Experimental Study On Drug-Loaded Targeted Collagen Phase Change Nanoparticles For Diagnosis And Treatment Of Myocardial Fibrosis

PART Ⅰ PREPARATION AND PHYSICOCHEMICAL PROPERTIES OF DRUG-LOADED TARGETED COLLAGEN PHASE CHANGE NANOPARTICLES CNA35/PFP/DIZE@NPSObjective:To prepare a liquid fluorocarbon lipid nanoparticle angiotensin converting enzyme2(ACE2)agonist diminazene aceturate(DIZE),which carries collagen specific binding protein(CNA35).(CNA35/PFP/DIZE@NPs)and study its physical and chemical properties.Methods:Phase-change drug-loaded liquid fluorocarbon lipid nanoparticles(DIZE/PFP/@NPs)were prepared by reverse rotary evaporation combined with acoustic vibration method,and CNA35 was connected to the surface by carbodiimide method to obtain drug-targeted phase Variable nanoparticles(CNA35/PFP/DIZE@NPs).The appearance,particle size,potential,connection rate,drug loading and encapsulation rate were measured.And observe the stability of nanoparticles in vitro and drug release ability.Nanoparticles were treated at different temperatures and ultrasonic intensities to observe their liquid-gas phase changes.Results:The prepared drug-loaded targeted phase-change nanoparticles CNA35/PFP/DIZE@NPs were spot-shaped and uniformly dispersed under a light microscope and a fluorescence microscope;the nanoparticles were spherical and uniform in size observed by transmission electron microscopy.The Malvern laser particle size instrument measures nanoparticles with a diameter of(304.68±4.21)nm and a potential of(-14±0.23)mV.Compared with non-targeted or non-drug-loaded nanoparticles,there is no statistically significant difference in particle size.The connection rate of CNA35 to the nanometer by flow cytometry was(75±0.34)%.In vitro stability tests showed no significant change in morphology and particle size of the nanoparticles within 24 hours at a temperature of 4℃.The entrapment efficiency of the nanoparticles measured by high performance liquid chromatography was(25.33±9.47)%,and the drug loading was(3.80±0.65)%.The in vitro drug release test showed that the nanometer drug release was faster within 12 hours,and the drug release was about 24%,36h drug release was greater than 50%,and then entered a slow-release plateau phase.The nanoparticles were irradiated with ultrasound with different intensities.As the sound intensity increased,the amount of drug released by the nanoparticles also increased significantly.The nanoparticles were heated at different temperatures,and no obvious changes were observed at 40℃under the microscope;at 45℃,some of the nanoparticles became larger in volume and showed microbubble-like changes;at 50℃,there were a large number of microbubbles;At 55℃,most of the nanoparticles were rapidly transformed into microbubbles,and some of them were broken instantly.Ultrasound irradiation nanoparticles with different parameters were observed to show that with the increase of the ultrasonic intensity and the irradiation time,the acoustically induced phase-change nanoparticles showed ultrasound-enhanced imaging with different intensities under the detection of ultrasonic instruments.The in vitro acoustic induced phase change found that the acoustic ultrasonic parameter was a condition of an intensity of 3W/cm~2 for an irradiation time of 3min,which could not only cause a large number of nanoparticle phase changes,but also achieve a more ideal ultrasound enhanced imaging.Conclusions:Liquid fluorocarbon lipid nanoparticles(CNA35/PFP/DIZE@NPs)carrying CNA35 antibodies and DIZE drugs have been successfully prepared.They have regular morphology,uniform size,stable physicochemical properties,and good liquid-gas phase change and drug release ability.PART Ⅱ STUDY ON IMAGING OF RABBIT MYOCARDIAL FIBROSIS WITH DRUG-LOADED TARGETED COLLAGEN PHASE CHANGE NANOPARTICLES CNA35/PFP/DIZE@NPSObjective: To detect the targeting of CNA35/PFP/DIZE@NPs nanoparticles to rabbit myocardial fibrosis in vitro and in vivo and the effect of ultrasound molecular imaging.Methods: This part of the experimental study is divided into three sections.In the first section,a rabbit myocardial fibrosis model was first established.The rabbit left coronary artery was ligated with a thoracotomy.The degree of myocardial fibrosis was examined by staining and FITCCNA35 staining.In the second section,frozen sections of rabbit myocardial fibrosis were prepared,and the myocardial fibrotic sections were treated with the prepared CNA35/PFP/DIZE@NPs nanoparticles.The confocal microscope was used to observe them in vitro targeting.In vivo targeting test: After intravenous injection of CNA35/PFP/DIZE@NPs nanoparticles for 10 min,the animals were sacrificed for frozen sections,and the in vivo targeting effect was observed under a laser confocal microscope.In the third section,after injecting CNA35/PFP/DIZE@NPs nanoparticles through the marginal vein of rabbit ears,the ultrasound imaging of myocardial fibrosis in vivo was observed with diagnostic ultrasound after irradiating with LIFU of 3W/cm~2 for 3minutes.Results: A model of myocardial fibrosis was established by ligation of the left coronary artery with thoracotomy,and staining of myocardial fibers with PSR,Masson,and CNA35 showed that the fibrosis range was(31.29±4.95)%,(32.31±5.49)%,and(27.56±5.35)%,The