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HA composite microspheres
Materials Letters 61 (2007) 1071 – 1076 www.elsevier.com/locate/matlet
Fabrication, characterization and long-term in vitro release of hydrophilic drug using PHBV/HA composite microspheres Yingjun Wang, Xudong Wang ⁎, Kun Wei, Naru Zhao, Shuhua Zhang, Jingdi Chen Biomaterials Research Department of Materials Science and Engineering, South China University of Technology, Guanghzhou, 510640, China The Key Laboratory of Specially Functional Materials and Advanced Manufacturing Technology, Ministry of Education, Guangzhou, 510640, China Received 31 October 2005; accepted 21 June 2006 Available online 12 July 2006
Abstract In this paper, we fabricated polyhydroxybutyrate-co-hydroxyvalerate (PHBV)/Hydroxyapatite (HA) composite microspheres as a long-term drug delivery system. The characterization and in vitro release properties of the system were investigated. It was observed that the PHBV/HA composite microspheres showed very low initial burst that could be neglected, owing to the high affinity and absorbability of nano-HA particles, and the sustained release lasted more than 10 weeks. The in vitro release rate was controlled by diffusion rate of drugs from polymer matrices, and the release profile can be expressed by the Higuchi equation. From our work, it indicated that PHBV/HA composite microsphere could be a promising long-term drug release system. © 2006 Elsevier B.V. All rights reserved. Keywords: Hydroxyapatite (nano-HA); polyhydroxybutyrate-co-hydroxyvalerate (PHBV); Nanoparticles; Composite microsphere; Controlled release
1. Introduction Hydroxyapatite (HA, Ca10(PO4)6(OH)2), which is a major inorganic component of bone, has been widely used as bioactive materials. Because of its excellent biocompatibility and osteoconductivity, HA has been used as a drug carrier in various drug delivery systems, especially in bone tissue treatment [1–3]. As plantable materials, porous hydroxyapatite blocks or granules can be used as drug scaffolds to delay drug release. Their pore structure can let drugs be impregnated into the pores under vacuum, and slow down drug release [4–6]. The main disadvantage of these delivery systems is that they are so large that they can only be implanted in particular tissues and their drug release profile is hard to control. Composite materials consisting of inorganic materials and polymers, combining the advantages of different materials, have attracted much attention from researchers [7–10]. In recent years, polymer/porous-HA composite microspheres have been studied as drug delivery ⁎ Corresponding author. Biomaterials Research Department of Materials Science and Engineering, South China University of Technology, Guanghzhou, 510640, China. Tel.: +86 20 87114645; fax: +86 20 85261559. E-mail address: [email protected] (X. Wang). 0167-577X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2006.06.062
systems [11,12]. Drugs were absorbed into the pores of these composites or on the surface of microspheres, and drug release lasted about three days. However, these composites were difficult to satisfy the need of long-term drug delivery and might lead to a sharp initial burst. The purpose of this study was to prepare a long-term drug release system by fabricating drug-loaded HA nanoparticles, followed by encapsulating the nanoparticles with biodegradable polymer, polyhydroxybutyrate-co-hydroxyvalerate (PHBV), to control drug release rate. The antibiotic gentamicin was used as a model drug. HA nanoparticles were prepared in our laboratory by the method we reported earlier [13]. And the in vitro release profile of drugs (gentamicin) was carried out in a PBS buffer (pH 7.4) at 37 °C. 2. Materials and methods 2.1. Materials Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) with hydroxyvalerate contents of 10% (molar) and with molecular weights of 23.5 kDa was purchased from ICI (England). Polyethylene glycol (PEG) with molecular weights of 19,000
