ECHO provides layer-specific insight of both myocardial deformation and microcirculation dysfunction in dilated cardiomyopathy patients: Clinical value of combined application of left ventricular layer-specific strain and myocardial contrast echocardiography.

AIM: To investigate the pattern of left ventricular (LV) function and myocardial perfusion and their relationship in dilated cardiomyopathy (DCM) patients using layer-specific speckle tracking imaging (STI) and layer-specific myocardial contrast echocardiography (MCE). MATERIAL AND METHODS: Thirty DCM patients and 30 controls were recruited and underwent STI and MCE examination. The peak values of longitudinal strain (LS), circumferential strain (CS) of each layer of LV were recorded and compared between groups. Additionally, cross-sectional area of a microvessel (A) and average myocardial microvascular lesion (ß) of each layer were measured, myocardial blood flow (MBF) was estimated using A × ß, above parameters were compared between two groups. RESULTS: The LS of endo- (LSendo ), mid- (LSmid ) and epicardium (LSepi ), as well as CS of endo- (RSendo ), mid- (RSmid ), (LSepi ) epicardium and LS endo/epi, CS endo/epi were significantly decreased in DCM patients. More importantly, DCM patients demonstrated decreased A, ß and A × ß in all three myocardium layers and A endo/epi, ß endo/epi, A × ß endo/epi compared to the controls. The time to peak and the cardiac cycle required to reach the peak were prolonged in DCM patients (p < 0.05). Longitudinal strain parameters of each layer had a negative relationship with perfusion parameter A and this relationship was strongest between LSendo and Aendo (r = 0.690, p < 0.01). CONCLUSIONS: The cardiac strain and, more importantly, coronary microcirculation perfusion was impaired in each layer in DCM patients. The longitudinal function of the LV myocardium was closely related to changes in myocardial microcirculation perfusion.

Optimization of the cotransfection of SERCA2a and Cx43 genes for myocardial infarction complications.

As our previous study showed, the therapeutic effect of two genes (SERCA2a and Cx43) on heart failure after myocardial infarction (MI) was greater than that of single gene (SERCA2a or Cx43) therapy for bone marrow stem cell (BMSC) transplantation. Based on previous research, the aim of this study was to investigate the optimal ratio of codelivery of SERCA2a and Cx43 genes for MI therapy after biotinylated microbubble (BMB) transplantation via ultrasonic-targeted microbubble destruction (UTMD). Forty rats underwent left anterior descending (LAD) ligation and BMSC injection into the infarct and border zones. Four weeks later, the genes SERCA2a and Cx43 were codelivered at different ratios (1:1, 1:2 and 2:1) into the infarcted heart via UTMD. Cardiac mechanoelectrical function was determined at 4 wks after gene delivery, and the hearts of the rats were harvested for measurement of MI size and detection of SERCA2a and Cx43 expression. Q-PCR analysis of the expression of Nkx2.5 and GATA4 in the myocardial infarct zone and measurement of neovascularization in infarcted hearts. After comparing the therapeutic effects of different cogene ratios, the SERCA2a/Cx43-1:2 group showed remarkable cardiac electrical stability and strengthened the role of anti-arrhythmia. In conclusion, the optimum ratio of the SERCA2a/Cx43 gene is 1:2, which is advantageous for maintaining cardiac electrophysiological stability.

Experimental study on the optimization of ANM33 release in foam cells.

Given the miR-33's mechanistic relationships with multiple etiological factors in the pathogenesis of atherosclerosis (AS), we investigated the therapeutic potentials of dual-targeted microbubbles (HA-PANBs) in foam cell-specific release of anti-miR-33 (ANM33) oligonucleotides, resulting in the early prevention of AS progression and severity. The intracellular localization, loading optimization, and therapeutic effects of HA-PANBs were examined in detail in a co-cultured cell model of phagocytosis. Compared with non-targeting nanobubbles (NBs) and single-targeted microbubbles as controls, HA-PANBs efficiently delivered the ANM33 specifically to foam cells via sustained release, exhibiting its clinical value in mediating RNA silencing. Moreover, when used at a dose of 12 µg/mL HA-PANBs per 107 cells for 48 h, a higher release rate and drug efficacy were observed. Therefore, HA-PANBs, effectively targeting early AS foam cells, may represent a novel and optimal gene therapy approach for AS management.

Pre-transplantation of Bone Marrow Mesenchymal Stem Cells Amplifies the Therapeutic Effect of Ultrasound-Targeted Microbubble Destruction-Mediated Localized Combined Gene Therapy in Post-Myocardial Infarction Heart Failure Rats.

