All chemicals were obtained commercially and used without additional purification. Human serum albumin (HSA), glutaraldehyde (GA), D,L-Sulforaphane (≥90%, HPLC), polyethyleneimine (PEI) with average M_w = 25,000 (all from Sigma-Aldrich), plasmid pF141 pAcGFP1 SOD1WT (Addgene, https://www.addgene.org/26402/), absolute ethanol (ET0016, 99.99%; Scharlau Chemie, Spain), phosphate buffer saline (PBS) and dimethyl sulfoxide (DMSO) (Reachim, Russia), and MilliQ water (18 mΩ×cm; Millipore). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Dulbecco’s Modified Eagle Medium (DMEM), and the 2',7'- dichlorofluorescein diacetate (DCFH-DA) dye. Commercial SOD assay kits were ordered from Sigma Aldrich. Hydrogen peroxide (30%) was purchased from Merck. Other chemicals used in the current study were of analytical and molecular biology grades. L-132 (Human Lung epithelial cells) cell line was obtained from National Centre for Cell Science, Pune, India. They were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum and 1% Penicillin-streptomycin maintained in a 37°C incubator with 5% CO₂ and 95% air.

The scheme of synthesis of HSA-PEI-SF-pSOD1 nanoparticles is presented in Figure 1. It is a coacervation procedure detailed in the study (Galisteo-González and Molina-Bolívar, 2014). Briefly, 100 mg of HSA was dissolved in 10 ml of water followed by the addition of 10 μL of sulforaphane solution in DMSO (100 mg/ml) and 100 μL of plasmid DNA solution (1490 ng/µL). Protein was transformed into NPs in a dropwise manner with the addition of 9.3 ml of absolute ethanol under strong stirring with the help of a magnetic stirrer. The solution turns into white color due to turbidity. To initiate cross-linking of NPs, after 5 min 150 μL of 8% glutaraldehyde water solution was added to the reaction mixture. Stirring of the mixture was carried out overnight at room temperature. During synthesis, color of the solution changes from white to light yellow, indicating the formation of cross-linked protein-based NPs. To eliminate the unreacted substances, the as-prepared HSA-SF-pSOD1 nanoparticles were washed by two-fold centrifugation at 15,000 g for 10 min with redispersion in water. By measuring the extinction of the supernatant after the first washing at a wavelength of 260 nm we found that no differences were observed for the HSA nanoparticles with DNA and without DNA. Thus, all added DNA was loaded into the HSA nanoparticles. To improve the colloid stability, nanoparticles were covered with a layer of polyethyleneimine (PEI) by the addition of PEI water solution to a final concentration of 1 mg/ml. After 24 h incubation under magnetic stirring, the resulting HSA-PEI-SF-pSOD1 NPs were washed by two-fold centrifugation at 15000 g for 10 min and redispersed in 10 ml of water. Unloaded HSA NPs and nanoparticles without SF or plasmid DNA were synthesized by a similar protocol.

The biocompatibility of NPs was determined by the cell viability assay, tetrazolium salt-based assays, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay. Briefly, L-132 cells were plated at a density of 1×10⁴ cells per well into 96-well plates until subconfluent (Corning, Costar, NY). Twenty four hours after seeding the cells, the cells were then incubated with different (low to high) concentrations (0.2, 0.6, 1.0, 1.5, 2.0, 2.5, 3.0 µg) of the blank HSA-PEI Np’s, HSA-PEI-SF Np’s, HSA-PEI-pSOD1 Np’s, and HSA-PEI-SF-pSOD1 Np’s up to 96 h. Sterile PBS wash was given to cells after 48 h incubation of nanoparticles, followed by the addition of fresh 90 µL DMEM media per well to the cells. 10 µL of MTT (Sigma-Aldrich, United States) reagent (5 mg/ml stock) was added per well, including controls, and incubated at 37°C for 3–4 h incubation. Cells were examined for the appearance of an intracellular punctate purple precipitate under an inverted microscope. Then the medium was discarded from each well and dimethyl sulfoxide (DMSO) i.e. 100 μL was added including controls further after the appearance of purple color precipitate to dissolve the formazan crystals produced. The plate was incubated in the dark for 20–30 min. Measured the absorbance of each well, including blank with the help of a multimode reader (Cytation3, Biotek) at 570 nm and the background control at 690 nm. The untreated cells were considered 100% viable, all the data were performed in triplicate.

