# Influence of Biological Medium pH on the Stability and Gene Delivery Efficiency of Chitosan/peGFP-C3 Polyelectrolyte Complexes

Jesrael Luz Elena Nando Rodríguez, Lizeth Montserrat Bravo Lozano, Edgar

Benjamin Figueroa Ochoa, Maite Renteria Urquiza, Eduardo Mendizabal

Mijares, Julien Rosselgong, Sylvain Tranchimand, Christophe Schatz, Pascal

Loyer, Lourdes Mónica Bravo-anaya

► To cite this version:

Jesrael Luz Elena Nando Rodríguez, Lizeth Montserrat Bravo Lozano, Edgar Benjamin Figueroa Ochoa, Maite Renteria Urquiza, Eduardo Mendizabal Mijares, et al.. Influence of Biological Medium pH on the Stability and Gene Delivery Efficiency of Chitosan/peGFP-C3 Polyelectrolyte Complexes. TOP 12th international conference, Jun 2025, ISCHIA, Italy. 10.1002/masy.70237. hal-05357589

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Submitted on 10 Nov 2025

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RESEARCH ARTICLE

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# Influence of Biological Medium pH on the Stability and Gene Delivery Efficiency of Chitosan/peGFP-C3 Polyelectrolyte Complexes

Jesrael Luz Elena Nando Rodríguez<fnref n="1" />,² | Lizeth Montserrat Bravo Lozano<fnref n="1" />,² | Edgar Benjamin Figueroa Ochoa<fnref n="1" /> | Maite Renteria Urquiza<fnref n="1" /> | Eduardo Mendizabal Mijares<fnref n="1" /> | Julien Rosselgong² | Sylvain Tranchimand³ | Christophe Schatz⁴ | Pascal Loyer⁵ | Lourdes Mónica Bravo-Anaya² ID

**Correspondence:** Lourdes Mónica Bravo-Anaya (lourdes-monica.anaya@univ-rennes.fr)

**Received:** 3 April 2025 | **Revised:** 4 April 2025 | **Accepted:** 24 October 2025

**Guest Editors:** Alberto D'Amore and Luigi Grassia

**Keywords:** chitosan/peGFP-C3 complexes | pH dependence | polyelectrolytes | stability | transfection efficiency

## ABSTRACT

Natural and synthetic polycations are commonly used in DNA- and RNA-based transfection for their ability to condense and protect nucleic acids, and the subsequent cellular uptake. While the formation of these complexes is well-documented, fewer studies have examined their stability under both extracellular and intracellular conditions. Chitosan, a biodegradable natural polysaccharide, has shown promise for gene delivery. However, its pKa of ~6.5 makes its ionization state highly sensitive to pH fluctuations around this value. In this study, we examined how pH-induced changes influence the stability of chitosan/peGFP-C3 complexes and their effects on cell viability, proliferation, and transfection efficiency in HEK293T cells. The highest transfection efficiency was achieved by transfecting the complexes at pH 6.5, followed by a medium replacement at pH 7.1, particularly with high molecular weight chitosans. Cell viability was found to be higher at pH 7.1, whereas acidic conditions (pH 6.5) affected proliferation, leading to S-phase accumulation, suggesting potential interference with cell cycle progression. These findings underscore the critical role of pH in optimizing gene delivery and maintaining favorable cell growth conditions.

## 1 | Introduction

Natural or synthetic polycations are widely used in nucleic acid-based therapies to form nanoparticles (complexes) that protect genetic material and regulate its cellular uptake. Chitosan, a biodegradable polysaccharide, shows strong potential for gene delivery due to its tunable physicochemical properties, particularly its pH sensitivity [1]. At pH lower than 6.5, chitosan carries a stronger positive charge, leading to interactions with nucleic acids and cell membranes [2–4]. However, its pKa is

below physiological pH, leading to precipitation under physiological conditions and ultimately resulting in lower transfection efficiencies compared to polyethylenimine (PEI). To address this limitation, we explored a pH modulation strategy aimed at improving the stability and efficiency of chitosan-based vectors. Since pH directly influences complex solubility, aggregation, and overall biological functionality, we investigated the stability of chitosan/peGFP-C3 complexes in different biological buffers (MES and HEPES), examining the effects of chitosan's molecular weight (MW) and charge ratio ($R = [N^+]/[P^-]$, where $[N^+]$

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly
cited.

