Toll-like receptors (TLRs) represent a fundamental component of the innate immune system with diverse roles in detecting and responding to microbial infections and tissue damage. They are strategically localized on the cell surface or within intracellular compartments like endosomes, allowing them to sense a wide range of pathogens as shown in Figure 2(Fig. 2). TLRs recognize specific molecular signatures associated with microbes, such as lipopolysaccharides (LPS) from bacteria (TLR4) or double-stranded RNA from viruses (TLR3). These receptors transmit signals via adapter proteins like MyD88 (utilized by most TLRs except TLR3) and TRIF (used by TLR3 and some others), initiating intricate signaling cascades. Once activated, TLR signaling converges on nuclear factor-kappa B (NF-κB) and interferon regulatory factors (IRFs), transcription factors that migrate to the nucleus and orchestrate the expression of various immune genes (Kawasaki and Kawai, 2014[32]). This results in the secretion of pro-inflammatory cytokines, chemokines, type I interferons, and other immune effectors. These molecules collectively shape the immune response by recruiting immune cells to sites of infection, enhancing antigen presentation by antigen-presenting cells (APCs), and establishing an antiviral state in neighbouring cells (O'Neill, 2006[51]). Beyond their immediate role in innate immunity, TLRs serve as crucial mediators connecting the innate and adaptive immune systems. Upon activation, TLRs stimulate APCs, particularly dendritic cells, to process and present pathogen-derived antigens to T lymphocytes (Barton and Medzhitov, 2003[10]). This interaction is pivotal for the initiation of adaptive immune responses, including the activation of T cells and B cells. Consequently, TLRs have profound implications not only in the defence against infections but also in vaccine development, where they are harnessed to enhance the efficacy of vaccines, and in immunotherapies, where they play a role in modulating immune responses against diseases like cancer.

Activation of TLRs can lead to modifications in histones, DNA methylation patterns and non-coding RNAs as well. For example stimulating TLR4 in macrophages has been shown to increase histone acetylation at promoters of genes encoding pro-inflammatory cytokines such as TNF-α and IL-6 which leads to boosting of their expression) (Hoden et al., 2022[23]). Similarly, TLR7 and TLR8 activation can alter microRNA levels, like miR-146a, which act to fine-tune the immune response and prevent excessive inflammation) (Hoden et al., 2022[23]). These changes can persist beyond the initial stimulus which gives rise to a type of innate immune memory, sometimes called trained immunity, which allows immune cells to respond more efficiently to subsequent infections.

TLR activation also reshapes cellular metabolism. Activated macrophages and dendritic cells often switch from relying primarily on oxidative phosphorylation to aerobic glycolysis, which provides rapid energy and the building blocks needed for cell growth and cytokine production. Changes in metabolism also affect mitochondrial activity, fatty acid processing, and the production of reactive oxygen species, all of which feed back into immune signaling and even influence epigenetic modifications. Metabolism and epigenetics are closely linked. Molecules like acetyl-CoA and S-adenosylmethionine, which are generated during metabolic processes, are essential for histone acetylation and DNA methylation. This means that the metabolic state of a cell can directly impact gene regulation while epigenetic changes can in turn affect genes that control metabolism.

Numerous research investigations have delved into the impact of nanoparticle size on their uptake within the respiratory system. Larger particles, typically with diameters surpassing 5 μm, exhibit a propensity to become trapped within the upper airways, whereas smaller particles within the range of 1-5 μm are better suited for deposition in the lower airways. Particles below the 500 nm size threshold can reach the alveoli via Brownian diffusion. Importantly, nanoparticles measuring less than 200 nm have exhibited a remarkable ability to effectively target respiratory epithelial cells, thus evading clearance by macrophages (Sung et al., 2007[74]).

Aerosol-based nanoparticle delivery to the lungs, aimed at precise treatment with reduced systemic toxicity, holds immense potential. A significant challenge arises from the airway's protective mucus layer, produced by airway goblet cells (Suk et al., 2014[73]).

When inhaled, nanoparticles encounter the swift mucociliary clearance process in the respiratory system, offering a limited timeframe for effective lung delivery. To address this, nanoparticles must first breach the outer mucus layer and then rapidly navigate the adherent internal layer. Larger particles and those interacting strongly with mucus are quickly cleared through mucociliary mechanisms. This underscores the importance of designing smaller nanoparticles, with recent evidence indicating that particles below 200 nm exhibit enhanced mucus penetration. Nanoparticles' ability to traverse the mucus barrier correlates with their size, favouring smaller counterparts (Mastorakos et al., 2015[45]). To minimize mucus interaction, nanoparticles with hydrophilic attributes and neutral surfaces, often incorporating materials like PEG, are preferred. A novel approach involves a brush-like triblock polymer coating comprising PEG and polypropylene glycol, were coating thickness and density impact interactions with mucus and lung surfactant (Tang et al., 2009[75]). Alongside PEG, alternative methods are emerging, including the use of mucus-penetrating peptides as nanoparticle coatings to improve mucus penetration and cellular uptake. Another strategy involves incorporating mucolytics into nanoparticle design. Agents that disrupt disulfide bonds, such as N-acetyl-cysteine, and mucolytic enzymes like papain and bromelain, show promise in nanoparticle formulations (Witten et al., 2018[86]). A recent innovation includes a "nano-into-micro" dry powder developed for ivacaftor delivery (Porsio et al., 2020[56]). This formulation integrates mannitol and cysteamine to alleviate viscosity and enhance mucus diffusion.

Before achieving targeted delivery to epithelial cells through the pulmonary route, addressing the crucial concern of nanoparticle clearance by airway and alveolar macrophages is important. Key factors influencing macrophage clearance encompass nanoparticle size, surface charge, and surface characteristics. Phagocytosis is the primary mode of clearance for nanoparticles spanning the range of 500 nm to 6 μm (Qie et al., 2016[57]). Consequently, meticulous nanoparticle design must consider size as a pivotal parameter. Surface charge, or potential, is equally significant to evade alveolar macrophage interactions. Positively charged (cationic) nanoparticles have a tendency to be more readily engulfed by alveolar macrophages, whereas negatively charged (anionic) nanoparticles are better at evading macrophage clearance. Importantly, cationic nanoparticles can trigger internalization by pulmonary dendritic cells, leading to their recruitment and maturation, whereas anionic nanoparticles typically do not elicit an immune response. Various polymers, such as PEG, polyvinyl alcohol, and zwitterionic polymers, have shown promise in reducing macrophage interaction when applied to nanoparticle surfaces. For instance, after PEG coating, there was a notable decrease in particle uptake by alveolar macrophages (Shen et al., 2015[67]). In nanoparticle synthesis, two primary strategies are employed to overcome both the mucus barrier and nonspecific macrophage uptake. The first strategy involves carriers like polyplexes, mesoporous nanoparticles, and liposomes, used to transport therapeutic agents to respiratory epithelial cells. The second strategy utilizes mucolytic agents to facilitate the penetration of nanoparticle carriers through the mucus barrier, ultimately targeting the airway epithelium upon pulmonary administration.

Once the obstacles of mucus and macrophage clearance have been overcome, nanoparticles reach the surfaces of epithelial cells. At this stage, the focus shifts to epithelial endocytosis, a crucial element in nanoparticle design. Typically, nanoparticles are engineered with specific ligands that engage with epithelial receptors or adhesion molecules. For instance, nanoparticles conjugated with Vitamin B12 exhibit increased cellular uptake by respiratory epithelial cells, owing to the presence of Vitamin B12 receptors on these cell surfaces (Fowler et al., 2013[20]). Likewise, cell-penetrating peptides offer an alternative approach to enhance nanoparticle uptake by respiratory epithelium. These peptides act as amphiphilic carriers for delivering proteins to airway epithelial cells. Innovations also extend to designing glycosaminoglycan-binding enhanced transduction peptides to augment cellular uptake (Abu-Awwad et al., 2017[1]). Nanoparticles chemical composition can be tailored for optimal epithelial cell uptake. Comparative studies have showcased varied uptake abilities among nanoparticles, highlighting the importance of composition. The non-invasive aerosol inhalation method has gained attention for epithelial targeting, with stabilized mRNAs delivered using hyperbranched poly-β-amino ester nanoparticles (PBAEs) (Wang et al., 2022[82]). Through nebulized delivery, these PBAE nanoparticles achieved substantial targeting of lung epithelial cells, offering promising clinical delivery systems. Intravenous injection can also be employed, provided nanoparticles feature specific modifications for heightened epithelial cell uptake. An example involves the creation of liposome-based nanoparticles that integrate nanobodies tailored for surfactant-associated protein (Balmert and Little, 2012[8]). These nanobody-linked liposomes efficiently transported therapeutic agents into precise lung epithelial cells, demonstrating promise in various disease scenarios.