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was purchased from Runjie (Shanghai, China). Gentamicin sulfate (powder) was a kind gift from Zhujiang Hospital (China). All other chemicals used were of analytical grade. 2.2. Methods 2.2.1. Preparation of hydroxyapatite nanoparticles The hydroxyapatite nanoparticles were prepared by an oil-inwater emulsion synthesis method using dodecylamine (DDA) as a template. First, DDA (12.435 g) was dissolved in mixed ethanol (140 g) and heptane (60 g). 500 ml of Ca(NO3)2 (0.3576 mol/l, pH 11.5) was then poured into the previous solution under vigorous stirring. And then 500 ml of (NH4)2HPO4 (0.1585 mol pH 8.5) was added rapidly in the mixed solution, followed by continuous stirring for 24 h at ambient temperature (25 °C). The products were filtered, washed with distilled water and ether several times, air dried overnight and calcined at 500 °C for 28 h. 2.2.2. Preparation of PHBV–HA composite microspheres encapsulating gentamicin 10 ml of gentamicin solution with two concentrations (100 mg/ ml and 200 mg/ml), respectively, was dropwise added in 1 g of HA nanoparticles, followed by magnetic stirring for 72 h at room temperature (25 °C). The resulting mixture was freeze-dried overnight and kept at 4 °C. A solid-in-oil-in-water (S/O/W) emulsion solvent evaporation method [14] was modified to prepare PHBV–HA composite microspheres. Briefly, 500 mg of various amounts of gentamicin-loaded HA nanoparticles was mixed by ball milling with polymer solution, produced by 3 g of PHBV in 30 ml of dichloromethane (DCM), with PEG at a w/w ratio of 1:2. PEG has been reported that it could improve the permeability of the polymer and surface property of microspheres [15–17]. The resulting mixture was poured into 500 ml of distilled water, containing 0.4% (w/v) methyl cellulose as an emulsifier and the
Fig. 2. XRD pattern of HA nanoparticles.
resulting emulsion was then stirred magnetically for more than 4 h to evaporate the solvent under ambient temperature (25 °C). The microspheres were centrifuged, filtered, washed and freezedried. The final products were kept in a desiccator at 4 °C. 2.2.3. Microsphere characterization Microsphere size and morphology were obtained by transmission electron microscope (TEM; EM430 ST Philips). The X-ray diffraction (XRD; PW18v5/20 Philips) analysis performed phase analysis of HA nanoparticles. FT-IR spectra of composite microspheres and original materials were recorded using an FT-IR spectrophotometer (FT-IR; Nicolet 360). Gentamicin loading in composite microspheres and encapsulation efficiency were determined using a UV–visible spectrophotometer (UV755B Cany). Briefly, 20 mg of the microspheres was dissolved in 1 ml DCM and 5 ml PBS buffer (pH 7.4) was then added. After the mixture was blended enough and demixed, the upper aqueous solution was collected. The gentamicin content in aqueous solution was analyzed after performing derivative reaction, using a UV-spectroscopy method at the wavelength of 356 nm [18]. The drug encapsulation efficiency was determined by comparing the quantity of drugs actually incorporated with that of initial drugs before encapsulated by PHBV. Gentamicin loading ð%Þ ¼ ð Weight of gentamicin loadedðgÞÞ= ð weight of microspheresðgÞÞ Encapsulation efficiencyð%Þ ¼ ð weight of gentamicin loadedðgÞÞ= ð weight of initial gentamicinðgÞÞ
Fig. 1. TEM image of HA nanoparticles.
2.2.4. In vitro release of gentamicin from microspheres Freeze-dried microspheres (90 mg) were dispersed in 2 ml PBS buffer (pH 7.4). At an interval of one day up to 70 days, at which point we halted the experiment, the medium of the samples was removed, and the drug content was analyzed by using the UV-spectroscopy as described above. Fresh PBS
Y. Wang et al. / Materials Letters 61 (2007) 1071–1076
Fig. 3. TEM image of the gentamicin-loading HA nanoparticles.
buffer was then supplied. Gentamicin release was calculated in terms of cumulative release (% w/w). 3. Results and discussions A TEM image of the HA nanoparticles was shown in Fig. 1. The size of the nod-like HA nanoparticles was about 20–60 nm in length. The X-ray diffraction pattern revealed characteristic peaks of HA and no secondary phases (Fig. 2) [19].
