Intranasal delivery became more prominent in the last decades because modern treatments focus on biopharmaceuticals and concurrently reveal opportunities, but also provide challenges. Intranasal delivery is a promising route to overcome general limitations such as the blood-brain barrier (BBB). In the treatment of neurological disorders, for example multiple sclerosis or Alzheimer’s disease. Future prospective treatments may also include not only proteins or antibodies, but also specialized particles as delivery vehicles, but the BBB is shielding the central nervous system (CNS) and the brain from the blood stream. Modern therapeutics, such as antibodies and proteins, are therefore banned through size exclusion and surface charge (Wolburg et al., 1994; Stützle et al., 2015), which is problematic for intravenous and oral administration in current therapeutic approaches. More than one billion people are currently affected by neurological diseases with a rising tendency (WHO 2006). Further, the global need displays a great demand for effective treatments of the upper airways, such as the nasal cavity. Covid-19 infections, as one prominent example, occur mainly from the initial contact to the nasal mucosa and can harm the brain (Yu et al., 2020; Layden et al., 2020). Therefore, to prevent greater damage, effective treatments should also occur along this route. As a consequence, the actual need for innovative intranasal delivery systems is rapidly increasing.

The nasal mucosa, as the first biological barrier in the nose to target the brain and the CNS, consists of respiratory and olfactory mucosa. The respiratory epithelium covers approximately 97% of the nasal cavity in humans and is necessary for respiration (Ugwoke et al., 2005). Further, rapid clearance of dust and other foreign substances occurs via the directed movement of millions of cilia (Menache et al., 1997). In contrast to this, the olfactory mucosa is characterized by fewer long cilia, specialized to detect odorants, initializing an electric signal in olfactory sensory neurons to maintain olfaction (Ugwoke et al., 2005). The olfactory sensory neurons are located between epithelial cells and underlying basal cells, forming neuronal bundles, which protrude through the ethmoid bone and therefore directly connect the olfactory mucosa to the brain and the CNS (Morrison and Costanzo 1990; Morrison and Costanzo 1992). Further, the olfactory mucosa consists of a lamina propria mainly built of fibroblastic cells. The olfactory epithelium covers 4–5 cm² in humans. Up to approximately 10 cm² of the olfactory mucosa can be excised from the dorsal part of the concha nasalis dorsalis in pigs (Getty 1975; Mistry 2009; Ladel et al., 2018). The olfactory epithelium can be distinguished from the respiratory epithelium due to fewer blood capillaries and hence brighter color (Mistry 2009; Stützle et al., 2015).

Polymeric nanoparticles embody convincing advantages such as stability, high loading capacity, and controlled drug release (Bourganis et al., 2018; Rabiee et al., 2020). Poly(lactic-co-glycolic acid) (PLGA) nanoparticles especially are widely known as promising delivery vehicles. PLGA, consisting of lactic and glycolic acid, is a biodegradable biopolymer already approved by the Food and Drug Administration (FDA) and the European Medicine Agency (EMA) (Danhier et al., 2012). Its release properties are tunable due to the mass ratio of lactic and glycolic acid. Exemplarily, a mass ratio of 50% each results in a controlled release over several days. Degradation occurs through hydrolysis and the decomposition products are further metabolized. U. Seju et al. (2011) reported PLGA nanoparticles as promising delivery systems for nose-to-brain delivery because drug release of olanzapine occurs over several days due to slow swelling of PLGA and long-term residence of the resulting gel at the site of action, further 8-fold drug concentrations, compared to the solute form, have been achieved (Seju et al., 2011). Muntimadugu et al. (2016) reported the transport of PLGA nanoparticles with diameters below 200 nm along axons within the nasal mucosa (Muntimadugu et al., 2016). Neuronal bundles within the olfactory mucosa being directly connected to the olfactory or trigeminal nerve is identified as one transport route to the brain and the CNS (Anderson et al., 2008; Stützle et al., 2015; Gänger and Schindowski 2018). PLGA and chitosan particles up to 250 µm were recently reported to be mainly transported via the olfactory route (Rabiee et al., 2020); however, uptake and distribution examinations of PLGA and chitosan particles in nasal mucosa samples are still rare.

