Effects of poly lactic-co-glycolic acid-Nogo A antibody delayed-release microspheres on regeneration of injured spinal cord in rats

BACKGROUND： Nogo A antigen is the major inhibiting factor blocking regeneration of the injured spinal cord. Neutralizing Nogo A antigens using Nogo A antibodies may help promote neurite regeneration and nervous function recovery. For successful regeneration, sustained release of the antibody from a biodegradable material loaded with Nogo A antibodies to the injury site is required. OBJECTIVE： To compare the therapeutic effects of poly lactic-co-glycolic acid （PLGA）-Nogo A antibody delayed-release microspheres and Nogo A antibody alone on spinal regeneration in Sprague-Dawley rats with complete transverse injury to the spinal cord. DESIGN, TIME AND SETTING： A randomized, controlled animal trial was performed at the Pharmacological Laboratory of West China Center of Medical Sciences, Sichuan University, between October 2007 and January 2008. MATERIALS： Goat anti-rat Nogo A monoclonal antibody was purchased from Santa, American; goat anti-rat neurofilament 200 monoclonal antibody was from Zhongshan Goldenbridge, Beijing, China; PLGA-Nogo A antibody delayed-release microspheres were provided by the College of Pharmacy, Sichuan University. METHODS： A total of 36 adult female Sprague Dawley rats were used to establish models of completely transected spinal cord injury, at T10. Animals were randomly divided into three groups （n=12）： model, Nogo A antibody alone, and Nogo A antibody delayed-release microsphere groups. After transverse injury of the spinal cord, 50 μ L normal saline solution, 50 μL normal saline solution containing 50μL g Nogo A antibody, and 50 μL normal saline solution containing 50 μg Nogo A antibody microspheres were administered to the respective groups at the injury site. MAIN OUTCOME MEASURES： The expression of Nogo A and neurofilament 200 in injured spinal cord was tested immunohistochemically, and motor function of rats was assessed by Basso-Beattie-Bresnahan （BBB） locomotor rating scale. RESULTS： Four weeks after injury, expression of Nogo A in microsphere group was significantly less than model and Nogo A antibody alone groups （P 〈 0.05）; while there was no significant difference between model and Nogo A antibody alone groups （P 〉 0.05）. Ten weeks after injury, microsphere group showed a significantly greater expression of neurofilament 200 than model and Nogo A antibody alone groups （P 〈 0.05）; while no significant difference was found between model and Nogo A antibody alone groups （P 〉 0.05）. At postoperative weeks 5 and 6, the score of BBB locomotor rating scale in microsphere group was significantly greater than the model group （P 〈 0.05）, and at postoperative weeks 7 10, the score was much greater than model and Nogo A antibody alone groups （P 〈 0.05）. CONCLUSION： Nogo A antibody delayed-release microspheres decreased Nogo A expression, increased neurofilament 200 expression in the injured spinal cord of rats, and promoted recovery of motor function through sustained drug release over a long-term period.

Poly(lactic acid)-aspirin microspheres prepared via the traditional and improved solvent evaporation methods and its application performances

Drug-loaded microspheres are significant for the development of modern pharmaceutical products. It is well known that the taken of aspirin for long-term increases the risk of serious gastrointestinal complications, therefore a controllable delivery of aspirin is of importance to lighten those side effects. In this work, poly(lactic acid)(PLA) was chosen as the carrier to prepare PLA-aspirin microspheres by using the traditional and the improved solvent evaporation methods. It was found that no matter which experimental condition was, the encapsulation efficiency of aspirin was higher by using the improved method than that of the traditional method. Specifically, when the concentration of polyvinyl alcohol = 1%(mass),the polymer concentration = 1:20, the oil/water rate = 1:2.5, PLA-aspirin microspheres were obtained via the improved method with a high yield of 82.83%(mass) and an encapsulation efficiency of 44.09%. PLAaspirin microspheres were then prepared continuously using the improved method, which further enhanced the encapsulation efficiency to 54.56%. Approximate 85% aspirin released from microspheres within 7 days. Obvious degradation which was represented by reduction on hardness was observed by soaking microspheres in PBS for 60 days. This work is of interest because it provides a continuous route to prepare PLA-aspirin microspheres continuously with a high drug encapsulation efficiency.

