Chitosan-DNA nanoparticles: synthesis and optimization for long-term storage and effective delivery

Plasmid vectors

The CMV-H2B-mScarlet plasmid (Addgene, Watertown, MA, USA), harbourings a monomeric red fluorescent protein (Arendzen et al., 2023), was extracted by a MaxiPrep Kit (Qiagen, Hilden, Germany). The plasmid was aliquoted and stored frozen in EP buffer in a concentration at least of 1 μg/μL until needed.

Chitosan/DNA nanoparticle formulation

H2B-mScarlet CsNPs were formulated using the coacervation method (Roy et al., 1999). Chitosan CL113 (Mw ± 150 kDa) with a 90% degree of deacetylation (Novamatrix, Drammen, Norway) was used at a working concentration of 1 wt/vol % diluted with 25 mM acetic buffer, and H2B-mScarlet DNA was diluted in 50 mM sodium sulfate. Both solutions were heated to 55 °C for 20 min, mixed, and then vortexed for 30 s. After preparation nanoparticles were left to stabilize at room temperature for 30 min for stabilization. For the different DNA concentrations (0.005, 0.0025, and 0.00125 wt/vol %) various concentrations of chitosan were adjusted according to the different N:P ratios from 1:1 to 4:1.

Lyophilization of nanoparticles

Freshly prepared H2B-mScarlet CsNPs were placed into a plastic 50 ml tube and were freeze-dried under vacuum at −55 °C by Lyotrap Freeze Dryer (LTE Scientific, Greater Manchester, UK) and stored at 4 °C until used. Before being used in transfection experiments, lyophilized nanoparticles were reconstituted in nuclease-free water to the original concentration and sonicated for 10 min using a sonicator from Fisher Scientific, Model FB120 (Thermo Fisher Scientific, Waltham, MA, USA). The conditions used were 120 Watts and 20 kHz with an amplitude of 80 (Gokce et al., 2014). Subsequently, reconstituted nanoparticles were used as described for the freshly prepared nanoparticles.

TEM

The Jem-1200EX TEM (JOEL, Tokyo, Japan) set to operate at 80 kV was used to evaluate the morphology of CsNPs. A Formvar coated carbon 300 mesh coated Cu ⁺⁺ grid (Ted Pella, CA, USA) was covered with a 10 μl solution of nanoparticles diluted 1:100 with nuclease-free water. The grid was further dried and viewed under TEM.

Particle size and zeta potential

The dynamic laser light scattering system (DLS) (Zetasizer, Malvern Instruments, Malvern, Worcestershire, UK) with a HeNe laser with a 635 nm wavelength was used to analyze the size and surface charge zeta potential (ZP) of nanoparticles. Scattered light was observed at a 90-degree angle. Nanoparticle samples were diluted in water to a 1:2,000 ratio and examined for 5 min, following the manufacturer’s instructions. Preparation and analysis of nanoparticles with various N:P ratios were conducted five times.

Nanoparticle stability

The integrity of the DNA released from nanoparticles was assessed by using gel electrophoresis. The 1% w/v agarose gel was stained with ethidium bromide (Sigma-Aldrich, Burlington, MA, USA). Loaded H2B-mScarlet CsNPs at 100 ng of DNA per well, and a single H2B-mScarlet DNA was used as control. Agarose gel was run for 45 min at 80 V and was visualized using a real-time UV transilluminator at 480 nm (Invitrogen, Carlsbad, CA, USA).

Transfection of HEK293 cells

HEK293 cells (ATCC, Manassas, VA, USA) were cultured in 24-well plates to 50% confluency with α-MEM, 10% fetal bovine serum, 1% Pen/Strep (Thermo Fisher Scientific, Waltham, MA, USA). HEK293 cells were transfected with CsNPs, DNA coupled to lipofectamine 2000 (LFN) (Invitrogen, Carlsbad, CA, USA) or unformulated DNA (-ve). For the transfection experiments, Opti-MEM (Gibco, Waltham, MA, USA) was used as the transfection medium, with adjusted to 6.5 using MES buffer (1 mM, pH 5.5) (Merck, Rahway, NJ, USA) (Ishii, Okahata & Sato, 2001).

