Bigger or Smaller? Size and Loading Effects on Nanoparticle Uptake Efficiency in the Nasal Mucosa

Abstract

PLGA nanoparticles hold great promise for nasal administration, but only with careful design will efficient, effective, and safe delivery systems be developed. To better understand the size-dependence of nasal epithelial uptake, PLGA nanoparticles (60 nm or 125 nm) loaded with-Nile Red were prepared, and their uptake into excised sections of bovine nasal respiratory or olfactory mucosa was measured for 30 or 60 min. The epithelial layer and the submucosal tissues were separated and the amount of Nile Red was used to calculate the number of nanoparticles in each tissue region. Both particle sizes were able to be internalized into the nasal tissues in as little as 30 min, but their total uptake represented less than 5% of the nanoparticles available. Nanoparticles were present in both the epithelial cells and in the submucosal tissues, and greater numbers of 60 nm particles were present in the submucosa than the epithelium while greater numbers of the 125 nm particles remained in the epithelial cell layer. The amount of Nile Red recovered from the mucosal tissues after exposure to 125 nm nanoparticles was at least 2-fold greater than from the 60 nm nanoparticles, however, due to the higher (~ 9-fold) capacity of the larger particles. The greater mass transfer of the Nile Red from the larger particles suggests that it may not be necessary to develop small nanoparticulate delivery systems for efficient drug delivery via the nasal mucosa. Well-designed nanoparticles with diameters > 100 nm show good uptake into the nasal epithelium and are capable of transfer to the submucosal tissues, near the location of significant populations of blood and lymphatic vessels.

Graphical Abstract

INTRODUCTION

Increasing interest in the development of nanoparticulate drug delivery systems has also extended to an interest in the nasal mucosa as an administration site for systemic nanoparticle delivery, for vaccine administration due to the local population of nasal-associated lymphoid tissue (NALT), and for potential direct delivery of these drug carriers to the brain (1, 2). While drug administration via the nasal mucosa has been shown to be an effective method for the administration of small-molecule drugs for both topical and systemic action, little is known about the ability of the nasal tissues to transfer nanoparticles beyond the mucosal surface, and even less is understood about the characteristics of nanoparticles that would provide effective and efficient delivery. Polymeric nanoparticles for intranasal application have been proposed to overcome some known limitations to efficient nasal delivery including reducing mucosal metabolism and mucociliary clearance (2, 3). Nasal administration of nanoparticles has also been proposed as a means to enhance drug delivery to the CNS (1, 2, 4). Owing to their small size, nanoparticles may provide improved targeting and transport through the nasal mucosa, and drug-loaded nanoparticles may enhance the delivery of drugs or vaccines via the intranasal route.

Nanoparticle diameter is reported to be a significant factor, along with surface charge, hydrophobicity, and the presence of targeting or permeation enhancing moieties, in determining nanoparticle uptake. In fact, nanoparticle size frequently determines the in vivo behavior of nanoparticulate delivery systems, especially regarding their circulation time, immunogenicity and internalization (5). It has been proposed that the smaller the particle diameter, the higher the nanoparticle uptake across epithelial barriers with resulting potential systemic distribution (6, 7). In a study by Brooking et al., the level of radioactivity detected in rat blood after intranasal administration of ¹²⁵I-radiolabeled sulphate-modified polystyrene nanoparticles was highest for the smallest nanoparticles, with the blood radioactivity level was in the order of 20 nm > 100 nm > 500 nm = 1000 nm (7). Several recent reviews have compiled information about the characteristics of nanoparticle delivery systems investigated using the nasal route (1, 2) and these reports describe nanoparticles with sizes ranging between 65–300 nm; typically, each investigation only describes the results for a single particle size.

Identifying the optimal nanoparticle characteristics, including size, that control nanoparticle uptake and transfer in the nasal mucosa will provide an improved understanding of nanoparticle trafficking and contribute to the design of new, effective particulate delivery systems. Nanoparticles made from biodegradable polymers, including poly-lactic-co-glycolic acid (PLGA), are of great interest for use in drug delivery systems due to their relative safety and ease of surface modification, and two different, relatively small PLGA nanoparticles were selected for investigation in these studies to further investigate the role of size in the uptake of nanomaterials by the nasal mucosa.

