Cross-linked cytochrome c-based nanoparticles for targeted and controlled cancer therapy

Abstract

Cancer is a major public health problem worldwide and is the second leading cause of death in the United States. The persistent need to develop cancer therapeutics with improved safety and efficacy provides constant fuel to drive the development and optimization of protein-based therapeutics. Proteins are an excellent natural building block for drug delivery systems (DDS) fabrication due to their unique advantages. They are naturally produced by the body and often well tolerated with poorly immunogenicity. Therefore, proteins that exhibit potent cytotoxic activities are also attractive substitutes for cytotoxic drugs because they are highly specific and less toxic than conventional small drug molecules. In this context, cytochrome c (Cyt c) has drawn attention to cancer research because it is non-toxic, and when delivered to the cytoplasm of cancer cells, it can kill them by inducing apoptosis. Various nano vehicles have been explored to protect the sensitive load and facilitate the intracellular delivery of protein therapeutics with different degrees of success. Recently, our research group overcame biocompatibility and off-target limitations commonly seen in anticancer therapeutics by designing a Cyt c-based DDS coated with a biodegradable polymer, PLGA-PEG-FA, which is 253 nm in size. However, this delivery system showed no cytotoxicity after an in vivo injection using a lung carcinoma immune-competent mouse model. For the in vivo application, it has been reported that spherical particles that are 100-200 nm in size have the highest potential for prolonged circulation because they are large enough to avoid uptake in the liver but small enough to prevent filtration to the spleen. In addition, the folate receptor alpha (FR) is overexpressed in 40% of human cancers, including non-small cell lung carcinoma (NSCLC), and can be utilized for active tumor targeting to afford more effective cancer therapies. Therefore, this dissertation aims to develop a redox-sensitive protein-based nanoparticle (NP) that uses Cyt c as a drug and carrier material for targeted and controlled cancer therapy.<br /> <br /> In Chapter 3, we substantially simplify our previously reported system by employing another strategy for preventing protein dissolution in buffer and blood, which uses a homo-bifunctional redox-sensitive cross-linking, dithiobis(succinimidyl propionate) (DSP). This cross-linker contains a disulfide bond that is reduced under intra-cellular conditions, thus affording the dissolution of the NPs in the cytoplasm of target cells. It is reported that the non-solvent nanoprecipitation method is an easy and reproducible technique to prepare Cyt c NPs. However, the size, size distribution, surface charge, and delivery properties of nanoparticles are highly influenced by the nanoprecipitation operation process conditions such as protein concentration, cross-linker concentration, and injection rate. Therefore, this chapter aims to optimize the nanoprecipitation method to establish a simpler and straightforward method for preparing cross-linked nanoparticles based on a single step with controllable size and distribution for delivery applications. Special attention has been dedicated to a systematic study to understand the effect of the operating parameters of the one-step nanoprecipitation method, such as cross-linker concentration, non-solvent/cross-linking injection rate, and method variation (i.e., one-step vs. two-step) on the physicochemical and delivery properties of the nanoparticles. To the best of our knowledge, this is the first work investigating the impact of the cross linking process on the preparation of Cyt c-based nanoparticles by the nanoprecipitation process. Our results demonstrated that an increase in cross-linker concentration led to an increase in NP size and a decrease in zeta potential. In addition, the diameter of cross-linked Cyt c NPs decreases as the cross-linking/nanoprecipitation rate increases. For Cyt c NPs cross-linked with the one-step method, the mean size was smaller (179 &plusmn; 4 nm) than the two-step method (189 &plusmn; 2 nm) (*p = 0.02). However, the two-step nanoprecipitation method demonstrated a more efficient release profile, with 71% of Cyt c released in the initial 24 h compared with the 40% of the one-step method. Finally, for both methods, the activity of the encapsulated Cyt c is mainly conserved after the cross-linking process. Therefore, it can be concluded that the non-solvent nanoprecipitation method using a one-step or two-step cross-linking approach presents an excellent opportunity for the smart delivery of Cyt c as a therapeutic protein for cancer treatment.<br /> <br /> In Chapter 4, we select the two-step method to prepare the folate-decorated cross-linked Cyt c NPs because they present a more efficient drug release profile than the one-step method. To achieve receptor-mediated internalization by FR-overexpressing cancer cells, we conjugated folate-poly(ethylene glycol) (FA-PEG) to the surface of the NPs. Cyt c nanoparticles (NPs, 169 &plusmn; 9 nm) were obtained by solvent precipitation with acetonitrile and then stabilized by reversible homo-bifunctional cross-linking to accomplish a Cyt c-based drug delivery system combines stimulus-responsive release and active targeting. Cyt c was released under intracellular redox conditions due to an S-S bond in the NPs linker, while NPs remained intact without any release under extracellular conditions. The NP surface was decorated with a hydrophilic folic acid&ndash;polyethylene glycol (FA-PEG) polymer for active targeting. The FA-decorated NPs specifically recognized and killed cancer cells (IC₅₀ = 47.46 &micro;g/mL) that overexpressed FR but showed no toxicity against FR-negative cells. Confocal microscopy confirmed the preferential uptake and apoptosis induction of our NPs by FR-positive cancer cells. In vivo experiments using a Lewis lung carcinoma (LLC) mouse model showed visible NP accumulation within the tumor and inhibited the growth of LLC tumors. Our data demonstrate a substantial improvement over our previous Cyt c delivery system both in vitro, using the Lewis lung carcinoma (LLC) cell line, and in vivo, using the LLC mouse model. This mouse model is a practical in vivo approach to studying drug safety and testing whether targeted NP therapies reach their target in the presence of a functional immune system.