| Objetive:To study preparation and characteristics of dexamethasone loaded PLGA magnetic microspheres,and observe its appearance,span,yield,encapsulation rate,drug load,magnetic responsiveness;to observe its metabolic models of released in vitro and stability;to observe its biocompatibility;to observe its effect on rat model of chronic inflammatory.Method:(1)Dexamethasone loaded PLGA magnetic microspheres is made by emulsion-solvent evaporation method.Its appearance and span are observed by optical microscope;its yield,encapsulation rate and drug load are tested by HPLC;its magnetic responsiveness is tested by 4000GS external magnetic field.(2)In vitro release test:to weight ratio is 1:3,1:4,1:5 dexamethasone PLGA magnetic microsphere 10mg precisely,and placed in the cork flask respectively,which is filled with PBS solution(PH=7.4).Put them in thermostatic water bath oscillometer.And water level above PBS solution plane,oscillation condition are 36-37?,100-120/min.Take the supernatant on 0.5h,1.0h,1.5h,2.0h,2.5h,3.0h in 1st day,and it was taken every 24h up to 14th day.it is 2ml sample taken away and stored samples in 4 ? frige.(3)Stability test:placed microspheres(n?500)with different ratio in EP tube,respectively.Then they will be stored them in 4 ? frige in 3months.To observe Physical characteristics(span and appearance)and the drug loadings.(4)Hemolysis test in vitro:Different concentration(lmg/ml,2mg/ml,4mg/ml)sample are blend with blank PLGA magnetic microspheres,defibrous red cell suspension and the physiological saline/distilled water,and incubate in a thermostatic water bath oscillometer with in 3h(water temperature:36 ?-37 ?).The above samples are centrifuged with 3000r/min in 5min,remove the supernatant and store them at room temperature in 30 minutes.The absorption value and hemolysis rate were obtained by the spectra of 540nm,which is taken supernatant of negative control as blank.(5)Blood compatibility in vivo:Take 3 SD rats and weigh them separately,The rats are injected with 0.8ml(high dose),0.5ml(medium dose)and 0.2ml(low dose)by samples respectively.The blood samples of rats were collected at 0.5h,6h,12h and 24h respectively before and after the injection,and they was collected in the anticoagulant tube(purple)for routine blood test.(6)Histocompatibility test:After inhalation of sevoflurane in rats,the hair on the left back was shaved,rats in the experimental group,lml of mother liquor was injected into the subcutaneous injection and 1ml of saline was injected into the control group.After 10 days and 30 days,HE staining will be done on the skin and subcutaneous tissue of the injection site.(7)Establishment of chronic inflammatory model of rats:40SD rats,220-250g,were injected on their left hind limbs with 0.1ml CFA solution respectively.On 2,4,6,8,10 days after injected,the feet appearance are observed,the mechanical pain threshold are measured with the Von Frey filaments,and pathological section of the foot was HE stained after anesthesia on the 10th day after injected.(8)Evaluation of the efficacy of microspheres in the treatment of model rats:The 36 model rats were divided into 6 groups,6 rats each group.And namely microspheres,microspheres + magnetic therapy group,ground rice group,dimeter + magnetic therapy group,positive control group,and magnetic therapy group.Model rats are injected by dexamethasone PLGA magnetic microspheres(1:4)/dexamethasone sodium phosphate injection respectively with or without magnetic therapy.On 2,4,6,8,10 days after injected,the feet appearance are observed,the mechanical pain threshold are measured with the Von Frey filaments,and pathological section of the foot was HE stained after anesthesia on the 10th day after injected.Results:(1)Dexamethasone PLGA magnetic microsphere,the feeding ratio is 1:3,1:4,1:5 microsphere average diameters(spans,S1)are(27.47±8.78)um,(29.66±10.02)um,(30.14±11.2)um respectively;the yields are(87.02±2.25)%,(87.25±3.14)%,(84.00±5.53)%respectively;the encapsulation rates(S3)are(76.44±3.11)%,(78.89±2.42)%,(71.33±5.60)%;the drug loadings(S4)are(33.09± 1.89)%,(30.33±2.01)%,(24.29±1.78)%;the sum values(S)are(168.55±3.62),(167.47±4.15),(149.48±4.21)respectively.The above microspheres have smooth and round surface,uniform size,good dispersion and high pass rate through needle.(2)In vitro release model of dexamethasone PLGA magnetic microspheres with feeding ratio of 1:3,1:4 and 1:5 was Q=22.313In(t1/2)+28.651(R2=0.949),Q=4.0844t1/2+ 6.6602,(R2=0.977),Q=5.212t1/2+ 12.481,(R2=0.975);the cumulative release rates are 79.12%,32.91%,40.33%on the 2nd day,and the cumulative release rate are 82.61%,62.74%,76.04%on the 7th day,and the cumulative release rate are 86.34%,66.99%and 97.85%on the 14th day.(3)Stability test:the microspheres have been in 4 to 8 ? frige for 3 months,the drug loadings and the spans has not changed obviously.(4)Hemolysis test in vitro:OD indexes of test samples(1 mg/ml,2mg/ml,4mg/ml)are 0.006,0.011,0.032,respectively.The hemolysis rate was 0.5%,1.2%and 3.3%respectively.(5)Blood compatibility in vivo:compared to the result before administration,rats blood routine changes are not statistically significant,whatever any dosage at 0.5h,6h,12h and 24h.(6)Histocompatibility:The pathological section(HE staining)of the rat injection sites have a small number of inflammatory cells on 10th days,and inflammatory cells are more than before on 30th day,and fibroblast proliferation and the wrapped are seen locally.However,there was no significant difference compared with the control group.(7)Establishment of chronic inflammatory model in rats:The appearance of the rats left hind feet are red and swollen,the mechanical pain thresholds are significantly decreased,and the pathological sections(HE staining)showed a large number of inflammatory cells infiltrating.(8)Evaluation of the efficacy of microspheres in the momdel rats:Comparison with microspheres(+ magnetic therapy)group and dexamethasone injection(+ magnetic therapy),the effects of mechanical pain threshold are no statistical difference on 2nd day,the mechanical pain threshold of(microsphere + magnetic therapy)group was significantly higher than that of other groups during the 4th-10th day.In addition to the magnetic therapy group,the inflammatory cells of the other groups were significantly decreased.The improvement are showed significantly in microsphere group and(microsphere + magnetic therapy)group.Conclusion:Dexamethasone PLGA magnetic microspheres have certain sustained release,good biocompatibility;and they can improve chronic pain which caused by chronic inflammation. |
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