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Fig. 3 showed a typical TEM image of drug-loaded HA particles. It is important to note that drug molecules were absorbed on the surface of HA nanoparticles, which tended to be sphere-like form in the organic dispersion medium and congregated together. Fig. 4 showed the FT-IR spectra of HA and gentamicin-loaded HA. The HA spectrum (Fig. 4a) showed two sharp peaks at about 3570 cm− 1 and 630 cm− 1, which can be due to the O–H bond presented in the HA molecules, and peaks at 565, 602, 962 and 1035 cm− 1 corresponding to the PO−4 3 ions [20]. While as shown in the gentamicin-loaded HA spectrum (Fig. 4b), the two sharp peaks disappeared or moved to lower frequency zone. And the latter analysis was more reasonable, because the peaks next to them turned a little broader. The FT-IR study indicated that an H bond might exist between gentamicin and HA, which might be one of the reasons why gentamicin-loaded HA particles congregated that was shown in Fig. 3. Microspheres were generally prepared by double or single emulsion solvent evaporation methods. The outer phase, which usually was aqueous phase, induced hydrophilic drugs like gentamicin to move out of the polymer phase. It resulted in comparatively lower encapsulation efficiency of gentamicin [14,21]. In this study, a kind of hybrid structure consisting of HA nanoparticles and polymers was fabricated to increase encapsulation efficiency. When the weight ratio of the initial drug to HA was 1:1, the drug concentration and drug encapsulation efficiency of the resulted composite microspheres (called microspheres sample a; MSa for short) were 6.31 wt.% and 88.4 wt.%, respectively. The comparatively higher encapsulation efficiency should be due to the high bond affinity and hydrophilicity of nano-HA particles. High hydrophilic drugs (gentamicin) were distributed over the HA phase, which reduced the amount of drugs that moved to aqueous phase. It was equivalent to increase the polymers' compatibility with drugs, and
Fig. 4. FT-IR spectra of HA (a) and gentamicin-loaded HA (b) particles.
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provided comparatively higher encapsulation efficiency. When the weight ratio of the initial drug to HA was increased to 2:1, drug concentration was only increased to 6.34 wt.%, while drug encapsulation efficiency of the resulted microspheres (called microspheres sample b; MSb for short) dropped to 66.6 wt.%. The marked decline of encapsulation efficiency, as well as the slight rise of drug concentration of MSb, indicated that the bond affinity and absorbability of HA nanoparticles could be saturable, and excess drugs migrated into the outer aqueous phase during the fabrication process. Fig. 5a and b revealed that the well-established spherical microspheres had smooth surfaces, and the drug-loaded HA particles were encapsulated inside the microspheres. TEM images indicated that the microspheres had hybrid structure with two phases: PHBV made of the polymer matrices and HA particles that distributed in the matrices formed separated phase. Owing to its highly hydrophilic character, gentamicin could be expected to be distributed over HA phase. As shown in Fig. 5a, the size of MSa was about 3–4 μm, which was relatively smaller compared to that of MSb as shown in Fig. 5b. Under the same preparation conditions, when the amount of initial gentamicin was increased, more gentamicin-loaded HA particles assembled together in separated oil droplet, which might lead to relatively larger oil droplet because of their interaction. While the oil phase was evaporated, excess drugs migrated into the outer aqueous
