Establishment Of Amiodarone Nanoliposomes Modified By Multifunctional Peptide And Study On Its Myocardial Targeting

Coronary atherosclerotic heart disease,referred to as coronary heart disease,has become one of the most fatal diseases in the world.Clinical treatment methods,including drug,interventional therapy and surgical treatment.Amiodarone(AMD)is a class ?(potassium channel blocker)antiarrhythmic drug,which can dilate coronary artery and improve myocardial blood supply.However,long-term use of the drug easily cause drug accumulation in non target organs,resulting in a series of adverse reactions,thus restricting the clinical application of amiodarone.Nanoliposome(NLPs)have a cell like structure,which can change the distribution of encapsulated drugs in vivo,improve the treatment index,reduce the dosage and toxicity of drugs.At present,AMD-NLPs or nanoliposome carrier system modified by single target are mainly used in cardiovascular disease,but there are still some limitations,such as:? the drug encapsulation rate is not ideal and the drug release is too fast;? the specific selectivity of tissues and organs is not satisfactied,which may increase the drug distribution of other tissues;? after the drug is presented into the heart,it is often difficult to further guide to the target organ,so that the treatment effect is greatly reduced and the effective drug concentration cannot be maintained for a long time.Therefore,more and more researchers are committed to the development of new drug carriers,and target modification of the carriers to make the drug more targeted and better efficacy.In this study,we aimed to construct a double targeting nanoliposome system,which is composed of two functional nanomaterials PCM-1(wlseagpvvtalrgtgsw,myocardial cell specificity target peptide)and TAT(ygrkrrqrr,cell penetrating peptide)with different targeting effects to construct amiodarone nanoliposome with double targeting effect in the common nanoliposome body system(PCM-1/TAT AMD-NLPs,referred to as PTA-NLPs).Using amiodarone as drug,PCM-1 and TAT double modified amiodarone nanoliposomes were prepared by high-pressure homogenization method.Myocardial cells were used to evaluate the cytotoxicity and uptake ability of PTA-NLPs.The ST segment number of ECG was detected by slow tail vein injection of dual target AMD nanoliposomes after establishing myocardial infarction model in rats.According to the data,the contents of serum creatine kinase(CK),lactate dehydrogenase(LDH),aspartate transaminase(AST)and malondialdehyde(MDA)were also measured,and the infarct area of rats were observed to evaluate whether the dual target has stronger myocardial targeting than the single target modified amiodarone liposome,and it is easier to concentrate in the myocardial area.Part ? preparation and characterization of PTA-NLPsObjective:to construct and prepare the PTA-NLPs modified by PCM-1 and TAT,and to analyze the characterization and stability of PTA NLPs.Methods:1.Using 60 mg of soybean phospholipid,30 mg of cholesterol,3 mg of DSPE-PEG2000-PCM-1 and 2 mg of DSPE-PEG2000-TAT added into the flask,5 mL of chloroform to dissolve them,another 2 mg of AMD in the flask,and add 2 mL of anhydrous ethanol was dissolved,and the ethanol solution was slowly added into the trichloromethane solution by syringe under the condition of constant temperature magnetic stirring at 50? to obtain the coarse nano suspension.Then,under the condition of ice bath,ultrasonic dispersion was carried out at a certain power(working for 4 S,intermittently for 2 s)to obtain uniform nanoliposome solution.Then,the organic solvent was removed by rotary evaporation,and uniform film was obtained.After vacuum drying for 2h,2mL phosphate buffer saline was added for hydration,and then AMD nanoliposome was obtained by freeze-drying.2.The light scattering particle size and zeta potential of nanoliposomes were measured by laser scattering particle size analyzer,and the sample was diluted in 3 mL of double distilled water.Take 0.5mL of nanoliposome solution and prepare the sample by negative staining with 1mL of 2%phosphotungstic acid.After mixing,cover one side of copper mesh with formavar supporting membrane on the dye drop,positive staining for 15-30min.After the copper mesh is naturally dried,observed the morphology of nanoliposome under transmission electron microscope.3.The prepared PTA-NLPs lyophilized powder was placed in aseptic vials,sealed and stored in refrigerators at 0-4?,and 10 mg of the sample was taken after 1,2 and 3 months of placement respectively.The particle size and encapsulation efficiency of the PTA-NLPs were measured,and the stability of the preparation was observed under the electron microscope.Result:1.The optimized prescription was selected by single factor method:AMD 2mg,soybean phospholipid 60mg,cholesterol 30mg,DSPE-PEG2000-PCM-1 