METHODS

Materials

PLGA polymer [Resomer^® 503, 50:50 with inherent viscosity 0.32 – 0.44 dl/g] was obtained from Evonik Industries AG (Darmstadt, Germany). Acetone and N, N dimethylformamide (DMF) were purchased from Fisher Chemicals (Fair Lawn, NJ, USA). Nile Red and Trypsin-EDTA solution were obtained from MilliporeSigma (St. Louis, MO, USA). SnakeSkin^® pleated dialysis tubing with a 7000 Da molecular weight cutoff was purchased from ThermoFisher Scientific (Rockford, IL, USA). Cellosolve^® Acetate (2-ethoxyethyl acetate) was obtained from AlfaAesar (Ward Hill, MD, USA). D-Glucose was obtained from Research Products International (RPI) (Mt. Prospect, IL, USA). Whatman filter paper was purchased from Global Life Sciences Solutions USA LLC (Pittsburgh, PA, USA).

Nanoparticle preparation

Nanoparticles were prepared using a surfactant-free nanoprecipitation method as previously described (8, 9). Various combinations of preparation conditions were investigated to identify the parameter combinations that would result in the desired size nanoparticles. Parameters investigated included the amount of polymer, organic solvent type, and temperature of the aqueous phase. Investigations of the effects of altering these parameters on nanoparticle diameter have also been previously described in other reports (10–12). The final compositions for the PLGA nanoparticles used in this study are listed in Table 1. To prepare the nanoparticles. To prepare the nanoparticles, PLGA was dissolved in DMF and a measured volume of Nile Red dye in acetone (stock solution of 100 μg/ml) was added to the polymer solution. This organic phase was added gradually into room temperature Nanopure^® water for the preparation of 125 nm PLGA nanoparticles with stirring (~ 300 rpm) for two hours for removal of DMF and loading of the nanoparticles. For the preparation of 60 nm nanoparticles, the temperature of the aqueous phase was increased to 40 °C and, following the addition of the organic phase, the temperature was decreased gradually (5 °C every 30 min until reaching room temperature). Residual solvent was removed from both the 60 and 125 nm particle dispersions by dialysis in SnakeSkin^® dialysis tubing against Nanopure^® water for an additional 40 hours. The nanoparticle dispersion within the dialysis tubing was removed and filtered through a 25 μm Whatman^® (# 541) filter paper to remove any large aggregates. The recovered nanoparticle dispersion was used immediately for uptake experiments.

Table 1.

Nanoparticle preparation parameters for 60 and 125 nm PLGA particles.

Solvent (ml)	PLGA Polymer (mg)	Nile Red solution in acetone (100 μg/ml)	Aqueous Phase (ml)	Temperature (°C)	Target Diameter (nm)
DMF (5 ml)	36	0.7 ml	30	40	60
DMF (2 ml)	70	1.4 ml	20	Ambient	125

Nanoparticle characterization

Nanoparticle size and surface charge were evaluated using a Malvern Nano-ZS Zetasizer (Malvern Instruments Limited, Worcestershire, UK). Additional evaluation of nanoparticle size and shape was performed using scanning electron microscopy on one control batch for each of the 60 nm and 125 nm nanoparticles.

Nanoparticle yield was evaluated after freeze-drying of three batches of the dialyzed dispersions, and the mass of nanoparticles after lyophilization was compared to the starting mass of PLGA polymer for the calculation of the percentage yield.

Preparation of Excised Bovine Nasal Tissues

Bovine nasal tissues were obtained from a local abattoir (Buds Custom Meats, Riverside, IA, USA). A longitudinal cut was made along the nasal septum and separate samples of respiratory and olfactory mucosae were harvested (13). The collected tissues were transferred into 5 % (w/v) ice-cold glucose and then kept on ice until reaching the laboratory. Tissues were stripped from the underlying cartilage with tweezers and the resulting tissues, which contained the single columnar epithelial cell layer, olfactory axons for the olfactory tissues, and the underlying lamina propria with associated vasculature, lymphatics and glands, was mounted in NaviCyte^® 1 ml vertical diffusion cells with aperture area of 0.64 cm² (Warner Instruments, LLC, Hamden, CT, USA) with the mucosal surface facing the donor chamber. Donor and receiver chambers were filled with 1 ml of pre-warmed (37 °C), 5% (w/v) glucose and equilibrated at 37 °C for 10 min. Each chamber was aerated with Carbogen^® gas (95 % oxygen plus 5 % carbon dioxide) at a rate of 3–4 bubbles per second to maintain oxygen concentration and provide mild mixing. Glucose (5 % w/v) was selected as a replacement for the typically used buffer solutions to avoid the nanoparticle aggregation observed when using buffer solutions while providing an isotonic environment with an energy source to maintain tissue viability. Nanoparticle size and surface charge in the glucose media was evaluated using dynamic light scattering (DLS). No significant changes in the hydrodynamic diameter or zeta potential were observed for any of the PLGA nanoparticles dispersed in 5 % glucose compared to PLGA nanoparticles dispersed in water (9).