Flow cytometry

FACS analysis was used to quantify H2B-mScarlet (ex/em wavelengths 569/594 nm) expression after CsNPs transfection. Briefly, HEK293 cells were washed thoroughly 24 and 48 hours (hrs) post-transfection three times with cold phosphate-buffered solution (PBS) (Sigma-Aldrich, Burlington, MA, USA) to remove unbound and surface-bound polyplexes. After the cells were trypsinized to detach cells (Therma Fisher, Waltham, MA, USA) for 3–5 minutes (mins) at 37 °C and then trypsin was neutralized with 10% Fetal Bovine Serum (FBS) Dulbecco’s Modified Eagle Medium (DMEM) media. Cells were collected and centrifuged and 4,000×g for 7 min and resuspended in PBS (Liang, Liu & Barman, 2019). Expression was analyzed using an Attune Nxt flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA). The samples of 300 μL were collected to a designated 30,000 events.

Cytotoxicity of CsNPs

The viability of HEK293 cells before transfection was confirmed with an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay (Thermo Fisher Scientific, Waltham, MA, USA). The protocol was adapted and modified (Riss et al., 2004). HEK293 cells were seeded in a 96-well plate (Thermo Fisher Scientific, Waltham, MA, USA) at a density of 2 × 10⁴ cells/mL in 100 μL of DMEM medium. The cells were incubated overnight at 37 °C in a 5% CO₂ atmosphere. After 24 h the cell medium was changed to H2B-mScarlet CsNPs in Opti-MEM and continued to be incubated for another 24 h. After adding 20 μL of 1× MTT solution to each well, the cells were incubated for an additional 4 h in an incubator (37 °C, 5% CO₂ atmosphere). Following this, 100 μL of 10% sodium dodecyl sulfate (SDS) was added per each well and incubated overnight at room temperature. The absorbance at 570/630 nm was measured and expressed as relative values compared to the untreated negative control, which was considered 100% viable.

Confocal microscopy

HEK293 at a density of 1 × 10⁵ cells per well were seeded in 8-well µ-Slide (Idibi, Bangkok, Thailand) and allowed to grow for 24 h. Four different ratios (1:1–4:1) of H2B-mScarlet CsNPs were then added to cells and incubated at 37 °C in a serum-free medium for 24 and 48 h. Images of cells expressing H2B-mScarlet were acquired using a Zeiss LSM980 confocal microscope (Zeiss, Oberkochen, Germany) and a 63× oil objective. Expression of H2B-mScarlet fluorescent emission was detected using an excitation of 569 nm with a multi-line Argon laser and detected through a 594 nm emission filter.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 10. Normality tests were used to check whether the data satisfied the requirements of a normal distribution assumption for a parametric test. Depending on the normality of the data, the differences between experimental groups were compared using both parametric and non-parametric tests. FlowJo software was used to analyze data output.

Size and morphology of CsNPs

The cellular uptake and internalization of nanoparticles are determined by their size, shape, surface charge, and surface functionalization, as well as the interactions among these factors. Therefore, achieving optimal cellular uptake efficiency requires attention to these factors at every stage of the process. The average particle size, PDI (polydispersity index), and PDI width of these CsNPs 0.0025 wt/vol % DNA (5 µg per 200 µl of nanoparticles) are listed in Table 1. DLS analysis showed that the size of CsNPs is dependent on the N:P ratio, the average particle size became larger with the increasing of chitosan concentration. The mean diameter of freshly prepared particles formulated with N:P ratios 1:1 and 2:1 was <300 nm (250 ± 12.9 and 290.6 ± 16.6 nm, respectively), while the higher N:P ratios of fresh particles (3:1 and 4:1) were sized at 349.5 ± 22.4 and 363.3 ± 32.8 nm, respectively.