A novel artificial nerve graft for repairing longdistance sciatic nerve defects:a self-assembling peptide nanofiber scaffold-containing poly (lactic-co-glycolic acid) conduit

In this study, we developed a novel artificial nerve graft termed self-assembling peptide nanofiber scaffold （SAPNS）-containing poly（lactic-co-glycolic acid） （PLGA） conduit （SPC） and used it to bridge a 10-mm-long sciatic nerve defect in the rat. Retrograde tracing, behavioral testing and histomorphometric analyses showed that compared with the empty PLGA conduit implantation group, the SPC implantation group had a larger number of growing and extending axons, a markedly increased diameter of regenerated axons and a greater thickness of the myelin sheath in the conduit. Furthermore, there was an increase in the size of the neuromuscular junction and myofiber diameter in the target muscle. These findings suggest that the novel artificial SPC nerve graft can promote axonal regeneration and remyelination in the transected peripheral nerve and can be used for repairing peripheral nerve injury.

Dorsal root ganglion-derived Schwann cells combined with poly(lactic-co-glycolic acid)/chitosan conduits for the repair of sciatic nerve defects in rats

Schwann cells, nerve regeneration promoters in peripheral nerve tissue engineering, can be used to repair both the peripheral and central nervous systems. However, isolation and puriifcation of Schwann cells are complicated by contamination with ifbroblasts. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. In this study, we collected dorsal root ganglia from neonatal rats from which we obtained highly puriifed Schwann cells using serum-free melanocyte culture medium. The purity of Schwann cells （〉95%） using our method was higher than that using standard medium containing fetal bovine serum. The obtained Schwann cells were implanted into poly（lactic-co-glycolic acid）/chi-tosan conduits to repair 10-mm sciatic nerve defects in rats. Results showed that axonal diameter and area were signiifcantly increased and motor functions were obviously improved in the rat sciatic nerve tissue. Experimental ifndings suggest that serum-free melanocyte culture medium is conducive to purify Schwann cells and poly（lactic-co-glycolic acid）/chitosan nerve conduits combined with Schwann cells contribute to restore sciatic nerve defects.

PREPARATION OF POLY(ETHYLENEGLYCOL-co-ACRYLIC ACID) MICROSPHERES WITH DIVINYLBENZNE AS CROSSLINKER BY DISTILLATION-PRECIPITATION POLYMERIZATION

Monodisperse poly（poly（ethyleneglycol） methyl ether acrylate-co-acrylic acid） （poly（PEGMA-co-AA）） microspheres were prepared by distillation-precipitation polymerization with divinylbenzene （DVB） as crosslinker with 2,2＇- azobisisobutyronitrile （AIBN） as initiator in neat acetonitrile without stirring. Under various reaction conditions, four distinct morphologies including the sol, microemulsion, microgels and microspheres were formed during the distillation of the solvent from the reaction system. A 2D morphological map was established as a function of crosslinker concentration and the polar monomer AA concentration, in comonomer feed in the transition between the morphology domains. The effect of the covalent crosslinker DVB on the morphology of the polymer network was investigated in detail at AA fraction of 40 vol%. The ratios of acid to ethylene oxide units presenting in the comonomers dramatically affected the polymer-polymer interaction and hence the morphology of the resultant polymer network. The covalent crosslinking by DVB and the hydrogen bonding crosslinking between two acid units as well as between the acid and ethylene oxide unit played key roles in the formation of monodisperse polymer microspheres.

