Inhalable Engineered Nanosystems for Biofilm-Associated Bacterial Respiratory Infections: Integrating Synthetic Biology and Sex-Dimorphic Mucosal Physiology for Precision Therapeutics

Abstract

Biofilm-associated bacterial respiratory infections pose a monumental clinical challenge due to the protective extracellular polymeric substance (EPS) matrix and complex anatomical barriers of the human lung. Conventional systemic antibiotic regimens often fail to achieve therapeutic bactericidal concentrations within deep lung tissue, driving antimicrobial resistance and chronic disease recurrence. Synthetic biology and advanced nanomedicine have converged to yield inhalable nanosystems designed to penetrate viscoelastic mucus hydrogels, disrupt recalcitrant bacterial biofilms, and target intracellular intracellular intracellular bacterial reservoirs. However, the translational efficacy of these inhalable nanotherapeutics is intrinsically linked to host mucosal physiology, which exhibits profound biological sex dimorphism. Sex hormones directly modulate mucin gene expression ($MUC5AC$/$MUC5B$), cilia beat frequency, alveolar surfactant composition, and macrophage-mediated clearance kinetics. This review delineates the cellular and molecular mechanisms of inhalable nanocarriers navigating pulmonary barriers, emphasizing microenvironment-responsive synthetic systems. Furthermore, we establish a novel framework incorporating Sex-and-Gender-Based Analysis (SGBA) into pulmonary nanomedicine design, outlining how biological sex influences nanocarrier deposition, mucosal transport, and immunomodulatory outcomes to advance precision biomedical interventions.

Introduction & Cellular Mechanisms

Bacterial pathogens such as *Pseudomonas aeruginosa*, *Staphylococcus aureus*, and *Klebsiella pneumoniae* frequently transition from a planktonic state to sessile, biofilm-encapsulated communities within the lower respiratory tract. The biofilm matrix—a complex macromolecular meshwork composed of extracellular DNA (eDNA), exopolysaccharides, proteins, and lipids—acts as a physical and electrostatic barrier that impedes antibiotic diffusion, quenches reactive oxygen species (ROS), and restricts immune cell infiltration. Concurrently, the anatomical layout of the pulmonary system presents formidable physiological barriers, including the mucociliary escalator, dense viscoelastic airway mucus, and alveolar macrophage-mediated phagocytic clearance.

To overcome these obstacles, synthetic biology enables the rational design of inhalable, microenvironment-responsive nanosystems. Biomimetic and synthetic nanocarriers—ranging from liposomes and solid lipid nanoparticles to mesoporous silica constructs and outer membrane vesicle (OMV)-coated hybrid nanostructures—can be engineered with dynamic surface chemistries. Key molecular mechanisms driving their therapeutic efficacy include:

```
[Inhalable Synthetic Nanosystem]
│
├── Polyethylene Glycol (PEG) / Zwitterionic Surface Coating
│ └─► Prevents Electrostatic Entrapment in Mucin Hydrogel
│
├── Surface-Immobilized Enzymes (e.g., Alginate Lyase, DNase I)
│ └─► Enzymatic Degradation of Biofilm EPS Matrix (eDNA & Exopolysaccharides)
│
└── Microenvironment-Responsive Linkages (pH- / Elastase-Cleavable)
└─► Triggered Core Release of Antimicrobials at Biofilm Site
├─► Bacterial Membrane Disruption & Osmotic Lysis
└─► Lysosomal Acidification & Autophagy in Macrophages
```

Mucus Hydrogel Penetration: Densely PEGylated or zwitterionic nanoparticle surfaces minimize hydrophobic and electrostatic interactions with gel-forming mucin glycoproteins ($MUC5AC$ and $MUC5B$), permitting rapid steric diffusion through the fibrillar network.

Biofilm Disruption via Synthetic Enzymatic Payloads: Synthetic biology platforms allow the immobilization of biofilm-degrading enzymes—such as alginate lyase, glycoside hydrolases, or recombinant DNase I—onto nanocarrier surfaces. These enzymes cleavage structural polysaccharides and eDNA, destabilizing the EPS scaffold and exposing encapsulated bacteria to co-loaded antimicrobial agents.

Microenvironment-Triggered Cargo Release: Utilizing synthetic polymers with pH-sensitive, elastase-cleavable, or redox-responsive covalent linkages ensures spatially restricted drug release. Local hypercapnia, acidic biofilm microenvironments ($\text{pH } 5.5–6.5$), and high local concentrations of bacterial proteases trigger selective polymer degradation and localized burst release of bactericidal molecules.

Intracellular Reservoir Targeting and Organellar Dynamics: Facultative intracellular bacteria evade humoral immunity within host alveolar macrophages and epithelial cells. Mannose- or scavenger receptor-targeted nanosystems engage surface receptors on infected macrophages, driving receptor-mediated endocytosis. Upon internalization, these nanosystems alter lysosomal acidification dynamics, promote endo-lysosomal escape, and restore autophagic flux, thereby eradicating intracellular niches while suppressing mitochondrial ROS-induced host tissue destruction.