3mg,DSPE-PEG2000-TAT 2mg and hydration medium 2mL.2.The results showed that the particle size of PTA-NLPs was(124.31 ± 13.62)nm,PDI was 0.21,encapsulation efficiency was(88.61 ± 4.23)%and zeta potential was(-17.53±1.91)mV.3.The transmission electron microscope data showed that the product was spherical in appearance,smooth in surface,without mutual aggregation and even in dispersion;the basic characteristics of PTA-NLPs,such as particle size and zeta potential,remained unchanged after being stored in cold storage for 3 months in dark.Conclusion:1.AMD nanoliposomes modified by PCM-1 and TAT were prepared by high-pressure homogenization,and the optimized formulation was selected by single factor observation.2.The particle size of PTA-NLPs was(124.31±13.62)nm,PDI was 0.21,encapsulation efficiency was(88.61±4.23)%,zeta potential was(-17.53 ± 1.91)mV,which indicated that PTA-NLPs was stable.3.According to the optimized formulation,PTA-NLPs showed spherical appearance,smooth surface and no aggregation phenomenon under transmission electron microscope.The basic characteristics of PTA-NLPs remained unchanged and stable up to 3 months of cold storage.Part ? in vitro evaluation of PTA-NLPsObjective:To evaluate the in vitro release rate of PCM-1 and TAT modified AMD nanoliposomes(PTA-NLPs).To evaluate the inhibitory effect of PTA-NLPs on the growth of MCs cells and the uptake of PTA-NLPs by MCs cells.Methods:1.The release characteristics of PTA-NLPs were observed by in vitro release and cell proliferation experiments.Accurately weigh and take PTA-NLPs 100 mg,put it into a dialysis bag with molecular weight of 8000-12000,tied the bag tightly,put it into a 200 mL PBS(pH=7.4)release medium,and carried out the release test in a 37?constant temperature water bath under 200 rpm oscillation,with 3 samples operated in parallel.At 0.5,1,2,3,4,6,8,10 and 12 h after the release,1 mL of medium was extracted and filtered with 0.45 mm microporous membrane.Meanwhile,the same dose of free AMD,A-NLPs,PA-NLPs and TA-NLPs were used as control.HPLC was used to determine the actual measured concentration of each sample in the medium at each time point,and the corrected concentration was calculated according to the formula.The cumulative release percentage was calculated with the corrected concentration.2.The MCs cells were cultured 96 well culture plated at 37? in 5%CO2 incubator for for 24 hours,then the culture solution was discarded,the cells were carefully washed twice with PBS,and then different amd preparations(free AMD,A-NLPs,PA-NLPs,TA-NLPs and PTA-NLPs)were added,the dosage was from 5 ?g/mL to 50 ?g/mL,and 60 ?L MTT(thiazole blue)solution(5 mg/mL)and 1mL were added The inhibition of AMD nanoliposomes on the growth of MCs cells was detected after incubation in a incubator for 4 hours.Suck out the supernatant and discard it.Add 150 ?L DMSO(dimethylsulfoxide)into each hole,and shake it on a flat shaking table for 5 minutes.The absorbance value(a)was measured at 570 nm with an enzyme reader.The cell inhibition rate was calculated=[(a blank control group-a administration group)/a blank control group]× 100%.3.Using coumarin-6(C6,concentration of 100mg/mL)as fluorescence probe,according to the preparation method of AMD nanoliposome,combined with film hydration method,C6 is embedded in the lipophilic core of nanoliposome,and then the AMD related nanoliposome with green fluorescence can be obtained.MCs cells were inoculated in a 12 well culture plate for 24 h.The culture medium was DMEM(Dulbeccos Modified Eagle Medium,the Mediim containing various amino acids and glucose)containing 10%bovine serum.The cell density was controlled at 4.0 × 105 cells/well.Then the AMD preparations(free AMD,A-NLPs,PA-NLPs,TA-NLPs and PTA-NLPs)of each group were incubated with MCs,respectively.The concentration of coumarin-6 was 100 mg/mL,and the incubation time was 2 hours,the cells were washed with PBS solution(pH 7.4)for three times,and the remaining fluorescent substances were removed.Finally,the distribution of coumarin-6 in the cells was observed by laser scanning confocal microscope.At the same time,after digesting MCs as a single cell suspension by trypsin,flow cytometry assay was used to detect the absorbed quantitatively of cells and then determine the fluorescence intensity.Results:1.PTA-NLPs was released to PBS solution for 12 hours,the cumulative release rate was only(53.3±0.3)%,which had a strong sustained-release performance.2.Using MTT test,the cytotoxicity of the related preparations in the experimental group(free AMD,A-NLPs,PA-NLPs,TA-NLPs,PTA-NLPs)was compared.Until the concentration reached 50 ?g/mL,the preparations in each group significantly inhibited the growth of cardiomyocytes,and there was significant statistical difference compared with the control group(P<0.05),but when the concentration was less