Characterization and stability of plasmid DNA calcium nanoparticles using a simple formulation for gene therapy

Abstract

Gene-based therapy is crucial in treating various disorders and diseases by conferring genetic material to target cells. Calcium phosphate (Ca-P) nanoparticles show promise as a non-viral vector for targeted gene delivery due to cost-effectiveness and established biocompatibility, which enhances their application in nanomedicine. Our previous pre-clinical studies in rats and monkeys exhibited the safety of our Ca-P nanoparticle formulation by local and systemic administration, with a simple preparation. However, the tendency of plasmid DNA-encapsulated Ca-P (pDNA/Ca-P) nanoparticles to aggregate during prolonged storage at inappropriate temperatures is a significant challenge. To investigate the effects of temperature and storage duration on the physicochemical properties and gene delivery efficiency of pDNA/Ca-P nanoparticles, we exposed the pDNA/Ca-P nanoparticles to varied temperatures ranging from − 80 °C to 65 °C for 7 days after formulation and measured physicochemical properties, including size, shape, surface charge, pH level, and pDNA delivery efficiency. The results indicated that pDNA/Ca-P nanoparticles stored at 4 °C and 29 °C exhibited the highest gene expression while maintaining the smallest particle size. Continuous storage at a similar temperature for 5 months demonstrated that the nanoparticles retained their favourable physicochemical properties. These findings showed that the proper long-term storage temperature at 29 °C of the pDNA/Ca-P nanoparticles, without the need for specialised instruments, paves the way for extensive use of Ca-P nanoparticles for gene therapy in pre-clinical and clinical studies in resource-limited areas.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-33265-3.

Introduction

Gene-based therapy shows promising potential for treating genetic disorders. It works by delivering nucleic acids that produce proteins to address abnormal conditions^1,2. Recently, advancements in nanoparticle platforms, such as polymeric and biomimetic nanoparticles, have enhanced the efficacy of nucleic acid therapies. While these delivery systems have made considerable progress in addressing important challenges like stability, cellular uptake, and controlled release³. They still present areas needing further attention, particularly regarding carrier compatibility^4–7. Despite these ongoing challenges in some nanoparticle platforms, especially calcium phosphate (Ca-P) nanoparticles, have garnered attention for non-viral gene delivery due to their biodegradable and biocompatible properties. Calcium and phosphate are naturally occurring substances in mammalian hard tissues, for example, bone and teeth. These characteristics confer Ca-P nanoparticles suitable for safe and effective gene therapy applications^8–10.

The formation of plasmid DNA-encapsulated in calcium phosphate (pDNA/Ca-P) nanoparticles occurs due to the chemically attractive force between the positively charged calcium ions and the negatively charged phosphate groups of the nucleic acids and other solutions, leading to co-precipitation^11,12. They establish ionic bonds to encapsulate biomolecules, e.g., plasmid DNA, and embed them on their surfaces, preserving the organic molecules until the nanocarriers reach their target. As a result, Ca-P nanoparticles have potential for use in genetic material delivery systems^13,14.

Research in this field has been exploring the potential of pDNA/Ca-P nanoparticles and their characterizations for therapeutic applications for various diseases for many decades^15–17. These investigations have led to developing of numerous nanoparticle formulations optimized for specific experimental settings, both in vitro and in vivo18²⁰. However, a thorough understanding of the physicochemical characteristics of Ca-P nanoparticles synthesized via the co-precipitation method remains crucial and ongoing. Several factors contribute to the physicochemical stability of this system, including the ratio between Ca/P, size, shape, surface charge or zeta potential, pH, and time of co-precipitation^21–23. The major challenge with drug delivery systems for gene therapy based on Ca-P nanoparticles is their physical instability. Ca-P nanoparticles have a natural tendency to aggregate rapidly after synthesis, causing the growth of Ca-P nanocrystals over time. The aggregation influences the effectiveness of the nanoparticles in gene delivery, leading to highlighting the need for strategies to maintain their stability and suitable storage conditions^24–26.

Suitable nanoparticle formulations have been developed for various experiments and laboratory settings to address this challenge. Sokolova et al. used co-precipitation to create pDNA-coated Ca-P nanoparticles and added a calcium phosphate layer to form multiple shells. These nanoparticles, with a 10–20 nm diameter, exhibited excellent transfection efficiency and could be easily stored for up to 14 days at 4°C²⁷. Bisht et al. modified pDNA by co-precipitating it with Ca-P nanoparticles in an anionic surfactant Dioctyl Sodium Sulfosuccinate (Aerosol-OT, AOT) microemulsion in hexane, resulting in high transfection efficiency in HeLa cells, comparable to transfection commercial²⁸. Liu and colleagues compared two methods for creating calcium phosphate (Ca-P) nanoparticles. They found that standard Ca-P nanoparticles rapidly aggregated over time, increasing in size from 30 nm to 100 nm within 7 days of synthesis at 4 °C, while protamine sulfate-coated calcium phosphate nanoparticles did not exhibit the same aggregation²⁹. In 2014, Zhao and colleagues developed a new technique for preparing Ca-P nanoparticles. They relied on calcium carbonate (CaCO₃) for this process. The technique involved co-precipitating Ca2 + ions and DNA and mixing the compound with carbonate and phosphate ions. The co-formulation of carbonate and phosphate slows down the process of crystallization. The results showed that this formulation enhanced gene delivery effectiveness without causing toxicity³⁰.