phase, and the HA particles remained in the harden microspheres, which might explain that the MSb was relatively larger than MSa even though the drug concentration was almost the same. Fig. 6 showed the FT-IR spectra of PHBV, gentamicin-loaded HA and PHBA/HA composite microspheres. The PHBV/HA composite microspheres spectrum shows similarity to the PHBV spectrum, while the absorption peaks of OH− 1 and PO−4 3 ions corresponding to HA were weakened. The FT-IR study indicated the presence of PHBV and HA in the composite microspheres, and the content of HA and the drug was comparatively lower in the whole microspheres. In vitro release profile of gentamicin from the composite microspheres was depicted in Fig. 7. The release profiles of the two samples were very similar. The initial release of the first day was very low, and the cumulative release amount of Fig. 7A was only ∼ 2.9 wt.%, while that of Fig. 7B, which was a little higher, was 5.1 wt.%. And then the drug sustained released, and the cumulative release amount as shown in Fig. 7A was increased from 2.9% to 16.6% (5.1% to 15.6% for Fig. 7B) over the period of day 1 to day 21, which might be explained as the release of the drug from the HA particles near the microspheres surfaces. After three weeks, the release rate became slower and slower, and the cumulative release amount was only increased from 16.6 to 23.8% (15.6% to 21.4% for Fig. 7B) over the period of day 21 to day 70. PHBV, as a biodegradable polymer, is generally degraded very slowly in vitro [22]. The rate of drug diffusion was substantially higher than that of polymer degradation, so the release profile was more dependent on drug diffusion rather than on polymer degradation. The hybrid structure of the microspheres consisted of HA nanoparticles and polymer matrices, as discussed above, allowing the drugs to impregnate in the polymer matrices, rather than on the surface of the microspheres. So the initial burst was so low that can be neglected, and then the drugs diffused slowly through the interspaces of the polymer matrices during 70 days. The diffusion of the drugs from spherical matrices can be expressed by the modified Higuchi equation as follows [23]: rffiffiffiffiffiffiffiffiffiffiffiffiffi Dt Mt =Ml ¼ k r2 Where Mt and M∞ are the cumulative amounts of drug release at time t and infinity, respectively, D represents the diffusion coefficient of the drug, and r is related to the size of the microspheres. It indicated that the cumulative release amount was proportional to t1 / 2. Fig. 8 showed the cumulative release (%) vs. square root time curves of microspheres sample a and b, which revealed the release plots from day 1 to day 70 were nearly in line after a little initial burst of first day. So the whole release rate, over the period of day 1 to day 70, was controlled by the diffusion rate of drugs. The slope of curve A was higher than that of B. Considering that the drug loading of sample a was close to that of sample b, while the size of sample a was smaller than that of b, the experiment results were in agreement with the Higuchi equation. Hydrophilic gentamicin distributed more toward the MSb surface because of its larger surface than that of MSa, which led to a more rapid release in the beginning for MSb. However, drugs distributed within the microsphere matrix of MSb, which had relatively smaller surface-to-volume ratio than MSa, were harder to diffuse due to longer diffusion distance. Therefore, comparing with MSa, hydrophilic drug gentamicin released faster from MSb at first but slower afterwards.
4. Conclusion Fig. 5. TEM image of a PHBV/HA composite microsphere. a for MSa; b for MSb.
HA nanoparticles were synthesized using dodecylamine as template by an O/W emulsion method. The size of the particles
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Fig. 6. FT-IR spectra of PHBV (a), gentamicin-loaded HA (b) and PHBV/HA microspheres encapsulating gentamicin (c).
was about 20–60 nm. The composite microspheres were fabricated by an S/O/W emulsion solvent evaporation method. The polymer formed the matrices and outer wall of the microsphere, and the drug-loaded HA particles were encapsulated in it, forming a kind of double phase hybrid structure. The initial burst of the composite microspheres was very low that could be neglected because of the interaction between HA nanoparticles and gentamicin, while the sustained release can last more than 10 weeks. The release rate during 10 weeks was controlled by the diffusion rate of the drugs and could be
expressed by the modified Higuchi equation. From our work, the potential prospect of the polymer/HA composite microspheres, as a long-term drug delivery system, can be highly expected.
Fig. 7. In vitro release profile of gentamicin from composite microspheres. A for MSa; B for MSb.
Fig. 8. Higuchi plot of the in vitro release profile of gentamicin from composite microspheres. A for MSa; B for MSb.
