Study On The Preparation And Application Of Bacterial γ-PGA/CS/Fe₃O₄ Nanoparticles

Firstly,we studied the production of high molecular weight poly-gamma-glutamic acid（γ-PGA）by using Bacillus licheniformis WBL-3 as a fermentation strain in a small fermentor,and degraded it into low molecular weightγ-PGA.Thenγ-PGA and chitosan oligosaccharide（CS）were cross-linked and coated on the surface of superparamagnetic ferric oxide（Fe₃O₄）to prepareγ-PGA/CS/Fe₃O₄ nanoparticles（PCN）.The anti-cancer active substance sulforaphane（1-isothiocyanate-4-methylsulfonyl butane,English name Sulforaphane,SFN）was extracted and purified from broccoli seeds.SFN/γ-PGA/CS/Fe₃O₄ nanoparticles（PCSN）were prepared and their therapeutic effects on human lung cancer A549 cells were studied.Finally,CLX/γ-PGA/CS/Fe₃O₄ nanoparticles（PCCN）and DOX/γ-PGA/CS/Fe₃O₄ nanoparticles（PCDN）loaded with PCN-loaded Cephalox（CLX）and Doxorubicin（DOX）)Were studied.Firstly,the activated strain WBL-3 was cultured in shaking flask.The growth curve and the yield curve ofγ-PGA were measured to determine the feeding time and fermentation time.After entering the fermentation tank,the growth curve and product curve were continuously measured to monitor the growth and yield trend of the strains.The crude products were collected and purified with dialysis bags after alcohol precipitation.The products were decomposed at pH3.5 and temperature 120℃for 1.5 h.The molecular weight of the small molecular weightγ-PGA was in the range of 40-80 KD.The CS andγ-PGA were dissolved in water.The two biomaterials,CS andγ-PGA,were joined by cross-linking method and coated on the surface of Fe₃O₄ to prepare PCN.The morphology of PCN was observed by transmission electron microscopy（TEM）.The average particle size was about 75 nm.After vacuum freeze-drying,the PCN was qualitatively detected by Fourier transform infrared spectroscopy（FTIR）.The results showed that the amino group of CS and the carboxyl group ofγ-PGA were successfully combined by cross-linking method.The method for determining the content of SFN was established by means of high performance liquid chromatography.SFN was extracted from broccoli seeds by extraction method and purified by silica gel column.The content of SFN was analyzed by HPLC,the fractions containing SFN were collected,and the purity of SFN was analyzed by HPLC.After rotary evaporation of SFN,vacuum freeze-drying was used for qualitative detection by FTIR.The results showed that the extract was SFN.The purity of SFN was 87.50%,and the yield of SFN was 5.91 mg per gram of broccoli seeds.PCSN was prepared by extracting and purifying SFN.The average particle size was about 75 nm,the drug loading was about 30%,the encapsulation efficiency was about 90%,the sustained release time was more than 72 hours,and the cumulative release rate was about 87.9%.Then MTT assay was used to detect the effect of PCSN on the growth of human lung adenocarcinoma cell A549,and Transwell assay was used to detect the effect of PCSN on the migration and invasion of human lung adenocarcinoma cell A549.The results showed that PCN had no cytotoxicity and PCSN could effectively inhibit the growth,migration and invasion of A549 cells.Finally,the ability of PCN to carry other drugs was explored by in vitro sustained release experiments.The drug loading,encapsulation efficiency and sustained release capacity of superparamagnetic nanoparticles（PCCN,PCDN）after carrying cephexin（CLX）and doxorubicin（DOX）were investigated.For CLX,the sustained release ability is poor and the cumulative release rate is about 40%,while carrying DOX shows excellent sustained release effect.