In 2022, our research team adjusted the formulation Zhao et al. created. We optimized the novel formulation of Ca-P nanoparticles and performed the studies in vitro and in vivo. The novel formula could be prepared without high-level equipment needs, and it showed positive transfection results in cell culture without cytotoxicity observed. A recent study using the pDNA of Box A of HMGB1/Ca-P nanoparticles in aging rats effectively rejuvenated multiple organs, including the brains of aged rats³¹. In addition, our in vivo study demonstrated that a system involving Ca-P nanoparticles can effectively deliver for a topical application. Diabetic rat wounds were administered with B1 siRNA/Ca-P nanoparticles through a topical route. The results showed that the siRNA-encapsulated Ca-P nanoparticles reduced DNA damage and accelerated wound healing³². Our laboratory has successfully utilized siRNA or pDNA/Ca-P nanoparticles in both in vitro and in vivo applications. Prior studies have mainly focused on short-term storage conditions. However, a critical knowledge gap exists in comprehending the characterization of the nanoparticles during long-term storage. Establishing proper storage conditions is essential for future large-scale storage. Our study takes a pioneering step by systematically evaluating the impact of storage temperature and duration on the physicochemical properties of the nanoparticles.

The study aims to investigate the long-term characterization of Ca-P nanoparticles under different storage temperatures and times suitable for their potential applications. We continuously monitored the nanoparticles for five months, with measurements taken at six time points: 1 day, 7 days, 30 days, 120 days, and 150 days. The focus was on understanding how extended storage impacted key physicochemical properties, including size, shape, surface charge (zeta potential), and solution pH. These properties influence nanoparticle stability and functionality. The transmission electron microscope (TEM) is used to measure the size and shape of particles. Zeta potential, which represents the electrical charge, is important for predicting and controlling the stability of nanoparticles. A long-term energy barrier is necessary to maintain particle stability and prevent flocculation and aggregation at the nanoscale. Maintaining proper pH levels is essential for sample stability. pH level can impact zeta potential, leading to coagulation and instability. Monitoring the solution’s pH is necessary to prevent adverse effects regularly. In the study on particle-mediated gene delivery, we used Ca-P nanoparticles to transport green fluorescent protein (GFP) pDNA into human embryonic kidney 293 FT cells (HEK293FT) at different temperatures and time intervals. We observed transfection efficiency using a fluorescence microscope and evaluated them via flow cytometry.

These findings set the groundwork for establishing optimal storage conditions with a simple process of pDNA-nanoparticle encapsulation, paving the way for large-scale applications of Ca-P nanoparticles in gene therapy in areas with limited resources.

Methods

Plasmid DNA preparation and purification

A plasmid carrying the GFP protein was utilized to produce calcium phosphate nanoparticles. The GFP was incorporated into the pLenti-P2A-tGFP backbone vector and cloned into a plasmid DNA driven by the cytomegalovirus (CMV) promoter (Origene Technologies, Inc, Rockville, MD, USA). This vector also contains the chloramphenicol-resistant gene element, which can act as a selective marker for the plasmid DNA. All plasmid DNA was then extracted from Escherichia coli (DH5-alpha) and purified by the Qiagen Plasmid Maxi kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. The Spectrophotometric A260/A280 ratios were detected, and their purification was between 1.8 and 2.0.

Preparation of calcium phosphate nanoparticle complexes

The GFP plasmid DNA/calcium phosphate nanoparticles were synthesized using the previously published method²⁶. Briefly, the synthesis of calcium phosphate nanoparticles from three different aqueous solutions consists of 0.5 M calcium chloride (CaCl₂), 0.01 M sodium carbonate (Na₂CO₃), and 0.01 M sodium dihydrogen phosphate monohydrate (NaH₂PO₄·H₂O). The optimal ratio for plasmid DNA delivery is 5 µg of plasmid in 100 µl of Ca-P nanoparticle solution for the in vitro experiment.

To prepare solution no. 1, 5 µg of plasmid DNA was gradually added into a 16 µl CaCl₂ solution and thoroughly mixed vigorously using a vortex. Once the addition was complete, the mixture was diluted by adding deionized water until the final volume reached 50 µl. To prepare solution no. 2, we conducted a meticulous and thorough process. Firstly, two separate aqueous solutions of Na₂CO₃ and NaH₂PO₄·H₂O were mixed at different molar ratios of CO₃²⁻/PO₃⁴⁻ at a ratio of 31:1. Then, 16 µl of solution no. 2 was mixed with sterile deionized water and adjusted to a volume of 50 µl. Finally, the conjugated solution (solution no. 1) was gradually added to solution no. 2 at 10 µl per 5 s while mixing vigorously using a vortex (Fig. 1a).

Fig. 1.

Co-precipitation method and Evaluation of optimal conditions for transfection in HEK293FT cells with pDNA/Ca-based nanoparticles at different storage temperatures at 7 days. (a) Co-precipitation method of calcium phosphate nanoparticles. (b) The panel shows fluorescence images of the expression of the GFP protein at different temperatures after 7 days of storage. (c) Flow cytometry analysis showed the GFP protein expression level of GFP pDNA/Ca-P nanoparticles in HEK293FT cells at different storage temperatures for 7 days. (d) The zeta potential values of pDNA/Ca-based nanoparticles were measured at different temperatures after 7 days of storage. The experiments were performed in triplicate; one-way ANOVA was used for statistical analysis where p < 0.01, p < 0.001, and p < 0.0001 represented as **, ***, **** respectively, and the error bar showed the SEM.

