| Cardiovascular disease has become one of the chief causes of death among residents in China,and ischemic heart disease has been the highest proportion.After the occurrence of myocardial infarction,timely and effective reperfusion treatment can still further cause myocardial ischemia and reperfusion injury.Studies have shown that transiently increased ROS levels in myocardial cells after vascular opening are one of the key mechanisms of reperfusion injury.Statins are the most commonly prescribed drugs for the treatment of dyslipidemia,and the efficacy and safety of statins have been confirmed by clinical practice for many years.In recent years,a large number of basic experiments have pointed out that statins have a non-cholesterol-lowering multi-effect myocardial protection pathway.However,multiple studies have also shown that oral administration of the acute phase of myocardial infarction is difficult to achieve effective concentrations in the myocardial site.The drug delivery system is expected to solve the clinical limitation of statins and provide valuable therapeutic strategies for clinical treatment of myocardial ischemia-reperfusion injury.Objective: To modify the surface of gold nanoparticles(AuNPs)with polyethylene glycol(PEG)in order to form functional gold nanoparticles.Under the combined action of electrostatic adsorption and chemical bond,Pravastatin was loaded to construct gold nanoparticle-mediated Pravastatin("AuNPs-Pra")drug composite transporter.The biocompatibility of "AuNPs-Pra" drug composite transporter was evaluated by in primary rat ventricular myocytes,and selected the optimal concentration which used in the next studies.The effect of this transporter whether it can decrease the level of ROS produced by oxygen–glucose deprivation/recovery in primary rat ventricular myocytes was further investigated.Methods:(1)Construct the optimal ratio of functionalized gold nanoparticles,and characterize the microstructure and surface charge of the material by transmission electron microscope and nanometer particle size and potential analyzer.(2)Pravastatin was loaded with functionalized gold nanoparticles to construct "AuNPs-Pra" drug transport composite carrier.Nanoparticle size and potential analyzer,UV-Vis,and infrared spectrum were used to characterize the particle size,surface charge,drug loading capacity,drug release rate,and linking groups of the transporter.(3)The biocompatibility of the drug complex transporter was evaluated by CCK-8 detection and Live-dead staining,and then the optimal concentration was been selected for subsequent experimental research.(4)Due to the different time and concentration of statins,their cardioprotective effects are also different.The optimal incubation concentration and time of pravastatin were determined by CCK-8 and DHE staining,and the optimal concentrations and incubation time was selected to used as a control group for subsequent experiments.The experiment was divided into four groups: blank control group,gold nanoparticle group(AuNPs),pravastatin group(Pra),and drug complex carrier transport group(AuNPs-Pra).After incubating for a certain period of time by adding appropriate concentrations of materials,construct in vitro oxygen–glucose deprivation/recovery model by used in primary rat ventricular myocyte,DHE staining and WB detection was used to evaluate intracellular ROS and anti-apoptotic levels in each treatment group.Results:(1)When functionalized gold nanoparticles(F-AuNPs)were constructed with polyethylene glycol(PEG)and gold nanoparticles at a mass ratio(W / W)of 3: 1,the surface of the particles was about + 7MV Potential.(2)The particle size range of bare gold nanoparticles measured by the nanometer particle size and potential analyzer is 5-15 nm,and the zeta potential is-12MV;the particle size range of F-AuNPs is concentrated around 10-20 nm,and the zeta potential is + 7MV;The particle size range of "AuNPs-Pra" drug transporter is concentrated around 25-45 nm,and the zeta potential is-6MV.The carrying capacity of pravastatin measured by UV-Vis is 56%.When it released under static conditions in vitro,the drug release plateau phase can be reached within 12 hours.The cumulative drug release rate for 48 hours is 73.26%.Infrared spectroscopy confirmed the successful construction of F-AuNPs and the polycondensation reaction between the amino group of polyethylene glycol and the carboxyl group of pravastatin on the nanocarrier on the pravastatin load.(3)Through the detection methods of CCK-8 and Live-dead,the suitable concentrations of drug complex carrier which is 0.05 mmol / l was selected,it has no obvious inhibitory effect and toxicity on primary rat ventricular myocyte proliferation,which was proved that it has good biocompatibility,and the difference is not statistically significant.(4)CCK-8 detected the effects of pravastatin concentration and incubation time on the cardiomyocyte proliferation.The results showed that when the concentration of pravastatin was 4 u M or less,there was no significant effect on the cardiomyocyte proliferation,and the difference was not statistically significant.Continuous incubation for 24 hours can inhibit cell proliferation and produce certain toxic and side effects.The difference is statistically significant.DHE staining for optimal antioxidant concentration and incubation time showed that the higher the concentration of pravastatin and the longer the incubation time,the better the antioxidant effect.Comprehensive CCK-8 detection,the concentrations of 4u M and continuous incubation for 12 hours was selected as the subsequent control group.(5)A blank control group and three experimental groups of different materials were established.After continuous incubation for a certain period of time,an in vitro model of oxygen–glucose deprivation/recovery was established.DHE staining showed that both AuNPs and Pra could significantly reduce intracellular ROS production,while the "AuNPs-Pra" drug composite carrier group had better antioxidant activity than the material group alone,and the difference was statistically significant.WB test results again confirmed that the "AuNPs-Pra" group had better oxidation resistance than the single material treatment group.Further detection of intracellular Capase-3 expression levels showed that "AuNPs-Pra" had superior anti-oxidation properties compared to the single material treatment group Apoptosis level.Conclusion:(1)When the mass ratio(W / W)of polyethylene glycol to gold nanoparticles is 3: 1,functionalized gold particles can maximize the loading of pravastatin.(2)Potential analyzer and infrared spectrum characterization show that the load of pravastatin may be combined by electrostatic adsorption and chemical bonding.The carrying capacity of pravastatin in the "AuNPs-Pra" drug composite transporter is 56%,and the plateau phase of drug release can be reached in 12 hours,and the cumulative drug release rate in 48 hours is 73.26%.(3)Compared with the effect of a single material,"AuNPs-Pra" composite material has the best oxidation resistance and anti-apoptotic,and the difference is statistically significant. |
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