Cell viability (%) was calculated as Cell   viability = ( A 570 - A 690 ) treated / ( A 570 - A 690 ) control × 100 ( % ) , where A₅₇₀ and A₆₉₀ are the absorbances at 570 and 690 nm, respectively.

The superoxide anion scavenging activity of blank HSA-PEI NP’s, HSA-PEI-pSOD1 Np’s, and HSA-PEI-SF-pSOD1 Np’s at different time intervals (10, 24, and 48 h) were evaluated with a commercially available SOD assay kit (Sigma Aldrich). Briefly, a sample solution (20 μL, i.e., 2 µg SF and 300 ng pSOD1) was added with 200 μL of WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2H-tetrazolium, monosodium salt) working solution in a 96 well plate. With the addition of 20 μL enzyme (xanthine oxidase) solution, the reaction was initiated and preceded at 37°C for 20 min. Finally, the inhibition activity was measured by taking the absorbance at 450 nm as compared to the control using a Cytation 3 cell imaging multi-mode plate reader (Biotek).

L-132 cells were seeded onto a 24-well plate at a density of 1×10⁴ cells/well 24 h before the transfection experiment. Transfection studies were started when cells reached 70–80% confluent by washing the L-132 cells twice with 1 ml of sterile PBS which was further incubated at 37°C in a serum-free culture medium. Nanoformulations were added into the well as follows: 1) blank HSA-PEI Np’s in serum-free medium at the same concentrations as in (2); 2) HSA-PEI-SF-pSOD1 Np’s containing 30 µL (460 ng) of plasmid DNA in serum-free medium, 3) 5000 ng of free pSOD1 in a serum-free medium; 4) 400 µL of serum-free culture medium as negative control. Treatments were terminated after a 6 h incubation and the cells were maintained in a complete medium for another 48 h. After incubation with nanoformulations and subsequent washing, the cells were visualized. The intracellular gene expression in each well was reflected by the fluorescent intensity of the images and that was examined by the EVOS cell imaging system (Life Technologies, United States).

L-132 cells were plated into six-well plates and grown to optimal confluence. Subsequently, the cells were incubated with 30 µL of HSA-PEI-SF-pSOD1 Np’s (460 ng pSOD1 & 3 µg SF) and blank HSA-PEI Np’s for different periods i.e. 12, 24, and 96 h. After incubation, the cells were washed 3 times with PBS followed by exposure to 200 μM H₂O₂ in a culture medium for 4 h at 37°C. The 2’,7'- dichlorofluorescein diacetate (DCFH-DA, Sigma Aldrich) dye was used to measure intracellular ROS production. Inside the cell, deacetylation of DCFH-DA into nonfluorescent 2’,7'-dichlorofluorescein (DCFH) is carried out by endogenous esterases, which is further transformed into dichlorofluorescein (DCF) compound that produces green fluorescence in response to ROS generation that was analyzed by fluorescence microscopy. The stock solution of DCFH-DA was prepared in dimethyl sulfoxide (DMSO, Sigma Aldrich). After the treatment, the cells were given a thorough PBS wash, followed by incubation with 20 μM DCFH-DA at 37°C for 30 min. At the end of incubation, the cells were thoroughly washed; the intracellular antioxidant activity of HSA-PEI-SF-pSOD1 Np’s was studied by the EVOS cell imaging system (Life technologies, United States). The intracellular ROS level in each sample was reflected by the green fluorescent intensity of the cells.

Data are presented as mean ± standard error of the mean and were analyzed by using Microsoft Excel and the GraphPad Prism 5 software (GraphPad, v. 6.1). Statistical analyses were performed using Student’s t-test for unpaired data or by two-way ANOVA whichever was applicable and p <0.05 was considered statistically significant.