Lyophilization of nanoparticles is an attractive strategy for storage and medical applications. However, our group and others (Dhadwar et al., 2010) reported a drop in transfection efficiency of lyophilized (lyo) nanoparticles of approximately 50%. In this study, we studied whether the sonication of lyo nanoparticles could improve transfection efficiency. Fresh CsNPs had a comparable size range as lyophilized sonicated (lyo S) nanoparticles at the N:P ratio 1:1, 2:1, 3:1 and 4:1 mean sizes were 246.1 ± 28.8, 276.2 ± 18.0, 354.6 ± 20.1 and 383.5 ± 26.2. In comparison, lyophilized unsonicated (lyo Us) nanoparticles had a size range 2–3 times higher, which increased beyond 915.1 ± 23.3 nm as the N:P ratio increased to 4:1. Subsequently, the PDI of fresh and lyophilized sonicated particles at different ratio were 0.169 ± 0.099, 0.261 ± 0.062, 0.263 ± 0.046, 0.303 ± 0.055 mV and 0.272 ± 0.099, 0.289 ± 0.028, 0.297 ± 0.043, 0.321± 0.044 mV, respectively (Table 1). As the same size, the PDI of lyophilized unsonicated nanoparticles was increased beyond 0.639 ± 0.013 (Table 1). Those results show the effect of ultrasonication on the mean size and polydispersity of nanoparticles.

Next, TEM was utilized to assess the morphology of nanoparticle formulations made with chitosan CL113 at 1:1, 2:1, 3:1, and 4:1 N:P ratios, and H2B-mScarlet plasmid generated with different ionic strengths. We assessed the shape and size of fresh, lyophilized sonicated and lyophilized unsonicated nanoparticles (Fig. 1). Both fresh and lyophilized sonicated particles appeared globular in shape, which were constant across the ratios 1:1 to 4:1. Nanoparticles which were lyophilized but unsonicated had physical aggregations in all N:P ratios (Fig. 1).

Stability of CsNPs and DNA integrity

The stability of CsNPs was analyzed by measuring ZP and mean size for 24, 48, and 72 h. ZP quantifies effective electric charge on the surface of the nanoparticle. The magnitude of the ZP correlates with the stability of particles in suspension (Othman et al., 2012). Nanoparticles with higher ZP indicate that particles have a strong electrostatic repulsion between them, which is crucial in enhancing their stability by reducing the aggregation (Pardeshi et al., 2023). DLS analysis showed that the ZP of CsNPs was less positive with an N:P ratio of 1:1 (11.41 mV) and 2:1 (21.336) due to the low concentration of cationic Cs. Indeed, the addition of more chitosan resulted in more cationic nanoparticles. The electric repulsion induced by high ZP (>30 mV) can contribute to avoiding the aggregation of nanoparticles. Moreover, ZP is a key parameter for the stability of NPs in aqueous media. The ZP value was observed to be higher than 30 mV for the N:P ratios 3:1 (32.19 mV) and 4:1 (33.36 mV) for both fresh and lyophilized sonicated nanoparticles (Table S1) at 72 h.

Secondly, stability also was assessed by the DNA binding capacity of CsNPs, and 1% gel electrophoresis was performed at 24, 48, and 72 h as well. The binding capacity and stability of the CsNPs are reflected by the presence of unbound DNA in the gels. At all three time points, unbound DNA was observed in the fresh nanoparticles at the N:P ratio 1:1, indicating the ability of chitosan to form a stable complex with DNA in the N:P ratio 2:1 and (Fig. 2). However, no unbound DNA was detected in all ratios of both lyophilized sonicated and unsonicated nanoparticles. Our results confirmed the stability of CsNPs across all tested ratios and formations (Fig. 2).

Transfection efficiency

The effect of the N:P ratio and DNA concentration on transfection efficiency was assessed by quantifying the level of H2B-mScarlet expression. HEK293 cells were transfected with nanoparticles formulated with H2B-mScarlet DNA using an equal volume of nanoparticles per 4 × 10⁵ cells per well, and H2B-mScarlet expression was quantified 48 h post-transfection via flow cytometry. Nanoparticles at 2:1 and 3:1 N:P ratios showed the highest gene expression in all tested concentrations (0.00125, 0.0025, and 0.005 wt/vol %). The difference between N:P ratios of 2:1 and 3:1 was not statistically significant (p > 0.01). Compared to the lowest (0.00125 wt/vol %) and highest concentrations (0.005 wt/vol %), cells transfected with the 0.0025 wt/vol % at all four N:P ratios also demonstrated the highest mean H2B-mScarlet expression levels. N:P ratios of 2:1 and 3:1 showed the highest levels of gene expression, achieving 27.10% and 25.98%, respectively.

