The Anti-Oxidant Curcumin Solubilized as Oil-in-Water Nanoemulsions or Chitosan Nanocapsules Effectively Reduces Helicobacter pylori Growth, Bacterial Biofilm Formation, Gastric Cell Adhesion and Internalization

Abstract

The bacterium Helicobacter pylori (H. pylori) represents a major risk factor associated with the development of gastric cancer. The anti-oxidant curcumin has been ascribed many benefits to human health, including bactericidal effects. However, these effects are poorly reproducible because the molecule is extremely unstable and water insoluble. Here we solubilized curcumin as either nanoemulsions or chitosan nanocapsules and tested the effects on H. pylori. The nanoemulsions were on average 200 nm in diameter with a PdI ≤ 0.16 and a negative zeta potential (−54 mV), while the nanocapsules were 305 nm in diameter with a PdI ≤ 0.29 and a positive zeta potential (+68 mV). Nanocapsules were safer than nanoemulsions when testing effects on the viability of GES-1 gastric cells. Also, nanocapsules were more efficient than nanoemulsions at inhibiting H. pylori growth (minimal inhibitory concentration: 50 and 75 μM, respectively), whereby chitosan contributed to this activity. Importantly, both formulations effectively diminished H. pylori’s adherence to and internalization by GES-1 cells, as well as biofilm formation. In summary, the demonstrated activity of the curcumin nanoformulations described here against H. pylori posit them as having great potential to treat or complement other therapies currently in use against H. pylori infection.

Introduction

Cancer is the second leading cause of deaths worldwide, and among the different types of cancer, gastric cancer is considered to have one of the highest mortality rates and the worst prognosis after diagnosis [1]. Various environmental and nutritional factors are involved in the development of this disease, including sedentary lifestyle, poor diet, tobacco and/or alcohol consumption, stress, and family history, among others. Moreover, several studies showed that some gastric cancers are not associated with these factors and that infection with pathogens (viruses or bacteria) can favor the development of this disease [2]. Indeed, one of the main risk factors associated with the development of gastric cancer is the presence of Helicobacter pylori (H. pylori) in the stomach [3]. H. pylori is a Gram-negative microaerophilic bacterium with helical-shaped flagella that particularly infects the gastric epithelium. In humans, it can be detected in more than 50% of the world population [4].

H. pylori is able to attach to and internalize into gastric epithelial cells. Several virulence factors permit the adherence to the membrane of epithelial cells, such as BabA, SabA, OipA, and HopQ, among others [5]. After attachment, H. pylori can translocate proteins from the bacteria into the cytoplasm of infected cells, through a mechanism involving the multi-component bacterial type IV secretion system (T4SS) and the protein CagA [6]. These components are encoded in the Cag-pathogenicity island (cagPAI), which is essential for T4SS function and CagA traslocation [6]. Once inside the host cells, H. pylori virulence factors increase the survival and proliferation rate of infected cells [7] and induce the production of pro-inflammatory cytokines, such as IL-8 [8], which promote cell transformation.
H. pylori persists as a chronic infection unless treated, likely due to the presence of survival mechanisms that permit adaption to the acidic stomach environment and the formation of biofilms [9]. The latter ability is important, because it reduces the efficiency of antibiotic therapy and the possibility of clearance by the host immune response [9]. In particular, biofilm-associated H. pylori have been shown to be more resistant in vitro to clarithromycin, one of the antibiotics commonly used to treat H. pylori infections. The authors noted a four-fold increase in the minimal inhibitory concentration (MIC) to prevent bacterial growth in biofilms, as compared to bacteria growing individually [10]. Additionally, an increased mutation rate was observed for biofilm-associated H. pylori, which in turn facilitates the development of clarithromycin-resistant strains [10].

To eradicate this bacterium, various antibiotics are used following standard regimens, referred to as triple and quadruple therapy [11]. Antibiotics employed in this context include amoxicillin, clarithromycin, and metronidazole, which in different combinations result in greater than 70% efficiency of eradication [12]. Subsequently, if this does not suffice to eliminate H. pylori, the patient is given quadruple therapy, which includes the use of a proton pump inhibitor (PPI), bismuth, metronidazole, and tetracycline, also for 14 days. Although this treatment currently represents the gold standard, its effectiveness has been seen diminished by factors such as resistance to antibiotics (due to the indiscriminate use of these substances) and because people do not apply the treatment protocol rigorously enough [13]. Indeed, it has been estimated that the recurrence rate worldwide after treatment is around 4.3% [14].
Several options are being considered to improve the efficacy of treatments against H. pylori infection and thereby prevent the possible long-term development of cancer. Some of these have been developed using pharmaceutical and nanotechnological approaches, whereby the basic idea is to generate formulations that either preserve antibiotic function over extended periods of time [15] or involve generating formulations including new molecules with antibacterial effects [16].

