The 1-(3-aminopropyl)-4-methyl piperazine (E7), 1,4-butanediol diacrylate (B4), 4-amino-1-butanol (S4), and Polyethylenimine (PEI) were obtained from Alfa Aesar (Beijing, China). Dimethyl sulfoxide (DMSO), N-hexane, dimethylformamide (DMF), Ethyl acetate (EtOAc), Dichloromethane (DCM), tetrahydrofuran (THF), and diethyl ether were purchased from Sigma-Aldrich (Beijing, China). Methoxy Polyethylene glycol-succinimidyl succinate (mPEG-SS) commonly referred to as PEG of 5 kDa was acquired from Shanghai Titan Technology Co., Ltd (Shanghai, China).

The cell culture media and all other reagents were used as received. Aqueous solutions of sodium acetate (NaAc, pH 5.1 ± 0.1 at 0.025 M), sodium chloride (NaCl, 150 mM), and sodium hydroxide (NaOH, 0.1 N) were prepared and sterilized. Plasmid DNA (pDNA) expressing green fluorescent protein (GFP) was prepared accordingly, while growth media and Hoechst dye were stored according to the manufacturer’s instructions.

Poly (β-amino ester)-447 (PβAE-447) was synthesized following a previously published procedure (Smith et al., 2017), with alterations as outlined in (Figure 1). In the first step, the monomers B4 and S4 were mixed at a 1:1 M ratio and stirred (1,000 rpm) overnight at 90°C to yield the polymer B4-S4 (Figure 1).

In the second step, the polymer B4-S4 was dissolved in THF at 100 mg/ml and then combined with E7 (polymer end-capping group) in THF (0.2 M) at 500 rpm for 2 hours at room temperature. The PβAE-447 was precipitated in cold diethyl ether and washed to remove the residual monomers. The polymer was dispersed again in diethyl ether to guarantee the removal of the remaining monomers. The polymer was vacuumed dried for 2 days to remove traces of solvents. The dry and clean polymer was dissolved in DMSO at 100 mg/ml and stored in small aliquots at −20 °C for further use.

The PβAE-447 was PEGylated. For this, PEG (2.05 M equivalent) and the prepared PβAE-447 solution were transferred into a glass vial, vacuumed, and then purged with nitrogen. The mixture was then reacted in anhydrous THF at room temperature overnight. The PEGylated PβAE was washed two times in cold diethyl ether, and then vacuumed dried. The PEGylated PβAE-447 was separately dissolved in DMSO at 100 mg/ml and stored at −20°C for further use.

The polydispersity and molecular weight (M_w) of the PβAE-447 were determined by gel permeation chromatography (GPC). The polymer stock solution in DMSO was diluted in THF (100%) at 5.5 mg/ml. The prepared polymer solution was filtered in a 0.2 μm polytetrafluoroethylene syringe filter before flowing through a waters 515 liquid chromatograph equipped with three styragel columns and 2,414 refractive index detector at a flow rate of 1.0 ml/min at 40°C and then analyzed with Breeze two software. The number average molecular weight (M_n) and M_w were determined using the polystyrene standard.

The purified and dried PβAE-447 was dissolved in deuterated chloroform (CDCl₃) at 10 mg/ml and then analyzed by ¹H NMR spectroscopy, using a Bruker instrument 400 MHz, Topspin 2.0 (Toronto, Canada).

Different aliquots of the PβAE-447 were mixed with NaAc buffer (25 mM), forming polymer suspensions (“milky” appearance). After sonication (15 min), the samples were kept in an orbital shaker at room temperature for 1 h. Exemplary, 1, 3, 5, 7, 10, and 20 mg of PβAE-447 were dissolved in NaAc (1 ml), and four wells of 96-well plate were used for each concentration. The absorbance of the systems (each well) was recorded with a plate reader at 620 nm. The polymer solubility was confirmed with the naked eye as well as by plotting the recorded absorbance at 620 nm. For this, the absorbance of the polymer systems was compared with the absorbance of references (NaAc and DMSO used to prepare the polymer systems) (Sunshine et al., 2011).

