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Objective To investigate the effect of polylactic-co-glycolic acid (PLGA)/graphene oxide (GO) nanofibers combined with brain derived neurotrophic factor (BDNF) on neural stem cells (NSCs) proliferation and differentiation as well as on the spinal cord injury repair.Methods PLGA/GO nanofibers were manufactured and absorbed with BDNF,and the microstructure of PLGA/GO nanofibers was observed by scanning electron microscope.The loading efficiency and release curve of BDNF on PLGA/GO nanofibers were measured by ELISA.NSCs were implanted on the surface of PLGA/GO and PLGA/GO/BDNF nanofibers.The absorbance values of each group were measured by MTT method,and the expression of Tuj-1 was observed by immunofluorescence and PCR.A total of 30 female SD rats were divided into control group (n =10),PLGA/GO group (n =10) and PLGA/GO/BDNF group (n =10) according to random number table.T9 spinal cord tissue was cut by Venus scissors to establish spinal cord hemisection injury model of rats.PLGA/GO and PLGA/GO/BDNF nanofibers were implanted onto the surface of injury site.BBB score was used to assess the motion functional recovery of the rats at 1,7,14 and 28 days after operation.Immunofluorescence staining of neuron specific nucleoprotein (NeuN)and glial fibrillary acidic protein (GFAP) were performed to observe the expressions of neurons and astrocytes at the injured site respectively one month after injury.Results The PLGA/GO nanofibers showed an irregular smooth fiber-like structure,and the average fiber diameter was (987.5 ± 176.3)nm.NSCs could differentiate into neurons on the nanofibers.The result of ELISA showed loading rate of BDNF on PLGA/GO nanofibers was about 47.5％.The release curve showed that BDNF was first released about 30％ on the first day and then about 60％ on the 21st day.The results of MTT and PCR showed that optical density value and Tuj-1 gene expression in the PLGA/GO/BDNF group were significantly higher than those in the PLGA/GO group (P ＜ 0.05).The animal experiment results showed that the BBB score of PLGA/GO/BDNF group was (15.3 ±0.7) points at 28 days after injury,which was significantly higher than that of the injury control group [(11.8 ± 0.8) points] and that of PLGA/GO group [(12.7 ±0.8) points] (P ＜ 0.05).Immunofluorescence results showed that the expression of NeuN in PLGA/GO/BDNF group was 13.7 ± 2.2,significantly higher than that in injury control group (4.3 ± 2.9) (P ＜0.05),and the expression of GFAP in PLGA/GO group was (25.6 ± 4.3) ％ significantly lower than that in injury control group [(38.5 ± 6.2) ％] and PLGA/GO group [(36.7 ± 7.3) ％] (P ＜ 0.05).Conclusion PLGMGO nanofibers combined with BDNF can effectively promote the proliferation and neuron differentiation of NSCs in vitro and repair spinal cord injury in vivo through orthotopic transplantation at the injury site.
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Objective To prepare a novel loaded NdFeB and Fe3O4 liquid-solid phase inversion poly (polylactic-co-glycolic acid,PLGA) in situ implant (PLGA 60％ NdFeB-20％ Fe3O4) for ultrasound-guided intratumoral injection,and to investigate its intensity of magnetism and therapeutic efficiency of nude mice bearing MDA-MB-231 breast cancer.Methods PLGA-60 ％ NdFeB-20 ％ Fe3O4 in situ implant was prepared.The microstructure of PLGA-60 ％ NdFeB-20 ％ Fe3O4 was tested with scanning electron microscope (SEM).Magnetometer was used to qualify the intensity of magnetism.For the in vitro assay,60 μl solid PLGA-60％ NdFeB-20％ Fe3O4 implant was put in the center of the electromagnetic induction heating coil.Infrared thermal video of the tubes was simultaneously recorded.The size of 2 cm × 2 cm× 2 cm fresh ex vivo bovine liver was prepared,and then 60 μl PLGA-60％ NdFeB-20％ Fe3O4 was injected into it.Similarly,ex vivo bovine liver was heated by the above intermittent time for 1,2,3,4 minutes,respectively,and the ablation volume was calculated.Finally,nude mice were equally divided into treatment group and control group.Mice in treatment group were discontinuously heated for 3 min after being injected with the above mentioned implant,while CEUS was performed before and after heating to observe the blood perfusion in the tumor.One nude mouse was executed on the next day in each group,then pathological sections and HE staining of tumor tissue were taken,whereas the growth of the remaining nude mice were observed daily.Results SEM showed the implant with rough and porous surface.The magnetism increased with the volume of material.The tube and bovine liver experiments showed that the PLGA-60 ％ NdFeB-20％ Fe3O4 generated heat efficiently.The bovine liver ablation experiment showed that the range of ablation of 60 μl PLGA-60 ％ NdFeB-20％ Fe3O4 implant reached (2.34±0.25)cm3 after 3 min heating.In vivo experiments showed that the tumor tissue began to form a scab on the 2nd day,and the scab began to desquamate on the 20th day.CEUS showed the original predominant enhancement disappeared significantly after the treatment.HE staining proved that cancer cells had coagulative necrosis,whereas tumors in control group became bigger.Conclusion PLGA-60％ NdFeB-20％ Fe3O4 in situ implant can produce obvious thermal effect under high frequency alternating magnetic field,therefore can be effectively ablated with nude mice MDA-MB-231 breast cancer during time interval heating.