Inhaled particles face diverse clearance pathways contingent on their attributes and regional distribution. Primarily, three mechanisms govern this process: muco-ciliary clearance, phagocytosis, and systemic uptake. Muco-ciliary clearance takes precedence in upper airways, orchestrated by the ciliated columnar epithelium. This epithelium produces mucus, capturing particles and setting them in motion through ciliary beats, eventually leading to expulsion through coughing or swallowing. Particles exceeding 5 μm in size are predominantly eliminated within the upper airways through this mechanism. In contrast, smaller particles settled deeper in the lungs experience reduced muco-ciliary action, leading to extended retention. Phagocytosis plays a diminished role in upper airways, with macrophages present but less dominant. Deep lungs entail more intricate clearance mechanisms determined by particle dissolution kinetics. Slower-dissolving or insoluble particles might interact with epithelial and immune cells, undergoing removal through muco-ciliary clearance, phagocytosis by alveolar macrophages, or endocytosis. Alveolar macrophages take the lead in deep lung clearance, internalizing particles, then either digesting them or exiting into the lymph or through muco-ciliary clearance. Phagocytosis chiefly accounts for particle clearance in the 1-5 μm range. Sub-200 nm particles often escape macrophage recognition, potentially due to size or rapid uptake by epithelial cells (Stuart, 1984[71]). A portion of nanoparticles can migrate into the systemic bloodstream via protein/receptor-mediated uptake, and some might enter systemic circulation through endocytosis via alveolar caveolae. For nanoparticles rapidly dissolving in the deep lungs, drug release occurs promptly, leading to systemic circulation absorption. The rate of absorption depends on drug characteristics, particularly lipophilicity and molecular weight. Low-molecular-weight lipophilic drugs tend to be absorbed most rapidly.

A significant concern surrounding nanoparticle therapeutics revolves around potential unforeseen adverse health impacts. Experiments conducted on rodents have suggested that when carbon nanoparticles are administered intratracheally, they can expedite vascular thrombosis (Medina et al., 2007[46]). Similarly, inhaled iridium particles have shown the potential to move from the lungs into the systemic circulation, giving rise to concerns regarding possible adverse vascular effects (Semmler et al., 2004[65]). Inhaled carbon nanoparticles have been observed to reach the brain, although the extent of their impact on the central nervous system remains uncertain. Intriguingly, recent research suggests that the cytotoxicity of metallic nanoparticles is comparable to that of metallic microparticles, implying that size might not be the primary determinant of toxicity. Nanoparticle toxicity remains a serious issue and necessitates thorough investigation. Beyond inherent nanoparticle toxicity, the materials employed for nanoparticle formulation could also shows toxic effects, potentially limiting their viability for therapeutic products. As an example, the harmful effects of polycyanoacrylates have been evidenced, with specific forms leading to an elevation in lactate dehydrogenase (LDH) activity within human pulmonary epithelial cells (Stone et al., 2006[70]). The toxicity level was influenced by the type of polycyanoacrylate, where shorter chain variants exhibited greater cytotoxicity. Similarly, polyethyleneimine (PEI) has displayed cytotoxicity in lung cells. Interestingly, studies indicate that particle toxicity in the lungs might hinge more on material selection than on particle size. For instance, comparisons between PLGA nanoparticles and polystyrene particles of comparable size suggest that the choice of material plays a crucial role in determining lung nanoparticle toxicity (Dailey et al., 2006[15]).

Nanoparticles are increasingly recognized not just for their ability to deliver drugs but also for their influence on epigenetic regulation in immune cells. By affecting DNA methylation, histone modifications and even non-coding RNA expression nanoparticles can change the expression of genes involved in inflammation and Toll-like receptor (TLR) signaling. For example, gold nanoparticles have been reported to reduce DNA methylation at promoters of pro-inflammatory genes in macrophages which can enhance cytokine production and support immune activation (Wang et al., 2022[82]). Also chitosan based nanoparticles have been shown to increase histone acetylation leading to higher expression of TLR4 and stronger downstream NF-κB signaling (Kumar et al., 2003[37]). PLGA nanoparticles loaded with TLR agonists can influence the expression of microRNAs such as miR-155 and miR-146a in dendritic cells, helping to balance pro- and anti-inflammatory signals and modulate adaptive immune responses (Dailey et al., 2006[15]).

In respiratory immunotherapy these epigenetic effects are particularly important. Nanoparticles can be designed to selectively influence TLR pathways in airway epithelial cells and alveolar macrophages which than enhances their protective immune responses while minimizing harmful inflammation. Understanding these epigenetic interactions can guide the design of nanoparticles with optimal size, composition, and surface chemistry to achieve targeted and safe immunomodulation.

