 2009;30:3532e41. [35] Terracciano R, Casadonte F, Pasqua L, Candeloro P, Di Fabrizio E, Urbani A, et al. Enhancing plasma peptide MALDI-TOF-MS proling by mesoporous silica assisted crystallization. Talanta 2010;80:1532e8. [36] Wang S, Bao H, Zhang L, Yang P, Chen G. Infrared-assisted on-plate proteolysis for MALDI-TOF-MS peptide mapping. Anal Chem 2008;80:5640e7. [37] Myasein KT, Pulido JS, Hateld RM, McCannel CA, Dundervill Robert F. Submicrolitre dialysis system to enable trace level peptide detection from volume-limited biological samples using MALDI-TOF-MS. Analyst 2007;132:1046e52. [38] Jiang G, Thanoo BC, DeLuca PP. Effect of osmotic pressure in the solvent extraction phase on BSA release prole from PLGA microspheres. Pharm Dev Technol 2002;7:391e9. [39] Jiang G, Woo BH, Kang FR, Singh J, DeLuca PP. Assessment of protein release kinetics, stability and protein polymer interaction of lysozyme encapsulated poly (D,L-lactide-co-glycolide) microspheres. J Controlled Release 2002;79: 137e45.