Evaluation of in vitro and in vivo immunostimulatory activities of poly(lactic-co-glycolic acid) nanoparticles loaded with soluble and autoclaved Leishmania infantum antigens: A novel vaccine candidate against visceral leishmaniasis

Objective: To prepare and characterize poly lactic-co-glycolic acid(PLGA) nanoparticles loaded with soluble leishmanial antigen or autoclaved leishmanial antigen and explore in vitro and in vivo immunogenicity of antigen encapsulated nanoparticles. Methods: Water/oil/water double emulsion technique was employed to synthesize PLGA nanoparticles, and scanning electron microscopy, Fourier transform infrared spectroscopy and Zeta-potential measurements were used to identify the characteristics of nanoparticles. Cytotoxicity of synthetized nanoparticles on J774 macrophage were investigated by MTT assays. To determine the in vitro immunostimulatory efficacies of nanoparticles, griess reaction and ELISA was used to measure the amounts of NO and cytokines. During the in vivo analysis, Balb/c mice were immunized with vaccine formulations, and protective properties of nanoparticles were measured by Leishman Donovan unit in the liver following the infection. Cytokine levels in spleens of mice were determined by ELISA. Results: MTT assay showed that neither soluble leishmanial antigen nor autoclaved leishmanial antigen encapsulated nanoparticles showed cytotoxicity against J774 macrophage cells. Contrary to free antigens, both autoclaved leishmanial antigen-nanoparticle and soluble leishmanial antigen-nanoparticle formulations led to a 10 and 16-fold increase in NO amounts by macrophages, respectively. Leishman Donovan unit calculations revealed that soluble leishmanial antigen-nanoparticles and autoclaved leishmanial antigen-nanoparticles yielded 52% and 64% protection against visceral leishmaniasis in mouse models. Besides, in vitro and in vivo tests demonstrated that by increasing IFN-γ and IL-12 levels and inhibiting IL-4 and IL-10 secretions, autoclaved leishmanial antigen-nanoparticles and soluble leishmanial antigennanoparticles triggered Th1 immune response. Conclusions: Both autoclaved leishmanial antigen-nanoparticles and soluble leishmanial antigen-nanoparticles formulations provide exceptional in vitro and in vivo immunostimulatory activities. Hence, PLGA-based antigen delivery systems are recommended as potential vaccine candidates against visceral leishmaniasis.

Poly(lactic-co-glycolic acid) conduit for repair of injured sciatic nerve A mechanical analysis

Tensile stress and tensile strain directly affect the quality of nerve regeneration after bridging nerve defects by poly（lactic-co-glycolic acid） conduit transplantation and autogenous nerve grafting for sciatic nerve injury. This study collected the sciatic nerve from the gluteus maximus muscle from fresh human cadaver, and established 10-mm-long sciatic nerve injury models by removing the ischium, following which poly（lactic-co-glycolic acid） conduits or autogenous nerve grafts were transplanted. Scanning electron microscopy revealed that the axon and myelin sheath were torn, and the vessels of basilar membrane were obstructed in the poly（lactic-co-glycolic acid） conduit-repaired sciatic nerve following tensile testing. There were no significant differences in tensile tests with autogenous nerve graft-repaired sciatic nerve. Following poly（lactic-co-glycolic acid） conduit transplantation for sciatic nerve repair, tensile test results suggest that maximum tensile load, maximum stress, elastic limit load and elastic limit stress increased compared with autogenous nerve grafts, but elastic limit strain and maximum strain decreased. Moreover, the tendencies of stress-strain curves of sciatic nerves were similar after transplantation of poly（lactic-co-glycolic acid） conduits or autogenous nerve grafts. Results showed that after transplantation in vitro for sciatic nerve injury, poly（lactic-co-glycolic acid） conduits exhibited good intensity, elasticity and plasticity, indicating that poly（lactic-co-glycolic acid） conduits are suitable for sciatic nerve injury repair.