Nanoparticles have been explored for delivering antiviral drugs and vaccines directly to the respiratory system to combat the SARS-CoV-2 pandemic. Several companies are vigorously pursuing the development of mRNA vaccines encoding viral proteins like the spike protein, encapsulated within nanoliposomes possessing physicochemical properties that resonate with immunization strategies aimed at specific tumour antigens (Dheyab et al., 2021[17]). Crafting such nanocarriers that can elude scavenger cell recognition while maintaining nontoxic and nonimmunogenic profiles presents a formidable challenge, necessitating substantial time before clinical availability. Moderna achieved a significant milestone on March 16, 2020, by commencing a Phase I clinical trial for an mRNA vaccine known as mRNA-1273, which was enclosed within lipid nanoparticles, a mere 63 days after the selection of the genetic sequence (Hodgson, 2020[24]). Collaborating with the Vaccine Research Center at the U.S. National Institutes of Health, the trial successfully enrolled its initial participants, marking swift progress. Similar initiatives are in progress by CureVac and BioNTech, with the latter in collaboration with Pfizer. The Pfizer/BioNTech partnership has embarked on Phase I/II trials for their vaccine candidates (Dheyab et al., 2021[17]). Inovio Pharmaceuticals' exploration of DNA plasmid vaccines yielded promising results in animal studies, prompting the initiation of Phase I human trials (Dheyab et al., 2021[17]).

Another Phase I candidate, developed jointly by the University of Oxford and AstraZeneca, hinges on a chimpanzee adenovirus vaccine vector (ChAdOx1) fused with the SARS-CoV-2 spike protein (Watanabe et al., 2021[85]). Promising results from animal studies indicate a reduction in disease severity upon exposure to the virus. Similarly, CanSino Biological Inc. and Beijing Institute of Biotechnology's adenoviral vector vaccine (Ad5-nCoV) is undergoing Phase I/II trials (Barajas-Nava, 2021[9]). While novel DNA and mRNA technologies hold promise, they remain unproven in human applications, with Moderna's existing mRNA-based vaccine programs still in Phase II. Recent data demonstrates that Moderna's mRNA-1273 prompts protective antibody titers in early-phase trials, accelerating progress towards Phase II (Bakhiet and Taurin, 2021[7]). Contrastingly, established protein-based vaccine platforms, including recombinant-protein, viral-vector, attenuated, or inactivated vaccines, have already demonstrated efficacy in various infectious diseases. In the fight against SARS-CoV-2, multiple approaches are being investigated, exemplified by China National Pharmaceutical Group's inactivated vaccine trials and the efforts of Sinopharm, Sinovac Biotech, and others.

Live-attenuated vaccines bear inherent immunogenic properties however it is crucial to conduct thorough safety assessments to prevent potential reversion to pathogenic forms. Robust research has been conducted on SARS-CoV live-attenuated vaccines, demonstrating their stability. Similarly, recombinant-protein and inactivated vaccines, though safer, might necessitate adjuvants for enhanced immunogenicity. The role of adjuvants, particularly in the context of SARS-CoV-2, is dual: they could bolster vaccine efficacy, especially in high-risk individuals, and optimize vaccine production scalability (Dheyab et al., 2021[17]). Advancements in adjuvant technologies include MF59, introduced in influenza vaccines for the elderly, and others like AS03, AF 03, AS 01, and AS04, each designed to enhance the immune response. These adjuvants could play a pivotal role in augmenting vaccine efficiency and streamlining production timelines, amplifying the arsenal against SARS-CoV-2.

Nanoparticles are currently under scrutiny for their potential in delivering gene therapies aimed at addressing genetic respiratory conditions like cystic fibrosis (Ruigrok et al., 2021[63]). The intricate biopolymeric structure of respiratory mucus can pose challenges for the dispersion of nanocomplexes, either through steric hindrance or interactions with the nanocomplexes themselves. Non-cross-linked macromolecules may adhere to nanocomplex surfaces, potentially leading to aggregation and hindrance of their mucus traversal. Interestingly, studies indicate that even in the absence of aggregation, the binding of extracellular components to nanocomplex surfaces can markedly diminish their gene transfection efficacy. Consequently, the journey of nanocomplexes towards epithelial cell surfaces hinges on several variables, including aspects such as their movement within mucus, the thickness of the mucus layer, and the rate at which mucus is cleared. When nanocomplexes interact with constituents found in mucus, a series of adverse effects can occur. These effects include their entrapment within the mucus matrix, neutralization of their surface charges leading to aggregation, release of their DNA cargo, and a reduction in cellular uptake. This decreased cellular uptake results from the masking of their positive charges or receptor ligands. In essence, the complex interaction between nanocomplexes and the intricate environment of respiratory mucus significantly impacts their movement, functionality, and potential for successful gene transfection.

