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Objective: To evaluate the feasibility of the chitosan-poly (lactide-co-glycolide) (PLGA) double-walled microspheres for sustained release of bioactive nerve growth factor (NGF) in vitro. Methods: NGF loaded chitosan-PLGA double-walled microspheres were prepared by emulsion-ionic method with sodium tripolyphosphate (TPP) as an ionic cross-linker. The double-walled microspheres were cross-linked by different concentrations of TPP [1%, 3%, 10% ( W/ V)]. NGF loaded PLGA microspheres were also prepared. The outer and inner structures of double-walled microspheres were observed by light microscopy, scanning electron microscopy, confocal laser scanning microscopy, respectively. The size and distribution of microspheres and fourier transform infra red spectroscopy (FT-IR) were analyzed. PLGA microspheres with NGF or chitosan-PLGA double-walled microspheres cross-linked by 1%, 3%, and 10%TPP concentration (set as groups A, B, C, and D respectively) were used to determine the degradation ratio of microspheres in vitro and the sustained release ratio of NGF in microspheres at different time points. The bioactivity of NGF (expressed as the percentage of PC12 cells with positive axonal elongation reaction) in the sustained release solution of chitosan-PLGA double-walled microspheres without NGF (set as group A1) was compared in groups B, C, and D. Results: The chitosan-PLGA double-walled microspheres showed relative rough and spherical surfaces without aggregation. Confocal laser scanning microscopy showed PLGA microspheres were evenly uniformly distributed in the chitosan-PLGA double-walled microspheres. The particle size of microspheres ranged from 18.5 to 42.7 μm. The results of FT-IR analysis showed ionic interaction between amino groups and phosphoric groups of chitosan in double-walled microspheres and TPP. In vitro degradation ratio analysis showed that the degradation ratio of double-walled microspheres in groups B, C, and D appeared faster in contrast to that in group A. In addition, the degradation ratio of double-walled microsphere in groups B, C, and D decreased when the TPP concentration increased. There were significant differences in the degradation ratio of each group ( P0.05); at 56 and 84 days of culture, the percentage of PC12 cells with positive axonal elongation reaction in groups C and D was significantly higher than that in group B ( P0.05). Conclusion: NGF loaded chitosan-PLGA double-walled microspheres have a potential clinical application in peripheral nerve regeneration after injury.
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Poly(lactide-co-glycolide acid) (PLGA) is an extraordinary well-described polymer and has excellent pharmaceutical properties like high biocompatibility and good biodegradability. Hence, it is one of the most used materials for drug delivery and biomedical systems, also being present in several US Food and Drug Administration-approved carrier systems and therapeutic devices. For both applications, the quantification of the polymer is inalienable. During the development of a production process, parameters like yield or loading efficacy are essential to be determined. Although PLGA is a well-defined biomaterial, it still lacks a sensitive and convenient quantification approach for PLGA-based systems. Thus, we present a novel method for the fast and precise quantification of PLGA by RP-HPLC. The polymer is hydrolyzed into its monomers, glycolic acid and lactic acid. Afterwards, the monomers are derivatized with the absorption-enhancing molecule 2,4′-dibromoacetophenone. Furthermore, the wavelength of the deri-vatized monomers is shifted to higher wavelengths, where the used solvents show a lower absorption, increasing the sensitivity and detectability. The developed method has a detection limit of 0.1 μg/mL, enabling the quantification of low amounts of PLGA. By quantifying both monomers separately, in-formation about the PLGA monomer ratio can be also directly obtained, being relevant for degradation behavior. Compared to existing approaches, like gravimetric or nuclear magnetic resonance measure-ments, which are tedious or expensive, the developed method is fast, ideal for routine screening, and it is selective since no stabilizer or excipient is interfering. Due to the high sensitivity and rapidity of the method, it is suitable for both laboratory and industrial uses.
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Objective: To evaluate the effect of bone morphogenetic protein 7 (BMP-7)/poly (lactide-co-glycolide) (PLGA) microspheres on in vitro proliferation and chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs).
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ABSTRACT The development and validation of a simple and efficient method for the quantification of ferulic acid in poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles coated with chitosan (CS) by reverse phase high performance liquid chromatography coupled to photodiode array detection was described. For the chromatographic analysis, a reverse phase C-18 column was used, mobile phase consisting of acetonitrile and 0.5% acetic acid (37:63, v/v), isocratically eluted at a flow rate of 1 mL/min. Drug determination was performed at 320 nm. The method was validated in terms of the selectivity, linearity, precision, accuracy, robustness, limits of detection and quantification. The method was linear in the range of 10 to 100 µg/mL (r=0.999) and presented limit of detection and quantification of 102 ng/mL and 310 ng/mL, respectively. The method was precise (intra and inter-day) based on relative standard deviation values (less than 3.20%). The recovery was between 101.06 and 102.10%. Robustness was demonstrated considering change in mobile phase proportion. Specificity assay showed no interference from the components of nanoparticles or from the degradation products derived from acidic and oxidative conditions. The proposed method was suitable to be applied in determining the encapsulation efficiency of ferulic acid in PLGA-CS nanoparticles and can be employed as stability indicating one.
