Intranasal and Pulmonary Lipid Nanoparticles for Gene Delivery: Turning Challenges into Opportunities

Introduction

Lipid nanoparticles (LNPs) and other lipid-based delivery systems have emerged as a groundbreaking modality for administering gene-based therapeutics and vaccines. These delivery systems are on a trajectory to become more dominant than the traditionally used viral vectors for delivering gene medicines owing to several advantages related to their safety profile, flexibility in formulation, and reduced immunogenicity, which collectively enhance their potential for widespread clinical application.^1–3 LNPs exhibit distinct physicochemical characteristics that facilitate the encapsulation of a wide range of genetic materials, including messenger RNA (mRNA) and deoxyribonucleic acid (DNA), while concurrently providing a protective barrier against enzymatic degradation. This attribute is particularly vital within the context of gene medicine, where the protection of delivery and the bioavailability of therapeutic agents are paramount for achieving optimal therapeutic results.^1–3

As the landscape of gene-based therapeutics continues to evolve, integrating LNPs into nasal delivery systems presents a promising avenue for enhancing therapeutic efficacy and patient compliance.⁴ Recent advancements in this field demonstrates the ability for LNPs to be fine-tuned, not only for optimal encapsulation but also targeted delivery to specific tissues such as the lungs. In this regard, this capability could revolutionize treatments for respiratory diseases by allowing localized action with minimal systemic exposure, thus reducing potential side effects associated with a broader distribution all the way through the fine bronchiolar labyrinth structure.^4–6

Intranasal (IN) delivery is a non-invasive and patient-centric approach that markedly enhances upper and lower respiratory tract bioavailability and potentially allows direct targeting of the central nervous system (CNS).^7–9 For the purposes of our review, we will use the term “intranasal delivery” to encompass nasal administration via the nasal route to include the upper and lower respiratory tract, and CNS targets accessed. Other applications for delivering genes through the nasal route include the treatment of lower respiratory pulmonary conditions, such as chronic obstructive pulmonary disease (COPD) and asthma,^10–12 respiratory infections,^13–15 and vaccination.^16–20 Although intranasal administration is characterized by clear, accessible anatomy, relative immune privilege, and low enzyme activity, a number of significant challenges remain to be addressed.^21–25

One of the primary challenges in optimizing LNPs for intranasal delivery is achieving precise control over its physicochemical properties to avoid administration-related degradation, reduce mucociliary clearance and control its tissue deposition site. Other considerations related to cellular barriers include low cellular uptake and endosomal escape rates. Nevertheless, innovations in formulation techniques, including the use of ionizable lipids and helper lipids with specific chain lengths, can potentially modulate transfection efficiencies and therapeutic outcomes.^21–25 Moreover, exploring additional formulation strategies, such as utilizing mucoadhesive agents that prolong retention time, may further improve the efficacy of LNP-mediated therapies by facilitating the sustained release of therapeutic cargo and enhancing local concentrations at the site of action.^26–28 As development in these areas improve, these advancements not only promise to refine existing applications but also pave the way for novel interventions targeting a broader spectrum of diseases through more sophisticated delivery mechanisms.

The primary aim of this review is to conduct a thorough examination of the contemporary landscape in intranasal applications of LNP technology. In short, we seek to elucidate not only the significant challenges that researchers encounter in the intranasal delivery space but also the innovative strategies being utilized to overcome these obstacles.