Additional nose-to-brain delivery routes are intracellular uptake and further transcytosis, as well as paracellular transport. Other research groups focused on the paracellular route in epithelial cell cultures and mucosal tissues (Sadeghi et al., 2008; Fazil et al., 2012; Sonaje et al., 2012; Rassu et al., 2016). Herein chitosan-based carriers have become one of the most promising and intensively-studied mucosal drug delivery systems (Garcia-Fuentes and Alonso 2012). This is attributed to their mild and simple preparation technique as well as their capacity to associate biologics and enable transport across mucosal barriers. Chitosan is a renewable biopolymer, which is gained from the naturally occurring chitin of crustaceans, extracted by deacetylation (Wenling et al., 2005; Yuan et al., 2011; Sarmento and Neves 2012). With at least 50% N-deacetylated chemical groups, it is referred to as chitosan. The origin, as well as the degree of deacetylation, affects the polymer properties, such as solubility, swelling, and adhesion. The interaction between the positive amino groups of chitosan and several negative groups in the mucosa enables mucoadhesion, which is further dependent on the molecular weight of the chitosan (Henriksen et al., 1996). The length of the chitosan polymer chains and their average molecular weight can be controlled by enzyme-mediated decomposition or acid hydrolysis (Ilium 1998). The chitosan utilized in this study, for example, has 80% deacetylated residues and is; therefore, mucoadhesive and well soluble in acidic solutions. Chitosan can open tight junctions (Fazil et al., 2012; Sonaje et al., 2012; Rassu et al., 2016), which are the characteristic intercellular connections present in epithelial cells and endothelial cells. One major component Zonula occludens protein 1 (ZO-1) was initially explored, in 1986, by Stevenson et al. (1986) in epithelial cells (Stevenson et al., 1986), it was later also documented in endothelial cells (Anderson et al., 1988) and fibroblasts (Itoh et al., 1991; Howarth et al., 1992). The above-mentioned research groups described the ability of chitosan to open tight junctions in cell culture, studying the impact of chitosan solutions on cellular interconnections. Further, other research groups have postulated that chitosan nanoparticles open tight junctions and; therewith, affect increased drug levels in the brain and the CNS, but did not directly prove the tight junction opening (Rassu et al., 2016; Bourganis et al., 2018; Rabiee et al., 2020). Further experimental investigations of tight junction opening are, therefore, still needed to fully clarify intranasal transport routes of chitosan particles and encapsulated active pharmaceutical ingredients.

Together with the application of particles to the nasal mucosa, it is important to consider particle distribution in the olfactory tissue, biocompatibility, biodegradation, and especially immune response. Nevertheless, these aspects are rarely reported in literature for intranasal delivery and more research is needed (Rabiee et al., 2020). The nasal mucosa, as a biological barrier directly exposed to the environment, is a well-known immune active tissue (Morrison and Costanzo 1990; Doty, 2015; Stützle et al., 2015; Bourganis et al., 2018; Ladel et al., 2018). Nasal-associated lymphoid tissue (NALT) includes immune active cells, either present between the epithelial layer and the lamina propria, infiltrated from blood vessels, or directly produced inside of lymphoid follicles located in the mucosa itself. Moreover, recent studies have revealed the still incomplete understanding of intranasal pathways and distribution routes of individual compounds, as well as formulation compositions (Rabiee et al., 2020; Keller et al., 2021). Which drug delivery system exactly triggers what specific pathway is mainly unclear up to now. Consequently, clarification of distribution routes of individual compounds as well as formulation composition, such as the herein investigated prominent particle materials PLGA and chitosan, are needed to develop suitable drug delivery systems in the future.

Within this study, we examined size- and time- dependent particle permeation studies on porcine olfactory mucosa. We compared the permeation of pure PLGA nanoparticles of different diameters up to 520 nm to spray-dried nano-in-micro particles (NiMPs) coated with chitosan to identify chitosan-mediated mechanisms important for intranasal delivery. Additionally, we utilized the unique benefits of the controlled release of PLGA nanoparticles over several days combined with the aforementioned abilities of chitosan. Moreover, we investigated organoid accumulation, such as in glands and neuronal bundles as well as the local initial immune response within the olfactory mucosa.

In this study, we successfully prepared poly(lactic-co-glycolic acid) PLGA nanoparticles of different sizes (80, 175, and 520 nm) using the precipitation and double emulsion method. Further, we produced specialized nano-in-micro particles (NiMPs) via spray drying, consisting of PLGA nanoparticles (520 nm), embedded into the matrix polymer chitosan.