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Chromatography, High Pressure Liquid/methods , Chitosan/pharmacology , Nanoparticles/classification , Coumaric Acids/pharmacology , Validation Study , Antioxidants/classificationABSTRACT
Background Choroidal neovascularization (CNV) is a common pathological basis of many ocular fundus diseases.Some treating methods are proved to be effective on CNV but there exist their own shortages.Celecoxib can inhibit experimental neovescularization.Sustained release drug of celecoxib and application approach can offer a basis for the therapy of CNV.Objective This study was to evaluate the sustained release ability of celecoxib-poly lactide-co-glycolide microsphere (CEL-PLGA-MS) in vitro and its inhibitory ability on experimental CNV in vivo.Methods CEL-PLGA-MS was prepared by Hebei Medical University and examined under the scanning electron microscope.The size of CEL-PLGA-MS was measured by Laser Particle Size Analyzer.The drugloading in vitro releasing was monitored by high performance liquid chromatograph (HPLC).Experimental CNV was induced by laser photocoagulation of retina in the right eyes of 72 male brown Norway (BN) rats and then were randomized into the CEL-PLGA-MS group,celecoxib group,blank PLGA group and PBS group.CEL-PLGA-MS with 320 μmol/L celecoxib,80 μmol/L celecoxib,blank PLGA microspheres solution and 0.01 mol/L PBS was intravitreally injected separately according to the grouping.CNV was assessed by fundus fluorescein angiography (FFA) on the 14th day after injection.The fibrovascular proliferation (FVP) thickness at photocoagulation spots was measured by OCT.The retinal pigment epithelium (RPE)-choroid-sclera sections were prepared for the histopathologieal examination of FVP.On the 7th and 28th day after intravitreal injection,the relative expression levels of VEGF mRNA and COX-2 mRNA in the photocoagulation area were detected by reverse transcription PCR (RTPCR).The use and feeding of the experimental animals were followed by the ARVO statement.Results CELPLGA-MS showed the spherical shape with the mean size of 2 467.9 nm and the drug-loading of 7.77％ and the drugrelease rate of 80.91％ in vitro for 45 days.It presented the controllable release characteristics.CEL-PLGA-MS agglomerated in vitreous body after injection.On the 14th day after intravitreal injection,the mean FVP thicknesses were (94.67±4.64),(98.56±4.72),(71.00±4.77),(50.44±3.01) μm in the blank PLGA microspheres group,PBS group,celecoxib group and CEL-PLGA-MS group,respectively,showing significant increases in mean FVP thickness in the blank PLGA microspheres group and PBS group compared with the celecoxib group and CEL-PLGAMS group (all at P＜0.01),and the CEL-PLGA-MS group appeared a lower mean FVP thickness value than the celecoxib group (P＜0.01).FFA revealed a large number of strong hyperfluorescences at the photocoagulation area in the rat eyes of the blank PLGA microspheres group and PBS group;while only weak hyperfluorescences were seen in the eelecoxib group and CEL-PLGA-MS group.Histopathological examinations verified the same results in the FVP thickness to OCT image.The relative expression levels of COX-2 mRNA and VEGF mRNA in the RPE-choroid-sclera were all significantly elevated in the blank PLGA microspheres group compared with the celecoxib group and CELPLGA-MS group both on the 7th and 28th day after intravitreal injection (all at P＜0.01).On the 7th day after injection,the relative expression levels of COX-2 mRNA were lower on the 7th day and the relative expression levels of COX-2 mRNA and VEGF mRNA were higher on the 28th day in the celecoxib group in comparison with the CEL-PLGA-MS group (all at P＜0.01).Conclusions CEL-PLGA-MSs are even in size with the spherical shape and controllable release characteristics in vitro.CEL-PLGA-MS can inhibit experimental CNV and was more durable effective than celecoxib after intravitrea] injection.