In contrast, cells transfected with nanoparticles at a 1:1 ratio exhibited the lowest gene expression 16.81% (Fig. 3). These results indicated a positive dose-response relationship, revealing a direct correlation between nanoparticle dose and transgene expression for the same formulation. Confocal microscopy confirmed visually that the CsNPs at ratios 2:1 and 3:1 have the highest level of transfection efficiency (Fig. S1).

Further optimization of CsNP synthesis

Following our previous findings, we focused our optimization efforts on lyophilized nanoparticles at this specific N:P ratio of 2:1, as a ratio with high efficiency and low NPs size. Figure 4A presents the transfection efficiency of nanoparticles lyophilized for different time intervals (12, 24 and 48 h), with the 24-h time point yielding the highest efficiency at 28.46% (p < 0.0001). Figure 4B illustrates the effects of varying volumes during lyophilization (1,500, 3,000, 4,500 µL), where the 3,000 µL volume resulted in the highest H2B-mScarlet expression at 28.10% (p < 0.0001). Figure 4C depicts the impact of sonication on lyophilized nanoparticles before transfection, showing that lyophilized sonicated nanoparticles achieved nearly double the H2B-mScarlet expression (27.49%) compared to lyophilized unsonicated nanoparticles (12.62%).

Following the optimization, lyophilized nanoparticles were compared with fresh and lyophilized unsonicated nanoparticles at various N:P ratios of 1:1, 2:1, 3:1, and 4:1. Lyophilized sonicated nanoparticles showed the highest expression levels at ratios 2:1 and 3:1, 26.22% and 24.32%, respectively. Visual assessment on Confocal microscopy demonstrated similar results (Fig. S2). The ratios 2:1 and 3:1 of lyophilized sonicated nanoparticles demonstrated significantly higher transfection efficiency than unsonicated lyophilized nanoparticles (p < 0.0001) (Fig. 5). However, there was no significant difference between fresh and lyophilized sonicated nanoparticles at ratios 2:1 and 3:1. Specifically, at N:P ratio 2:1 the expression level of lyophilized unsonicated nanoparticles (12.05%) is approximately half that of the fresh (25.56%) and sonicated lyophilized particles (26.22%).

Cytotoxicity

The toxicity of CsNPs was examined by MTT assay that included the transfection of HEK293 cells with different ratios of fresh and lyophilized sonicated nanoparticles. The results reveal that all formulations in both states (fresh and lyo S) showed high cell viability at 72 h (Fig. 6). In ratios with the highest transfection efficiency, 2:1 and 3:1, the viability of fresh and lyophilized sonicated nanoparticles was 93.46% and 95.87% for the 2:1 ratio, 90.02% and 85.97% for the 3:1 ratio, respectively (Fig. 6A). We also tested different nanoparticles made with different concentrations of DNA at 2:1 N:P ratio (Fig. 6B). No significant toxicity of nanoparticle complexes for HEK293 cells was observed at concentrations of DNA under 0.005 wt/vol %, with viability of 83.65 % in fresh and 85.52% in lyophilized sonicated nanoparticles. For higher concentrations of DNA, viability was above 58.68% in both fresh and lyophilizedsonicated nanoparticles (Fig. 6B). The effect of N:P ratio and DNA concentrations on cell viability was also tested at 24 and 48 h (Figs. S3A and S3B). Similarly, at 72 h cell viability depends on the ratio and concentration of nanoparticles. In other words, the ratios from 1:1–4:1 at concentration 0.0025 wt/vol % were safe and non-toxic for HEK293 cells at 24, 48, and 72 h (Figs. S3A and S3B).