Department Of Pharmaceutics, college of Pharmaceutical Sciences, Govt. Medical College, Kozhikode
The delivery of therapeutic agents to the central nervous system (CNS) remains a major challenge due to the restrictive nature of the blood–brain barrier (BBB), which limits the penetration of most small molecules and biologics. Conventional systemic approaches often fail to achieve adequate brain concentrations while increasing the risk of systemic toxicity. In response to these limitations, Trojan horse strategies have emerged as a promising approach to facilitate drug transport across the BBB by exploiting endogenous transcytosis mechanisms. These strategies primarily rely on receptor-mediated transcytosis and adsorptive-mediated transcytosis pathways, enabling nanocarriers to mimic physiological substrates and gain controlled access to the brain. Recent advances in nanotechnology have enabled the design of polymeric nanoparticles, lipid-based carriers, mesoporous silica systems, and vesicular nanoplatforms capable of integrating targeting ligands, charge modulation, and stimulus-responsive elements to enhance BBB transport efficiency. In parallel, the intranasal route has attracted increasing attention as a non-invasive alternative for brain delivery by bypassing vascular barriers through olfactory and trigeminal neural pathways. The convergence of Trojan-horse targeting principles with nose-to-brain delivery strategies represents a promising paradigm for improving CNS drug delivery. However, several translational challenges remain, including receptor saturation, mucociliary clearance, variability in nasal deposition, safety concerns, and large-scale manufacturing limitations. This review provides a comprehensive overview of the biological basis of BBB transport, the mechanistic principles underlying Trojan-horse nanocarriers, and emerging nanotechnological strategies designed to enhance nose-to-brain drug delivery. Understanding the interplay between nanoparticle physicochemistry, endothelial transport mechanisms, and nasal anatomical pathways will be essential for advancing Trojan-based systems toward clinically viable brain-targeting therapies.
Effective delivery of therapeutic agents to the central nervous system (CNS) remains one of the most formidable challenges in pharmaceutical science. The principal obstacle is the presence of the blood–brain barrier (BBB), a highly specialised and dynamic neurovascular interface that tightly regulates molecular exchange between systemic circulation and the brain. Structurally composed of brain endothelial cells interconnected by tight junctions and supported by pericytes, astrocytes, and basement membrane components, the BBB maintains neuronal homeostasis while severely restricting drug entry into the brain parenchyma[1] it is estimated that over 95% of small molecules and nearly all biologics fail to adequately penetrate the BBB, significantly limiting therapeutic efficacy in CNS disorders[2]
Conventional systemic strategies, including dose escalation or transient barrier disruption, often result in systemic toxicity, heterogeneous drug distribution, or safety concerns[1] Consequently, biologically inspired transport strategies have gained attention. Among these, Trojan horse approaches have emerged as a rational method to exploit endogenous transport mechanisms such as receptor-mediated transcytosis (RMT) and adsorptive-mediated transcytosis (AMT)[3] By mimicking natural ligands or engaging physiological receptors—including transferrin, insulin, and low-density lipoprotein receptors—engineered nanocarriers can undergo controlled endothelial uptake and translocation across the BBB[4] Recent advances further demonstrate that ligand affinity, valency, and surface density critically influence transcytosis efficiency, as excessive receptor binding may lead to endothelial retention rather than effective brain delivery[5]
Parallel to vascular targeting strategies, the intranasal route has gained increasing attention as a non-invasive alternative capable of bypassing the BBB. The anatomical connectivity of the nasal cavity with the CNS through the olfactory and trigeminal pathways enables direct nose-to-brain transport, reducing systemic exposure and potentially enhancing therapeutic selectivity. However, physiological barriers such as mucociliary clearance, enzymatic degradation, limited olfactory surface area, and variability in deposition patterns continue to constrain delivery efficiency, necessitating advanced formulation approaches. Nanocarrier-based systems have therefore been extensively investigated to overcome both endothelial and epithelial barriers. Polymeric nanoparticles, lipid-based carriers, vesicular systems, and surface-functionalised nanosystems offer tunable physicochemical properties that influence cellular uptake, membrane interaction, and transcytosis behaviour[1]. Importantly, recent studies highlight that nanoparticle size, geometry, surface charge, stiffness, ligand density, and protein corona formation significantly modulate BBB interaction and transport outcomes[1] In addition to RMT, transporter-mediated mechanisms and carrier-mediated pathways are also being explored to improve brain targeting efficiency[2] Despite promising preclinical findings, the clinical translation of Trojan-based nanocarrier systems remains limited. Species-dependent differences in receptor expression, variability in in vitro BBB models, protein corona unpredictability, immunogenicity concerns, and large-scale manufacturing challenges continue to impede progress[1,3] These translational barriers underscore the need for a deeper mechanistic understanding of nano–bio interactions and the development of rational design frameworks grounded in biological transport principles.
This review provides a comprehensive and mechanistically focused analysis of Trojan horse strategies and intranasal nanocarrier systems for brain targeting. Emphasis is placed on biological uptake pathways, nano–bio interface interactions, physicochemical design determinants, and translational considerations. By integrating vascular and nasal delivery paradigms within a unified mechanistic framework, this review aims to clarify current advances, critically examine limitations, and outline rational design principles for next-generation CNS drug delivery systems.