© 2025 The Author(s). *Macromolecular Symposia* published by Wiley-VCH GmbH

<footnote n="1">¹Universidad De Guadalajara, Blvd. M. García Barragán #1451, Guadalajara, Jalisco 44430, México | ²Université de Rennes, ISCR, CNRS, UMR 6226, Campus de Beaulieu, Bat 10A, Rennes Cedex 35042, France | ³Université de Rennes, École Nationale Supérieure de Chimie de Rennes, CNRS, ISCR, UMR 6226, Université de Rennes, Rennes F-35000, France | ⁴Université de Bordeaux, CNRS, Bordeaux INP, LCPO, UMR 5629, Pessac F-33600, France | ⁵Université de Rennes, INSERM, INRAE, Institut NUMECAN, UMR-A 1341, UMR-S 1317, Plateforme SynNanoVect, Rennes F-35000, France</footnote>
<figure><img src="image_3.png" /><figcaption>FIGURE 1 | Intensity-averaged size distribution obtained by DLS for chitosan/peGFP-C3 complexes (R = 5) prepared in water with MMW chitosan (86.75 kDa, DA = 0.16), in the presence of a) MES and b) HEPES buffers, supplemented with culture media with and without serum (CM+, CM-, respectively).</figcaption></figure>

corresponds to the molar concentration of positively charged amino groups and [P⁻] to the negatively charged phosphate groups in DNA). While acidifying the culture medium helps maintain chitosan solubility and complex stability, it is not compatible with proper cellular function. To overcome this limitation, we proposed a two-step approach: first, facilitating the internalization of chitosan/DNA complexes under mildly acidic conditions to preserve their stability, followed by a controlled increase in intracellular pH to promote DNA release and enhance transfection efficiency. To evaluate this strategy, we assessed cell viability by measuring ATP production in HEK293T cells incubated with chitosan/peGFP-C3 complexes and conducted proliferation studies to examine their impact on cell growth under different pH conditions. Finally, transfection efficiency was determined by assessing the ability of the complexes to deliver plasmid DNA and promote its expression in HEK293T cells.

## 2 | Results and Discussion

### 2.1 | Chitosan/peGFP-C3 Stability in Presence of Biological Media

Figure 1a,b shows the intensity-averaged size distribution of chitosan/peGFP-C3 complexes (R = 5), measured after preparation and exposure to MES (pH 6.5) and HEPES (pH 7.1) buffers, respectively, with culture media with and without serum (CM+, CM-). The complexes showed a slight size increase in both biological media, with a slightly greater effect in DMEM-HEPES, likely due to its higher pH. This size increase is attributed to the presence of salts, which reduce electrostatic attraction between the oppositely charged polyelectrolytes. The addition of serum led to the formation of multiple size populations, corresponding to free proteins for the smallest ones and to aggregates of polyplexes and proteins for the largest ones.

### 2.2 | Chitosan/peGFP-C3 Transfection Efficiency and ATP Determination in HEK293T Cells

Transfection efficiency was found to be highly influenced by the pH of the culture medium (Figure 2).

The highest efficiencies were observed under conditions combining DMEM-MES (pH 6.5), followed by DMEM-HEPES (pH 7.1), particularly when using high MW chitosans (86.75 and 417.6 kDa) at R = 10. ATP quantification indicates that the culture medium affects cell viability and/or proliferation index (Figure 3a). Cells grown in DMEM-HEPES (pH 7.1) exhibited higher ATP levels, suggesting greater viability or lower impact of cell proliferation, whereas those in DMEM-MES (pH 6.5) had significantly lower ATP levels, indicating reduced viability or major impact on cell proliferation. Cell proliferation varied with chitosan type, with 45 kDa chitosan supporting higher cell counts, while the R had no significant effect on ATP levels.

Finally, cellular DNA content analysis suggested that pH influences the cell cycle. In DMEM-HEPES (pH 7.1), cells exhibited a normal phase distribution, whereas in DMEM-MES (pH 6.5), an accumulation in the synthesis phase was observed, suggesting that acidic pH may interfere with cell cycle progression (Figure 3b). Our results highlight pH's crucial role in stability, cell viability, DNA content, and transfection efficiency, emphasizing the need for optimization and further study on cell proliferation.

## 3 | Conclusions

This study examines the influence of biological media pH on the stability, transfection efficiency, and cellular effects of peGFP-C3/chitosan complexes. Complex size slightly increased in MES and HEPES buffers, while serum induced size heterogeneity. Transfection was most efficient in DMEM-MES (pH 6.5), followed by DMEM-HEPES (pH 7.1), particularly with high MW chitosans. Cell viability was higher at pH 7.1, whereas acidic conditions (pH 6.5) disrupted the cell cycle, leading to S-phase accumulation. These findings underscore the critical role of pH in optimizing chitosan-based gene delivery.

