(E-197) Investigation of Thermoresponsive and Biodegradable Nanogel Systems for RNA based Therapeutics

Introduction:: RNA based therapeutics have attracted significant interest for gene editing, vaccines and immunotherapy. The main bottleneck for gene therapy is the effective delivery of genetic material. Nanocarriers show great promise for transporting drugs across biological barriers, reducing drug clearance, and improving the bioavailability of drugs at the target. Polymer-based nanocarriers have evolved as promising systems for the delivery of various therapeutics due to their tunability and robustness. Hydrophobicity and surface charge of nanoparticles are two of the main factors that affect the size, composition and shape that affect permeation through biological membranes and drug release. The objective of this work is to show the robustness and the tunability of polymeric systems to control the size, permeability, cellular uptake and release of nucleotides for gene therapy.

Materials and Methods:: Thermoresponsive and biodegradable nanogels, based on dextran-hydroxyethylmethacrylate with either polycaprolactone (DEX-PCL-HEMA) and N-isopropylacrylamide (NIPAAM) monomer were synthesized using Irgacure® 2959 as an UV initiator. To render nanogels negatively, positively or not charged, 0, 2, 5 and 10 mol% of acrylic acid (AA) or 2 amino-ethyl methacrylate (AM) with respect to NIPAAM was added to the reaction mixture. Cy3 labeled siRNA was added to the mixture following proper procedure to prevent degradation. The nanoparticles were characterized with respect to size, zeta-potential, yield. The toxicity of nanoparticles synthesized with different charges was assessed in ARPE-19 cells by an MTT assay at concentrations from 0.1 to 2 mg/mL. The in vitro permeability of nanogels across the ARPE-19 cell membrane on a transwell plate was tested. Fluorescein isothiocyanatate (FITC) labeled 4k-dextran was used as a control. ARPE-19 cells were incubated with FITC-labeled nanogels for 2, 4, 6 and 24 h. Hoechst 33342 was used to stain the nucleus of ARPE-19 cells. After washing several times with PBS, the cells were analyzed via Leica DMi8 microscope and SH800 Cell Sorter to quantitively and qualitatively observe cellular association. For release study, nanogels loaded with nucleotides were resuspended in phosphate buffer at 20 mg/mL and released materials were measured via fluorescent plate reader and Nanodrop.


Results, Conclusions, and Discussions:: Results: The nanogels showed a z-average diameter of 95-250 nm, zeta potential of -12-31 mV.. MTT assay showed that the nanogels were not toxic to ARPE-19 cells at concentration up to 2 mg/mL. The neutral nanogels and nanogels with 2% and 10% acrylic acid were more permeable than 4k-dextran across the ARPE-19 cell in 4 hours. More cells associated with negatively charged and neutral nanogels than with positively charged nanogels, but after 1 day, cell association for all nanogels increased above 95% except 5% AM nanogel. siRNA was successfully loaded into nanogels and it was released for more than 3 months.

Discussion: The size of the nanogels got smaller with increase in charge, because higher charge causes stronger repulsion, reducing aggregation. The bigger size for the positively charged nanogels in general could be caused by the bigger size of 2-aminoethyl methacrylate than acrylic acid. Size may have had bigger role in affecting the rate of cellular uptake, because the negatively charged nanogels were taken up mostly by 6 hours, while the positively charged nanogels took 1 day to be taken up as much as the negatively charged nanogels. The 10% AM nanogel again may have been taken up faster than other positively charged nanogels, because of its size. The same could be said for the permeability across the ARPE-19 cell membrane. In the span of 4 hours, negatively charged and neutral nanogels were able to cross the membrane significantly higher than the control. The release of the drugs from the nanogels was also controlled by the charge and hydrophobicity. Based on previous studies, 10% AA nanogel was used for this release study and it was able to release siRNA for more than 3 months.

Conclusion: The thermoresponsive and biodegradable nanogels showed no sign of toxicity and the surface charges played an important role in the size, permeability, cellular uptake of nanogels and the release duration of nucleotides.

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