Nanotechnology-Based Drug Delivery to Control Bacterial-Biofilm-Associated Lung Infections

Nanotechnology-Based Drug Delivery to Control Bacterial-Biofilm-Associated Lung Infections: History

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Subjects: Infectious Diseases

Contributor:

Yutong Guo

Zeyuan Mao

Fang Ran

Jihong Sun

Jingfeng Zhang

Guihong Chai

Jian Wang

Airway mucus dysfunction and impaired immunological defenses are hallmarks of several lung diseases, including asthma, cystic fibrosis, and chronic obstructive pulmonary diseases, and are mostly causative factors in bacterial-biofilm-associated respiratory tract infections. To combat bacterial biofilm in the respiratory tract, researchers have developed various strategies, including a pipeline of new antibiotics, biofilm biomatrix disruption, quorum sensing inhibition, biofilm dispersion promotion, or combinations of these. Among the strategies, nanoparticle-based drug delivery systems have received increasing attention for delivering antibiotics to biofilm sites or enhancing anti-biofilm activity through the nanoparticles themselves.

chronic lung infections
bacterial biofilm
mucosal barriers
nanoparticle-based drug delivery

1. Nanotechnology-Based Diverse Antimicrobials for Bacterial Biofilm Control

Nanotechnology-based antimicrobials represent promising arsenals that have been used to deliver antibacterial agents, effectively targeting planktonic bacteria, antibiotic-resistant strains, and biofilm-forming bacteria. When encapsulated within nanoparticles, antibiotics not only retain their antibacterial activity but also leverage the unique properties of nanoparticles, further amplifying anti-biofilm actions. Such antibiotic-loaded nanoparticles can be further optimized to enhance their anti-biofilm efficacy. Typically, the size of these nanoparticles is smaller than the size of the airway mucus network, enabling them to effortlessly penetrate both the airway mucus and biofilm structures. The new insight into the application of nanoparticles against bacterial biofilm will be reviewed as follows in terms of their types, design strategies, and biofilm-combating mechanisms.

1.1. Physicochemical Properties of Anti-Biofilm Nanoparticles

The physicochemical properties of nanomedicines can significantly influence their efficacy in biofilm control. Biofilms often possess a complex three-dimensional matrix that can hinder nanoparticles from reaching embedded bacteria [97]. To ensure effective penetration, the size of nanoparticles should be smaller than the dimensions of the water-filled channels within biofilms. Multiple studies have suggested that the ability of nanoparticles to penetrate biofilms diminishes as their diameters increase [118,119]. Nonetheless, for intravenous administration, nanoparticles with a diameter below 5 nm might be readily cleared from the body. Importantly, factors such as nutrient conditions and fluid shear presence can lead to variations in the structure of the biofilm matrix, even within the same bacterial species. Additionally, biofilm matrix components differ among various bacterial types. Generally, for effective biofilm control, a nanoparticle diameter below 200 nm is recommended.

Regarding the systemic administration of nanomedicines, the surface charge of the nanoparticles plays a pivotal role in ensuring effective delivery to infectious biofilms. Positive surface charges can enhance bacteria-killing effects through contact-mediated lethal membrane damage and subsequent cell death. Nevertheless, nanoparticles with positively charged surfaces can become targets for opsonization and immune cells recognition, complicating their journey to infected sites via blood circulation [120]. A recent trend suggests using charge-reversible nanoparticles, which balance the stealthy transport properties in blood while retaining their bacteria-killing capabilities.

Beyond size and surface properties, the shape of the nanoparticles also impacts their anti-biofilm effectiveness. Certain shapes enable nanoparticles to adhere more tightly to bacteria, boosting the efficacy of positively charged nanoparticles. Those with sharp edges can compromise bacterial membrane integrity, leading to cellular content leakage and eventual cell death [121,122]. Furthermore, the shape of nanoparticles affects their in vivo behavior, including hemorheological dynamics, cellular uptake, and blood circulation half-life [120].

1.2. Inorganic Antimicrobial Nanoparticles

Inorganic materials, with their distinct physiochemical properties, have been harnessed for centuries. Notably, specific types of inorganic nanoparticles demonstrate potent antimicrobial actions, primarily through ROS generation, DNA damage, and cell membrane destruction. Enhancing these nanoparticles with surface functionalization, via polymers, small ligand molecules, and charged moieties, allows for drug encapsulation and controlled release.

Inorganic nanoparticles are typically prepared by top–down and bottom–up approaches. The top–down approach involves converting larger particles into their nano-sized counterparts, whereas the bottom–up approach constructs nanoparticles from atoms or molecules [17]. Concerning synthesis, these approaches can be categorized into physical, chemical, and green synthesis techniques. Physical methods, including laser ablation, ball milling, ion sputtering, and ultraviolet radiation [123,124], primarily use the top–down approach and often necessitate intricate apparatus and specific conditions. On the other hand, chemical methods such as hydrothermal methods, electrochemical synthesis, sol-gel synthesis, the co-precipitation method, and microemulsion synthesis [125] are quicker and more adaptable. However, the potential toxicity and challenging removal of reagents involved limit their applicability.