than 50 ?g/mL,there was no significant effect on myocardial cell activity in all groups(P>0.05).3.The uptake of PTA-NLPs by flow cytometry was significantly better than that of PA-NLPs and TA-NLPs.Conclusion:1.The cumulative release rate of PTA-NLPs in vitro for 12 hours was only(53.3±0.3)%.PTA-NLPs has a better sustained-release performance.The slow release characteristics of PTA-NLPs were conducive to the stability and targeting of drug delivery system in vivo,so as to change the drug distribution behavior in vivo.2.When the concentration of PTA-NLPs was less than 50 ?g/mL,there was no significant cytotoxicity.3.The uptake ability of PTA-NLPs by MCs was significantly better than that of PA-NLPs and TA-NLPs,indicating that the combined modification of PCM-1 and TAT can play a synergistic effect and increase the ability of AMD drugs to enter cardiomyocytes.Part ? pharmacokinetics and tissue distribution of PTA-NLPsObjective:1.HPLC(high performance liquid chromatography)was used to detect the concentration of drugs in AMD and to study the pharmacokinetics and tissue distribution of PTA-NLPs system.Methods:1.HPLC was used to detect the concentration of AMD in vivo.Chromatographic conditions:column:dikma diamond C18(5 ?m,200 mm × 4.6 mm);mobile phase:acetonitrile-100 mmol/L sodium dihydrogen phosphate(pH value of phosphoric acid adjusted to 3.0)(55:45);flow rate:1.2 ml/min;detection wavelength:241 nm;column temperature:30?,Take 0.3mL blank plasma,add different amount of AMD stock solution,prepare standard blood samples with concentration of 10,50,100,500,2500 and 5000 ng/ml,repeat each concentration for three times,and conduct linear regression with drug peak area ratio(a)to drug concentration(c),and obtain standard curve formula(A=7.276C-1.283)the drug peak area of rats was determined by HPLC after intravenous injection,and the concentration of AMD in vivo was calculated by the standard curve.2.Thirty healthy Sprague Dawley rats were divided into five groups.Each group(AMD solution,A-NLPs,PA-NLPs,TA-NLPs and PTA-NLPs)was given 5 mg/kg through tail vein respectively.And 0.6 mL of blood was collected through ophthalmic vein at 0.5,1,2,4,6,8,10,12 and 24 h before and after administration respectively.The blood was collected in polypropylene centrifuge tube with heparin and centrifuged for 10 min at 12000 r/min.the plasma was separated and stored in refrigerator at-18? for blood concentration analysis.3.One hundred and eighty healthy Kunming mice were divided into 5 groups.Each group(AMD solution,A-NLPs,PA-NLPs,TA-NLPs and PTA-NLPs,respectively)was given 5 mg/kg through tail vein respectively.About 0.6 mL of blood was extracted from eyeball at 0.5,1,2,4,8 and 12 h after administration,and placed in a centrifuge tube with heparin before administration.Plasma was separated after centrifugation for 10 min at 12000 r/min.Six mice in each group were repeated at each time point.All samples were stored at-20? for drug concentration in the tissues.At the same time,the mice heart,liver,spleen,lung and kidney,washing them with proper amount of normal saline,then sucking up the plasma through filter paper,and observing the pathological changes of each organ.Results:1.By the HPLC method,AMD has symmetrical peak shape,steep peak,good resolution and no interference of impurity peak near the main peak.The recovery rate of the prepared plasma and tissue samples in different concentrations was showed between 90%and 105%,and the recovery rate of extraction was showed more than(70±3)%,RSD(Relative Standard Deviation)within and between days was significantly different(P<0.05).2.After administration,the concentration of AMD solution group was the highest(313.3 ng/mL),but then the concentration decreased rapidly(elimination half-life was short,2.6 h),and completely metabolized at 10 h.When AMD enters the body in the form of NLPs,its peak plasma concentration drops significantly,maintaining at 130-160 ng/mL,but the elimination half-life is significantly prolonged.The double modified AMD-NLPs can reach 7.2 hours,30.70 ng/mL at 10 hours and 11.16 ng/mL at 24 hours.3.In the experimental group(PTA-NLPs),there were significant differences in the different tissues,especially in the heart.In the PTA-NLPs group,the concentration of AMD in the myocardium of mice is reached 354ng/g in one hour,which was significantly higher than other tissues and plasma,with significant statistical difference(P<0.05).Conclusion:1.HPLC assay in this study is suitable for the determination of AMD in vivo.It has the characteristics of good specificity,high sensitivity,simple and fast.2.Studies on the pharmacokinetic and histological distribution of PTA-NLPs system have shown that PTA-NLPs system can not only delay the release of drugs in the body and has a long circulation effect,but also concentrate more on the heart without the histological