A pH-shift preparation of lipoprotein complex conjugated mesoporous silica nanocomposite contributed to synergistic antibacterial therapy of drug-resistant bacteria

Abstract

Nowadays, the emerging multidrug-resistant bacteria with increasing infectious diseases and rising mortality rates remain serious global public health threats, which prompts a continuous search for alternative antibacterial strategies capable of restoring antibiotics susceptibility. Here, a synergistic antibacterial nanoplatform was proposed using mesoporous silica nanoparticles (MSN) as carrier to incorporate two membrane targeting agents, including the well-known lipoprotein complex (BAMLET) formed by milk derived α-lactalbumin bound to oleic acid and a naturally occurring chalcone of isobavachalcone (IBC).

It was found that a pH-shift preparation of BAMLET (termed HBAMLET at pH = 3 and NBAMLET at pH = 8) may cause observable conformational changes of lipoprotein complex stabilized on MSN, pointing to distinctive nanocomposite named as HBMSN (pH = 3) and NBMSN (pH = 8), respectively. According to previous studies, the preparation of BAMLET cannot be made in an acidic environment while we proposed a straightforward method to synthesize a stable and homogeneous lipoprotein complex of HBMSN at pH = 3 using MSN as a carrier. Moreover, both in vitro and in vivo assays demonstrated that compared to NBMSN, acidic conditions-synthesized HBMSN exhibited superior antibacterial effect to kill effectively methicillin-resistant Staphylococcus aureus and restoring its susceptibility to aminoglycoside antibiotics.

The underlying mechanistic studies revealed HBMSN as well as hydrophobic IBC compound can disrupt bacterial membrane permeability while inhibit biofilm formation, which may be ascribed to more exposure of hydrophobic interiors of HBMSN through the acidic synthesis endowing its ability to passively target bacterial membranes. Therefore, the constructed pH-responsive lipoprotein complex decorated silica mesopores with synergistic bactericidal activities may provide a manipulable yet efficient nanotherapeutics to combat drug-resistant bacteria.

Introduction

With the accelerating emergence of bacterial resistance, common infections may become difficult to treat as a result of antibiotic therapy failure, which has posed serious threats to global public health. According to a recent report, it was estimated that drug-resistant infections may result in 8.2 million deaths worldwide by 2050 from the global burden of antibacterial resistance across 204 countries and regions from 1990 to 2021 [1]. In particular, the mortality associated with antibacterial resistance among the elderly (> 70 years) has increased by more than 80%, and this trend is exacerbated by the rapid ageing of the population [2]. Among the drug-resistant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) has been identified as the leading cause of mortality related to antibiotic resistance in both clinical and environmental settings [1], [3]. MRSA was listed as a high-priority bacterial pathogen by World Health Organization (WHO) [4]. According to 2023 data from the China Antimicrobial Resistance Surveillance Network, MRSA remained a prevalent multidrug-resistant bacterium in both environmental and healthcare settings. To meet the challenging global antibacterial resistance, it was requesting to develop alternative antibacterial strategies including non-antibiotic and combination therapies with minimal and complementary antibiotics uses [5].

The lipoprotein complexes HAMLET or BAMLET, formed by human or bovine α-lactalbumin (α-LA) and oleic acid, were well known for their anticancer and antibacterial activities [6], [7]. Particularly, it was recently reported that than can also function as antibiotic adjuvants to restore the susceptibility of drug-resistant bacteria to conventional antibiotics [5], [8]. Nevertheless, the labor-intensive preparation process may limit the translational biomedical applications. Meanwhile, owing to the remarkable drug-loading capacity, easy surface chemistry, and superior biocompatibility, mesoporous silica nanoparticles (MSN) held promise for the delivery of therapeutic compound such as anticancer and antibacterial agents [9], [10], [11]. In our previous studies, we have successfully developed a simplified preparation method of lipoprotein complexes conjugated MSN nanocomposite as antibiotics adjuvant [5].

Previous studies have indicated that different pH conditions may determine the assembly conformation of oleic acid bound α-linolenic acid complex [12]. As the pH decreased, the tryptophan residues of α-LA was gradually exposed to a more hydrophilic environment. At the acidic pH of 3, α-LA populated mainly adopting a stable secondary structure while exposing multiple hydrophobic clusters [12]. Under acidic conditions, a homogeneous complex of BAMLET cannot be formed, and MSN is required as a carrier to achieve a stable conformation. It has been demonstrated that the secondary structure of proteins on MSN surfaces is altered by pH changes and that the protein corona is modulated by protein-nanoparticle interactions [13].

