Functionalized locked nucleic acid system for targeted inhibition of hepatitis B virus C gene in transgenic models

2.1. Chemicals and measurements

All solvents and reagents used in this study were of analytical grade and utilized without further purification. The infrared (IR) spectra of the synthesized complexes were recorded on a Nicolet Impact 410 spectrometer in the range of 400–4000 cm⁻¹ using KBr pellets. Thermogravimetric analyses (TGA) were carried out on a PerkinElmer thermogravimetric analyzer under a nitrogen atmosphere from room temperature to 700°C, with a heating rate of 20 K min⁻¹. The specific surface areas and pore size distributions of the samples were determined by nitrogen adsorption–desorption measurements at 77 K using a Micromeritics ASAP 2460 surface area analyzer, and the Brunauer–Emmett–Teller (BET) method was applied to calculate the surface area.

2.2. Extraction and purification of compound 1

Dried pomegranate peel powder (100 g) was defatted with petroleum ether (1 L) under stirring at room temperature for 2 h, then filtered and dried. The residue was extracted twice with 70% ethanol (1.5 L) under reflux for 1.5 h each time, and the combined filtrates were concentrated by rotary evaporation. The concentrate was hydrolyzed with 1 L of 1.2 mol L⁻¹ HCl aqueous solution in a water bath at 85°C for 2 h to generate the target lactone, then cooled and allowed to crystallize. The precipitate was collected by filtration and extracted three times with ethyl acetate (3 × 500 mL), followed by washing with 0.5% NaHCO₃ solution and acidification to precipitate the product. The resulting solid was dissolved in a DMF: water (1:4) mixture at 60°C, recrystallized upon cooling, and vacuum-dried at 40°C to afford compound 1 (0.5 g, yield 0.5%).

2.3. Synthesis and characterization of polymer-encapsulated CS-1@LNA Complex

Compound 1 (0.1 mmol) and chitosan (CS, 0.05 g) were first dissolved in 20 mL anhydrous acetonitrile under a nitrogen atmosphere. DCC (0.12 mmol) and DMAP (0.05 mmol) were then added as coupling reagents, and the reaction mixture was stirred at room temperature for 24 h to obtain the modified chitosan (CS-1) (Scheme 1). After removal of dicyclohexylurea by filtration, the filtrate was concentrated, dialyzed (MWCO 3500 Da) for 48 h, and lyophilized to yield CS-1 as an orange solid.

Close

Scheme 1. Synthetic route to CS-1.

Export to PPT

For preparation of the CS-1@LNA nanocomplex, LNA (0.33 µg/OD) was dissolved in water to obtain a 0.33 µg/µL solution. Solution I was prepared by mixing 100 µL of LNA solution with 100 µL of 10% glucose and adjusting the total volume to 200 µL with H₂O. Solution II was prepared by mixing 100 µL of 10% glucose with CS-1 polymer (0.16 µL per µg LNA) and adjusting to 200 µL with ddH₂O. The two solutions were then combined, gently vortexed, and briefly centrifuged (356 × g, 30 s) to allow electrostatic self-assembly, resulting in the formation of a CS-1@LNA nanocomplex (final volume 400 µL, 5% glucose), which was stored at 4°C for immediate use.

2.4. Breeding and blood collection of HBV transgenic mice

The Affiliated Hospital of Youjiang Medical University for Nationalities provided a clean animal room where HBV transgenic mice were bred. All mouse operations were in compliance with Covance requirements and the “Guide for the Care and Use of Laboratory Animals”, and were approved by the Ethics Committee of the Affiliated Hospital of Youjiang Medical College for Nationalities (Ethics Approval Number: YYFY-LL-2023-121), with free access to sterile deionized water and high-pressure sterilized ordinary mouse feed. The food was replenished, and the water and bedding were changed daily between 17:00 and 18:00. Simultaneously, the health status of the mice was observed and recorded. The 30 HBV transgenic mice were numbered and grouped using a completely randomized design. Blood was collected from the orbital venous plexus of the mice using a capillary glass tube, and the serum HBV markers were quantitatively detected.