Although stem cell transplantation and single-gene therapy have been intensively discussed separately as treatments for myocardial infarction (MI) hearts and have exhibited ideal therapeutic efficiency in animal models, clinical trials turned out to be disappointing. Here, we deliver sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) and connexin 43 (Cx43) genes simultaneously via an ultrasound-targeted microbubble destruction (UTMD) approach to chronic MI hearts that have been pre-treated with bone marrow mesenchymal stem cells (BMSCs) to amplify cardiac repair. First, biotinylated microbubbles (BMBs) were fabricated, and biotinylated recombinant adenoviruses carrying the SERCA2a or Cx43 gene were conjugated to the surface of self-assembled BMBs to form SERCA2a-BMBs, Cx43-BMBs or dual gene-loaded BMBs. Then, the general characteristics of these bubbles, including particle size, concentration, contrast signal and gene loading capacity, were examined. Second, a rat myocardial infarction model was created by ligating the left anterior descending coronary artery and injecting BMSCs into the infarct and border zones. Four weeks later, co-delivery of SERCA2a and Cx43 genes to the infarcted heart were delivered together to the infarcted heart using the UTMD approach. Cardiac mechano-electrical function was determined 4 wk after gene transfection, and the infarcted hearts were collected for myocardial infarct size measurement and detection of expression of SERCA2a, Cx43 and cardiac-specific markers. Finally, to validate the role of BMSC transplantation, MI rats transplanted or not with BMSCs were transfected with SERCA2a and Cx43, and the cardiac mechano-electrical function of these two groups of rats was recorded and compared. General characteristics of the self-assembled gene-loaded BMBs were qualified, and the gene loading rate was satisfactory. The self-assembled gene-loaded BMBs were in microscale and exhibit satisfactory dual-gene loading capacity. High transfection efficiency was achieved under ultrasound irradiation in vitro. In addition, rats in which SERCA2a and Cx43 were overexpressed simultaneously had the best contractile function and electrical stability among all experimental groups. Immunofluorescence assay revealed that the levels of SERCA2a and/or Cx43 proteins were significantly elevated, especially in the border zone. Moreover, compared with rats that did not receive BMSCs, rats pre-treated with BMSCs have better mechano-electrical function after transfection with SERCA2a and Cx43. Collectively, we report a promising cardiac repair strategy for post-MI hearts that exploits the providential advantages of stem cell therapy and UTMD-mediated localized co-delivery of specific genes.

Experimental Study on the Compatibility and Characteristics of a Dual-Target Microbubble Loaded with Anti-miR-33.

OBJECTIVE: To prepare a new type of dual-target microbubble loaded with anti-miR-33 (ANM33). METHODS: Carrier core nanobubbles (NBs) were prepared by thin film hydration, and microbubbles loaded with PM1 (PCNBs) were prepared by grafting DSPE-PEG2000-maleimide-PM1 onto the NB surface. ANM33 was connected via electrostatic adsorption and covalent bonding, and hyaluronic acid (HA) was covalently connected. PM1 and HA were the targets, and ANM33 was the intervention drug. To evaluate the general physical and chemical properties of the prepared dual-target microbubbles loaded with ANM33 (HA-PANBs), we observed their morphology, particle size and surface potential while monitoring their stability and in vitro imaging ability, evaluated their toxic effect on cells and verified their ability to target cells. RESULTS: HA-PANBs had a regular morphology and good stability. The average particle size measured by a Malvern potentiometer was 1421.75±163.23 nm, and the average surface potential was -5.51±1.87 mV. PM1 and ANM33 were effectively connected to the NBs. The PM1, ANM33, and HA binding reached 89.0±1.1%, 65.02±5.0%, and 61.4±3.5%, respectively, and the maximum binding reached 2 µg, 5 µg, and 7 µg/108 microbubbles, respectively. HA-PANBs had no obvious toxic effects on cells, and their ability to continuously enhance imaging in vitro persisted for more than 15 minutes, obviously targeting foam cells in the early stage of AS. CONCLUSION: HA-PANBs are ideal ultrasound contrast agents. The successful, firm connection of PM1 and HA to the NBs significantly increased the amount of carried ANM33. When microbubbles prepared with 2:4:7 PM1:ANM33:HA were used as a contrast agent, they had a high ANM33 carrying capacity, stable physical properties, and significantly enhanced imaging and targeting of foam cells in the early stage of AS.

Early evaluation of survival of the transplanted ovaries through ultrasound molecular imaging via targeted nanobubbles.

Ovarian survival after transplantation is key to determining the success and efficacy of ovarian tissue cryopreservation and transplantation (OTCP). However, non-invasive monitoring of ovarian survival in the early stages of ovarian transplantation remains a great challenge. Anti-Müllerian hormone (AMH) is a survival factor that can promote the growth of follicles and has been recognized as an ovarian tissue-specific marker. In this study, we developed AMH-targeted nanobubbles (NBAMH) by integrating an AMH antibody onto the surface of NBs. The resulting NBAMH exhibited a high affinity for ovarian granulosa cells in vitro and a significantly enhanced ultrasound signal in transplanted ovaries relative to the non-targeted NBs. Notably, the difference in enhanced ultrasonic signals became more significant with the increase in time after transplantation from 3 to 10 days, indicating a gradually enhanced AMH expression along with the increase in transplant time. These results were further confirmed by immunohistochemical staining and western immunoblotting analyses. In conclusion, our study offers a promising non-invasive tool to monitor ovarian survival in the early stages following transplantation.

Biosynthetic nanobubbles for targeted gene delivery by focused ultrasound.

Ultrasound-targeted microbubble destruction (UTMD) has recently drawn considerable attention in biomedicine applications due to its great potential to locally enhance gene delivery. However, conventional microbubbles have a microscale particle size and polydisperse particle size distribution, which makes it difficult for them to directly come into contact with tumor cells and to efficiently deliver therapeutic genes via ultrasound cavitation effects. In the current study, we developed a kind of novel cationic biosynthetic nanobubble (CBNB) as an ultrasonic gene delivery carrier through coating PEI on the surface of these biosynthetic nanobubbles (BNBs). The BNBs, produced from an extremely halophilic archaeon (Halobacterium NRC-1), possess a nanoscale size and can produce stable contrast signals both in vitro and in vivo. Surface modification with PEI polymer greatly increased the DNA loading capability of BNBs, leading to significantly improved gene transfection efficiency when combining with ultrasound. To our knowledge, this is the first report to apply biosynthetic bubbles as non-viral gene carriers which can effectively deliver genes into tumor cells with the aid of ultrasound cavitation. Our study provides a powerful tool for image-guided and efficient gene delivery using biosynthetic nanoscale contrast agents.