Assessment of Polymer Wafer Aided Gene Transfection. Efficiency and Cell Viability

Transcription

1 Assessment of Polymer Aided Gene Transfection Efficiency and Cell Viability Noah Nathan, Samuel Hanson, Chun Wang Polymeric Materials Laboratory Department of Biomedical Engineering University of Minnesota Summer 2018

2 1. Introduction DNA transfection to eukaryotic nuclei has been accomplished through chemical, non-chemical and viral methods. Polymer-mediated DNA vaccines have been proposed as an alternative method for DNA transfection, because of their high protective capabilities and biocompatibility. In this study, the transfection efficiency and cell viability of different water soluble neutral mucoadhesive polymers were explored. Plasmid DNA and polyethylenimine (PEI) polyplexes were embedded in the polymers polyvinylpyrrolidone (PVP), hydroxypropyl cellulose (HPC), polyethylene oxide (PEO) and polyvinyl alcohol (PVA) for transfection, which was tested on both murine fibroblast (NIH3T3) and dendritic (DC2.4) cells. 1.1 Methodology The four different polymers were freeze-dried and compressed into wafers pre-loaded or admixed with the PEI/DNA polyplexes (at N:P ratios of 11:1 or 8:1). Transfection experiments were performed for 4 h in either 10% serum-containing medium (DMEM-high glucose, 10% FBS, Pen/Strep) or serum-free medium (DMEM-high glucose), followed by removal of polyplexes and polymer by wash and replacement with fresh media. Transfected cells were analyzed using a BD Accuri C6 flow cytometer and fluorescence microscope. Furthermore, cell viability upon exposure to PEI/DNA polyplexes, blank polymer wafers, and PEI/DNA loaded polymer wafers was measured via MTT colorimetric assay. 2. Results Data of all transfection experiments of NIH3T3 cells were averaged and plotted in Figures 1-8. The data was collected using FlowJo and was further analyzed using Excel. The negative control was defined as the untreated cells, which were not subjected to any polymer or PEI/DNA polyplexes. The standard value of 0.2% GFP fluorescence (FL-1) was used as control due to the baseline fluorescence of murine cells. Figures 1-4. Transfection efficiencies of NIH3T3 cells using PVA, PEO, HPC and PVP wafers in 10% serum-containing medium 14.0% 12.0% 1 8.0% 6.0% 4.0% 2.0% 1. 10% Serum PVA Transfection of NIH3T3 Cells (4 replicates) (PEI/Luc) Solution Polymer atop Polymer 1 9.0% 8.0% 7.0% 6.0% 5.0% 4.0% 3.0% 2.0% 1.0% 2. 10% Serum PEO Transfection of NIH3T3 Cells (4 replicates) (PEI/Luc) Solution Polymer atop Polymer

3 4.0% 3.0% 3. 10% Serum HPC Transfection of NIH3T3 Cells (2 replicates) 0.8% 0.7% 0.6% 4. 10% Serum PVP Transfection of NIH3T3 Cells (1 replicate) 0.5% 2.0% 0.4% 0.3% 1.0% 0.2% 0.1% (PEI/Luc) Solution Polymer atop Polymer (PEI/Luc) Solution Polymer atop Polymer Figures 5-8. Transfection efficiencies of NIH3T3 cells using PVA, PEO, HPC and PVP wafers in serumfree medium Serum Free PVA Transfection of NIH3T3 Cells (4 replicates) (PEI/Luc) Solution Polymer atop Polymer 45.0% % % % 1 5.0% 6. Serum Free PEO Transfection of NIH3T3 Cells (4 replicates) (PEI/Luc) Solution Polymer atop Polymer 8 7. Serum Free HPC Transfection of NIH3T3 Cells (2 replicates) 45.0% 8. Serum Free PVP Transfection of NIH3T3 Cells (1 replicate) % % % % (PEI/Luc) Solution Polymer atop Polymer HEPES HEPES Polymer HEPES + HEPES Polymer (PEI/Luc) Solution Polymer atop Polymer

