Effect of Side Chain Conjugation on the Cytotoxicity and siRNA Transfection Efficiency of 6‑Amino-6-deoxy Curdlan

Abstract

Cationic curdlan is a promising siRNA delivery platform because it protects siRNA, promotes intracellular siRNA release through charge reduction, prolongs intracellular retention, and supports sustained gene-silencing. However, its relatively high cationic charge can induce cytotoxicity and limit its therapeutic potential. In this study, to reduce toxicity while enhancing siRNA delivery efficiency, we conjugated short ethylene glycol side chains to 6-amino-6-deoxy curdlan and compared the siRNA transfection efficiency of the resulting polymers with that of the parental polymer. Conjugation of ethylene glycol side chains significantly improved both the biocompatibility and in vitro siRNA delivery efficiency, yielding polymers designated MEG1 and MEG2. Notably, conjugation of a short ethylene glycol side chain terminated with a lysine residue (designated CegK1) resulted in markedly enhanced biocompatibility and siRNA delivery efficiency in multiple human glioblastoma cell lines, including U251, U87-MG, and T98G. These findings demonstrate that short ethylene glycol side-chain modification is an effective strategy to optimize cationic curdlan as a safe and efficient siRNA delivery carrier for glioblastoma therapy.

1. Introduction

RNA interference (RNAi) is a highly conserved post-transcriptional gene silencing mechanism in eukaryotes. − The core process goes like this: the Dicer enzyme chops long double-stranded RNA into small interfering RNAs (siRNAs) that are 21–23 nucleotides long. Then, the siRNAs team up with proteins like Argonaute to form the RNA-induced silencing complex (RISC). The RISC uses the guide strand to match up with the target mRNA, and the Argonaute protein (mainly AGO2) cuts and degrades the target mRNA, precisely silencing specific genes. − Under optimized conditions, the in vitro silencing efficiency of siRNA with well-designed sequences can exceed 95%. It is highly specific and effective, making it an important tool for studying gene function and developing nucleic acid drugs. Since siRNA is a negatively charged double-stranded RNA molecule, it is easily broken down by serum nucleases and enzymes in lysosomes. Plus, it has a large molecular weight, is highly hydrophilic, does not penetrate membranes well, and has no active targeting ability, so it is hard for it to accumulate in target tissues and be taken up effectively by cells. − That is why we need to develop an efficient delivery system.

Cationic polymers are a key type of carriers for siRNA delivery and are popular because of their tunable chemical structures, plenty of functionalization sites, and high loading potential. − They neutralize the negatively charged siRNA through electrostatic interactions, forming stable nanoscale polyplexes that protect nucleic acid drugs from nuclease degradation while helping them get into target cells and efficiently deliver gene silencing. Right now, classic cationic polymers like polyethylenimine (PEI), cyclodextrin (CD) derivatives, and chitosan are widely used for in vivo siRNA delivery research. − However, these traditional cationic polymers still commonly face issues like insufficient stability in the body, lack of tissue targeting, and potential cell toxicity, which seriously limit their clinical translation. −

Natural polysaccharides, because of their excellent biocompatibility, low immunogenicity, and biodegradability, have become important backbone materials for building new cationic delivery carriers. Curdlan is a natural linear glucan connected by β-1,3-glycosidic bonds, featuring good water solubility and modifiability, and its abundant hydroxyl sites provide plenty of room for chemical functionalization. − Our research group previously used curdlan as a backbone and introduced cationic groups to get the derivative 6AC-100, preliminarily proving its feasibility as an siRNA delivery carrier. However, this carrier still has key issues like poor stability in circulation, insufficient accumulation in target tissues, and potential toxicity. So, developing novel curdlan-based carriers that are highly efficient, precisely targeted, and low in toxicity remains a major focus in the field.

On the basis of this, we introduced different functional groups onto a gel polysaccharide backbone and designed and synthesized three new cationic derivatives: MEG1, MEG2, and Cegk1. We systematically characterized their chemical structures, degree of substitution, and in vitro cell activity and screened for the most effective siRNA delivery carrier, laying the groundwork for subsequent in vivo evaluation and clinical translation. Ethylene glycol side-chain conjugation significantly enhanced both biocompatibility and in vitro siRNA delivery efficiency. MEG1 and MEG2, bearing one and two ethylene glycol units, respectively, demonstrated improved performance with MEG2 outperforming MEG1. Notably, CegK1, featuring a short ethylene glycol side chain terminated with a lysine residue, markedly improved the biocompatibility and siRNA transfection efficiency across multiple human glioblastoma cell lines, including U251, U87-MG, and T98G.

2. Materials and Methods

2.1. Synthesis of MEG1, MEG2, and Cegk1

Synthesis of 6AC-100: 0.2 g of dried Curdlan was taken in a 100 mL flask, 8 mL of dry DMF was added, and the mixture was heated with stirring in an 80 °C oil bath for 1 h to dissolve the Curdlan in DMF. Then, 640 mg of sodium azide was added and the reaction was stirred for 3 h. After the reaction was complete, the mixture was cooled to room temperature, 867 mg of triphenylphosphine was added, the mixture was stirred for 10 min, and then, 1133 mg of carbon tetrabromide was added under nitrogen protection and away from light, letting it react for 48 h. After the reaction was finished, the nitrogen balloon was removed, 60 mL of anhydrous methanol was added, and the mixture was stirred for 30 min to observe the formation of a white flocculent precipitate. Centrifugation was performed at 3000 rpm for 5 min to obtain the white precipitate; then, it was washed sequentially with anhydrous methanol twice, anhydrous ethanol three times, and distilled water three times. Finally, the precipitate was washed with anhydrous ethanol to get the intermediate product. It was dried at room temperature for 2 days, then transferred to a 50 °C vacuum drying oven for 1 day, and set aside. The fully dried sample was weighed, ground, and placed in a 100 mL flask. 8 mL of dry DMSO was added and stirred until the intermediate was completely dissolved; then, the corresponding amount of sodium borohydride was added. It was reacted at 60 °C for 8 h. After the reaction, the mixture was cooled to room temperature, 60 mL of anhydrous ethanol was added, and the mixture was stirred for 30 min to produce a white precipitate, which was washed and centrifuged with anhydrous ethanol 8 times. It was dried at room temperature for 2 days and then placed in a 50 °C drying oven for 1 day. The fully dried product was ground and dissolved in water, the pH was adjusted to 5.0 with dilute hydrochloric acid, and then, it was transferred into a dialysis bag with an 8000 MWCO for 3 days, changing the distilled water every 4 h. After dialysis, the sample was lyophilized to get the final product as a white sponge-like solid. It was characterized by NMR and stored sealed for future use.

Synthesis of MEG1: 20 mg of 6AC-100 was taken in a flask, 1 mL of DMSO was added, and the mixture was stirred at 60 °C until completely dissolved. The solution was cooled down to 40 °C; 36 μL of DIPEA and 46 mg of potassium carbonate were added and mixed well; then, 29.5 mg (21 L) of 2-methoxyethyl bromide was added; and the reaction was run under nitrogen for 24 h. 3 mL of ethyl acetate was added to precipitate and then centrifuged to remove the supernatant with ethyl acetate 3 times and ethanol 2 times (centrifugation was performed at 3000 rpm for 5 min each). After a white precipitate was obtained, it was dried. It was dissolved in 0.1 M HCl, put it into a dialysis bag with a 3500 molecular weight cutoff, and dialyzed for 3 days, changing the distilled water every 4 h. After dialysis, the sample was freeze-dried to get the final product (denoted by MEG1) as a white sponge-like solid.

Synthesis of MEG2: 20 mg of 6AC-100 was taken in a flask, 1 mL of DMSO was added, and the mixture was stirred at 60 °C until completely dissolved. The solution was cooled down to 40 °C; 36 μL of DIPEA and 46 mg of potassium carbonate were added and mixed well; then, 30 μL of 1-bromo-2-(2-methoxyethoxy)­ethane was added; and the reaction was allowed to proceed under nitrogen protection for 24 h. 3 mL of ethyl acetate was added to precipitate; then, the mixture was centrifuged 3 times with ethyl acetate and 2 times with ethanol to remove the supernatant (centrifugation was performed at 3000 rpm for 5 min), and the obtained white precipitate was dried. It was dissolved in 0.1 M HCl, placed in a dialysis bag with a molecular weight cutoff of 3500, and dialyzed for 3 days, changing the distilled water every 4 h. After dialysis, the sample was lyophilized to get the final product (denoted by MEG2) as a white sponge-like solid.