2.5. Quantitative detection of mouse serum HBV DNA by PCR

Nucleic acids were extracted using a nucleic acid release reagent. HBV DNA levels were quantified by real-time PCR using primers targeting the HBV S gene (forward: 5′-GTGTCTGCGGCGTTTTATCA-3′; reverse: 5′-ACAAACGGGCAACATACCTT-3′). PCR reactions were performed using TaqMan FastVirus One-Step Master Mix (Thermo Fisher Scientific) in a total volume of 50 µL containing 50 µL serum-derived template. Amplification was conducted on an ABI 7500 Fast Real-Time PCR System under the following conditions: 50°C for 5 min, 95°C for 20 s, followed by 45 cycles of 95°C for 3 s and 60°C for 30 s. Quantification was based on standard curves generated using HBV quantitative standards, and data were analyzed using the instrument software with appropriate baseline and threshold settings. The assay validity was confirmed by positive and negative controls, with standard curve correlation coefficients (R²) > 0.99.

2.6. Quantitative detection of mouse serum HBsAg and HBeAg

Serum levels of HBsAg and HBeAg were measured using an automatic chemiluminescence analyzer (A2000Plus, Autobio) according to the manufacturer’s instructions. Mouse serum samples were thawed to room temperature and diluted with physiological saline (50-fold for HBsAg and 5-fold for HBeAg) prior to analysis. Quantitative results were expressed as IU/mL for HBsAg and PEI U/mL for HBeAg. Instrument calibration and quality control were performed using standard calibrators, and intra- and inter-assay coefficients of variation were maintained below 15% and 10%, respectively.

2.7. Detection of mouse serum HBV subtypes

HBV viral DNA was extracted from mouse serum using a centrifugal column kit (TIANamp Virus DNA/RNA Kit, Tiangen Biotech). Primers targeting conserved regions of the HBV C and S genes were designed based on the reference sequence (GenBank: U95551.1) using Primer Premier 5.0 and Oligo 6.0, and verified by BLAST analysis. PCR products were analyzed by agarose gel electrophoresis and subsequently subjected to high-throughput sequencing (Sangon Biotech). HBV subtypes were determined by comparing the obtained sequences with reference sequences in the database. The primer sequences were as follows: HBV C gene, forward 5′-GACCGACCTTGAGGCATACT-3′ and reverse 5′-TCCCCACCTTATGAGTCCAA-3′; HBV S gene, forward 5′-CTGCCTCTCCCTTATCGTCA-3′ and reverse 5′-TGGCAAGGACCCATAACTTC-3′.

2.8. HBV C coding strand CS-1@LNA design and synthesis

Obtain the complete HBV gene sequence (U95551.1; GI:2182117) from the NCBI gene database. Using the principles of antisense oligonucleotides, employ RNA structure software to design sequences targeting the HBV C coding strand at nucleotide positions 2404-2418. Screen sequences with lower free energy using the Walk function, and after basic local alignment search tool (BLAST) homology analysis, obtain the sequence: 5’-CGA*CGCGGCGA*T*TGA*-3’ (* represents the modified position). Send the sequence to Shanghai Sangon for synthesis, and synthesize antigen LNA (antisense LNA) (5’-CGA*CGCGGCGA*T*TGA-3’) targeting the HBV C gene, antisense LNA (anti-LNA) (5’-T*GG*TA*CGT*TG*A-3’), and unrelated sequence (5’-ATACCTGTAGCTCGTA-3’).

2.9. Agarose gel electrophoresis identification of CS-1@LNA stability

For the stability assay, CS-1@LNA and the unmodified unrelated sequence (5′-ATACCTGTAGCTCGTA-3′) were incubated with single-strand-specific nuclease (S1 nuclease) under controlled conditions (37°C for 30 min in the recommended reaction buffer). Samples without nuclease treatment were used as controls. After incubation, the reactions were terminated according to the manufacturer’s protocol, and all samples were analyzed by agarose gel electrophoresis (1% agarose gel, stained with nucleic acid dye) to evaluate nuclease resistance based on band integrity and degradation patterns.