Controlled release of cisplatin and cancer cell apoptosis with cisplatin encapsulated poly(lactic-co-glycolic acid) nanoparticles

The goal of the present study is to utilize cis-diamminedichloroplatinum (cisplatin) loaded polymer nanoparticles (NPs) to give a controlled, extended, and local drug therapy for the treatment of cancer. We have used biodegradable and biocompatible poly(lactic-co-glycolic acid) (PLGA) to prepare the NPs by adjusting the double emulsion technique using poly(vinylalcohol) as a surface active agent. The PLGA NPs were characterized for particle size and shape, controlled release of cisplatin, and degradation. Cisplatin solubility in deionized water was increased up to 4 mg/mL by simply changing the solution parameters. Cisplatin encapsulated NPs were incubated in phosphate buffered saline (PBS) at 37?C to study the release kinetics of cisplatin. Cisplatin was released in a sustained manner with less than 20% release during a 3-day period followed by 50% release during a 21-day period. A degradation study of PLGA NPs demonstrated the loss of spherical shape during a 21-day period. We also examined the cisplatin sensitive A2780 cell apoptosis when cells were incubated with cisplatin encapsulated PLGA NPs. A large number of cell apoptosis occurred as a result of cisplatin release from the PLGA NPs. These results suggest that cisplatin encapsulated PLGA NPs can be used to treat the cancer cells by injecting them into a localized site minimizing the side effects.

A porous poly(lactic-co-glycolic acid) scaffold induces innervation in a rabbit model of disc degeneration following annular injury

BACKGROUND： A degradable poly（lactic-co-glycolic acid） （PLGA） scaffold has been used to construct a degradable porous scaffold. This template can simulate the in vivo microenvironment and promote tissue formation. OBJECTIVE： To observe the histopathological changes during degeneration and regeneration of the intervertebral disc, and to analyze the effects of a PLGA scaffold on nerve fiber ingrowth into the lesion in vivo. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed at the Orthopaedic Laboratory, Clinic Medical Research Institution, Sir Run Run Shaw Hospital, Zhejiang University, from December 2007 to July 2008. MATERIALS： PLGA （China Textile Academy）; growth-associated protein-43 （Life-span, USA）; and protein gene product 9.5 antibody （AbD, United Kingdom） were used in this study. METHODS： Three consecutive segments of the intervertebral disc of thirty-two healthy adult male New Zealand rabbits were exposed, comprising L3-4, L4-5 and L5-6. Experimental intervertebral disc （L4-5 and L5-6） models were established by two different methods. In the test （trephine ＋ scaffold） group, a 5-mm deep hole was drilled into the annulus fibrosus using a 3-mm diameter trephine, and the PLGA scaffold was implanted into the hole. In the acupuncture group, the remaining experimental intervertebral disc annulus fibrosus was damaged using a 16G needle at a depth of 5 mm. The L3-4 disc served as a control. MAIN OUTCOME MEASURES： Intervertebral disc degeneration was assessed using radiography, magnetic resonance imaging, and histological examination at various time points post-surgery. Nerve fiber ingrowth into the degenerated intervertebral disc was observed using immunohistochemical staining for growth-associated protein-43 and protein gene product 9.5. RESULTS： Compared with the normal controls, the heights of the damaged intervertebral discs were decreased, and T2 signal intensity was decreased in the test and acupuncture groups 2 weeks post-surgery. Intervertebral disc degeneration was faster in the test group than in the acupuncture group. PLGA was coated with newly formed tissue, gradually degraded, and absorbed, and could induce tissue ingrowth deep into the annulus fibrosus. Results of immunohistochemical staining showed that nerve fibers were distributed in newly formed tissue in the test group, and in the superficial layer or surrounding scar tissue in the acupuncture group. CONCLUSION： A porous PLGA scaffold provides an important biological channel to induce nerve fiber ingrowth deep into the degenerated intervertebral disc.