2.The Blood–Brain Barrier: Structure and Transport Limitations
The blood–brain barrier (BBB) is a highly specialised dynamic interface that regulates molecular exchange between systemic circulation and the central nervous system (CNS). While essential for maintaining neuronal homeostasis and protecting neural tissue from toxins and pathogens, the BBB represents the principal obstacle to effective brain drug delivery.
2.1 Structural Organisation of the BBB
The BBB is formed primarily by brain microvascular endothelial cells (BMECs), which are interconnected by continuous tight junctions. These endothelial cells are supported by pericytes, astrocytic end-feet, and basement membrane components, collectively forming the neurovascular unit (NVU)[6].
Unlike peripheral capillaries, BBB endothelial cells exhibit:
This unique architecture severely restricts passive diffusion of hydrophilic and high-molecular-weight compounds.
2.2 Transport Mechanisms Across the BBB[7]
The blood–brain barrier (BBB) is a highly specialized endothelial interface that tightly regulates molecular transport between the systemic circulation and the central nervous system. Brain endothelial cells are interconnected by tight junction complexes composed of proteins such as claudins, occludin, and zonula occludens, which severely restrict paracellular diffusion. As a result, the passage of most hydrophilic molecules through intercellular spaces is extremely limited, allowing only very small solutes to diffuse between endothelial cells.
Small lipophilic molecules may cross the BBB through transcellular diffusion, which occurs across the endothelial plasma membrane. However, this passive route is largely restricted to compounds with favorable lipophilicity and low molecular weight, excluding the majority of therapeutic macromolecules.
To maintain brain homeostasis, the BBB expresses several carrier-mediated transport (CMT) systems that facilitate the uptake of essential nutrients. These include glucose transporter-1 (GLUT1), large neutral amino acid transporters (LAT1), and monocarboxylate transporters (MCTs). Such carriers enable selective entry of physiological substrates required for neuronal metabolism.
In addition to nutrient carriers, the BBB also utilizes receptor-mediated transcytosis (RMT) to transport larger biomolecules. Endogenous ligands such as transferrin and insulin bind to their respective receptors on the luminal surface of endothelial cells, triggering vesicular internalization and transcytotic transport across the endothelial layer. This pathway has become a key target for Trojan-horse drug delivery strategies designed to shuttle therapeutic cargo into the brain.
Another transport mechanism is adsorptive-mediated transcytosis (AMT), which is driven by electrostatic interactions between positively charged molecules and the negatively charged glycocalyx present on the endothelial surface. This pathway allows the uptake of certain cationic proteins and nanoparticles through nonspecific endocytic processes.
2.3 Efflux Transporters: The Major Limitation[8]
Even when therapeutic molecules successfully enter endothelial cells, ATP-dependent efflux transporters actively pump many compounds back into the bloodstream, significantly limiting drug accumulation in the brain. Among these, P-glycoprotein (P-gp, ABCB1) is the most extensively studied and is responsible for the extrusion of numerous xenobiotics and pharmaceutical agents. Other important transporters include breast cancer resistance protein (BCRP) and members of the multidrug resistance-associated protein (MRP) family.
These efflux pumps are strategically localized on the luminal membrane of brain endothelial cells and serve as a protective mechanism against potentially harmful substances. However, they also represent a major barrier for central nervous system drug delivery, as many therapeutic compounds are recognized as substrates for these transport systems. Consequently, overcoming efflux-mediated clearance remains a critical challenge in the development of effective brain-targeted drug delivery strategies.
2.4 Physicochemical Constraints on Drug Penetration[7,9]
The permeability of therapeutic molecules across the blood–brain barrier is strongly influenced by their physicochemical properties. Due to the restrictive nature of the endothelial tight junctions and the lipid-rich cellular membranes of the BBB, only molecules possessing favourable physicochemical characteristics can readily penetrate into the brain. Generally, compounds with molecular weights below 400–500 Da are considered more suitable for passive diffusion across the BBB. In addition, lipophilicity plays a crucial role, with optimal brain penetration typically observed for molecules with a log P value between 1 and 3, which facilitates diffusion through endothelial lipid membranes.
Other structural features also significantly influence BBB permeability. Molecules with low polar surface area (PSA) and limited hydrogen-bonding capacity tend to exhibit enhanced membrane permeability. In contrast, compounds with high polarity or extensive hydrogen-bonding potential often show poor BBB penetration due to limited ability to traverse the lipid bilayer of endothelial cells. Consequently, many biologics, peptides, and hydrophilic drugs fail to satisfy these physicochemical requirements, resulting in inadequate brain delivery following systemic administration. These limitations have stimulated the development of advanced delivery strategies, including nanoparticle-based Trojan-horse systems, designed to bypass or exploit endogenous BBB transport pathways.
2.5 Disease-Induced BBB Modulation
Pathological conditions such as tumours, inflammation, and neurodegeneration can alter BBB permeability. However, such disruptions are heterogeneous and unpredictable. In glioblastoma, the blood–tumour barrier may allow partial penetration, but intact regions still restrict drug entry[11].