Acknowledgments This study was supported by the National Nature Science Foundation of China (59932050, 50272021, 50572029) and postdoctoral Fund (2004036103).
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Fabrication, characterization and long-term in vitro release of hydrophilic drug using PHBV/HA composite microspheres Yingjun Wang, Xudong Wang ⁎, Kun Wei, Naru Zhao, Shuhua Zhang, Jingdi Chen Biomaterials Research Department of Materials Science and Engineering, South China University of Technology, Guanghzhou, 510640, China The Key Laboratory of Specially Functional Materials and Advanced Manufacturing Technology, Ministry of Education, Guangzhou, 510640, China Received 31 October 2005; accepted 21 June 2006 Available online 12 July 2006
Abstract In this paper, we fabricated polyhydroxybutyrate-co-hydroxyvalerate (PHBV)/Hydroxyapatite (HA) composite microspheres as a long-term drug delivery system. The characterization and in vitro release properties of the system were investigated. It was observed that the PHBV/HA composite microspheres showed very low initial burst that could be neglected, owing to the high affinity and absorbability of nano-HA particles, and the sustained release lasted more than 10 weeks. The in vitro release rate was controlled by diffusion rate of drugs from polymer matrices, and the release profile can be expressed by the Higuchi equation. From our work, it indicated that PHBV/HA composite microsphere could be a promising long-term drug release system. © 2006 Elsevier B.V. All rights reserved. Keywords: Hydroxyapatite (nano-HA); polyhydroxybutyrate-co-hydroxyvalerate (PHBV); Nanoparticles; Composite microsphere; Controlled release
1. Introduction Hydroxyapatite (HA, Ca10(PO4)6(OH)2), which is a major inorganic component of bone, has been widely used as bioactive materials. Because of its excellent biocompatibility and osteoconductivity, HA has been used as a drug carrier in various drug delivery systems, especially in bone tissue treatment [1–3]. As plantable materials, porous hydroxyapatite blocks or granules can be used as drug scaffolds to delay drug release. Their pore structure can let drugs be impregnated into the pores under vacuum, and slow down drug release [4–6]. The main disadvantage of these delivery systems is that they are so large that they can only be implanted in particular tissues and their drug release profile is hard to control. Composite materials consisting of inorganic materials and polymers, combining the advantages of different materials, have attracted much attention from researchers [7–10]. In recent years, polymer/porous-HA composite microspheres have been studied as drug delivery ⁎ Corresponding author. Biomaterials Research Department of Materials Science and Engineering, South China University of Technology, Guanghzhou, 510640, China. Tel.: +86 20 87114645; fax: +86 20 85261559. E-mail address: [email protected] (X. Wang). 0167-577X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2006.06.062
systems [11,12]. Drugs were absorbed into the pores of these composites or on the surface of microspheres, and drug release lasted about three days. However, these composites were difficult to satisfy the need of long-term drug delivery and might lead to a sharp initial burst. The purpose of this study was to prepare a long-term drug release system by fabricating drug-loaded HA nanoparticles, followed by encapsulating the nanoparticles with biodegradable polymer, polyhydroxybutyrate-co-hydroxyvalerate (PHBV), to control drug release rate. The antibiotic gentamicin was used as a model drug. HA nanoparticles were prepared in our laboratory by the method we reported earlier [13]. And the in vitro release profile of drugs (gentamicin) was carried out in a PBS buffer (pH 7.4) at 37 °C. 2. Materials and methods 2.1. Materials Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) with hydroxyvalerate contents of 10% (molar) and with molecular weights of 23.5 kDa was purchased from ICI (England). Polyethylene glycol (PEG) with molecular weights of 19,000