Green synthesis, which utilizes plants, bacteria, fungi, or their derivatives as raw materials, is gaining traction due to its sustainable nature and the absence of toxic byproducts [17]. Various plant components, such as polysaccharides, amino acids, enzymes, phenols, and saponins, act as reducing and stabilizing agents, giving rise to inorganic nanoparticles of diverse shapes and sizes. For example, various plant parts, including seeds, fruits, flowers, and roots, can be used for Ag NPs synthesis [126]. Beyond plant sources, microorganisms like algae, fungi, bacteria, and yeast are also employed extensively for nanoparticle synthesis. This microbial synthesis can either be intracellular, where metal ions attach to cell walls via electrostatic forces and undergo enzymatic reduction, or extracellular, which involves microbial metabolites (e.g., lipids, proteins, pigments) serving as capping and reducing agents, simplifying subsequent purification processes [127,128]. A study by Kashyap et al. [129] demonstrated that treating Scenedesmus sp. MT636554 cells with AgNO₃ (0.5 mM and 1 mM) led to the biosynthesis of intracellular Ag/AgCl nanoparticles ranging from 10–50 nm, exhibiting strong antibacterial activity against four bacterial strains.

1.2.1. Ag Antimicrobial Nanoparticles

Ag nanomaterials have been recognized as a promising antimicrobial agent for decades. Their antimicrobial mechanisms include the following: (1) disruption of the bacterial cell wall and membrane structure; (2) damage to subcellular structure [130,131]. Ag NPs have been shown to interact with peptidoglycan on the bacterial cell wall, either directly via individual NPs or through the Ag⁺ ions released from the NPs, leading to bacterial wall disruption [132]. In addition, Ag NPs or the released Ag⁺ ions were found to interact with the biomolecules on the bacterial membrane, notably lipopolysaccharides, causing membrane damage and subsequently cell lysis [133]. Once Ag NPs are endocytosed into the bacterial cytoplasm, the subsequently released Ag⁺ ions emerge as the key factor in driving the antimicrobial activity [134]. Reactions causing oxidation on the Ag NPs’ surface initiate the release of Ag⁺ ions, subsequently leading to the generation of reactive oxygen species (ROS). Elevated levels of Ag⁺ ions and ROS have been implicated in damaging DNA, proteins, and other subcellular structures, which disrupts bacterial metabolism and leads to bacterial lysis [130].

The physiochemical properties of Ag NPs, including their size, oxidation states, and surface coating, influence the release of Ag⁺ ions. Smaller-sized Ag NPs are thought to release Ag⁺ ion quicker than their larger counterparts; this characteristic is attributed to their larger surface area [134]. Therefore, Ag nanoclusters (Ag NCs), defined as ultrasmall NPs with a core of “countable” Ag atoms shielded by surrounding organic ligands, have been developed as new generation of Ag antimicrobial agents [130]. Typically, Ag NCs have a particle size ranging between 1 and 2 nm, a trait that significantly bolsters their antibacterial and antibiofilm properties. Haidari et al. designed an ultrasmall, uniform, and polycationic Ag NC variant for biofilm eradication. Notably, the high percentage (>50%) of Ag⁺ nano-reservoirs on these clusters grants an enhanced ability to Ag NCs to penetrate the bacterial cell membrane and significantly delay the onset of bacterial resistance compared to similarly sized negatively charged counterparts or conventional antibiotics [135]. Although Ag NPs and Ag NCs showed great potential in combating bacterial biofilm, their intrinsic toxicity remains a major obstacle to their clinical application. Amyloid, a recently identified target for bacterial biofilm, has mechanisms of inhibition that remain elusive [136,137,138]. Huma et al. explored the potential of Ag NPs and Ag NCs at sub-bactericidal concentrations to hinder functional amyloid formation, thereby curtailing biofilm genesis. Their findings suggest that both agents hold promise for development as effective anti-biofilm materials.

1.2.2. Au Antimicrobial Nanoparticles

The antimicrobial mechanisms of Au nanomaterials include the following: (1) binding to the bacterial membrane to alter its membrane potential; (2) reducing the ATP level by inhibiting ATPase activity, leading to declined metabolism; (3) preventing the binding of tRNA with ribosome, potentially inhibiting biological processes [139,140]. Unlike other metal nanoparticles, Au NPs do not induce ROS generation, rendering them a much safer metal antimicrobial agent [140]. Additionally, functionalization can further amplify their antimicrobial and antibiofilm efficacy. Inspired by the selective carbohydrate-based recognition of the key virulence factors of P. aeruginosa, namely LecB (fucose-specific lectin) and LecA (galactose-specific lectin), Zhang et al. designed fucose-based (Fuc-AuNPs) and galactose-based (Gal-AuNPs) glycoconjugate Au NPs [141]. Both Fuc-AuNPs and Gal-AuNPs exhibited notable bacterial targeting efficiency. When loaded with ceftazidime, these nanoparticulate carriers exhibited both photothermal and photodynamic therapeutic effects, achieving impressive biofilm eradication.

