Assessment of the Biological Effect of Oral Ginkgolic Acid Liposome Nanoparticles for Treating Refractory Helicobacter pylori Infection

Introduction

Helicobacter pylori is a Gram-negative bacillus that can survive under highly acidic conditions and is primarily transmitted from person to person through the oral–oral route.¹ The global infection rate of H. pylori is approximately 44.3%, with both the number of new cases and deaths (primarily from gastric cancer) increasing yearly.² Antibiotic resistance is one of the main reasons for the failure of current treatment of H. pylori infection. Although the types of resistance have not changed (primarily clarithromycin, levofloxacin, and metronidazole), the resistance rate is increasing, leading to a poor effect for traditional quadruple therapy.^3,4 In recent years, the recurrence rate of H. pylori has increased, increasing with time after eradication.⁵ There is an urgent need to develop new anti-H. pylori drugs to address this problem. Compared with Western medicine, the advantages of traditional Chinese medicine in treating H. pylori infection include a lower likelihood of drug resistance development, fewer adverse reactions, and lower toxicity.⁶

Ginkgo biloba L., a traditional medicinal plant in China, has a long history of application. Its extracts are widely used for treating inflammatory and cardiovascular diseases. Ginkgolic acid (GA) is derived from the abundant substances in the outer seed coat of ginkgo. It is a derivative of 6-alkyl or 6-alkenyl salicylic acid; the number of carbon atoms in its side chain can vary from 13 to 17, and the number of double bonds in the side chain ranges from zero to two. Currently, five main types of GA have been discovered: GA C13:0, GA C15:1, GA C17:2, GA C15:0, and GA C17:1.⁷ GA not only has various biological toxicities but also a variety of pharmacological activities, such as anti-tumor, antibacterial, insecticidal, and anti-inflammatory effects, showing a wide range of application prospects. GA can inhibit oral cancer and pulmonary fibrosis by blocking the TGF-β/SMAD4 pathway,^8,9 as well as inhibit the formation of Escherichia coli and Staphylococcus aureus biofilms.¹⁰ However, there are few research reports on the inhibition of H. pylori by GA, and its mechanism of action remains unclear.

As an organic nanomaterial, liposomes can be integrated with a variety of materials as well as be prepared into drug carriers suitable for various administration routes.¹¹ After liposomes enter the body, they are subject to specific opsonization and non-specific hydrophobic interaction with cells in the reticuloendothelial system, resulting in a short circulation time in the body.

To overcome this problem in this study, distearoyl ethanolamine–polyethylene glycol-maleimide (DSPE-PEG2000), an intermediate in long-circulating targeted liposomes, was used. PEG can form a coating on the surface of liposomes, reducing their capture of hydrophobic mucus.¹² Positively charged DSPE-PEG2000 can electrostatically adsorb mucus, enhancing carrier stability and circulation time in the body.¹³ Trans-activator of transcription (TAT) is a cell-penetrating peptide from the human immunodeficiency virus-1 that can efficiently enter the cytoplasm and nucleus, making it an important tool in drug delivery.¹⁴

Our team designed a multifunctional liposome nanoparticle (GA-TAT-DSPE-PEG2000, TLM@GA) with the mucus-penetrating ability of PEG and the transmembrane effect of TAT; this was used to efficiently load GA. We assessed whether TLM@GA can prolong nanodrug action time in the stomach and significantly enhance its anti-H. pylori activity.

Materials and Methods

Experimental Materials

Compounds such as GA were purchased from Chengdu Ruifensi. TNF-α Elisa kit, IL-6 Elisa kit, and IL-1β Elisa kit were purchased from Abclonal. Levofloxacin, metronidazole, polymyxin B, bacitracin, amoxicillin, and vancomycin were purchased from Macklin. Omeprazole was purchased from Yuekang Pharmaceutical Group Co., Ltd. LB broth medium and nutrient agar medium were purchased from Huankai Biotechnology. H&E staining kit, Diaminobenzidine (DAB) chromogenic kit, hematoxylin stain, 96-well plates, 50 mL centrifuge tubes, and PBS buffer were purchased from Servicebio. Polyethylene glycol and dimethyl sulfoxide were purchased from Shanghai Aladdin.