A Study of Surface Modification of Poly(lactic-co-glycolic) Acid Using Argon Ion Irradiation

The effect of Argon ion irradiation to the surface properties of poly(lactic-co-glycolic) acid (PLGA) was studied. A beam of 170 keV Argon ions was implanted at different fluencies (1 × 1012, 1 × 1013, 1 × 1014, and 1 × 1015 ions/cm2). X-ray photoelectron spectroscopy (XPS) was used to analyze the evolution of the bonding microstructure of PLGA due to irradiation. Surface morphology was monitored using atomic force microscopy (AFM). AFM analysis shows that film roughness increased to maximum at the dose of 1 × 1014 ions/cm2 where the formations of hillocks were also detected. Hydrophilicity of PLGA is important for their applications in biomedicine such as bioscaffolds. Hydrophilicity was monitored using water contact angle measurements for both unmodified and ion-modified PLGA. It was observed that hydrophilicity of PLGA changes with the ion irradiation. This demonstrates that ion irradiation can be an alternative approach to control hydrophilicity of PLGA. PLGA scaffolds modified with ion irradiation could therefore be more suitable for the biomedical applications.

Preparation of poly(lactide-co-glycolide)microspheres and evaluation of pharmacokinetics and tissue distribution of BDMC-PLGA-MS in rats

The aim of the present study was to develop a novel long-acting Poly(lactic-co-glycolic acid)(PLGA)-based microspheres formulation of Bisdemethoxycurcum(BDMC) by emulsionsolvent evaporation method. Meanwhile, the effects of the volume ratio of the dispersed phase and continuous phase, the concentration of PLGA and PVA, the theoretical drug loading and stirring speed were investigated. The mean diameter of the microspheres was 8.5 μm and the size distribution was narrow. The encapsulation efficiency(EE) and drug loading efficiency(DLE) of BDME loaded PLGA microspheres(BDMC-PLGA-MS) was 94.18% and 8.14%,respectively. In an in vitro study of drug release, it can be concluded that the BDMC-PLGAMS exhibited sustained and long-term release properties for 96 h. Stability studies suggested that the microspheres we prepared had a very good stability. Furthermore, the results of an in vivo study indicated that the BDMC-PLGA-MS had sustained release effect and was mainly distributed in the lung tissue, and less distribution in other tissues, which indicated that microspheres could be an effective parenteral carrier for the delivery of BDMC in lung cancer treatment.

Preparation of Tolterodine Metabolite Loaded Biodegradable PLGA Microspheres

Biodegradable polymer poly（lactic-co-glycolic acid）（PLGA） was used to encapsulate the pharmacological activity metabolite of tolterodine by means of O/W emulsion solvent evaporation method via homogenization in the emulsification process. The influences of preparation parameters were investigated. The results indicate that increa- sing PLGA concentration from 15% to 40% made the encapsulation efficiency of 5-hydroxymethyl derivative of tol- terodine（5-HMT） increased from 55.39% to 76.32%, and the particle size increased from 34.33 μm to 70,78 lain. In addition, when homogenization speed increased from 850 r/min to 2300 r/min, both particle size and encapsulation efficiency of microspheres decreased. An increase in the volume of aqueous phase led to higher encapsulation efficiency and bigger particle size. Increasing temperature made encapsulation efficiency and particle size change significantly. While reaction temperature increased from 20 ℃ to 50 ℃, the encapsulation efficiency decreased from 70.44% to 24.07%, and particle size increased from 38.66 μm to 69.38 μm. High reaction temperature（over 40 ℃） may lead to porous surface of microspheres. Porous surface, encapsulation efficiency and particle size influenced on the in vitro release of 5-HMT together.