Thus, relying on disease-associated BBB breakdown is insufficient for consistent therapeutic delivery.
2.6 Limitations of Conventional Systemic Strategies
Traditional strategies such as:
All of these may transiently increase permeability but carry risks including neurotoxicity, oedema, and inflammatory responses.[12]
Given these limitations, non-invasive alternative routes such as intranasal delivery have gained attention.
2.7 Rationale for Alternative Routes
Because systemic administration faces structural, enzymatic, and efflux-based barriers, bypass strategies are increasingly investigated. The intranasal route offers the potential to circumvent BBB endothelial restrictions by exploiting direct neuronal connections between the nasal cavity and CNS structures.[13]
This anatomical opportunity forms the foundation for advanced nanocarrier-based targeting strategies discussed in subsequent sections.
3. Trojan Horse Strategies for Brain Targeting
The delivery of therapeutics across biological barriers, particularly the blood–brain barrier (BBB), remains a major challenge in central nervous system (CNS) drug delivery. To address this limitation, the “Trojan horse” strategy has emerged as a biologically inspired approach in which drug carriers are engineered to exploit endogenous transport pathways, thereby enabling controlled cellular uptake and translocation across endothelial barriers. At its core, the Trojan horse concept relies on the interaction of nanocarriers with naturally occurring biological systems. Upon exposure to biological fluids, nanoparticles rapidly adsorb proteins to form a dynamic protein corona, which significantly influences their biological identity, cellular interaction, and transport behaviour[14]. This corona can act as a “biological disguise,” modulating receptor engagement and potentially facilitating transcytosis across the BBB. However, the composition of the protein corona is highly dependent on nanoparticle surface chemistry, size, and charge, introducing variability in targeting efficiency[15] In addition to passive protein adsorption, active targeting through surface functionalisation represents a more controlled Trojan strategy. Functional ligands such as folic acid can be conjugated to nanoparticle surfaces to exploit receptor-mediated uptake pathways. Folic acid-decorated nanocarriers have demonstrated enhanced cellular internalisation due to overexpression of folate receptors in certain cell types[16]. More broadly, receptor-mediated transcytosis (RMT) strategies commonly target transferrin, insulin, or low-density lipoprotein receptors to induce endothelial uptake and vesicular trafficking across the BBB[17–19]. While such ligand–receptor interactions improve specificity, recent evidence suggests that ligand affinity and surface density critically determine whether nanoparticles undergo successful transcytosis or become retained within endothelial compartments[5]. Nanocrystal-based systems further illustrate the Trojan paradigm by combining high drug loading capacity with surface-engineered targeting[16]. These systems provide near-complete payload encapsulation while maintaining nanoscale properties compatible with cellular uptake mechanisms. Similarly, vesicular systems, including liposomes and other nanoscale carriers, can encapsulate therapeutic agents and be modified with targeting ligands to enhance membrane interaction and intracellular delivery[20]. Importantly, vesicle deformability, surface hydration, and lipid composition influence their interaction with endothelial membranes and subsequent trafficking routes[1]. Trojan strategies frequently leverage endocytic pathways, including receptor-mediated and adsorptive-mediated mechanisms. In adsorptive-mediated transcytosis (AMT), electrostatic interactions between cationic nanoparticles and negatively charged endothelial glycocalyx components promote membrane wrapping and internalisation [14]. Surface charge modulation therefore plays a pivotal role in determining uptake efficiency. However, excessive positive charge may increase nonspecific interactions and cytotoxicity, underscoring the need for careful optimisation[15].
Beyond biochemical recognition, emerging evidence suggests that mechanobiological determinants—such as nanoparticle size, geometry, stiffness, and ligand spatial arrangement—substantially influence endothelial membrane deformation and vesicle formation[19]. Optimal particle sizes for transcytosis are generally reported within the 20–50 nm range, although this remains context-dependent[15]. Moreover, excessive ligand density can induce receptor clustering and recycling dynamics that paradoxically reduce effective brain delivery[5,18].
Recent studies have further challenged the assumption that intentional ligand conjugation is always necessary for BBB transport. Ligand-free nanoparticles may acquire targeting capability through selective adsorption of endogenous apolipoproteins within the protein corona, thereby engaging lipoprotein receptors at the BBB[21,22]. This “biomimetic Trojan” mechanism highlights the complex interplay between nanoparticle surface engineering and in situ biological modification. Despite promising preclinical findings, several challenges limit the clinical translation of Trojan-based nanocarriers. These include species-dependent differences in receptor expression, overestimation of transcytosis in simplified in vitro models, receptor saturation effects, and variability in pathological BBB integrity[15,23]. Furthermore, intracellular trafficking may favour lysosomal routing rather than transcytotic exocytosis, reducing effective brain accumulation[5]. These considerations underscore that successful Trojan design requires integration of receptor biology, intracellular trafficking kinetics, and nanoparticle physicochemical optimisation.
