Scotti, Lorena (2021) Self-assembling nanoparticles for drug delivery in tumors: insights for novel applications and industrial development. [Tesi di dottorato]

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Hybrid self-assembling nanoparticles (SANPs) have been previously designed as novel drug delivery system, with an easy scale-up, that overcome stability issues following long-term storage. This system has been successfully used to deliver anionic-charged agents, e.g. bisphosphonates, in different types of tumors, such as glioblastoma. Here, further insights have been provided for SANPs to promote the technology transfer and to found novel potential applications for this technology. More in details, the Ph.D. activity has been organized in two different directions. The first one aimed to support the industrial development of SANPs encapsulating zoledronic acid. More in details, following a deep characterization of each component, namely PEGylated liposomes, human transferrin, calcium-phosphate nanoparticles and zoledronic acid solution, the preparation protocol of SANPs encapsulating zoledronic acid was modified to adapt the formulation to the preparation in the plant of the company owner of a license agreement on the SANPs technology. Finally, a first kit of the formulation, named as EDROMA, was entirely prepared by the company and assembled to obtain the first "industrial" batch of EDROMA. Thus, this part of the work demonstrated the industrial transferability of the SANPs technology. In the second phase of this first part of the Ph.D. activity, the development of a formulation strategy, able to simplify the preparation protocol of the product, was investigated. In particular, the study was focused on the development of a strategy to freeze-dry SANPs-containing zoledronic acid. More in details, a thermodynamic study was carried out to select the cryoprotectants able to preserve the product during the lyophilization process. The dried product was analyzed in term of physical-chemical characteristics. Moreover, in vitro and in vivo studies were set up to demonstrate that SANPs encapsulating zoledronic acid, reconstituted following freeze-drying, maintained the ability to inhibit glioblastoma (GBM) cell viability and tumor regression. A second direction of the Ph.D. thesis has been focused on the research of novel applications for SANPs. In particular, in the first project of this second part of the Ph.D. activity, the SANPs technology has been optimized for encapsulation and delivery of miRNA. To this aim, SANPs with different lipids were prepared and characterized, in terms of size, polydispersity index, zeta potential, miRNA encapsulation, stability in bovine serum albumin (BSA), serum and hemolytic activity. Then, SANPs were tested in vitro on two different cell lines of GBM. Finally, miRNA biodistribution was tested in vivo in an orthotopic model of GBM. The majority of the formulations showed good technological characteristics and were stable in BSA and serum with a low hemolytic activity. Cell cloture studies showed that SANPs allowed enhance the miRNA intracellular delivery. Finally, in vivo biodistribution studies demonstrated that the optimized SANP formulations were able to deliver miRNA in different organs, e.g. the brain. Finally, in the last part of the Ph.D. activity, the modification of SANPs surface with hyaluronic acid (HA) for targeting of CD44-overexpressing cells was investigated. More in details, SANPs including different molecular weight HA were prepared by two different protocols and with or without PEGylated lipid. Zoledronic acid was encapsulated as model drug. SANPs were fully characterized and the selected formulations were investigated on cell culture to investigate the CD44 targeting.

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