Electrospun and woven silk fibroin/poly(lactic-coglycolic acid) nerve guidance conduits for repairing peripheral nerve injury

We have designed a novel nerve guidance conduit（NGC） made from silk fibroin and poly（lactic-co-glycolic acid） through electrospinning and weaving（ESP-NGCs）. Several physical and biological properties of the ESP-NGCs were assessed in order to evaluate their biocompatibility. The physical properties, including thickness, tensile stiffness, infrared spectroscopy, porosity, and water absorption were determined in vitro. To assess the biological properties, Schwann cells were cultured in ESP-NGC extracts and were assessed by morphological observation, the MTT assay, and immunohistochemistry. In addition, ESP-NGCs were subcutaneously implanted in the backs of rabbits to evaluate their biocompatibility in vivo. The results showed that ESP-NGCs have high porosity, strong hydrophilicity, and strong tensile stiffness. Schwann cells cultured in the ESP-NGC extract fluids showed no significant differences compared to control cells in their morphology or viability. Histological evaluation of the ESP-NGCs implanted in vivo indicated a mild inflammatory reaction and high biocompatibility. Together, these data suggest that these novel ESP-NGCs are biocompatible, and may thus provide a reliable scaffold for peripheral nerve repair in clinical application.

Synthesis,Characterization and Application of Poly(lactic-co-glycolic acid)with a Mass Ratio of Lactic to Glycolic Segments of 52/48

Poly(lactic-co-glycolic acid)(PLGA)is one of the most representative degradable copolymers and promising drug carriers.In the current paper,the PLGAs with a lactic acid/glycolic acid(LA/GA)molar ratio of 52/48 and various molecular weights were prepared by a melting method.The molecular weight,molecular weight distribution,and thermal stability were determined by 1H NMR and thermogravimetric analysis methods.The results demonstrated that PLGAs with the fixed LA/GA molar ratio(52/48),different molecular weights,and narrow molecular weight distribution could be obtained by solely altering the reaction time.The PLGA films were prepared,and their properties including micro-structure,mechanical property,in-vitro cytotoxicity,and biodegradability were characterized.In combination with the homogeneous microstructures and mechanical properties,the drug-loading and releasing properties of PLGA3.2 films were investigated.The results show that PLGA3.2 film with an LA/GA molar ratio of 52/48 is a promising curcumin carrier.

A combination of chondroitinase ABC,glial cell line-derived neurotrophic factor,and Nogo A antibody delayed-release microspheres for the treatment of spinal cord injury

The purpose of this study was to evaluate the effect of poly（lactide-co-glycolic acid） delayed-release microspheres,which were prepared using glial cell line-derived neurotrophic factor（GDNF）,on the delayed-release,controllability,and protection of GDNF activity.The present study is the first to combine chondroitinase ABC,GDNF,and Nogo A antibody delayed-release microspheres for the treatment of spinal cord injury.Results show that the combined therapy of chondroitinase ABC,GDNF,and Nogo A antibody microspheres can increase the immunoreaction of neurofilament 200 in the injured spinal cord,and this therapeutic effect was better than chondroitinase ABC,GDNF,or Nogo A antibody microspheres administered singularly.

Dexamethasone-Loaded PLGA Microspheres Incorporated PLLA/PLGA/PCL Composite Scaffold for Bone Tissue Engineering

The combination of micro-carriers and polymer scaffolds as promising bone grafts have attracted considerable interest in recent decades.The poly(L-lactic acid)/poly(lactic-co-glycolic acid)/polycaprolactone(PLLA/PLGA/PCL)composite scaffold with porous structure was fabricated by thermally induced phase separation(TIPS).Dexamethasone(DEX)was incorporated into PLGA microspheres and then loaded on the PLLA/PLGA/PCL scaffoldtopreparethedesiredcompositescaffold.The physicochemical properties of the prepared composite scaffold were characterized.The morphology of rat bone marrow mesenchymal stem cells(BMSCs)grown on scaffolds was observed using scanning electron microscope(SEM)and fluorescence microscope.The resultsshowedthatthePLLA/PLGA/PCLscaffoldhad interconnected macropores and biomimetic nanofibrous structure.In addition,DEX can be released from scaffold in a sustained manner.More importantly,DEX loaded composite scaffold can effectively support the proliferation of BMSCs as indicated by fluorescence observation and cell proliferation assay.The results suggested that the prepared PLLA/PLGA/PCL composite scaffold incorporating drug-loaded PLGA microspheres could hold great potential for bone tissue engineering applications.