Accordingly, when complexes are synthesized with MSN at different pH levels, varying degrees of unfolding and hydrophobic exposure are induced in the surface lipoproteins. Note that the increase in hydrophobic domains on the protein surface can significantly strengthen its interaction with bacterial cell membranes and thereby enhancing the protein’s ability to translocate across the cell membrane. It was reported that antibacterial peptides with higher hydrophobicity can more readily penetrate bacterial membranes, leading to cell death through disruption of membrane permeability [14]. Nevertheless, despite extensively mechanistic studies on HAMLET or BAMLET lipoprotein complex from the therapeutic perspective, it remained elusive how the conformational changes of lipoprotein complexes were correlated with the biological functions such as antibacterial activities.

Isobavachalcone (IBC), a natural flavonoid derived from wide-distributed Psoralea corylifolia in south Asia, which was used as an important herb in Chinese medicine involving diverse pharmacological activities including anticancer, antimicrobial, and anti-inflammatory effects [15], [16], [17]. An earlier study indicated that IBC can target the cell membrane of MRSA, highlighting its potential as a candidate antibiotic against drug-resistant bacteria [15]. However, the poor water solubility and low bioavailability of IBC may limit its therapeutic uses. To overcome the shortcomings of single use of antibacterial agent, the combination therapy showed advantages from the practical purposes. For example, when two drugs concurrently target the bacterial membrane, one agent may enhance membrane permeability or compromise its integrity, thereby promoting the intracellular accumulation of the other and yielding a synergistic antibacterial effect [18]. Alternatively, the utilization of MSN as drug carrier to enhance therapeutic efficiency has also been extensively explored [19].

In our previous studies, it was found that size-optimized MSN represented an ideal delivery vehicle for hydrophobic drugs [5], [20], [21]. Therefore, in this study, we set out to explore the combination use of lipoprotein complexes coated MSN and IBC for synergistic antibacterial applications. Specifically, a pH-shift preparation of lipoprotein complex, NBAMLET and HBAMLET was made under basic (pH = 8) and acidic (pH = 3) conditions, respectively, which was further conjugated into MSN to produce lipoprotein-based complexes of NBMSN and HBMSN. The antibacterial activity of various formulations against S. aureus and MRSA was assessed by comparing antibiotic monotherapy with combination therapies under different preparation pH conditions. The obtained nanocomposites under distinct pH was characterized by a set of physicochemical methods. It was found that varying the synthesis pH may induce pronounced conformational changes of surface-conjugated lipoprotein on the siliceous support with different exposure of protein hydrophobic interiors, which may facilitate passive adsorption onto bacterial membranes contributing to improved antibacterial activity of the formulated complex [22]. The synergistic antibacterial effects between lipoprotein complexes and IBC were studied using IBC-loaded formulations targeting bacterial membranes, which was further validated based on the analysis of bacterial morphology, biofilm formation, and membrane function. In a murine subcutaneous infection model, HBMSN co-administered with antibiotics and IBC effectively killed bacteria at infection sites, reduced antibiotic-associated toxicity, and attenuated local inflammation. The present study indicated that HBMSN prepared under acidic conditions can restore aminoglycoside susceptibility in resistant strains and act as delivery vehicle for lipoproteins and hydrophobic compounds like IBC.

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Materials

Cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), sodium hydroxide (NaOH), hydrochloric acid (HCl), crystal violet, and anhydrous ethanol were obtained from Sinopharm Chemical Reagent (Shanghai, China). Isobavachalcone (IBC) was from Chengdu Herbal Reference Standards Biological Technology Co., Ltd. Bovine α-lactalbumin (BLA, calcium depleted, ≥85% purity) was purchased from Sigma Aldrich. Triethanolamine (TEA), 3-aminopropyltriethoxysilane (ATPES), oleic acid, gentamicin sulfate, kanamycin, 8-anilinonaphthalene-1-sulfonic acid (8-ANS), and Triton X-100 were obtained from Shanghai Aladdin biochemical technology. Cell Counting Kit-8 (CCK-8), DiSC3(5) fluorescent probe was from MedChemExpress. Propidium iodide (PI) was from Beijing Solarbio Science & Technology Co., Ltd. Experimental reagents were prepared using deionized water, and all other chemicals and reagents were of analytical purity. All vessels and reagents used were sterilized prior to the test.

Yuqing Li, Ling Cai, Jinhuan Li, Jiazi Luo, Minghui Ji, Jin Chen, Yanqiang Huang, A pH-shift preparation of lipoprotein complex conjugated mesoporous silica nanocomposite contributed to synergistic antibacterial therapy of drug-resistant bacteria, Chemical Engineering Journal, 2026, 178294, ISSN 1385-8947, https://doi.org/10.1016/j.cej.2026.178294.

Read also our introduction article on Mesoporous Silica here: