Tramontano, Chiara (2022) Targeting cancer with inorganic multifunctional nanoparticles. [Tesi di dottorato]

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Abstract

Treating cancer disease is an enormous challenge, especially when metastases outbreak the primary tumor site spreading to many different organs, either sequentially or synchronously. The majority of the currently available anti-cancer drugs target physiological pathways that are upregulated in cancer, among whom the transforming growth factor (TGF) and protein death (PD) receptor/ligand-mediated signaling. These proteins are actively involved in physiological functions, such as cell proliferation, differentiation, migration, adhesion, and apoptosis. The attack of physiological pathways by traditional anti-cancer drugs often results in unspecific and distressing treatments, painful side effects, and poor quality of life. Therefore, it is of utmost importance to develop novel treatments targeting the cancer microenvironment and improving therapeutic outcomes. Over the years, nanoparticles (NPs) emerged with great potential to carry and deliver anti-cancer drugs (i.e., small molecules, proteins, oligonucleotides) in a precisely tuned and controllable manner. The encapsulation of NPs in polymers exhibiting a pH and stimuli-responsive dissolution has been shown to enable delivering drugs upon exposure to specific stimuli in cancer cells. Among the inorganic nanocarriers, diatomite NPs (DNPs) have secured their seat in the race of nanomedicine due to their porosity, cost-effective production from diatoms, biocompatibility and, nonetheless, ease of surface functionalization. DNPs with a mean size of 400 nm are obtained by ultrasonication of the diatom frustules, which are composed of mesoporous biosilica considered safe by the food and drug administration. Thus, the increasing knowledge of DNPs’ surface chemistry has contributed to the development of hybrid DNPs exhibiting multifunctional features, among whom drug delivery and biosensing are the main purposes. The combination of the plasmonic features of gold NPs (AuNPs) with the drug-loading capacity of DNPs allows for the design of hybrid nanosensors for the real-time quantification of intracellular drug release and dose-response correlation studies. The opportunity to attach antibodies on the surface of drug-loaded DNPs allows for targeting cancer cells expressing specific surface antigens, thus increasing the local accumulation of the drug and reducing the likelihood of side effects. Furthermore, the blast of microfluidic approaches already used for the mass production of nanotherapeutics has shed a light on the potentiality of DNPs as oral nanocarriers for the treatment of colorectal cancer. The following dissertation describes the development of DNPs designed ad hoc to load and deliver anti-cancer drugs in cancer cells with release profiles controlled by stimuli-responsive polymeric coatings. The successful delivery of the TGF- inhibitor galunisertib from gelatin-covered DNPs is demonstrated in multiple CRC cell lines, showing the enhanced antimetastatic effect of the delivered drug compared to conventional approaches. The decoration of DNPs with AuNPs allows for the quantification of released galunisertib in CRC cells with a sub-femtogram scale resolution by surface-enhanced Raman scattering, outlining the potentiality of hybrid DNPs as both nanocarriers and nanosensors. Furthermore, the plasmonic properties of hybrid DNPs are exploited for the design of a theoretical model for the estimation of polymer coating thicknesses on the DNPs, exceeding the shortcomings of costly microscopy equipment. Herein, polymeric coatings made of gelatin and derivatized cellulose are shown to affect both the drug-loading efficiency of DNPs and release kinetics, offering unprecedented solutions to the delivery of drugs with a fast degradation rate. Throughout this work, DNPs are also shown as efficient nanocarriers of immunological checkpoint-targeting peptide nucleic acids (PNAs), of whom the DNPs enhance cell uptake and membrane permeability. We demonstrate the ability of redox-sensitive DNPs to release PNAs in breast and lung cancer cells upon specific exposure to altered levels of glutathione, which is the main character of the redox balance in human beings. Moreover, the outstanding advantages of microfluidics over bulk mixing are exploited for the development of gastro-resistant DNPs targeting metastatic cells for the oral treatment of colorectal cancer. For the first time, the oral administration of DNPs is considered a valid route to overcoming the challenges of intravenous injection, including the risk of vessel clogging. The vast subset of functionalization described herein (polymer coatings, antibody grafting, decoration with AuNPs, loading of different drugs) demonstrates the possibility to design DNPs targeting the cancer microenvironment specifically, improving both drug pharmacokinetics and efficacy. The reported results and in vitro investigations in multiple cell lines, both in monolayers and three-dimensional 3(D) structures, suggest that DNPs hold great promise as nanocarriers of various drugs and for the treatment of different diseases, among whom cancer has been thoroughly exploited.

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