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was purchased from Runjie (Shanghai, China). Gentamicin sulfate (powder) was a kind gift from Zhujiang Hospital (China). All other chemicals used were of analytical grade. 2.2. Methods 2.2.1. Preparation of hydroxyapatite nanoparticles The hydroxyapatite nanoparticles were prepared by an oil-inwater emulsion synthesis method using dodecylamine (DDA) as a template. First, DDA (12.435 g) was dissolved in mixed ethanol (140 g) and heptane (60 g). 500 ml of Ca(NO3)2 (0.3576 mol/l, pH 11.5) was then poured into the previous solution under vigorous stirring. And then 500 ml of (NH4)2HPO4 (0.1585 mol pH 8.5) was added rapidly in the mixed solution, followed by continuous stirring for 24 h at ambient temperature (25 °C). The products were filtered, washed with distilled water and ether several times, air dried overnight and calcined at 500 °C for 28 h. 2.2.2. Preparation of PHBV–HA composite microspheres encapsulating gentamicin 10 ml of gentamicin solution with two concentrations (100 mg/ ml and 200 mg/ml), respectively, was dropwise added in 1 g of HA nanoparticles, followed by magnetic stirring for 72 h at room temperature (25 °C). The resulting mixture was freeze-dried overnight and kept at 4 °C. A solid-in-oil-in-water (S/O/W) emulsion solvent evaporation method [14] was modified to prepare PHBV–HA composite microspheres. Briefly, 500 mg of various amounts of gentamicin-loaded HA nanoparticles was mixed by ball milling with polymer solution, produced by 3 g of PHBV in 30 ml of dichloromethane (DCM), with PEG at a w/w ratio of 1:2. PEG has been reported that it could improve the permeability of the polymer and surface property of microspheres [15–17]. The resulting mixture was poured into 500 ml of distilled water, containing 0.4% (w/v) methyl cellulose as an emulsifier and the
Fig. 2. XRD pattern of HA nanoparticles.
resulting emulsion was then stirred magnetically for more than 4 h to evaporate the solvent under ambient temperature (25 °C). The microspheres were centrifuged, filtered, washed and freezedried. The final products were kept in a desiccator at 4 °C. 2.2.3. Microsphere characterization Microsphere size and morphology were obtained by transmission electron microscope (TEM; EM430 ST Philips). The X-ray diffraction (XRD; PW18v5/20 Philips) analysis performed phase analysis of HA nanoparticles. FT-IR spectra of composite microspheres and original materials were recorded using an FT-IR spectrophotometer (FT-IR; Nicolet 360). Gentamicin loading in composite microspheres and encapsulation efficiency were determined using a UV–visible spectrophotometer (UV755B Cany). Briefly, 20 mg of the microspheres was dissolved in 1 ml DCM and 5 ml PBS buffer (pH 7.4) was then added. After the mixture was blended enough and demixed, the upper aqueous solution was collected. The gentamicin content in aqueous solution was analyzed after performing derivative reaction, using a UV-spectroscopy method at the wavelength of 356 nm [18]. The drug encapsulation efficiency was determined by comparing the quantity of drugs actually incorporated with that of initial drugs before encapsulated by PHBV. Gentamicin loading ð%Þ ¼ ð Weight of gentamicin loadedðgÞÞ= ð weight of microspheresðgÞÞ Encapsulation efficiencyð%Þ ¼ ð weight of gentamicin loadedðgÞÞ= ð weight of initial gentamicinðgÞÞ
Fig. 1. TEM image of HA nanoparticles.
2.2.4. In vitro release of gentamicin from microspheres Freeze-dried microspheres (90 mg) were dispersed in 2 ml PBS buffer (pH 7.4). At an interval of one day up to 70 days, at which point we halted the experiment, the medium of the samples was removed, and the drug content was analyzed by using the UV-spectroscopy as described above. Fresh PBS
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Fig. 3. TEM image of the gentamicin-loading HA nanoparticles.
buffer was then supplied. Gentamicin release was calculated in terms of cumulative release (% w/w). 3. Results and discussions A TEM image of the HA nanoparticles was shown in Fig. 1. The size of the nod-like HA nanoparticles was about 20–60 nm in length. The X-ray diffraction pattern revealed characteristic peaks of HA and no secondary phases (Fig. 2) [19].