NiMPs have in this study been produced via spray drying. In contrast to other innovative methods for nano- and microparticle production, e.g. electrospraying or supercritical CO₂ assisted electrohydrodynamic processes (Jaworek and Sobczyk 2008; Baldino et al., 2019a; Baldino et al., 2019b), spray drying is an even commercially used formulation process. Especially the pharmaceutical industry (Stahl 1980; Lee et al., 2002) benefits from high throughput production of spray drying, concurrently resource-friendly, as well as from high resulting encapsulation efficiencies of up to 100% (Walz et al., 2018). Spray drying of chitosan has been already reported in the literature; however, ineffective process parameters or inhomogeneous particles have mainly been documented (He et al., 1999; Tokárová et al., 2013). To achieve a robust production process and reproducible particles, with homogenous size distribution and morphology, it is necessary to choose suitable process parameters, such as inlet temperature, feed rate, and gas flow (Paudel et al., 2013). The crucial factors in this process are droplet atomization followed by feed evaporation. To generate homogenous particles, atomization should be uniform, resulting in fine droplets at the first stage. These droplets then need to evaporate continuously in the second step, to achieve homogenous particles with smooth surface. Droplets dry from the outer to the inner phase, meaning that the remaining liquid in the center of the particle needs to evaporate through the already partly dried matrix. When evaporation takes place too fast at inlet temperatures above 100°C, either hollow particles are formed through the so-called Rush-hour effect (Paudel et al., 2013), or they even burst and empty shells remain. Contrary to common knowledge, wrinkled surfaces of spray-dried chitosan particles do not occur from the polymeric properties of chitosan itself, but mainly from the process parameters of spray-drying. When particles cool down at the end of the process, they shrink and characteristic wrinkles occur. Hence, it is possible to prepare reproducible smooth chitosan particles utilizing suitable spray drying parameters like the ones reported herein. With moderate evaporation rates, dense and homogenous particles were achieved. Consequently, we developed a robust spray drying process. The morphology of all particulate samples in this study was spherical, with smooth surfaces because we optimized the spray drying process performed with a Büchi B-290 Mini-Spray dryer. An inlet temperature of 100°C was identified, as a crucial parameter, which resulted in an outlet temperature of 45 ± 1°C. This allowed the polymer droplets to continuously evaporate, ultimately resulting in homogenous and smooth particles.

For the chitosan:PLGA polymer ratio of NiMPs, a higher chitosan ratio resulted in increased particle diameters determined via image analysis of Scanning Electron Microscopy images and static light scattering; however, NiMPs tend to agglomerate in suspension. They were split up during static light scattering after an additional ultrasonic treatment within the measurement cell, which then revealed a bidisperse size distribution with a nanoparticle and a microparticle fraction. A higher chitosan ratio mainly influenced the nanoparticle fraction, which is attributed to the formation of additional, and slightly bigger pure chitosan nanoparticles during spray drying. Generally, due to the higher PLGA content about seven PLGA nanoparticles with a mean diameter of 519.7 nm were found in one chitosan microparticle with a mean diameter of 6.61 µm for low chitosan ratio, whereas, high chitosan ratio during spray drying resulted in an encapsulation of about two 519.7 nm-PLGA-nanoparticles per chitosan microparticle with a mean diameter of 5.75 µm. The achieved encapsulation efficiency of NiMPs was either 39 ± 19% (1/3 chitosan:2/3 PLGA) or 51 ± 16% (2/3 chitosan:1/3 PLGA); and therefore, not significantly different. Although, 1/3 more chitosan as matrix material was present, the encapsulation efficiency did not significantly increase. This is again attributed to the formation of additional pure chitosan nanoparticles, which were determined via static light scattering measurements after additional ultrasonic treatment. Consequently, spray drying is a very efficient method to encapsulate nanoparticles up to a mass ratio of 2/3, covering them successfully with the added matrix polymer, even more beneficial when using less matrix material. The polymer ratio variation had no impact on the following ex vivo experiments.