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OBJECTIVE:To optimize the formulation and technology of poly(lactide-co-glycolide)(PLGA)microspheres con-taining total alkaloids of Panzeria alaschanica,and to prepare microspheres and conduct quality investigation. METHODS:PLGA microspheres containing total alkaloids of P. alaschanica (PTPM) was prepared by double emulsion-solvent evaporation method. The formulation of microspheres was optimized by L9(34) orthogonal design using mass concentration of PLGA,PVA concentra-tion,ratio of water phase to oil phase as factor,drug-loading amount,encapsulation efficiency,yield as index. The morphology, particle size and drug release of microspheres were all investigated. RESULTS:The optimal formulation was as follows as the mass concentration of PLGA 200 mg/ml,the concentration of PVA 2%,and the water phase-oil phase ratio 1:5. In validation test,aver-age encapsulation efficiency was(83.2±2.4)%,average drug-loading amount(4.16±0.17)%,average yield(86.7±3.6)%,and comprehensive score (95.7 ± 4.4)%;all RSDs were lower than 5.0%(n=3). Prepared microspheres were spherical with smooth surface and uniform particle size. The average particle size was(22.3±2.4)μm and accumulative release rate of 24 h was(82.3± 3.5)%,which conformed to first-order release model(r=0.972 4). CONCLUSIONS:Optimized technology is stable. Prepared mi-crospheres show good sustained-release property,and fit to quality requirements.
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Objective To establish a capillary gas chromatography( GC)method for the determination of residual organic solvents in poly( lactic-co-glycolide)( PLGA),including methanol,acetone,dichloromethane,and toluene. Methods A capillary GC method was carried out. DB-624 capillary column(30 m× 0. 32 mm,1. 80 μm)with programmed temperature chromatography was employed. The initial temperature was kept at 40 ℃ for 8 min. Then the temperature was raised to 200 ℃ at a rate of 10 ℃·min-1 . The injection port temperature was 180 ℃,and the split ratio was 10:1. The carrier gas was nitrogen. The temperature of FID was set at 250 ℃,and the sample volume was set at 3 μL. Results Four residual organic solvents consisting of methanol,acetone,dichloromethane and toluene in PLGA were completely separated. The linear range of concentration of methanol,acetone,dichloromethane and toluene was within 10. 0-50. 0( r=0. 999 8 ),16. 7-83. 3( r=0.9998),2.0-10.0(r=0.9993),and3.0-14.8(r=0.9997)μg·mL-1,respectively.Therecoveryofmethanol,acetone, dichloromethane and toluene was 99. 9%( RSD=1. 5%),100. 8%( RSD=0. 9%),100. 1%( RSD=1. 1%),and 99. 5%(RSD=0. 6%),respectively. Six batches of samples met the requirements. Conclusion The method is proven to be sensitive and accurate after the validation. It is suitable for the determination of residual organic solvents in PLGA.
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OBJECTIVE: To prepare and evaluate donepezil sustained-release microspheres by ultrasound technique. METHODS: Preparation technology of donepezil biodegradable microspheres by ultrasound technique was established and optimized. In vitro evaluation of donepezil microspheres was carried out. The pharmacokinetics of donepezil microspheres was investigated by LC-MS/MS after subcutaneous injection in rabbits at a dose of 30.0 mg · kg. RESULTS: Donepezil microspheres with drug loading of 12.1% and mean particle size between 40 to 130 μm were successfully prepared by ultrasound technique. The donepezil microspheres displayed a one-month sustained-release character in vitro. The pharmacokinetic parameters were as follows: pmax 40.63 μg · L-1, tmax 2.47 d, MRT 14.81 d, and AUC0→∞ 646.96 μg · d · L-1. CONCLUSION: Ultrasound technique is successfully applied in the preparation of donepezil sustained-release microspheres.
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Objective: To prepare a self-designed three-dimensional woven scaffold using poly lactide-co-glycolide acid (PLGA), and to observe the influence of the prepared scaffold on the growth of Schwann cells in vitro and its in vivo degradation. Methods: The 3D scaffolds were prepared by means of melt spinning, extension, weaving, and other procedures. The alignment of micro-tubules was observed under the scanning electronic microscope (SEM). The size of the micropores was also measured. Primary cultured Schwann cells were seeded on the 3D scaffolds, and the growth, adherence, proliferation, and apoptosis of Schwann cells were observed under inverted phase contrast microscopy and SEM; the results were compared between Schwann cells cultured in collagen sponge and culture dish. The scaffold carrying Schwann cells was implanted into the paraspinal muscle in rats, and H-E staining was used to observe the in vivo degradation and the inflammation responses. Results: The external diameters of the scaffold and micro-tubules were 3 mm and 100 μm, respectively, and the micro-tubules were arranged in an even and parallel manner. The adherence rates and proliferation rates of Schwann cells were similar between scaffold group and collagen sponge group, but both groups were significantly lower than the culture dish group (P<0.05). The 3D scaffold degraded completely within 12 weeks, with no visible inflammatory cells around. Conclusion: The self-designed 3D scaffold has no harmful effect on the growth of Schwann cells, and it can degrade in vivo, showing a satisfactory biocompatibility.