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Fig. 3 showed a typical TEM image of drug-loaded HA particles. It is important to note that drug molecules were absorbed on the surface of HA nanoparticles, which tended to be sphere-like form in the organic dispersion medium and congregated together. Fig. 4 showed the FT-IR spectra of HA and gentamicin-loaded HA. The HA spectrum (Fig. 4a) showed two sharp peaks at about 3570 cm− 1 and 630 cm− 1, which can be due to the O–H bond presented in the HA molecules, and peaks at 565, 602, 962 and 1035 cm− 1 corresponding to the PO−4 3 ions [20]. While as shown in the gentamicin-loaded HA spectrum (Fig. 4b), the two sharp peaks disappeared or moved to lower frequency zone. And the latter analysis was more reasonable, because the peaks next to them turned a little broader. The FT-IR study indicated that an H bond might exist between gentamicin and HA, which might be one of the reasons why gentamicin-loaded HA particles congregated that was shown in Fig. 3. Microspheres were generally prepared by double or single emulsion solvent evaporation methods. The outer phase, which usually was aqueous phase, induced hydrophilic drugs like gentamicin to move out of the polymer phase. It resulted in comparatively lower encapsulation efficiency of gentamicin [14,21]. In this study, a kind of hybrid structure consisting of HA nanoparticles and polymers was fabricated to increase encapsulation efficiency. When the weight ratio of the initial drug to HA was 1:1, the drug concentration and drug encapsulation efficiency of the resulted composite microspheres (called microspheres sample a; MSa for short) were 6.31 wt.% and 88.4 wt.%, respectively. The comparatively higher encapsulation efficiency should be due to the high bond affinity and hydrophilicity of nano-HA particles. High hydrophilic drugs (gentamicin) were distributed over the HA phase, which reduced the amount of drugs that moved to aqueous phase. It was equivalent to increase the polymers' compatibility with drugs, and
Fig. 4. FT-IR spectra of HA (a) and gentamicin-loaded HA (b) particles.
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provided comparatively higher encapsulation efficiency. When the weight ratio of the initial drug to HA was increased to 2:1, drug concentration was only increased to 6.34 wt.%, while drug encapsulation efficiency of the resulted microspheres (called microspheres sample b; MSb for short) dropped to 66.6 wt.%. The marked decline of encapsulation efficiency, as well as the slight rise of drug concentration of MSb, indicated that the bond affinity and absorbability of HA nanoparticles could be saturable, and excess drugs migrated into the outer aqueous phase during the fabrication process. Fig. 5a and b revealed that the well-established spherical microspheres had smooth surfaces, and the drug-loaded HA particles were encapsulated inside the microspheres. TEM images indicated that the microspheres had hybrid structure with two phases: PHBV made of the polymer matrices and HA particles that distributed in the matrices formed separated phase. Owing to its highly hydrophilic character, gentamicin could be expected to be distributed over HA phase. As shown in Fig. 5a, the size of MSa was about 3–4 μm, which was relatively smaller compared to that of MSb as shown in Fig. 5b. Under the same preparation conditions, when the amount of initial gentamicin was increased, more gentamicin-loaded HA particles assembled together in separated oil droplet, which might lead to relatively larger oil droplet because of their interaction. While the oil phase was evaporated, excess drugs migrated into the outer aqueous
phase, and the HA particles remained in the harden microspheres, which might explain that the MSb was relatively larger than MSa even though the drug concentration was almost the same. Fig. 6 showed the FT-IR spectra of PHBV, gentamicin-loaded HA and PHBA/HA composite microspheres. The PHBV/HA composite microspheres spectrum shows similarity to the PHBV spectrum, while the absorption peaks of OH− 1 and PO−4 3 ions corresponding to HA were weakened. The FT-IR study indicated the presence of PHBV and HA in the composite microspheres, and the content of HA and the drug was comparatively lower in the whole microspheres. In vitro release profile of gentamicin from the composite microspheres was depicted in Fig. 7. The release profiles of the two samples were very similar. The initial release of the first day was very low, and the cumulative release amount of Fig. 7A was only ∼ 2.9 wt.