Lipid Nanoparticles; an Introduction

The journey in LNP development for nucleic acid delivery began over six decades ago. Early studies on liposomes, small vesicles made up of one or more lipid bilayers that can encapsulate and transport drugs and other molecules to specific sites in the body, led to the regulatory approval of several lipid-based drug formulations, including the antifungal agent, Abelcet²⁹ and chemotherapeutic agents Myocet,³⁰ and Marqibo.³¹ These formulations paved the way for more advanced LNP systems, particularly in the context of gene delivery. Indeed, the first US Food Drug Administration (FDA) approved for LNP product was granted in 2018 for Onpattro (patisiran), an LNP encapsulated small interfering RNA (siRNA) therapeutic for treating a genetic liver disease called transthyretin-induced amyloidosis.³² LNPs gained further traction in the field of mRNA vaccines, exemplified by the rapid development and deployment of COVID-19 vaccines, which showcased its potential for delivering genetic material effectively and safely.³³

A typical formulation for LNPs consists of a carefully curated blend of four essential lipid components; ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol (PEG)-lipids.^34,35 The ionizable cationic lipids are the primary functional components that facilitate electrostatic interactions with negatively charged nucleic acids, enabling efficient encapsulation. At physiological pH, these lipids maintain a near-neutral charge, minimizing toxicity and undesired interactions while becoming positively charged in the acidic environment of endosomes to promote endosomal escape.³⁶ Phospholipids, such as distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylethanolamine (DOPE), act as structural lipids that stabilize the LNP architecture and influence membrane fusion properties. Cholesterol enhances the rigidity and stability of the lipid bilayer, reduces permeability, and helps maintain the structural integrity of LNPs during storage and administration. PEG-lipids serve multiple functions: they stabilize LNPs during formation by preventing aggregation, extend circulation time by creating a hydrophilic shield that reduces recognition by the immune system, and influence cellular uptake and biodistribution patterns.^37–40

The optimal composition of LNPs remains a topic of ongoing debate. Notably, formulations that include an ionizable lipid at a molar ratio of 33% have demonstrated remarkable mRNA transfection efficiency, both in vitro and in vivo.⁴¹ The intricate interplay between the types and ratios of these lipids plays a crucial role in determining LNP performance. Modifications to the lipid composition, whether through altering the types of lipids used, adjusting their proportions, introducing or omitting specific components, can significantly impact nanoparticle physicochemical properties. For instance, LNP size and surface charge can be tailored to optimize its interaction with cellular membranes, thereby altering cellular uptake efficiency. Furthermore, these adjustments directly influence the release kinetics of the encapsulated therapeutic agents, as well as the overall stability of the nanoparticles.^34,35,41 The efficacy of the LNPs in delivering its payload is intricately linked to these factors, as is its safety profile in therapeutic applications. Understanding the nuanced dynamics of lipid interactions and its effects on LNP behavior is essential for the development of effective and safe delivery systems for gene medicines.

The clinical utility of LNPs for gene delivery has been demonstrated through several successful applications that have either received regulatory approval or are in advanced clinical trials. The first FDA-approved LNP-based therapeutic, Onpattro (patisiran), marked a significant milestone in 2018 for the treatment of hereditary transthyretin-mediated amyloidosis (hATTR) by delivering siRNA to silence the production of transthyretin protein in the liver.^32,42 This success was followed by the unprecedented rapid development and deployment of mRNA-LNP COVID-19 vaccines (BNT162b2 by Pfizer/BioNTech and mRNA-1273 by Moderna), which demonstrated remarkable efficacy and safety profiles, further validating the clinical potential of LNP delivery systems.⁴³

Beyond these approved therapies, numerous LNP-based gene delivery systems are advancing through clinical trials for various indications. For instance, Moderna’s mRNA-1944, an LNP-formulated mRNA encoding an anti-Chikungunya virus antibody, has shown promising results in Phase 1 trials with successful antibody expression in humans.^44,45 Additionally, Intellia Therapeutics’ NTLA-2001, a CRISPR-Cas9-based therapy delivered using LNPs, has entered clinical trials for transthyretin amyloidosis (Clinical Trials NCT05697861, NCT04601051, and NCT06128629). This represents an important advancement as one of the first in vivo CRISPR genome editing therapies to reach clinical testing.