The average diameters of the synthesized HSA nanoparticles and their polydispersity indices were measured using the dynamic light scattering method and are included in Table 1. A TEM image of unloaded HSA nanoparticles is presented in Figure 2A. An important feature of our synthesis protocol is the fact that loading with the target substance occurs during the synthesis of nanoparticles. In the synthesis of other nanocontainers, such as mesoporous silica, nanoparticle synthesis, and drug loading are usually two distinct steps. In our synthesis, on the contrary, there is a simultaneous synthesis of nanoparticles and loading with the target substance. Therefore, the final size of nanoparticles differs depending on what substances were loaded into them during the synthesis (Table 1). The extinction spectrum of the synthesized unloaded HSA nanoparticles is shown in Figure 2B. The nanoparticle loading with sulforaphane and plasmid DNA as well as PEI coating did not cause noticeable changes in their extinction spectrum (data not shown). The extinction spectrum is typical for non-absorbing particles, which scatter light. This is because sulforaphane, plasmid pSOD1, and PEI do not show significant interaction with light from this wavelength range (from 350 to 1100 nm). The PEI coating of the HSA nanoparticles led to a change of the zeta potential of the nanoparticles from negative (for uncoated HSA particles) to positive (Table 1) to enhance the colloidal stability of particles. In addition, the PEI coating noticeably increases the hydrodynamic radius of the nanoparticles, which is shown in Table 1. After the PEI coating, the particle size always increases. However, the data of DLS measurements convincingly indicate that there is no aggregation of nanoparticles in this case. The point is that the relatively small value of PdI is still preserved, which indicates the absence of aggregation. HSA is conjugated with polyethyleneimine (PEI) to improve the effect of PEI on gene transfection (Baker et al., 1997). The positive surface charge due to the PEI coating helps in efficient cell targeting.

Surface characteristics, physical stability, and particle size play a very vital role in nano-drug delivery (Langer et al., 2003). As particle size and zeta potential of the nanoparticles ensure maximum efficacy to reach the target site. The size ranges from 10 to 100 nm easily get entry into the systemic circulation and undergo clearance (Longmire et al., 2008), whereas the size ranging from 250 to 1000 nm are encountered phagocytosis and taken up by macrophages a subsequently untaken by the reticuloendothelial system (RES). Through the process of opsonization, macrophage removes the nanoparticles/particles from blood circulation (Harashima et al., 1994; Longmire et al., 2008). A lower surface curvature of the nanomaterials also decreases the possibility of opsonization (Owens and Peppas, 2006).

The MTT assay demonstrates the cytotoxicity which is based on the conversion of MTT dye to insoluble formazan, which was further detected by spectrophotometry (Li et al., 2009). The cytotoxicity of blank HSA-PEI NPs, HSA-PEI-SF Np’s, HSA-PEI-pSOD1 Np’s, and HSA-PEI-SF-pSOD1 NPs was determined by incubating L-132 cells at concentrations ranging from 2-3.0 µg for up to 96 h. The viability of HSA Np’s-treated cells was examined using the MTT assay. The results demonstrated that blank HSA-PEI Np’s, HSA-PEI-SF Np’s, HSA-PEI-pSOD1 Np’s, and HSA-PEI-SF-pSOD1 Np’s were found to be nontoxic to L-132 cells up to a concentration of 30 µL (i.e., 3.0 µg- SF).

As shown in Figure 3, the percentage of the viable cell was more than 90% up to 96 h at nanoparticle concentrations up to 3.0 µg. This confirms that the HSA nanocomposites are safe in nature.

The short-lived radical superoxide anion (O₂ ⁻) is produced by an electron addition to oxygen. When environmental determinants such as UV light, environmental pollutants, cigarette smoke, and γ-radiation or oxidases such as NADPH oxidase or xanthine oxidase are exposed to cells, these free radicals such as superoxide anion (O₂ ⁻) are formed. Once formed, O₂ ⁻ attacks the cellular components and leads to damage to the cell membrane, lipids, proteins, and DNA. Endogenously ROS is produced due to metabolic reactions taking place in a body or response to external factors. In the respiratory tract, it is produced through the activation of phagocytes (Ho et al., 2001). ROS is important for the initiation and promotion of cells to neoplastic growth (Ho et al., 2001).

The damage caused by free radicals leads to the development of certain diseases, including cancer, rheumatoid arthritis, atherosclerosis, diabetes, liver damage, and central nervous system disorders.