Synthesis of Nanocarriers

First, an intermediate was synthesized using β-alanine and N-(tert-butoxycarbonyl) ethanolamine as raw materials. Subsequently, through the amination reaction of PEG2000, followed by the addition of acetol acetonide and stearoyl chloride for esterification, and the desilylation condensation reaction using Tetrabutylammonium fluoride (TBAF), the crude DSPE was obtained after oxidation and removal of the tert-butoxycarbonyl (de-Boc) group. Next, the maleimide group in DSPE-PEG2000-Mal was covalently linked to the sulfhydryl group (-SH) in the TAT peptide to form the desired TAT-DdrugSPE-PEG2000 (TLM). Finally, the traditional Chinese medicine monomer GA was efficiently loaded to synthesize the GA-TAT-DSPE-PEG2000 nanoliposomes (TLM@GA). The surface of the polymer nanocarrier was modified using a nucleophilic substitution reaction.

The characterization methods of TLM@GA liposome nanoparticles: Dynamic Light Scattering (DLS) for particle size, PDI, and zeta potential analysis; Transmission Electron Microscopy (TEM) for morphological observation; UV-Vis Spectrophotometry/HPLC for determining encapsulation efficiency and drug loading; FTIR/XRD for structural characterization.

Strain Cultivation

Clinical H. pylori strains 26695, G27, NSH57, and BHKS159 were provided by Professor Bi from Nanjing Medical University. Clinical H. pylori strains HPBS001–HPBS007 were provided by the Research Center for the Prevention and Treatment of Drug-Resistant Microbial Infections in Guangxi Universities. The susceptibility of Clinical H. pylori strains to antibiotics (eg, clarithromycin, amoxicillin, and metronidazole) was determined using the Agar dilution method. The clinical isolates were classified as “resistant” or “sensitive” based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (version 13.0, 2023). Specifically, the MIC threshold for clarithromycin resistance was defined as >0.25 mg/L, and for metronidazole as >8 mg/L. The H. pylori strains containing the cryopreservation solution were taken out from the −80°C freezer and before inoculation onto selective media, the Clinical H. pylori strains were recovered in brain-heart infusion (BHI) broth containing 10% calf serum. Then cultured on Columbia agar plates in a microaerobic environment (85% N₂, 5% O₂, 10% CO₂) at 37°C for 3–4 days. A single colony was picked and cultured in brain-heart infusion medium containing 10% calf serum under microaerobic conditions at 37°C for 2–3 days to obtain the H. pylori bacterial suspension for subsequent experiments.

Microdilution Method

The Minimum Inhibitory Concentration (MIC) was determined using the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. The microdilution method was used to detect the Minimum Inhibitory Concentration (MIC). The drug-containing medium solution was added to a sterilized 96-well polystyrene plate, 100 μL per well. The drug solution was diluted in a two-fold manner to make the drug concentration range from 0.5 to 128 μg/mL. Then, the bacterial suspension (10 μL) with density 1 × 10⁷ CFU/mL was added to each well. A negative control well containing only the medium was set. After culturing under microaerobic conditions at 37°C for 3–4 days, the minimum drug concentration in the well without bacterial growth was defined as the MIC. The experiment was repeated three times to verify the stability of the results.

In-vivo Safety Evaluation of TLM@GA

In this study, the in-vivo drug safety evaluation was carried out under the premise of complying with the ethics of animal experiments. The mice were housed in a Specific Pathogen Free (SPF)-grade environment and randomly divided into the TLM@GA group and the negative Infected control group. The drug-administration group was intragastrically administered with a TLM@GA concentration 5 times the therapeutic dose (140 mg/kg) once a day for 7 consecutive days, while the negative Infected control group received PBS buffer solution with the same administration time and dose as the TLM@GA group. The body weight changes of the mice were continuously monitored for 7 days. On the third day after stopping the drug, the mice were weighed again, and the average body weight of each group was calculated. After collecting the blood from the eyeballs of the mice, the blood was allowed to stand for 1 h, centrifuged, and the supernatant was taken and stored in a −80°C freezer for later use. Subsequently, pathological sections of the stomach, liver, spleen, and kidney of the mice were prepared, and HE staining was used for the observation and analysis of the tissue structure.