Au NPs have been widely used in photothermal therapy (PTT) for treating cancer and infection, attributed to their exceptional light-thermal conversion efficiency under external light source illumination. The PTT strategy can curb the proliferation of drug-resistant bacteria and disrupt the structure of biofilm, positioning it as a potent strategy for biofilm control [142]. Cui et al. designed a near-infrared (NIR) light-driven nano-swimmer (HSMV) with asymmetrically embedded Au NPs. This innovation efficiently self-propels and penetrates S. aureus biofilm within 5 min under NIR light irradiation [143]. The thermal-triggered release of co-loaded vancomycin from this HSMV achieved an efficient combination of photothermal therapy and chemotherapy in one system. While some reports stated that Au nanomaterials can attain temperatures close to 70 °C under illumination [144,145,146], posing a challenge for photothermal therapy, more recent research suggests that the localized temperature for such therapy can be maintained at around 45 °C [143,147]. This optimization ensures effective bacterial treatment while minimizing harm to surrounding tissues.

1.2.3. Metal Oxide Antimicrobial Nanoparticles

Metal-oxide-based antimicrobial nanoparticles typically comprise materials like ZnO [148], CuO [149], Fe₃O₄ [150], etc. Their antimicrobial mechanisms can be summarized as follows: (1) disruption of bacterial cell wall and membrane structure, which leads to the leakage of intracellular substances and subsequent bacterial death; (2) generation of ROS, inducing oxidative stress; (3) releasing of toxic metal ion [139]. Armijo et al. investigated the inhibitory concentration and susceptibility of iron–oxide (Fe₃O₄) NPs, both in combination with and without tobramycin, against P. aeruginosa. Their findings highlighted that the capping agent of the Fe₃O₄ NPs critically influences its bactericidal capabilities. For instance, particles capped with PEG showed no susceptibility, while those capped with alginate demonstrated enhanced dispersibility in alginate-rich biofilms, leading to improved diffusion through bacterial biofilm barriers [151]. The influence of the magnetic field on the performance of Fe₃O₄ NPs was further investigated in an in vitro model that utilized an artificial biofilm (alginate layer) and mucus (ager layer). Under an external magnetic field, the bactericidal effect of the Fe₃O₄ NPs, both with and without alginate capping, was enhanced. Moreover, while the susceptibility of tobramycin against the bacteria diminished over time, the susceptibility of Fe₃O₄ NPs remained consistent, suggesting their potential as promising antimicrobial agents.

In summary, metal nanoparticles offer great potential as anti-biofilm agents through various mechanisms, including metal ion release, bacterial structure disruption, and ROS generation. However, it is crucial to also consider the limitations of these antimicrobial nanoparticles. For instance, Ag NPs catalyze electron transfers that consume oxygen molecules, triggering a cascade of radical reactions and ROS generation. This process can subsequently lead to oxidative stress and cellular malfunctions in organs such as the kidneys, liver, lungs, brain, and spleen [152]. Specifically, Ag NPs of 20 nm in size have been shown to induce lung eosinophil and neutrophil inflammation, paired with bronchial hyper-responsiveness [152]. Furthermore, emerging adaptions and cross-resistances in bacteria and their biofilms to inorganic antimicrobial NPs are concerning. Bacteria in the biofilm have been found to evolve a reduced sensitivity to Ag NPs due to the evolution of persist bacteria within these structures. Cross-resistance between Ag and antibiotics, as seen in gentamicin-resistant P. aeruginosa biofilm [153], has also been observed. Therefore, before transitioning these nanomedicines to clinical applications, it is imperative to delve deeper into their toxicity and the bacterial resistance mechanisms they may induce.

1.3. Polymeric Antimicrobial Nanomaterials

Polymeric antimicrobial nanoparticles, including lipid and polymer nanoparticles, are viewed as powerful platforms for overcoming biofilm resistance. These particles can enhance the delivery of antibiotics to biofilm-residing bacteria, thus amplifying the efficacy of biofilm eradication. In this section, we provide a comprehensive overview of various types of lipid and polymer nanoparticles that have been developed for combating bacterial biofilm caused infections. Moreover, we also delve into functionalization and targeting strategies crafted to enhance their antimicrobial prowess.