Mucosal uptake of PLGA nanoparticles and NiMPs was investigated, with porcine olfactory mucosa specimens, in specialized side-by-side cells, mimicking the upside-down orientation and physiological conditions within the nasal cavity at 35°C and 90% humidity. Overall, a size- and time-dependent uptake of PLGA nanoparticles was observed. Smaller PLGA nanoparticles, with 80 nm diameter, were taken up immediately deep into the lamina propria 5 min after application to the epithelium. Fewer 175 nm particles penetrated the epithelial and the basal layer within 5 min. For 520 nm PLGA nanoparticles, slower uptake was observed, reaching the outer ∼150 µm of the lamina propria within 10 min. Additionally, 80 and 175 nm particles were documented to directly associate with cell nuclei within 5 min, which implies intracellular uptake. In contrast, 520 nm particles have not initially been observed near nuclei, but after 15 min, and to lower amounts, compared to the smaller nanoparticles. The occurring decelerated penetration due to increasing particle size is already reported in the literature (Mistry et al., 2009; Bourganis et al., 2018). Further, reported critical particle size enabling transcellular transport after intranasal administration is 200 nm (Bourganis et al., 2018; Rabiee et al., 2020). However, Musumeci et al. (2018) reported that PLGA nanoparticles, up to 300 nm, reach and accumulate in the brain of rats, but did not clarify the occurring transport mechanism (Musumeci et al., 2018). Nanoparticles with diameters greater than 500 nm have not yet been reported for nose-to-brain (N2B) targeting (Bourganis et al., 2018; Rabiee et al., 2020), but size-dependent intracellular uptake is possible also for 200, 500 and even 1,000 nm particles in epithelial cells (Caco-2) (Win and Feng 2005).

Remarkably, we reported herein co-localization of 520 nm PLGA particles with nuclei, especially accumulated in neuronal bundles, which implies intracellular uptake in neuronal axons and could enable transcellular transport within the olfactory, or trigeminal nerve pathway to reach the brain and the central nervous system (CNS) (Gänger and Schindowski 2018; Bourganis et al., 2018; Rabiee et al., 2020; Keller et al., 2021). The diameter of axons in humans ranges from 100 to 700 nm (Morrison and Costanzo 1990; Morrison and Costanzo 1992; Mistry et al., 2009). It is broadly reported that directed transport along with neuronal cells and posterior neuronal bundles is one of the most promising routes for nose-to-brain delivery. To further investigate this phenomenon, additional experiments visualizing the exact cellular uptake and transport mechanism could be helpful. Hence, live imaging or in vivo experiments with particle sizes above 200 nm should again also be taken into account.

Contrasting the observations of 520 nm pure PLGA particles to the similar 520 nm PLGA particles embedded into chitosan, NiMPs were taken up faster and deeper in the lamina propria. A quantitative analysis was herein not possible because sample sizes differed between PLGA nanoparticle suspension and NiMP powder samples. The effect of applied polymer amount and sample condition needs hence to be investigated in future studies. Within 15 min high amounts of NiMPs were detected spread over the whole olfactory mucosa section, reaching the collagen tissue at the basolateral side, where none of the purely applied PLGA nanoparticles were found. Furthermore, chitosan has been detected associated with the penetrated PLGA nanoparticles within the olfactory mucosa. Especially after 2 h, several 520 nm particles have been found together with chitosan in the outer zone of lymphoid follicles, where usually T-cells and B-cells are present in the porcine olfactory mucosa (Ladel et al., 2018). Small pure PLGA nanoparticles (80 and 175 nm) have also been detected in this area after approximately 5 min. In contrast to this, purely applied 520 nm PLGA particles were not found associated with lymphoid follicles. Ladel et al. (2018) showed CD3⁺, CD20⁺, and CD14⁺ cells in the outer area of the porcine olfactory epithelium-associated lymphoid follicles (Ladel et al., 2018). This finding further matches the subepithelial dome more prominent in intestinal Peyer’s patch but also found in young adults and sheep.

Also within this study, we consequently examined the general initial immune reaction, which is mediated by CD14⁺ immune cells, namely monocytes and macrophages. It takes place within the first few hours after contact with a biomaterial (Anderson et al., 2008). We further monitored the presence of CD14 around lymphoid follicles. In our study, PLGA was not found co-localized with CD14⁺ cells, thus seems not to be recognized by CD14. Therefore, it is assumed that PLGA nanoparticles can pass the olfactory epithelium reaching for example, neuronal bundles, and could then be further transported to the brain and the CNS. Nevertheless, 80 nm PLGA nanoparticles especially have been documented associated with lymphoid follicles, which should be further clarified. Although this result is not attributable to the recognition by monocytes or macrophages, an interaction mechanism with other present immune cells could still be possible and should be explored in future studies.

