TPGS Modified Inorganic Nano Drug Delivery System For Drug-resistant Bacterial Infection Therapy

The problems of antibiotic resistance are widespread worldwide,and the emergence of antibiotic resistance and multi-drug resistant bacteria have greatly reduced the effectiveness of antibiotic treatment and limited antibiotic treatment options for drug-resistant bacterial infections,thus prolonging hospital stays and costing more medical expenses further lead to higher morbidity and mortality,eventually placing a huge burden on public health systems and society.As one of the most important conditioned pathogens in the clinic,Acinetobacter baumannii can be widely spread in the hospital,especially in intensive care units,causing hospital acquired pneumonia,abdominal infection,bloodstream infection,urinary tract infection and skin and soft tissue infection,and the detection rate of multi-drug resistance,extensive drug resistance(only sensitive to tigecycline and polymyxin)and pan drug resistant Acinetobacter baumannii increased year by year.Tigecycline(TIG)is a new generation of broad-spectrum glycylcycline antibacterials that can be used in the treatment of common pathogens and widespread Acinetobacter infections.TIG has good antibacterial activity against multi-drug resistance and extensive drug-resistant Acinetobacter baumannii,and has become the first-line therapeutic drug and the "final choice" for clinical application.However,with the widespread use of TIG in clinical practice and the gradual prevalence of the pan drug resistant Acinetobacter baumannii,the resistance of Acinetobacter baumannii to TIG has gradually attracted public attention.The resistance mechanism of TIG-resistant Acinetobacter baumannii is closely related to the low permeability of cell membranes to antibiotics and the overexpression of efflux pumps such as AdeABC and AdeIJK.These drug efflux mechanisms lead to lower TIG plasma concentrations in patients with TIG-based treatments,which may result in treatment failure,and increased treatment may increase adverse events in patients.In this study,TIG was used as a model drug to construct a nano drug delivery system to improve drug efficacy and reduce drug side effects.Silver nanoparticles(AgNPs)can be antibacterial by various mechanisms of action.Combined AgNPs with antibiotics can greatly improve their antibacterial properties and it can be used as an excellent carrier material for antibacterial research in nano drug delivery systems.D-a-Tocopherol polyethylene glycol 1000 succinate(TPGS)is an amphiphilic and saf:e nonionic surfactant.The polyethylene glycol structure can prolong the in vivo circulation of the TPGS modified nano drug delivery system and prolong the half life of the drug,furthermore,the tocopherol structure in TPGS can increase the permeability of the bacterial membrane structure and inhibit the efflux pump activity in resistant bacteria.In this study,TPGS was firstly applied to the treatment of drug-resistant bacterial infections.TPGS was used as a stabilizer to successfully synthesize a uniform distribution of AgNPs with a hollow structure of 9.84±0.86 nm.TPGS/AgNPs can utilize its hollow structure to effectively load the antibiotic TIG,the drug loading amount can reach 1.80%,the particle size of drug-loaded nanoparticle(TPGS/AgNPs/TIG)is determined to be 10.15±0.73 nm,and the drug released behavior can be prolonged to 48 h.Using human normal liver cells(LO2)as a model cell,the cytotoxicities of TPGS/AgNPs and TPGS/AgNPs/TIG were investigated.The results showed that the cell viability was still higher than 80%when the concentration of nanoparticles was 0.4 mg/mL,indicating that the TPGS/AgNPs/TIG has low cytotoxicity.The sensitive and resistant Escherichia coli and Acinetobacter baumannii were used as bacterial models to investigate the antibacterial effect of TPGS/AgNPs/TIG.The results showed that TPGS/AgNPs/TIG could achieve stronger antibacterial effect through the synergistic action of TIG and AgNPs,and effectively inhibit resistant bacteria.The uptake behavior of TPGS/AgNPs in these strains was investigated.The results indicated that the TPGS/AgNPs can be internalized more to the interior of bacteria due to the small particle size and good dispersibility.The change of efflux pump gene expression levels in drug-resistant bacteria and the activity of efflux pumps were next investigated,and the results showed that the modification of TPGS can inhibit the expression of bacterial resistance efflux pump genes adeB and adeJ and effectively inhibit the efflux of substrate and increase the accumulation of antibacterial drugs in bacteria,thus achieving bacterial resistance overcoming.Female ICR mice were used as model animals.TIG-sensitive and TIG-resistant Acinetobacter baumannii were used as model strains.The acute peritonitis model was established by intraperitoneal injection of bacteria.After intraperitoneal administration,TPGS/AgNPs/TIG can achieve a significantly enhanced anti-infective effect in vivo by synergistic antibacterial action of AgNPs and TIG,small particle size advantage and the mechanism of TPGS inhibiting bacterial efflux pumps.The enhanced anti-infective effect could eventually result in the highest survival rate of TPGS/AgNPs/TIG group in all groups.The number of colonies in the peritoneal cavity of each group of model animals after treatments was examined by spread plate method.After treatment with TPGS/AgNPs/TIG,the number of peritoneal colonies in the mice inoculated