The pDNA/Ca-P nanoparticle complex solution was equally divided and stored at 4 °C and 29 °C for different periods, including 24 h, 7 days, 30 days, 120 days, and 150 days, before being used for characterization and transfection efficacy studies.

Cells and cell culture

HEK293FT cells were supplied by the American Type Culture Collection (ATCC, VA, USA). Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, MA, USA) and 1% antibiotic-antimycotic (Thermo Fisher Scientific, MA, USA) used for the cell culture under 5% CO₂ and humidified atmosphere at 37 °C.

Electron microscopy and flow cytometry for GFP protein detection

The transfection efficiency level was evaluated using electron microscopy and flow cytometry analysis. The HEK293FT cells were seeded in 6-well plates at a density of 2.5 × 10⁵ with a cell confluence of approximately 80% and were transfected with nano complex solution (500 µl with 25 µg plasmid DNA) in each well. The transfected HEK293FT cells were observed using an inverted microscope under magnification of 10X at 48 h post-transfection. To determine the expression level of GFP protein, the culture medium was removed, and the cells were collected using trypsinization and washed twice with phosphate-buffered saline solution before fixing with the fixative solution. The GFP-expressing cells, defined as positive cells, were enumerated by flow cytometry (Beckman Coulter CytoFLEX, USA), whereas the non-transfected cells were gated and designated as a negative control group. The experiments were performed at least three times for statistical calculation.

Transmission electron microscopy

The characteristic of the GFP pDNA/Ca-based nanoparticle complex was captured by Transmission Electron Microscopy (TEM) (JEM-1400, JEOL, Tokyo, Japan) after storage at 4 °C and 29 °C for different periods, for 24 h,7 days, 30 days, 120 days, 150 days, and 365 days. In addition, the analysis by TEM was performed in the experiment of temperature variation, including − 80 °C, -20 °C, 4 °C, 29 °C, 37 °C, 50 °C, and 65 °C at seven-day storage timepoint. To perform the analysis, 100 µL of nanoparticle solution, containing 5 µg of plasmid DNA, was aliquoted 10 µL and dropped onto the carbon/Formvar^® coated grid, which was stabilized with evaporated carbon film and self-dried at room temperature. The processed samples on the grids were identified under TEM.

Zeta potential and pH assessment

Nanoparticle solutions at different storage times and temperatures were determined by a Zetasizer Nano ZSP (Malvern Instruments). Before measurement, 1 mL of nanoparticle solution containing 25 µg plasmid DNA was diluted with 4 mL of distilled H₂O until the final volume of nanoparticles was 5 mL. The measured zeta potential results were given three independent measurements collected in each condition. The nanoparticle solution’s pH was measured with Fisherbrand™ accumet™ AE150 Benchtop pH meter (ThermoFisher Scientific, USA).

Encapsulation efficiency (EE) of pDNA into Ca-P nanoparticles

After pDNA encapsulation into Ca-P nanoparticles, the pDNA-nanoparticle solution (5 µg of pDNA/100 µL of Ca-P nanoparticle solution) was aliquot and incubated at -80 °C, -20 °C, 4 °C, RT °C, 37 °C, 50 °C, and 65 °C for 24 h. To investigate the EE, after the incubation period, the pDNA stock solution and the Ca-pDNA nanoparticle solution, containing the same amount of plasmid DNA (100 ng) per lane, were loaded into 1% agarose gel stained with 2 µL of 10,000X SYBR^® Safe (Invitrogen; cat. no. S33102) and using a 1 kb DNA ladder (Promega; cat. no. 6941, USA). The samples were electrophoresed at 80 volts for 60 min. The bands were captured using Omega Fluorplus (Gel Documentation System; Aplegen Inc. CA, USA). The band intensities were analyzed by the Fiji program (https://imagej.net/software/fiji/downloads) and utilized to calculate the %EE using the following formula. The %Encapsulation Efficiency (%EE) = (Band intensity of the encapsulated pDNA/Band intensity of the total loading pDNA) x100, as shown in Supplementary Figure S2 and Supplementary Table 1.

Statistical analysis

The data were collected from replications of the experiment. GraphPad Prism 10 (GraphPad Software) was utilized to analyze data using one-way ANOVA followed by Sidak’s test to determine differences more than two groups. The significant differences between the two groups were obtained by unpaired t-test. All values are given as mean ± standard error of the mean (SEM). In addition, ImageJ software was used for size and shape of nanoparticle analysis.

Results

The effects of storage temperature on the mammalian cell transfection efficiency, surface charge, and size of pDNA/calcium-based nanoparticles