An acid-base titration was performed to evaluate the buffering capacity of PβAE-447. The polymer from its stock solution in DMSO (100 mg/ml) was diluted to 1 mg/ml in NaCl (2.0 ml, 150 mM), and then NaOH (0.1 M) was used to adjust the pH to 10. The pH of the solution (2.0 ml) was reduced to 3 with HCl (0.1 N). The pH alteration was continuously monitored (Gong et al., 2018). Distilled water was titrated in the same way to compare the pH changes as the polymer was titrated. The pH values of the polymer solution were recorded each time after the repeated addition of HCl. A pH meter (pH 211 microprocessor pH meter, HANA Instruments, Seoul, South Korea) was used to measure the pH constantly. The slope of the line in the plot for pH and the concentration of HCl used show the intrinsic buffering ability. The proton buffering capacity of the polymer was calculated through Buffering capacity = Δ V HCl   ×  C HCl m (1) where ΔV_HCl is the HCl volume, C_HCl is the concentration of HCl, and m is the polymer mass (Hwang et al., 2014).

The swelling property of the PβAE-447 was evaluated by immersing dried polymer disks (0.1 g) in DMSO, THF, DCM, and EtOAc at room temperature for a defined time. The dried mass for each polymer disk was called W_o, and the swollen polymer disk mass was labeled W_s. The polymer disks were taken out from the solvents at calculated times. The solvents in excess on the disks were gently removed with filter papers before measuring W_s (Biswal et al., 2011). The swelling degree (SD) was determined at different times after contact with the solvents. The SD (%) measurements were determined through . S D = W S − W 0   W 0   ×   100 (2)

The relative cytotoxicity of pure and PEGylated PβAE-447 was separately investigated by using the 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Human embryonic kidney (HEK-293), bronchial epithelial (BEAS-2B), and lung adenocarcinoma epithelial (A549) cells were incubated in 96-well plates with DMEM (100 μL) for 24 h. When the cells achieved almost 70% confluence, the old media was removed. The new media containing various concentrations of pure and PEGylated PβAE-447 were added to each well. The 96 well plates were incubated for 24 h, and then 25 μL MTT solution (5.0 mg/ml) in PBS was added to each well. After 4 h of incubation, the MTT solution and DMEM were aspirated, and 150 μL DMSO was added to dissolve the formazan crystals. The plates were placed on a shaker for 10 min before recording the absorbance at 570 nm by an ELISA microplate reader (Bio-Rad, California, United States). The percentage of cell viability was calculated by using Eq. (3) Cell viability  ( % ) = ( A sample A control )   ×   100 (3) where A_sample is the absorbance from the treated cells and A_control is the absorbance from untreated cells.

Moreover, the cell viability of PEI 25 KDa was also determined.

The pDNA and PβAE-447 were diluted to 0.06 and 3.6 μg/ml in NaAc (25 mM, pH 5.0) at room temperature, respectively. The polymer concentration was 60-fold higher than the pDNA concentration (PβAE-447/pDNA weight ratio equal to 60).

For preparing the PβAE-447/pDNA nanocomplexes, the diluted polymer (50 µL) was added to 50 µL pDNA and gently mixed by pipetting. The prepared suspension was kept for 10 min under rest to facilitate the self-assembling of the nanocomplexes. The as-prepared nanoparticle suspension was characterized by dynamic light scattering (DLS). Hydrodynamic radius and Zeta potential measurements were determined in a Malvern Zetasizer Nano (ZS) instrument (NanoSight Ltd, United Kingdom) at room temperature (Kamat et al., 2013).