%, while that of Fig. 7B, which was a little higher, was 5.1 wt.%. And then the drug sustained released, and the cumulative release amount as shown in Fig. 7A was increased from 2.9% to 16.6% (5.1% to 15.6% for Fig. 7B) over the period of day 1 to day 21, which might be explained as the release of the drug from the HA particles near the microspheres surfaces. After three weeks, the release rate became slower and slower, and the cumulative release amount was only increased from 16.6 to 23.8% (15.6% to 21.4% for Fig. 7B) over the period of day 21 to day 70. PHBV, as a biodegradable polymer, is generally degraded very slowly in vitro [22]. The rate of drug diffusion was substantially higher than that of polymer degradation, so the release profile was more dependent on drug diffusion rather than on polymer degradation. The hybrid structure of the microspheres consisted of HA nanoparticles and polymer matrices, as discussed above, allowing the drugs to impregnate in the polymer matrices, rather than on the surface of the microspheres. So the initial burst was so low that can be neglected, and then the drugs diffused slowly through the interspaces of the polymer matrices during 70 days. The diffusion of the drugs from spherical matrices can be expressed by the modified Higuchi equation as follows [23]: rffiffiffiffiffiffiffiffiffiffiffiffiffi Dt Mt =Ml ¼ k r2 Where Mt and M∞ are the cumulative amounts of drug release at time t and infinity, respectively, D represents the diffusion coefficient of the drug, and r is related to the size of the microspheres. It indicated that the cumulative release amount was proportional to t1 / 2. Fig. 8 showed the cumulative release (%) vs. square root time curves of microspheres sample a and b, which revealed the release plots from day 1 to day 70 were nearly in line after a little initial burst of first day. So the whole release rate, over the period of day 1 to day 70, was controlled by the diffusion rate of drugs. The slope of curve A was higher than that of B. Considering that the drug loading of sample a was close to that of sample b, while the size of sample a was smaller than that of b, the experiment results were in agreement with the Higuchi equation. Hydrophilic gentamicin distributed more toward the MSb surface because of its larger surface than that of MSa, which led to a more rapid release in the beginning for MSb. However, drugs distributed within the microsphere matrix of MSb, which had relatively smaller surface-to-volume ratio than MSa, were harder to diffuse due to longer diffusion distance. Therefore, comparing with MSa, hydrophilic drug gentamicin released faster from MSb at first but slower afterwards.
4. Conclusion Fig. 5. TEM image of a PHBV/HA composite microsphere. a for MSa; b for MSb.
HA nanoparticles were synthesized using dodecylamine as template by an O/W emulsion method. The size of the particles
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Fig. 6. FT-IR spectra of PHBV (a), gentamicin-loaded HA (b) and PHBV/HA microspheres encapsulating gentamicin (c).
was about 20–60 nm. The composite microspheres were fabricated by an S/O/W emulsion solvent evaporation method. The polymer formed the matrices and outer wall of the microsphere, and the drug-loaded HA particles were encapsulated in it, forming a kind of double phase hybrid structure. The initial burst of the composite microspheres was very low that could be neglected because of the interaction between HA nanoparticles and gentamicin, while the sustained release can last more than 10 weeks. The release rate during 10 weeks was controlled by the diffusion rate of the drugs and could be
expressed by the modified Higuchi equation. From our work, the potential prospect of the polymer/HA composite microspheres, as a long-term drug delivery system, can be highly expected.
Fig. 7. In vitro release profile of gentamicin from composite microspheres. A for MSa; B for MSb.
Fig. 8. Higuchi plot of the in vitro release profile of gentamicin from composite microspheres. A for MSa; B for MSb.
Acknowledgments This study was supported by the National Nature Science Foundation of China (59932050, 50272021, 50572029) and postdoctoral Fund (2004036103).
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