2.10. Screening the optimal CS-1@LNA dosing, route, and mode in HBV transgenic mice

A total of n = 6 mice were used for each group, which was determined based on preliminary experiments and commonly reported sample sizes in similar in vivo HBV studies, providing sufficient statistical power to detect biologically relevant differences while minimizing animal use in accordance with ethical guidelines. Mice were anesthetized with isoflurane (2%–4% for induction, 1%–2% for maintenance) and euthanized by cervical dislocation following established protocols. LNA was administered to mice via tail vein injection at doses of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 µg/g. This dose range was selected based on preliminary exploratory experiments and relevant literature on nucleic acid delivery in vivo, aiming to cover a gradient from low to potentially effective doses while maintaining biosafety and minimizing potential toxicity. Orbital venous blood was collected to measure serum HBV DNA, HBsAg, and HBeAg levels using PCR fluorescence probe and magnetic particle chemiluminescence methods, and inhibition rates were calculated to determine the optimal CS-1@LNA dose. Subsequently, CS-1@LNA (0.5 µg/g) was administered via different routes, including tail vein, intraperitoneal, and subcutaneous injection. Serum HBV markers were measured using the same methods, and inhibition rates were compared to evaluate the optimal administration route.

2.11. Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Data are expressed as mean ± standard deviation (mean ± SD). Comparisons between two groups were conducted using Student’s t-test, and multiple-group comparisons were analyzed using one-way analysis of variance (ANOVA) followed by appropriate post hoc tests. A P value < 0.05 was considered statistically significant. Inhibition rates of HBV DNA, HBsAg, and HBeAg were calculated as: (pre-treatment value − post-treatment value)/pre-treatment value × 100%. Gray values of polymerase chain reaction (PCR) electrophoresis bands and relative protein expression levels in Western blot assays were quantified using ImageJ software and normalized to the internal reference.

3.1. Characterization of compound 1

The ¹H NMR spectrum of compound 1 (Figure 1a) exhibited a characteristic downfield singlet at δ 12.84 ppm, assigned to the strongly hydrogen-bonded phenolic proton (–OH). Multiple aromatic proton signals appeared at δ 7.12, 6.95, 6.73, 6.44, 6.26, and 5.95 ppm, corresponding to different positions on the substituted aromatic rings. The methylene and methine protons were observed at δ 3.43, 3.28, and 3.12 ppm, while signals at δ 2.61, 2.50, and 2.18 ppm were attributed to methyl/methylene protons adjacent to the aromatic system. The ¹³C NMR spectrum (Figure 1b) showed resonances at δ 193.40 and 180.08 ppm, characteristic of carbonyl carbons, while signals in the range of δ 150–165 ppm were assigned to oxygenated aromatic carbons. Aromatic carbons were observed between δ 100–140 ppm, with methoxy carbons at δ 55.93 and 55.56 ppm, and methyl carbons at δ 21.91 and 21.11 ppm. These NMR results are consistent with the proposed structure of compound 1, confirming its successful synthesis.

Close

Figure 1. (a) ¹H NMR and (b) ¹³C NMR spectra of compound 1 in DMSO.

Export to PPT

By combining the 1D and 2D NMR spectra, the structural features of compound 1 were confirmed. In the correlation spectroscopy (COSY) spectrum Figure 2(a), strong correlations were observed in the δ 6.2–7.5 ppm region, indicating coupling among aromatic protons, while cross-peaks in the δ 2.0–3.5 ppm region were attributed to methylene or methine groups. The hydroxyl proton at δ 12.8 ppm showed no correlations, consistent with a free hydroxyl group. In the heteronuclear single quantum coherence (HSQC) spectrum Figure 2(b), aromatic protons at δ 6.2–7.2 ppm correlated with aromatic carbons at δ 100–130 ppm, protons at δ 3.2–3.4 ppm correlated with carbons around δ 55 ppm, confirming the presence of –OCH₂– or –NCH₂– groups, and protons at δ 2.1–2.6 ppm correlated with carbons at δ 20–22 ppm, further supporting the assignment of methyl or methylene groups. In the heteronuclear multiple bond correlation (HMBC) spectrum Figure 2(c), aromatic protons (δ 6.5–7.5 ppm) exhibited long-range correlations with carbonyl carbons at δ 160–180 ppm, confirming hydroxyl and carbonyl substitution on the aromatic rings. Meanwhile, methylene protons at δ 3.2 ppm showed correlations with both aromatic and carbonyl carbons, indicating their linkage to the aromatic backbone and carbonyl groups, while δ 2.2 ppm protons also correlated with aromatic carbons, suggesting their attachment to the aromatic framework. Collectively, the 2D NMR data are highly consistent with the 1D spectra, fully verifying the multi-substituted aromatic skeleton and functional groups of compound 1, and confirming the successful synthesis and structural rationality of the target compound.