The chitosan penetration gradient within the olfactory mucosa was found in our study to be proportional to a gradient of the initial immune response. Chitosan, therefore, seemed to cause a migration of CD14⁺ cells to the particles underlying the lamina propria within 15 min after particle application to the epithelium. The membrane located glycoprotein CD14 forms together with the lipopolysaccharide-binding protein (LPB), the lipopolysaccharide-receptor (LPR), which recognizes bacterial endotoxin lipopolysaccharides lipopolysaccharide-receptor present on the surface of Gram-negative bacteria (Wright et al., 1990). Lipopolysaccharide-induced upregulation of the CD14 membrane receptor is known as an initial immune reaction in mucosal tissues, such as the intestine (Funda et al., 2001); (Frolova et al., 2008). Otterlei et al. (1994) initially described chitosan-recognition by CD14 on monocytes (Otterlei et al., 1994). To the contrary, Qiao et al. (2010) reported the reduction of pro-inflammatory cytokines through chitosan oligosaccharides (Qiao et al., 2010). Recently, Chang et al. (2019) differentiated between low molecular (below 7.1 kDa) and high molecular (above 72 kDa) chitosan in the macrophage-binding mechanism. They reported that only low molecular weight chitosan up to 7.1 kDa would bind CD14 and interpreted it as an activation of a pro-inflammatory cascade. Studies describing the immune reaction of applied chitosan microparticles are still rather poor. Solely, S. Davis et al. (2018) proved chitosan particles with a high degree of deacetylation with sizes of 1–10 µm causing less M1-macrophage activation than similar chitin particles (Davis et al., 2018). Consequently, either the pro- or anti-inflammatory ability of chitosan particles is dependent on molecular weight, particle size, and degree of deacetylation. Taking this into account, we assume that in our study the observed CD14⁺ signal should be anti-inflammatory inducing a wound healing cascade, because the herein utilized chitosan has a molecular weight of 150–300 kDa. This should hence not be critical because Chang et al. (2019) reported for chitosans with molecular weights above 72 kDa solely anti-inflammatory reactions. Further, a high degree of 80% deacetylation and lastly a particle size ranging through agglomerates from approximately one micron up to 100 µm supports this reasoning. Nevertheless, the herein investigated CD14 immunohistochemical staining does not allow us to distinguish between M1- and M2-macrophages, which should be further evaluated in cell culture experiments, because, for example, a quantitative cell assay with altering cytokine concentrations would be needed as a reference (Zarif et al., 2016). This was not possible with the ex vivo samples of this study.

Moreover, the chitosan coating of NiMPs revealed several other interesting findings different from the purely applied identical PLGA nanoparticles. Chitosan was found to penetrate the olfactory mucosa approximately ∼150 µm within 15 min. Further, it accelerated the penetration of coated nanoparticles significantly and enables transport over the whole transverse tissue section within 15 min as described above. This is attributed to the swelling of chitosan on the top of the mucosa because the naturally occurring pH value in the nose of healthy individuals ranges around pH 6 (England et al., 1999). Unmodified chitosan does not completely dissolve at physiological pH values in the nose due to its pKa value of ∼6.5 but it is swelling and single chitosan polymer chains can move into the tissue. One explanation of enhanced uptake and accelerated permeation into the olfactory mucosa could be attributed to the well-known ability of chitosan to open tight junctions or more general cell-cell junctions enabling paracellular transport. The membrane protein Zonula occludens protein 1 (ZO-1) is part of tight junctions within the epithelial cell layer as well as endothelial cells, and adherens junctions within the lamina propria (Steinke et al., 2008; Wolburg et al., 2008). When tight junctions are opened due to chitosan treatment, the membrane protein ZO-1 immunohistological staining fades. V. Dodane et al. previously reported this in 1999 in the cell culture of Caco-2 cells. The underlying mechanism caused by the internalization of this specific protein from the membrane to the cytoskeleton was later clarified by Smith et al. (2004) (Smith et al., 2004). This ability in cell culture has been proved by several research groups in the past decades (Dodane et al., 1999; Rassu et al., 2016); nevertheless, the chitosan application onto tissues is rarely reported. The opening mechanism in epithelial cells, previously reported by Dodane et al. (1999) to take place after 1 h and regenerate within 24 h also observed through fading and recovery of ZO-1 staining (Dodane et al., 1999), however, they investigated acidic solutions at low chitosan concentrations. Smith et al., 2004 further described a concentration-dependent internalization of ZO-1 on Caco2-monolayers within 1 h. M. Fazil et al. (2012) additionally showed increased brain uptake caused by chitosan particles but did not show ZO-1 staining as reference (Fazil et al., 2012). We, herein, showed for the first time that this is also true in the naturally occurring environment of porcine olfactory mucosa as a prominent biological barrier. The ZO-1 discoloring is attributed to internalization and opening of cell-cell junctions occuring in the ex vivo setting within only 15 min, including swelling of the dry chitosan-coated particles, which was never previously reported. To better understand which dosage of chitosan is most effective to promote paracellular transport, further experiments with different concentrations, particle amounts, as well as shorter timeframes would be interesting.

