Dual Core-Shell Loaded Lipid-Polymer Hybrid Nanoparticles as Combination Anti-Infective Delivery Platforms

Abstract

Background/Objectives: The growing threat posed by antimicrobial resistance to worldwide public health highlights the urgent need not only for new anti-infective candidates, but also for innovative formulation strategies capable of mediating effective delivery of anti-infective compounds. The current study, therefore, aimed to demonstrate the feasibility of formulating lipid-polymer hybrid nanoparticles (LPHNPs) with dual loading of both core and shell compartments for combination anti-infective delivery.

Methods: LPHNPs containing the antibiotic cefotaxime within a chitosan polymer core and the novel antimicrobial peptide RN7IN6 within a bacteria-mimicking lipid shell were produced by microfluidic mixing, and optimized with respect to parameters including total flow rate, flow rate ratio, and lipid concentration. Minimum inhibitory concentrations of cefotaxime and RN7IN6 co-incorporated in LPHNPs were assessed as a preliminary indicator of antibacterial efficacy.

Results: Uniformly nanosized LPHNPs were produced, with maximized loading of cefotaxime and RN7IN6 within particle cores and shells, respectively. Empty LPHNPs showed an appreciable antibacterial activity, particularly against the Gram-negative bacterium Escherichia coli, while RN7IN6 was indicated to enhance cefotaxime activity against E. coli when both actives were incorporated in LPHNPs.

Conclusions: The current findings clearly demonstrate the feasibility of formulating LPHNPs for core-shell co-encapsulation and delivery of anti-infectives. The promising antibacterial efficacy of co-loaded LPHNPs warrants further in-depth investigation to determine the extent of co-loaded LPHNP applications as combination anti-infective delivery platforms.

Introduction

Antimicrobial resistance (AMR) is a major and growing threat to public health worldwide, with significant effects on global morbidity, mortality, and healthcare costs. Unless impactful measures can be implemented, drug-resistant infections have been projected to result in 10 million deaths globally by 2050 [1]. Bacterial AMR, of particular concern, has already been reported to have contributed to 4.95 million deaths worldwide in 2019 alone [2], with bacterial cell envelope structures often acting as significant barriers to effective anti-infective delivery even in wild-type organisms [3]. As a further problem, diminishing flow through the antibiotic pipeline means that the shrinking pool of effective antibiotics is not being refilled by additional or novel treatment options [4,5]. This escalating global crisis draws attention to an urgent need not only for new anti-infective candidates with alternative targets and/or modes of action to traditional antibiotics, but also importantly, for innovative strategies to facilitate the effective delivery of novel and traditional anti-infective therapies into and across bacterial cell envelope structures to reach their targets.

Lipid-polymer hybrid nanoparticles (LPHNPs) consisting of a robust polymer core surrounded by a membrane-like lipid shell have been widely investigated to date for the delivery of cancer therapeutics [6,7,8], and are emerging as a delivery strategy of promise in the context of infection research. LPHNPs combine the benefits (and may circumvent the limitations) of polymeric nanoparticles and liposomes as separate delivery systems—the LPHNP polymeric core provides a high structural stability and capacity for drug loading, while the lipid shell confers an enhanced biocompatibility, and the potential to prevent premature escape or even affect controlled release of drug payload [9,10]. In an innovative step, the lipid shell component can be further engineered to consist of native bacterial cell membranes or synthetic bacteria-relevant lipid mixtures, leading to the fabrication of bacteriomimetic LPHNPs possessing the potential for enhanced interaction with and improved anti-infective delivery to target bacterial cell envelopes [11,12,13].

The distinct two-compartment structure of LPHNPs further creates the possibility for co-encapsulation of hydrophilic and lipophilic actives within a single carrier structure. While core-shell loading of LPHNPs with chemotherapeutic agents has been previously trialed [8,14], to the best of the authors’ knowledge, incorporation of anti-infectives separately into core and shell structures of LPHNPs has not been extensively investigated. This is of significant interest in the context of combination therapy, which involves the simultaneous use of anti-infectives of differing but complementary modes of action. Delivering combinations of anti-infectives may lead to an enhanced antibacterial activity compared to single-agent use, in turn facilitating a reduction in therapeutic drug dose and mitigating resistance development [15,16]. The considerable potential for individual actives within a combination to have differing physicochemical properties (including water solubility/lipophilicity), however, creates practical challenges for co-administration, and as such may present a hurdle to effective translation of certain combination approaches. This renders the ability to incorporate and deliver combinations of anti-infectives via a single nanocarrier platform an attractive prospect.

The current work was, therefore, designed to serve as an in-depth proof-of-concept study, with the aim of exploring the feasibility of formulating LPHNPs as dual-compartment platforms for co-encapsulation and delivery of anti-infective compounds. To this end, a process for the production of LPHNPs consisting of a chitosan polymer core encapsulating the traditional antibiotic cefotaxime and a bacteria-relevant lipid shell incorporating the novel antimicrobial peptide RN7IN6 was established. With a view to creating clear possibilities for scale-up and translation, a microfluidic mixing technique was employed for this purpose, and a detailed investigation of the impact of varying parameters such as flow rate ratio (FRR), total flow rate (TFR), and component concentrations was conducted. This process was optimized to produce uniformly nanosized LPHNPs, with maximal anti-infective co-loading. Co-loaded LPHNPs were then screened in a preliminary antibacterial efficacy study to gauge the extent of their future promise as combination anti-infective delivery platforms. The study demonstrates a considerable potential for co-loaded LPHNPs as platforms for combination anti-infective delivery, and highlights a number of key focal points for further, in-depth investigation to fully explore the extent of this potential.

Download the full article as PDF here Dual Core-Shell Loaded Lipid-Polymer Hybrid Nanoparticles as Combination Anti-Infective Delivery Platforms

or continue reading here

Materials

Mueller–Hinton Broth 2 (MHB2), nutrient agar, and resazurin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chitosan hydrochloride (MW 30–400 kDa, degree of deacetylation 80–95%) was obtained from Heppe Medical Chitosan GmbH (Halle, Germany). Phosphate-buffered saline (PBS, pH 7.4) tablets were obtained from Oxoid Ltd. (Basingstoke, UK). 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (POPG), and 1′,3′-bis [1,2-dioleoyl-sn-glycero-3-phospho]- glycerol (sodium salt) (tetraoleoyl cardiolipin, CL) were purchased from Avanti Polar Lipids (Merck, Rahway, NJ, USA). HPLC-grade acetonitrile and methanol were purchased from Thermo Fisher Scientific (Waltham, MA, USA). N,N-dimethylformamide (DMF) was bought from Acros Organics (Bridgewater, NJ, USA). 1-propanol and formic acid were purchased from VWR (Radnor, PA, USA). Trifluoroacetic acid (TFA) was purchased from Fisher Scientific (Hampton, NH, USA). Cefotaxime sodium salt (CTX, ≥95%) was obtained from Enzo Life Sciences (Exeter, UK). The antimicrobial peptide (AMP) RN7IN6 (amino acid sequence FLGGLIKWWPWRR-NH2, MW 1709.078 g/mol [17] purity > 95%) was synthesized and purified in-house [18], as detailed in the Supplementary Materials.

Carini, V.; Scagnetti, G.; Foulkes, J.; Evans, K.; Saleem, I.; Gordon, S. Dual Core-Shell Loaded Lipid-Polymer Hybrid Nanoparticles as Combination Anti-Infective Delivery Platforms. Pharmaceutics 2026, 18, 13. https://doi.org/10.3390/pharmaceutics18010013

Read also out interesting article:

World Antimicrobial Resistance Awareness Week 2025