A potentially attractive candidate, in the latter case, is curcumin, a polyphenolic anti-oxidant molecule (formula: 1,7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), found in plants growing in India and Southeast Asia [17]. For centuries, these plants and their alcoholic extracts have been used for medicinal purposes, because of their antibacterial [18] and healing (favoring the formation of an extracellular matrix) [19] properties. In addition, curcumin has been shown to have anti-cancer and anti-inflammatory properties [20], as well as act as an antioxidant [21], amongst others. However, in clinical trials, it has been reported that at least 12 g/day of curcumin is required to obtain any notable therapeutic effects [22], essentially due to the low water solubility and bioavailability of this active molecule [23]. Therefore, when this molecule is consumed orally, only a small fraction is absorbed by the intestine and much of it is rapidly metabolized (about 75%), mainly via sulfation and glucuronidation, to be subsequently eliminated in the feces and urine [24]. In addition, curcumin is extremely sensitive to light and oxygen [25], and, importantly, exposure to either significantly reduces the concentration of the bioactive compound. Ideally, therefore, a formulation is required that permits simultaneous solubilization, dispersing in aqueous/biological medium and stabilizing the curcumin in order to develop its maximum therapeutic potential [26]. Several examples of nanotechnological formulations containing curcumin are available which permit exploiting its beneficial effects in different settings [27]. Among them, the combination of curcumin and other natural agents, such as garlic extract, have been evaluated. Using this formulation, strong anti-inflammatory effects were observed in vivo in diabetic rats as a model [28]. Alternatively, another study showed that chitosan/curcumin nanoparticles had strong cytotoxic effects in vitro in colorectal cancer cells [29].
The antimicrobial effects of curcumin have been observed for a wide variety of bacterial species. Using curcumin included by poly(lactic-co-glycolic acid (PLGA)-nanocapsules, antimicrobial effects were reported for Staphylococcus aureus (Gram-positive), Bacillius subtilis (Gram-positive), Escherichia coli (Gram-negative), and Pseudomonas aeruginosa (Gram-negative) [17]. In addition, in vivo approaches showed that curcumin nanoparticles coated by chitosan-tripolyphosphate displayed high antimicrobial activity (over 75%) against Staphylococcus aureus and Pseudomonas aeruginosa infections [30]. For H. pylori, the antimicrobial effect of curcumin was demonstrated, in vitro and in vivo, using clinical isolates from Indian patients [31]. Furthermore, curcumin was shown to reduce the inflammation induced by H. pylori in infected mice, as well as the biofilm formation ability [32]. A similar effect on biofilm formation was observed for curcumin together with a decrease in the adherence to gastric Hep-2 cells [33].
In previous studies, we showed that curcumin could be solubilized and stabilized as a nanoemulsion, with potent cancer cell cytotoxicity in vitro and the ability in vivo, in a mouse melanoma model, to prevent re-incident and metastatic tumor growth [34]. These studies indicated that such nanoformulations, including curcumin, had tremendous potential for biomedical applications. However, whether they would serve to eliminate H. pylori was not clear. In our current study, we compared the previously described nanoemulsion of curcumin with a chitosan-based nanocapsule in terms of their ability to solubilize curcumin, dispersing the molecule in aqueous/biological media, and elicit bacteriostatic and bacteriotoxic effects towards H. pylori. We observed that the nanoencapsulated presentation of curcumin was more effective in reducing H. pylori growth, adhesion to gastric cells, bacterial internalization, and biofilm formation.

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Materials

Curcumin (from curcuma longa, CAS# 458-37-7, purity 75%, product number C1386, Sigma-Aldrich, Burlington, MA, USA), low-molecular-weight chitosan (CAS# 9012-76-4, 84% deacetylation, 94 cps, product number 448869, Sigma-Aldrich, USA), Miglyol 812 (neutral oil formed by esterification of caprylic fatty acids, CAS# 73398-61-5, 57.2% of C8 fatty acids and 42.4% of C10 fatty acids, Sasol, Houston, TX, USA), Epikuron 145 V (soy lecithin fraction enriched with phosphatidylcholine, CAS 8002-43-5, ≥97% phospholipids, Cargill, Düsseldorf, Germany), acetone, and ethanol were from Merck (Darmstadt, Germany).

Hidalgo, A.; Bravo, D.; Soto, C.; Maturana, G.; Cordero-Machuca, J.; Zúñiga-López, M.C.; Oyarzun-Ampuero, F.; Quest, A.F.G. The Anti-Oxidant Curcumin Solubilized as Oil-in-Water Nanoemulsions or Chitosan Nanocapsules Effectively Reduces Helicobacter pylori Growth, Bacterial Biofilm Formation, Gastric Cell Adhesion and Internalization. Antioxidants 2023, 12, 1866. https://doi.org/10.3390/antiox12101866

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