For respiratory applications specifically, several LNP-based gene therapies are under clinical investigation. Translate Bio’s MRT5005, an inhaled mRNA therapy encoding cystic fibrosis transmembrane conductance regulator (CFTR) protein, entered clinical trials for cystic fibrosis treatment (Clinical Trial NCT03375047). Similarly, Arcturus Therapeutics has developed ARCT-032, another inhaled mRNA therapy for cystic fibrosis that has advanced to clinical evaluation (Clinical Trial NCT05712538). These respiratory-focused therapies are particularly relevant to intranasal delivery approaches, as they utilize similar administration routes and face comparable physiological barriers.

Beyond respiratory conditions, LNP-mRNA therapeutics are being evaluated for metabolic disorders, including Moderna’s mRNA-3704 for methylmalonic acidemia (Clinical Trial NCT03810690) and mRNA-3927 (Clinical Trial NCT04159103) for propionic acidemia.⁴⁶ Arcturus Therapeutics’ ARCT-810, which delivers ornithine transcarbamylase (OTC) mRNA for OTC deficiency, has also entered clinical development (Clinical Trial NCT05526066). These examples highlight the versatility of LNP delivery systems across various therapeutic applications and administration routes.

In this review, we focus on inhalable LNPs due to its many advantages and unique features. For instance, inhalable LNPs reduce systemic toxicity and can achieve higher localized drug cargo concentrations in the lungs⁴⁷ while also having the potential to modify the LNP surface with specific ligands that target particular cell types within the respiratory tract.⁴⁸ Indeed, these LNPs have been shown to protect its therapeutic cargo from enzymatic degradation^46,49 and improve its cellular uptake.⁵⁰ Lastly, these inhalable LNPs can be tailored to provide a therapeutic effect distal to the lungs, with the potential to extend its therapeutic effects in the brain for the treatment of neurological disorders.^7–9,51,52

Challenges and Opportunities for Intranasal LNP Delivery

Delivering LNPs intranasally provides opportunities such as direct access to the lungs, avoiding first-pass metabolism by minimizing systemic exposure and the potential for brain delivery. However, the process of administering nanoparticles intranasally, in addition to the complex pulmonary environment, poses many challenges for LNPs to effectively deliver its cargo. Understanding the challenges and barriers is essential for designing an effective and safe intranasal formulation. Here, we will categorize these challenges into pre-cellular and cellular barriers.

Formulation Stability and Integrity

The administration of pharmacological agents through the pulmonary pathway (eg, via aerosolization) necessitates that these substances endure the mechanical stresses applied to these agents during the procedure. This includes factors such as shear forces, turbulence, and the potential for aggregation.^53,54 This is of special concern in the case of LNPs due to its high surface energy, especially in suspension form, which could promote its aggregation by Ostwald ripening and recrystallization.⁵⁵ LNP instability during aerosolization poses a significant barrier to achieving successful pulmonary delivery. Several strategies could be explored to overcome these issues, including optimization of particle size, surface charge, and the choice of lipid composition.⁵⁶ For instance, the inclusion of PEG-lipids was shown to be essential in preventing LNP aggregation. However, fine tuning the ratio of its composition is essential to improve the colloidal stability whilst not impacting its cellular uptake.⁵⁷ Other strategies to improve the colloidal stability include charge-assisted stabilization (CAS) by utilizing a peptide-lipid conjugates,⁵⁸ employing stabilizing excipients such as trehalose, dextran, and leucine,⁵⁷ and the use of advanced atomization techniques, such as residual free atomizer.⁵⁹ To further study and improve the stability of LNPs, techniques such as single particle automated Raman trapping analysis (SPARTA) and small-angle X-ray and neutron scattering (SAXS/SANS) have been utilized to link LNP composition to its internal structure.⁶⁰ Aside from PEG, the role of the helper lipids was found to be critical in modulating its transfection efficiency in airway epithelial cells using unsaturated lipids, such as those derived from oleyl groups, as opposed to their saturated counterparts.⁶¹