4 As shown in the Figures 1-8, HPC and PVP samples had only 2 and 1 replicates respectively, due to poor cell harvesting post-transfection. This indicated that the presence of polymer wafers, PEI/DNA polyplexes or the combination of both was potentially toxic to cells. MTT colorimetric assay was performed in order to assess the viability of cells subjected to PEI/DNA polyplexes, blank polymer wafers, and PEI/DNA loaded polymer wafers. Table 3 shows the averaged data for all 3 conditions. All the data was normalized to the viability of untreated cells. Table 3. Cell viability data for NIH3T3 Cells upon exposure to polyplexes and PVA, PVP, HPC and PEO wafers in 10% serum-containing and serum-free medium 10% Serum Serum Free Conditions PEI/DNA polyplex Blank 100 ± /Polyplex 100 ± Blank + Polyplex 100 ± ± 9.35 Blank 100 ± /Polyplex Blank + Polyplex 100 ± ± ± 3.81 PVA PVP HPC PEO ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Discussion As hypothesized, the water soluble neutral mucoadhesive polymer wafers PVA, HPC, PEO and PVP increased the transfection efficiency of PEI/GFP polyplexes into NIH3T3 fibroblast cells compared to untreated cells. Some of the polymer delivery systems had a better transfection efficiency compared to polyplexes delivered in HEPES buffer. Flow cytometry data indicates that the best transfection method was either polyplexes in HEPES atop polymer wafers or polyplexes HEPES added separately from the polymer wafers, in serum free conditions. This might be due to the absence of serum proteins, which can interact with the wafer/polyplex complex, blocking its entrance to the cell and nucleus. The highest transfection efficiency of 64.30% was obtained using polyplexes in HEPES buffer added separately from the HPC wafers. Also, PEI/GFP loaded PVA wafers yielded a transfection efficiency of 45.6% in serum free conditions, also higher than polyplexes delivered in only HEPES buffer. In 10% serum-containing medium, the only two polymers that effectively increased the transfection efficiency were PVA and PEO. Structurally, PVA and PEO are linear polymers; whereas, PVP and HPC contain a five member pyrrolidone side group and cellulose six membered rings, respectively. It is possible that the more linear and biocompatible polymers PVA and PEO have an increased protective ability compared to polymers with ring groups; however, further studies should be performed in order to confirm this idea. Although transfection efficiency is increased, the mechanism of action of these polymers is still unknown. According to Brooks et al, neutral polymers such as dextran increase the dielectric constant surrounding human erythrocyte cells. In this case, if the dielectric constant is increased around the NIH3T3 fibroblasts

5 (when exposed to polymer wafers), this might electrostatically attract PEI/DNA polyplexes, which have a net positive charge. In order to explore this idea, zeta potential measurements of NIH3T3 cells treated with blank polymer wafers and PEI/DNA polymer wafers could be performed. On the other hand, MTT assay revealed why smaller cell counts were obtained for some of the delivery methods. Blank polymer wafers did not affect cell viability in 10% serum-containing or serum-free medium, as the averages and standard deviations are within the range of the values of untreated NIH3T3 cells. Although some polymer wafers such as HPC and PVA had high transfection efficiencies, they also lowered the cell viability significantly in both 10% serum-containing or serum-free medium. It is still not known which interactions are the PEI/DNA polyplexes, serum proteins and polymers establishing. Determining whether there are new intermolecular or covalent interactions could reveal if any cytotoxic species are being formed. This might be also related to the ph of the systems studied, as polymers could not longer be neutral under those conditions. In the future, flow cytometry analysis of transfected DC2.4 cells should be performed, as fluorescence microscopy did not reveal any significant transfection (see Appendix). Furthermore, it would be relevant to test different DNA cargos to determine the size capacity of the polymer wafers. 4. Conclusions In conclusion, the transfection efficiencies of four different neutral water-soluble mucoadhesive polymers (PVA, PEO, HPC, PVP) carrying PEI/DNA polyplexes were determined for NIH3T3 murine fibroblast cells in 10% serum-containing and serum-free medium. Moreover, cell viability of NIH3T3 cells in a combination of PEI/DNA polyplexes, blank polymer wafers, and PEI/DNA loaded polymer wafers were determined through MTT assay in 10% serum-containing and serum-free medium. Furthermore, the initial transfection experiments of DC2.4 dendritic murine cells were performed. It was found that NIH3T3 murine fibroblasts were transfected with polyplexes very efficiently (in terms of ) using polyvynil alcohol (PVA) and hydroxylpropyl cellulose (HPC) wafers, with HEPES buffer. The polymer wafer and HEPES delivery vehicle was even more effective than HEPES buffer alone. In addition, it was found that cationic polyplexes lowered the viability of NIH3T3 cells. Of all the delivery vehicles, PVA and PEO wafers were the less toxic to murine fibroblasts. Finally, based on fluorescence microscopy, DC2.4 cells were not transfected efficiently by the delivery methods explored.

6 5. Appendix PVA Transfection Images of NIH3T3 cells in 10% serum -containing medium PVA PVA Transfection Images of NIH3T3 cells in serum-free medium PVA PVA Transfection Images of DC2.4 cells in 10% serum-containing medium PVA

7 PVA Transfection Images of DC2.4 cells in serum-free medium PVA 6. Sources 1. Li, F., Zhu, A., Song, X., Ji, L., & Wang, J. (2013). The internalization of fluorescencelabeled PLA nanoparticles by macrophages. International Journal of Pharmaceutics, 453(2), doi: /j.ijpharm Zhang, M., Hong, Y., Chen, W., & Wang, C. (2016). Polymers for DNA Vaccine Delivery. ACS Biomaterials Science & Engineering, 3(2), doi: /acsbiomaterials.6b Chawla, Vinay & Saraf, Shubhini & Saraf, Shailendra. (2011). Gene Delivery: The Non-Viral Vector Advantage. Biotechnology Research Communications Schulze, J., Hendrikx, S., Schulz-Siegmund, M., & Aigner, A. (2016). Microparticulate poly(vinyl alcohol) hydrogel formulations for embedding and controlled release of polyethylenimine (PEI)-based nanoparticles. Acta Biomaterialia, 45, doi: /j.actbio Brooks, D.E, and G.V.F Seaman. "The Effect of Neutral Polymers on the Electrokinetic Potential of Cells and Other Charged Particles". Journal of Colloid and Interface Science, vol 43, no. 3, 1973, pp Elsevier BV, doi: / (73)90413-x.