Construction of an Acute Gastritis Model of H. pylori Infection in C57BL/6 Mice

The SPF-grade C57BL/6 mice used in this experiment were provided by the Experimental Animal Center of Inner Mongolia Medical University, with an age of 6–8 weeks, half male and half female. The license number for the use of SPF-grade animals was SYXK (Meng) 2020–0003, and the ethical approval for the animal experiments in this study was granted by the Animal Experimental Ethics Committee of the Affiliated Hospital of Inner Mongolia Medical University (Approval No. 2022MS08031). All procedures were performed in strict accordance with the national guidelines and regulations for the care and use of laboratory animals. The H. pylori suspension of HPBS001 bacteria (1 × 10⁹ CFU/mL in PBS) was administered to the SPF-grade 6–8- week-old mice via intragastric gavage using a sterile curved feeding needle. Prior to each inoculation, mice were fasted for 12 hours with free access to water. To neutralize gastric acid and enhance bacterial colonization, 0.2 mL of 5% NaHCO₃ was administered 20 minutes before the bacterial challenge. Each mouse received 0.2 mL of the suspension every other day for a total of three doses. After verifying the successful model construction, the mice were randomly divided into six groups (n = 10 per group): PBS group (not treated after infection), the OPZ + AC dual-therapy group (omeprazole 138.2 mg/kg + amoxicillin 28.5 mg/kg), the high-concentration TLM@GA group (28 mg/kg), the low-concentration TLM@GA group (7 mg/kg), and the high-concentration GA group (28 mg/kg). Ten normal mice were used as the negative Infected control group. The treatment was administered continuously for 3 days. After stopping the drug, half of the gastric tissue was taken under sterile conditions (Gastric tissues, including the fundus, body, and antrum, were collected for subsequent experiments.), weighed, ground, diluted, and spread on Columbia plates containing antibiotics. After culturing under microaerobic conditions for 3–4 days, the gastric colonization amount of H. pylori was calculated.

Statistical Analysis

The experimental data were plotted and statistically analyzed using GraphPad Prism 8. The results were expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using the t-test, and p < 0.05 was considered statistically significant. All experiments were performed in triplicate (n = 3), and results are reported as mean ± standard deviation (SD).

Results

Synthesis and Optimization of TAT-DSPE-PEG2000 Nanocarriers

With the modification of TAT on DSPE-PEG2000, a protein absorption peak appeared at 268 nm in the ultraviolet absorption spectrum that was significantly different from that of DSPE-PEG2000 (Figure 1A). By comparing and analyzing DSPE-PEG2000 and TAT-DSPE-PEG2000 in the Fourier transform infrared spectrum, corresponding peaks of functional groups appeared in TAT-DSPE-PEG2000 and showed shifts (Figure 1B). These findings indicated that TAT had been successfully loaded into DSPE-PEG2000, demonstrating that the TAT-DSPE-PEG2000 nanocarrier was constructed.

Figure 1 (A) Full-wavelength scanning of TAT covalently coupled to DSPE-PEG2000 molecules shows a protein absorption peak at approximately 268 nm. (B) The corresponding peak shift in the infrared spectrum proves the successful coupling.

Preparation and Characterization of TLM@GA

The particle size distribution and morphology of LM, TLM, and TLM@GA were analyzed. Through scanning electron microscopy, the particle sizes of the three materials were found to be uniform with regular morphologies; the morphologies of the three differed, indicating the successful preparation of each (Figure 2A–C). In the Zeta potential analysis, the potential of TLM@GA was around −17 mV, with the highest absolute potential value (Figure 2D); it was stable between 35 and 45 nm (Figure 2E).

Figure 2 Preparation and characterization of targeted liposome nanoparticles. (A) Particle size distribution and morphology of liposome nanoparticles (LM) composed of DSPE-PEG2000. (B) Particle size distribution and morphology of liposome nanoparticles (TLM) composed of TAT-conjugated DSPE-PEG2000. (C) Particle size distribution and morphology of TLM after loading GA. (D) Charge distribution of lipid nano-micelles in different formulations. (E) Particle size changes of TLM@GA during different storage times. (F) Cumulative drug release of TLM@GA under different pH conditions.

The amount of drug released from TLM@GA in environments with pH 7.4 and 4.5 was detected. At around 4 hours, the cumulative drug release efficiency of TLM@GA at pH 4.5 was higher than that at pH 7.4. When the release time reached 24 hours, the cumulative drug release efficiency of TLM@GA at pH 4.5 was approximately 80% (Figure 2F). In addition, we also detected and analyzed the drug-loading efficiency of TLM@GA, finding it to be between 56.24% and 62.87%. Overall, TLM@GA had good drug-loading capacity and efficiency (Table 1).

Table 1 Determination of TLM@GA Drug Loading Efficiency

In vitro Evaluation of TLM@GA Anti-H. pylori Activity

The MIC of GA against sensitive and resistant H. pylori strains was 16–32 µg/mL, while that of TLM@GA was 0.25–0.5 µg/mL, indicating its antibacterial activity was 32–128 times higher than that of GA (Table 2).

Table 2 Minimum Inhibitory Concentrations (MIC: μg/mL) of TLM@GA Against Sensitive and Resistant H. pylori Strains

In vivo Safety Evaluation of TLM@GA

There was no significant change in mouse body weight during the continuous 1-week detection (Figure 3A), and no obvious pathological damage was observed in the stomach, liver, spleen, or kidneys (Figure 3B).