Anatomical and Administration Considerations

The optimal size range for the effective deposition of intranasally delivered particles in the pulmonary regions of the lungs is between 1–5µm in diameter.⁶² While larger particles tend to be predominantly retained within the oropharyngeal region of the upper respiratory tract,⁶³ nano-sized particles are often easily expelled from the respiratory system upon inhalation due to the effects of Brownian motion, which facilitates its suspension and movement within the respiratory pathways.⁶² One potential strategy for alleviating this concern is to encapsulate these nanoparticles within microparticles that are designed to release its nanoparticle contents upon contact with the mucus or fluid present in the lung environment, thereby enhancing its deposition and therapeutic efficacy.⁶⁴ The strategy of the nano-embedded microparticles (NEM) holds promise and has been successfully used to improve the intranasal delivery of LNPs.^65–70 Further, to ensure the effective and uniform delivery to the intended site within the pulmonary route, the selection of an appropriate delivery device is critical.^61,71–73 Various applicator devices have been utilized for the intranasal administration of LNPs, each offering unique advantages and limitations. These devices include inhalers, nebulizers, and other applicators.

Inhalers

Inhalers are widely used as an effective method for delivering therapeutic agents directly to the pulmonary tract. Its popularity stems from their ease of use, portability, and ability to deliver precise doses of medication. In the context of LNP delivery, inhalers have been explored for their potential to provide targeted treatment for various respiratory conditions. The three main types of inhalers include pressurized metered dose inhalers (pMDIs), dry powder inhalers (DPIs), and soft mist inhalers (SMIs).

Pressurized Metered Dose Inhalers (pMDIs)

Pressurized Metered Dose Inhalers (pMDIs) are one of the most common inhaler types used for delivering medications to the lungs. These devices utilize a propellant to disperse a specific dose of the medication in aerosol form, which patients inhale through a mouthpiece. The primary advantage of pMDIs is its ability to deliver a consistent and precise dose of medication, making it highly reliable for patients requiring regular dosing. The aerosolization process in pMDIs involves the rapid expansion and atomization of a formulation through a nozzle, which can potentially impact the stability of LNPs. However, studies have shown that with careful formulation design, the structural integrity and functionality of LNPs can be preserved. This typically involves optimizing the propellant, surfactants, and other excipients to ensure that LNPs are not destabilized during aerosolization. For instance, the use of hydrofluoroalkane (HFA) propellants, which are less likely to disrupt lipid structures, has been found to be beneficial in maintaining the integrity of LNPs.^{19,47,74–76} Furthermore, the actuator nozzle and surrounding conditions significantly impact the atomization process, which in turn affects the droplet size distribution and deposition of the drug in the respiratory tract. Computational fluid dynamics (CFD) simulations have shown that different nozzle designs can influence drug deposition, with twin nozzles increasing deposition in the mouth-throat region, potentially affecting the delivery of LNPs.^62,77,78

Soft Mist Inhalers (SMIs)

SMIs represent a newer category of inhalers that provide a unique method of delivering medication to the lungs. SMIs do not use a propellant; instead, they generate a slow-moving mist using a mechanical action. This mist is finer and slower compared to aerosols produced by pMDIs, resulting in a longer duration of mist of the inhaler airstream, which allows more time for the patient to inhale the medication.

SMIs offer several advantages over pMDIs, particularly in terms of inhalation technique and deposition efficiency. Notably, they were found to be one of the most effective applicators for inhalable mRNA-LNPs, that when coupled with a trehalose buffer system, can withstand the shear forces of aerosolization and effectively protect and deliver their cargo.⁵⁶ However, they have higher cost compared with other inhalers, and limited availability of medications in soft mist formulation.⁷⁹

Dry Powder Inhalers (DPIs)

DPIs offer an alternative to pMDIs, particularly for patients who find it difficult to coordinate inhalation with actuation. DPIs deliver medication in a dry powder form that is aerosolized by the patient’s inhalation effort rather than a propellant.¹² This design eliminates the need for synchronization, making DPIs more user-friendly for individuals with limited dexterity or lung capacity. In addition, dry form may be more suitable for delivering LNPs due to their superior physical stability.^80,81 Administering LNPs using this approach primarily targets the nasal cavity but may also result in some deposition in the lungs if particles are too fine.⁸² It is important to note here that the DPI approach has been effectively used to overcome the blood-brain barrier.⁸³ However, the drying process can potentially affect the stability and integrity of LNPs.^80,84

