Environmental and anthropogenic drivers of AR

In addition to clinical misuse, subtherapeutic environmental antibiotic exposure is a major driver of AR. Widespread antibiotic application in medicine, veterinary practice, agriculture, and aquaculture, combined with insufficient wastewater treatment and pharmaceutical manufacturing effluents, has resulted in the continuous release of antimicrobial residues into soil and aquatic environments [6, 20, 21]. These conditions create persistent selective pressure that favors resistant microorganisms and facilitates the horizontal transfer of antibiotic resistance genes (ARGs).

Figure 2 summarizes the main anthropogenic and natural sources of environmental antibiotic contamination. Agricultural runoff, livestock and aquaculture practices, improper medical-waste disposal, and pharmaceutical production introduce antibiotics into soil and water, where they interact with diverse microbial communities. Environmental compartments—soil, water, and biofilms—act as reservoirs and transfer media, facilitating the spread of resistance genes across ecosystems and their eventual re-entry into clinical settings.

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Figure 2. Environmental and anthropogenic drivers of antibiotic resistance. Key sources—including agriculture, pharmaceutical production, human and animal antibiotic overuse, aquaculture, and contaminated water—create selective pressure, promoting bacterial adaptation, horizontal gene transfer, and consequences such as therapy failure, prolonged illness, hospitalization, and occasionally death. The figure was created in BioRender. Ruano, M. (https://BioRender.com/30itgss) is licensed under CC BY 4.0.

Mechanisms of bacterial resistance

Bacteria employ multiple, often synergistic mechanisms to evade antibiotic action [1]. These include:

• 1.

  Enzymatic inactivation or modification, such as β-lactamase-mediated hydrolysis of β-lactam antibiotics;

• 2.

  Target modification, involving mutations or chemical alterations that reduce drug binding to ribosomes, enzymes, or cell wall components;

• 3.

  Active efflux, where membrane-embedded transporters expel antibiotics and reduce intracellular concentrations;

• 4.

  Reduced permeability, resulting from alterations in membrane porins or cell envelope composition; and

• 5.

  Horizontal gene transfer, via conjugation, transformation, or transduction, enables rapid dissemination of resistance traits across species.

These mechanisms are often enhanced by chronic exposure to low antibiotic concentrations in environmental matrices, accelerating the emergence of MDR and XDR phenotypes. Understanding these resistance pathways is critical for the rational design of advanced biomaterials and delivery systems that can bypass or suppress conventional resistance mechanisms.

Stagnation in antibiotic discovery and emerging solutions

Despite advances in microbiology and medicinal chemistry, antibiotic discovery has stagnated over the past three decades. No fundamentally new antibiotic classes have reached the market in this period; most recently approved agents are structural derivatives of existing drugs [22, 23]. As a result, monotherapy has become increasingly ineffective for severe infections, necessitating combination regimens, as exemplified by multidrug-resistant tuberculosis [24].

In response, a range of complementary strategies is under investigation, including antimicrobial stewardship to reduce selective pressure, development of antibiotics with novel molecular targets, bacteriophage and probiotic therapies, and gene-editing approaches such as CRISPR-based interventions. Advanced drug delivery systems (DDSs), including nanocarriers, lipid nanoparticles, and bio-inspired materials—have emerged as powerful tools to enhance antibiotic efficacy, improve bioavailability, reduce off-target toxicity, and overcome bacterial defence mechanisms [17, 25]. These material-enabled strategies shift the paradigm from purely pharmacological solutions toward integrated bio-material–microbe interface engineering.

Mechanisms of antibacterial nanoparticle action

NPs exert antibacterial effects via multiple, often concurrent mechanisms, reducing the likelihood of resistance compared with conventional antibiotics [31]. The known antibacterial mechanisms include:

• 1.

  Physical membrane disruption: Direct interactions of sharp NP edges with bacterial cell walls compromise membrane integrity, leading to leakage of cytoplasmic contents [32].

• 2.

  Generation of ROS: Oxidative stress overwhelms bacterial defences, even in dark conditions [33, 34].

• 3.

  Bacterial trapping/immobilization: NP aggregation can physically trap bacteria, limiting nutrient uptake and proliferation [35].

• 4.

  Redox imbalance: NPs disrupt cellular redox homeostasis, damaging proteins, lipids, and DNA [36].

• 5.

  Metabolic interference: NPs may inhibit glycolysis or energy production, impairing bacterial survival [37].

• 6.

  DNA damage: Interaction with genetic material can induce strand breaks and replication errors [38].

• 7.

  Electric charge transfer: Electron exchange between NPs and bacteria can disrupt membrane potential and enzymatic function [39].

• 8.