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Objective To study the best formulation and technology of quercetin-loaded polylactic-co-glycolic acid-D-α-tocopheryl polyethylene glycol 1000 succinate(PLGA-TPGS) nanoparticles(QPTN) with QT as model drug and PLGA-TPGS as polymer materials by orthogonal tests,and to investigate the in vitro stability of QPTN. Methods To ensure the best formula-tion and technology for preparing QPTN,single-factor test was established to determine the influence of the ratio of quercetin to PLGA-TPGS,the concentration of TPGS as emulsifiers,the ultrasonic power and ultrasonic time to the particle size,drug loading (DL) and entrapment efficiency(EE).According to single-factor test,the factor levels of nanoparticles were set to select the best prescription and technology of QPTN by orthogonal test.The stability of QPTN was examined using the effecting factor test,accel-eration test and long-term test. Results The best formulation and technology of QPTN was that the ratio of quercetin to PLGA-TPGS was 3:10 (W:W) with 0.05% TPGS as emulsifier,the mixed solution was sonicated for 6 min at 200 W.The average particle size,DL and EE of QPTN prepared under the conditions described above were (155.4 ± 2.7) nm,(21.6 ± 1.5)% and (93.7±2.9)% (n=6),respectively.In the in vitro stability test,QPTN showed a good stability at high temperature,high humidity and strong light condition. Conclusion The best formulation and preparation technology of QPTN was selected.QPTN got small particle size,high DL and EE,and good in vitro stability.
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OBJECTIVE: To develop the accelerated release test method of docetaxel capsule tension ring and optimize its formulation. METHODS: The effects of ethanol concentration in the medium (0%, 5%, 10%) and temperature (37 and 45 ℃) on the release rate of docetaxel capsule tension ring were investigated, and the correlation regression equation between the accelerated release rate and the long-term release was established. Then, the best prescription was screened out with the accelerated release test method. RESULTS: The drug release rate of these preparations could be increased by four times under the accelerated conditions, i.e., using pH 7.4 phosphate buffer solution containing 5% ethanol as the release medium and operating at (45±0.5)℃. The accelerated release and long-term release were fitted the best using binomial model (y=0.004 6x2+0.437 4x+9.683 7, r2=0.998 4). The preparation using PLGA5050 (60K-100K=1:1, W/W) with drug-loading of 30% released drug stably and sustainedly for 30 d, and its release behavior was consistent with Peppas equation drug release model. CONCLUSION: Accelerated release testing can be employed as a rapid screening test to facilitate the formulation optimization of docetaxel capsule tension ring. This preparation has been proven to have sustained-release characteristics, suggesting a good clinical application prospect.