Nanoparticle Uptake by Nasal Tissues

Nasal tissues were equilibrated in the NaviCyte^® chambers using 1 ml, pre-warmed 5 % (w/v) glucose in the donor and receiver chambers for 10 min. Donor and receiver volumes were removed, and the receiver side was replaced with fresh, pre-warmed 5 % glucose. The donor side was replaced with 1 ml of pre-warmed nanoparticle dispersion followed by incubation for either 30 or 60 min. At the end of the incubation, the receiver solution was collected, the exposed area of the tissues rinsed with Nanopure^® water, and the exposed tissue region was trimmed free from the remaining tissues and transferred to a 15 ml polypropylene centrifuge tube containing 2 ml trypsin-EDTA (0.25%) solution in order to separate the outer epithelial cell layer from the underlying submucosal tissue. Enzymatic removal of epithelial layer was performed based on the procedures previously described for nasal and other epithelial tissues (14–17).

After a 2 hr incubation with trypsin-EDTA, the remaining submucosal tissue was transferred into a separate 15 ml polypropylene tube containing 1 ml of the organic solvent, Cellosolve^® acetate, in order to disrupt and solubilize the sub-mucosal tissues and to dissolve the entrapped nanoparticles to allow for quantification of the Nile Red content. The epithelial cells remaining in the trypsin-EDTA solution were also treated with 1 ml Cellosolve^® acetate. In addition, 1 ml of Cellosolve^® acetate was added to the receiver solution which was removed and placed in another 15 ml polypropylene tube in order to quantify any nanoparticles translocated from the mucosal tissue into the receiver solution. Each sample (epithelial cell, submucosal layer, and receiver solution) was incubated with Cellosolve^® acetate for > 6 hr (overnight) in order to fully dissolve the nanoparticles (9). The amount of dissolved Nile Red dye was quantified using a fluorescence microplate reader (SpectraMax M5; Ex.: 520 nm – Em.: 620 nm, Molecular Devices, Sunnyvale, CA, USA). The amount of dissolved Nile Red dye was quantified using a fluorescence microplate reader (SpectraMax M5; Ex.: 520 nm – Em.: 620 nm, Molecular Devices, Sunnyvale, CA, USA). The approximate number of nanoparticles transferred into the nasal mucosa was estimated using Eqn 1 (13, 18, 19):

$$Number\mathit{\text{of}}NP=\frac{W}{\rho \ast r^3\pi }$$ Equation 1

where W represents the mass of nanoparticles in the final preparation (g); ρ is the density of PLGA polymer used (mg/cm³); and r is the average NP diameter obtained from DLS (cm). The fluorescence intensity measured following Nile Red extraction from the tissue sample was used to determine the mass of Nile Red in the sample, and the percentage loading of the nanoparticles was used to determine “W”, the mass of nanoparticles in the sample.

Data Analysis

Results were analyzed for statistical significance and plotted using GraphPad Prism 8 (GraphPad Software, San Diego, California USA) and Excel software (Microsoft Inc., Seattle, WA, USA). Unpaired, two-tailed t-tests were used to compare between tissue exposure times; respiratory and olfactory tissues; or nanoparticle sizes. A sample size of 3 was available for each group and a p-value < 0.05 was used to evaluated statistical significance.

RESULTS

Preparation and Characterization of Nanoparticles

Dynamic light scattering showed nanoparticles with a narrow size distribution and an average diameter of 60 or 125 nm (Table 2). SEM analysis also showed spherical nanoparticles with diameters similar to the measurements obtained from DLS. The diameter of the nanoparticles in the SEM sample were reasonably uniform which supported the PDI < 0.15 values. Nanoparticle yield was ~ 75 % of the starting amount of PLGA polymer, and the Nile Red loading was ~ 26% of the amount added during nanoparticle preparation.