Numerous research studies have delved into the influence of sputum, particularly from individuals with cystic fibrosis (CF), on the structure and transfection efficiency of nanocomplexes. In one study, lipoplexes composed of 1,2-Dioleoyloxy-3-(trimethylammonium) propane (DOTAP)/DOPE were exposed to varying amounts of mucin, linear DNA, or albumin (Di Gioia et al., 2015[18]). The interaction between charged mucus components and cationic nanocomplexes resulted in a decrease or reversal of the nanocomplexes' surface charge. Increased levels of albumin and linear DNA created an anionic shield around lipoplexes, enhancing protection against aggregation. Mucin-coated lipoplexes, while avoiding substantial aggregation or dissociation, exhibited reduced transfection efficacy. This observation suggests that mucins may influence the intracellular trafficking of complexes during transfection.

The albumin-coated PEI polyplexes exhibited superior gene transfer efficiency compared to unmodified PEI, even in the presence of CF sputum (Di Gioia et al., 2015[18]) (Aguilar et al., 2020[2]). The presence of endogenous DNA within mucus is another crucial factor contributing to the decreased transfection efficiency of nanocomplexes. Experiments conducted by Alton and colleagues using cell cultures covered with diluted CF sputum revealed that gene delivery, mediated by both lipoplexes and adenoviral vectors, experienced a reduction due to CF sputum DNA binding to the epithelial cell surface (Dheyab et al., 2021[17]). In fact, a significant decline in gene transfer occurred in cells pre-coated with highly diluted sputum, as well as in those subjected to sputum removal before transfection. The transfection capability of both vectors was partially restored following pre-treatment of cells with recombinant human deoxyribonuclease (rhDNase). These findings underscore the intricate interplay between nanocomplexes and mucus components, shedding light on the multifaceted dynamics that shape transfection outcomes.

The development of an effective non-viral gene delivery system relies on its capacity to facilitate efficient cell delivery while minimizing interactions with extracellular components like mucus and avoiding detection by macrophages. In the domain of nanocomplex-based gene delivery, maintaining an overall positive surface charge of polyplexes is a common prerequisite to ensure stable complexes and achieve high transfection rates. But this positive surface charge presents challenges in interactions with extracellular anionic components. Particularly in gene transfer to the lungs, obstacles arise due to interactions with mucus components such as proteoglycans, glycosaminoglycans, and, to some extent, constituents of the surfactant layer like phospholipids. To tackle these challenges, various shielding strategies have emerged to counteract the self-aggregation of non-viral gene carriers and mitigate interactions with extracellular components. Among these strategies, the incorporation of hydrophilic, uncharged polymers like PEG (polyethylene glycol) stands out as a critical factor in facilitating effective pulmonary gene delivery. The inclusion of PEG results in more stable polyplexes while concurrently reducing interactions with sialic acid, a key component of mucus (Nguyen et al., 2008[50]). The concept of using PEG to modify drugs, proteins, and particles is not novel; its conjugation has been extensively explored to extend the circulation time of various agents, ranging from drugs to particulate drug carriers, following intravenous administration. In essence, these strategies represent innovative approaches to surmount the challenges posed by mucus interaction and cellular recognition, thereby advancing the potential for successful non-viral gene delivery to the lungs. Two distinct strategies have been employed to create nanocomplexes featuring inert polymers on their surface. The initial approach involves covalently linking the non-viral cationic carrier with the protective polymer and then mixing it with DNA. But in this method, the presence of shielding polymers might hinder the self-assembly process between the cationic carrier and DNA. The second strategy entails covalently attaching the shielding polymer to pre-formed non-viral nanocomplexes. Since PEGylation could potentially impede endosomal escape, several research groups are actively developing strategies wherein nanocomplexes shed their PEG chains either outside the cell or within acidifying endosomal compartments. Prominent among the successful nanocomplexes for lung gene delivery are synthetic cationic lipids like GL67. PEGylated GL67/DOPE lipoplexes exhibited uncompromised gene transfer efficiency even in the presence of certain factors (Sanders et al., 2002[64]).

Nanoparticles are used to deliver immune checkpoint inhibitors or antigens to lung cancer cells, enhancing the immune response against tumors. ARAC, an immunotherapy based on nanoparticles, amplifies the effectiveness of PD-L1 inhibitors in advanced non-small cell lung cancer (NSCLC) (Reda et al., 2022[59]). This approach simultaneously delivers a PLK1 inhibitor (volasertib) and a PD-L1 antibody, targeting the overexpression of PLK1 in cancer. In a metastatic lung tumor model, ARAC lowers the required doses of volasertib and PD-L1 antibody by a factor of five, demonstrating efficacy in another lung tumor model as well. This study highlights the need for superior immunotherapy strategies.