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Objective:To use polylactic-co-glycolic acid(PLGA) as vector material to prepare the compound total flavonoids of Rhizoma Drynariae(TFRD) and total flavonoids of Chrysanthemum(TFC) sustained release microcapsule TF-PLGA microcapsules,and to investigate the the best preparation technique of TF-PLGA microcapsules and their sustained release characteristics in vitro.Methods:The TF-PLGA microcapsules were prepared with TFRD,TFC,and PLGA by emulsifying-solvent evaporation technique under certain conditions.With the encapsulation efficiency(EE) as the evaluation indicator,the optimal formulation was verified by single factor experiment and orthogonal design;the general morphology,the particle size and distribution of the microcapsules were observed by light microscope(LM) and scanning electron microscope(SEM);the cumulative drug release rate of TF-PLGA microcapsules was detected by constant temperature commotion method in vitro and the release curves of the TF-PLGA were drawn.Results:The optimal prescription was as follows:the concentration of PLGA was 140 g·L-1,oil phase volume was 1.4 mL,emulsifying speed was 900 r·min-1,emulsifying time was 5 min,the average EE was(83.89±2.30)%,and the average drug loading rate(DL) was(5.90±0.07)%.The LM and SEM resluts showed that the TF-PLGA microcapsules presented as round ball,the average particle size was(44.34±14.68)μm,and the distribution was relatively narrow.The drug release in vitro results showed that the initial drug release rate(24 h)was about 40%,and the cumulative drug release rate was over 90% after 50 d.Conclusion:The TF-PLGA sustained release microcapslue has better drug-loaded and sustained-release effects with simple preparation technique and better repeatability.
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OBJECTIVE:To study the inhibitory effects of sinomenine(SIN)-loaded polylactic-co-glycolic acid-D-α-tocopherol polyethylene glycol 1000 succinate(PLGA-TPGS)nanoparticles(SPTN)on the proliferation of HCa-F cells in lymph tubes and ec-topic transplantation tumors in mice. METHODS:HCa-F cell suspension were incubated with normal saline,5-fluorouracil(FS), sinomenine solutions (SS),sinomenine PLGA nanoparticles (SPN) and SPTN,with concentration of 80 μg/ml. The cells were marked with CFSE,and then were injected with suspension 50 μl via one footpad of mice. Inhibitory effect of above suspensions on the proliferation of HCa-F in lymph tubes of mice was observed by fluorescence inverted microscope at 3,6,9,12,24 h(n=15). Mice were divided into normal control group,blank PLGA-TPGS nanoparticles (EPTN) group,normal saline group,SPTN group,SPN group,SS group and FS group with 10 mice in each group. The latter 5 groups were injected with relevant medicine 15 mg/kg,once a day,via tail vein for consecutive 10 days after the model of HCa-F cells-bearing ectopic transplantation tumor mice was established. The serum content of ALT,AST,γ-glutamyltransferase(γ-GT),ALB and T-BIL were determined;solid tu-mors were taken,measured and weighed,and the inhibitory rate of tumor was also calculated. RESULTS:The inhibitory effect of above solution on the proliferation of HCa-F cells in descending order was as follows:SPTN>SPN>FS>SS>normal saline. Com-pared with normal saline group,the serum levels of ALT,AST,γ-GT and TBIL of SPTN group,SPN group,SS group and FS group decreased,while ALB level increased(P<0.05);the amount of tumor volume increase and tumor weight in SPTN group, SPN group and FS group decreased significantly (P<0.05). The inhibitory rate of tumor in 3 groups were 49.62%,40.53% and 33.90%. CONCLUSIONS:SPTN can inhibit the proliferation of HCa-F cells in lymph tubes of mice,and can improve HCa-F cells-bearing ectopic transplantation tumor in mice. It is better than SPN and FS.
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Objective To observe the protective effect of brain-derived neurotrophic factor-polylactic-co-glycolic acid (BDNF-PLGA) sustained release microspheres on the peripheral nerve injury of rats. Methods BDNF loaded PLGA microspheres were prepared by double emulsion solvent evolution method. A total of 48 adult SD rats were divided into sham operation group , model group , BDNF group and BDNF-PLGA sustained release microspheres group (n=12); the model of sciatic nerve ring clamp injury was established in the latter three groups. After operation BDNF(30 0g/mL , 1 mL/kg) was injected into the local nerve injury sites for BDNF group and BDNF-PLGA sustained release microspheres (BDNF 30 0g/mL , 1 mL/kg) were injected for BDNF-PLGA group , once a week for four weeks. The general status , pace , and joint motion of each group were observed after operation; and the changes of neurological scores , nerve conduction velocity (NCV) , amplitude , latency period , compound muscle action potential (CMAP) and histopathology were observed 4 weeks after operation. Results The general status , pace , and joint motion of the sciatic nerve injury in BDNF-PLGA group were significantly improved compared with the model group and BDNF group. The BDNF-PLGA sustained release microspheres effectively improved the functions of injured peripheral nerve , which basically restored the normal level after four weeks , and the recovery was rapid than the BDNF group. The NCV , amplitude and latency period , CMAP amplitude , and recovery rate of CMAP in the BDNF-PLGA group was significantly improved compared with the model group(all P$0. 01) , and the improvements were also significantly greater than those in group BDNF (P$0. 05). The BDNF-PLGA sustained release microspheres also significantly improved the pathology changes oi sciatic nerve injury, including those oi the myelin sheath, axon and vacuole. Conclusion The BDNF- PLGA sustained release microspheres have prominent protective effect against the peripheral nerve injury in rats.
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Objective To identify the effective results of ultrasound in degradation of polymeric nanoparticles released DNA .Polymeric nanoparticles was made by dehydration of polyacetylglutamicacid (PLGA, polylactic-co-glycolic acid)solution. Method Green Fluorescent Protein (GFP) was enclosed by PLGA. Different kinds of ultrasound mode and different duct cycle and power ones were used to radiate PLGA solution for 90 s, 9 min, 20 min separately after the solution prepared for 2 hrs,then putted the solution on centrifugal machine at 13000 r/m. Using Choloroform to get rid of fat-soluble impurity,then applied nanodrop to survey the releasing rate of DNA. Finally the effect of cell expression were observed by fluorescent microscope. Results The amount of DNA released from PLGA in groups which were exposed to ultrasound were significantly different from the groups which were not exposed to ultrosound. The releasing amount of former groups had upper limitation. The releasing rate was increased with the increment of the irradiation time,frequency of ultrasound;The effect of the DNA releasing and PLGA degradation by continuous-wave irradiation was stronger than pulsed-wave ultrasound. Conclusion Ultrasound can promote the degradation of PLGA, and do help in DNA releasing and expression in vitro.
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Objective To construct bone marrow stem cell sheets and to investigate its effects in the process of osteogenesis.Methods BMSCs were differentiated into osteoblasts and then seeded into a temperature responsive culture dish to construct BMSC sheets.PLGA scaffolds in which both BMSC suspension and BMSC sheets were added,were implanted into the left side of the dogs' mandible.In the other side,PLGA scaffolds that were not wapped with BMSC sheets were implanted as control.At 16 weeks,the samples were processed for radiological analysis and histological examination.Results Cells in the BMSC sheets grew well.In the experimental side,the optical density of the samples was higher than that of the control side (P＜0.05) and plenty of lamellar bones and Haversian system were observed.Conclusions The formation of lamellar bones can be promoted by PLGA scaffolds and BMSC sheets in the process of tissue engineering bone reconstrution.
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Background Seed cells and scaffold material are the important aspects of corneal tissue engineering research.Adipose-derived stem cells(ASCs) are becoming the focus of seed cells research because of their wide source,powerful proliferation and differentiation abilities.As biodegradable polymer,polylactic-co-glycolic acid(PLGA) has successfully build multiple tissues and organs.Objective Present study was to ascertain the biological characteristics of the rabbit ASCs and their biocompatibility with PLGA scaffold in vitro and to provide groundwork for further study on the reconstruction of tissue engineered corneal stroma.Methods Adipose cells were isolated from lipoaspirate of New Zealand white rabbit using collagenase Ι.The cells were cultured and passaged.The generation 4 cells were inoculated to culture plate with 6 holes at the density of 3×104/cm2,3×104/cm2,3×106/cm2 respectively and cultivated in ossification inducing medium,lipoblast inducing medium and chondroblast inducing medium to identify the characteristics of the cells.The multilineage differentiated cells were identified by alizarin red staining,oil red O staining and immunoinfluorescene technique.The generation 4 cells were re-suspended with DiO influorescence fluid at the density of 1×107/ml and seeded on PLGA scaffold to fabricate cell-PLGA constructs.Quantitative analysis of cell proliferation on PLGA was detected by Hoechst DNA assay.The attachment and growth of adipose-derived stem cells on the scaffold were observed under the scanning electron microscope(SEM) and confocal microscopy in 1 day,3,7 days after seeding for the evaluation of biocompatibility between cells and PLGA.Results Primarily cultured cells reached 80%-90% confluence after 7-8 days with the fibroblast-like appearance.Adipose-derived stem cells of rabbits differentiated into osteoblast,adipocyte and chondroblast successfully,showing the positive stain for alizarin red staining,oil red O staining and immunoinfluorescene technique respectively.Proliferation of cells on PLGA scaffold went into plateau phase at 7 days after culture.SEM and confocal microscopy revealed the well-attached,spread cells along the scaffold and abundant excellular matrix both on the surface and interior pore of scaffold.Conclusion Cultured rabbit adipose cells have the ability of potential multilineage differentiation and good biocompatibility with PLGA scaffold,which could be used to construction of tissue engineered corneal stroma.
