Grafting MSI-78A onto chitosan microspheres enhances its antimicrobial activity

Abstract

MSI-78A (Pexiganan A) is one of the few antimicrobial peptides (AMPs) able to kill Helicobacter pylori, a pathogenic bacterium that colonizes the gastric mucosa of half of the world's population. Antibiotics fail in 20–40% of H. pylori-infected patients, reinforcing the need for alternative treatments. Herein, a bioengineered approach was developed. MSI-78A with a C-terminal cysteine was grafted onto chitosan microspheres (AMP-ChMic) by thiol-maleimide (Michael-addition) chemistry using a long heterobifunctional spacer (NHS-PEG₁₁₃-MAL). Microspheres with ∼4 µm diameter (near H. pylori length) and stable at low pH were produced by spray drying using a chitosan solution with an incomplete genipin crosslinking. A 3 × 10⁻⁵ µg AMP/microsphere grafting was estimated/confirmed by UV/Vis and FTIR spectroscopies. AMP-ChMic were bactericidal against H. pylori J99 (highly pathogenic human strain) at lower concentrations than the free peptide (∼277 µg grafted MSI-78A-SH/mL vs 512 µg free MSI-78A-SH/mL), even after pre-incubation in simulated gastric conditions with pepsin. AMP-ChMic killed H. pylori by membrane destabilization and cytoplasm release in a ratio of ∼10 bacteria/microsphere. This can be attributed to H. pylori attraction to chitosan, facilitating the interaction of grafted AMP with bacterium membrane. Overall, it was demonstrated that the peptide-microsphere conjugation chemistry did not compromise the MSI-78A antimicrobial activity, instead it boosted its bactericidal performance against H. pylori.

Statement of significance

Half of the world's population is infected with Helicobacter pylori, a gastric bacterium that is responsible for 90% of non-cardia gastric cancers. Therefore, H. pylori eradication is now advocated in all infected individuals. However, available antibiotic therapies fail in up to 40% patients. Antimicrobial peptides (AMPs) are appealing alternatives to antibiotics, but their high susceptibility in vivo limits their clinical translation. AMP immobilization onto biomaterials surface will overcome this problem. Herein, we demonstrate that immobilization of MSI-78A (one of the few AMPs with activity against H. pylori) onto chitosan microspheres (AMP-ChMic) enhances its anti-H. pylori activity even at acidic pH (gastric settings). These results highlight the strong potential of AMP-ChMic as an antibiotic alternative for H. pylori eradication.

Introduction

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that colonizes the gastric mucosa of half of the world's population [1]. It is the etiological agent of several gastrointestinal diseases, such as gastritis, peptic and duodenal ulcer [2]. Chronic infection with this gastric pathogen leads to non-cardia gastric cancer development, which accounts for 90% of all gastric cancer cases worldwide [3,4]. Therefore, H. pylori eradication is now advocated in all infected individuals [5]. The available therapy is based on a combination of at least two antibiotics and a proton pump inhibitor, but it fails in 20 to 40% of the patients [5], [6], [7], [8]. Several factors contribute to the decline in therapy effectiveness, such as failure in compliance due to a complex therapeutic scheme with severe side effects, but H. pylori resistance to antibiotics arises as the most significant aspect [9]. The World Health Organization (WHO) considers H. pylori one of the 16 antibiotic-resistant bacteria that pose the greatest threat to human health [10], urging the need for alternative treatments.

Antimicrobial peptides (AMPs) are mostly small (< 50 amino acids), cationic and amphipathic peptides [11] with a broad spectrum of activity against several microorganisms, including multi-drug resistant (MDR) bacteria [12], [13], [14], [15]. AMPs are reported to have low toxicity towards mammalian cells at bactericidal concentrations [16], but they can be toxic in vivo due to the high concentrations needed to overcome their self-aggregation and proteolytic degradation [11,17,18]. One way to overcome this problem is by their immobilization onto the surface of biomaterials, namely nanoparticles and microspheres [11,19,20]. AMPs described to have a strong bactericidal effect against H. pylori are very scarce, being MSI‐78 (pexiganan; GIGKFLKKAKKFGKAFVKILKK) and its analogs among the few reported examples [21]. Furthermore, H. pylori did not develop resistance against this AMP after 15 serial passages in vitro, in contrast to the tested antibiotics: metronidazole, clarithromycin and amoxicillin [22]. The encapsulation of MSI-78 into a drug delivery system (chitosan-alginate polyelectrolyte complex nanoparticles) enhanced the clearance of H. pylori infection in vivo (mice model) when compared to MSI-78 administered in suspension [22]. However, this strategy did not prevent self-aggregation or proteolytic degradation of the AMP after delivery.

We have previously demonstrated that MSI-78A (Pexiganan A; GIGKFLKKAKKFAKAFVKILKK), a derivative of MSI-78 obtained by substitution of one glycine by one alanine, possesses higher bactericidal effect than MSI-78 against H. pylori J99 (minimal inhibitory concentrations (MIC) of 64 µg/mL vs 256 µg/mL) [20]. This improvement was associated with the enhancement of the α-helical tendency observed for MSI-78A as compared to MSI-78 [23]. Furthermore, we demonstrated that MSI-78A maintains its antimicrobial performance when immobilized onto model surfaces (self-assembled monolayers - SAMs) - proof-of-concept study [20]. Herein, we translated the knowledge obtained with 2D model surfaces to biocompatible chitosan microspheres suitable for clinical application. MSI-78A was grafted onto chitosan microspheres – ChMic (∼ 4 µm in diameter) using a chemo-selective reaction (thiol-maleimide coupling) and a long chain spacer of polyethylene glycol – PEG₁₁₃ (5000 Da). A shorter PEG linker (PEG₂; 425.39 Da) was included as control to evaluate the influence of the spacer length on the reaction yield and AMP's bactericidal activity. The selected chemistry allows to control peptide orientation, concentration, and exposure, therefore tuning the AMP bactericidal activity against the gastric pathogen. ChMic were chosen due to their mucoadhesive properties and their ability to bind H. pylori, as previously described by us using larger microspheres (∼40 µm to ∼170 µm in diameter) [24], [25], [26], [27], [28]. ChMic were produced by spray drying technique, using solutions of chitosan partially crosslinked with genipin, to avoid the aggregation of these small microspheres, which would occur if crosslinking was performed after their preparation as described for larger microspheres [24], [25], [26], [27], [28]. The developed microspheres were tested in vitro against H. pylori J99, a highly pathogenic strain in humans [29], at different time points, and in a range of concentrations (1 × 10⁵, 1 × 10⁶ and 1 × 10⁷ microspheres/mL) and after microspheres pre-immersion in simulated gastric conditions in the presence of pepsin, a gastric enzyme that is able to cleave the MSI-78A at 5 different positions at pH 2 (amino acids 4, 5, 11, 15, 19 – Peptidecutter, ExPASy software).