difference was not statistically significant(P>0.05).Targeted detection in vivo and in vitro showed that FITC-CNA35 was conjugated to NPs and showed green under a laser confocal microscope,while the Di I-labeled lipid membrane was red and the DAPI-labeled nuclei showed blue fluorescence.From frozen sections,a large amount of red and green fluorescence can be clearly observed in the blue-stained nuclei,and the two fluorescences overlap well.It is proved that a large amount of collagen is deposited in the intercellular substance after myocardial fibers,and the type I collagen-specific binding protein CNA35 can well target the type I collagen deposited in the intercellular substance after myocardial fibrosis.Conclusions: The prepared CNA35/PFP/DIZE@NPs nanoparticles have type I collagen targeting myocardial fibrosis after myocardial infarction.PART Ⅲ THE STUDY OF DRUG-LOADED TARGETED COLLAGEN PHASE CHANGE NANOPARTICLES CNA35/PFP/DIZE@NPS IN THE TREATMENT OF RABBIT MYOCARDIAL FIBROSISObjective: To observe the study of CNA35/PFP/DIZE@NPs nanoparticles in the treatment of myocardial fibrosis.Methods: A rabbit model of myocardial infarction was established.One week later,it was randomly divided into 4 groups of 5 animals,each of which was: myocardial infarction(MI)group;the phase change nanoparticles CNA35/PFP@NPs(C-NPs)group was targeted;Drug-only(DIZE)group;Drug-loaded targeted phase change nanoparticles CNA35/PFP/DIZE@NPs(CD-NPs)group.Before each group,conventional echocardiography was performed to evaluate the cardiac function of each group.The DIZE group and the C-D-NPs group were treated with 0.5mg/kg of DIZE through the ear marginal vein;the MI group and the C-NPs group were injected with the same amount of normal saline.After injection,all rabbits were irradiated with a low-power focused ultrasound therapy instrument(LIFU)with a power of 3W/cm2.The irradiation time was 3 minutes,and the myocardial echo intensity was observed with diagnostic ultrasound.Treatment was performed every three days for a total of 4 treatments.Times.Four weeks later,echocardiography was performed to evaluate cardiac function in each group.Animals were sacrificed and myocardial infarction tissues were taken for myocardial collagen staining;RT-PCR was used to examine the expression levels of angiotensin-converting enzymes ACE2 and ACE genes in myocardial tissues;ELISA was used to determine collagenⅠ and vascular tone in myocardial tissues Angiotensin Ⅱ(Ang Ⅱ)and Ang(1-7);Western blot was used to detect the expression of angiotensin Ⅱ type 1 receptor(AT1R)and Mas R receptor protein in cardiac tissue.Results: Echocardiographic examination results showed that the left ventricular systolic function of each group was reduced by about 46% before treatment,and there was no significant difference between the groups.However,after different treatments,the cardiac function of the CD-NPS group and the DIZE group was significantly recovered compared with the other two groups,and the difference was statistically significant.The recovery of ejection fraction EF in the CD-NPs group was also better than that in the DIZE group(P<0.05).The PSR and Masson collagen staining results showed that collagen staining in the MI group and C-NPs group was significantly more extensive than that in the DIZE and C-D-NPs groups.There was no significant difference in collagen staining between MI group and C-NPs group,DIZE group and C-D-NPs group.The results of RT-PCR showed that the expression of ACE2 in the CD-NPs group was significantly higher than that in the MI group,while the ACE was lower than that in the MI group.The difference was statistically significant(P<0.05).Compared with the DIZE group,the expression of ACE2 was statistically different.Significant(P<0.05),but there was no significant difference in the expression of ACE between the two groups(P>0.05).The correlation between the amplification factors of the two genes showed that the ACE2/ACE in the C-D-NPs group was significantly higher than the other three groups,and the difference was statistically significant(P<0.05).ELISA showed that the content of Ang(1-7)in the CD-NPs group was significantly higher than that in the MI group,while Ang Ⅱ and Collagen I were lower than those in the MI group,and the difference was statistically significant(P<0.05);Ang(1-7)and Collagen I had statistically significant differences(P <0.05),while Ang Ⅱ had no significant difference between the two groups(P>0.05).Western blot semi-quantitative analysis showed that the expression of AT1 R in the CD-NPs and DIZE groups was significantly lower than that in the MI and C-NPs groups,while the expression of Mas R was significantly higher than the other two groups,the difference was statistically significant;The difference in Mas R expression was statistically significant(P<0.05),but the difference in AT1 R expression was not statistically significant(P>0.05).Conclusions: A liquid-gas phase angiotensin-converting enzyme 2 agonist triazine-containing fluorocarbon nanoparticle with CNA35 molecular probe targeting myocardial collagen was successfully developed,and combined with ultrasound irradiation can treat myocardial fibrosis,Provides a new method for the accurate diagnosis and treatment of myocardial fibrosis.