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OBJECTIVE: To prepare Polylactic-co-glycolic acid)(PLGA) microspheres loaded with Chinese herbal extract cucurbitacin B.METHODS: Cucurbitacin B loaded by PLGA microspheres were prepared by modified emulsification-solvent evaporation method.Central composite design-response surface method was applied to optimize the formulation with PVA concentration and ratio of drug to polymer as independent variables,and with yield of microspheres(Y1),drug loading amount(Y2),encapsulation efficiency(Y3),mean particle diameter(Y4),and the cumulative percentage of the drug release in 24 hr(Y5) as indexes to conduct multiple linear regression and second-order polynomial equation fitting.In vitro release test was performed by modified immediate release method.RESULTS: The results showed that all response variables were greatly fitted by a second-order polynomial equation.The optimal formulation was proved to be as follows: PVA concentration was 0.014 and ratio of drug to polymer was 0.066 5.The microspheres prepared in the optimal formulation were spherical and had smooth surface.Y1,Y2,Y3,Y4,and Y5 were 79.9%,7.83%,80.5%,56.18 ?m and 6.98%,respectively.The cumulative release from microspheres within 35 days reached 86.73%.CONCLUSION: Cucurbitacin B-PLGA microspheres are characterized by prolonged action and sustained-release.Furthermore,the established model has satisfactory predictability.
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PURPOSE: To compare the antibiotic release kinetics of the implant coated with antibiotic-impregnated polymers MATERIALS AND METHODS: Authors used polylactic acid (PLA) and polylactic-co-glycolic acid (PLGA) as the biodegradable carriers, gentamicin sulfate as the antibiotic and Steinmann pin as the implant. Ten Steinmann pins were coated with gentamicin of each 10, 20 and 30% mixture of PLA or PLGA for the elution kinetics study. In the elution study, total 60 coated implants were incubated in 10 mL of phosphate buffered saline (PBS) at 37 delta C and sampled at 6 hrs, 1, 3, 6, 9, 12, 15, 20, and 25 days. Assays were performed with fluorescence polarization immunoassay. Statistical analysis was done with SAS release 2.01. RESULTS: Released concentration of GM decreased with time. Minimum inhibitory concentration was maintained until 6th day on PLA 10% subgroup, 9th day in the 20 and 30% subgroups, until 6th day on PLGA 20% subgroup, and 3rd day in the 10 and 30% subgroups. Released concentrations were significantly higher in all PLA subgroups than in PLGA as a parameter of sampled time (all p<0.05). There was no statistical difference between PLA 20 and 30% subgroup after 12th sampled day (p=0.2636). CONCLUSION: PLA-GM group showed higher effective concentration for longer time than PLGA-GM group. 20 and 30% subgroups of PLA-GM showed prolonged maintenance of minimum inhibitory concentration compared with 10% subgroup, but there was no difference between the two groups.
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Fluorescence Polarization Immunoassay , Gentamicins , Kinetics , Microbial Sensitivity Tests , PolymersABSTRACT
Objective To study the preparation of polylactic-co-glycolic acid(PLGA) nanoparticles carrying arsenic trioxide(As_(2)O_(3)).Methods Polymer PLGA was used to prepare arsenic trioxide nanoparticles with w/o/w double-emulsion evaporation technique,which was optimized by uniform design test.Results The shape of As_(2)O_(3)-loaded PLGA nanoparticle was round,the size was well-distributed,the mean diameter was 178.2 nm,the average encapsulation ratio reached 53.19%,and the average drug loading was 0.64%.Conclusion The technique to prepare nanoparticles is simple and the quality can be easily controlled.