An as-prepared nanoparticle suspension was mixed with sucrose (30 mg/ml in 25 mM NaAc), performing a 1:1 v/v nanoparticle/sucrose mixture to assess the nanoparticles’ stability over time. The mixture was frozen for 2 h at −80°C, lyophilized for 2 days, and stored at 4°C. The lyophilized nanoparticles were re-dispersed in water (30 mg/ml) for further DLS analysis (Zeta potential and hydrodynamic radius). These nanoparticles were called lyophilized nanoparticles.

The stability of the PβAE-447/pDNA and PEGylated PβAE-447/pDNA nanoparticles was evaluated in fetal bovine serum (FBS) and NaCl. The polyplexes were prepared the same way as mentioned above. Then FBS (10% w/v) and NaCl (300 mmol) were separately added to the nanoparticle’s suspensions. The size and Zeta potential of the resulting suspensions were measured after 4 h of incubation at room temperature (Zeng et al., 2017).

The agglutinating activity of PβAE-447/pDNA formulated at 60 wt/wt was examined in a 96-well plate. Briefly, fresh mice blood was centrifuged at 1000 rpm for 15 minutes. The supernatant containing plasma and buffy coat were removed. The erythrocytes were washed three times with PBS. After each cycle, the supernatant was carefully discarded. The red blood cells obtained in this method were found to be pure from cell debris and leucocytes. The erythrocytes were diluted in PBS to a final concentration of 2 % (v/v). 50 μL from this dilution was transferred to a 96-well microplate. The PβAE-447/pDNA complexes were added to the RBC suspension (1:1) and incubated for 30 minutes at room temperature. After incubation, hemagglutination was observed with the naked eye as well as under an optical microscope. The experiment was performed in triplicate.

HEK-293, BEAS-2B, and A549 cells) cell lines were grown at a density of 12,500 cells/well in 100 μL media in three separate 96-well plates for 24 h to allow cell adhesion. pDNA was dissolved in NaAc (25 mM, pH 5.0) to 0.06 μg/μL. Polymer (stock solution in DMSO) was diluted in NaAc (25 mM, pH 5.0) to 3.6 μg/μL (60 polymer/p-DNA weight ratio). The diluted pDNA (30 μL) and diluted polymer (30 μL) solutions were mixed for 5 seconds with a vortex mixer. The mixture was kept at rest for 10 minutes to promote the formation of the nanoparticles. Then, 20 μL nanoparticle suspension was added to 100 μL of the cell growth media.

Before adding the polymer/pDNA nanoparticles (as-prepared, lyophilized, and pegylated PβAE-447/pDNA nanoparticles) over the seeded cells, the media was aspirated, and new media containing mentioned nanoparticles were used for transfection. After 4 h of incubation, the media containing nanoparticles was poured out from each well, and an equal volume of fresh and prewarmed media was added. The 96-wells plates were kept in an incubator at 37°C under 5% CO₂ and examined for cell transfection after 48 h.

Control experiments with PEI (MW 25,000) were also performed as above. PEI/pDNA complexes were formulated at a 3:1 PEI to pDNA weight ratio in NaCl (25 mM, pH 5.0). PEI/pDNA suspension was shaken vigorously and then incubated for 15 min at room temperature. PEI/pDNA was added to cells for the final concentration of pDNA 600 ng/well.

A semi-quantitative analysis was performed on the images captured by fluorescent microscope (Carl Zeiss MicroImaging GmbH, Germany) to calculate the mean fluorescent intensity of GFP-expressed cells using the ImageJ (http://rsb.info.nih.gov/ij).

Four different experiments were performed to investigate the effect of the incubation period of the PEGYlated PβAE-447/pDNA nanoparticles on cellular uptake.

