טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLetko Khait Nitzan
SubjectTargeted Cell-Derived Nano-Ghosts as Drug Carriers to
Inflammatory Tissues
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Marcelle Machluf


Abstract

Vast efforts have been invested in recent years in the development of nanoparticle (NP)-based therapeutics, with a growing interest in biomimicry and bioinspired drug delivery systems. One such promising approach for biomimicry involves the use of cell membrane-derived NPs, intended to deliver therapeutics while exploiting the functions of the natural membranes. Our laboratory has developed membrane-based nano-vesicles produced from the plasma membranes of mesenchymal stem cells (MSCs), termed Nano-Ghosts (NGs). As part of their natural role in wound healing and immunomodulation, MSCs are known to home in on diverse sites of inflammation and injuries, including solid tumors, and were tested for these reasons in multiple clinical trials.

Inflammation is a common cause and consequence of many highly disabling conditions whose prevalence is constantly growing due to population aging. Drugs that treat inflammation have been available for decades. However, despite their potency, most anti-inflammatory drugs are associated with severe side effects, especially in light of the long lasting administration periods required. Formulation with targeted drug delivery systems have been often suggested to reduce off-target effects and improve the efficacy of anti-inflammatory drugs, yet no such formulation are available in the clinic.

The aim of this study was to modify and optimize the NGs system to target inflammation.

NGs were produced and characterized, and as previously published, they were found to be spherical, nano-sized vesicles partly retaining MSCs membrane proteins. A robust method to track NGs in vitro and in vivo was established, based on the radiolabeling of the MSCs before NGs production. Radiolabeling did not affect NGs’ characteristics, which remained stable at storage in terms of size and radioactivity. Targeting results of radiolabeled-NGs correlated with fluorescent labeling techniques and provided further support to our presumed active and selective NGs targeting mechanisms, and bioavailability studies revealed new information on NGs’ kinetics.

The interactions of the NGs with macrophages, key players in inflammatory responses, were studied. NGs were shown to modulate the inflammatory response of stimulated macrophages, demonstrating the importance of cell-cell contact in the MSC mechanism of action. In addition, an anti-inflammatory drug was encapsulated inside the NGs and further enhanced the immunomodulatory effect.

A myocardial infarction (MI) model was applied to test NGs targeting potential towards an inflamed tissue. The NGs specificity was improved by the addition of a targeting peptide on their surface, by means of genetic engineering or direct conjugation. Specific interactions with cardiomyocytes were enhanced by both means in vitro. Finally, in vivo studies in a rat MI model revealed a substantial NGs accumulation in the inflamed infarcts, while hardly no NGs were found in the healthy heart tissue.

I n conclusion, the versatile NG system is capable of carrying therapeutics, in vitro and in vivo tracking, modifications according to the desired purpose, and the potential to accumulate in inflamed tissues. Our results, along with previously published data on the NGs abilities to treat solid tumors in mice, clearly demonstrated out the NG s system as a promising nano-carrier platform.