Design of Experiments to Achieve an Efficient Chitosan-Based DNA Vaccine Delivery System

Abstract

In current times, DNA vaccines are seen as a promising approach to treat and prevent diseases, such as virus infections and cancer. Aiming at the production of a functional and effective plasmid DNA (pDNA) delivery system, four chitosan polymers, differing in the molecular weight, were studied using the design of experiments (DoE) tool. These gene delivery systems were formulated by ionotropic gelation and exploring the chitosan and TPP concentrations as DoE inputs to maximize the nanoparticle positive charge and minimize their size and polydispersity index (PDI) as DoE outputs. The obtained linear and quadratic models were statistically significant (p-value < 0.05) and non-significant lack of fit, with suitable coefficient of determination and the respective optimal points successfully validated. Furthermore, morphology, stability and cytotoxicity assays were performed to evaluate the endurance of these systems over time and their further potential for future in vitro studies. The subsequent optimization process was successful achieved for the delivery systems based on the four chitosan polymers, in which the smallest particle size was obtained for the carrier containing the 5 kDa chitosan (~82 nm), while the nanosystem prepared with the high molecular weight (HMW) chitosan displayed the highest zeta potential (~+26.8 mV). Delivery systems were stable in the formulation buffer after a month and did not exhibit toxicity for the cells. In this sense, DoE revealed to be a powerful tool to explore and tailor the characteristics of chitosan/pDNA nanosystems significantly contributing to unraveling an optimum carrier for advancing the DNA vaccines delivery field.

Modulation of Chitosan-TPP Nanoparticle Properties for Plasmid DNA Vaccines Delivery

“…Moreover, reducing the CS volume from 4 to 3 mL, a decrease in PDI value is observed from 0.34 to 0.25, as shown in Figure 2 C. When the TTP concentration is reduced, the same effect is noticed, a decrease from 0.39 to 0.32 in PDI values, which means more monodisperse nanoparticles are obtained. The negative impact of higher TPP concentration on PDI was also observed by Rodolfo and colleagues, which concluded that this parameter should be controlled because it is directly related to aggregation phenomena [ 31 ].…”

“…The same pattern was observed by Rodolfo and co-authors, since with the increase in TPP concentration and volume, the zeta potential decreased in all CS polymers used. Their work showed that TPP could be responsible for decreasing the zeta potential values due to the increment of the global negative charge of the system [ 31 ]. The positive surface charge of carriers is a very important requirement to improve the cell uptake, and consequently reach an efficient gene delivery system.…”

“…The assay was performed at three different incubation times (0, 2 and 6 h) to evaluate the behavior of naked pDNA and NPs in contact with DMEM-F12 medium supplemented with 10% of serum (FBS) and trypsin. These incubation conditions simulated the in vitro cell transfection and in vivo extracellular conditions to ensure the success of cellular transfection of these systems [ 31 ]. The agarose electrophoresis was used to analyze the supernatant of each sample, after the formulation and after the incubation time ( Figure 4 ), to verify the presence of free plasmid, which indicates low EE during the formulation or the NPs decomplexation after the incubation.…”

Nasal Nanovaccines for SARS-CoV-2 to Address COVID-19

“…In particular, chitosan is soluble in acidic environments and has adhesive properties. The excellent adhesion of chitosan reduces the nasal clearance of the vaccine [ 58 , 59 , 60 ]. Chitosan can prolong the retention time of drugs or vaccines and improve their efficacy.…”

Materiomically Designed Polymeric Vehicles for Nucleic Acids: Quo Vadis?

“…Response surface methodology and statistical experimental design approaches, such as Box–Behnken designs, are well-suited to identify the optimal polyplex assembly parameters for obtaining uniform, narrowly sized polyplexes that encapsulate their payloads efficiently . Other groups have also investigated the roles of the pH value of the formulation buffer chosen, , cationic polymer molecular weight, and polymer concentration within a DoE framework to rapidly optimize polyplex assembly conditions. Manickam and co-workers optimized self-assembly parameters for polyplexes comprising pDNA and diblock copolymers and found that the pDNA dose and N/P ratio were critical in ensuring efficient delivery of apoptosis-inducing factors to glioblastoma cells …”