Collectively, Trojan horse strategies represent a convergence of nanotechnology, surface engineering, and vascular biology. By rationally exploiting endogenous transport mechanisms while accounting for mechanistic limitations, these systems offer a promising yet complex platform for targeted CNS drug delivery.
Having introduced the general Trojan framework, the following subsections detail major receptor-mediated pathways: -
3.1 Receptor-Mediated Transcytosis (RMT)
3.1.1 Transferrin Receptor Targeting
The transferrin receptor (TfR) remains the most extensively investigated BBB shuttle. Functionalisation of nanoparticles with transferrin or TfR-specific ligands significantly enhances transport across in vitro BBB models.
Zhang et al. [17] demonstrated that transferrin-functionalised porous silicon nanoparticles exhibited size- and ligand-density-dependent transport across hCMEC/D3 monolayers and BBB-on-chip systems. Notably, intermediate ligand density resulted in superior transcytosis compared to maximal conjugation, highlighting receptor saturation and internalisation kinetics as limiting factors.
Similarly, Esparza et al.[18]engineered a pH-sensitive TfR-binding nanobody incorporating histidine mutations to promote dissociation in acidic endosomes (pH ~5.5). Enhanced endosomal release significantly improved brain retention, demonstrating that intracellular release kinetics critically influence Trojan efficiency.
These findings indicate that RMT efficiency is governed by:
Over-functionalisation may paradoxically reduce brain delivery due to receptor recycling dynamics and lysosomal trafficking.
3.1.2 LRP1 and Alternative Receptor Pathways
Low-density lipoprotein receptor-related protein 1 (LRP1) represents another major transcytotic pathway. Ligands such as Angiopep-2 and receptor-specific D-peptides have been incorporated into polymeric nanobioconjugates to enhance BBB penetration.
Israel et al. [19]demonstrated that LRP1-mediated permeation varied depending on pathological context. While tumour-bearing brains exhibited enhanced permeation, neurodegenerative models showed reduced efficiency, likely due to receptor dysregulation. This underscores a key translational insight: Trojan strategies are disease-context dependent.
Lactoferrin receptor (LfR) targeting has also shown promising results. Song et al. reported that lactoferrin-conjugated silica nanoparticles (~25 nm) achieved superior transport compared to larger constructs, reinforcing the combined importance of receptor engagement and nanoscale optimisation.
Collectively, these studies demonstrate that RMT is highly effective but must be tailored to receptor biology and disease-specific vascular remodelling.
3.2 Ligand Density, Vector Synergy, and Membrane Mechanics
Receptor engagement is not solely determined by ligand presence but is strongly influenced by ligand density and spatial arrangement. Israel et al.[19] observed that dual-vector systems did not necessarily enhance permeation; excessive ligand loading introduced steric hindrance and potential receptor competition.
Interestingly, mechanistic analysis suggested a two-step process:
This indicates that polymeric platforms themselves may modulate endothelial membrane mechanics, facilitating receptor exposure. Thus, Trojan systems operate through both biochemical recognition and biophysical membrane interactions.
3.3 Adsorptive-Mediated Transcytosis (AMT)
In contrast to receptor-dependent mechanisms, AMT exploits electrostatic interactions between nanoparticles and the negatively charged endothelial glycocalyx.
Zhang et al.[24] demonstrated through computational and experimental modelling that positively charged nanoparticles exhibited dramatically higher permeability across endothelial monolayers compared to neutral counterparts. Electrostatic attraction enhanced membrane wrapping and reduced energetic barriers for vesicle formation. Neutral particles showed up to 100-fold lower permeability.
Similarly, PepH3-modified nanocarriers exhibited temperature-dependent uptake mediated by surface charge interactions, confirming the active nature of AMT-based transport.
While AMT avoids receptor saturation issues, it must be carefully controlled to minimise nonspecific interactions and systemic toxicity.
3.4 Ligand-Free and Protein Corona–Mediated Trojan Mechanisms
Recent studies challenge the assumption that intentional ligand conjugation is mandatory for BBB translocation.
Chen et al.[22] demonstrated that negatively charged mesoporous silica nanoparticles (~50 nm) penetrated the BBB in zebrafish models without receptor decoration. Proteomic analysis revealed adsorption of apolipoprotein E and basigin within the protein corona, suggesting that endogenous serum proteins may confer secondary targeting capabilities.
Similarly, Chen et al.[21]reported enhanced BBB penetration of ligand-free PEGylated mesoporous silica nanoparticles (~25 nm), with LC-MS/MS analysis identifying an apolipoprotein-enriched corona.
These findings support an evolving “biomimetic Trojan” paradigm in which surface-engineered nanoparticles exploit in situ protein corona formation to mediate BBB interaction.
3.5 Stimuli-Responsive Trojan Systems
Advanced Trojan strategies integrate transport with stimulus-triggered release.
Singh et al.[23] developed protease-responsive nanogels functionalised with diphtheria toxin receptor ligands. After RMT-mediated BBB crossing, nanogels degraded in matrix metalloproteinase-rich tumour environments, enabling controlled intracellular radiopharmaceutical release.
This dual-stage design — BBB transport followed by environment-specific activation — represents a refined evolution of Trojan systems toward precision therapy.