Nebulizers

Nebulizers are devices that convert liquid formulations into fine mists, making them particularly suitable for delivering LNPs to the lungs.⁸⁵ These devices are advantageous for patients who have difficulty using inhalers, such as children or the elderly. Nebulizers have shown promise in delivering significantly more mRNA packaged in LNPs to the lungs compared to conventional LNP formulations optimized for systemic delivery. Despite their advantages, nebulizers can be cumbersome, requiring longer administration times and regular maintenance.^86,87 Most importantly, without extensive optimization, LNPs tend to break up during nebulization, impacting its delivery efficiency. LNPs aerosolized using a nebulizer results in an increase in particle size and decreased encapsulation, which is attributed to shear forces impacting the particle.^3,4,88,89 More recently, Kim, Jozićet al developed a microfluidic aerosolization platform (MAP) specifically for LNPs, offering advantages such as preserving the structural and physicochemical integrity of lipid nanoparticles, avoiding aggregation, and improving cell transfection.⁹⁰

Other Intranasal Delivery Devices and Formulations

A variety of other intranasal applicators have been developed to improve deposition patterns in the nasal cavity. Traditional liquid nasal spray pumps are simple and convenient but tend to deposit a large portion of the dose in the anterior, non-ciliated region, with relatively little reaching the upper/posterior nasal cavity.^91,92 This limits drug access to deeper regions (eg the olfactory cleft or nasopharynx). Liquid droppers can achieve slightly deeper delivery beyond the nasal valve, but are less patient-friendly and often yield inconsistent dosing ^93,94 Dry powder nasal insufflators (solid dose devices) avoid the need for preservatives and can improve stability, but the powder must be well dispersed to coat the mucosa uniformly. Recent innovations like bi-directional nasal delivery systems (eg Exhalation Delivery Systems) use the patient’s exhaled breath to propel formulations into the nasal passages.^95–97 These systems create a closed soft palate and a positive pressure airflow, resulting in significantly greater deposition in the superior and posterior regions (including the olfactory area) compared to conventional sprays. Notably, bi-directional devices can deliver drug broadly across the nasal cavity while minimizing lung deposition, which is advantageous for both sinus therapies and nose-to-brain applications.

Mucociliary Clearance

Mucociliary clearance (MCC) is an important physiological procedure that aids the expulsion of inhaled molecules and pathogens from the respiratory tract. This process has a significant impact on the retention and efficiency of LNPs when inhaled.^98,99 Many factors, such as nanoparticle size, surface composition, and the physical properties of the mucus and the respiratory tract, play a role in determining the interactions between the mucus and LNPs. The mucus layer acts as a shield that stops particles from reaching the epithelial surface. This shield mainly consists of a semi-liquid substance composed of water and glycoproteins, which have a direct influence on the penetration and retention of nanoparticles.^98,99 Although some studies claim that the mucociliary clearance of nanoparticles does not depend on its size, shape, or charge,^100,101 a considerable body of evidence suggests otherwise.^102–108 For instance, particles larger than 500 nm were found to be rapidly trapped and immobilized in mucus, while ones under 300nm had significantly better diffusion rates.^102,109 The surface charge of nanoparticles also plays a critical role in determining its fate in the respiratory tract. Mucus is rich in negatively charged groups, which readily interact with positive nanoparticles, resulting in its adhesion. This could be beneficial to some extent to increase nanoparticle retention time.¹⁰⁶ However, slightly negatively charged particles tend to have better mucus penetration properties.^105,110,111 In summary, neutral net charge was found to perform the best in terms of overall efficacy.¹¹² Hence, LNPs based on ionizable lipids may provide an opportunity for improving the therapeutic potential of these delivery systems in the pulmonary tract.