Adjuvants imitate molecules resembling those produced by infectious pathogens, which are identified by pattern recognition receptors (PRRs), and act as immune response boosters. Frequently utilized adjuvants in cancer immunotherapy include compounds such as 3-O-desacyl-4'-monophosphoryl lipid A (MPLA), lipopolysaccharide (LPS), CpG oligodeoxynucleotides (CpG ODNs), polyinosinic: polycytidylic acid (poly I:C), and stimulator of IFN genes (STING) agonists (Park et al., 2018[54]). These adjuvants facilitate heightened anti-cancer immune responses when taken up by antigen-presenting cells (APCs) along with tumor antigens. As a result, various approaches have been devised to efficiently transport adjuvants into antigen-presenting cells (APCs) using nanoparticles. For example, adjuvants such as CpG ODNs, poly I:C, and STING agonists, which possess a negative charge, can establish electrostatic complexes with positively charged nanoparticles or polymers (Alvarez et al., 2021[4]). This enables their internalization by APCs through endocytosis, consequently bolstering the promotion of anti-cancer immunity. The utilization of nanoparticles for adjuvant delivery, combined with the direct delivery of antigens into the cytoplasm of APCs, significantly contributes to the stimulation of potent, antigen-specific T-cell responses. The integration of nanoparticle-mediated cancer antigen delivery with immune checkpoint blockade approaches can further enhance anti-cancer immune responses. Thus, nanoparticle-based strategies hold considerable promise for addressing various blood and solid cancers.

Regulatory T cells (Tregs) are a subset of T cells with immunosuppressive capabilities that can hinder the actions of anti-tumor T-effector cells (Gao et al., 2022[21]). While Tregs serve to prevent autoimmune diseases by promoting immune tolerance to self-antigens, their presence within the cancer context can impede immune responses against tumors by dampening anti-cancer immune activities within the tumor microenvironment (TME). To initiate a potent anti-tumor immune response, tactics can be utilized to actively restrain or eliminate Tregs. A prevalent strategy in lung cancer immunotherapy focuses on modulating Treg activity via checkpoint blockade. This is illustrated by substances designed to target molecules such as CTLA-4 (Gao et al., 2022[21]). Engineered nanoparticles hold promise in selectively targeting Tregs and facilitating their removal from the tumor microenvironment, thereby contributing to enhancing anti-cancer immune responses.

Nanoparticles can deliver anti-inflammatory agents to mitigate symptoms of asthma and allergic reactions in the respiratory system. Si-RNA-based therapy has potential for treating asthma and COPD by reducing toxicity and immunogenicity in airway inflammatory cells (Perl et al., 2010[55]). Kumar and colleagues showcased the potential of chitosan interferon-γ-plasmid DNA nanoparticles in mitigating allergen-induced airway inflammation and hyperresponsiveness (Kumar et al., 2003[37]). There have been advancements in the delivery of inhalable siRNA using poly (amidoamin) dendrimer carriers and dendrimer-based nanoparticles for in vivo applications.

Research is focusing on developing drug nanocarriers to deliver asthma therapeutics to inflammation sites. The anti-inflammatory effects of theophylline were enhanced through intranasal delivery using thiolated chitosan nanoparticles (Lee et al., 2006[39]). Muco-adhesive nanoparticles and chitosan-hyaluronic acid nanoparticles loaded with heparin have demonstrated potential in addressing asthma-related conditions (Ahmad, 2022[3]). Heparin-loaded chitosan-cyclodextrin nanoparticles have exhibited significant improvements in drug efficacy, making them promising candidates for asthma treatment.

Nanoparticles loaded with steroids have shown remarkable therapeutic benefits in comparison to free drug formulations. Research has indicated that betamethasone phosphate-loaded polymeric nanoparticles accumulate at sites of airway inflammation and exert anti-inflammatory effects (Howard et al., 2014[25]). Self-assembling nanoparticles containing dexamethasone specifically target the lungs, reducing allergic lung inflammation and airway hyperresponsiveness. Combining therapeutic peptides and dexamethasone (Dexa) within the same nanocarrier has been effective in reducing neutrophilic pulmonary inflammation (Kim et al., 2011[34]). Efforts are also underway to develop oral dispersible tablets containing prednisolone-loaded chitosan nanoparticles for paediatric asthma management.

