Preparation and characterization of bioartificial polymeric nanoparticles for microRNA release

Currently, myocardial infarction (MI) is the cause of around 50% of the deceases among cardiovascular diseases (CVDs). The massive loss of cardiomyocytes (CMs) due to MI can lead to pathological remodeling of the left ventricle (LV), resulting in deterioration of heart structure and function. Moreover, the adult myocardium has a limited capacity for endogenous regeneration and repair because adult cardiomyocytes are terminally differentiated cells without replication ability after injury. To date, transplantation remains the only therapeutic option in the clinics for endstage heart failure, while the stem cell-based regenerative medicine strategies moderately improve cardiomyocyte survival, but do not allow cardiomyogenesis. This has prompted the need for new regenerative medicine approaches. MicroRNAs (miRNAs), small noncoding RNAs, have been shown to have a key role in cardiac development. Moreover, a combination of four miRNAs (miR-Combo) has been reported to promote the direct conversion of mouse cardiac fibroblasts into cardiomyocytes, using viral vectors [1]. However, a safer administration of such reprogramming agents requires their encapsulation inside specific vehicles. In this contest, several viral and nonviral systems have been proposed for miRNA delivery. Despite viral vectors allow high transfection efficiency of the genetic material into the target cells, their application in the clinics is limited by some disadvantages, including high cost and the risk for off-target effects, immunogenicity and insertional mutagenesis when using integrating vectors. Non-viral systems could represent safer systems for miRNA release. In this work, bioartificial polymeric nanoparticles based on Chitosan (CS) and poly(lactic-co-glycolic acid) (PLGA) were produced by nanoprecipitation technique for the entrapment and release of miRNAs. CS was chosen for the presence of positively charged amino groups able to electrostatically interact with the negative phosphate groups of the miRNA, forming complexes. PLGA was used to give stability and protection to CS/miRNA complexes, moreover it is biocompatible and biodegradable polymer approved by FDA (Food and Drug Administration). An initial protocol for nanoparticle preparation was derived from literature [2] as described in Material and Methods section (“First Method). The nanoparticles realized with this method shown a size of about 210 nm and PDI 0.18 as regard blank nanoparticles and 220 nm and PDI of 0.17 as regard miRNA-loaded nanoparticles. The percentage Yield was around 37% and 50% for blank nanoparticles and miRNA-loaded nanoparticles respectively. However, the problems seen with this method, including the presence of surfactant at the end of the process, toxicity of the solution and probably miRNA damage because homogenization phases, forced us to modify the protocol. In order to avoid the formation of more nanoparticle population, the surfactant used was replaced with a PLGA-COOH which in any case guarantees the stability of suspended nanoparticles. The chitosan solution used was replaced with a higher pH solution (5.4), in order to solve the toxicity problem. Finally, the homogenization phase has been removed. With the new method we studied the interaction between the positive ammine groups (N) of chitosan and the negative phosphate groups (P) of the miRNA in order to find an interaction strong for complexes formation but enough to allow a miRNA release. Has been investigated six different N/P ratios (0.7, 1.75, 3, 5, 8 and 15), obtained by varying the amount of chitosan and maintaining that of miRNA fixed. Subsequently the PLGA-COOH solution has been added. This work of thesis focused on evaluation of nanoparticles in terms of Size and PDI, Zeta potential, miRNA Entrapment Efficiency, Release, Morphology and Stability (in water and in DMEM-FBS). On the basis of these studies, considerations and choices have been made in order to choose the best condition, trying to optimize the process. The sample that showed the most promising features at the end of this work was the nanoparticles with N/P ratio of 8. In details, the nanoparticles realized in the last step show an average size ~200 nm, low PDI (<0.2) and positive zeta potential of about 28 mV. The entrapment efficiency exhibited excellent results (~90%) and the release was around 70% from 7th day to 14th day. The morphology study, made through SEM, confirmed the nanoparticle spherical shape and their nanometric dimensions. Finally, these nanoparticles were found to be stable for 7 day in water but only 1 in DMEM-FBS because of their interaction with the protein in culture medium. In conclusion, the developed bioartificial polymeric nanoparticles, after further improvements, could be tested in the future as delivery system for reprogramming agents (miRNA) for cardiac reprogramming.