3.6 Integrative Design Determinants
Across studies, several converging determinants govern Trojan efficiency as given in Table 1:
Table 1: Key physicochemical determinants influencing Trojan nanocarrier transport across the blood–brain barrier.
|
|
Reference |
||||||||||
|
Optimal range ~20–50 nm |
[17,25] |
||||||||||
|
Surface charge |
Moderate positive or controlled negative influences uptake |
[1,26] |
||||||||||
|
Ligand density |
Intermediate density superior to maximal loading |
[17] |
||||||||||
|
Endosomal release |
|
[18] |
||||||||||
|
Protein corona |
May confer unintended but beneficial targeting |
[22,27] |
||||||||||
|
Disease context |
Receptor expression varies in tumour vs neurodegeneration |
[19] |
Advanced BBB-on-chip and triculture models further reveal differences not observed in static 2D systems, underscoring the importance of physiologically relevant validation platforms.
Several Trojan-horse approaches have been explored to improve nanoparticle transport across the blood–brain barrier by utilizing endogenous cellular transport mechanisms. These include receptor-mediated transcytosis, adsorptive-mediated transcytosis, and emerging ligand-independent strategies such as protein corona-mediated targeting. Each strategy presents distinct advantages and limitations depending on nanoparticle design and biological context. A comparative overview of these Trojan transport strategies is presented in Table 2.
Table 2: Comparison of Trojan strategies used for nanoparticle transport across the blood–brain barrier
|
Trojan Strategy |
Mechanism |
Key Advantages |
Limitations |
Example Targets |
Reference |
|
Receptor-mediated transcytosis (RMT) |
Nanocarriers bind to endothelial receptors triggering vesicular transport across BBB |
High specificity and efficient transport |
Receptor saturation and competition with endogenous ligands |
Transferrin receptor, insulin receptor, LRP1 |
[24,28] |
|
Adsorptive-mediated transcytosis (AMT) |
Electrostatic interaction between positively charged nanoparticles and negatively charged endothelial membranes |
Does not require specific ligands |
Low selectivity and possible systemic toxicity |
Cationic proteins, cell-penetrating peptides |
[29] |
|
Ligand-functionalised nanoparticles |
Surface ligands mimic endogenous substrates enabling receptor recognition |
Improved targeting and uptake |
Complex formulation and stability issues |
Transferrin, lactoferrin, angiopep-2 |
[30] |
|
Protein corona-mediated targeting |
Adsorbed plasma proteins on nanoparticle surface facilitate BBB interaction |
No need for synthetic ligand conjugation |
Difficult to control corona composition |
Apolipoproteins |
[22] |
|
Stimuli-responsive Trojan systems |
Nanocarriers release drug after BBB transport in response to pH, enzymes, or tumour microenvironment |
Targeted drug release |
More complex formulation design |
Enzyme-responsive nanogels |
[23] |
3.7 Emerging Mechanobiological Perspective
The Trojan-horse paradigm is shifting from a simplistic ligand–receptor framework toward an integrative mechanobiological model. Effective BBB translocation appears to require:
Future translational success will depend on harmonising receptor biology, endothelial membrane mechanics, and nanoparticle physicochemistry into unified design principles.
4. Anatomical and Cellular Basis of Nose-to-Brain Transport
The intranasal route offers a unique method for delivering therapeutics to the central nervous system (CNS) by bypassing the restrictive blood–brain barrier (BBB) and exploiting direct neural connections between the nasal cavity and the brain. The human nasal cavity contains distinct anatomical regions — the olfactory epithelium and the respiratory epithelium — each with different roles in nasal absorption and brain targeting[31]
4.1 Nasal Cavity Architecture and Functional Regions
The nasal cavity is divided into several regions, but the two most relevant for nose-to-brain delivery are:
Nanoparticles deposited on the olfactory mucosa can interact with the underlying neurons and supporting cells, allowing them to take advantage of these direct anatomical routes into CNS tissues.
4.2 Olfactory Pathway: Direct Neural Route
The olfactory pathway provides a direct conduit between the nasal cavity and the brain via the olfactory nerve. Molecules can access the CNS through two proposed mechanisms:
Intranasal administration of nanoparticles has been shown to result in measurable distribution within the olfactory bulb within minutes in experimental systems, supporting the relevance of rapid extracellular routes[31]
4.3 Trigeminal Pathway: Secondary Access Route
The trigeminal nerve provides an additional anatomical pathway for nose-to-brain transport. Its branches innervate both the olfactory and respiratory epithelium and extend into the brainstem and other deeper structures [33]. This route may deliver therapeutic agents to brain regions beyond the olfactory bulb and cerebrospinal fluid (CSF), broadening the spectrum of CNS targets accessible via intranasal administration.