1.3.1. Lipid-Based Antimicrobial Nanoparticles

Lipid-based nanoparticles, such as liposomes, solid lipid nanoparticles, etc., have been employed in drug delivery for decades due to their excellent biocompatibility and safety. Certain cationic lipid materials, such as 1,2-dioleoyl-trimethylammonium-propane (DOTAP), can be integrated into lipid–polymer hybrid nanoparticles (LPNs). This allows the LPNs to attach to the surfaces of both Gram-positive and Gram-negative bacteria, regardless of their planktonic or biofilm lifestyles, thus demonstrating enhanced bactericidal activities. Traditional techniques for producing lipid-based nanoparticles include methods such as thin-film hydration, the freeze–thaw method, high-pressure homogenization, membrane extrusion, reverse-phase evaporation, and solvent injection method, among others. In terms of scaling up production, the microfluidic technique has been employed in nanomedicine manufacturing. This process creates nanoparticles by skillfully handling minuscule fluid amounts within microchannels, significantly bolstering the control and consistency of the nanoparticles [154]. Baek et al. designed a non-toxic LPNs delivery system, consisting of a polymer core (PLGA) and cationic lipid shell (DOTAP). These LPNs, with their uniform particle size, encapsulated over 95% of the antibiotic [155]. Impressively, these LPNs decreased biofilm activity by more than 95% at concentrations 8–32 times lower than free antibiotics, attributed to the targeted and prolonged release of antibiotics at the biofilm site. This suggests that LPNs could be an effective nanocarrier to augment biofilm treatment. By fusing DOTAP-modified polymeric NPs with zwitterionic unilamellar vesicles, Wan et al. designed lipid-bilayer-enveloped lipid–polymer hybrid nanoparticles aimed at treating P. aeruginosa biofilm caused lung infections [156]. These lipid–polymer hybrid NPs, composed of a polystyrene core and modified with the positively charged DOTAP and poly-L-lysine (PLL) as the shell, were then enveloped in zwitterionic unilamellar vesicles made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000). The inclusion of DSPE-PEG2000 in the outer membrane enhanced the mucus penetration capabilities of these particles, ensuring efficient access and penetration into the biofilm. Additionally, DOPC played a pivotal role in aiding these particles to merge with bacterial membranes, positioning this nanocarrier as a potent solution to tackle the persistent challenges of mucus and biofilm barriers.

1.3.2. Chitosan Antimicrobial Nanoparticles

Chitosan is a biocompatible and biodegradable polysaccharide composed of glucosamine and N-acetyl-glucosamine residues. The amine groups of glucosamine give chitosan its cationic properties, enabling it to interact with the negatively charged components on bacterial membrane, such as the teichoic acid in Gram-positive bacteria and the lipopolysaccharide of Gram-negative bacteria. This interaction can lead to cell membrane damage and leakage of intracellular components. Moreover, chitosan has been found to bind with bacterial DNA, consequently inhibiting mRNA transcription and protein synthesis [157]. The prevalent techniques for preparing chitosan nanoparticles include methods like ionic gelation, microemulsion, polyelectrolyte complexation, emulsification solvent diffusion, and the reverse micellar method [158]. Ma et al. encapsulated curcumin into positively charged chitosan nanoparticles, and these curcumin-loaded nanoparticles exhibited strong antibacterial activity against biofilm formed by planktonic bacteria or fungi, irrespective of whether they were single or polymicrobial organisms [159]. Rhamnolipid, a natural glycolipid known for its antimicrobial, anti-adhesive, and biofilm-disrupting activities, was employed to prepare antimicrobial nanoparticles in conjunction with chitosan [160]. The incorporation of rhamnolipid resulted in nanoparticles of smaller and more uniform size, exhibiting a significantly positive surface charge and enhanced stability. The antibacterial efficacy of these chitosan/rhamnolipid NPs against both planktonic and biofilm-state Staphylococcus strains was superior to that of either rhamnolipid or chitosan alone, indicating that these hybrid NPs might present a formidable approach for biofilm control.

1.3.3. Dextran Antimicrobial Nanoparticles

Dextran, characterized by α(1→6) glucose-linked polysaccharides with a high degree of linkage variability and branching, is a polymeric material extensively used in drug delivery [161]. Its strong bio-affinity with the polysaccharides in biofilm EPS enhances the penetration of dextran nanoparticles into the biofilm matrix [162]. The abundant active hydroxyl groups and its narrow molecular weight distribution allow dextran to be chemically tailored to suit the needs of nanoparticulate drug delivery more effectively [163]. Barros et al. integrated curcumin into nanoparticles, using PLGA as the hydrophobic core and dextran as the hydrophilic shell [164]. These curcumin-loaded nanoparticles outperformed the free drug in terms of penetration into Pseudomonas putida biofilm, resulting in significantly heightened antibiofilm activity. Recognizing dextran’s affinity for the polysaccharides in the biofilm matrix, Li et al. employed cationic dextran to disrupt the intrinsic electrostatic interaction in the biofilm matrix. The biofilm matrix, maintained by the positively charged Pel (positively charged polysaccharide) and eDNA, underwent a gel-to-sol phase transition when disrupted by the cationic dextran, leading to a collapse of the biofilm’s structural integrity [165]. This particular form of cationic dextran showed enhanced antibacterial capability against P. aeruginosa biofilms, positioning it as a promising cationic substance to combat bacterial biofilm through inducing phase transitions.

