Optimization of peptide-plasmid DNA vectors formulation for gene delivery in cancer therapy exploring design of experiments

Abstract

The field of gene therapy still attracts great interest due to its potential therapeutic effect towards the most deadly diseases, such as cancer. For cancer gene therapy to be feasible and viable in a clinical setting, the design and development of a suitable gene delivery system is imperative. Peptide based vectors, in particular, reveal to be promising for therapeutic gene release. Following this, two different peptides, RALA and WRAP5, have been investigated mainly regarding their ability to form complexes with a p53 encoding plasmid (pDNA) with suitable properties for gene delivery. To address this issue, and after an initial screening study focused on the dependence of pDNA complexation capacity with the nitrogen to phosphate groups (N/P) ratio, a design of experiments (DoE) tool has been employed. For each peptide/pDNA system, parameters such as, the buffer pH and the N/P ratio were considered the DoE inputs and the vector size, zeta potential and pDNA complexation capacity (CC) were monitored as DoE outputs. The main goal was to find the optimal experimental conditions to minimize particle sizes, as well as, to maximize the positive surface charges of the formulated nanosystems and maximize the pDNA CC. Through the DoE method applied, the optimal RALA/pDNA and WRAP5/pDNA formulations were revealed and show interesting features related to peptide structure and pDNA complexation ability. This work illustrates the great utility of experimental design tools in optimizing the formulation of peptide/pDNA vectors in a minimum number of experiments providing relevant knowledge for the development of more suitable and efficient gene delivery systems. The new insights achieved on these carriers clearly instigate deeper research on gene therapy.

Chitosan Derivatives and Their Application in Biomedicine

“…Trans transcriptional activator/ poly(N-3-benzyloxycarbonyl-lysine) chitosan p53, DOX Cancer treatment Improve transfection efficiency and drug delivery efficiency [188] Genes can regulate signaling networks and promote tumor suppression through chemotherapy, which is important for the treatment of tumors [189]. However, naked nucleic acids cannot cross cell membranes and are easily degraded by nucleases [190]. TMC can be further modified to protect genes from degradation by nucleases in serum [191][192][193].…”

Development of Peptide-Based Nanoparticles for Mitochondrial Plasmid DNA Delivery

“…In line with this aim, micelles, polymers, lipid-and peptide-based nanoparticles are among the most studied systems for gene release [25][26][27][28]. In particular, cell-penetrating peptides (CPP) offer exceptional properties to be explored as gene delivery vehicles for successful gene therapy [28][29][30][31][32]. CPPs are short peptides, usually of fewer than 30 amino acids, and can be separated into arginine-rich and amphipathic peptides.…”

Synthesis and Characterization of Mannosylated Formulations to Deliver a Minicircle DNA Vaccine

“…This can, however, induce higher cytotoxicity [ 41 ]. Nevertheless, the results obtained confirm that the increase of the amine group content will result in a greater degree of mcDNA condensation, resulting in smaller complexes [ 32 ]. The high charge density and the great condensing capacity of both PEI and R8 enhance the strong interaction with the mcDNA, leading to the formation of smaller particles.…”

“…Its ability to bind, interact, and consequently condense DNA has been explored via the functionalization of systems for the delivery of genetic materials to eukaryotic cells [ 30 , 31 ]. Additionally, the PEI polymer has been widely applied in the formation of DNA-based systems, since it strongly interacts with DNA, condensing it and showing the capacity to efficiently deliver DNA both in vitro and in vivo [ 32 ]. PEI is a synthetic polymer that is highly soluble in water, positively charged, and whose cationic amines reduce the negative charge of DNA after complexation, causing its condensation [ 33 ].…”