Finally, local particle clearance was observed above surface-located bowman glands. NiMPs have been cleared completely within 30 min, which could be attributed either to accelerated particle uptake within glands or ongoing mucus production resulting in continuously pushing away the overlaying particle layer. Ongoing mucus production even in ex vivo setting was already early described to last at least 4–5 h for frog palate mucosa (Sadé et al., 1970; Aspden et al., 1995), later also for human specimens (Aspden et al., 1997) and ferret trachea with velocities up to 9.5 ± 3.5 mm min⁻¹ (Abanses et al., 2009; Jeong et al., 2014). Further, chitosan was found to reduce mucociliary transport rate (MTR) proportionally to its molecular weight (Aspden et al., 1995), which was attributed only to its physical interaction with mucins not harming the mucosa itself (Aspden et al., 1997). Unfortunately, no further tracking of particles was possible in this study because no particles have been detected directly associated with the gland ensheathing cells, or the underlying cells of the lamina propria, and continuous monitoring was not possible due to the sampling procedure. As Raber et al. (2014) also described enhanced uptake of chitosan-coated PLGA nanoparticles in glandular ducts of hair follicles (Raber et al., 2014), mucosal glands could also function as uptake enhancing mucosa components. For the clarification of the role of surface-associated glands, it would be interesting to continuously monitor the site of action. It would be beneficial to monitor organoids, such as glands, using a complex model of the olfactory mucosa. For example an organ-on-a-chip model, a so-called nose-on-a-chip.

Within this study, we successfully developed specialized Nano-in-Micro particles (NiMPs) consisting of PLGA nanoparticles embedded into chitosan microparticles via spray drying. We evolved a robust spray-drying process resulting in homogenous microparticles with high encapsulation efficiency, uniform size distribution, smooth surface, and dense pores. We demonstrated, that not only 80 and 175 nm PLGA nanoparticles purely applied to the olfactory mucosa can be taken up intracellularly within only 5 min, but also 520 nm PLGA particles are associated with nuclei and neuronal fibers after 15 min, which can imply transcellular transport within the olfactory epithelium and intracellular uptake into neuronal cells following transport along those to the olfactory or trigeminal nerve, which finally enables targeting of the brain and the central nervous system (CNS). These findings were further proved to be size-time-proportional, resulting in smaller particles moving faster (within only 5 min) and to a higher extent into the lamina propria. Chitosan-coated NiMPs were identified as even more interesting nanoparticle carriers and are auspicious delivery vehicles themselves. Their chitosan coating is subsequently swelling on the mucosal barrier; therefore, single chitosan polymer chains penetrate within 15 min approximately 150 µm into the olfactory mucosa. With this, the chitosan penetration affects the opening of tight junctions and, therefore, can additionally enable paracellular transport through the olfactory epithelium. Consequently, the accelerated uptake of chitosan-coated nanoparticles was examined.

PLGA nanoparticles, as well as, NiMPs were found in lymphoid follicles; however, only NiMPs were co-localized after 15 min with the immunoreactivity signal of membrane protein CD14 present on monocytes and macrophages. Hence, we assume any other interaction for PLGA nanoparticles e.g., T- or B-cell mediated binding, which needs to be investigated in future studies. NiMPs herein showed the ability to initialize an anti-inflammatory cascade within 15 min after particle application, due to the beneficial properties of the herein utilized chitosan (molecular weight, degree of deacetylation, and particle size). However, CD14⁺ macrophages could be either pro-inflammatory M1-macrophages or anti-inflammatory M2-macrophages effecting wound healing, which could not be fully clarified within this study because therefore cytokine levels are needed as a reference, which should be investigated in cell culture. Additionally, particle clearance of gland overlaying particles was herein documented, but could not be fully enlightened, because the sampling procedure was limited and continuously monitoring was not possible.

In conclusion, within this study, we demonstrated the possibility for PLGA nanoparticles as well as for chitosan NiMPs to take all three prominent pathways to the brain and the CNS, namely transcellular, intracellular via neuronal cells, and paracellular transport. Overall, although polymeric particles are broadly reported as promising drug delivery systems, not only for intranasal application the usage in the clinic is still lacking. Up to now, no nano-based system has entered advanced clinical trials. Consequently, still more research is needed to collect additional data and unravel the complex phenomena within the nasal mucosa, to clarify the different transport mechanisms and the role of special organoids, like bowman glands. Further, verifying specific influences of particle material, other formulation components, and particle sizes.