The different cell lines (HEK-293, BEAS-2B, and A549) were cultured in 96-wells for 24 h for cell adhesion. Nanocomplexes in culture media were added to the attached cells for different time periods. After the predefined incubation time intervals, the media containing the nanoparticles were replaced by new media, and the plates were incubated for 48 h. Afterward, the culture media was removed, and the cells were incubated for 10 min in the Hoechst dye 33,342 solutions. Immediately before confocal imaging, the media from the wells were aspirated, and the cells were rinsed twice with PBS(1X). Confocal microscopy images were recorded with a confocal laser scanning microscopy (GmbH Wetzlar, Germany).

All the experiments were performed in triplicate (n = 3), and the results were expressed as the mean ± standard deviations.

In this study, PβAE-447 was synthesized following the Michael addition reaction mechanism by polymerizing 1,4-butanediol diacrylate “B4” and 4-amino-1-butanol “S4” stoichiometrically. The reaction formed a polymer base that was end-capped with 1-(3-aminopropyl)-4-methyl piperazine (E7). The yielded polymer obtained from the monomers “B4”, “S4”, and the end-capping reagent “E7” is named as 1-(3- aminopropyl)-4-methyl piperazine end-modified poly (1,4-butanediol diacrylate-co-4-amino-1- butanol) (PβAE-447). This nomenclature depends on four carbon atoms between the acrylate groups in “B4” and four carbon atoms across the amine and the alcohol groups in” S4” (Li et al., 2013). The polymerization of PβAEs can be carried out in a wide range of solvents. However, DMSO was preferred in the synthesis because it is a commonly used solvent in bio-assays including cellular-based assays.

The PβAE-447 was characterized by gel permeation chromatography (GPC) to determine the molecular weight and polydispersity index (PDI) (Figure 2A). The GPC chromatogram shows the presence of a single polymer with Mn of 5,354, Mw of 9,575, MP of 4,934, and PDI of 1.7.

The chemical structure of PβAE-447 was analyzed by ¹H NMR (Figure 2B), and the spectrum matches with previously published results (Chemical shift analysis Supplementary Figure S2). The PβAE-447 ¹H NMR spectrum shows peaks assigned to hydrogen atoms found on the monomers B4 and S4 and the disappearance of the acrylate peaks at the end of both sides of ppm shows completion of the end-capping reaction.

The well-established method for investigating the carrier solubility is adding an extra quantity of carriers to a fixed buffer volume at a set pH. A 96-well plate absorbance assay was used to quantitatively evaluate the PβAE-447 solubility at 620 nm. The data (not shown) shows that PβAE-447 was completely soluble at 10 μg/μL in a NaAc buffer. The gene release depends on the PβAE-447 solubility inside the cells. The water-soluble and biodegradable cationic polymer has hydrolyzable ester bonds in its backbone (Agarwal et al., 2012). The polymer degradation should support the release of genetic materials in an active form, resulting in better gene expression. Other strategies can also be used to modify the hydrophilic PβAE nature, including its N-quaternization and pegylation. These processes should increase the aqueous solubility and enhance pDNA release at physiological pH.

For evaluating the buffering capacity of PβAE, direct polymer titration with HCl was performed. For comparison, the polymer titration was repeated with distilled water. It is pretty clear from (Figure 3) that approximately 0.013 mmol HCl is needed to change the pH from 7.4 to 5.1 (i.e., endosomal pH range). PβAEs with the end-capping group E7 have high buffering capacities because the E7 comprises two tertiary amines. Similarly, PβAEs with the end-capping E6 would have high buffering capabilities than E4 and E6 groups. It has been hypothesized that the nanoparticles remain in the endosomes if the carriers have low buffering capacities (Sunshine et al., 2012). It suggests no relationship between cell uptake and buffering capacity. Still, there is a strong relationship between transfection efficiency and buffering capacity.

Additionally, the nanoparticles formulated at a 75 w/w PβAE-447/pDNA ratio would have a higher buffering capacity than at 60 w/w, because of the high polymer content at 75 w/w. The PβAE-447 can have different proton buffering capacities depending on its concentration. Moreover, the monomer ratio used to synthesize the PβAE-447 could also influence the proton buffering capacity because the amino group content in the polymer structure depends on the S4 concentration.