Nanoparticles are being explored for targeted delivery of vasodilators to treat pulmonary hypertension. Pulmonary arterial hypertension (PAH) is a severe cardiopulmonary condition resulting from the remodeling of pulmonary arteries (Rubin, 2006[62]). Existing treatments aim to alleviate PAH symptoms, but a co-delivery system has been developed to target p53 and baicalein, showing promise in mitigating the disease (Teng et al., 2022[76]). This system effectively homes in on pulmonary arterial smooth muscle cells (PASMCs) in vitro, allowing for gene transfection, apoptosis induction, and inflammation suppression. In in vivo experiments, it successfully reverses PAH induced by monocrotaline, reducing pulmonary artery pressure, downregulating TNF-α, and inhibiting pulmonary arteries and right ventricular remodeling. The robust efficacy may be linked to the activation of the Bax/Bcl-2/Cas-3 signaling pathway.

TLRs play a pivotal role in triggering immune responses by identifying patterns associated with pathogens (PAMPs) and patterns associated with danger (DAMPs). TLR agonists can activate immune cells and enhance their responses against infections, tumours, and other diseases. However, direct administration of TLR agonists can lead to systemic side effects and limited targeting to the desired site. Nanoparticles provide an attractive solution to these challenges due to their unique properties. They enable targeted delivery to immune cells in the respiratory system, ensuring efficient TLR agonist stimulation with minimal side effects. The controlled release of agonists from nanoparticles extends their presence at the site of action, preventing rapid clearance. Encapsulation shields agonists from degradation, preserving their potency during transport. Facilitating cellular uptake, nanoparticles enhance immune activation. In essence, nanoparticles provide a multi-faceted strategy to address challenges, optimizing therapeutic outcomes.

The choice of TLR agonists and their compatibility with nanoparticle formulations is crucial for effective immunotherapy. Several TLR agonists have been investigated for respiratory applications:

TLR4 Agonists: Lipopolysaccharides (LPS) derived from bacteria are TLR4 agonists. Their strong pro-inflammatory responses and potential toxicity make them challenging candidates. Modified LPS or other TLR4 ligands with reduced toxicity may be considered. Lipopolysaccharide (LPS) serves as a potent immunomodulatory agent, activating TLR-4 in Gram-negative bacteria. Its clinical utility is restricted due to its associated toxicity. Monophosphoryl lipid A (MPL), derived from Salmonella minnesota R595, induces similar cytokine profiles to LPS but with reduced toxicity, making it a subject of extensive investigation in clinical applications (Michaud et al., 2013[47]). MPL vaccines have been assessed for their capacity to boost systemic and mucosal immune responses against hepatitis B surface antigens, tetanus toxoid, and influenza. Both MPL and MPL-mimetics show potential as therapeutic agents in modulating the innate immune response to provide short-term resistance against infectious challenges. Co-administering Francisella tularensis-LPS with MPL or MPL alone has been found to protect mice from F. tularensis infection (Richard et al., 2017[61]). Fimbriae H protein (FimH) acts as a ligand for TLR-4 and induces pulmonary changes in mice, thereby reducing mortality and morbidity associated with influenza infection. Synthetic mimetics of lipid A known as AGPs require TLR-4 recognition. Prophylactic treatment of mice with AGPs prior to inhalation of F. novicida resulted in reduced bacterial burden in the lung, liver, and spleen upon challenge, along with lower mortality rates compared to PBS-treated mice.

TLR3 Agonists: TLR-3 agonists, such as polyinosinic:polycytidylic acid (PIKA) and polyinosinic-polycytidylic acid (Poly IC:LC), have potent anti-viral actions and are associated with viral infection (Mifsud et al., 2014[48]). PIKA is a synthetic agonist that recruits macrophages, neutrophils, and plasmacytoid DCs into the lungs, reducing viral burden in mice infected with various influenza viruses. Poly IC:LC, a chemically stabilized variant of polyinosinic IC, activates TLR-3, providing defense against lethal challenges from avian influenza viruses (Mifsud et al., 2014[48]). Administering Poly IC:LC twice daily after infection enhances bacterial replication and leads to extensive lung necrosis in treated mice. The research conducted by Antonelli et al. advises caution in the use of immunomodulatory agents that stimulate Type 1 interferon (IFN) in regions where tuberculosis is prevalent (Antonelli et al., 2006[6]). In mice, intranasal delivery of two doses of Poly IC has been observed to increase the bacterial burden following infections with S. pneumoniae and methicillin-resistant S. aureus. Type 1 IFNs do not aid in bacterial clearance, and elevated levels of Type 1 IFNs render mice more vulnerable to S. pneumoniae infection. These interactions between viruses and bacteria are pertinent to human infections, as viral infections can predispose individuals to secondary bacterial infections, especially in cases of influenza A infections.