changes of organs,laying a foundation for the follow-up clinical efficacy studies.Part ? pharmacodynamic study of PTA-NLPsObjective:to establish a rat model of myocardial infarction and evaluate the pharmacodynamics of PTA-NLPs.Methods:90 SD rats were randomly divided into 9 groups,including sham operation group,model group and AMD group,A-NLPs group,PA-NLPs group,TA-NLPs group,PTA-NLPs(low dose)group,PTA-NLPs(medium dose)group and PTA-NLPs(high dose)group.SD rats treated with 3%pentobarbital sodium(30 mg/kg)by intraperitoneal injection of line,lie on your back is fixed in the animal operating table,after anesthesia endotracheal intubation after connection animal breathing machine,rats are recorded with a standard of ? lead electrocardiogram(ecg),along the left edge sternum is 2?4 open thoracic rib clearance,exposed to the heart,cut happy capsular,using noninvasive sutures under left from 2 to 3 mm at the aortic root with a 6-0 thread through a small bunch of cardiac muscle and the anterior descending coronary artery ligation(the control group only thread ligation),electrocardiogram lead ? marking ST-elevation myocardial infarction model preparation of success.According to the grouping,different preparations were slowly injected into the caudal vein 5min after coronary artery ligation.Rat electrocardiograph was used to monitor the electrocardiogram,and ST segment changes at 30,60,90 and 120min after administration were recorded in each group.The abdominal cavity was opened and the abdominal aortic blood was extracted with a sterile syringe for about 5mL.The serum was centrifuged after resting for 5 min to determine the contents of creatine kinase(CK),lactate dehydrogenase(LDH),aspartate transaminase(AST)and malondialdehyde(MDA).Extraction after abdominal aortic blood quickly cut open chest gather the heart,the heart was washed with PBS to remove blood along the coronary sulcus resection of the right ventricle,the left ventricle was preserved,the ventricle was cut into 4-5 pieces parallel to the ligation line along the direction from the apex to the bottom of the cardiac base,and then incubated in 0.1%NBT(Nitro-Blue Tetrazolium)dye solution in 37? for 10 min,and then taken out.The filter paper was used to absorb the water.The Image ProPlus 6.0 was used to process the image,calculate the myocardial infarction area and left ventricular area,and calculate the ratio of myocardial infarction area(myocardial infarction area ratio=infarction area/left ventricular area).Results:1.Changes of ST segment:during the observation period of 120min,the ST segment elevation of rats in the model group was maintained at a relatively high level with no downward trend.The ST segment of the rats receiving different AMD preparations decreased to different degrees.The ST segment of the high-dose dual-target AMD nanoliposomes group decreased the most significantly,reaching(0.09±0.07)mV,which was statistically significant compared with other groups(p<0.05).2.Changes in biochemical indicators:compared with the model group,the serum levels of CK,LDH,AST and MDA in the acute myocardial infarction model rats of each AMD group decreased,and the effect of the double-target modified nanoliposome was significantly better than that of the single-target nanoliposome,with the largest reduction range of PTA-NLPs(CK:(3289.50±1102.90)U/L.LDH:(1854.60±191.30)U/L;AST:(583.50±142.70)U/L;MDA:(8.20±2.10)U/L;P<0.05).3.Myocardial infarct area:the ratio of myocardial infarct area in the model group was about 38%.After administration of different AMD preparations,the ratio of myocardial infarct area in each group decreased to different degrees.The ratio of myocardial infarct area in the high,middle and low dose dual target AMD nanoliposomes group significantly decreased,and the ratio of myocardial infarct area in the high dose PTA-NLPs group(13%)decreased mostly significantly.Conclusion:1.The ST segment elevation of all the AMD groups in the rat model of acute myocardial infarction decreased,with the mostly significant decrease in the high-dose PTA-NLPs group,indicating that the double-target amiodarone nanoliposomes(especially at the high dose)decreased the ST segment of the model rats more significantly than the single-target amiodarone nanoliposomes.2.Compared with single-target amiodarone nanoliposomes,double-target amiodarone liposomes(especially at high dose)significantly reduced the serum levels of CK,LDH,AST and MDA in myocardial infarction model rats.3.The myocardial infarction area ratio of different AMD preparation groups decreased at different extent,with the mostly significant decrease in the high-dose PTA-NLPs group,indicating that the double-target amiodarone nanoliposomes(especially at high dose)significantly reduced the myocardial infarction area of model rats than the single-target amiodarone nanoliposomes.