4.4 Lymphatic and Perivascular Pathways
In addition to direct neuronal pathways, several studies indicate that lymphatic and perivascular spaces contribute to nose-to-brain transport. These extracellular conduits may allow substances to reach CNS tissues via bulk flow or diffusion. The perivascular exchange connects nasal submucosal spaces with the CSF and perineural channels, facilitating movement of molecules over short timescales.[34]
4.5 Physiological Barriers and Uptake Mechanics
Despite the potential of neural routes, several physiological barriers influence nose-to-brain delivery:
Nanoparticles can interact with the nasal mucosa using mucoadhesive polymers (e.g., chitosan, hyaluronic acid) that enhance residence time and promote epithelial contact, increasing the likelihood of transcellular uptake[35]
4.6 Nanoparticle Interaction with Nasal Tissue
Nanoparticles influence both the deposition and uptake processes at the nasal epithelium. Those smaller than ~100 nm penetrate deeper into mucosal layers and are less susceptible to rapid mucociliary clearance, while surface modifications (e.g., positive charge or ligands) enhance interactions with membranes and receptors on epithelial cells[35]
Moreover, nanoparticle surface functionalisation with bioadhesive polymers or targeting ligands can increase the probability of uptake. This is particularly relevant for Trojan strategies, where surface design not only facilitates transport across the BBB but also improves epithelial internalisation.
4.7 Integration of Pathways and Trojan Strategy Relevance
The olfactory and trigeminal routes form the primary anatomical backbone for nose-to-brain delivery, while lymphatic and perivascular channels provide supplementary pathways. The relative contribution of these pathways depends on formulation characteristics, such as particle size, surface charge, and mucoadhesive properties.
Designing Trojan nanocarriers that consider both epithelial uptake and downstream neural transport is critical. For example, ligands that engage receptor-mediated uptake at the nasal epithelium may enhance internalisation, providing better entry into neuronal channels that ultimately reach CNS targets. Integrating Trojan mechanics at both the epithelial and BBB levels offers a holistic approach to maximizing intranasal drug delivery efficiency.
5. Nanocarrier Platforms for Nose-to-Brain Drug Delivery
Nanocarrier-based systems have emerged as pivotal platforms for enhancing intranasal drug delivery to the central nervous system (CNS). These systems are designed to overcome nasal physiological barriers such as mucociliary clearance, enzymatic degradation, limited epithelial permeability, and restricted residence time. By modulating particle size, surface charge, mucoadhesive properties, and targeting ligand density, nanocarriers can significantly improve brain bioavailability following intranasal administration.
5.1 Polymeric Nanoparticles
Polymeric nanoparticles are among the most extensively studied systems for nose-to-brain delivery due to their biodegradability, tunable surface chemistry, and controlled drug release characteristics.
Polymers such as PLGA, chitosan, and PEG-based copolymers have demonstrated enhanced nasal retention and improved brain accumulation in preclinical models[36]
Chitosan-based nanoparticles, in particular, enhance mucoadhesion and transiently open tight junctions, facilitating transcellular transport across nasal epithelium[37]
Moreover, cationic polymeric nanoparticles can exploit adsorptive-mediated uptake mechanisms at the nasal epithelium, increasing epithelial internalisation.
5.2 Lipid-Based Nanocarriers
Lipid-based systems such as solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) offer improved biocompatibility and enhanced permeation due to their lipidic nature, which facilitates interaction with biological membranes.
SLNs have demonstrated enhanced brain targeting efficiency when administered intranasally, improving drug retention and reducing systemic exposure[38]
NLCs further improve drug loading capacity and reduce crystallinity issues associated with SLNs
5.3 Nanoemulsions and Microemulsions
Nanoemulsions are thermodynamically or kinetically stable dispersions that enhance solubility of poorly water-soluble drugs and improve mucosal permeation.
Their small droplet size (typically <200 nm) enables deeper mucosal penetration and prolonged nasal retention. Intranasal nanoemulsion systems have demonstrated rapid brain uptake and enhanced pharmacodynamic effects in various CNS models[39]
5.4 Mesoporous Silica Nanoparticles
Mesoporous silica nanoparticles (MSNs) provide high surface area and tunable pore size, enabling high drug loading and controlled release. Surface functionalisation allows targeted delivery and modulation of surface charge to optimise epithelial uptake.
Recent advancements suggest MSNs can be engineered for mucoadhesion and receptor-mediated uptake in nasal applications [40]
5.5 Vesicular Systems (Liposomes, Transfersomes, Niosomes)
Liposomes and deformable vesicular systems enhance membrane fusion and facilitate transcellular uptake. Transfersomes, due to their elasticity, may penetrate mucosal barriers more effectively than conventional liposomes.
Intranasal liposomal systems have demonstrated enhanced brain targeting and reduced systemic distribution
5.6 Mucoadhesive and In Situ Gelling Systems
Mucoadhesive polymers prolong nasal residence time, increasing drug absorption probability. Thermosensitive in situ gels transition from liquid to gel at nasal temperature, reducing mucociliary clearance.
Poloxamer-based thermosensitive systems have demonstrated improved brain bioavailability in intranasal formulations[41]
5.7 Surface Functionalisation and Targeting Strategies
Surface modification with ligands such as transferrin, lactoferrin, peptides, or cell-penetrating moieties enhances epithelial uptake and subsequent neuronal transport.
Nanoparticles functionalised with targeting ligands have demonstrated improved uptake and transport across nasal epithelium, providing a mechanistic link between Trojan strategies and intranasal delivery[1]
5.8 Comparative Perspective
Each nanocarrier system offers distinct advantages as given in table 3.