This study explored pDNA/Ca-based nanoparticle’s long-term storage stability for gene delivery into mammalian cells. The GFP protein expression was employed using fluorescence microscopy and flow cytometry to indicate transfection efficiency. We characterized the Ca-P nanoparticles by examining the level of GFP protein expression and analyzing zeta potential, particle size, and shape by TEM. The study evaluated the effectiveness of GFP pDNA/Ca-P nanoparticles after being stored at various temperature ranges for seven days, defined as short-term storage, including − 80 °C, -20 °C, 4 °C, 29 °C, 37 °C, 50 °C, and 65 °C. The nanoparticles were stored at the designated temperatures before transfecting into HEK293FT cells. The cells were transfected, and GFP protein expression was assessed 48 h after transfection using fluorescence microscopy and flow cytometry analysis. Our findings exhibited that the storage temperatures significantly impacted the transfection efficiency of pDNA/Ca-based nanoparticles. We found two storage temperatures showing transfection efficiencies higher than 80%, including 4 °C and 29 °C (Fig. 1b-c). Expectedly, the highest transfection capacity by flow cytometry analysis was observed at 4 °C and 29 °C, with no significant difference between the two temperatures after 7 days of storage (Fig. 1c). In contrast, the lower and higher storage temperatures of 4 °C and 29 °C exhibited significant decreases in %transfection efficiencies, including − 80 °C, -20 °C, 37 °C, 50 °C, and 65 °C, which emphasized that they were inappropriate for short-term storage of pDNA/Ca-based nanoparticles. These results indicate that the pDNA/Ca-based nanoparticles retain functionality when stored at temperatures (4 °C and 29 °C) typically used for pharmaceutical storage and transport. The 4 °C and 29 °C storage offers a practical advantage and convenience for effective delivery after temperature changes during storage or transport.

Additionally, this study measured the zeta potential of pDNA/Ca-based nanoparticle solution stored at various temperatures after 7 days. The zeta potential showed the negative surface charge of the nanoparticles. The results uncovered that at 4 °C, 29 °C, and 50 °C, the zeta potential values were measured as -13.9 mV, -14.1 mV, and − 13.5 mV, respectively. The lower zeta potential values were observed at -80 °C, -20 °C, 37 °C, and 65 °C, including − 10.8 mV, -11.63 mV, -11.4 mV, and − 3.95 mV, respectively. The study explained that the lower and the higher temperatures of 4–29 °C had a noticeable impact on the zeta potential of the pDNA/Ca-based nanoparticle formula (Fig. 1d).

Besides, transmission electron microscopy (TEM) was utilized to evaluate the size and shape of the GFP pDNA/Ca-based nanoparticles. The TEM images show the almost spherical shape of the GFP pDNA/Ca-based nanoparticles after seven days of storage at different temperatures (Supplementary Figure S1). The images also provide data on the varied size of the pDNA/Ca-based nanoparticles. At -80 °C and − 20 °C storage conditions, the particle size ranged from 91 to 390 nm and 50 to 370 nm, respectively. At 4 °C, 29 °C, and 37 °C storage conditions, the particle sizes ranged from 20 to 170 nm, 20 to 100 nm, and 75 to 430 nm, respectively. The highest variation of the GFP pDNA/Ca-based nanoparticle size was found at 50 °C and 65 °C storage conditions, varied from 76 to 260 nm and 78 to 643 nm, respectively (Fig. 2). To further evaluate the encapsulation efficiency (EE) of the pDNA into Ca-based nanocomplexes, we performed gel electrophoresis of the encapsulated-pDNA nanocomplexes compared to the same amount of plasmid DNA (100 ng) without encapsulation. After 24 h of storage at the desired temperature, we analyzed the percentage of EE (%EE) using gel electrophoresis analysis and visualization (see Materials and Methods and Supplementary Figures S2 and S3). We calculated the %EE of the Ca-based nanoparticle solution, and the finding exhibited the encapsulation efficiency ranged from 44% to 61.5% at 24 h of the storage. The two highest EE were observed at -80 °C and 29 °C storage, showing 61.5% and 58.6%, respectively. Moderate range of EE was detected at 4 °C, 37 °C, and 50 °C, including 53.3%, 56.2%, and 48.6%, respectively, and the lowest EE was 44.0% at -20 °C storage. However, no estimated band was detected when the pDNA was stored at 65 °C after 24 h. The data is shown in Supplementary Table 1. Therefore, the finding unveiled that the encapsulation efficiency using the gel electrophoresis analysis did not confer the correlated outcomes compared to transfection efficiency by the GFP expression in Fig. 1b-c. The results demonstrated the most appropriate storage temperatures, including 4 °C and 29 °C, by showing the highest transfection efficiency but not related to their encapsulation efficiencies.

Fig. 2.

The particle size alteration of the GFP pDNA/Ca-P nanoparticles after seven-day storage at different temperatures. After GFP pDNA was encapsulated into the Ca-based nanoparticle solution in the same formulation batch, the GFP pDNA/Ca-P nanoparticle solution was equally separated and stored for 7 days at -80 °C, -20 °C, 4 °C, 29 °C, 37 °C, 50 °C, and 65 °C before nanoparticle size analysis using TEM.

It can be concluded that the most suitable temperature for short-term storage was 29 °C, and the storage temperature had a substantial effect on the mammalian cell transfection efficiency, surface charge, and size of pDNA/calcium-based nanoparticles.

Calcium-based nanoparticles-mediated gene delivery capability after storage in varied time and temperature

Following an initial investigation to identify proper storage temperatures for long-term storage, we focused on two storage temperatures that showed the highest transfection efficiencies after short-term storage; 4 °C and 29 °C, based on the %GFP expression by flow cytometry analysis. We further evaluated the effects of long-term storage (1 month, 4 months, and 5 months) at 4 °C and 29 °C on calcium phosphate nanoparticles (Ca-P nanoparticles) encapsulated with GFP plasmid DNA (pDNA/Ca-P nanoparticles), compared to short-term storage at 1 day and 7 days. This comprehensive analysis encompassed several key nanoparticle characteristics, including gene expression, particle shape and size, zeta potential, and pH levels. Subsequently, the HEK293T cells were transfected with pDNA/Ca-P NPs stored at the temperatures above and at different time points to assess the impact of storage temperature and duration on protein expression efficiency.