Table 2.

Characteristics of PLGA nanoparticles dispersed in water. (SD describes between batch replicates (n=3))

Target Diameter	Measured Diameter (nm) (SD)	Polydispersity Index (SD)	Zeta Potential (SD)	Yield % (SD)	Nile Red Loading (SD)
60 nm	60 (5)	0.11 (0.05)	−28.0 (5.0) mV	75 (1)	26 (2)
125 nm	126 (2.6)	0.08 (0.04)	−43.9 (3.3) mV	76 (1.4)	29 (2.7)

Nanoparticle Uptake

Uptake of 60 nm Nanoparticles

Nanoparticle uptake into the full thickness respiratory mucosa was approximately ~ 2.5% and ~ 5.1% of the total number of nanoparticles placed in the donor medium after 30 min and 60 min of incubation, respectively (Figure 1). Nanoparticles were detected in both the epithelial and submucosal layers, and a very limited number (< 1% of the number measured in the tissues) of the nanoparticles traversed the full thickness of the tissue and were transferred into the receiver medium. In the respiratory mucosa, the translocated nanoparticles were distributed within the epithelial layer (~ 27–30%) and the submucosal layer (~ 65–70%) at both 30 and 60 min (Figure 1).

Figure 1.

PLGA nanoparticle (NP) uptake (60 nm) by the respiratory and the olfactory nasal mucosa. Nanoparticle uptake was time dependent and a greater number of NP were located in the sub-mucosal region (n=3). Numbers above the bars represent the percentage of particles transferred compared to the starting number of particles in the donor medium.

In the olfactory mucosa, nanoparticle uptake was also time dependent and the total nanoparticle uptake was ~ 2.5% and ~ 4.3% of the number of nanoparticles present in the donor medium after 30 min and 60 min of incubation, respectively. The distribution of particles between the epithelial cells and sub-mucosal tissues was similar to the respiratory tissues (Figure 1).

Uptake of 125 nm Nanoparticles

Studies of the uptake of the larger, 125 nm diameter, nanoparticles across the nasal tissues revealed that these nanoparticles were also able to translocate into both the nasal respiratory and olfactory mucosae. The nanoparticles in the respiratory mucosa were distributed nearly equally between the epithelial cell layer and the submucosal layer. Similar results were observed for the 125 nm particles in the olfactory mucosa (Figure 2).

Figure 2.

PLGA nanoparticle (NP) uptake (125 nm) by the respiratory and olfactory nasal mucosa. Numbers above the bars represent the percentage particles transferred compared to the starting number of particles in the donor medium.

Effect of Nanoparticle Size on Uptake within the Nasal Mucosa

The number of 60 nm nanoparticles measured in the full thickness (epithelial + submucosal) tissues was significantly (3.4 – 4.4 times, p<0.038) higher than for the 125 nm nanoparticles (Figure 3), and the number of 60 nm nanoparticles detected in the epithelial cells was 2 −3 times higher than the 125 nm nanoparticles (Figure 3). The number of nanoparticles transferred to the submucosal layer (in both tissue layers) was also significantly higher (5–7 fold, p< 0.035) for the 60 nm particles.

Figure 3.

Effect of nanoparticle diameter on distribution of internalized nanoparticles between epithelial and submucosal regions in the nasal respiratory mucosa. Smaller diameter nanoparticles (60 nm) were internalized to a greater extent in each mucosal region (n=3, bars represent mean ± SD).

Due to the higher mass carrying capacity of the 125 nm particles, however, the total amount of Nile Red quantified in the nasal tissues was greater following incubation with the 125 nm nanoparticles as compared to the 60 nm nanoparticles. For example, the amount of Nile Red measured in the full thickness respiratory mucosa from the 125 nm nanoparticles was 1.9 times greater than the amount of Nile Red measured in the tissues following a 30 min incubation with 60 nm particles (Figure 4). In the olfactory mucosa, the amount of Nile Red measured following incubation with 125 nm nanoparticles was ~2 times greater than the amount from the 60 nm nanoparticles after a 30 min exposure (Figure 4).