Table 3: Comparison of major nanocarrier platforms investigated for nose-to-brain drug delivery
|
Nanocarrier system |
Key advantages for nose-to-brain delivery |
Limitation |
Reference |
|
Polymeric NPs |
Controlled drug release, tunable surface modification, ligand conjugation possible |
Potential polymer toxicity if not optimised |
[36] |
|
Solid lipid nanoparticles (SLNs) |
High biocompatibility, enhanced mucosal penetration |
Limited drug loading capacity |
[38] |
|
Nano emulsions |
Improved solubility of lipophilic drugs, rapid brain uptake after intranasal delivery |
Physical instability during long-term storage |
[39] |
|
MSNs |
High surface area and drug loading capacity, tunable pore structure |
Long-term biocompatibility concerns |
[40] |
|
Carbon Nanotubes |
Characteristic large surface area |
Toxicity and immune response |
[42] |
|
Vesicular systems (liposomes/transfersomes) |
Enhanced membrane interaction and transcellular transport |
Possible drug leakage and stability issues |
[43] |
|
In-situ gels |
Prolonged nasal residence time and reduced mucociliary clearance |
Possible nasal irritation with repeated use |
[41] |
Therefore, optimal nose-to-brain systems require integrated design considering deposition pattern, epithelial uptake, residence time, and downstream neuronal transport.
6. Intracellular Trafficking and Endosomal Escape Mechanisms in Nose-to-Brain Nanocarrier Systems
While successful deposition and epithelial uptake are essential for intranasal drug delivery, the intracellular fate of nanocarriers critically determines their ultimate therapeutic efficacy. After internalisation into nasal epithelial cells or brain endothelial cells, nanoparticles undergo complex vesicular trafficking pathways that dictate whether cargo reaches the cytosol, undergoes transcytosis, or is degraded within lysosomes. Understanding these intracellular mechanisms is therefore fundamental for designing effective Trojan-based nose-to-brain systems.
6.1 Endocytic Pathways Involved in Nanoparticle Uptake[44]
Nanoparticle internalisation typically occurs through one or more of the following mechanisms:
Clathrin-mediated pathways are receptor-dependent and are commonly involved in transferrin- or ligand-targeted systems. Caveolae-mediated pathways, in contrast, may facilitate transcytosis without lysosomal degradation.
These pathways are influenced by particle size, surface charge, ligand density, and membrane lipid composition.
6.2 Endosomal Maturation and Lysosomal Degradation
Following internalisation, nanoparticles are typically trafficked from early endosomes to late endosomes and ultimately lysosomes. The progressive acidification of vesicles (pH 6.5 → 5.5 → 4.5) activates hydrolytic enzymes capable of degrading both carrier and payload.
Many nanocarriers fail at this stage due to lysosomal entrapment.[45]
Therefore, effective Trojan systems must not only cross biological barriers but also avoid intracellular degradation.
6.3 Strategies for Endosomal Escape
To enhance cytosolic delivery or facilitate transcytosis, several strategies have been developed:
1. Proton Sponge Effect[46,47]
Cationic polymers (e.g., polyethyleneimine, chitosan derivatives) buffer endosomal pH, causing osmotic swelling and membrane rupture.
2. pH-Responsive Materials
pH-sensitive polymers undergo conformational changes or membrane destabilisation in acidic endosomes, promoting release.[48]
3. Membrane-Disruptive Peptides
Cell-penetrating peptides (CPPs) and fusogenic peptides can destabilise endosomal membranes, enhancing escape efficiency.[49]
6.4 Transcytosis vs Cytosolic Delivery
For nose-to-brain systems, intracellular fate differs depending on the intended mechanism:
Caveolae-mediated pathways are often considered more favorable for transcytosis because they may bypass lysosomal compartments[50]
Understanding which pathway predominates is essential when designing targeting ligands or surface modifications.
6.5 Intracellular Trafficking in Nasal Epithelium
In nasal epithelial cells, nanoparticles may undergo:
Studies suggest that mucoadhesive and cationic nanoparticles enhance uptake but may increase lysosomal entrapment if not properly engineered [51]
Thus, formulation balance is required between adhesion strength and intracellular trafficking efficiency.
6.6 Design Implications for Trojan Nose-to-Brain Systems
Effective Trojan systems must integrate:
Failure to consider intracellular trafficking often results in high epithelial uptake but low CNS bioavailability.
Therefore, intracellular fate should be considered a central design parameter in next-generation nose-to-brain nanocarrier systems
7. Translational Challenges and Clinical Outlook of Trojan-Based Nose-to-Brain Delivery
Despite extensive preclinical evidence supporting nanocarrier-mediated nose-to-brain delivery, translation into clinical practice remains limited. Several biological, technological, and regulatory challenges must be addressed before Trojan-based intranasal systems can achieve therapeutic reliability and commercial viability.
7.1 Anatomical and Deposition Variability
Efficient nose-to-brain delivery critically depends on deposition within the olfactory region, which constitutes only 5–10% of the total nasal surface area in humans. Variability in nasal anatomy, airflow patterns, and administration technique significantly influence drug deposition efficiency[52].