with these two strains was significantly decreased,and there was no significant difference in the number of peritoneal colonies between these two groups of mice inoculated with sensitive and resistant strains,indicating that the antibacterial activity of antibiotics was significantly enhanced after being loaded,and high-efficiency anti-infective treatment can be achieved in drug-resistant bacterial infections.H&E staining showed that after TPGS/AgNPs/TIG treatment,the pathological changes of the acute peritonitis mice were the most significant.The alveolar septum of the lungs returned to normal,the inflammatory cells decreased,and the size of lymph nodes in the spleen tissues returned to normal.The number of inflammatory cells in the peritoneal tissues basically returned to normal levels.Klebsiella pneumoniae is one of the main pathogens in the hospital,which can cause multiple infections in patients with low immunity.With the widespread use of antibacterial drugs,drug-resistant Klebsiella pneumoniae has also gradually increased.The alternative drugs for multi-drug resistant Klebsiella pneumoniae infection are also limited.TIG is one of the preferred antibacterial drugs for domestic and international applications.The emergence of TIG-resistant Klebsiella pneumoniae poses a great challenge to the clinical treatment of drug-resistant bacterial infections.The resistance mechanism of TIG-resistant Klebsiella pneumoniae was correlated with the high expression of the RND-type efflux pump gene AcrAB and the upstream binding site ramA.In this study,Cr3+ doped ?-Ga2O3 nanoparticles were used as a safe and effective nanocarrier for in vivo fluorescence imaging.TPGS was used to inhibit the drug-resistant bacterial efflux pump.At the same time,ICAM1 monoclonal antibody was used as a target for modification,and the targeted distribution of the nano drug delivery system was achieved by the specific binding of the ICAM1 antibody and the ICAM1 intercellular adhesion molecule with high expression at the infection site,so as to improve the target tissue distribution of the drug and improve the drug efficacy and reduce systemic adverse reactions.The gallium oxide(?-Ga2O3:Cr3+)nanoparticles with particle size of 14.79±4.62 nm were successfully synthesized by hydrothermal method and high temperature calcination method.TPGS was used as a stabilizer to prepare monodisperse ?-Ga2O3:Cr3+nanoparticles with a particle size of 15.16±1.75 nm.TPGS-COOH was activated by ring-opening reaction and applied as stabilizer to prepared TPGS-COOH modified gallium oxide nanoparticles.I-TPGS modified gallium oxide nanoparticles(I-TPGS/Ga2O3)were then prepared by grafting ICAM1 monoclonal antibody to the nanoparticles by amide reaction.I-TPGS/Ga2O3/TIG nanoparticles with a particle size of 15.33 ± 1.26 nm with good dispersibility were prepared by effectively adsorbing TIG.The drug loading amount was up to 8.46±0.51%,and the drug released behavior can be prolonged to 72 h.Human umbilical vein endothelial cells(HUEVC)were used as model cells.LPS was used to stimulate the overexpression of ICAM1 on the surface of cells to mimic cells under inflammatory pathology after bacterial infection.The high expression of ICAM1 adhesion factor on cell surface after LPS stimulation was confirmed by Western blotting.I-TPGS/Ga2O3 nanoparticles can be internalized more by LPS-stimulated cells through the specific binding of ICAM1 monoclonal antibody to ICAM1,which is highly expressed on the cell surface.After ICAM1 was blocked,the uptake of I-TPGS/Ga2O3 nanoparticles was reduced.Female ICR mice were used as model animals.The sensitive and TIG-resistant Klebsiella pneumoniae strains were used as model strains.An acute pneumonia mouse model was constructed by micro-tracheal injection.After the modeling,the IC AM1 of the lung tissue was found highly expressed.After the tail vein administration,the I-TPGS/Ga2O3 nano drug delivery system can specifically bind to ICAM1 which was highly expressed in the infected site,thereby achieving targeted distribution at the infected site and significantly improve the survival rates(100%)in both models.I-TPGS/Ga2O3/TIG can effectively inhibit the growth of sensitive bacteria and TIG-resistant bacteria in vivo,and significantly improve the in vivo antibacterial activity of the antibacterial drug TIG.The pathological changes of lungs and spleens in mice were observed by H&E staining.After I-TPGS/Ga2O3/TIG administration,the histopathological changes in these two models were significantly improved.The alveolar septum and spleen tissue of lung tissue were improved and the size of the middle lymph nodes returned to normal.The H&E staining observation of liver and kidney of the mice showed no obvious pathological changes and damage,indicating the effectiveness and safety of I-TPGS/Ga2O3/TIG.After administration by I-TPGS/Ga2O3/TIG,the main cells and parameters in the blood of mice did not change significantly,indicating the biosafety of the I-TPGS/Ga203/TIG.In conclusion,these two functional TIG nano drug delivery systems constructed in this study all have small particle sizes.The modification of TPGS increases the particle dispersibility and thus increases the bacterial internalization.At the same time,the inhibition of bacterial efflux pump gene overexpression by TPGS reduced the efflux of antibacterial drugs,thus enhancing the antibacterial activity of antibacterial drugs and achieving efficient treatment of drug-resistant bacterial infections.