Various other strategies to improve mucus penetration have also been tested. For instance, PEGylation, the process of attaching PEG chains to nanoparticles, was found to play a crucial role in enhancing nanoparticle mucus penetrating ability.^113,114 PEG layers shield the particles from electrostatic and hydrogen bonding interactions with mucin, a major component in mucus, and, as a result, act as a steric barrier preventing adhesion to the mucin network.^103,113,115 It is also notable that denser PEG coatings achieve higher efficiency in penetration.^103,116 Other modifications, such as incorporating rhamnolipids,¹¹⁷ pulmonary surfactant lipids, such as DPPC,^118–121 modifying with cell-penetrating peptides,^122,123 and using mucoadhesive agents,^124–126 were also found to improve the mucus penetrating properties of nanoparticles.

As mentioned earlier, the physicochemical properties of mucus greatly influence the ability of nanoparticles to reach their cellular targets. Hence, it is safe to assume that modulating the mucus layer may be a viable option to facilitate the cellular uptake of LNPs in the pulmonary tract. One such strategy is the use of mucolytic agents, such as N-acetylcysteine (NAC), to reduce the viscosity of the mucus, allowing deeper penetration of the particles.¹²⁷ Pre-adjustment of the mucus pH levels in the pulmonary tract may also facilitate the cellular uptake of some particles.¹¹³ Finally, variations in parameters related to mucus rheological characteristics as well as ciliary movements may affect the speed of mucus transport and, as a result, the time nanoparticles spend within the respiratory tract.^109,128

The Lung Surfactant Problem

The pulmonary surfactant is a critical component of the respiratory system, which is produced by type II alveolar epithelial cells.^129–131 It is a complex mixture composed predominantly of lipids (about 90%) and proteins (approximately 10%), with phosphatidylcholines—particularly dipalmitoylphosphatidylcholine (DPPC)—being the main lipid constituents. The surfactant also contains specific proteins, namely surfactant protein A (SP-A), SP-B, SP-C, and SP-D, which play vital roles in its function.^129,132 This surfactant layer, with a thickness of about 0.2–0.5 μm, lines the alveolar surface and is essential for normal respiratory mechanics.^133–135

The primary function of the lung surfactant is to reduce surface tension at the air-liquid interface within the alveoli, thereby preventing alveolar collapse during exhalation and facilitating lung expansion during inhalation.^135–138 By lowering and regulating the surface tension, the surfactant improves lung compliance and enhances oxygenation, which is crucial for efficient gas exchange.^135,137,139 Additionally, surfactant proteins (SP), particularly SP-A and SP-D, contribute to the innate immune defense by recognizing and binding to pathogens and toxins, thus playing a role in host defense mechanisms.^140–142 The surfactant also acts as a biological barrier that influences the retention time of inhaled particles and determines pulmonary drug bioavailability. Its presence can significantly impact the deposition, absorption, and clearance of inhaled therapeutics, including nanoparticles.¹²⁹ For instance, surfactant proteins and lipids can adsorb onto the surface of nanoparticles (especially those >100 nm), forming a “corona” that alters their physicochemical properties and biological fate. This corona can affect the nanoparticles’ mucoadhesive properties, mucus permeability, cellular uptake, retention in bronchoalveolar lavage fluid, lung tissue absorption, systemic exposure, and extrapulmonary distribution.^138,143,144 Moreover, these interactions can impact the physicochemical properties of the surfactant itself, potentially affecting surfactant metabolism, particle clearance, and its biophysical functions.^132,139 This could lead to disruptions in surfactant activity, affecting lung function and potentially leading to adverse effects.