Conventional nasal sprays often deposit drugs primarily in the anterior respiratory region, limiting direct neuronal access. Advanced delivery devices are therefore required to improve targeting of the upper posterior nasal cavity.
7.2 Mucociliary Clearance and Residence Time
Rapid mucociliary clearance limits nasal residence time to approximately 15–30 minutes. Although mucoadhesive polymers and thermosensitive gels prolong retention, excessive adhesion may impair patient comfort or alter normal mucosal function.[53]
Achieving a balance between retention and permeability remains a central formulation challenge.
7.3 Receptor Saturation and Targeting Efficiency
Trojan-based systems relying on receptor-mediated uptake may encounter receptor saturation or competition with endogenous ligands. For example, transferrin receptor targeting must compete with physiological transferrin concentrations in systemic circulation.[54]
Furthermore, receptor expression can vary across disease states, age groups, and inflammatory conditions, reducing predictability of targeting efficiency.
7.4 Safety and Immunogenicity[55]
Repeated intranasal administration of nanocarriers raises concerns regarding:
Surface charge, polymer composition, and particle size significantly influence toxicity profiles.
Regulatory agencies increasingly demand comprehensive nanotoxicological profiling, particularly for CNS-targeted systems.
7.5 Scale-Up and Manufacturing Complexity
Laboratory-scale nanoparticle synthesis often lacks reproducibility when translated to industrial scale. Parameters such as particle size distribution, zeta potential, ligand density, and encapsulation efficiency must be tightly controlled[56].
Batch-to-batch consistency is particularly critical for targeted Trojan systems where surface functionalisation directly influences biological performance.
7.6 Regulatory Considerations[57]
Currently, few intranasal nanocarrier systems for brain targeting have advanced beyond early clinical trials. Regulatory pathways for nanomedicine remain complex due to:
Clear regulatory frameworks specific to nanocarrier-based intranasal systems are still evolving.
7.7 Future Directions
To improve translational success, future research should focus on:
Emerging technologies such as AI-guided nanoparticle optimisation and biomimetic surface engineering may further refine Trojan strategies for CNS delivery.
CONCLUSION
The delivery of therapeutics to the central nervous system remains one of the most formidable challenges in pharmaceutical science, largely due to the structural and functional complexity of the blood–brain barrier (BBB). While systemic approaches have traditionally dominated brain-targeting strategies, their success is often limited by efflux transporters, tight junction integrity, enzymatic degradation, and off-target systemic toxicity. In this context, Trojan-based strategies and intranasal delivery systems have emerged as promising alternatives capable of circumventing conventional vascular barriers.
Trojan horse approaches, particularly those exploiting receptor-mediated and adsorptive-mediated transcytosis, provide a mechanistically rational framework for enhancing nanoparticle transport across endothelial and epithelial barriers. By leveraging endogenous transport pathways, these systems aim to improve brain accumulation without compromising barrier integrity. However, successful translation requires careful consideration of receptor saturation, ligand density optimisation, and intracellular trafficking dynamics.
Parallel to this, the intranasal route offers a non-invasive anatomical bypass to the brain via olfactory and trigeminal neural pathways. Nanocarrier platforms—including polymeric nanoparticles, lipid-based systems, nanoemulsions, vesicular carriers, and in situ gelling formulations—have demonstrated the ability to enhance nasal retention, epithelial uptake, and neuronal transport. The integration of Trojan targeting principles with nose-to-brain delivery thus represents a synergistic strategy that combines biological exploitation with formulation engineering.
Nevertheless, significant translational barriers remain. Variability in nasal deposition, rapid mucociliary clearance, potential immunogenicity, large-scale manufacturing challenges, and evolving regulatory expectations continue to limit clinical progression. Future advancements must therefore prioritise rational design guided by mechanistic insight, reproducible manufacturing processes, and comprehensive safety evaluation.
In summary, Trojan-based nanocarrier systems for nose-to-brain delivery represent a scientifically compelling and clinically relevant frontier in CNS therapeutics. Continued interdisciplinary research integrating neurobiology, nanotechnology, and pharmaceutical engineering will be essential to transform experimental promise into viable therapeutic solutions.[58]
REFERENCES
Muhammed Danish Haneefa, Dr. Jisha Mohanan, Aparna E., Ashik T. N., Trojan Horse Strategies for Nose-to-Brain Drug Delivery: Exploiting Biological Uptake Mechanisms Beyond the Blood-Brain Barrier., Int. J. of Pharm. Sci., 2026, Vol 4, Issue 3, 1622-1642. https://doi.org/Muhammed Danish Haneefa, Dr. Jisha Mohanan, Aparna E., Ashik T. N., Trojan Horse Strategies for Nose-to-Brain Drug Delivery: Exploiting Biological Uptake Mechanisms Beyond the Blood-Brain Barrier., Int. J. of Pharm. Sci., 2026, Vol 4, Issue 3, 1622-1642. https://doi.org/10.5281/zenodo.19045361
10.5281/zenodo.19045361
