Study On Antibiotic-loaded Nanocomposites Synthesis And Their Anti-intracellular Bacteria Effects

Intracellular bacterial infection is a difficult problem in clinical anti-infection treatment.Intracellular parasitic bacteria invade eukaryotic cells to survive and reproduce,which can not only avoid the killing effect of antibiotics and humoral immunity,but also lead to new infections with the spread of host cells.At present,the clinical treatment strategy of intracellular bacterial infection is long period,high dose of antibiotics.However,nearly two-thirds of the existing antibiotics cannot effectively enter cells,and there are problems such as weak intracellular accumulation capacity and low activity.Long-term use of large doses of antibiotics has large adverse reactions,and intracellular infections cannot be effectively treated.How to make antibiotics efficiently enter eukaryotic cells to exert their efficacy has become an urgent scientific problem to be solved.Transporting antibiotics into cells via vectors is one solution.Many research groups at home and abroad have synthesized nanometer drug carriers through experimental methods,and loaded antibiotics into cells to improve the intracellular antibacterial activity of antibiotics.Graphene(Gr)is an emerging nano-material.It is a two-dimensional material that is composed of sp~2 hybrid carbon atoms with honeycomb structure and closely connected lamellar structure.It has excellent nano properties,large specific surface area,and both sides of the nanosheet can adsorb drugs.As a carrier of nanometer drugs,graphene has great application value.It loads drugs on the lamellar structure by means of covalent bond or non-covalent bond,which has the advantages of efficient drug loading capacity and controllable release.Graphene oxide(GO)in addition to keep the family of graphene materials of fine nano features,compared to graphene,its surface has many oxygen-containing groups,make it have good solubility in water,modifiability and lower biological toxicity,have better biological application prospects,has become an important candidate carrier of drug transport research direction.However,in physiological solution,because the charge between the lamellas is shielded by ions in the solution,GO is prone to aggregation and precipitation,which results in the decrease of the active surface and greatly limits its biological application.In this study,GO was functionalized by polyethylene glycol(PEG),an amphiphilic macromolecular polymer with good solubility,to solve the problems of its aggregation and precipitation in physiological solution.In addition,in view of polyethyleneimine(PEI)is often used for gene transfection and drug transport,wear R8,MPG,hpp10membrane peptides can also be functional macromolecule material into the cell,this study combines the four material respectively and GO,can filter out the collaborative PEI in improving the efficiency of nanometer carrier intracellular delivery of components,evaluation was prepared by conditions optimization and comparison may payload of antibiotics into the cell of graphene polymer-nanoparticle carrier GO-PEG-PEI,enhance removal ability of antibiotics on intracellular.Polyethyleneimine(PEI)is often used for gene transfection and drug delivery,peptide R8,MPG,hpp10wear membrane can also be functional macromolecule material into the cell,this study combines the four material respectively and GO,can filter out the collaborative PEI in improving the efficiency of nanometer carrier intracellular delivery of components,evaluation was prepared by conditions optimization and comparison may payload of antibiotics into the cell of graphene polymer-nanoparticle carrier GO-PEG-PEI,enhance removal ability of antibiotics on intracellular.In terms of strain selection,the previous study found that the difference in cell wall structure between Gram-positive bacteria and Gram-negative bacteria would lead to the difference in sensitivity to nanomaterials.To avoid because of the different structures may lead to influence on nanometer carrier function,this study selected the MRSA and salmonella typhimurium respectively as gram-positive bacteria and gram-negative bacteria of representative strains,respectively established the MRSA and parasitic bacteria salmonella typhimurium cellular model,load of antibiotics nanocomposites are investigated two kinds of intracellular bacteria removal effect.In the first part,GO was functionalized with PEG to solve the problem that GO was prone to polymerization in physiological solution.Then,four kinds of nanocomposites of GO-PEG-R8,GO-PEG-MPG,GO-PEG-HPP10 and GO-PEG-PEI were prepared by connecting membrane penetrating peptide(R8,MPG,HPP10)and PEI functional molecules respectively.Combining with the structure-activity relationship,comparison and analysis for the influence of different experimental factors on nanomaterials characterization,identified the first synthetic GO-PEG,decorate in membrane peptides,PEI solution,again through can deliver green fluorescent protein(EGFP)plasmid into intracellular expression of four kinds of nano composite material into the cell effect compare screening,the final three connection in membrane peptide nanocomposite failed to deliver EGFP expression plasmid into the cell,and success will GO-PEG-PEI after EGFP plasmid delivery to EGFP expression in the cell.As a gene transfection reagent,PEI itself was modified into GO,which not only reduced the amount and toxicity of PEI,but also played the advantage of high GO load.The size of the synthesized GO-PEG-PEI was 155.10±4.16 nm,and the potential was 41.82±2.61m V.The fluorescence microscopy confirmed that the synthesized GO-PEG-PEI could deliver EGFP plasmid into cells,which had a high application value of nano-carriers.In the second part,vancomycin(VAN)loaded nanocomposite GO-PEG-PEI synthesized in the first part was used to carry out the intracellular bacteria scavenging experiment.MRSA was used as the intracellular model bacterium to establish the intracellular MRSA model,and the scavenging effect of the synthesized nanocomposite was evaluated.The experimental results showed that the drug loading and encapsulation rate of GO-PEG-PEI nanocomposite for vancomycin reached 20%and 25%.The clearance rate of intracellular MRSA in vancomycin-loaded GO-PEG-PEI group(80.45%and 80.19%for 2 h and 4 h,respectively)was significantly higher than that in vancomycin group(55.78%and 58.12%for 2 h and 4 h,respectively).The results of cytotoxicity test showed that GO-PEG-PEI/VAN loaded with vancomycin had low cytotoxicity and good biocompatibility.In the third part,the synthetic nano-carrier GO-PEG-PEI was used to carry out the scavenging experiment of intracellular bacteria.Salmonella typhimurium was used as the intracellular model bacterium to establish the intracellular model of Salmonella typhimurium,and the scavenging effect of the synthesized nanocomposite was evaluated.The experimental results showed that GO-PEG-PEI nanocomposite had a 7%drug loading capacity and 7.5%encapsulation efficiency of gentamicin.The scavenging rate of Salmonella typhimurium in the GO-PEG-PEI group(73.45%and 72.67%for 2 h and 4 h,respectively)was significantly higher than that in the gentamicin-treated group(38.05%and 19.33%for 2 h and 4 h,respectively).The results of cytotoxicity test showed that GO-PEG-PEI/GEN loaded with gentamicin had low cytotoxicity and good biocompatibility.In summary,the GO-PEG-PEI nanocarrier was synthesized in this study,which has a good function of drug delivery and gene entry and play a role,with low cytotoxicity.Based on GO-PEG-PEI nanomaterials,the antibiotic composite nanomaterials were prepared by carrying vancomycin and gentamicin respectively,which significantly improved the scavenging effect of intracellular MRSA and Salmonella typhimurium,and provided research ideas and methods for future research and application of anti-intracellular bacterial infection.