After a 48-hour incubation post-delivery, the percentage of GFP-positive cells was quantified using fluorescence microscopy and flow cytometry analysis. We found that at 4 °C showed the highest levels of GFP expression after 1 day and 7 days of storage (short-term storage) and significantly decreased at 30 days (73.77%), 120 days (67.26%), and 150 days of storage when compared to day 1 (Fig. 3, left panel and Fig. 4, golden bars). No statistically significant differences existed in the percentage of cells exhibiting GFP protein expression between temperatures of 4 °C and 29 °C after 1 day of synthesis. Both conditions yielded comparable results with 84.28% and 84.81% GFP-positive cells, respectively. The maintenance of transfection efficacy for seven consecutive days suggests that the gene delivery system was successful, and the transfection efficiency remained comparable at the initial time with a percentage of more than 80% (Fig. 4). As expected, the GFP expression after 1 day and 7 days of storage (short-term storage) at 29 °C displayed the maximum levels and no significant difference compared to storage at 4 °C (Fig. 4). Surprisingly, we observed the constant GFP expression levels at 30 days (81.47%) and 120 (84.59%) days of storage at 29 °C with no significant difference when compared with day 1 after encapsulation (Fig. 3, right panel and Fig. 4, green bars). Extended the solution held for 5 months was transfected into the HEK293FT cells, and we found that GFP protein was still expressed in the cell culture. However, the expression levels significantly dropped compared to one day of pDNA/Ca-P encapsulation. The calculation of the percentage of GFP-positive cells at 4 °C was 65.57%, while at 29 °C, it was 73.33% (Fig. 4). The results have demonstrated that the variance in temperature has a measurable impact on the gene expression levels. Specifically, at a temperature of 4 °C, we have observed a gradual but significant decrease in GFP gene expression levels after 30, 120, and 150 days (long-term) of storage, compared to the one-day (short-term) storage solution. In conclusion, the optimal temperature for long-term storage of pDNA/Ca-P nanoparticle solution is 29 °C. The stability of Ca-P nanoparticles at 29 °C storage prolongs for 5 months with the GFP gene expression efficiency of more than 80%.

Fig. 3.

GFP fluorescence imaging indicated that GFP plasmid DNA-encapsulated calcium phosphate nanoparticles successfully transfected HEK293FT cells. The samples were stored at different temperatures and times before being imaged 48 h after delivery. The left panel shows the GFP protein expression of sample storage at 4 °C, and the solution storage at 29 °C is displayed on the right panel.

Fig. 4.

The detection of GFP expression using flow cytometry after the nanoparticle solutions were stored at 4 °C and 29 °C over time. The data is the representative dataset from three independent experiments presented as the mean ± SEM. Statistical analysis was performed using one-way ANOVA. Statistical analysis was performed where p < 0.01, p < 0.0001 is represented as **, **** respectively.

The findings emphasize the significance of proper temperature control for preserving genetic material during long-term storage of biological samples. Further research is needed to investigate the mechanisms of gene expression changes under various storage conditions.

Influence of storage time and temperature on the zeta potential of calcium phosphate nanoparticles

The zeta potential of the calcium phosphate nanoparticles was measured to assess their stability and surface electrical charge under different storage conditions. The initial measurement of the freshly prepared nanoparticle solution showed an average zeta potential of -12.97 mV, indicating a negative surface charge. Subsequent measurements were taken on samples stored at 4 °C and 29 °C for various durations: 24 h, 7 days, 1 month, 4 months, and 5 months. The zeta potential remained consistently negative in all storage conditions, indicating a well-maintained electrostatic value between the nanoparticles. After 1 day of storage, the average zeta potential values were − 13.93 mV at 4 °C and − 15.4 mV at 29 °C. This minimal variation suggests a negligible influence of temperature during this short period. On day 7, the evaluations showed sustained negative zeta potential values at both temperatures. The nanoparticles showed a zeta potential of -13.81 mV and − 13.61 mV at 4 °C and 29 °C, respectively. There was no significant difference between the two temperature groups. This trend continued over a month, with the stored nanoparticles maintaining a negative charge of -14.98 mV and − 14.56 mV at 4 °C and 29 °C, respectively. Notably, the zeta potential remained consistent even after extended storage periods.

After four months of storage, the values were − 14.5 mV at 4 °C and − 13.12 mV at 29 °C. After five months, the measurements were − 14.17 mV at 4 °C and − 15.16 mV at 29 °C. The study revealed no significant difference in the zeta potential between the groups stored at 4 °C and 29 °C for each storage time. However, it is worth noting that only the samples stored at 4 °C for four months showed higher values than at 0 h post-co-precipitation (Fig. 5).

Fig. 5.

The zeta potential of plasmid DNA encapsulated Ca-P Nanoparticles from different storage times and storage temperatures. The zeta value of pDNA/Ca-P Nanoparticles at 4 °C and 29 °C storage temperatures and times compared with a freshly prepared solution. The data is presented as mean ± SEM. Significance levels are denoted as *p < 0.05 and ***p < 0.001 based on a one-way ANOVA.