1.3.4. Amphiphilic Cationic Copolymer Antimicrobial Nanoparticles

Amphiphilic block copolymers are composed of two or more polymer fragments with distinct solubility properties. These copolymers can spontaneously form various nanostructures in aqueous solutions, such as polymeric micelles, polymersomes, etc. [166] Inspired by the structure of antimicrobial peptides, which consist of hydrophobic and positively charged amino acids, amphiphilic cationic polymers containing hydrophobic segments and positively charged hydrophilic segments were designed as nanomaterials for antimicrobial and antibiofilm treatments. The cationic segments of the polymer are designed to enhance binding to anionic bacterial membranes through electrostatic interactions, while the hydrophobic segments drive the polymer chains to insert into the hydrophobic bacterial membrane, leading to bacterial membrane disruption. Additionally, cationic amphiphilic polymers have been found to inhibit biofilm formation and destroy already-formed biofilm architecture [167]. Takahashi et al. [168] synthesized amphiphilic methacrylate homopolymers PE0 and co-polymers PE31 using reversible addition-fracture chain transfer (RAFT) to control streptococcus mutans biofilm. The minimum biofilm inhibitory concentrations (MBIC) of PE31 and PE0 were 6.3 μg/mL and 8.3 μg/mL, respectively. For established biofilms, the polymer concentration at 1000 μg/mL reduced bacterial biofilm biomass by 80–85%. Zhao et al. synthesized a series of PEG-blocked amphiphilic cationic polymers consisting of hydrophobic alkyl groups and quaternary ammonium salts. They investigated the relationship between the polymer structure and its antibacterial activity against Escherichia coli and Staphylococcus aureus [169]. The polymers were screened based on PEGylation, the length, and content of hydrophobic alkyl chains, among other factors. The optimized cationic polymer showed excellent antibacterial activity against both S. aureus (MIC, 4 μg/mL) and E. coli (MIC, 8 μg/mL). Notably, these polymeric nanoparticles can even eradicate bacteria within the biofilm. Vishwakarma et al. [170] synthesized a class of peptidomimetic polyurethanes for bacterial biofilm disruption. These polymers utilized arginine or lysine mimics as cationic groups and phenylalanine and alanine mimics as hydrophobic side groups. These polyurethanes can disrupt biofilms of P. aeruginosa, S. aureus, and E. coli, even those resistant to conventional antibiotics. Moreover, these polyurethanes prevent bacterial attachment to surfaces and enhance bacterial motility, inhibiting biofilm formation in both Gram-positive and Gram-negative bacteria at sub-inhibitory concentrations.

1.3.5. Antimicrobial Peptides Loaded Nanoparticles

Distinct from inorganic NPs and antibiotics, antimicrobial peptides (AMPs), also known as host defense peptides, consist of 12–100 amino acid fragments and exhibit promising antimicrobial efficacy [171]. Found in various organisms, including insects, animals and plants, AMPs serve as a vital defense mechanism in nature. In the lung, AMPs are released by epithelial cells and immune cells into the airway mucus, playing important roles in both innate and acquired immunity [31,172]. Their cationic and amphiphilic nature endows AMPs with broad-spectrum activity against bacteria, fungi, and viruses. The positively charged AMPs can bind to and disrupt negatively charged bacteria cell membranes through electrostatic interaction, leading to intracellular component leakage and subsequent cell death [173]. Therefore, this non-specific bactericidal mechanism positions AMPs as promising antimicrobial agents with a reduced likelihood of resistance development. While over 3000 AMPs have been identified, only seven have receive FDA approval for clinical use [174]. The limited clinical translation might be due to their sensitivity to protease degradation, potential systemic toxicity, and rapid renal clearance post systemic administration. Hence, novel treatment regimens need to be developed to mitigate these limitations.

Inhalation can achieve high drug concentrations locally in the lung, while minimizing systemic drug exposure. This results in decreased toxicity and enhanced efficacy, making it a viable route for AMPs in treating biofilm-associated lung infections. Several nanoparticulate systems, including inorganic and polymeric nanoparticles, have been explored to augment the therapeutic effects and safety profile of AMPs [175,176]. Casciaro et al. used PLGA NPs to encapsulate Esc peptides, AMPs derived from the frog skin, with the objective of enhancing peptide transport through CF mucus and bacterial extracellular matrix [177]. These Esc-peptides-loaded PLGA NPs can be efficiently administered using liquid jet nebulizers available to patients and exhibited enhanced efficacy in inhibiting P. aeruginosa growth both in vitro and in vivo. This suggests the potential of PLGA NPs as a reliable delivery system for AMPs targeting the lungs. Additionally, a synthesized branched antimicrobial peptide resistant to biological fluid degradation, has demonstrated efficacy in vitro against numerous Gram-negative multidrug and extensively drug-resistant clinical isolates. Falciani et al. encapsulated SET-M33 in dextran NPs and aerosol-administered these nanoparticles to healthy rat lungs [178]. The findings revealed that these SET-M33-loaded dextran NPs significantly extended the peptide’s lung residence time and effectively managed pulmonary infections in a mouse model afflicted by P. aeruginosa induced pneumonia. Collectively, nanoparticulate drug delivery systems show significant promise in delivering AMPs to the lungs to combat both bacteria and the biofilm.