To overcome the detrimental effect of the lung surfactant on the successful delivery of nanoparticles, it is important to consider the charge, shape, and composition of nanoparticles. For instance, neutral-to-negative surface charged nanoparticles with moderate surface polarity were all found to be important factors to facilitate nanoparticle transit across the lung surfactant barrier.⁸² Additionally, molecular dynamics simulations have shown that the shape of the nanoparticles significantly affects their ability to penetrate the pulmonary surfactant layer. While spherical nanoparticles have been the primary focus in past studies, recent research highlights that non-spherical nanoparticles exhibit different translocation behaviors. Hydrophilic nanoparticles smaller than 5nm can penetrate the surfactant layer regardless of shape, while larger particles show shape-dependent translocation, with certain geometries like tetrahedral and cylindrical NPs causing less disruption compared to cubic and spherical shapes.^85,145

Recent studies have shown promising results with engineered LNPs that include β-sitosterol, to enhance transfection potency and substantial expression in the airway and alveolar epithelium,⁸⁵ and conditioning the surface properties with grafting polymers.¹⁴⁶ Understanding the role of the lung surfactant as a barrier has also led to innovative strategies to enhance nanoparticle delivery, such as the development of nanoparticles that mimic the composition and properties of the lung surfactant. Pre-coating nanoparticles with pulmonary surfactant components, such as commercial surfactant preparations like Curosurf^®, was found to improve nanoparticle stability and promote intracellular delivery of therapeutic agents like small interfering RNAs (siRNA) in the lung, both in vitro and in vivo.^147–149 The incorporation of surfactant proteins, particularly SP-B, can facilitate efficient cytosolic delivery of encapsulated nucleic acids into target cells, enhancing therapeutic efficacy.^135,150

Safety of Administering LNPs Intranasally

LNPs are relatively immunologically inert, hence why it is acceptably safe in humans. However, several considerations should be considered when designing a formulation for intranasal use. Taking mRNA-based vaccines as an example, the type and dose of the mRNA itself, the produced antigen, and the delivering LNPs, could result in adverse events.²⁰

It is evident that the safety of IN delivery of LNPs has not yet been examined extensively, and only a few studies focused on assessing the composition of the LNPs. For instance, Polyethylenimine (PEI) was shown to elevate the levels of the proinflammatory cytokine, interleukin (IL)-6, and result in weight loss in animals.^151,152 In another study by Andries, De Filette,¹⁵³ LNPs containing GL67, DOPE, and DMPE-PEG5000 led to an increase in pro-inflammatory cytokines (eg, tumour necrosis factor (TNF) α and IL-6), suggesting the need to avoid these charged and surface-active molecules when designing IN LNPs.¹⁵³

One of the most debated components of any LNP formulation is PEG. Anti-PEG IgE and IgM antibodies generated in patients administered with LNP formulated COVID-19 vaccines were shown to be responsible for serious side effects related to LNPs, such as acute organ toxicity, allergies, and the accelerated blood clearance (ABC) phenomenon mediated by IgM.¹⁵⁴ Approaches to limit these adverse events include shortening the length of the PEG lipid to allow easy separation from the LNP surface^155,156 and using a reduced PEG molecular weight to reduce circulation times.^157–159 Lastly, substituting PEG with other molecules, such as cleavable PEG-cholesterol derivatives or polysarcosine (PSar) is also being researched.^160–162

Conclusions

LNP-based systems have emerged as a leading delivery platform for gene medicine that was shown to be effective, safe, and versatile. IN administration of these LNPs holds several advantages: it is patient-friendly, non-invasive, and has the potential to provide targeted delivery to the respiratory tract and even to the central nervous system (CNS). Although progress in this field is evident, several critical challenges, knowledge gaps, and regulatory hurdles must be addressed before IN LNP formulations can reach their full clinical potential.

One of the most significant challenges for this route is the biological barriers within the nasal and pulmonary environments. While recent advances have led to improved LNP compositions, including the use of ionizable lipids and PEGylation strategies, the precise mechanisms by which nanoparticles interact with and traverse the nasal epithelium remain incompletely understood. Studies employing pharmacological inhibitors and advanced imaging techniques have started exploring the endocytic pathways involved in LNP uptake.¹⁶³ Such insights can guide the rational design of nanoparticles to exploit specific cellular entry routes, thereby enhancing transfection efficiencies and therapeutic outcomes.