  Metal-ion release: Release of ions (e.g., Ag⁺) interferes with enzymes and membrane integrity [40].

• 9.

  Nanobubble formation/explosion: Localized cavitation events can mechanically damage bacterial structures [41].

• 10.

  Charge polarity effects: Surface charge polarity of NPs influences bacterial adhesion and membrane disruption [42].

The multimodal action of NPs underpins potent activity against multidrug-resistant pathogens and biofilms, where single-target antibiotics often fail (Figure 3).

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Figure 3. Multimodal antibacterial mechanisms of nanoparticles (NPs). NPs disrupt bacterial membranes, induce ROS and redox stress, impair metabolism, damage DNA, release toxic ions, and alter membrane potential. These combined actions enhance bactericidal efficacy and limit the development of conventional resistance [43]. The figure was created in BioRender. Ruano, M. (https://BioRender.com/yx7ilj1) is licensed under CC BY 4.0.

Comparative landscape of antimicrobial nanocarriers

Various nanocarrier systems differ in biocompatibility, toxicity, stability, and translational potential (Table 1). Metallic NPs (Ag, Au, Fe₃O₄) show strong antibacterial activity but risk oxidative toxicity and accumulation [44]. Metal oxide NPs (ZnO, TiO₂, CeO₂) act as potent antibiotic enhancers, though biodegradability can be limited [12]. Polymeric NPs (PLGA, chitosan) provide controlled release but may present monomer toxicity and batch variability [45]. Carbon-based nanomaterials are effective due to high surface area and membrane interactions but face regulatory hurdles [46]. LNPs combine high biocompatibility, biodegradability, and scalability, making them highly translational for antimicrobial applications [10].

Lipid nanoparticles as a privileged antimicrobial platform

LNPs uniquely balance antibacterial efficacy with biocompatibility, offering minimal systemic toxicity, low immunogenicity, and limited long-term accumulation. Composed of physiologically compatible lipids, they exhibit minimal systemic toxicity, low immunogenicity, and limited long-term accumulation [51, 53, 54]. LNPs encapsulate both hydrophilic and hydrophobic antimicrobials, protecting labile agents from enzymatic degradation and enabling controlled or stimuli-responsive release [52, 54, 55].

LNP systems include non-vesicular platforms (solid lipid nanoparticles, nanostructured lipid carriers, cubosomes, lipid–drug conjugates) and vesicular systems (liposomes, lipid–polymer hybrids, niosomes, bilosomes) [56–58]. Cubosomes, for instance, enhance antimicrobial peptide stability and achieve rapid bactericidal activity, outperforming free peptides [59]. Similarly, cubosome-encapsulated norfloxacin improved local deposition by ~150% in otitis externa models [60]. Clinically, lipid-based nanocarriers such as AmBisome^®, Abelcet^®, and Epaxal^® exemplify their translational readiness [61].

Clinical advantages and future potential of lipid nanoparticles

LNPs exhibit high biocompatibility, superior drug loading, robust colloidal stability, and scalable manufacturing, outperforming many polymeric and inorganic systems (Table 2) [10, 57]. Their surfaces can be functionalized for targeted delivery, enhancing biodistribution for personalized medicine [62].

These attributes position LNPs as a leading platform for targeted antimicrobial delivery, reduction of systemic toxicity, facilitation of combination therapies, and support of personalized medicine approaches.

Relevance of nanoparticles to the chicken embryo model

The CEM provides a vascularized and cost-effective system. It is ethically favorable for evaluating nanoparticle biodistribution, toxicity, and therapeutic efficacy. By enabling injection into the chorioallantoic membrane (CAM) or yolk sac, the CEM allows assessment of NP–host and NP–pathogen interactions, infection progression, and mid-throughput screening prior to mammalian studies [63, 64].

In vitro screening of nanoparticle therapeutics

In vitro assays provide the first step in NP evaluation, offering controlled conditions, high throughput, and cost efficiency. They are commonly used to assess cytotoxicity (e.g., MTT or Alamar Blue™), cell viability, metabolic activity [67], and mechanistic endpoints such as cytokine release, oxidative stress, and gene expression [68]. Antimicrobial activity is typically evaluated using disc diffusion, broth microdilution, or metabolic readouts, enabling direct comparison with conventional antibiotics [44, 68, 69].

The main advantages of in vitro screening are experimental control, scalability, and rapid identification of lead candidates (Table 3). These assays also align with the 3Rs principle, reducing reliance on animal testing while providing mechanistic insight into NP–cell and NP–pathogen interactions. Limitations include the absence of systemic factors (immune response, metabolism, multicellular interactions), oversimplified tissue models, and limited predictive power for long-term toxicity, chronic exposure, or in vivo efficacy. Advanced commercial models, such as organotypic intestinal systems, developed by Alimetrics, improve physiological relevance but cannot fully replace in vivo validation.

In vitro assays are indispensable for early-stage NP screening but must be complemented by intermediate and mammalian models to assess systemic safety and therapeutic performance.

In vivo and ex vivo screening models

In vivo studies capture complex organism-level interactions, including pharmacokinetics, tissue distribution, immune responses, efficacy, and toxicity. Ex vivo models complement these studies by preserving tissue or organ physiology under controlled conditions, enabling mechanistic investigation of NP–host interactions.

Model selection balances ethical considerations, cost, throughput, and translational relevance. Mammalian models (mice, rabbits, non-human primates) provide detailed pharmacokinetic, immunological, and toxicological data [70] but are constrained by high cost, ethical requirements, and low throughput (Table 4). Non-mammalian models, such as Caenorhabditis elegans [45, 71–73], Drosophila melanogaster [74], Galleria mellonella [75–78] and zebrafish [79–81] provide cost-effective, ethically favorable alternatives for higher-throughput screening, though species differences limit predictive power for humans.

Ex vivo platforms bridge the gap between in vitro and in vivo studies, maintaining tissue architecture and mechanistic insight while lacking systemic integration.

Overall, in vivo models remain indispensable for comprehensive safety and efficacy evaluation, while non-mammalian and ex vivo systems support early decision-making and mechanistic insight. A tiered screening strategy combining these models is therefore essential to balance translational relevance, ethical responsibility, and development efficiency.

Avian immune development in the CEM

The CEM exhibits a rapidly maturing immune system with structural and functional parallels to humans, enabling translationally relevant studies of host–pathogen interactions and immunomodulatory interventions (Figure 4).

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Figure 4. Comparative immune development in humans and chickens. Human immune maturation occurs over ~40 gestational weeks (embryonic weeks, EW), whereas chicken immune development is completed within 21 embryonic days (ED). Key primary and secondary lymphoid organs are shown, including the thymus, spleen, bone marrow (humans), and the bursa of Fabricius (chickens), as well as mucosa-associated lymphoid tissue (MALT) in humans and cecal tonsils in chickens, highlighting conserved functional immunity despite differences in developmental timing and MHC diversity. The figure was created in BioRender. Ruano, M. (https://BioRender.com/d52c6jx) is licensed under CC BY 4.0.

Primary lymphoid organs develop in a defined sequence: the thymus supports T-cell differentiation (γδ T cells precede αβ T cells), the bursa of Fabricius mediates B-cell maturation (functionally analogous to human bone marrow), and the spleen acts as a secondary lymphoid organ in both species. Innate immunity is highly conserved; Toll-like receptors (TLRs) detect pathogen-associated molecular patterns, triggering comparable inflammatory signaling in chickens and humans.

Antigen presentation occurs through the major histocompatibility complex (MHC), which, despite lower genetic diversity in chickens (~46 genes versus > 200 in humans), enables effective T-cell activation. Early emergence of macrophages, dendritic cells, and NK cells, together with embryonic cytokine expression (IL-1β, IL-8, IFN-γ), allows functional evaluation of immune responses pre-hatch. The rapid, functional immune maturation makes the CEM a versatile platform for screening nanocarriers and immunomodulatory therapies, bridging mechanistic insight with potential clinical translation.

Methodological approaches in the CEM

The CEM enables multiple experimental configurations for nanomaterial evaluation (Figure 5). In ovo cultivation preserves physiological conditions and is used for toxicity, survival, and biodistribution studies [86]. Ex ovo cultivation allows direct access to the CAM for angiogenesis assays and imaging-based analyses, with higher risk of dehydration and contamination [87].

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Figure 5. Experimental workflow of the CEM. Overview of in ovo and ex ovo cultivation, nanoparticle delivery routes (CAM injection, yolk sac injection, topical CAM application), and analytical readouts, including CAM angiogenesis assays, fluorescence/bioluminescence imaging, histological analysis, and toxicity assessment, for integrated evaluation of nanocarrier biodistribution and efficacy. CAM: chorioallantoic membrane; CEM: chick embryo model; HET-CAM: Hen’s Egg Test – Chorioallantoic Membrane; HET-CAV: Hen’s Egg Test – Chorioallantoic Vasculature. The figure was created in BioRender. Ruano, M. (https://BioRender.com/v51f276) is licensed under CC BY 4.0.

NP administration is performed via CAM injection [87], yolk sac injection [88], or topical CAM application [89], depending on the intended exposure profile. CAM injection enables localized assessment of vascular interactions, angiogenic responses [90], and antimicrobial efficacy. Yolk sac injection provides systemic exposure, supporting evaluation of NP circulation and organ distribution. Topical CAM application allows confined delivery to the membrane surface and is particularly suited for localized antibacterial or anti-angiogenic studies. These standardized routes, combined with multimodal readouts [82, 91], support reproducible and translational screening of LNPs in the CEM.