Synthesis of Cegk1: 500 mg of Boc-Lys­(Boc)-OH was dissolved in 2 mL of THF and cooled to −10 °C under N₂ protection. Then, 205 mg of N,N-diisopropylethylamine and 197 mg of isobutyl chloroformate were added sequentially and stirred at −10 °C for 40 min, forming a white solid. The white solid was washed 3 times with 5 mL of THF each time (2–3 mL per washing) and filtered under vacuum. The combined filtrate was cooled to −10 °C, and 81.9 mg of sodium borohydride was added in batches, reacting for 30 min. Water was then added; the mixture was warmed to room temperature and stirred for 1 h. Water was added, and the mixture was extracted 3 times with ethyl acetate. The mixture was washed 3 times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. An elution solvent of ethyl acetate/n-hexane = 2/1 was prepared for column chromatography purification. After removing the solvent by rotary evaporation, the product was placed in a drying oven for over 12 h to obtain the alcoholized Boc-Lys­(Boc)-OH. Alcoholized Boc-Lys­(Boc)-OH was dissolved in dichloromethane, 207 mg of EDC and 132.9 mg of DMAP were added, and finally, 223.2 mg of Bis-PEG-acid was added. The reaction was protected with nitrogen for 24 h. The mixture was dissolved in dichloromethane and extracted 3 times with citric acid, 3 times with water, and once with saturated brine to remove impurities and then dried over anhydrous sodium sulfate. An elution solvent of dichloromethane/methanol = 8/1 was prepared for column chromatography purification. After removing the solvent by rotary evaporation, the product was placed in a drying oven for over 12 h to obtain the intermediate.

31 mg of 6AC-100 was taken in a flask, 1 mL of DMSO was added, and the mixture was stirred at 60 °C until completely dissolved. The solution was cooled to 40 °C; then, 30 mg of EDC, 22 μL of TEA, and 82 mg of the intermediate product were added, and the reaction was allowed to proceed under nitrogen protection for 24 h. 3 mL of ethyl acetate was added to precipitate; then, the mixture was centrifuged 8 times to remove the supernatant (centrifugation was performed at 3000 rpm for 5 min each time) until a white precipitate was obtained; then, it was dried. It was dissolved in 0.1 M HCl, placed in a dialysis bag with a molecular weight cutoff of 3500, and dialyzed for 3 days, changing the distilled water every 4 h. After dialysis, the sample was freeze-dried to get the final product (denoted by Cegk1) as a white sponge-like solid.

2.2. Characterization of MEG1, MEG2, and Cegk1

MEG1, MEG2, and Cegk1 (1 mg/mL) and siRNA (10 μM) were mixed at different ratios (N/P ratio; N represents C6-NH2 in polymer; P represents phosphate in siRNA), and the polymer were incubated at RT for 20 min. The zeta potential and particle size distributions of Cegk1/siRNA were measured by a Zetasizer Nano Series instrument (Malvern Instrument, U.K.).

Using a series of standard amino acid solutions (2, 4, 6, 8, 10 μg/mL) as the x-axis and the corresponding absorbance at 570 nm as the y-axis, we plotted a standard curve and got the linear regression equation: y = 0.0107x + 0.0083 (R² = 0.9938). Amino acid content was measured using the ninhydrin colorimetric method. 0.30 mL of the sample was taken; then, 0.45 mL of ninhydrin working solution and 0.15 mL of vitamin C working solution sequentially were added, mixed well, heated in an 80 °C water bath for 20 min, and cooled in an ice-water bath; then, 2.1 mL of 60% ethanol was added. Using a blank as zero, the absorbance s measured at 570 nm. Amino acid standards (2–10 μg/mL) were measured to draw a standard curve and calculate the total free amino acid content in the sample using the formula AA = C × N; then, finally, the degree of substitution was determined.

2.3. Cell Culture and Cytotoxicity Assay

U251, U87-MG, and T98G human glioblastoma cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). The cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well and incubated overnight. The following day, three different samples were complexed with siRNA (100 nM) at an N/P ratio of 4 and added to the cells at final concentrations of 5, 10, 30, 50, and 100 μg/mL. After 24 h, cell viability was measured using an MTT kit according to the instructions.

Fresh blood was collected from the mouse’s eye socket, washed with PBS, and then centrifuged, and the supernatant was discarded to collect the red blood cells. The red blood cells were resuspended in PBS at a 9:1 volume ratio, and 100 μL was taken to add into a 96-well plate. Then, samples were added to reach final concentrations of 100, 200, 300, 400, and 500 μg/mL, and incubation was performed at 37 °C for 2 h. The mixture was centrifuged to collect the supernatant, and a microplate reader was used to measure the hemolysis rate.

2.4. Transfection Efficiency

The three types of cells were plated in a 48-well plate at 1 × 10⁴ cells per well. The next day, the three samples were mixed with FITC-siRNA (100 nM) at an N/P ratio of 4, allowed to sit at room temperature for 20 min, and then added to the cells. Culture was performed in a 37 °C incubator with 5% CO₂ for 8 h; then, the supernatant was discarded and the cells were washed once with PBS. Digestion was carried out with trypsin; culture medium was added; the mixture was centrifuged at 4 °C for 5 min, washed once with FACS buffer, and then analyzed using a flow cytometer (Beckman Coulter).

2.5. Knock Down and Western Blot Analysis

To measure gene silencing efficiency, three types of cells were seeded at 5 × 10⁴ cells per well in 12-well plates and cultured for 24 h until they reached 70%–80% confluence before transfection. Each sample was mixed with siPLK1­(100 nM) at an N/P ratio of 4, left at room temperature for 20 min to form complexes, and then diluted with Opti-MEM. After removing the old medium, the mixture was added to the cells and incubated at 37 °C for 4 h, followed by replacement with complete medium for continued culture. After 24 h of transfection, total RNA and protein were separately extracted. For RNA extraction using the Trizol method, cells were washed with PBS and then lysed with a 500 μL Trizol reagent (Invitrogen). 100 μL chloroform was added and vortexed and then centrifuged at 4 °C 12,000 rpm for 15 min. The upper aqueous phase was collected, precipitated with isopropanol, washed with 75% ethanol, dried, and dissolved for quantification. RT-qPCR was performed using SYBR Green Supermix (Bio-Rad) with human PLK1 primers (forward: 5′-cacagtgtcaatgcctcca-3′, reverse: 5′-ttgctgacccagaagatgg-3′, Takara) and human β-actin reference primers (forward: 5′-ccaaccgcgagaagatga-3′, reverse: 5′-ccagaggcgtacagggatag-3′), with relative expression calculated using the 2-ΔΔCt method.

Protein extraction used RIPA lysis buffer (Solebao, containing PMSF and phosphatase inhibitors). Protein was quantified by the BCA method (Thermo), and 35 μg total protein was run on a 12% SDS-PAGE gel (80 V for 30 min, then 120 V for 30 min). After wet transfer to a membrane, it was blocked with 5% BSA at room temperature for 1 h, washed with TBST, and incubated overnight at 4 °C with primary antibodies anti-PLK1 (Absin) and anti-GAPDH (Thermo). The next day, membranes were washed three times with TBST, incubated with secondary antibody (Cell Signaling) at room temperature for 1 h, washed with TBST, and developed with ECL chemiluminescence. Images were collected and analyzed using a gel imaging system. The final concentrations of MEG1, MEG2, and Cegk1 mixed with siRNA are 8.5 μg/mL and 100 nM, respectively, meaning the carrier concentration is 8.5 μg/mL and the siRNA concentration is 100 nM.

2.6. Analysis of Endosome Escape of siRNA/Cegk1 Polyplex

2 × 10⁴ U87-MG cells per well were seeded in a 24-well plate and cultured for 24 h until the cells reached 70%–80% confluence; then, cellular uptake was measured. The three samples were mixed separately with FITC-siRNA (100 nM) at an N/P ratio of 4, allowed to sit at room temperature for 20 min to form complexes, and then added to the cells. Incubation was performed at 37 °C for 4 h; then, the mixture was replaced with complete medium, and culturing was continued for 4, 8, or 24 h. After culturing, the cells were washed with PBS, Lyso-Tracker (Beyotime) was added, and incubation was carried out at 37 °C for 30 min; then, washing was performed with PBS three times. Fixation was done with 4% paraformaldehyde at room temperature for 15 min, and the cells were washed with PBS three times. Permeabilization was executed with 0.2% Triton X-100 at room temperature for 5 min, and the cells were washed with PBS once. The nuclei was stained with DAPI (MCE) for 5 min and washed with PBS twice. Finally, mounting was performed with anti-fade mounting medium, and the samples were observed under a confocal microscope to capture images.

2.7. Cell Apoptosis

The cells were seeded at 5 × 10⁴ per well in a 96-well plate and cultured for 24 h. The three samples were mixed separately with siPLK1­(100 nM) at an N/P ratio of 4, allowed to sit at room temperature for 20 min to form complexes, and then added to the cells. The MTT kit was used to check cell viability at 24 and 48 h, and cell apoptosis was assessed. After 48 h, live/dead staining (MCE) was performed: the dye was mixed with assay buffer according to the instructions, added to the cells, incubated at 37 °C for 30 min, washed with PBS, and observed, and photos were taken under a fluorescence microscope. For nuclear apoptosis detection, cells were seeded at 2 × 10⁴ per well in a 24-well plate, and 48 h after transfection, staining was performed with DAPI to observe changes in nuclear morphology.

2.8. Statistical Analysis

All results were expressed as the mean ± standard deviation. Student’s t test was used for statistical analysis between two groups, and one-way ANOVA was used for a multiple comparison test.

3. Results

3.1. Synthesis and Characterization of MEG1, MEG2, and Cegk1

To optimize the cationic polysaccharide carrier 6-amino-6-deoxy curdlan (denoted by 6AC-100) that our research group developed earlier, we designed and synthesized three new derivatives: MEG1, MEG2, and Cegk1 by introducing different functional groups. Their chemical structures are shown in Scheme . Among them, MEG1 has a methoxyethyl amino group, MEG2 has a methoxydiethanol amino group to boost hydrophilicity, and Cegk1 has a diamino structure to increase charge density. We also systematically explored how chain length, hydrophilicity, and charge density affect the carrier’s performance.

1. Synthetic Route of MEG1, MEG2, and Cegk1.

Next, the three polymers were systematically characterized in terms of their structure and physicochemical properties. ¹³C-NMR spectroscopy was used to confirm the chemical structures of MEG1, MEG2, and Cegk1, and the characteristic carbon signals of each derivative matched the target structures, confirming the successful introduction of functional groups, as shown in Figure . The results of gel permeation chromatography (GPC) are shown in Table . The weight-average molecular weight (Mw) and number-average molecular weight (Mn) of the three derivatives are fairly consistent, indicating that the polymerization reaction conditions are well-controlled. The products have a narrow molecular weight distribution and good uniformity, which ensures batch-to-batch reproducibility and stable performance later on. We also measured the degree of amino substitution (Degree of Substitution, DS) of the three polymers using the ninhydrin colorimetric method, with results shown in Table . MEG1 has the highest substitution degree at 66%, followed by MEG2 at 48%, and Cegk1 is relatively lower at 39%. It is worth noting that although Cegk1 has the lowest substitution degree, each substituted site carries a diamino structure, giving it a significantly higher overall charge density than MEG1 and MEG2. This structural feature lays the molecular foundation for its excellent siRNA binding ability and gene transfection efficiency later on.

1.

¹³C-NMR spectrum of MEG1, MEG2, and Cegk1 polymers in D₂O.

1. Molecular Weight, Degree of Substitution, PDI, and Zeta Potential of 6AC-100, MEG1, MEG2, and Cegk1.

	Polymers			Polyplex
Name	Mn	Mw	DS	PDI	Zeta potential
6AC-100	50000	65000	100%	0.3	+3
MEG1	8500	9200	66%	0.2	–3
MEG2	9000	9900	48%	0.3	+2
Cegk1	56000	74000	39%	0.2	–6

We further examined the binding capabilities of the three derivatives with siRNA. The fully complexed N/P ratio (protonable amino group mole amount/phosphage group mole amount) was determined using an agarose gel electrophoresis migration assay, and the results are shown in Figure A. At an N/P ratio of 4, all three derivatives could completely bind to siRNA to form stable nanocomplexes, with no obvious free siRNA bands on the gel, indicating that this N/P ratio is the optimal complexation condition for subsequent experiments.

2.

Binding and encapsulation characteristics of MEG1, MEG2, and Cegk1 polymers with siRNA. (A) Agarose gel electrophoresis analysis of the electrostatic binding ability of the three polymers with siRNA at different N/P ratios (polymer/siRNA), with complete complexation achieved at an N/P ratio of 4. (B) Dynamic light scattering (DLS) measurement of the particle size distribution of the three siRNA/polymer complexes at an N/P ratio of 4.

Based on this, we performed systematic nanoscale characterization of the siRNA/derivative complexes formed at an N/P ratio of 4. As shown in Figure B, dynamic light scattering (DLS) analysis revealed that all three complexes exhibited an average particle size of approximately 100 nm with good size distribution, falling within the optimal nanoscale range (50–200 nm) for cellular uptake. As shown in Table , the polydispersity index (PDI) of each sample is below 0.4, indicating a good particle size distribution and high uniformity. Zeta potential anaylsis revealed that all three complexes exhibited a near-neutral surface charge (0 ± 6 mV). This charge level suggests that the surface charge density of the complexes is moderate, allowing electrostatic attraction with negatively charged cell membranes to enhance cellular uptake without causing strong cytotoxicity or nonspecific adsorption of serum proteins due to excessive positive charge, thus achieving a good balance between uptake efficiency and biocompatibility. Additionally, the near-neutral surface charge helps improve colloidal stability in physiological conditions, reducing aggregation and precipitation caused by electrostatic repulsion.