MMAE-loaded PLGA nanomedicine with improved biosafety to achieve efficient antitumor treatment

Monomethyl auristatin E(MMAE)is a derivative of the marine peptide Dolastatin 10,which has therapeutic effects against various cancers according to its antimitotic activity in multiple clinical trials.The antibody drug conjugate(ADC)of MMAE is currently used in clinical practice.However,the safety issues of MMAE-based ADC,such as high drug toxicity and poor bioavailability,still exist when using it for anticancer therapy.A sustained release of drug delivery approach should be used to reduce toxicity and achieve sufficient anticancer effects.Herein,PLGA-b-PEG 2000 with excellent biocompatibility and slow degradation ability was adopted to construct MMAE-loaded nanoparticles for safe and effective chemotherapy.The sustained release effect and the immunogenic cell death(ICD)effect of PLGA-MMAE nanoparticles were assessed by in vitro experiments.The PLGA-MMAE nanoparticles effectively accumulated in the tumor through the enhanced permeability and retention(EPR)effect,inducing cell apoptosis and causing a certain degree of immune response.The sustained drug release of PLGA-MMAE improved the bioavailability and effectively reduced the toxicity and development of the tumor compared to the effect of free MMAE or ADC.Overall,this study provides a safe and effective chemotherapeutic approach,as well as a simple and effective synthetic process for MMAE-based nanoparticles,improving their therapeutic efficacy and safety.

运载双层抗原蛋白的CS-PLGA制剂制备及其表征鉴定

目的制备运载双层抗原的CS-PLGA微球制剂,并对其进行相关表征鉴定。方法通过复乳法结合溶剂挥发法制备得到多孔微球后,在4℃水浴条件下装载抗原,利用抗原浓度梯度介导的物理扩散促进抗原进入微球内部,并通过静电作用结合在微球外表面,形成内外双层抗原运载。根据聚乳酸-羟基乙酸共聚物(PLGA)材料玻璃转化温度这一特性,促进多孔微球表面孔道在48℃条件下发生愈合,使其形成闭合的微球制剂,再将得到的微球制剂与壳聚糖溶液混悬镀层,进行阳离子修饰,逆转微球表面负性电位。通过扫描电子显微镜、动态光散射粒度仪等检测微球形态、粒径分布和电位变化,采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定抗原是否装载至微球制剂中,采用荧光标记的BSA和荧光标记的PLGA材料进行激光共聚焦显微镜观察抗原装载后的分布情况,通过BCA法检测微球制剂的包封率和载药率。结果扫描电镜和光学显微镜结果显示多孔微球成孔良好,粒径大小为(73.94±0.81)nm,多分散性指数为0.038±0.004。Zeta电位由负转正说明壳聚糖已被成功镀层至微球表面。经过SDS-PAGE、激光共聚焦显微镜等证实BSA已被成功运载。经micro BCA试剂盒检测后多孔微球包封率为(3.01±0.04)%,载药率为(1.50±0.02)%。结论成功制备得到运载双层抗原的CS-PLGA制剂,为后续缓控释制剂研究提供新思路。