Figure 3 (A) Change of body weight in mice. (B) H&E staining of the stomach, liver, spleen, and kidneys in mice. Scale bar = 200 μm. (n = 10 for each group; data are presented as mean ± SD).

In vivo Evaluation of TLM@GA Anti-Clinical H. pylori Activity

An acute gastritis model was constructed by inducing the adaptive colonization of the clinical strain H. pylori HPBS001 (resistant to clarithromycin, levofloxacin, and metronidazole) in C57BL/6 mice to evaluate the in vivo efficacy of TLM@GA against H. pylori (Figure 4A). H&E staining showed that neutrophils were significantly infiltrated in the gastric mucosa of mice in the PBS group (untreated after infection). After treatment with TLM@GA, the infiltration area was significantly reduced, with the high-concentration treatment having a better effect. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining showed that the apoptotic cells in the PBS group were tan, while the nuclei in the TLM@GA (28 mg/kg) group were blue, indicating that high-dose TLM@GA reduced the apoptosis of gastric epithelial cells (Figure 4B).

Figure 4 (A) Mouse model of acute gastritis. (B) Repair of gastric mucosal tissue in mice with acute gastritis after treatment with TLM@GA. Scale bar = 200 μm. (C) Colonization of H. pylori in mice with acute gastritis after treatment with TLM@GA. (D) Detection of TNF-α by ELISA. (E) Detection of IL-1β by ELISA. (F) Detection of IL-6 by ELISA. Results are shown as mean ± SD (n = 3). (n = 10 for each group, data are presented as mean ± SD; ns > 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Treatment with TLM@GA (both high and low doses) significantly suppressed H. pylori colonization in the gastric mucosa compared to the PBS group (P < 0.01), similar to the results observed in the positive control (omeprazole plus amoxicillin). Notably, no inhibitory effect was observed for the GA monomer group. High-dose TLM@GA showed a significantly stronger inhibitory effect than dual therapy (P < 0.01) (Figure 4C). To evaluate the immunomodulatory effects of TLM@GA, we quantified the levels of IL-6, TNF-α, and IL-1β, which are hallmark pro-inflammatory cytokines in the progression of Stomach Cancer. Compared with the Infected control group, the protein expression of IL-6, TNF-α, and IL-1β in the PBS group was increased. After treatment with TLM@GA, the expression of these proteins decreased (P < 0.01), with the high-dose group (28 mg/kg) having a better therapeutic effect (Figure 4D–F). Notably, there were no significant differences between the TLM@GA group and the normal group (P > 0.05). This lack of significant change indicates that the TLM@GA liposomes possess excellent biocompatibility and minimal systemic toxicity at the administered dose. This suggests that the TLM@GA can effectively reprogram the inflammatory microenvironment, hereby suppressing the overactivation of the immune response.

Discussion

H. pylori was newly added as a definite carcinogen in the 15^th edition of the Report on Carcinogens issued by the National Toxicology Program under the U.S. Department of Health and Human Services.¹⁵ Mahady et al evaluated the effects of 15 plant extracts on H. pylori strains; among them, the MIC of the methanol extract of Ginkgo biloba leaves was >100 mg/mL. Aside from this, there are few reports on the antibacterial activity of GA against H. pylori.¹⁶

DSPE-PEG2000 is a nanomaterial formed by the combination of phospholipids and PEG. It can be used as a drug carrier, improving stability and efficacy.^17,18 TAT is a cell-penetrating peptide derived from the human immunodeficiency virus-1 that can efficiently penetrate the cell membrane to enter the cytoplasm and nucleus.¹⁹ This transmembrane transport does not depend on specific ligand-receptor binding and has no saturation, making it an important tool in the field of drug delivery.²⁰ The outer DSPE-PEG layer of TLM@GA makes it hydrophilic and negatively charged, allowing it to effectively avoid being captured by mucus. When the PEG outer layer is removed, the TAT cell-penetrating peptide and the positively charged core are exposed; the particle can then effectively penetrate the gastric mucosal epithelium and release GA. The acid-resistant TLM@GA was able to significantly enhance the anti-H. pylori activity of GA.

In this study, we randomly selected H. pylori strains from different sources and with different sensitivities to test the in vitro antibacterial activities of GA and TLM@GA. The in vitro antibacterial activity of TLM@GA was 32–128 times higher than that of GA. The particle size of TLM@GA did not change significantly within 48 hours, and Zeta potential analysis indicated that it had good stability. TLM@GA had high drug release under acidic conditions. We also found that TLM@GA had good drug-loading capacity and efficiency.