2. Nanotechnology-Based Nanoparticle Fabrication Strategies for Bacterial Biofilm Control

Distinct from bacterial biofilms that adhere to substratum, the biofilms in the lung exhibit unique properties. The biofilms of chronic lung infections are typically entrenched in highly viscous mucus or even tenacious sputum, rather than directly on epithelial cell surfaces. Beyond the challenges presented by biofilm barrier, airway mucus also hinders antibiotic-loaded nanoparticles’ access to the biofilm sites. Consequently, airway mucosal barriers should be factored into the design of nanoparticulate drug delivery systems intended for biofilm control. Mucus-penetrating particles serve as an illustrative example of surmounting these mucus barriers, which will be elaborated on in this section. Additionally, the intricate biofilm structure impedes the penetration of antibiotics or their encapsulated nanomedicines. To tackle this challenge, biofilm-microenvironment-responsive nanoparticles have been developed, offering a promising approach for combating biofilm infections. These biofilm-microenvironment-responsive NPs enable targeted drug delivery, enhance biofilm penetration, and amplify the antibiofilm efficacy of antibiotic agents while reducing their side effects. In this section, we will further delve into the design strategies for bacterial biofilm-microenvironment-responsive NPs and elucidate their operational paradigm.

2.1. Mucus-Penetrating Particles or Muco-Inert Particles

Contrary to common knowledge, local drug delivery through inhalation is an optimal method to address lung infections, as antibiotics or antibiotic-loaded NPs can be directly delivered to the infection sites [210]. However, bacterial biofilm is usually embedded in mucus, which hinders these antibiotics or their nanoparticles from accessing the biofilm. The barrier features of airway mucus result from adhesive and steric interactions within the mucus network [211]. The constituents in the airway mucus can engage with nanoparticles through hydrophobic and electrostatic interactions, inhibiting their deeper penetration into the mucus. Moreover, the entangled mucus network, primarily formed by eDNA and disulfide-bond-linked mucin, creates steric obstructions for particle penetration [52]. In inflammatory and infectious airway diseases, the physicochemical properties of the airway mucus usually differ from those in healthy individuals, complicating the penetration of nanoparticles. Moreover, nanoparticles adhering to the superficial mucus gel layer can be swiftly eliminated by mucociliary clearance [28]. For effective mucus penetration, nanoparticles must resist adhesion by mucus constituents and be adequately small to bypass the dense mucus meshwork. Addressing this, mucus-penetrating particles (MPPs) or muco-inert particles have been designed, showing promise in enhancing drug and gene delivery to mucosal tissues [28,29,212]. Typically, MPPs surfaces are densely coated with hydrophilic polymers, like poly(ethylene glycol) (PEG) or Pluronic 127 (F127), allowing them to minimize adhesion to airway mucus constituents. For instance, dense PEG modification offers a neutral, highly hydrophilic surface which minimizes electrostatic and hydrophobic interactions. This has been shown to improve mucus penetration [213,214]. Ernst et al. designed tobramycin encapsulated polyester-based particles using PLGA-PEG di-block polymer to overcome mucus and biofilm barriers, enhancing biofilm eradication [215]. The effectiveness of tobramycin against P. aeruginosa and B. cepacia biofilms was dramatically enhanced when encapsulated under both fluidic and static experimental conditions in artificial mucus. Compared to either free tobramycin or the bulk mixture of tobramycin and blank particles, the MIC of tobramycin-loaded PLGA-PEG NPs against biofilm-embedded P. aeruginosa and B. cepacia was reduced by more than 1000-fold. Furthermore, PEG-coated particles can be further equipped with moieties to facilitate targeting or cellular uptake. For instance, Tat, a well-researched cell-penetrating peptide, was fabricated onto PEGylated mucus-penetrating nanoparticles for pulmonary delivery of ivacaftor to patients with CF. This dual aim targeted enhancing ivacaftor delivery to airway epithelial cells by rapid diffusion through mucus while simultaneously promoting ivacaftor uptake by the lung epithelial cells [216]. The findings have shown that the presence of Tat on the surface of the MPPs strongly enhanced their uptake by lung epithelial cells. In summary, PEGylated NPs, owing to their excellent biocompatibility, muco-inert nature, and stealth character, are well-documented as a potential strategy to facilitate diffusion through mucosal barriers.