The SOD mimetic activity of blank HSA-PEI Np’s, HSA-PEI-SF Np’s, HSA-PEI-pSOD1 Np’s, and HSA-PEI-SF-pSOD1 Np’s was studied in L-132 cell line against H₂O₂ mediated oxidative stress for 6-hour incubation were shown in Figure 4. We observed an elevation in the SOD mimetic activity which is time-dependent on HSA-PEI-pSOD1 Np’s and HSA-PEI-SF-pSOD1 Np’s, which correlates with the enhanced concentration of antioxidants with dual loading of the SOD1 gene and antioxidant sulforaphane drug in the HSA Np’s, which further promotes an increased level of antioxidants in the surrounding cytosolic medium which is when released from the core of HSA Np’s. During the first 10 h, due to the initial burst release of SOD1 and drug from HSA Np’s, a significant increment in the SOD activity was observed as an effect of the higher concentration of expression of gene SOD1 and drug in the surrounding medium. On the other hand, we did not notice any antioxidant activity in blank HSA Np’s, which further confirms the expression of the SOD1 gene and sulforaphane drug. In our results, we observed that HSA Np’s loaded with SOD1 gene and sulforaphane showed higher antioxidant activity at 48 h as compared to the initial 10 h of incubation, this is maybe due to subsequent expression of SOD1 gene in cells after 48 h. After 24 and 48 h, the antioxidant activity of the three types of loaded nanoparticles is approximately at the same level, the so-called saturation effect is observed at a level of about 80 and 100%, respectively. During reaction time, free radicals were generated due to the enzymatic activity of xanthine oxidase, which further converts WST-1 working solution into end product i.e., water-soluble colored formazan dye, which exhibits maximum absorbance at 450 nm. The addition of HSA-PEI-pSOD1 Np’s and HSA-PEI-SF-pSOD1 Np’s inhibits the color formation by actively removing the superoxide radicals that lead to the reduction in the absorbance value (Singh et al., 2010; Bhushan and Gopinath, 2015).

We demonstrated the in vitro transfection efficiency of pSOD1-SF loaded PEI-HSA Np’s by incubating human lung cancer cells (L-132) with blank HSA-PEI Np’s, naked plasmid pSOD-1, and HSA-PEI-SF-pSOD1 Np’s for 6 h, in serum-free medium followed by 48 h incubation in complete medium. Figure 5 illustrates negligible transfection efficiency for naked plasmid at a pSOD1 dose of 5000 ng, suggesting that the transfection potential of plasmid alone is insignificant. The results signify that blank HSA-PEI nanoparticles also showed 1–2% fluorescence, probably due to the autofluorescence of human serum albumin nanoparticles cross-linked with glutaraldehyde, whereas only 2–3% transfection efficiency was obtained with naked pSOD-1, which is insignificant as compared to the pSOD1-SF loaded PEI-HSA Np’s’ transfection efficiency. The developed NCs demonstrated, on the other hand, HSA-PEI-SF-pSOD1 Np’s results in 66% transfection efficiency, containing 460 ng pSOD1, were indicating that the HSA Np’s’ formulation has high transfection efficiency even at very low concentrations. It is reported that the HSA nanoparticles with the unmodified surface have lower transfection efficiency due to the presence of their negative surface charge, even the negatively charged HSA nanoparticles could not interact efficiently with the negatively charged phosphate backbone of plasmid DNA, and also affects or impedes the cellular uptake of nanoparticles (Torrano et al., 2013). Thus, to overcome these problems, researchers synthesized the cationized HSA nanoparticles surface (Fischer et al., 2001). The HSA nanoparticles with polyethyleneimine (PEI) showed an endosomolytic effect that led to significant gene expression in vitro in HEK293 cell lines. Therefore, to enhance cellular uptake and gene transfection efficiency, they did the surface modification with PEG-NH₂ (Rhaese et al., 2003). In our study, we modified the HSA nanoparticles’ surface with PEI, which exhibits high transfection efficiency i.e. 66% as compared to bare HSA nanoparticles i.e. only 2–3%, this suggested that nanoparticles enter the L-132 cells via the caveolae or clathrin-mediated pathway, showed endosomal escape, and further sustain plasmid release occurs in nucleolus without degradation in the endosomal compartment.

The ROS scavenging potential of HSA-PEI-SF-pSOD1 Np’s was further assessed in L-132 cells where we have used hydrogen peroxide as positive control which mediates oxidative stress in cells. As depicted in Figure 6, when cells were exposed to HSA-PEI-SF-pSOD1 Np’s before H₂O₂ exposure, they were not exhibiting high reactive oxygen species (ROS). Whereas, in Figure 6, we have noticed considerable changes in the level of ROS species at longer incubation time as compared to the initial hours of incubation of nanoparticles. After 96 h the intensity of DCF decreases, this is ascribed may be due to drug internalized inside the cytosolic compartment, and the subsequent expression of SOD1 transgene occurs in 96 h. On the other hand, L-132 cells exhibit high DCF fluorescence intensity, where H₂O₂ exposure was given without HSA-PEI-SF-pSOD1 Np’s preincubation, suggesting removal of ROS species by nanoparticles was successfully achieved.