Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TKSR39 mutants), yeast cytosine deaminase: uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose-response relationship studies in animals. Considering the limitations imposed by the prodrugs’ bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems.

Over the past couple of decades, the sequences of several cationic and anionic pH-responsive amphiphile fusogenic peptides (FPs) have been reported in the literature. Due to their endosome membrane disrupting activity, these peptides have been routinely used for enhancing the efficacy of drug/gene delivery systems. However, no accurate comparative study has been performed to establish the precise correlation between FP sequence and its impact on enhancing drug/gene delivery efficiency. Therefore, there has been no clear rationale for selecting one FP over another in the past, and it is still unclear which FP is the most suitable and efficient construct for use in drug/gene delivery system design. To address this shortcoming, we examined the use of a recombinant biopolymeric platform as a tool to assess the pH-dependent membrane disruption activity, cell toxicity and impact on gene transfer efficiency of the five most widely used cationic and anionic pH-responsive FPs, INF7, GALA, KALA, H5WYG, and RALA. We first developed specific expression methods for the production of five identical recombinant biopolymers that were different only in FP sequence in their structures. Through the use of physicochemical and biological assays, the biopolymers were characterized and compared in terms of DNA condensation ability, cell toxicity, pH-dependent cell membrane disruption activity, and gene transfer efficiency. Overall, our data suggests that, among the tested constructs, GALA is the most suitable pH-responsive FP for enhancing the efficiency of gene delivery systems due mostly to its efficient endosomolytic activity and negligible cell toxicity. Most importantly, this study demonstrates the application of an effective biopolymeric tool that facilitates reliable evaluation of the physicochemical and biological activities of any pH-responsive FP independent of its charge. Therefore, whether artificially designed or inspired by nature, the FPs can be screened for their efficacy with a higher degree of accuracy in the future.

The identification of the sequence of an ELP-based peptide that does not induce IgG response opens the door to more focused in-depth immunotoxicological studies which could ultimately lead to the production of safer and more effective drug/gene delivery systems such as liposomes, micelles, polymeric nanoparticles, viruses and antibodies.

In the past decades, numerous types of nanomedicines have been developed for efficient and safe delivery of nucleic acid-based drugs for cancer therapy. Given that the destination sites for nucleic acid based drugs are inside the cancer cells, delivery systems need to be both targeted and shielded in order to overcome the extracellular and intracellular barriers. One of the major obstacles that has hindered the translation of nanotechnology-based gene delivery systems into the clinic has been the complexity of the design and assembly process resulting in non-uniform nancarriers with unpredictable surface property and efficiency. Consequently, no product has reached the clinic yet. In order to address this shortcoming, we genetically engineered a multifunctional targeted biopolymer in one step; therefore, eliminating the need for multiple chemical conjugations. Then by systematic modulation of the ratios of the targeted recombinant vector to PEGylated peptides of different sizes, a library of targeted-shielded viral-mimetic nanoparticles (VMNs) with diverse surface properties was assembled. Through use of physico-chemical and biological assays, targeted-shielded VMNs with remarkably high transfection efficiency (>95%) were screened. In addition, the batch-to-batch variability of the assembled targeted-shielded VMNs in terms of uniformity and efficiency were examined and in both cases the coefficient of variation was calculated to be below 20%, indicating a highly reproducible and uniform system. Our results provide design parameters for engineering uniform targeted-shielded VMNs with very high cell transfection rate that exhibit the important characteristics for in vivo translation. These design parameters and principles could be used to tailor-make and assemble targeted-shielded VMNs that could deliver any nucleic acid payload to any mammalian cell type.

Methotrexate-human serum albumin conjugates were developed by a simple carbodiimide reaction. Methotrexate-human serum albumin conjugates were then crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) to form nanoparticles. The size of nanoparticles determined by laser light scattering and TEM was between 90–150 nm. Nanoparticles were very stable at physiologic conditions (PBS pH 7.4, ) and after incubation with serum. The effect of amount of EDC used for crosslinking on the particle size and free amino groups of nanoparticles was examined. The amount of crosslinker showed no significant effect on the size of nanoparticles but free amino groups of nanoparticles were decreased by increasing the crosslinker. The physicochemical interactions between methotrexate and human serum albumin were investigated by differential scanning calorimetry (DSC). Nanoparticles were more cytotoxic on T47D cells compared to free methotrexate. Moreover, methotrexate-human serum albumin nanoparticles decreased the IC50 value of methotrexate on T47D cells in comparison with free methotrexate.