As alternatives, several water-soluble polymers, such as polysarcosine, polyglycydol, poly(vinyl alcohols) (PVA), poly(2-alkyl-2-oxazolines), zwitterionic polymers, and certain hydroxyl-containing polymers, have been explored in their potential in aiding diffusion through mucosal barriers [217]. Characterized by low molecular weight, high hydrophilicity, and non-charged nature, these polymers are promising. Hu et al. developed a PLGA-based platform with various surface modifications, including PEG, PVA, F127, and polydopamine (PDA), and systematically evaluated their mucus penetration ability and cellular uptake [218]. Findings revealed that PLGA-PEG and PLGA-F127 NPs showcased superior mucus penetration, while the PDA-modified PLGA NPs excelled in both mucus penetration and cellular uptake. With advancements in controlled polymerization, we can anticipate the emergence of well-defined, low-molecular-weight muco-inert polymers to further the design of cutting-edge mucus-penetrating drug delivery systems.

2.2. Enhance Mucus Penetration of Particles by Mucus Disrupting Agents

In addition to the mucus-penetrating particles, mucus-disrupting agents can modify the mucus network, facilitating easier penetration of particles into the mucus. Typically, this strategy employs mucolytic agents, such as using N-acetyl-L-cysteine (NAC), to break the disulfide bond between mucins and to utilize DNA enzymes to degrade extracellular DNA, thereby thinning the mucus. Airway mucus treated with these agents exhibits reduced viscoelasticity and larger mucus mesh pore size, enhancing the penetration of particles into the mucus.

Enzymes can degrade components of airway mucus and have found clinical applications. Among mucus constituents, DNA stands out as a predominant component, entangling with mucin fibers and other mucus ingredients [23]. Elevated DNA levels are observed in inflammatory and infectious lung diseases, primarily due to the necrosis of recruited neutrophils. DNase is routinely used in clinics to treat patients with CF. Recently, DNase-encapsulated nanoparticles have been designed to diminish the crosslinking and viscoelasticity of mucus, thereby aiding particle penetration [219,220,221]. Deacon et al. designed a combination of tobramycin and Dornase alfa (recombinant human deoxyribonuclease Ⅰ, DNase) to concurrently degrade thick DNA-rich mucus and enhance NP penetration into CF sputum [221]. These nanoparticles merge two commonly prescribed CF drugs into a singular nanoparticulate formulation. This represents an innovative approach to surmount the sputum barrier, amplify local drug concentrations, avert systemic side effects, and optimize outcomes for lung infections in patients with CF. In a different study, Suk et al. fabricated a nonviral gene carrier composed of poly-L-lysine conjugated with a 10 kDa PEG segment, either use alone or in combination with mucolytic agents [220]. This synthetic nanoparticulate gene carrier demonstrated superior effectiveness in crossing the mucus/sputum barriers, especially when paired with adjuvant mucolytic treatment using NAC or a combination of NAC and rhDNase.

NAC, a prevalent mucolytic agent, cleaves the disulfide bonds of mucin fibers, leading to decreased mucus viscoelasticity, and promotes drug penetration and that of their nanoparticulate formulations [23]. Dry powder inhalers containing NAC and three distinct antibiotics have been formulated. Research indicates that this combined delivery system of antibiotics and NAC either maintains or boosts antibiotic efficacy, showing significant potential in inhibiting P. aeruginosa biofilm formation [222]. Lipid nanoparticles encapsulating NAC were designed with D-amino acids to target and disrupt bacterial biofilms. These NAC-loaded nanoparticles not only showcased a safe profile than their unloaded counterparts but also exhibited notable biomass and bacterial viability reduction in P. aeruginosa biofilms when paired with moxifloxacin. This suggests that such nanoparticulate formulations could serve as potential treatment strategies against P. aeruginosa biofilms, either as standalone treatments or in tandem with other antibiotics [223]. Besides NAC, numerous mucolytic agents, including methacholine and thiol-based drugs, have been utilized to enlarge the mucus mesh through disulfide bond disruption, offering potential avenues to bolster nanoparticle mucus penetration [224,225,226]. Thiol-based drugs, in addition to their mucolytic properties, function as antioxidants, either directly via free sulfhydryl groups or by replenishing intracellular glutathione levels [226]. They can also hinder bacterial adherence to respiratory epithelial surfaces and inhibit biofilm formation, thus enhancing antibiotic therapy efficacy. Additionally, a variety of mucolytic enzyme-decorated carrier systems (MECSs) have demonstrated efficiency in cleaving mucus structures, facilitating their journey deeper into the mucus [227]. Studies, both in vitro and in vivo, have highlighted the fact that nanoparticles loaded with these mucolytic enzymes surpass nanocarriers lacking encapsulated enzyme in mucus penetration. Crucially, these mucolytic-enzyme-loaded NPs disrupt mucus structure locally without compromising the overall protective function of the mucosal barrier, hinting at the potential for long-duration treatment using these systems.