Characterization of particle size and dispersion of GFP pDNA/Ca-P nanoparticles after storage in varied temperatures and times

This study investigated the characteristics of GFP pDNA/Ca-P nanoparticles after storage at 4 °C and 29 °C. Transmission electron microscopy (TEM) was used to analyze the particle size and shape. Changes during storage were monitored at different time points. The TEM images showed that the shape of pDNA/Ca-P nanoparticles remained approximately spherical under all storage conditions (Fig. 6).

Fig. 6.

The morphology of calcium phosphate nanoparticles post-encapsulation at different storage temperatures and times. The upper panel represents the storage temperatures at 4 °C, and the lower panel represents the storage temperatures at 29 °C. The images from left to right illustrate the storage time of pDNA-Ca-based nanoparticles at days 1, 7, 30, 120 and 150, respectively. The scale bar for 1 day of storage is 200 nm, whereas the scale bar for 7-150 days of storage is 500 nm. Morphological detection was performed using transmission electron microscopy (TEM) (JEOL JEM-1400 TEM, JEOL, Tokyo, Japan) at an accelerating voltage of 80 kV.

The initial assessment was conducted 24 h after the formulation to establish a baseline. The TEM images of Ca-P nanoparticles stored at 4 °C showed a size distribution ranging from 27 to 110 nm. Similarly, the images at 29 °C revealed an average diameter of 20 to 100 nm. After being stored for seven days in a temperature-controlled environment, the particle size at a storage temperature of 4 °C exhibited a size distribution around 20 to 170 nm. Meanwhile, the nanoparticles stored at 29 °C showed that the particles had a diameter ranging from 30 to 130 nm. As our research progressed, we continuously observed the Ca-P nanoparticles at one month of storage. At 4 °C, the particle size distribution ranged from 40 to 190 nm. Similarly, at 29 °C, the nanoparticle solution exhibited a size range of 20 to 170 nm. Following storage for 4 months, the TEM images of the particles were examined to monitor the Ca-P nanoparticles at a temperature of 4 °C. The diameter of the nanoparticles showed a size distribution ranging from 40 to 200 nm. To characterize the nanoparticles at 29 °C, a TEM image revealed an average size ranging from 20 to 120 nm. Finally, we completed our comprehensive study by monitoring the characterization of nanoparticles at two different temperatures over 5 months. At 4 °C, the nanoparticles explored an average diameter ranging from 30 to 200 nm. At 29 °C, the nanoparticles also revealed an average diameter ranging from 20 to 200 nm (Fig. 7).

Fig. 7.

The graph illustrates the particle size at different temperatures and times ranging from 0 to 200 nm.

pH stability evaluation

Assessing pH stability in plasmid DNA-coated Ca-P NPs is a critical aspect that warrants thorough evaluation for gene therapy because Ca-P nanoparticles can preserve stability at pH 7.4 and dissolve at low pH. The pH values were analyzed under different storage conditions to investigate whether pH values affect Ca-P NPs. Cell transfection solutions were partitioned into 3 mL portions and stored to determine the effect of long-term storage on pH. The pH values were recorded under various conditions and presented in Table 1. The results indicate that the measured pH values exhibit a relatively consistent pattern; no statistically significant difference was observed. These findings suggest that plasmid DNA-coated Ca-P nanoparticles demonstrate pH stability under the evaluated storage conditions.

Table 1.

The pH of Ca-P nanoparticles at the different storage temperatures and storage times. The experiment was performed in triplicate, and an unpaired sample t-test was used for statistical analysis.

Storage times	pH of Ca-P nanoparticles
	4 °C	29 °C
Fresh preparation	N/A	7.40 ± 0.01
1 day	7.39 ± 0.01	7.38 ± 0.01
7 days	7.41 ± 0.01	7.39 ± 0.01
30 days	7.37 ± 0.03	7.41 ± 0.01
120 days	7.40 ± 0.00	7.40 ± 0.01
150 days	7.39 ± 0.01	7.40 ± 0.01

Discussion

This study investigated the storage temperature of Ca-P nanoparticles for plasmid DNA delivery. In previous research, we successfully formulated Ca-P nanoparticles to treat and prevent diseases in rats^31,32.Therefore, this study aimed to determine the most appropriate storage temperature and time necessary to maintain optimal transfection efficiency. Transfection efficiency was measured at -80 °C, -20 °C, 4 °C, 29 °C, 37 °C, 50 °C, and 65 °C. Our findings revealed that the proper storage temperature is 29 °C, allowing four months of storage time to confer the stability of transfection efficiency of the pDNA/Ca-P NPs at higher than 80%.

Comparative analysis of Ca-P nanoparticles with lipid-based nanoparticles and polymer-based vectors showed the advantages and limitations for drug delivery in terms of biomedical applications. Ca-P nanoparticles, including our formulation, are outstanding for the lowest cost because of the use of natural materials and simple synthesis methods¹⁴. On the other hand, the lipid-based vectors revealed that high costs form complex synthesis and specific materials^6,7. Therefore, the cost-efficiency of the Ca-P platform makes it an attractive option for large-scale production¹⁴, especially in resource-limited settings. Furthermore, our Ca-P formulation demonstrates excellent storage stability for over four months, outperforming general Ca-P formulations (1 week-stability)²⁹, lipid-based vectors requiring cold chain storage and specialized suppliers¹⁴, and variable stability in polymer-based vectors⁴. Because of the excellent biocompatibility, derived from natural-based calcium and phosphate ions, presents a safer profile^33,34, compared to potential inflammation from lipid-based vectors^6,7,35 or toxicity concern with cationic polymer^4,5. Ca-P nanoparticles induced an increase in intracellular calcium³⁶. However, no adverse effects were reported in preclinical studies involving rats and monkeys with our formulation^31,32,37 In terms of manufacturing, the simple synthesis of Ca-P nanoparticles facilitates easy scale-up and potential field synthesis without complex equipment³⁸,making them applicable for production in resource-limited areas. Ca-P nanoparticle application is an interesting advantage over lipid-based vectors, which require complicated production processes^7,38 and use advanced protocols. At the same time, polymer-based vectors also have complex manufacturing steps and strict purity requirements, which can limit their scalability and increase production costs^4,5. The advantages of Ca-P nanoparticles position them as an upcoming and accessible delivery platform, particularly when affordability, ease of manufacturing, and safety are priorities (Supplementary Table 2).

