Polyphenol-assisted Facile Assembly Of Bioactive Nanoparticles For Targeted Therapy Of Heart Diseases

Heart diseases（HD）remain the leading cause of morbidity and mortality worldwide.As for conventional formulations,their efficacies are generally compromised as a result of short retention time and low distribution of drug molecules in heart tissues.Delivery via either intramyocardial or epicardial injection presents unsatisfactory effectiveness of clinical treatment due to invasiveness and nonuniform distribution.Increasing evidence has demonstrated that nanotherapies are promising for the treatment of heart diseases.However,facile engineering of heart-targeting nanotherapies with high delivery efficiency,good quality control,desirable manufacturing reproducibility and scalability,and relatively low cost remains highly challenging.Taking advantage of the high affinity of polyphenols to myocardium cells and tissues,we found that nanoparticles can be facilely engineered from different types of carrier materials by polyphenol-assisted nanoprecipitation/self-assembly.Both in vitro and in vivo studies showed that nanoparticles with potent bioactivity were obtained.Functional nanoparticles showed considerable accumulation in the injured heart and afforded excellent therapeutic effects in heart diseases,Methods1.Synthesis and characterization of a reactive oxygen species（ROS）-responsiveβ-CD material and a broad-spectrum ROS-eliminating materialOCD was obtained functionalizingβ-CD with 4-（Hydroxymethyl）phenylboronic acid pinacol ester（PBAP）.TPCD was synthesized by sequentially conjugating 4-Hydroxy-Tempol（Tpl）and PBAP ontoβ-CD.Different materials were confirmed by Fourier transform-infrared¹H NMR,and MALDI-TOF.2.Elimination of ROS by different polyphenols and carrier materialsThe radical-eliminating tests,H₂O₂-scavenging activity and superoxide anion-scavenging capability of different materials were assessed.3.Fabrication and characterization of different nanoparticles in the presence of polyphenolsGallic acid（GA）,catechin（CAT）,epigallocatechin gallate（EG）,or tannic acid（TA）was dissolved in deionized water to obtain a water phase,while poly（lactide-co-glycolide）（PLGA）,OCD,or TPCD was dissolved in a specific organic solvent to give rise to an organic phase.Nanoparticles（NPs）were prepared by a polyphenol-assisted nanoprecipitation/self-assembly method.By optimizing different parameters,nanoparticles were prepared.The size,size distribution profiles,andζ-potential values of different NPs were measured and the stability of NPs was determined.The presence of TA on different NPs was directly indicated by incubation with Ag NO₃,FT-IR spectra,BCA assay and X-ray photoelectron spectroscopy.4.In vitro ROS-responsive hydrolysis and ROS-scavenging activity of different NPsROS-responsive hydrolysis was calculated by measured the hydrolysis degree and the particle size distribution profiles of the corresponding samples after dispersed in H₂O₂.Hydroxyl radical,superoxide anion,and H₂O₂ scavenging activity by different NPs was tested by corresponding kit.5.Cellular uptake of functional nanoparticlesH9C2 cells were incubated with medium containing Cy5-labeled TPTD-TA NP（Cy5/TPTN）and incubated for different time.Confocal laser scanning microscopy（CLSM）and flow cytometry was used to test phagocytosis.Similarly,cellular uptake in hypoxic/ischemic injury cells and cardiac hypertrophy cells determined by flow cytometry.6.In vitro therapeutic effects of TPTN in hypoxic/ischemic injury or hypertrophy cellsAfter sequential treatment with different doses of TPTN and Co Cl₂ or Isoprenaline hydrochloride（ISO）,intracellular ROS generation of H9C2 was compared.The levels of H₂O₂ and malondialdehyde（MDA）of H9C2 cell lysates and the levels of tumor necrosis factor（TNF）-αand cardiac troponin（c Tn）-I in the supernatant of culture medium were analyzed.7.In vivo heart targeting of TPTN in rats with ventricular fibrillationThe VF rat model and cardiac hypertrophy mice model was successfully established.The fluorescence intensities of Cy7.5/TPTN in hearts collected from different groups were analyzed by the Living Imaging software.8.In vivo therapeutic effects of TPTN in rats with ventricular fibrillationAfter establishment of ventricular fibrillation cardiac arrest and resuscitation,animals were randomly divided into five groups.During treatment,arterial pressure（MAP）and left ventricular ejection fraction（LVEF）were recorded at hourly intervals after resuscitation for 5h with an echocardiography system.The neurological deficit score and adverse events were examined at 96 h and the myocardial sections were prepared.In addition,at 6 h after resuscitation,the levels of H₂O₂,MDA,TNF-αand Interleukin（IL）-6 were separately measured.c Tn-I and creatine kinase（CK）in serum were detected with the related ELISA kits.9.In vivo therapeutic effects of TPTN in mice with myocardial hypertrophyAfter ISO administration,mice were randomized into four experimental groups.Echocardiography