Pathogen studies using the CEM

The CEM is widely applied for infection modeling and antimicrobial evaluation, leveraging CAM accessibility, high vascularization and favorable ethical status. The model supports controlled investigation of pathogen invasion, host responses, and nanocarrier-mediated therapeutic efficacy across multiple microbial classes.

CEM-based studies encompass bacterial, fungal, parasitic, and probiotic systems, including clinically and agriculturally relevant pathogens. Bacterial models include Klebsiella pneumoniae, Escherichia coli K-12, Salmonella Typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium perfringens, Enterococcus cecorum, Chlamydia psittaci, and Chlamydia abortus [92]. Fungal infections have been established using Candida albicans [93], Aspergillus fumigatus [94], and Cryptococcus gattii [90], while parasitic infections such as Neospora caninum [95] have also been reported. In parallel, probiotic interventions (e.g., Enterococcus faecium, Bacillus subtilis) have been evaluated against enteric pathogens including Campylobacter jejuni [96].

Antimicrobial assessment in the CEM typically follows a tiered screening strategy. Initial in vitro assays (disc diffusion, broth microdilution, metabolic viability assays) define minimal inhibitory concentrations and baseline antimicrobial activity. Subsequent in vivo/ex vivo infection studies involve pathogen inoculation at defined embryonic stages (commonly ED5–ED10) via CAM, yolk sac, allantoic cavity, or amniotic routes, followed by quantification of embryo survival, pathogen burden, histopathology, and immune markers. Topical CAM application further enables localized therapeutic evaluation while limiting systemic exposure.

Pathogen-dependent outcomes are consistently observed in the CEM. Highly invasive extracellular bacteria induce rapid vascular dissemination and embryo lethality, whereas intracellular pathogens display delayed progression and tissue-restricted infection. Fungal pathogens show species-specific virulence profiles reflected in survival kinetics, inflammatory responses, and CAM vascular remodeling.

Methodological heterogeneity limits reproducibility. Variability in inoculation route, embryonic day, pathogen dose, cultivation mode, and relative humidity (RH) significantly affects infection outcomes and complicates cross-study comparison. Ex ovo cultivation is associated with increased baseline mortality, while RH fluctuations markedly influence embryonic susceptibility (Table 6), underscoring the need for standardized infection protocols.

Applications of the CEM in NP research

The CEM provides a rapid, translational platform for NP evaluation: rapid organogenesis (21 days) [13], low ethical constraints before ED15 [108], human-relevant physiology, and highly vascularized CAM for real-time NP tracking. Physiological and immune parallels with humans (Figure 4) support mechanistic insights, while the highly vascularized CAM allows real-time assessment of NP biodistribution and efficacy [109].

Limitations of the CEM

The CEM provides a cost-effective, ethically favorable platform for infection and therapeutic studies, but several biological and technical constraints must be considered. Table 7 summarizes the main limitations.

Remaining gaps include standardized pathogen inoculation and LNP dosing, limited systemic immune evaluation, and few comparative studies with mammalian models to validate translational relevance. Complex phenomena such as wound healing or scaffold dynamics remain challenging to assess in this model.

Proposed framework for standardization of CEM-based studies

To enhance reproducibility and comparability, we propose the following standardized framework in Table 8.

Future research directions

Despite the promise of LNPs and the CEM, the field currently faces key gaps: lack of standardized protocols across infection models, limited comparative studies of LNPs versus other nanocarriers and insufficient translational validation between CEM and mammalian systems.

Future studies should focus on systematic, mechanistically guided, and standardized investigations to maximize the CEM’s predictive power and accelerate clinical translation of nanoparticle-based therapeutics and key areas for further development include:

• 1.

  Standardization of experimental parameters: harmonized SOPs for inoculation, LNP dosing, temperature, and RH to reduce variability.

• 2.

  Integration of advanced readouts: use of fluorescence lifetime imaging, whole-embryo tomography, and transcriptomic profiling to better understand LNP biodistribution and infection mechanisms.

• 3.

  Improved immune modelling: use immune modulators, cell transplants, or immune-reporter pathogens to evaluate host responses.

• 4.

  Pathogen-specific frameworks: tailor inoculation days, RH, and disease markers to major bacterial, fungal, and parasitic species.

• 5.

  Rational LNP optimization: screen LNP formulations varying in lipid composition, surface chemistry, or targeting ligands.

• 6.

  Bridging to mammalian models: benchmark CEM outcomes against rodents to validate translational predictivity.