The study measured particle size and shape using TEM, zeta potential, and pH to understand their impact on transfection efficiency. The results showed that particle size strongly correlated with their transfection efficiencies. Our data identified nanoparticle size as the most critical factor impacting transfection efficiency. TEM images revealed that the most effective nanoparticles between 20 and 200 nm were stored at either 4–29 °C. This size range aligns with the previous findings demonstrating that calcium phosphate nanoparticles within the 50–200 nm range showed optimal cellular uptake and endosomal escape^39,40. Interestingly, our observation that nanoparticles stored at 29 °C maintained smaller size distributions than those at extreme temperatures also reported in the previous study because this temperature preserved the amorphous nature of calcium phosphate nanoparticles by prevention of crystallization and growth of nanoparticles⁴⁰. The stability at 29 °C exhibited the contrast of conventional pharmaceutical storage, which commonly needs cold chain maintenance. This finding has markedly advantage for gene therapy applications in resource-limited areas where cold chain maintenance is challenging. The increased particle size observed at storage temperatures below 0 °C (-20 °C and − 80 °C) can be attributed to freeze-thaw cycles causing ice crystal formation and subsequent nanoparticle aggregation, as previously established^41,42.

One intriguing finding from our study is the remarkable stability of pDNA/Ca-P nanoparticles at room temperature (~ 29 °C), with maintained transfection efficiency compared to traditional 4 °C storage. One possible reason behind this finding is that the calcium phosphate system at 29 °C reaches a more favorable thermodynamic equilibrium between dissolution and reprecipitation processes, maintaining nanoparticle integrity while preventing significant growth or aggregation. A previous study has demonstrated that calcium phosphate crystallization kinetics have temperature-dependent optima that can vary based on composition⁴². In addition, 29 °C promotes stronger electrostatic interactions between the negatively charged phosphate groups of pDNA and the calcium ions in the Ca-P nanoparticle matrix. This enhancement could create a more stable protective layer around the nanoparticles, preventing aggregation and maintaining the Ca-P nanoparticle size and transfection competence⁴³.

Calcium phosphate exists in various crystalline phases with different solubilities and stabilities. At lower temperatures (4 °C), slow phase transformation to more crystalline forms might occur over time, potentially affecting DNA binding and release. At 29 °C, the system may maintain a metastable phase optimal for transfection⁴⁴. In addition, the hydration layer surrounding nanoparticles plays a crucial role in their stability. At 29 °C, it may provide optimal dynamics for the hydration shell, preventing excessive hydration (which might occur at lower temperatures) and dehydration (at higher temperatures), thus maintaining an ideal interface for cellular interactions and DNA protection⁴⁰. Residual enzymatic activity in the preparation, including potential DNA degradation, may also be optimally balanced at 29 °C, which is not high enough to degrade the plasmid DNA or disrupt the nanoparticle structure⁴¹.

It was observed in this study that pH measurement remained consistent regardless of temperature and time. The ionic strength and pH of the storage and administration buffer significantly affect nanoparticle stability through modulation of the electrical double layer and particle-particle interactions⁴². Higher ionic strength environments can compress the double layer around charged nanoparticles, potentially reducing electrostatic stabilization and leading to aggregation. In our in vivo studies, we address this ionic strength challenge by using a normal saline solution (0.9% NaCl, ~ 154 mM ionic strength) for intravenous injection in a long-term safety study in non-human primates. (Manuscript No. 7733-J entitled “Long-term safety pharmacology and musculoskeletal changes of HMGB1 Box A gene therapy in middle-aged monkeys”, accepted 22 Apr 2025, In Vivo) In this previous study, the pDNA/Ca-P NPs were pre-mixed with sterile normal saline immediately prior to administration to ensure isotonic conditions were compatible with intravenous delivery into the monkeys. This approach allows us to maintain the storage stability benefits of our optimized buffer system while ensuring physiological compatibility as suggested in the previous studies⁴⁵. Although calcium phosphate (Ca-P) nanoparticles are widely recognized for their biocompatibility and potential in gene delivery, theoretical concerns have been raised regarding their ability to elevate intracellular calcium levels, which in excess could trigger undesirable physiological responses^46,47. However, in our recent animal studies, involving both rodents and monkeys, we reported that no adverse effects or abnormal physiological changes occurred throughout the study period^31,32,37. These data alleviate safety concerns raised in theory and show the evidence to support the continuous development of Ca-P nanoparticles for future medical applications.