was used to assess ventricular wall thickness and cardiac function.After treatment,myocardial sections were prepared and the levels of H₂O₂,MDA,TNF-α,and IL-6of heart tissues were separately measured.Also,serum levels of c Tn-I and B-type natriuretic peptide（BNP）were quantified.10.Preliminary safty evaluation of functional nanoparticlesCCK-8 assay and hemolysis test were examined to prove the safety of TPTN in vitro.In addition,weight changes and organ indices of typical major organs were observed during the treatment of cardiac hypertrophy in mice.Further quantification of biomarkers related to hepatic and kidney functions were measured after treatment with different doses of TPTN.Results1.Successful preparation of OCD was confirmed by FT IR and ¹H NMR as well as MALDI-TOF.Calculation according to related proton signals in the ¹H NMR spectrum of OCD revealed approximately 5 PBAP was conjugated in eachβ-CD molecule.The expected structure of TPCD was affirmed by FT IR and ¹H NMR spectra.Based on the corresponding¹H NMR spectrum,approximately 2 Tpl and 5 PBAP were covalently linked withβ-CD.2.Well-defined NPs with tailorable particle sizes could be successfully and efficiently prepared based on polyphenol-assisted nanoprecipitation/self-assembly of different materials.By optimizing different parameters,such as the weight ratio between carrier materials and polyphenols,the type of polyphenols,and the organic phase,well-defined nanoparticles with desirable physicochemical properties can be easily prepared.3.We confirmed the presence of TA on different NPs and the colloidal stability to nanoprecipitated NPs mainly via hydrogen-bonding interaction between carrier materials and TA.Also,ROS-scavenging and responsive capabilities of different NPs obtained in the presence of TA were excellent.4.TPTN can be efficiently internalized by H9C2 cells in both normal and injury states.TPTN can effectively inhibit oxidative stress,attenuate inflammatory response,and prevent cell injury in cardiomyocytes with induced hypoxic-ischemic injury or cardiac hypertrophy.5.Ventricular fibrillation rat model and myocardial hypertrophy mice model were established.Ex vivo imaging revealed significantly higher fluorescent signals in hearts isolated from model group,compared to those of the sham group.Also,fluorescence observation was conducted after i.v.administration of Cy5/TPTN.These results unambiguously demonstrated that TPTN delivery by i.v.injection can efficiently target the heart.6.TPTN is a promising nanotherapy for targeted treatment of post-cardiac arrest myocardial dysfunction,by effectively accumulating in the cardiac injury site,attenuating oxidative stress（H₂O₂ and MDA）,inhibiting local inflammation（TNF-αand IL-6）,and protecting myocardiocytes from ischemia-reperfusion injury（c Tn-I and CK）.Accordingly,treatment with TPTN notably increased the survival rate,MAP and LVEF of rats with ventricular fibrillation.Moreover,both TPTN and TPTN+TH groups exhibited no significant collagen deposition revealed by Masson’s trichrome-staining.7.In mice with induced hypertrophy,therapy with TPTN dose-dependently reduced left ventricular wall thickness,improved ejection fraction,and ameliorated the heart indices.Also,TPTN effectively reduced the levels of oxidative mediators,pro-inflammatory cytokines and serum markers of myocardial injury.TPTN can also effectively suppress ISO-induced cardiac fibrosis in mice.8.High cell viability was measured regardless of various doses of TPTN after incubation for different time.Negligible hemolysis was found after incubation with various concentrations of TPTN.In addition,no significant weight loss or abnormal changes in behaviors were observed during the treatment of cardiac hypertrophy in mice.Conclusion1.We have successfully developed a polyphenol-assisted nanoprecipitation/self-assembly strategy for facile engineering of NPs for targeted therapy of heart diseases.By optimizing different parameters,such as the weight ratio of carrier materials and polyphenols,polyphenol types,and organic phase,well-defined NPs with desirable physicochemical properties can be easily prepared.2.In combination of a bioactive cyclodextrin-based material TPCD with TA,a multifunctional nanotherapy TPTN was successfully engineered,which exhibited excellent ROS-sensitivity and ROS-scavenging activity.Cellularly,TPTN protected rat cardiomyocytes from hypoxic-ischemic injury after internalization into cells.3.After i.v.injection,TPTN effectively accumulated at the heart injury site of rats resuscitated from ventricular fibrillation cardiac arrest.Correspondingly,TPTN efficaciously ameliorated post-cardiac arrest myocardial dysfunction in rats,by attenuating oxidative stress,inhibiting inflammatory responses,and mitigating myocardial injury.Heart targeting and therapeutic benefits of TPTN were demonstrated in mice with induced pathological myocardial hypertrophy.4.Preliminary in vitro and in vivo experiments revealed good safety of TPTN.