Study On Targeting For Cardiomyocytes Of Surface-Conjugated Polylactic Acid Nanoparticles Loaded Hyperoside

Objective:To prepare polylactic acid nanoparticles loaded hyperoside(HPN) and conjudate its surface for targeting. Study on its shape, preparation process, drug release behavior in vitro., targeting mechanism for myocardial cells in vitro. and targeting in vivo. We studied pharmacokinetics in rats to evaluate targeting.Methods:1. To prepare HPN and targeting conjudated nanoparticles by emulsification-high pressure homogenization method. Detecting average particle size, particle size distribution and Zeta potential by Laser particle size analyzer, the shape of nanoparticles were observed by SEM.2. To establish of comprehensive evaluation by uniform design method and research of the best preparation process of nanospheres. Using differential scanning calorimetry to evaluate the preparation effect of nanospheres.3. To establish a method for the conten determination of Hyperoside which from nanospheres, cell lysates, rat’s plasma and tissue homogenates by HPLC.4. Discussing the drug release pattern in vitro of nanoparticles by dialysis method. Data were treated by zero-order model, monoexponential function, biexponential function, Weibull prediction model and Higuchi model respectively, to estimate the goodness of curve fitting, according by AIC and R2.5. To establish incorporation detect method of ligand targeting modified by HPLC, and validate the methodology.6. Using cardiomyocytes as targeting cells and the uptake rate of drug for cell protein as index to study targeting of nanosphere in vitro. Using known β1-blocker (atenolol) as a tool drug to study its mechanism of targeting in vitro cardiomyocytes were used as targeting cells to study the difference of protection effect in hypoxia cardiomyocytes between HPN and nanosphere which was modified by surface targeting ligand.7. Injecting PAC-HPN,HPN,Hyp respectively though tail vein of rat to investigate the process of Drug-Time and the distribution of major organs in rats, we used Ce,Re and te to evaluate myocardium targeted of nanosphere.Results:1.Using scanning electron microscope to observed the morphological characteristics of HPN and PAC-HPN which had prepared, its spherical and crude slightly. Laser particle analyzer analyse the average particle size and Zeta potential, HPN average particle size is (159 ± 43.7) nm, range of particle size is 106.2～226.5nm. The average Zeta potential is (-12.7 ± 0.23) mV. PAC-HPN average particle size is (200.7 ± 44) nm, range of particle size is 146.6-268.3nm. Average Zeta potential is (4.57 ± 0.24) mV.2. To confirm the optimum conditions by uniform design method, the rate of charge for polylactic acid and drugs is 10%, oil-water volume ratio is 15%, number of homogeneous is 6 times, pressure is 15000psi. The results of differential scanning calorimetry showed that melting peak of PLA was disappeared in HPN and PAC-HPN’s atlas, melting peak of Hyp was disappeared completely at the same time. Indicating Hyp was exist as amorphous in HPN and PAC-HPN, or was packaged by PLA.3. To establish a method for the conten determination of Hyperoside which from nanospheres, cell lysates, plasma and tissue homogenates by HPLC. The result of precision, recoverry, stability and repeatability were correspond the requirements of testing. The method is simple, accurate and reliable.4. The results of nanoparticles drug releasing experiments in vitro show that the release rate of HPN and PAC-HPN were 5.66% and 4.26% respectively in the first day. The data of cumulative release rate during 15 days were 56.89% and 54.03% respectively with significant slow-release feature. Releasing in vitro of HPN and PAC-HPN accord with Higuchi model after the data were simulated by various of model, its release curve was y=0.1678t1/2-0.039, y= 0.1672t1/2-0.0548 respectively, and T50 were 10.32 days,11.01 days respectively.5. To establish incorporation detection method of ligand targeting conjugated by HPLC. The average incorporation ratio of PAC is (5.33± 0.11)%. Incorporation ratio is not proportional to the adding amount which remained relatively stable.The methodology which study of precision, recovery, stability, repeatability accord with testing requirements, the method is simple, accurate and reliable.6. The results of targeting in vitro show that the total uptake of HPN and PAC-HPN was 0.141 ng/μg,0.032ng/μg, which uptake of phagocytosis was 0.088 ng/μg and 0.005 ng/μg.Uptake of PAC-HPN for myocardial cell was inhibited significantly (P<0.001) after injecting β1-bloker (Atenolol), while has no change inHPN(P>0.05). it means that the uptake of PAC-HPN for cardiac myocytes was competitively inhibited after injecting atenolol, PAC-HPN has significant targeting for myocardial cell.The testing results of hypoxic myocardial cell uptake shows that myocardial uptake rate of PAC-HPN were 3.72,4.73,4.67 times respectively to comparing with HPN which has cultured for 2,4,24 hours under normoxia condition. While myocardial uptake of PAC-HPN were 4.32,6.27,42.5 times respectively to comparing with HPN under hypoxic condition. The multiple of increasing was higher than normoxia condition explain that the number of β1-bloker in surface of myocardium was increased significantly because of hypoxia, the targeting of hypoxia myocardial cells are more prominent. And the targeting is more and more prominent with sustaining hypoxia.7. Injecting PAC-HPN, HPN, Hyp respectively though tail vein of rat, the dose of administration calculated by Hyp is 12 mg/kg. The data of pharmacokinetics were fitting process by PKSolver, t1/2, Tmax, Cmax, AUC of PAC-HPN, HPN, Hyp in plasma were 4.78h,1.0h,18.88μg/ml,59.66μg/ml*h,3.04h, 1.0h, 14.15μg/ml,50.28μg/ml*h; 2.07h,0.5h,28.22μg/ml,50.16μg/ml*h respectively.The targeting parameters were compared PAN-HPN with HPN in plasma, heart, liver, spleen, lung, kidney tissues, its Ce were 1.33,1.68,0.55,0.58, 0.37,1.19 respectively. Re were 1.19,1.69,0.63,0.53,0.29,1.02; respectively. And compared with Hyp, its Ce were 0.67,1.22,0.65,0.69, 0.42,1.14 respectively, Re were 1.19,3.68,1.58,1.70,0.85,1.24 respectively. The data of heart group show that it has a significant difference (P <0.01), which indicated has good targeting for myocardial.Conclusion:Polylactic acid Nanoparticle loaded Hyperoside(HPN) which we had prepared has good targeting of myocardium with sustained-release in vivo, and expected to become a targeted long-time therapeutic of injured myocardial cells.