3.2. Cytotoxicity of siRNA/MEG1, siRNA/MEG2, and siRNA/Cegk1 Polyplexes

First, we systematically evaluated the cytotoxicity of three new cationic polysaccharide derivatives: MEG1, MEG2, and Cegk1. We chose human glioblastoma cell lines U251, U87-MG, and T98G for in vitro experiments, with the classic cationic carrier PEI and our previous carrier 6AC-100 as positive controls. Cell viability after 24 h treatment with different concentrations of the samples was measured using the MTT assay, as shown in Figure A. The results indicated that within the tested concentration range, the toxicity of the three derivatives to the glioma cells was significantly lower than that of PEI, and they also showed lower cytotoxicity compared to 6AC-100. MEG2 and Cegk1, in particular, demonstrated excellent cell compatibility, suggesting that the PEG chain modification and the design of the diamine structure help reduce the carrier’s inherent cytotoxicity.

3.

Cytotoxicity measurement and hemolysis assay. (A) MTT assay to test the toxicity of MEG1, MEG2, and Cegk1 at different concentrations on three human glioblastoma cell lines: U251, U87-MG, and T98G. (B) Mouse red blood cell hemolysis test to evaluate the hemolytic activity and blood compatibility of the three polymers at concentrations ranging from 100 to 500 μg/mL. ***, p value is <0.005; ****, p value is <0.0001.

To further assess the blood compatibility of the three derivatives, we conducted a hemolysis assay using mouse red blood cells. Samples were prepared at concentrations of 100, 200, 300, 400, and 500 μg/mL, incubated with red blood cell suspensions at 37 °C for 2 h, and then centrifuged to measure hemoglobin release in the supernatant, as shown in Figure B. The results showed that the hemolysis rates of the three derivatives at all tested concentrations were significantly lower than PEI and also performed better than 6AC-100 in terms of blood compatibility, with Cegk1 showing the lowest hemolysis rate at 500 μg/mL, indicating good administration safety. In summary, the three new gel polysaccharide derivatives outperform the positive control PEI and our previous carrier 6AC-100 in both cytotoxicity and blood compatibility, demonstrating good biosafety.

3.3. Analysis of Cellular Uptake of siRNA/MEG1, MEG2, and Cegk1 Polyplexes

Next, we used flow cytometry to evaluate the siRNA transfection efficiency of three derivatives: MEG1, MEG2, and Cegk1, in different glioma cells. Using FITC-siRNA, we qualitatively observed the cellular uptake of siRNA mediated by each carrier under a fluorescence microscope, as shown in Supporting Information, Figure S1. The results are presented in Figure A–C: in U251 cells, Cegk1 showed the highest transfection efficiency, followed by MEG2, with MEG1 being relatively weak; in U87-MG and T98G cells, Cegk1 also showed the best transfection efficiency, MEG2 performed well, while MEG1 had relatively weak fluorescence signals. These results suggest that the high dual-amino charge density design of Cegk1 significantly enhances its ability to complex with siRNA and its cellular uptake efficiency. The introduction of PEG chains in MEG2 maintains decent transfection efficiency while improving the hydrophilicity of the carrier, whereas MEG1, having shorter chains and insufficient steric hindrance, has a relatively limited transfection efficiency. To further quantitatively compare the transfection efficiency of the three derivatives, the proportion of FITC-positive cells in each group was statistically analyzed, as shown in Figure D. In all three cell types, the transfection efficiencies of MEG2 and Cegk1 were similar, with no significant difference, but both were significantly higher than MEG1. Overall, both MEG2 and Cegk1 have excellent siRNA delivery capabilities, with MEG2 showing a better balance between transfection efficiency and biocompatibility, while Cegk1 achieves the strongest cellular uptake because of its high charge density. Interestingly, T98G cells exhibited substantially higher MFI values compared with the other two cell lines. This discrepancy is likely attributable to intrinsic biological differences among these glioblastoma cell lines, including variations in endocytic activity, membrane fluidity, and cell surface properties.

4.