Figure 4.

Amount of Nile Red quantified in the nasal mucosa following incubation with 60 nm or 125 nm nanoparticles in NaviCyte vertical diffusion chambers. The amount of Nile Red was measured following the dissolution of the PLGA particles entrapped within the tissue segment. (n=3, bars represent mean ± SD).

DISCUSSION

A surfactant-free nanoprecipitation method was used for the preparation of two different size nanoparticles, 60 and 125 nm, to investigate the effect of size on their uptake in the nasal mucosa. The PLGA nanoparticle characteristics were modified by controlling the preparation parameters, especially the concentration of the polymer used and the temperature of the aqueous phase. Dimethylformamide (DMF) was selected because of its high water miscibility, which aids in the production of smaller nanoparticles (10). Optimization of these methods are able to provide PLGA nanoparticles with diameters ranging from at least 50 −150 nm with good reproducibility and yield percent. However, the preparation procedure includes an ~40 hr dialysis step, so this method may not be suitable for the encapsulation of hydrophilic materials.

Nile Red was selected as a marker compound for incorporation into the PLGA nanoparticles due to its highly lipophilic nature and its ready quantification using fluorescence detection methods. There was no release of the Nile Red from the nanoparticles during the 60 min experimental time period, so the resulting Nile Red tissue concentrations result entirely from the encapsulated Nile Red rather than a combination of free dye and loaded nanoparticles. Unlike previous studies that measured nanoparticle transfer based on the fluorescence signal in culture or tissue media, blood, or tissues (20–22), these studies quantified the nanoparticle transfer into the nasal tissues by releasing all of the Nile Red by dissolving the PLGA matrix in Cellosolve acetate. This approach allowed for the evaluation of the nanoparticles present in discrete regions (epithelial, submucosal) of the nasal mucosa and allowed for the estimation of the actual number of nanoparticles present in those tissues. This new approach provides more detailed information about nanoparticle transfer and tissue distribution and enabled further interpretation of the size and mass-loading effects of the nanoparticles on tissue exposure.

PLGA nanoparticle translocation into the nasal respiratory and olfactory tissues was observed to be time dependent with a greater number of nanoparticles detected in the nasal tissues after longer (60 min) incubation times. This time-dependent increase in transfer suggests that the nanoparticles are being internalized into the nasal tissues rather than simply being associated with the surface of the tissues. Previous investigations using excised porcine olfactory tissues (21), and other tissue and cell culture models (23), have reported the significant accumulation of nanoparticles at the apical surfaces of the tissues. We have seen similar behaviors in our studies, where the increase in Nile Red concentrations within the tissues over time, especially within the submucosal tissue region, indicates that the PLGA nanoparticles were being continuously taken up by the nasal mucosal tissues and trafficked both within the epithelial cell layer and in the lamina propria. These results suggest that the nanoparticles can distribute within the nasal mucosal tissues leading to further distribution to distant tissues via intracellular and intercellular pathways. Lochhead et al. described the transfer of fluorescently-labeled dextrans along the perivascular pathways in the nasal mucosa with further transfer to the brain (24), and in a later report showed the involvement of extraneuronal pathways in the transfer of insulin from the nasal cavity to the brain (25). In another recent study reported by Godfrey et al., microparticles of clustered, peptide-loaded chitosan nanoparticles were observed to enhance peptide delivery to the rat brain, with the nanoparticle components being observed in the perineuronal and perivascular spaces of the thalamus and cortex along with other locations within the brain parenchyma (26).

Using excised tissues, virtually no nanoparticles were detected in the receiver cell fluids which suggests that nanoparticle escape from the primarily collagen-based matrix of the submucosa is limited due to the lack of elimination mechanisms from this tissue surface. Unlike cell culture monolayer systems, the lack of transfer into the receiver fluids from a multi-layer tissue should not be interpreted as suggesting limited access to the vascular or lymphatic systems in the associated tissue. Instead, the presence of a large number of nanoparticles in the submucosal tissues suggests that these nanoparticles are able to reach the vascular and lymphatic vessels resident in those regions.

