The Establishment Of The Myocardial Targeted Nanoparticles And Preliminary Evaluation Of Its Functional Effects

BackgroundSepsis,infected by bacteria,fungi,viruses or other factors,could lead to systemic inflammatory response.The mainly characteristics of sepsis are srvere,complex,dangerous.It can often cause SIRS?Systemic Inflammatory Response Syndrome??MODS?Multiple Organ Dysfunction Syndrome?and even MOF?Multiple Organ Failure?of the body,which put threats on people' life.It has become one of the most common cause of the death in the critically ill patients.A study showed that more than 25% sepsis patients suffered from cardiovascular complications,and myocardial injury is one of the most important symbol of poor prognosis in sepsis patients.At present,there is no specific clinical measures to alleviate myocardial injury,so the mainly significance lies at how to protect cardiac function,improve the critically ill patients' physical condition and survival rates.NF-?B,as one of the hottest spot in the contemporary,plays a critical role in the excessive inflammatory reaction process.Recent studies confirmed that the activity of the NF-?B increased significantly once the sepsis occurred.If we use the siRNA to inhibit the P65 subunit of NF-?B,the inflammatory response can be suppressed effectively.The traditional delivery way is not suitable for siRNA,because siRNAs in the body are susceptible to be degraded.However,nanotechnology can not only provide siRNA with effectively protection,,but also gain active targeting function after chemical modification.In human,?₁-adrenaline receptors??₁-AR?are mainly distributed in the surface of myocardial cells.When the myocardial injury occurred,the number and sensitivity of the receptors were significantly higher,which could be used as the target of nanoparticles.As reported,the polymer PAC and ?₁-AR blocker Esmolol have similar chemical structure,and the previous studies showed that the PAC modified liposome had stronge myocardial targeting property.So we can use the siRNA and nanotechnology to build a PAC-P65-NF-?b-siRNA nanoparticles,and study its effection on the myocardial protection.We could transfer the nanoparticles into mice via the tail vein,then establish the sepsis model and observe the distribution and effecting of nanoparticles in mice,thus we aim to reduce myocardial injury,protect myocardial function and increase the survival rates of sepsis treatment.Methods1.The synthesis of Esmolol analogue--PACBy analyzing literature and cooperating with relative department,we begun with 3-?4-hydroxy phenyl?propionic acid,after several chemical reaction steps,and finally got our aim-PAC.The route is stable and easy to be achieved.2.The design,synthesis and screening of siRNADesigned and syhthesis three different kinds of siRNA,one negative siRNA and P65 primers,after that,we cultivated myocardial cell line?HL-1?in vitro.Then we used lipo2000 to transfect the cells respectively,then added LPS 24 hours after.Finally,we extracted the RNA and performed PCR to examine the expression of gene,so that we could select the best one.3.The synthesis of nanoparticles.By referring to the previous methods and discussing with relative researchers,we got a pathway by using siRNA,PLGA,PEG and TPGS.Then we tried to optimize the reagent ratio and operating steps,and used Laser Particle Size and Zeta Potential Analyzer,Scanning Electron Microscope?SEM?,Ultraviolet Spectrophotometer to examine the characteristics.4.The cell experiment of nanoparticles.We cultivated myocardial cell line?HL-1?in vitro,then used fluorescence microscope to observe the expression of receptor ?₁-AR.Added these nanoparticles into the cell cultures,and used the fluorescence microscope to observe the dispersion of nanoparticles and siRNA respectively,then performed fluid cytology.Added LPS into cells,and operating PCR after 24 hours to examine the expression of gene,5.Animal experiments of nanoparticles.We made sepsis injury model by celiac injection of LPS,then we respectively collected bood to detect the change of CK,LDH and HBDH after 24 h.And took heart tissue slice to observe the pathological change.Results1.The PAC we got had high purity,stable properities,non-biological toxicity,and can be used for further synthesis.2.The cell induced by si435 had the lowest level expression of mRNA,so that it means the si435 had the strongest inhibitory effect towards P65.Finally,we choosed si435 as the core of myocardial targeted nanoparticles.3.Compared with different ratio and condition,we finally choose the 875 ul 5% PEG + 125 ul siRNA as the internal phase,80 mg PLGA+15mg TPGS+5mg PAC solubing in 8 ml of acetone as the organic phase,100 ml 0.03%TPGS as the external phase.Under this circumstance,the nanoparticles we got had a homogeneous size,a well dispersibility,a centralized potential,a steady encapsulation rate,drug-loading capacity and release curve.4.The surface of membrane showed the expression of ?₁-AR receptor.The survival rates of HL-1 cells were not influenced by co-culturing with blank nanoparticles,it could prove that the nanoparticles had no toxicity towards HL-1 cells.We used the fluorescence microscope to examine the distribution of nanoparticles in cells,and found it could distribute equally and could lead to obviouse inhibitory effect.5.Celiac injection of LPS?10 mg/Kg?in mice could produce significant myocardial injury,but the degree of myocardial injury in nanoparticles group were not obvious.Myocardial enzyme spectrum showed when sepsis occurred,the level of CK,LDH and HBDH were significantly increased,however,the expression in si435 nanoparticles group was significantly reducedConclusions:1.The PAC we got had high purity,stable properities,non-biological toxicity,and can be used for further synthesis.2.We choosed si435 as the core of myocardial targeted nanoparticles,because it had the strongest inhibitory effect towards P65.3.The nanoparticles we got had a homogeneous size,a well dispersibility,a centralized potential,a steady encapsulation rate,drug-loading capacity and release curve.4.The nanoparticles had no toxicity towards HL-1 cells,and it could distribute equally and could lead to obviouse inhibitory effect.5.The nanoparticles could help to lessen the degree of myocardial inflammation,so that it could help to protect heart against myocardia injury caused by sepsis.