Close

Figure 2. 2D NMR spectra of Compound 1: (a) COSY, (b) HSQC, and (c) HMBC.

Export to PPT

The relative molecular mass of compound 1 was characterized by high-resolution mass spectrometry. As shown in Figure 3, the experimental molecular ion peak was observed at m/z 552.14, which is in perfect agreement with the theoretical value of 552.14, with negligible deviation. This result strongly confirms that the molecular weight of the target compound matches the expected structure, further verifying its successful synthesis.

Close

Figure 3. Mass spectrometric characterization of compound 1.

Export to PPT

3.2. Structural characterization of CS-1@LNA composite

The structural and physicochemical properties of CS-1 and CS-1@LNA were comprehensively characterized to verify the successful conjugation of LNA and assess the carrier’s stability (Figure 4). The FT-IR spectra (Figure 4a) of CS-1@LNA displayed new characteristic absorption peaks at 1710 cm⁻¹ and 1235 cm⁻¹, corresponding to phosphate and N–O stretching vibrations of the nucleic acid backbone, confirming successful LNA incorporation. Nitrogen adsorption–desorption isotherms (Figure 4b) showed type IV hysteresis loops typical of mesoporous structures, with a slight decrease in specific surface area and pore volume after LNA loading, suggesting partial pore occupation. The pore size distribution (Figure 4c) remained mainly within 2–4 nm, indicating structural integrity was preserved. Thermogravimetric analysis (Figure 4d) revealed enhanced thermal stability of CS-1@LNA compared with CS-1, attributed to the introduction of the LNA component. Collectively, these results confirm that LNA was successfully grafted onto the chitosan-based carrier (CS-1) without compromising its porous framework, providing a stable and biocompatible platform for nucleic acid delivery.

Close

Figure 4. Characterization of CS-1 and CS-1@LNA: (a) FT-IR spectra; (b) N₂ adsorption–desorption isotherms; (c) Pore size distribution; (d) TGA curves.

Export to PPT

3.3. Preparation and stability assessment of CS-1@LNA targeting HBV C gene

Serum analysis of HBV transgenic mice revealed high viral activity, with average HBV DNA, HBsAg, and HBeAg levels of 11.75 × 10⁴ IU/mL, 10,403.12 IU/mL, and 7.39 PEI U/mL, respectively, showing no significant sex-related differences (Table S1). PCR amplification produced 760 bp and 830 bp fragments corresponding to the HBV C and S genes, consistent with theoretical predictions, and sequence alignment confirmed high homology with the ayw subtype reference strain (Figure S1). These findings indicate that the HBV transgenic mice possess robust viral replication activity and belong to the ayw subtype.

The design and synthesis of the HBV C gene-targeting antisense oligonucleotide CS-1@LNA were performed as described in the Methods section, with the overall workflow illustrated in Figure S2(a). The process involved sequence screening using RNA structural analysis and free energy filtering, followed by homology verification and locked nucleic acid (LNA) modification of the selected target site.

The stability assessment results are shown in Figure S2(b). The distinct and well-defined electrophoretic bands indicate that unmodified sequences were rapidly degraded by nucleases, whereas the LNA-modified CS-1@LNA maintained structural integrity and exhibited strong resistance to enzymatic digestion. These results confirm the enhanced stability of CS-1@LNA, supporting its suitability for subsequent in vivo delivery and antiviral studies.