2.3. Biofilm Microenvironment Responsive Nanoparticulate Systems

Drug-loaded nanoparticles traversing the airway mucus barrier will subsequently encounter the bacterial biofilm and must penetrate the biofilm matrix or release their payloads in a timely manner to achieve a bactericidal effect. Biofilm-microenvironment-responsive NPs have been proven to be a promising strategy for combating biofilm infections [228]. Increasing studies have confirmed that biofilm-microenvironment-responsive NPs enhance biofilm penetration, improve drug targeting efficiency, and enable timely drug release, augmenting the antibiofilm efficacy of the therapeutic agents while minimizing off-target side effects. Characteristics of the biofilm microenvironment, such as acidic pH, overexpression of hydrolases, and hypoxia, can guide the design of these nanoparticles. In addition, nanocarriers leveraging synergistic effects via biofilm-microenvironment-triggered features have proven effective in combating biofilm infections.

Acidic-pH-responsive NPs: Bacterial biofilms typically exhibit an acidic microenvironment (pH 4.5–6.5) due to the accumulation of lactic and acetic acid derived from sugar fermentation. This can guide the design of pH-responsive NPs for site-specific antibiotic release [103,229,230,231]. For instance, Liu and colleagues designed a type of mixed-shell–polymeric micelle (MSPM), comprising a hydrophilic PEG–shell and a pH-responsive poly (β-amino ester). These micelles exhibit a negative charge at physiological pH but become positively charged at pH 5.0 [232]. The stealth properties of the PEG–shell combined with surface charge reversal enable MSPMs to penetrate and accumulate in staphylococcal biofilms. Upon adherence to bacterial surfaces, bacterial lipases degrade nanoparticles, releasing the loaded antibiotics to kill bacteria within the biofilm. Similarly, Yin et al. fabricated ciprofloxacin-conjugated gold nanorods with acidic-induced surface-charge-switchable activity and lipase-triggered drug release properties to combat multidrug-resistant bacterial infections and their biofilms [233]. Gao et al. designed size and charge adaptive azithromycin-conjugated clustered nanoparticles (AZM-DA NPs) for treating bacterial biofilms [103]. These particles were formed by electrostatic complexation between azithromycin-conjugated amino-ended PAMAM dendrimer and 2,3-dinethyl maleic anhydride modified PEG-block-polylysine. These particles disintegrate in the acidic biofilm microenvironment, leading to the release of smaller, positively charged AZM-conjugated PAMAM NPs. This release mechanism augments biofilm penetration and bacterial uptake, demonstrating potent anti-biofilm activity.

Biofilm enzymes-responsive NPs: Bacteria excrete various enzymes, such as lipases, phosphatases, phospholipases, and hyaluronidases. These biofilm-specific enzymes offer potential stimuli for designing drug delivering nanocarriers to enhance selective accumulation at microbial infection sites. Although nanoparticles with hydrophilic surfaces enhance their mucus and biofilm matrix penetration, lipophilic surfaces enable bacterial membrane attachment [234]. Wan et al. devised biofilm microenvironment-adaptive NPs with a PLGA core and an enzymatically cleavable TPGS shell to target azithromycin delivery to bacterial biofilms through inhalation [235]. The hydrophobic vitamin E of TPGS enhances biofilm interaction and acts as a prolonged antibiotic release depot, boosting antibiofilm efficacy. As highlighted, lipase can degrade nanoparticles, releasing antibiotics to kill biofilm bacteria after an acidic pH triggers a surface charge switch, collaboratively enhancing biofilm elimination [233].

ROS-responsive NPs: Pro-inflammatory immune cell responses to bacterial biofilm infections results in elevated reactive oxygen species (ROS) levels at infection sites [236]. In P. aeruginosa biofilm-associated chronic lung infections, polymorphonuclear leukocytes (PMNs) surround the biofilm, consuming oxygen and producing ROS [16]. The overproduction of ROS at the bacterial biofilm sites serves as a stimulus for designing ROS-triggered nanoparticles. Wang et al. synthesized 4-(hydroxymethyl) phenylboronic acid pinacol ester-modified α-cyclodextrin (Oxi-αCD) as ROS-responsive material to encapsulate moxifloxacin (MXF) for treating pulmonary bacterial infections [237]. With the coating of DSPE-PEG-folic acid, these nanoparticles penetrate sputum easily and target the inflamed tissues. ROS abundance then triggers drug release, displaying enhanced antibacterial efficacy over free drugs and non-targeted counterparts. Ye et al. employed stimuli-responsive nanoparticles loaded with rifampicin to address bacterial resistance [238]. These rifampicin-loaded NPs were composed of dextran as the hydrophilic shell and a biodegradable poly (β-amino ester)–guanidine–phenylboronic acid (PBAE-G-B) polymer as the hydrophobic core that can encapsulate the drug. The PBAE-G-B polymer responds to both acidic pH and elevated ROS at the biofilm sites, releasing rifampicin and the cationic polymer. Both agents work synergistically against antimicrobial resistant pathogens. The safety and efficacy of these NPs have been validated in animal models of both Gram-positive and Gram-negative bacterial biofilm infections.

This entry is adapted from the peer-reviewed paper 10.3390/pharmaceutics15112582

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