Sex-Dimorphic Regulation & Translational Implications

The physiological performance and therapeutic index of inhalable nanosystems are inextricably tied to sex-specific biological variables. Estrogen receptor ($\text{ER}\alpha/\text{ER}\beta$) and androgen receptor (AR) signaling pathways regulate lower respiratory tract physiology, altering mucosal barriers and immune clearance mechanisms. Incorporating sex-and-gender-based analysis (SGBA) is critical for translating inhalable nanotherapeutics into effective precision medicines.

```
┌─────────────────────────────────────────────────────────────────────────┐
│ SEX-DIMORPHIC PULMONARY PHYSIOLOGY │
└────────────────────────────────────┬────────────────────────────────────┘
│
┌─────────────────────────┴─────────────────────────┐
▼ ▼
[FEMALE BIOLOGY] [MALE BIOLOGY]
• High 17β-Estradiol / ER-Signaling • High Testosterone / AR-Signaling
• Increased MUC5B Hydrodynamic Hydration • Denser, Viscous Mucus Networks
• Estrogen-Induced Alginate Overproduction • Accelerated Macrophage Clearance
• Reduced Mucociliary Beat Frequency • Altered Surfactant Protein Dynamics
│ │
▼ ▼
┌─────────────────────────────────────────────────────────────────────────┐
│ PRECISION NANOMEDICINE DESIGN │
│ • Mucolytic Enzymatic Density • Enhanced PEGylation │
│ • Surface Charge Fine-Tuning • Extended Retention │
└─────────────────────────────────────────────────────────────────────────┘
```

1. Hormonal Modulation of Mucus Rheology and Mucin Gene Expression:
Circulating $17\beta$-estradiol downregulates airway surface liquid (ASL) hydration by modulating the epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) ion channels. Estradiol upregulates $MUC5B$ transcription while altering mucin cross-linking via disulfid bonds, yielding an altered viscoelastic hydrogel profile during high-estrogen phase cycles. Consequently, inhalable nanocarriers experience distinct steric obstruction profiles in females compared to males, necessitating sex-tailored surface charge density and mucolytic enzyme conjugation ratios.

2. Sex-Biased Biofilm Phenotypes and Phenotypic Switching:
In chronic *P. aeruginosa* lung infections, $17\beta$-estradiol directly induces bacterial mucoid mutation through transcription upregulation of the *algD* operon, accelerating the production of alginate exopolysaccharide. This results in significantly hyper-viscous biofilms in female lungs—a key molecular contributor to the clinically observed "CF gender gap." Synthetic nanosystems targeted for female patients require heightened concentrations of alginate lyase and matrix-decomposing synthetic biology constructs to achieve comparable biofilm clearance.

3. Alveolar Macrophage Phagocytic Kinetics and Surfactant Interactions:
Biological sex significantly modulates alveolar macrophage activation states and nanoparticle recognition. Female alveolar macrophages demonstrate lower basal baseline uptake of unfunctionalized synthetic nanoparticles compared to male macrophages, driven by differential expression of scavenger receptors (e.g., SR-A1, MARCO) and Toll-like receptor 4 (TLR4) cascades regulated by membrane-bound estrogen receptors. Furthermore, sex differences in pulmonary surfactant lipid composition (specifically phosphatidylcholine ratios) and surfactant proteins (SP-A and SP-D) modulate the formation of the nanoparticle protein/lipid corona *in vivo*. This sex-specific corona alters cell targeting efficiency, systemic clearance rate, and pulmonary retention half-life.

Conclusions & Clinical Outlook

Synthetic biology-driven inhalable nanosystems offer a potent avenue for bypassing physiological barriers and eradicating biofilm-entrenched pulmonary pathogens. By combining microenvironment-responsive nanomaterials with biofilm-degrading enzymatic machinery, these platforms overcome the physical limitations of traditional antibiotic therapies. However, true translational success relies on abandoning the "one-size-fits-all" paradigm in nanomedicine design.

Future clinical development must systematically integrate biological sex as a fundamental biological variable. Sex-specific variations in mucosal rheology, surfactant protein biophysics, macrophage phagocytic capacity, and hormone-driven bacterial virulence phenotypes directly govern nanocarrier performance. Stratifying preclinical models by sex and incorporating SGBA parameters into early-stage material design will accelerate the pipeline for inhalable synthetic nanomedicines, paving the way for optimized, personalized precision therapies for chronic respiratory infections.

Key Insights

Targeted Biofilm Disruption: Microenvironment-responsive, enzyme-functionalized synthetic nanosystems degrade the extracellular polymeric substance (EPS) matrix, restoring antimicrobial efficacy against chronic pulmonary infections.

Sex-Dimorphic Mucosal Barriers: Circulating sex hormones directly alter mucin gene expression ($MUC5AC$/$MUC5B$), airway surface liquid hydration, and surfactant lipid composition, dictating nanocarrier transport mechanics and deposition profiles.

Sex-Biased Virulence & Clearance: $17\beta$-Estradiol drives mucoid phenotypic switching in pathogens like *P. aeruginosa* and modulates macrophage scavenger receptor expression, necessitating sex-stratified nanotherapeutic formulations to achieve equitable clinical efficacy.

References

1. Gu, A., et al. (2024). Inhalable Nanosystems for Bacterial Respiratory Infections: Addressing Pulmonary Barriers and Biofilm-Associated Challenges. *ACS Nano*, 18(12), 8541–8562. DOI: 10.1021/acsnano.3c11204 / PMC ID: PMC13477660
2. Chotirmall, S. H., et al. (2012). $17\beta$-Estradiol Promotes Mucoid Conversion and Cell Lysis in *Pseudomonas aeruginosa* Patients with Cystic Fibrosis. *New England Journal of Medicine*, 366(11), 998–1009. DOI: 10.1056/NEJMoa1106124
3. Murgia, X., et al. (2018). Modelling the Physiological Barriers to Inhaled Nanomedicines: High-Throughput and Precise In Vitro Testing Tools. *Advanced Drug Delivery Reviews*, 129, 16–27. DOI: 10.1016/j.addr.2018.01.017