Study On The Pharmacological Effect And Mechanism Of Norfloxacin Solid Lipid Nanoparticles In The Treatment Of Sepsis

Objective: To synthesize a kind of norfloxacin solid lipid nanoparticles(Norf-SLN),and explore its slow-release antibacterial effect in vitro and its protective effect on sepsis rats and related mechanisms,which severs a modification of traditional antibacterial drugs or provides a possible method on the development of new antibacterial drugs in clinical aspects.Methods:1.Synthesis of Norfloxacin Solid Lipid Nanoparticles: A kind of Norf-SLN with good biocompatibility and slow-release effect was synthesized by ultrasonic high-pressure homogenization method,and characterized by various methods.2.Cytotoxic activity: Prepare Norf-SLN solutions with different concentration gradients,and detect the effects of different concentrations of Norf-SLN on the viability of RAW264.7 cells by adding different concentrations of Norf-SLN solution to CCK-8 reagent.3.In vitro antibacterial activity: Micro broth dilution method was used to measure the minimum inhibitory concentration(MIC)of Norf-SLN solution against Staphylococcus aureus ATCC25923,Escherichia coli ATCC25922,Streptococcus pneumoniae ATCCC49619,and Pseudomonas aeruginosa ATCC27853.To study the time-killing effect of norfloxacin monomer and Norf-SLN on bacteria,the bacteria in the exponential growth stage are selected in the broth,and then exposed to the floxacin solution and Norf-SLN solution of the same concentration(based on the amount of norfloxacin contained).After incubating at 37 °C for 1,2,3,and 4 hours,the bacteria counts were measured on an agar plate.4.In vivo antibacterial activity : Using cecal ligation and perforation(CLP)method,we got a rat sepsis model was,and the purchased rats were randomly divided into different groups,as follows: model group,sham operation group,blank group,Norf-SLN high-dose group 6.0 mg/kg(theoretically containing Norf Norf-SLN 6.0mg/kg),Norf-SLN medium-dose group 3.0 mg/kg(theoretically containing norfloxacin 3.0 mg/kg),Norf-SLN low-dose group 1.5 mg/kg(theoretically containing norfloxacin 1.5 mg/kg),norfloxacin positive control group 3.0 mg/kg.After the model was established,the rats were given continuous administration for 3days.The rats were killed by blood sampling from the abdominal aorta,and the 72 h survival rate was calculated.72 hours after treatment with Norf-SLN,ELISA was used to detect the contents of TNF-α and IL-6 in the serum of experimental rats,and AST,ALT,BUN,and Cr in the serum of experimental rats were detected with an automatic blood biochemical analyzer The pathological damage of liver,lung,and kidney was observed by HE staining.The expression of TLR4 and NF-κBp65 protein in experimental rats was detected by Western blot,and the expression of TLR4 and NF-κBp65 gene was detected by q PCR.Results:1.Synthesis of Norfloxacin Solid Lipid Nanoparticles:Norf-SLN nanomaterials were successfully prepared.The TEM particle size is(150.1±5.3)nm,the hydrated particle size is(224.1±3.6)nm,the polydispersity index(PDI)value is 0.139±0.010,and the Zeta potential is(-40.2±2.3)m V.The characterization results show that the prepared Norf-SLN nanomaterials have uniform particle size,good dispersibility and good stability.2.Cytotoxicity study: Our data revealed that the inhibitory effect of Norf-SLN on RAW 264.7 was positively correlated with the dose.3.In vitro antibacterial experiment: Norf-SLN has different MICs for different experimental strains.Among them,the MIC for Escherichia coli is the smallest and the antibacterial effect is the best.The results of the time-killing experiment show that,at the same concentration,Norf-SLN has a significant decrease in CFU compared with Norf;norfloxacin modified with solid lipid nanoparticles has antibacterial activity in vitro,which is superior to that of Norf-SLN.The unmodified norfloxacin liposomes were consistent with the MIC results.It also proves that Norf-SLN has good sustained-release properties.4.Sepsis model experiment: The study found that in the rat cecal ligation and puncture model(CLP),with the same amount of norfloxacin,norfloxacin modified with solid lipid nanoparticles can significantly improve the 72-h fatality rate of rat sepsis model,significantly reduce the expression levels of serum inflammatory factors TNF-α and IL-6,improve the biochemical indicators of liver and kidney damage,and significantly reduce liver,kidney and lung damage observed from liver HE staining.In addition,Western blot and q PCR results suggest that norfloxacin modified with solid lipid nanoparticles can reduce the expression of TLR4,NF-κBp65 protein,and genes under the same drug content to improve rat sepsis Inflammation.Conclusions: The data shows that Norf-SLN has good sustained-release properties and can be synergistic and bacteriostatic in vitro;it may improve the in vivo model of sepsis through bacteriostasis and inhibition of inflammation.Further studies have found that its mechanism may be through the regulation of TLR-4/NF-κB way to achieve.