TLR7/8 Agonists: Imidazoquinoline compounds such as Resiquimod and Imiquimod are TLR7/8 agonists. They activate immune responses and have shown promise in cancer immunotherapy (Vasilakos and Tomai, 2013[79]). TLR-7, a receptor recognizing single-stranded RNA fragments within endosomes, is commonly activated during viral infections. Imiquimod, a potent TLR-7 agonist, triggers innate and adaptive immune pathways by inducing pro-inflammatory cytokines. Initially FDA-approved for genital wart treatment in 1997, Imiquimod's efficacy was demonstrated in a guinea pig model, reducing HSV-2 lesion frequency. Infectious agents like viruses and bacteria can contribute to cancer, with human papilloma viruses linked to basal cell carcinomas. Topical 5% Imiquimod cream displayed enhanced clearance rates for these conditions, highlighting immunostimulants' potential in cancer treatment (Vasilakos and Tomai, 2013[79]). Although extensively used for dermatologic ailments, Imiquimod can cause side effects, limiting its utility; improved variants could mitigate these concerns.

TLR9 Agonists: CpG oligonucleotides are TLR9 agonists that mimic bacterial DNA motifs. They induce robust immune activation and are being explored for various respiratory diseases. CpG, activating the innate immune system through TLR-9, mimics bacterial DNA's effects via non-methylated CpG motifs (Vollmer and Krieg, 2009[81]). Administering CpG-ODN shifts the Th2-biased response in Balb/c mice infected with Leishmania major towards a protective Th1 profile, rendering them resistant. This protection extends to intra-cellular pathogens like L. major and F. tularensis with durable effects independent of inoculation route, suggesting therapeutic potential. TLR-9's significance in countering HSV-2 infections is evident, as CpG-ODN administration limits viral replication and enhances survival without direct viral inhibition, instead promoting immune responses (Dar et al., 2009[16]). The CpG-ODN's efficacy surpasses other TLR ligands due to its induction of IFN-β, essential for protection. In a study comparing CpG-ODN and Resiquimod (R-848) for HSV-2 treatment, CpG-ODN's localized cytokine response proves superior, while R-848 triggers systemic chemokine release without virus control (Tomai et al., 2007[78]). Repetitive CpG-ODN administrations for therapeutic use raise concerns, as prolonged TLR activation may lead to organ pathology and harmful effects, necessitating a balanced approach for effective immunotherapies.

The compatibility between selected TLR agonists and nanoparticle formulations must consider physicochemical properties, such as solubility, charge, and hydrophobicity, to ensure stable encapsulation and release characteristics.

Some nanoparticles have been designed to enhance immune responses, making them valuable in cancer immunotherapy, vaccine adjuvants, and for treating infectious diseases. Nanoparticles can be engineered to increase the production of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-1β, and IFN-γ) and enhance antigen presentation. They can also activate Toll-like receptors (TLRs), particularly TLR4, and stimulate the function of immune cells like dendritic cells (DCs), macrophages, and T cells. However, overstimulation of these pathways can increase the risk of cytokine storms, a severe inflammatory response that can lead to tissue damage and systemic toxicity.

Conversely, some nanoparticles can exert immunosuppressive effects, making them useful in eating autoimmune diseases or in reducing inflammation in chronic conditions. These nanoparticles can reduce the release of pro-inflammatory cytokines and promote the secretion of anti-inflammatory cytokines (e.g., IL-10, TGF-β). They may also induce regulatory T cells (Tregs) and impair the maturation of dendritic cells, which dampens the overall immune response. Nanoparticles can also inhibit the function of certain immune cells, including macrophages and neutrophils, which helps in preventing excessive immune reactions.

Nanoparticles that strongly stimulate immune responses pose a risk for inducing a cytokine storm, particularly if they activate pathways involving TLRs and pro-inflammatory cytokine production. This phenomenon has been observed in some viral infections and immunotherapies and can lead to uncontrolled inflammation. Nanoparticles that activate TLR4, in particular, have been shown to induce a cascade of cytokine release, potentially leading to a cytokine storm. Proper formulation and surface modification are crucial to mitigate this risk while maintaining the desired therapeutic effects.

Authors would like to acknowledge the APC support of through UTS institutional agreement with journal through CAUL.

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Conceptualization; ASR, KD, KPR. Data curation; ASR. Formal analysis; ASR, KD. Investigation; ASR, KD. Methodology; ASR, KD. Project administration; ASR. Resources; ASR. Supervision; KD, KRP. Validation; KD. Visualization; ASR. KD. Roles/Writing - original draft: ASR, AV, SP, MBN and Writing - review & editing: KRP, ASR, AV, SP, MBN, KD.

None.

No artificial intelligence tools were used in the preparation, writing, editing, or analysis of this manuscript.