In in vivo experiments, we infected mice with the clinically isolated HPBS001 strain (resistant to levofloxacin, clarithromycin, and metronidazole) to construct an acute gastritis model to evaluate the efficacy of TLM@GA. After treatment with TLM@GA, the colonization amount of H. pylori in the stomach of mice was significantly reduced.

A large number of studies have shown that H. pylori infection leads to the secretion of various cytokines by gastric epithelial cells, including IL-6, TNF-α, IL-1β, and various chemokines. The release of these cytokines not only promotes the inflammatory response but also affects the immune response.^21–23 Cytotoxin-associated gene A (CagA)-positive strains can significantly enhance the inflammatory response of the gastric mucosa and promote the occurrence and development of gastric cancer.²⁴ In addition, vacuolating cytotoxin A (VacA) and neutrophil-activating protein A (NapA) also enhance the inflammatory response and oxidative stress.²⁵

H. pylori infection is also associated with inflammatory cytokines such as TNF-α. The expression of TNF-α in H. pylori -positive samples is significantly increased, which is positively correlated with chronic inflammation.^26–28 In patients infected with H. pylori, TNF-α expression is also significantly positively correlated with the number of Th1/Th17/Th22 lymphocytes.²⁸ The increase in IL-8 is also positively correlated with chronic inflammation and neutrophil infiltration caused by H. pylori,²⁹ and gastric mucosal epithelial cells produce IL-1β when infected with H. pylori, which is highly correlated with mucosal inflammation.²¹ IL-1β can also stimulate the production of reactive oxygen species, which further drives nuclear factor kappa beta signaling. This process is also associated with the upregulation of the expression of C-X-C motif chemokine ligand 8, which promotes inflammation and enhances the recruitment and invasion of gastric cancer cells through the activation of mitogen-activated protein kinase.³⁰

During H. pylori infection, IL-6 expression in the gastric mucosa is significantly up-regulated. Piao et al found that H. pylori infection activates the signal transducer and activator of transcription 3 (STAT3) signaling pathway through reactive oxygen species-mediated increase in IL-6 expression, promoting gastritis.²³ Guo et al also found that the IL-6 mRNA level in gastric cancer cells is significantly increased.³¹ However, Baek et al found that GA effectively reduces the phosphorylation of STAT3 induced by IL-6;³² in addition, GA also significantly promotes the expression of phosphatase and tensin homolog and src homology 2 domain-containing phosphatase-1, which play a key role in inhibiting STAT3 signaling. Zhang et al’s research results showed that GA significantly alleviates the oxidative and inflammatory responses induced by oxidized low-density lipoprotein by inhibiting nuclear factor kappa beta signaling, showing good anti-inflammatory effects.³³ In conclusion, H. pylori infection causes an increase in the expression levels of inflammatory cytokines IL-6, TNF-α, and IL-1β. TLM@GA can effectively reduce the levels of these key pro-inflammatory factors, further alleviating the inflammatory response.

After treating mice with gastritis with TLM@GA in this study, H&E and TUNEL staining showed that TLM@GA treatment effectively alleviated cell apoptosis and inflammatory responses in the gastric mucosa, improving the pathological state of gastritis; subsequent immunological testing supported this conclusion. After treatment, the expression levels of major inflammatory factors such as IL-6, TNF-α, and IL-1β were significantly reduced, indicating that TLM@GA could effectively alleviate the inflammatory response. TLM@GA was not only effective in reducing inflammation but also showed good results in treating infections caused by multidrug-resistant strains.

The safety and effectiveness of drugs are equally important. GA has long been considered a safe, natural ingredient and is well-tolerated in humans and animals. The pharmacopoeias of China, Europe, and the United States all require the GA concentration in Ginkgo biloba extract to be <5 mg/kg.^7,34 TLM@GA was found to also have low toxicity; in vivo, a dose of 140 mg/kg did not cause damage to the stomach, liver, spleen, or kidneys of mice, nor did it cause weight loss or mental abnormalities. It must be acknowledged that this study is a preliminary pilot study conducted in a mouse model. While the results are promising, caution should be exercised before extrapolating these findings to human clinical applications.

Conclusion

In summary, this study successfully developed a novel TLM@GA liposome nanoparticle system with excellent stability and drug-loading efficiency. TLM@GA demonstrated significantly enhanced antibacterial activity against both sensitive and multi-drug resistant H. pylori strains while maintaining a high safety profile. It effectively reduced gastric inflammation and mucosal apoptosis in vivo, suggesting its potential as a lead candidate for treating refractory H. pylori infections.