Bovine Milk-Derived Exosomes as a Drug Delivery Vehicle for miRNA-Based Therapy.

MicroRNAs (miRNAs) are small non-coding RNAs with a known role as mediators of gene expression in crucial biological processes, which converts them into high potential contenders in the ongoing search for effective therapeutic strategies. However, extracellular RNAs are unstable and rapidly degraded, reducing the possibility of successfully exerting a biological function in distant target cells. Strategies aimed at enhancing the therapeutic potential of miRNAs include the development of efficient, tissue-specific and nonimmunogenic delivery methods. Since miRNAs were discovered to be naturally transported within exosomes, a type of extracellular vesicle that confers protection against RNase degradation and increases miRNA stability have been proposed as ideal delivery vehicles for miRNA-based therapy. Although research in this field has grown rapidly in the last few years, a standard, reproducible and cost-effective protocol for exosome isolation and extracellular RNA delivery is lacking. We aimed to evaluate the use of milk-derived extracellular vesicles as vehicles for extracellular RNA drug delivery. With this purpose, exosomes were isolated from raw bovine milk, combining ultracentrifugation and size exclusion chromatography (SEC) methodology. Isolated exosomes were then loaded with exogenous hsa-miR148a-3p, a highly expressed miRNA in milk exosomes. The suitability of exosomes as delivery vehicles for extracellular RNAs was tested by evaluating the absorption of miR-148a-3p in hepatic (HepG2) and intestinal (Caco-2) cell lines. The potential exertion of a biological effect by miR-148a-3p was assessed by gene expression analysis, using microarrays. Results support that bovine milk is a cost-effective source of exosomes which can be used as nanocarriers of functional miRNAs with a potential use in RNA-based therapy. In addition, we show here that a combination of ultracentrifugation and SEC technics improve exosome enrichment, purity, and integrity for subsequent use.

Navigating the landscape of RNA delivery systems in cardiovascular disease therapeutics.

Cardiovascular diseases (CVDs) stand as the leading cause of death worldwide, posing a significant global health challenge. Consequently, the development of innovative therapeutic strategies to enhance CVDs treatment is imperative. RNA-based therapies, encompassing non-coding RNAs, mRNA, aptamers, and CRISPR/Cas9 technology, have emerged as promising tools for addressing CVDs. However, inherent challenges associated with RNA, such as poor cellular uptake, susceptibility to RNase degradation, and capture by the reticuloendothelial system, underscore the necessity of combining these therapies with effective drug delivery systems. Various non-viral delivery systems, including extracellular vesicles, lipid-based carriers, polymeric and inorganic nanoparticles, as well as hydrogels, have shown promise in enhancing the efficacy of RNA therapeutics. In this review, we offer an overview of the most relevant RNA-based therapeutic strategies explored for addressing CVDs and emphasize the pivotal role of delivery systems in augmenting their effectiveness. Additionally, we discuss the current status of these therapies and the challenges that hinder their clinical translation.

Cardiac tissue engineering for myocardial infarction treatment.

Myocardial infarction is one of the major causes of morbidity and mortality worldwide. Current treatments can relieve the symptoms of myocardial ischemia but cannot repair the necrotic myocardial tissue. Novel therapeutic strategies based on cellular therapy, extracellular vesicles, non-coding RNAs and growth factors have been designed to restore cardiac function while inducing cardiomyocyte cycle re-entry, ensuring angiogenesis and cardioprotection, and preventing ventricular remodeling. However, they face low stability, cell engraftment issues or enzymatic degradation in vivo, and it is thus essential to combine them with biomaterial-based delivery systems. Microcarriers, nanocarriers, cardiac patches and injectable hydrogels have yielded promising results in preclinical studies, some of which are currently being tested in clinical trials. In this review, we cover the recent advances made in cellular and acellular therapies used for cardiac repair after MI. We present current trends in cardiac tissue engineering related to the use of microcarriers, nanocarriers, cardiac patches and injectable hydrogels as biomaterial-based delivery systems for biologics. Finally, we discuss some of the most crucial aspects that should be addressed in order to advance towards the clinical translation of cardiac tissue engineering approaches.

Development of an injectable alginate-collagen hydrogel for cardiac delivery of extracellular vesicles.

Extracellular vesicles (EVs) are nanosized particles with attractive therapeutic potential for cardiac repair. However, low retention and stability after systemic administration limit their clinical translation. As an alternative, the combination of EVs with biomaterial-based hydrogels (HGs) is being investigated to increase their exposure in the myocardium and achieve an optimal therapeutic effect. In this study, we developed and characterized a novel injectable in-situ forming HG based on alginate and collagen as a cardiac delivery vehicle for EVs. Different concentrations of alginate and collagen crosslinked with calcium gluconate were tested. Based on injectability studies, 1% alginate, 0.5 mg/mL collagen and 0.25% calcium gluconate HG was selected as the idoneous combination for cardiac administration using catheter-based systems. Rheological examination revealed that the HG possessed an internal gel structure, weak mechanical properties and low viscosity, facilitating an easy administration. In addition, EVs were successfully incorporated and homogeneously distributed in the HG. After administration in a rat model of myocardial infarction, the HG showed long-term retention in the heart and allowed for a sustained release of EVs for at least 7 days. Thus, the combination of HGs and EVs represents a promising therapeutic strategy for myocardial repair. Besides EVs delivery, the developed HG could represent a useful platform for cardiac delivery of multiple therapeutic agents.