Nano-Ghosts: Biomimetic membranal vesicles, technology and characterization.

Currently, nano-carriers for anti-cancer drug delivery are complex systems, which struggle with immunogenicity and enhanced permeability effect (EPR)-related problems that halt the clinical translation of many therapeutics. Consequently, a rapidly growing field of research has been focusing on biomimetic nano-vesicles (BNVs) as an effective delivery alternative. Nevertheless, the translation of many BNVs is limited due to scalability problems, inconsistent production process, and insufficient loading efficiency. Here we discuss the process of our previously published BNVs, termed Nano-Ghosts (NGs), which are produced from the membrane of mesenchymal stem cells. We demonstrate the flexibility of the process, while alternating physical methodologies (sonication or extrusion) to produce the NGs while preserving their desired characteristics. We also show that our NGs can be labeled using multiple methods (fluorescence, radiolabeling, and genetic engineering) for tracking and diagnostic purposes. Lastly, we demonstrate that the loading efficiency can be improved by using electroporation to accommodate a range of therapeutics (small molecules, peptides and DNA) that can be delivered by the NGs. Our results emphasize the robustness of the NGs technology, its versatility and a vast range of applications, differentiating it from other BNVs and leading the way towards clinical translation.

Pretreating Mesenchymal Stem Cells with Cancer Conditioned-Media or Proinflammatory Cytokines Changes the Tumor and Immune Targeting by Nanoghosts Derived from these Cells.

Nanoghosts (NGs) are nanovesicles reconstructed from the cytoplasmic membranes of mesenchymal stem cells (MSCs). By retaining MSC membranes, the NGs retain the ability of these cells to home in on multiple tumors, laying the foundations, thereby, for the development of a targeted drug delivery platform. The susceptibility of MSCs to functional changes, following their exposure to cytokines or cancer-derived conditioned-media (CM), presents the opportunity to modify the NGs by conditioning their source cells. This opportunity is investigated by comparing the membrane protein composition and the tumor uptake of NGs derived from naïve MSCs (N-NG) against conditioned NGs made from MSCs pre-treated with conditioned-media (CM-NG) or with a mix of the proinflammatory cytokines TNF-α and IL-1ß (Cyto-NG). CM-NGs are found to be more targeted towards immune cells than Cyto- or N-NGs, while Cyto-NGs are the most tumor-targeted ones, with similar immune-targeting capacity as N-NGs but with a higher affinity towards endothelial cells. Proteomic variations were wider in the CM-NGs, with exceptionally higher levels of ICAM-1 compared to N- and Cyto-NGs. From a translational point of view, the data show that the tumor-targeting ability of the NGs, and possibly that of other MSC-derived extracellular vesicles, can be enhanced by simple conditioning of their source cells.

Radiolabeling of cell membrane-based nano-vesicles with 14C-linoleic acid for robust and sensitive quantification of their biodistribution.

The rapid development of biomimetic cell membrane-based nanoparticles is still overshadowed by many practical challenges, one of which is the difficulty to precisely measure the biodistribution of such nanoparticles. Currently, this challenge is mostly addressed using fluorescent techniques with limited sensitivity, or radioactive labeling methods, which rarely account for the nanoparticles themselves, but their payloads instead. Here we report the development of a robust method for the innate radioactive labeling of cells and membrane-based nanoparticles and their consequent sensitive detection and biodistribution measurements. The preclinical potential of this method was demonstrated with Nano-Ghosts (NGs), manufactured from the cytoplasmic membranes of mesenchymal stem cells cultured with radioactively-labeled linoleic acid and achieving a cell labeling efficiency of 36%. Radiolabeling did not affect the physiochemical properties of the NGs, which stably retained their radiolabels. Using radioactivity measurements, we are now able to determine precisely the amount of NGs uptaken by tissues and cells, thereby providing further support to our presumed active NG targeting mechanisms. Biodistribution studies comparing radiolabeled NGs to fluorescently-labeled ones have validated our method and revealed new information, which could not be obtained otherwise, regarding the NGs' unique kinetics and rapid clearance, supporting their excellent safety profiles. The reported approach may be expanded to other membrane-based entities to facilitate and hasten their preclinical development and be used in parallel with other labeling methods to provide different and additional information.

Biohybrid cardiac ECM-based hydrogels improve long term cardiac function post myocardial infarction.

Injectable scaffolds for cardiac tissue regeneration are a promising therapeutic approach for progressive heart failure following myocardial infarction (MI). Their major advantage lies in their delivery modality that is considered minimally invasive due to their direct injection into the myocardium. Biomaterials comprising such scaffolds should mimic the cardiac tissue in terms of composition, structure, mechanical support, and most importantly, bioactivity. Nonetheless, natural biomaterial-based gels may suffer from limited mechanical strength, which often fail to provide the long-term support required by the heart for contraction and relaxation. Here we present newly-developed injectable scaffolds, which are based on solubilized decellularized porcine cardiac extracellular matrix (pcECM) cross-linked with genipin alone or engineered with different amounts of chitosan to better control the gel's mechanical properties while still leveraging the ECM biological activity. We demonstrate that these new biohybrid materials are naturally remodeled by mesenchymal stem cells, while supporting high viabilities and affecting cell morphology and organization. They exhibit neither in vitro nor in vivo immunogenicity. Most importantly, their application in treating acute and long term chronic MI in rat models clearly demonstrates the significant therapeutic potential of these gels in the long-term (12weeks post MI). The pcECM-based gels enable not only preservation, but also improvement in cardiac function eight weeks post treatment, as measured using echocardiography as well as hemodynamics. Infiltration of progenitor cells into the gels highlights the possible biological remodeling properties of the ECM-based platform. STATEMENT OF SIGNIFICANCE: This work describes the development of new injectable scaffolds for cardiac tissue regeneration that are based on solubilized porcine cardiac extracellular matrix (ECM), combined with natural biomaterials: genipin, and chitosan. The design of such scaffolds aims at leveraging the natural bioactivity and unique structure of cardiac ECM, while overcoming its limited mechanical strength, which may fail to provide the long-term support required for heart contraction and relaxation. Here, we present a biocompatible gel-platform with custom-tailored mechanical properties that significantly improve cardiac function when injected into rat hearts following acute and chronic myocardial infarction. We clearly demonstrate the substantial therapeutic potential of these scaffolds, which not only preserved heart functions but also alleviated MI damage, even after the formation of a mature scar tissue.