The titration curve for the water decreased promptly in the pH range between 4 and 10, and the PβAE-447 curve displayed a delayed pH reduction, suggesting that PβAE has a high buffering capability (Figure 3). Thus, the buffer capacity should accelerate endosomal escape and enhance the transfection toward treated cells that have already taken up the particles.

The PβAE-447 SD was investigated in conventional solvents such as DMSO, DCM, THF, and EtOAc at room temperature for 0, 100, 250, and 360 min (Figure 4). The PβAE-447 swelling increases as time rises, achieving the equilibrium condition. The maximum swelling is reached in approximately 2 h in DCM, THF, and EtOAc, whereas in DMSO, the maximum swelling occurs in around 5 h. The polymer SD reduces in the followed order DMSO > DCM > THF > EtOAc. The swelling is rationalized with the solvent-polymer interaction theory that predicts polymer solubility (Biswal et al., 2011). Solvent polymer interactions have a critical role in polymer synthesis. Maximized polymer-solvent interactions may lead to chain expansion, which is important to optimize material’s processing; for example, resultant mechanical properties (Ferrell et al., 2017). PβAE-447 demonstrated an ability to absorb conventional solvents and increased in size, as revealed by the swelling behavior stated in Figure 4. The PβAE-447 could have a transition in the size through solvents absorption as a stimulus which shows that entrapped therapeutics would take some time to diffuse out. Summarizing the above results, it can be observed that PβAE-447 showed significant swelling degree with respect to its dry discs.

HEK-293 cell line is commonly used as a first-level screening host to evaluate new transfection vectors. The MTT assay is widely used as a first-level indicator of cytotoxicity. MTT assays help to determine the influence of the added substances on cell proliferation and metabolism. PβAE-447 cytotoxicity in different dosages (10, 20, 30, 40, 50, 60, 75, 90, and 100 μg/ml) was investigated against HEK-293, BEAS-2B, and A549 cells through MTT assay.

As shown in Figure 5, cells incubated with pure (Figure 5A) and PEGylated PβAE-447 (Figure 5B) show a cell viability sketch. For example, the cell viability of only PβAE-447 is higher than 80% for A549, 77% for BEAS-2B, and 76% for HEK-293 cells. The polymer is cytocompatible toward the investigated cell lines. We did not observe any significant difference in the cytotoxicity results by the conjugation of shielding polymer (PEG) to PβAE-447. PEG has been known as a safe, inert, and non-immunogenic synthetic polymer (Richter et al., 2021). The results are significantly better than the data obtained from the cells population treated with PEI (Mw ≈ 25 k) (Figure 5C), as a positive control as well as to facilitate the comparison. The PEI results revealed a dose-dependent decrease in cells viability. One of the remarkable decreases in cell viability was noted in the PEI treated group at a concentration of 100 µg/ml. Overall, PEI has a higher cytotoxic effect than PβAE-447. The cell viability percentage was (14 ± 5%) for PEI and (86 ± 4%) for PβAE-447 at 100 μg/ml concentration. High molecular weight PEI has a strong positive charge and is more cytotoxic than low molecular weight PEI (Valente et al., 2021).

On the other hand, PβAE-447 showed a dose-dependent decrease in cell viability. However, the cell viability was slightly reduced as the PβAE-447 concentration was raised; despite, the cell viability was higher than 75% for all 3 cell lines, confirming that the PβAE-447 is cytocompatible. Disulfide bonds in the PβAE support biodegradability, influencing vector cytotoxicity and pDNA release (Liu et al., 2019).