3.4. Dose and delivery effects of CS-1@LNA on HBV Inhibition

To determine the optimal therapeutic conditions for CS-1@LNA, HBV transgenic mice were administered different doses (0.1–0.8 µg/g) via tail vein injection. Serum levels of HBV DNA, HBsAg, and HBeAg were measured on days 1, 3, 5, and 7 post-treatment to calculate inhibition rates. As shown in Figure 5(a), the inhibitory effects increased in a dose-dependent manner and reached a maximum at 0.5 µg/g. At this dose, inhibition rates on day 7 were 57.09 ± 3.69% for HBV DNA, 49.29 ± 4.31% for HBsAg, and 71.72 ± 2.23% for HBeAg, indicating that 0.5 µg/g represents the optimal therapeutic dose.

Close

Figure 5. Effects of different CS-1@LNA doses and administration routes on antiviral activity in HBV transgenic mice. (a) Inhibition rates of HBV DNA, HBsAg, and HBeAg at various CS-1@LNA doses. (b) Comparison of antiviral effects via different administration routes. Data are presented as mean ± SD (n=3).

Export to PPT

To further evaluate delivery efficiency, mice were treated with CS-1@LNA (0.5 µg/g) via different administration routes, including tail vein, intraperitoneal, and subcutaneous injection. As shown in Figure 5(b), the tail vein group exhibited markedly higher inhibition rates than the other groups, reaching 56.61 ± 1.24% (HBV DNA), 48.65 ± 3.59% (HBsAg), and 69.95 ± 2.72% (HBeAg) on day 7. In contrast, intraperitoneal and subcutaneous administration resulted in minimal inhibitory effects. These results indicate that tail vein injection enables more efficient systemic delivery and hepatic uptake of CS-1@LNA, leading to enhanced antiviral activity.

3.5. Antiviral effects of CS-1@LNA on HBV markers and gene expression

To assess the antiviral efficacy of CS-1@LNA, serum levels of HBV DNA, HBsAg, and HBeAg were monitored on days 1, 3, 5, and 7 post-treatment. As shown in Figure 6(a), both the anti-LNA group (without chitosan encapsulation) and the CS-1@LNA group exhibited significant inhibitory effects on all three viral markers compared with the blank and negative control groups (P < 0.05), indicating that LNA-based antigen treatment effectively suppresses viral activity in vivo.

Close

Figure 6. Antiviral activity of CS-1@LNA in HBV transgenic mice. (a) Inhibition rates of serum HBV DNA, HBsAg, and HBeAg in different treatment groups (n = 3). (b) Relative mRNA expression of the HBV C gene after treatment (n = 3). Data are presented as mean ± SD. **P < 0.01; ***P < 0.001.

Export to PPT

Notably, CS-1@LNA demonstrated the strongest suppression, achieving the highest inhibition rates for HBV DNA, HBsAg, and HBeAg on day 7, and outperforming both lamivudine and unmodified antisense LNA. This enhanced effect suggests that chitosan-mediated delivery improves the stability and cellular uptake of LNA, thereby increasing its antiviral efficacy. In contrast, the unrelated sequence and blank groups showed negligible inhibition, confirming the sequence-specific antiviral activity of CS-1@LNA.

To further investigate its mechanism of action, the mRNA expression of the HBV C gene was quantified by RT-PCR. As shown in Figure 6(b), electrophoresis and grayscale analysis revealed that CS-1@LNA significantly downregulated HBV C gene mRNA compared with the blank, NC, and lamivudine groups (P < 0.05), while unmodified antisense LNA produced a moderate effect. This reduction in mRNA levels is consistent with transcriptional inhibition at the DNA level.

These results indicate that CS-1@LNA effectively suppresses HBV replication and antigen expression through sequence-specific inhibition of HBV C gene transcription, demonstrating its strong potential as a novel antigen therapeutic strategy.

3.6. Biocompatibility and histopathological evaluation of CS-1@LNA

Histopathological examination of liver and kidney tissues showed that CS-1@LNA treatment did not induce observable structural damage. As shown in Figure S3(a), hepatic lobules, sinusoids, and interstitial structures remained intact, with hepatocytes arranged in regular cords and exhibiting normal morphology without signs of degeneration, necrosis, or inflammation, indicating minimal hepatotoxicity. Similarly, Figure S3(b) shows that renal glomeruli, basement membranes, and mesangial matrices were well preserved, with no evidence of inflammatory cell infiltration or pathological alterations, suggesting that renal function was not adversely affected.

