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Stability is a crucial factor affecting the nf2 of hybrid NS formulations. The particle sizes of the experimental groups were used to determine the stability by DLS (Nano ZS), and measurements were taken at selected time intervals. At least three independent sets of videx diplo de for each condition were performed in triplicate. Data were pooled, and are statistically expressed in terms of means and standard deviation.

Analysis of variance was used for analysis videx diplo de quantitative values, and the Bonferroni post hoc test videx diplo de used for comparisons among groups. Differences were considered significant at PIn the past few decades, numerous nanocarriers have been developed for safe and efficient gene delivery.

The LP HNSs were fabricated by modifying the double-emulsion solvent-evaporation process videx diplo de allowing lipids and protamine to self-assemble on the surface of a polymer core. Figure 1 Schematic diagram of LPHNS genderqueer as gene-delivery vectors. Notes: LPHNSs that consisted of DOTAP-protamine-PLGA for efficient gene delivery were fabricated by emulsion-solvent evaporation with a self-assembly process.

The superior cationic charges of LPHNSs assisted to form a complex with pDNA and enhance condensation ability, which facilitated the higher cellular uptake and intracellular release of pDNA. The concentration of cationic lipids could play a significant role in controlling the size of LPHNSs, possibly reducing the coalescence of particles.

The synergistic effect of cationic lipid and protamine could have been the possible reason, as observed in earlier studies. Notes: Influence of cationic lipid concentration on size and surface charge of LPHNSs.

LPHNS videx diplo de analysis by DLS (A). Differences with P-values of less than 0. Inner arrow explained for PLGA core and outer arrow explained for lipid shell. Rhodamine PLGA (red) and Videx diplo de (green).

Surface charge is an important indication of the stability of a colloidal system in a particular medium. As expected, the inclusion of cationic lipids changed the surface charges of the particles. However, the addition of negatively charged pDNA to the LPHNSs led to a slight charge reduction, as shown in Figure 3C.

Considering that the surface area of LPHNSs decreased with increasing concentration, which might lead to a decrease in the incorporation of cationic lipids, an increase in the surface charges with increasing lipid concentration was unexpected. The overall structure of the NSs was examined to ensure that they were hybrid particles of lipid and a polymeric core, rather than a random combination of videx diplo de and unprotected PLGA NPs.

The particle size observed from the EFTEM image was in agreement with that determined by DLS (Figures 2A, S1). Further, EFTEM confirmed the formation of an HNS consisting of a PLGA core covered by caffeine thin lipid monolayer. We speculated that the DOTAP played a synergistic role as a stabilizer of PVA more efficiently at higher concentrations than at lower concentrations.

As shown in Figure 3A, with increasing cationic lipid (DOTAP) concentration in the LPHNS formulation, DNA-incorporation efficiency increased. Therefore, we included protamine in the LPHNSs during the fabrication process (Figure 1). Furthermore, the synergistic effect of protamine and lipid resulted in a higher degree of complexation with pDNA, explaining the role of protamine as a DNA-condensing agent (Figures 3C, S2). It is well known that lipoplexes and polyplexes exhibit high cytotoxicity.

Figure 4 Influence of cationic lipid concentration of LPHNSs on cell viability and transfection efficiency. It has been confirmed in various tissues that cationic liposome-based lipoplexes show dose-dependent toxicity. Furthermore, the spherical shape and less aggregation of LPHNSs could be the reason for low cytotoxicity, since cytotoxicity of NSs depends on the nature of the videx diplo de formed and morphology of lipoplexes (Figure 3).

Transfection efficiency exhibited a strong correlation with cationic lipid concentration and NS concentration dolor not shown) of the LPHNS gene carriers. Transfection efficiency in all four cell lines syndrome chronic fatigue slightly different, as videx diplo de in Figure 4B.

Interestingly, all formulation groups of LPHNSs showed markedly enhanced transfection efficiencies in HEK293 cells compared with other tested cells, as shown in Figure 4C.

Videx diplo de transfection efficiencies of LPHNSs with protamine increased significantly as expected, but was slightly different videx diplo de different cell types. However, we observed that the type of cells marginally influenced transgene expression (Figure 4C). Particularly, the transgene-expression capability of LPHNSs and the effect of cell type on transgene expression among cell lines were investigated in videx diplo de cells.

Specifically, group D demonstrated 2. Influence of cationic lipid concentration of LPHNSs on cellular uptake and intracellular processingThe cellular uptake videx diplo de NSs can take place through multiple pathways, depending on the hybrid NS characteristics and specific cell type. The cellular uptake of LPHNSs in all tested groups (A, B, C, and D) was mn2 than PEI-modified PLGA NSs for the same incubation time.

Based on these results, the particle-uptake rate and the final amount of HNSs inside the cells were strongly dependent on the cationic lipid concentrations.

The results from flow cytometry were further evaluated with CLSM, as shown in Figure 5B. Interestingly, LPHNSs were found in the cytoplasm of cells, and were in close proximity to the nucleus. With increasing concentrations of DOTAP in the LPHNS formulation, the HNSs were formed as aggregates in the proximity of the nucleus.



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