To study the particle size and zeta potential as potential variables, the nanoparticles were characterized as: 1) nanoparticles formed with 10% FBS; 2) nanoparticles formed with 300 mmol NaCl; 3) lyophilized nanoparticles; 4) PEGYlated nanoparticles in FBS, and 5) PEGYlated nanoparticles with 300 mmol NaCl. The obtained results were compared with the fresh nanoparticle properties (the as-prepared material). All these results were evaluated with the nanoparticles prepared at a 60:1 polymer: pDNA weight ratio.

The size of the as-prepared material was 111.1 nm while the lyophilized nanoparticle’s size remained 129.9 nm after 4 months of storage (Figure 6A and Supplementary Figure S3). This size range is suitable for cellular uptake. It has been reported that nanoparticles between 20 and 200 nm can internalize in cells, acting as target nanocarriers. In this size range, the nanoparticles can prevent filtration and quickly penetrate the cells (Zhao et al., 2014).

The Zeta potential of the as-prepared material was 9.51 mV while the lyophilized nanoparticle’s Zeta potential remained 9.45 mV nm after 4 months of storage. The Zeta potential is directly associated with the surface charge. So, it is critical for nanoparticle stability in physiological media and responsible for the initial nanoparticle adsorption on the cell membrane. After adsorption, the endocytic uptake rate is influenced by the particle’s size (Green et al., 2008; Rasmussen et al., 2020). These statements indicate that nanoparticle sizes affect cellular internalization, transfection efficiency, and biodistribution in vivo.

The PβAE/pDNA nanoparticles incubated with FBS and NaCl were aggregated, leading to large-sized particles with low Zeta potentials (Figure 6A,B and Supplementary Figure S3).

The FBS accelerates the nanoparticle aggregation, indicating that the PβAE/pDNA nanoparticle zeta potential is quickly altered by the adsorption of serum proteins. The FBS adsorption increased the average nanoparticle size to 567 nm after 4 h (Figure 6A). For preventing or reducing nanoparticle aggregation, the PβAE-447 was PEGylated before nanoparticle formulation. PEGylated polymers have a lower affinity to blood proteins because PEG is not charged (Kim et al., 2015). Compared to PβAE/pDNA nanoparticles in FBS and NaCl, the PEG adsorption reduced the PEGylated-PβAE/pDNA nanoparticles hydrodynamic radius to 184 nm in FBS and 175.1 nm in NaCl after 4 h (Figure 6). The size and Zeta potential of the PEGylated-PβAE-447/p-DNA nanoparticles in FBS and NaCl after 4 h were different compared to the as-prepared PβAE-447/p-DNA nanoparticles, confirming strong evidence of successful PEG coating. The PEGylated polymer reduced and prevented nonspecific interactions between the nanoparticles and FBS, avoided aggregation.

Non-PEGylated PβAE-based nanoparticle suspensions are unstable in NaAc solution, forming aggregates during long-term storage at room temperature (Wilson et al., 2019). For overcoming this disadvantage, the PβAE/pDNA nanoparticles were lyophilized and stored at 4 °C for 4 months. These nanoparticles were re-suspended in distilled water, and their average size and Zeta potential were measured and compared with the properties found for the fresh nanoparticles (as-prepared materials). The lyophilization and storage process at 4 °C for 4 months did not influence the nanoparticle features. The average sizes and Zeta potentials of the lyophilized and fresh nanoparticles are similar (Figure 6). These properties are desirable for gene delivery and cellular uptake. Therefore, the formulations that developed significant aggregations were eliminated for further considerations as candidates for gene delivery. The as-prepared PβAE-447/pDNA nanoparticles, lyophilized, and PEGylated nanoparticles were selected for further studies.

Various cationic non-viral gene vectors have shown agglutination activities (Kurosaki et al., 2009). Hemagglutination assay with mice erythrocytes was performed to investigate the agglutinating activity of PEGylated PβAE-447/pDNA complexes at a 60 wt/wt ratio. Subsequently, 2% erythrocytes suspension was selected because it supports best agglutination observation in the 96-well plate (Mrázková et al., 2019). Before using PEGylated PβAE-447/pDNA complexes for clinical applications, it is necessary to investigate its biocompatibility with blood components. The hemagglutination assay results ensure the PEGylated PβAE-447/pDNA complexes are cytocompatible with blood cells, as no RBC disruption was observed (Figure 7).