基质细胞衍生因子1修饰左旋聚乳酸多孔微球促进软骨细胞增殖和组织形成

背景:二维培养条件下的软骨细胞增殖及表型维持受限,多孔微球作为支架材料可提供三维培养环境,以更好地模拟体内生长条件。基质细胞衍生因子1是有强趋化效力的稳态细胞因子,能够促进细胞的黏附与增殖。目的:明确接枝基质细胞衍生因子1左旋聚乳酸多孔微球对软骨细胞生物学特性及软骨组织形成的影响。方法:(1)体外验证不同质量浓度基质细胞衍生因子1对兔软骨细胞增殖、迁移、表型维持的影响。(2)采用复乳法制备左旋聚乳酸多孔微球,利用碳二亚胺法将基质细胞衍生因子1接枝于左旋聚乳酸多孔微球上,通过酶联免疫吸附实验及孵育基质细胞衍生因子1特异荧光抗体验证接枝情况。(3)将兔软骨细胞分别接种于左旋聚乳酸多孔微球、接枝基质细胞衍生因子1左旋聚乳酸多孔微球上,检测细胞增殖与黏附。(4)在裸鼠背部皮下分别植入甲基丙烯酰胺基明胶-软骨细胞复合体(对照组)、左旋聚乳酸多孔微球-甲基丙烯酰胺基明胶-软骨细胞复合体(多孔微球组)、接枝基质细胞衍生因子1左旋聚乳酸多孔微球-甲基丙烯酰胺基明胶-软骨细胞复合体(多孔微球修饰组),8周后取材,分别进行组织学染色与成软骨相关基因qRT-PCR检测。结果与结论:(1)相较于0,1 000 ng/mL基质细胞衍生因子1,500 ng/mL基质细胞衍生因子1可促进软骨细胞的增殖与迁移,提升软骨细胞内Ⅱ型胶原、弹性蛋白、增殖细胞核抗原、Bcl-2 mRNA表达;(2)基质细胞衍生因子1成功接枝于左旋聚乳酸多孔微球上,接枝率为93.75%;(3)相较于左旋聚乳酸多孔微球,接枝基质细胞衍生因子1左旋聚乳酸多孔微球可促进软骨细胞的增殖、黏附;(4)裸鼠皮下植入8周后,相较于对照组、多孔微球组,多孔微球修饰组具有更明显的软骨陷窝结构、更丰富的软骨特异性基质和Ⅱ型胶原沉积,弹性蛋白、Ⅱ型胶原、增殖细胞核抗原、Bcl-2 mRNA表达升高。结果表明:接枝基质细胞衍生因子1左旋聚乳酸多孔微球有利于软骨细胞的黏附、增殖、表型维持以及体内软骨组织形成。

万古霉素缓释微球的制备及其质量评价

目的制备质量稳定、安全有效的载万古霉素缓释微球,评价其质量特征。方法采用复乳溶剂挥发法将聚乳酸(polylactic acid,PLA)和聚乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid),PLGA]按3种不同比例混合制备万古霉素微球进行筛选;利用单因素实验,研究不同浓度聚乙烯醇(polyvinyl alcohol,PVA)、内外水相比例对微球粒径、包封率、载药量的影响后并制备出优选的载万古霉素的PLA/PLGA缓释微球;对其体内外抗多重耐药菌耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)进行药效学研究。采用单因素方差分析。结果内外水相比和PVA浓度对万古霉素的包封率和载药量影响不大(P>0.05),但微球中PLA和PLGA质量比对万古霉素的包封率和载药量差异有统计学意义(P<0.0001),纯PLA时微球平均粒径[(41.95μm)]、包封率[(28.87±0.32)%]和载药量[(17.80±0.21)%]最大。万古霉素微球对MRSA 252的抗菌活性高于其水溶液(最小抑菌浓度0.391 ng/L比0.781 ng/L)(P<0.0001)。万古霉素微球治疗受损皮肤感染MRSA 252比相同浓度的万古霉素水溶液具有更满意的杀菌活性(P<0.05)。万古霉素微球对感染MRSA 252小鼠的伤口治愈效果比万古霉素水溶液更好。结论通过优选获得的微球处方,制备的PLA/PLGA缓释微球可实现万古霉素药物的局部可控释放,通过调节PLA和PLGA在共混物中的比例提高药物包封率和载药量、定制降解速率、改变药物释放,是有效对抗金黄色葡萄球菌感染的制剂。