The transfection effects of MEG1, MEG2, and Cegk1 in different glioma cells. (A) is the flow cytometry plot for U251 cells; (B) is for U87-MG cells; (C) is for T98G cells; (D) shows the quantitative analysis of transfection efficiency in all three cell types using flow cytometry. ***, p value is <0.005; ****, p value is <0.0001.

3.4. Gene Silencing Efficacy of siRNA/MEG1, siRNA/MEG2, and siRNA/Cegk1 Polyplexes

Next, to systematically evaluate the gene silencing effects mediated by the three derivatives, tests were carried out at both the RNA and protein levels. RT-qPCR was used to quantify PLK1 mRNA expression, and the results are shown in Figure A: at siRNA concentrations of 5, 50, and 100 nM, Cegk1 showed the best gene silencing efficiency, with a clear concentration-dependent effect; MEG2 was next, and MEG1 showed relatively weak silencing. These results indicate that Cegk1’s high charge density design significantly enhances intracellular siRNA release and gene silencing ability.

5.

MEG1, MEG2, and Cegk1 siRNA delivery and gene silencing efficiency in different glioblastoma cells. (A) RT-qPCR was used to quantify the knockdown effect of the three siRNA/polymer complexes on PLK1 mRNA in U251, U87-MG, and T98G cells. The results showed a clear concentration-dependent effect, with Cegk1 having the best gene silencing efficiency, reaching up to 70%. (B) Western blot confirmed that the Cegk1/siPLK1 complex effectively reduced PLK1 protein expression. *, p value is <0.05; **p value is<0.01.***, p value is <0.005; ****, p value is <0.0001.

Based on this, Cegk1 was further selected for Western blot validation, as shown in Figure B. Compared to the control, the Cegk1/siPLK1 complex significantly reduced PLK1 protein levels, and the silencing efficiency matched the RNA results, confirming that Cegk1 has excellent siRNA delivery and gene silencing capabilities, effectively suppressing the target gene at the translation level.

3.5. Analysis of Endosome Escape of siRNA/Cegk1 Polyplex

Next, to explore the intracellular transport mechanism of Cegk1/siRNA polyplexes, we used laser confocal microscopy to dynamically track their lysosomal escape ability. FITC-labeled siRNA (green) and Lyso-Tracker-labeled lysosomes (red) were used, and the subcellular distribution of siRNA at different time points was analyzed via fluorescence co-localization. Observations were made at 4, 8, and 24 h post-transfection, and the experimental results are shown in Figure . At 4 h posttransfection, FITC-siRNA was mainly distributed in the cytoplasm, with partial colocalization with lysosomes; at 8 h, the yellow colocalization signal became much stronger, indicating that most of the Cegk1/siRNA polyplexes had been taken up and trapped in lysosomes; by 24 h, the green fluorescence separated from the red lysosome signal and was dispersed in the cytoplasm, suggesting that the siRNA had successfully escaped from the lysosomes. These results show that Cegk1 has good lysosomal escape ability, and its double-amino high-charge-density design might mediate lysosome membrane disruption through the proton sponge effect, promoting intracellular release of siRNA and providing a key guarantee for subsequent gene silencing effects.

6.

Representative confocal microscope images showing the cellular uptake and intracellular distribution of Cegk1/FITC-siRNA polyplexes by U87-MG cells at different time points.

3.6. Cell Apoptosis

Next, to evaluate the anti-tumor effects of the Cegk1/siPLK1 complex, we used an MTT assay to check apoptosis in cells under different treatments. After 48 h of transfection, we measured the survival rates of U251, U87-MG, and T98G glioma cells, as shown in Figure A. The results showed that U251 and U87-MG cells are very sensitive to PLK1 silencing, with apoptosis significantly increasing after Cegk1/siPLK1 treatment. This inhibitory effect is clearly time-dependent, with the effect at 48 h being more pronounced than that at 24 h. In contrast, T98G cells are much less sensitive to PLK1 downregulation, so their apoptotic response is weaker compared with the other two cell lines. These findings suggest that different glioma cell lines have varying degrees of dependence on PLK1.

7.

Dose effect of the Cegk1/siPLK1 polyplexes on inducing apoptosis in three glioma cell lines. (A) Cell viability detected by MTT assay after treatment with different concentrations of siPLK1 (5, 50, 100 nM) for 48 h. (B). Live/dead cell double staining (Calcein-AM/PI) at 48 h, with green fluorescence indicating live cells and red fluorescence indicating dead cells.