The transfection efficiency of the nanoparticles was investigated toward three different cell lines (HEK-293, BEAS-2B, and A549). HEK-293 cells are preferred as an easily transfectable cell line, while BEAS-2B and A549 cells are selected as relevant target cell lines for transfection. These cells were seeded in 96-well plates at 12,500 cells/well in 100 μL of media and were incubated overnight for adherence. The pDNA concentration (0.06 μg/μL) in each well was kept constant.

As a model system, the same protocols were followed in each transfection experiment by using pGFP as a reporter gene. The cells were incubated for 4 h with the nanoparticles (as prepared, lyophilized, and PEGlayted) and cellular uptake was investigated after 48 h. The transfection efficiency was calculated from the microscopic images by analyzing the fraction of stained cells (green color).

Fluorescence images demonstrate that transfection efficiency considerably depends on the cell line (Figure 8). The A549 and HEK-293 cells show more GFP expression than the BEAS-2B. The A549 cells have been well transfected by PβAE-447-based nanoparticles, followed by the HEK-293 cells. However, the BEAS-2B cells are less transfected. Overall, PEI was found to have slightly lower transfection efficiency than PβAE-447-based nanoparticles. These results are consistent with the sunshine et al., findings. Their optimal formulation of PβAE showed better transfection in retinal pigment epithelium cells than PEI (25 kDa) (Sunshine et al., 2012). In contrast to PEI, Poly (β-amino ester)s have demonstrated higher transfection efficacy in vitro and in vivo than many commercially available transfection reagents. PβAEs can deliver various pDNA to multiple tumor models with improved survival outcomes (Wilson et al., 2019). The transfection efficiency is not solely dependent on the PβAE nanoparticles. Additional parameters such as the number of cells, transfection method, and transfection reagents influence the transfection efficiency of a DDS.

No significant difference in the transfection efficiency is observed among the lyophilized PβAE-447/pDNA, PEGlated PβAE-447/pDNA, and as-prepared PβAE-447/pDNA nanoparticles (Figure 9). The highest transfection efficiency (72%) was shown by the as-prepared PβAE-447/pDNA nanoparticles toward A549 cells, followed by the HEK-293 cells (56%) at a 60 PβAE-447/pDNA weight ratio. Interestingly, we found that PEI has slightly high transfection in BEAS-2B cells. We assume that different levels of transfection of the same reagents may be due to variant specificity toward target cells.

This experiment explores the capability of PβAE-447 to deliver pDNA to the nucleus of the cells. HEK-293, BEAS-2B, and A549 cells were treated only with PEGYlated PβAE-447/pDNA nanoparticles at different times to evaluate the endocytosis and internalization. Endocytosis is the essential cellular process in which the extracellular materials and nanoparticles are internalized in cells (Rennick et al., 2021). The confocal images show that PEGYlated PβAE/pDNA nanoparticles are taken up by the evaluated cell lines.

The results demonstrate that the short incubation period (at least 2 h) did not show significant cellular uptake, while long incubation periods (6 and 8 h) led to high cytotoxic effects. This indicates that polymer has a harmful effect on cells if exposed for a longer time. This toxic effect may depend on the charge of the PβAE-447. Meantime, when PβAE-based nanoparticles were incubated with the cells for 4 h, the nanoparticles are found primarily distributed in the cytoplasm and nuclei region (Figure 10). It indicates that PβAE-based nanocomplexes can be used to target the nuclei effectively. However, long exposure times provide cytotoxic effects, because the strong influence of high charges may lyse the membranes. Long time disrupting the membrane cell walls fluidity due to the increased cell permeability and degeneration (Jeong et al., 2017).