## 4 | Experimental Section

### 4.1 | Materials and Solutions Preparation

The peGFP-C3 plasmid (MW = 3,100,912 g/mol), encoding GFP, was amplified in *E. coli* and purified using the NucleoBond PC 1000 kit. Five chitosan samples were used: MMW (86.75 kDa, DA = 0.16), HMW (417.60 kDa, DA = 0.22), and samples #1 (540 kDa, DA = 0.20), #6 (160 kDa, DA = 0.05), and #45 (45 kDa, DA = 0.23). MMW and HMW were from Sigma-Aldrich, and #1 and #6 from Primex. Chitosan solutions (3.33 mg/mL) were prepared in 0.1 N HCl, stirred for 16 h, and refrigerated. Chitosan/peGFP-C3 complexes were formed by mixing chitosan (1 mg/mL) with peGFP-C3 (0.034 mg/mL) at charge ratios of 3, 5, and 10 using the “one shot and vortex” method [5]. HEK-293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM 1X, 11960044, Gibco) [6]. Two buffer solutions were used: MES (pH 6.5) and HEPES (pH 7.1), adjusted with NaOH.

### 4.2 | Chitosan/peGFP-C3 Complexes Characterization and Stability in Biological Media

The size and overall charge of complexes were analyzed by Dynamic Light Scattering (DLS) and ζ-potential measurements at
<figure><img src="image_4.png" /><figcaption>**FIGURE 2** | Transfection efficiency of HEK293T cells was assessed using chitosan (45 kDa, MMW-86.75 kDa, HMW-417.60 kDa, #1-540 kDa, #6-160 kDa) in complexes with peGFP-C3 at charge ratios (R) of 3, 5, and 10, along with Lipofectamine 3000/peGFP-C3 lipoplexes. Transfections were performed in DMEM-MES and DMEM-HEPES buffers, with a third condition where DMEM-MES medium was replaced with DMEM-HEPES at 24 h. All transfections were conducted in triplicate, and efficiencies were analyzed at 48 h post-transfection by flow cytometry.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**FIGURE 3** | a) Amount of ATP produced by HEK293T cells after transfection of peGFP-C3/chitosan complexes under different experimental conditions (buffer variation, pH, chitosan MW, charge ratio) (Same as in Figure 2). b) Cell proliferation study for: Chit #6 and lipofectamine, both in HEPES and in MES buffers.</figcaption></figure>

25°C using a Zetasizer Nano ZS (Malvern) working at a detection angle of 173°. Stability was assessed by DLS in different media: formulations (100 µL) were mixed with 900 µL of 1X buffer, with size and PDI measured immediately and after 30 min. Then, measurements were carried on in the presence of 500 µL DMEM and 100 µL serum.

## 4.3 | HEK293T Cell Transfection

In vitro studies were conducted using HEK293T cells, cultured in DMEM with 10% FBS, L-Glutamine, and antibiotics. Cells were seeded at $2 \times 10^4$ cells/cm² and transfected using two media: HEPES-buffered DMEM (pH 7.1) and a homemade MES-buffered DMEM (pH 6.5). On the day of transfection, the medium was replaced, and 100 µL of each formulation was added. After 2 h, the medium was changed to remove excess chitosan polyplexes. A third condition involved initial transfection in MES-buffered medium, followed by replacement with HEPES-buffered DMEM after 24 h. Transfection efficiency was assessed 48 h post-transfection. Cells were washed with PBS, observed by fluorescence microscopy, then detached and analyzed by flow

cytometry. Viable cells were gated, GFP fluorescence was measured, and transfection efficiency was assessed using FlowLogic software.

## 4.4 | ATP and Cell DNA Content Analysis

Cell viability was assessed using the CellTiter-Glo 2.0 assay (Promega) by measuring intracellular ATP levels at 24 and/or 48 h post-transfection. Luminescence was recorded at 540 nm with a Polarstar Omega microplate reader. DNA content was analyzed using the BD Cycle Test assay and detected via flow cytometry (BD LSRFortessa X-20). Cell cycle phases (G0/G1, S, G2/M) were determined by gating populations based on Phycoerythrin fluorescence.

### Acknowledgements

J.L.E. Nando-Rodríguez acknowledges CONAHCYT (CVU 1174879), the University of Guadalajara, and the University of Rennes for their support. Lourdes Mónica Bravo-Anaya acknowledges the French National Agency
of Research (ANR) for the grant (ANR-21-CE06-0027-01), as well as the
AES 2021 and the AIS Allocation 2023 founded by Rennes Métropole.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available on request
from the corresponding author. The data are not publicly available due to
privacy or ethical restrictions.

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