To further visually confirm the apoptosis, live/dead cell staining (Calcein-AM/PI) was performed and observed under a fluorescence microscope, as shown in Figure B. The Cegk1/siPLK1 group had a significantly higher proportion of dead cells (red) compared with the blank and negative control groups. Its ability to induce apoptosis was comparable to that of the positive control PEI/siPLK1 and notably better than that of the previously used 6AC-100/siPLK1 vector. These results suggest that Cegk1 has efficient gene delivery and anti-tumor activity, showing great potential for PLK1-targeted therapy in glioma.

3.7. Phenotypic Changes in Apoptotic Cells

Finally, to intuitively verify the apoptosis effect induced by siPLK1, we used DAPI staining to observe the nuclear morphology changes in each treatment group, and the results are shown in Figure . Compared with the negative control (NT), the nuclei in the PEI/siPLK1 and Cegk1/siPLK1 groups showed obvious apoptotic features, such as nuclear fragmentation, indicating that PLK1 gene silencing can effectively activate the apoptosis pathway. In contrast, the nuclear changes in the 6AC-100/siPLK1 group were relatively weak and the apoptotic features were not obvious, suggesting limited gene delivery efficiency and subsequent apoptosis induction. These results further confirm that Cegk1 has excellent siRNA delivery and gene silencing capability, and the PLK1 downregulation it induces can significantly trigger apoptosis in glioma cells, with effects comparable to the positive control PEI and better than the previous carrier 6AC-100 used by our team.

8.

DAPI stained confocal microscopy images showing nuclear morphology changes in three glioma cells after Cegk1/siPLK1 induced apoptosis.

4. Discussion

In this research, using the cationic polysaccharide carrier 6AC-100 previously developed by our research group as a framework, three new derivatives, MEG1, MEG2, and Cegk1, were designed and synthesized through molecular structure optimization strategies. The goal was to create a new siRNA delivery carrier that combines low cytotoxicity, high transfection efficiency, and good biocompatibility. The influence of different functional groups on the carrier performance was systematically investigated.

On the basis of the experimental results, the three derivatives showed clear structure–activity relationships in terms of biosafety and gene delivery efficiency. Regarding cytotoxicity, Cegk1 forms more stable nanocomplexes with siRNA, reducing damage caused by free carriers to cells, showing the lowest cytotoxicity and the best blood compatibility. MEG2, with the introduction of PEG chains, maintained relatively low toxicity while improving the carrier hydrophilicity and spatial stability. MEG1, due to its shorter chain, had slightly higher toxicity but was still significantly better than the positive control PEI. In terms of transfection efficiency, Cegk1 showed the best siRNA delivery capacity among the three glioma cell lines (U251, U87-MG, and T98G), with efficiency comparable to MEG2 but significantly higher than MEG1. It could also effectively mediate lysosomal escape and achieve efficient silencing of target gene PLK1. Further functional verification showed that Cegk1/siPLK1 complexes could significantly downregulate PLK1 protein expression, effectively inducing apoptosis in glioma cells; its apoptosis-inducing ability was comparable to that of the positive control PEI and significantly better than that of 6AC-100.

In summary, Cegk1’s efficient cellular uptake despite its low zeta potential likely arises from a combination of multivalent membrane binding (attributable to its high molecular weight), non-electrostatic interactions (e.g., hydrophobic and hydrogen-bonding effects), and alternative endocytic pathways that are less dependent on strong surface charge. Meanwhile, MEG2 also demonstrates promising potential for specific applications as an alternative candidate. Collectively, this study offers a novel strategy for designing natural polysaccharide-based cationic gene delivery carriers, underscoring the critical roles of charge density regulation and hydrophilic group modification in optimizing carrier performance.

Data will be made available on request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c08587.

• Fluorescence microscopy images of three transfected cell lines (Fig. S1) (PDF)

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R.W., Y.Z., S.S., and X.Y. authors contributed equally. R.W.: data curation and formal analysis. Y.Z.: methodology, data curation, formal analysis, and writingoriginal draft. X.Y.: methodology, investigation, and resources. S.S.: validation, visualization, and writingreview and editing. D.Y.: investigation and resources. X.H.: investigation, resources, project administration, and writingoriginal draft. H.B. (Huricha Baigude): conceptualization, funding acquisition, methodology, supervision, writingoriginal draft, and writingreview and editing. H.L.: funding acquisition, supervision, and review and editing. All authors read and approved the final article.

The authors declare no competing financial interest.