Synthesis and Evaluation of Nanoparticles as Drug Delivery Systems in Cancer Immunotherapy

This PhD thesis describes the formulation of different nanoparticles as drug delivery systems in cancer immunotherapy. Chapter 1 provides a general introduction to the relevance of nanotechnology in biomedical applications. In Chapter 2, the influence of coating variations on the physicochemical interactions of nanoparticles with the biological environment is examined. More precisely, mixed self-assembled monolayers (SAMs) were prepared on the gold nanoparticle (AuNP) surface by combining synthesized alkyl-PEGylated thiols with alkyl-polyglycerol-based thiols, both negatively charged and non-charged. Indeed, while PEGylated coatings are extensively applied in drug delivery to offer prolonged circulation times, lower degradation rates by metabolic enzymes, and reduced immunogenic responses to proteins, investigating the use of polyglycerol-based polymers as alternative coatings represents a challenging research area. Therefore, the influence of coating composition on the uptake of nanoparticles by immune cells was evaluated by exposing the synthesized AuNPs formulations to mouse macrophages. Moreover, the same SAMs were prepared on gold flat surfaces, and their protein adsorption was studied using 10% human serum diluted in phosphate-buffered saline (PBS). The results obtained in this chapter evidence a relation between charge variations in the coating and cell internalization levels, and show similar behavior for both PEG and polyglycerol AuNPs. Regarding protein interactions, fully negatively charged coatings exhibit the highest protein adsorption. Interestingly, the introduction of half of a non-charged polyglycerol thiol makes protein adsorption reversible. In Chapter 3, AuNPs are designed as a drug delivery system. The selected drug was the commercially available resiquimod, characterized by potent antitumor and immunostimulatory effects. Considering that this drug exerts its activity by binding to toll-like receptors 7/8, which are located in the endosomes of human macrophages and dendritic cells, it was necessary to prepare coatings that promote the internalization of AuNPs by antigen-presenting cells (APCs). Therefore, referring to the high internalization level detected in Chapter 2, negatively charged alkyl-PEG thiols are used as a coating and are mixed with alkyl-PEG-NH2 thiols. This amino group will be relevant for conjugating resiquimod to AuNPs; it has been reported in the literature that encapsulating this drug into NPs mitigates the side effects related to its direct injection into humans. Therefore, this chapter describes the novel functionalization strategy of this drug, which involves the selective functionalization of its tertiary alcohol with a synthesized linker molecule, thereby allowing for binding to AuNPs without compromising the resiquimod pharmacological activity. The resulting resiquimod-AuNPs, whose loading capacity was estimated at 30 molecules of drug/AuNP, represent an important step in the development of therapeutic cancer vaccines. Starting from such resiquimod-conjugated AuNPs, this system can be made more convenient for biomedical applications by incorporating alternative nanocarriers. In particular, nanoparticles synthesized via polymerization-induced self-assembly (PISA) have emerged in recent years as highly suitable for biomedical applications. Indeed, PISA is a powerful synthetic strategy that combines polymerization and self-assembly into a single, efficient, and scalable process, allowing the formation of nanostructures with well-defined morphologies, high solid contents, and narrow molecular weight distributions. Therefore, in Chapter 4, resiquimod is formulated with PISA-NPs. More precisely, two different approaches are examined: the pure resiquimod in situ encapsulation and, by exploiting the knowledge about resiquimod reactivity developed in the previous chapter, the drug is modified to be copolymerized into the hydrophilic block, which is then chain-extended with a hydrophobic monomer by reversible-addition chain transfer (RAFT) living polymerization. The resulting resiquimod-bound amphiphilic diblock copolymer self-assembles into micellar nanoparticles. The drug loading and encapsulation efficiency of the obtained resiquimod-NPs formulations were evaluated, and the resiquimod release kinetics was studied simulating the endosomal environment conditions. The results obtained in this chapter demonstrate that PISA-NPs are a suitable system for delivering resiquimod and for future biomedical applications.