Biochemical analyses further supported the biosafety of CS-1@LNA. As summarized in Tables S2 and S3, no significant differences were observed in hematological parameters or liver and kidney function markers (ALT, Alb, BUN, Crea) compared with the blank control (P > 0.05), confirming the absence of systemic toxicity.

Western blot analysis revealed that CS-1@LNA significantly downregulated the pro-apoptotic proteins Cleaved-Caspase-3 and Bax while upregulating the anti-apoptotic protein Bcl-2 relative to the NC group (P < 0.05, Figures 7a and b). This expression pattern suggests a reduction in apoptosis, which may contribute to the preservation of hepatocyte integrity during antiviral treatment.

Close

Figure 7. CS-1@LNA suppresses apoptosis in hepatic tissue cells. (a) Western blot analysis of Cleaved-caspase-3, Bax, and Bcl-2 expression in liver tissues. (b) Quantification of relative protein levels. Data are presented as mean ± SD. ***P < 0.05; ns, not significant.

Export to PPT

Together, these findings indicate that CS-1@LNA exhibits favorable biocompatibility and may protect hepatocytes from apoptosis while exerting its antiviral activity.

3.7. Antiviral Mechanism and Therapeutic Implications of CS-1@LNA

Chronic hepatitis B remains a major global health challenge, often progressing to cirrhosis and hepatocellular carcinoma. Although antiviral drugs such as lamivudine can effectively suppress viral replication, complete viral clearance is limited by the persistence of covalently closed circular DNA (cccDNA) and the emergence of drug resistance. In recent years, nucleic-acid-based strategies, including antisense oligonucleotides and RNA interference, have been explored for HBV treatment; however, these approaches primarily act at the RNA or protein level and often result in transient effects. In contrast, antigen therapy directly targets viral DNA transcription, offering a potentially more durable mechanism for inhibiting HBV replication. In particular, locked nucleic acid (LNA)-based systems exhibit high binding affinity, strong nuclease resistance, and improved sequence specificity, making them promising candidates for antigen applications.

In this study, HBV transgenic mice with the ayw subtype were used as an in vivo model to evaluate the antiviral efficacy of a cationic polymer–mediated antigen system, CS-1@LNA, designed to target the conserved 2404–2418 nt region of the HBV C gene. Sequence verification and serological analysis confirmed high viral activity, supporting the suitability of the model. The CS-1@LNA complex exhibited strong nuclease resistance and favorable stability, facilitating efficient in vivo delivery. Following tail vein administration, CS-1@LNA significantly reduced serum HBV DNA, HBsAg, and HBeAg levels in a time- and dose-dependent manner, outperforming lamivudine in short-term suppression. This enhanced efficacy is likely related to rapid systemic distribution and efficient liver targeting via intravenous delivery, which promotes accumulation of the nanocomplex in hepatocytes. In contrast, intraperitoneal and subcutaneous administration showed limited antiviral effects, possibly due to reduced bioavailability and slower absorption.

Furthermore, CS-1@LNA effectively suppressed HBV C-mRNA expression and viral antigen production in hepatic tissue without inducing observable liver or kidney toxicity. These findings indicate that CS-1@LNA exerts sequence-specific inhibition of HBV replication and gene expression, likely through direct antigen-mediated transcriptional blockade. Compared with conventional RNA-targeting strategies, this DNA-level inhibition may contribute to improved durability of antiviral effects. In addition, the use of a functionalized chitosan carrier enhances the stability and delivery efficiency of LNA, representing a key advantage of this system.

Nevertheless, several limitations should be considered. The incomplete suppression of viral replication suggests challenges such as limited nuclear delivery efficiency, the persistence of integrated or episomal HBV DNA, and potential host-related variability. Moreover, although LNA exhibits favorable stability and binding properties, issues such as delivery efficiency, long-term safety, and potential off-target effects require further investigation. Overall, CS-1@LNA represents a safe and effective antigen-based therapeutic strategy with advantages over conventional RNA-targeting approaches. Future studies will focus on improving nuclear targeting efficiency, optimizing the delivery system, and exploring multi-target or combination strategies to further enhance antiviral efficacy and support clinical translation.