The difficulty in translating preclinical findings to humans is hindered by anatomical and physiological differences between animal models and human nasal passages.^124,164 To bridge this translational gap, more research has to be done to develop physiological and disease models that better correlate with human nasal anatomy and conditions. These approaches can improve the predictability of deposition patterns, absorption kinetics, and eventual clinical performance of IN formulations.

Formulation stability during aerosolization is another critical concern. LNPs must withstand shear forces, turbulence, and potential aggregation as they transition from a liquid formulation to an inhalable aerosol. The use of hybrid nanoparticles—blending lipids for biocompatibility and polymers for enhanced binding affinity—can improve stability under these mechanical stresses, ensuring the LNPs maintain integrity, avoid premature release of their genetic cargo, and achieve efficient gene expression at the target site.⁸⁹ Strategies such as employing nano-embedded microparticles (NEM) and carefully selecting excipients also help achieve optimal aerodynamic properties and sustained release profiles.^19,57,69,70

Device selection is integral to successful IN delivery. Pressurized metered-dose inhalers, soft mist inhalers, dry powder inhalers, and nebulizers each offer unique advantages and challenges in terms of droplet size distribution, particle stability, and deposition efficiency.^71,80,84,165 Computational modelling, high-throughput screening, and structure-function analyses are increasingly employed to match LNP formulations with the most suitable delivery devices. Moreover, applying Quality by Design (QbD) principles can help streamline product development, ensuring that each step—from lipid selection to device engineering—is optimized to meet regulatory standards and maximize patient acceptance.^166,167

Beyond formulation and device parameters, overcoming innate lung defenses is critical. Mucociliary clearance, mucus viscosity, and lung surfactant layers all influence nanoparticle fate.^{82,98,105,168} While smaller or neutral-to-slightly negative particles and PEGylated formulations show improved mucus penetration, these design features must be balanced against the need for robust cellular uptake and sufficient residence time. Further modulation of mucus properties through mucolytics and adjusting pH can also improve nanoparticle transport to underlying epithelial cells.^109,113,114

Safety and immunogenicity remain areas that require closer scrutiny. Although LNP-based products have demonstrated acceptable safety profiles, potential inflammatory responses resulted from certain lipid components or PEG-related adverse effects highlights the need for thorough toxicological assessments. Designing LNPs that minimize inflammatory cytokine release and exploring alternatives to PEG can alleviate safety concerns. Such considerations become even more critical when developing intranasal gene therapies that may require repeated dosing.^154–156

It is important to acknowledge that while this review comprehensively addresses the challenges and opportunities of LNP delivery systems via the intranasal route, there remains a significant knowledge gap regarding the specific effects of these challenges on gene delivery efficiency and effectiveness. The field currently lacks sufficient research directly correlating LNP formulation parameters and delivery barriers with quantitative gene expression outcomes in intranasal applications. This knowledge gap presents an important opportunity for future studies to establish clearer connections between the physicochemical properties of LNPs, their interactions with biological barriers in the nasal cavity and respiratory tract, and the resulting gene expression levels in target tissues.

Despite promising advances, relatively few nanoparticle-based intranasal products have reached the market, and an integrated approach is needed to improve the outcomes. In looking ahead, addressing knowledge gaps in nanoparticle uptake, refining hybrid nanoparticle platforms, and developing human-relevant in vitro models will be critical. Advances in computational modelling and high-resolution imaging can expedite our understanding of nanoparticle transport across respiratory barriers. Additionally, leveraging new biomaterials, novel ligands, and surfactant-mimicking coatings may further improve nanoparticle targeting, reduce clearance, and increase the therapeutic index. These efforts, coupled with ongoing innovations in formulation science, device engineering, and regulatory science, are set to reshape the landscape of intranasal LNP-based gene delivery.