Zeta potential, a measure of surface charge, was also investigated as a factor influencing transfection efficiency. Meanwhile, zeta potential provides additional information on the nanoparticle stability and surface properties that can affect transfection outcomes. This study revealed that the zeta potential of GFP pDNA/Ca-P nanoparticles remained consistently negative across all storage temperatures (4 °C to 65 °C). Although the zeta potential varied from positive to negative depending on their compositions, it was reported in other studies⁴⁸. More negative zeta potential at 4 °C might be due to compositional changes that could influence transfection by affecting nanoparticle-cell interactions. The relative stability of zeta potential at 4 °C and 29 °C (-13.9 mV and − 14.1 mV, respectively) over extended storage periods indicates preservation of the electrical double layer, critical for preventing aggregation through electrostatic repulsion. At 65 °C, a decrease in negative zeta potential suggested nanoparticle aggregation, which could hinder cellular uptake. These findings highlight the pivotal interplay between zeta potential, nanoparticle properties, and transfection efficiency after long-term storage at 4 °C; however, no effect was observed when long-term stored at 29 °C, regarding to transfection efficiency. In addition, the solution’s pH remained constant throughout the experiment, indicating that pH did not directly impact transfection efficiency in our study. pH stability is crucial for maintaining calcium phosphate solubility, as dramatic pH shifts could trigger either excessive dissolution (at lower pH) or uncontrolled precipitation (at higher pH)⁴⁹. The previous observation reported that calcium phosphate phases exhibit pH-dependent stability, and our results of consistent pH values across storage conditions suggests effective buffering within our formulation. However, it is necessary to be concerned with isotonic and electrostatic conditions and the physiological compatibility of the Ca-P nanoparticles before preclinical and clinical applications.

To improve the stability of pDNA/Ca-P nanoparticles during long-term storage in further study. We suggest using lyophilization (freeze-drying) as an effective approach to prevent nanoparticle aggregation⁵⁰. The technique involves removing water from the nanoparticle solution at low temperatures and reduced pressure, resulting in a dry formulation that significantly minimizes the risk of aggregation and degradation over time⁵¹. Additionally, lyophilization confers transportation and storage easier under ambient conditions⁵². This aspect is crucial for use in resource-limited areas. However, the viability of lyophilization as a pDNA/Ca-P system will be investigated in our future research, together with the investigation of formulation parameters and the assessment of the effectiveness of gene delivery upon reconstitution.

Our findings indicated that pDNA/Ca-P nanoparticles could maintain their physicochemical properties and showed enhanced gene expression when stored at both 4 °C and 29 °C. While 4 °C is a typical storage temperature for biopharmaceuticals and vaccines, our novel finding showed that the solution stored at 29 °C showed prolonged stability. However, maintaining the precise temperatures presents a significant challenge in some resource-limited areas due to fluctuations in ambient temperatures. This variability can compromise product efficacy. According to the WHO Cold Chain Guidelines, a cold chain is essential for preserving vaccine potency, requiring controlled low-temperature storage and transport throughout the supply chain, from central stores to outreach sites. Building on our current findings, our subsequent study will focus on evaluating the stability of pDNA/Ca-P nanoparticles at controlled room temperature. If we find comparable stability, it will significantly simplify storage and transport logistics in challenging environments, reducing the reliance on complex electrical systems. These criteria align with the WHO’s practical supports for cold boxes and vaccine carriers, many of which offer 2 to 8 days of cold storage environment without electrical power⁵³. Achieving room temperature stability of pDNA vaccines is able to be a major step towards broader accessibility of this technology in underserved areas.

Our study suggests that optimal condition of the Ca-P nanoparticles stores within the temperature range of 4 °C to 29 °C. Our findings indicate that both temperatures can maintain conformational stability without compromising the transfection efficiency of the nanoparticles. Our study suggests the optimal temperature for storing the Ca-P nanoparticles might be ranged from 4 °C to 29 °C for short-term storage. Both temperatures can maintain conformational change without loss of transfection efficiency. For long-term storage (5 months), our study suggests that 29 °C is the most appropriate temperature for the pDNA/Ca-P nanoparticle storage by showing the highest gene transfer efficiency.

Conclusion

This study has investigated the impact of storage temperature on the stability and transfection efficacy of Ca-P nanoparticles for gene delivery. Meanwhile, the Ca-P nanoparticle formulation displayed promising stability for up to five months between 4° and 29 °C. Interestingly, 29 °C is closer to the synthesis environment than the others. The results emphasize the relationship between storage conditions and nanoparticle properties, highlighting the need for thorough characterization during storage. Our results align with the research suggesting that the physicochemical properties of nanoparticles, including size, shape, and surface charge, can be influenced by storage conditions. This underscores the importance of considering long-term storage effects when developing Ca-P nanoparticle formulations for gene therapy and drug delivery applications. Future studies should explore the impact of extended storage durations and broader temperature ranges to establish storage protocols that maintain optimal nanoparticle functionality. Our findings can be used as a prototype of Ca-based NP formulation with an optimal storage protocol to support and enhance the study and the accessibility of gene therapy and nanomedicine in many areas with limited resources worldwide.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

SY and AM conceived the study and designed the analysis; SY designed the methodology and investigation and wrote the original draft of the article; PC performed the experiments, analyzed the data and visualization; SY, PC and SP wrote the original draft of the article; SY and SP performed the additional experiment, visualized, and analyzed the data of the encapsulation efficiency. SY supplied grant support; SY, AM, and VB reviewed and edited the article; SY, AM, and VB were involved in supervision; and all authors read and approved the final manuscript.

Data availability

The datasets used and/or analyzed during the current study are in the supplementary document and available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.