The increased uptake of smaller diameter PLGA nanoparticles has been previously reported in other epithelial cell systems. Qaddoumi et al. showed that 100 nm PLGA nanoparticles had higher uptake than particles with diameters of 800 nm or 10 μm in primary cultured rabbit conjunctival epithelial cells (27). Similarly, in Caco-2 cells, Desai et al. showed greater uptake of 100 nm PLGA nanoparticles than 500 nm, 1 μm or 10 μm particles (19). A greater number of smaller (60 nm) nanoparticles were translocated into both the nasal respiratory and olfactory mucosa, especially in the submucosal tissue regions, compared to the larger, 125 nm nanoparticles. This is consistent with many previous reports regarding the size-dependency of nanoparticle uptake, but the nanoparticles used in these studies were smaller than most previous studies which evaluated particles in the 100–200 nm size range (1, 2, 21). The observed increased uptake of the even smaller diameter (60 nm) nanoparticles may be the result of their access to additional endocytic processes that have reported maximal size capacities (e.g. the caveolae-mediated endocytic pathway with a reported vesicle diameter of 60–90 nm) (28, 29). The increased uptake may also be the result of the probability of an increased number of interactions with the epithelial cell surface due to the increased number of nanoparticles in the exposure medium and the greater number of contacts made with the epithelial surface (30).

Despite the higher absolute number of 60 nm nanoparticles present in the nasal tissues, their smaller size significantly limits their total drug-payload carrying capacity. The results from these studies show that the higher Nile Red carrying capacity of the 125 nm particles, approximately 9 times the mass loaded in the 60 nm PLGA nanoparticles despite similar loading efficiencies, compensates for the lower number of 125 nm particles transferred into the tissues. As a result, the larger particles were able to deliver a greater total amount of Nile Red into the tissues, despite their absolute lower particle numbers. These results show that the mass-load carrying capacity of nanoparticle delivery systems, along with the temporal release pattern of the drug from the particles, should be considered along with the currently considered size and surface characteristics in the design and evaluation of nanoparticle-based delivery system. For nasally administered nano-systems, several different cellular targets and temporal release patterns have been proposed for effective drug delivery. These delivery patterns include: (1) nanoparticles entrapped in the mucus layer at the epithelial surface with free drug being released and absorbed by the epithelial cells; (2) nanoparticles taken up by the epithelial or neuronal cells with the particles releasing drug within the cell for local effect or for further distribution to other cells and tissues; (3) nanoparticles are taken up by the epithelial or neuronal cells and are trafficked as particles to distant tissues, including the brain, where they subsequently release their drug payloads (2). The optimal particle size, drug loading and drug release pattern would be quite different for each of these scenarios, and the design of nanoparticles capable of accomplishing any one of these delivery goals requires careful design and assessment of the delivery system to accomplish effective, safe and efficient nanotherapeutics for administration via the nasal mucosa.

CONCLUSIONS

Both 60 nm and 125 nm PLGA nanoparticles were able to be internalized into excised nasal mucosal tissues in as little as 30 min, but their total uptake represented less than 5% of the available nanoparticle load. The limited overall uptake (<5 %) of the PLGA nanoparticles by the nasal mucosa suggests a potential limitation to the development of efficient nanoparticle delivery systems, yet nanoparticle systems may enable targeted delivery or may reduce the impact of other limitations to nasal administration, including mucociliary clearance and mucosal metabolism. Uptake was observed to be size-dependent, where the smaller diameter nanoparticles were transferred in higher numbers compared to the larger nanoparticles. Both size particles were found in measurable quantities in the epithelial cells and in the underlying submucosal tissues. Transfer of the nanoparticles beyond the epithelial cells suggests that further distribution of the nanoparticles beyond the epithelial layer is possible from this administration site. Interestingly, since the larger, 125 nm particles carried a greater amount of encapsulated Nile Red compared to the smaller nanoparticles, the 125 nm particles provided a greater total tissue exposure to Nile Red despite the reduced absolute number of nanoparticles present in the tissues. This highlights the importance of careful nanoparticle design along with an understanding of the nano-bio interactions at the epithelial surfaces controlling uptake of the nanomaterials into epithelial tissues. Based on the results of these studies, the increased difficulties associated with the production and stabilization of very small nanoparticles may not be necessary to accomplish effective delivery via the nasal mucosa, yet smaller nanoparticles may still offer an advantage if distribution to specific non-epithelial locations within the mucosa is desired.