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OBJECTIVE: To optimize the preparation technology of poly(lactide-co-glycolide) (PLGA) microspheres loaded with ropivacaine (ROP-PLGA-MS) and study their powder particle characteristics and in vitro release characteristics. METHODS: ROP-PLGA-MS was prepared with PLGA as carriers using the water-in-oil-in-water(W/O/W) emulsion solvent evaporation method. The micromeritic characteristics of ROP-PLGA-MS,such as the particle size,loading and entrapment efficiency were taken as indexes for evaluation,and the preparation technology was optimized by orthogonal design. Its dissolution characteristics in vitro were studied. RESULTS: The prepared microspheres were spherical with smooth surfaces,with an average particle size of (2.525?0.047) ?m and over 80% falling in the range of 1.8～5.0 ?m. The encapsulation efficiency was (58.05?1.169)% and the drug loading efficiency was (6.067 ?0.312)%. The in vitro release curves of ropivacaine microspheres could be fitted with Higuchi equation. The accumulated release percentage of which was 82 % in 192 h,and t1/2=60.16 h. CONCLUSION: ROP-PLGA-MS has obvious sustained release.
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To modify the surface property of poly lactide co-glycolide (PLGA) by biomimetic mineralization to construct a new kind of artificial bone. PLGA films and 3 diamensional (3-D) porous scaffolds hydrolyzed in alkaline solution were minerilized in SBF for 14 days. The morphology and composition of the mineral grown on PLGA were analyzed with SEM, FTIR and XRD. The porosity of the scaffolds was detected by using the liquid displacement method. The compressive strength of the scaffolds was detected by using a Shimadzu universal mechanic tester. An obvious mineral coating was detected on the surface of films and scaffolds. The main component of the mineral was carbonated hydroxyapatite (HA) similar to the major mineral component of bone tissues. The porosity of the un-mineralized and mineralized porous scaffolds was (84.86±8.52) % and (79.70±7.70) % respectively. The compressive strength was 0. 784±0. 156 N/mm2 in un-mineralized 3-D porous PLGA and 0. 858±0. 145 N/mm2 in mineralized 3-D porous PLGA. There were no significant differences between the mineralized and un-mineralized scaffolds (P＞0. 05) in porosity and biomechanics. Biomimetic mineralization is a suitable method to construct artificial bone.
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The aim of this in vitro study was to evaluate the suitability of using chitosan, poly (lactide-co-glycolide) (PLGA), and polymethyl methacrylate (PMMA) to control the release of chlorhexidine digluconate (CHX) from a prototype of controlled release drug device (CRD) for root canal disinfection. Four different prototypes with different formulations were prepared. Group A (n = 12); The device (absorbent paper point) was loaded with CHX as control. Group B (n = 12); same as group A, but the device was coated with chitosan. In Groups C and D, the device was treated in the same way as group A and then coated three times with 5% PMMA (Group C, n = 12), or coated three times with 3% PLGA (Group D, n = 12). The devices were randomly allocated to experimental groups of 12 each. All CRD prototypes were soaked in 3 mL distilled water. The concentrations of CHX were determined using a UV spectrophotometer. The surface characteristics of each prototype were observed using a scanning electron microscope. The result showed that release rate of CHX was the greatest in the non-coated group, followed by the chitosan-coated group, the PLGA-coated group, and the PMMA-coated group (P < 0.05). Pores were observed on the surface of the prototypes that were coated with PLGA and PMMA. When the pore size was smaller, the release rate was lower. This data indicate that polymer coating can control the release rate of CHX from the CRD prototypes.
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Chitosan , Chlorhexidine , Dental Pulp Cavity , Disinfection , Polymers , Polymethyl Methacrylate , WaterABSTRACT
Objective To prepare Paracetamol (APP) multiloculated implant loaded with poly-lactide-co-glycolide acid (PLGA) and to study the drug release profile in vitro. Methods APP multiloculated implant was fabricated by micro-electro-mechanical system (MEMS), and high-performance liquid chromato graphy (HPLC) measurement was used to investigate in vitro drug release profile. HPLC analysis was carried out by employing C18 column and a mixture of methanol-water (15∶85) as mobile phase. The detection wavelength was 215nm and flow rate was 0.8mL/min. Results With different multiloculated shape, the rate of the drug release in vitro was varied significantly. Moreover, the releasing of APP multiloculated implant with ecto-tetragonum ento-hexagon in vitro conformed to Higuchi equation. Conclusion The technology of the preparations is feasible, and the structural and morphological characteristics of the multiloculated implant have a significant impact on the release speed of the drug delivery system.