Polymeric nanoparticles as a platform for nose-to-brain delivery

Abstract

The treatment of brain disorders via nose-to-brain delivery (NtBD) is a promising non-invasive strategy to bypass the blood–brain barrier. In this study, we developed and characterized polymeric nanoparticles (NPs) based on polylactic acid (PLA) and poloxamers (P188 and P407), synthesized via one-step or two-step nanoprecipitation methods. All formulations resulted in homogeneous, negatively charged NPs with a diameter of 110 nm, compatible with efficient brain delivery. Dissipative particle dynamics simulations confirmed the structural organisation of NPs and highlighted the role of poloxamers in forming a hydrophilic surface. NPs modified with poloxamers demonstrated improved colloidal stability in artificial mucus and cerebrospinal fluid, unlike bare PLA NPs. In vitro assays on human nasal epithelial cells and differentiated neuronal cells showed no cytotoxicity and cellular uptake in both cell types. In vivo fluorescence imaging and immunostaining revealed the presence of NPs in the olfactory bulb as early as one hour post-administration. These results support NP-PLA-P188 as a promising formulation for effective and safe NtBD of drugs targeting neurological diseases.

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Introduction

The treatment of neurodegenerative diseases continues to represent a major challenge in the healthcare field. The effectiveness of the treatments is greatly compromised by the selectivity of the blood–brain barrier (BBB). The tight junctions of this barrier prevent large and hydrophilic molecules from reaching the brain. Therefore, bypassing this barrier becomes crucial to targeting the central nervous system. To avoid passing through the BBB, direct administration into the brain (in the parenchyma, in the spine, or in the cerebral ventricles) is possible but is highly invasive.¹ Nose-to-brain delivery (NtBD) therefore appears to be a very promising approach for overcoming these obstacles.² Indeed, thanks to the proximity of the olfactory bulb and neuronal olfactory projections in the nasal cavity, it is possible for drugs to pass directly from the nasal cavity to the neuronal cells. Besides bypassing the BBB by using direct neuronal transport, NtBD also bypasses the intestinal barrier and the hepatic metabolism. Moreover, NtBD is non-invasive, patient-friendly, and effective, and ensures a rapid onset of action.³ Despite these numerous advantages, several challenges remain, including overcoming the mucociliary clearance and the mucus barrier that eliminates molecules before even reaching the nasal epithelium.³ Plus, the nasal cavity is characterised by a low pH and the presence of various enzymes that can degrade drugs before they even cross the mucosa.⁴ To overcome these limitations, the design of an effective delivery system is essential to facilitate penetration through the nasal mucosa and a balance must be found between efficacy and toxicity.⁵

Nanoparticles (NPs), and more specifically polymeric NPs, represent a promising tool to optimize NtBD and to target the brain. Indeed, several drug delivery systems have been developed for the NtBD, with NPs composed of lipids⁶ or inorganic NPs.⁷ These inorganic NPs (such as iron oxide or gold NPs) have many advantages, particularly because of their intrinsic properties (magnetism, surface plasmon, resonance etc.).⁸ For these reasons, they could also be used for the NtBD. However, polymeric NPs offer distinct advantages over these systems, in particular their high biocompatibility, biodegradability and easily modulated physicochemical properties, which makes them particularly suitable for mucosal administration, and NtBD.⁹ Polymeric NPs can protect the drug from enzyme degradation, as well as increase the contact time with the nasal mucosa.¹⁰ They can also improve drug solubility and penetration, therefore promoting delivery to the brain and increasing the therapeutic effect of the drug.^6,11

Designing polymeric NPs is a delicate process where optimisation of the carrier efficiency relies on every parameter.¹² The ideal properties for NtBD are not yet described but it has been mentioned that smaller NPs (around 100 nm) had better brain penetration than larger NPs.¹³ Plus, it has been demonstrated that positively charged NPs are not able to penetrate efficiently in the mucin layer of the nasal cavity.¹⁴ It seems also relevant to find a balance between adhesion to the nasal mucosa, penetration through the mucus layer and the nasal epithelium, and stealth to prevent detection from the immune system of the nasal cavity.

Polylactic acid (PLA) is widely used for the preparation of NPs, as it is biocompatible, biodegradable, and non-toxic. Plus, its hydrophobicity makes it a great candidate for NP synthesis and the entrapment of hydrophobic drugs. PLA NPs have also been used to deliver hydrophilic drugs by functionalising their surface, such as peptides or even proteins.^15,16

Poloxamers are non-ionic surfactants, characterised by a hydrophilic central block flanked by two hydrophobic blocks. Incorporating surfactants in the NP synthesis allows the enhancement of both stability and stealth, as the hydrophobic part remains in contact with the core of the NPs, whereas the hydrophilic part is at the surface of the particle. Moreover, modifying NPs with surfactants can also prevent their aggregation.¹⁷ Among these poloxamers, poloxamer 188 (P188) and poloxamer 407 (P407) are the most commonly used. Poloxamer 188 has a lower molecular weight and is more hydrophilic than poloxamer 407 which has a higher molecular weight and a higher viscosity. Poloxamer 188 is therefore used more for emulsification and stabilisation, and poloxamer 407 is more likely to be used for topical drug delivery. Both these poloxamers are biocompatible, biodegradable, and non-toxic.¹⁸

We aimed to design polymeric NPs for the NtBD and to determine if they were able to overcome the limitations of the nasal cavity. Our study compared two different types of synthesis: one in one step with the NP composed of a mixture of PLA and poloxamer, and the other one in two steps, first the synthesis of PLA NPs and then the addition of a coating of poloxamer onto the preformed NP. Our goal was to determine if the addition of poloxamers within the formulation of polymeric NPs was beneficial for the NtBD and evaluate if the organisation of poloxamers (either mixed with PLA or as a surface coating) was critical for their transport to the brain. The evaluation of the potential stabilizer effect of the poloxamer was evaluated using artificial mucus and cerebrospinal fluid. Comparison of their localisation in the nasal cavity and in the whole body was performed to determine the best candidate for further studies.

Experimental

Nanoparticle syntheses

NP-PLA synthesis. NP-PLA were prepared using a nanoprecipitation process previously described.¹⁹ Briefly, poly-D,L-lactic acid (PLA) was dissolved in acetone (10 mg mL⁻¹) and added dropwise in 5 mM sodium carbonate buffer under magnetic stirring (250 rpm) at room temperature. Solvents were removed from the solution under reduced pressure and controlled temperature (30 °C) using a rotary evaporator (Rotavapor® R-300, Buchi, France). Before use, formulations were centrifuged for 20 minutes at 16 000 rpm and resuspended in water.

NP-PLA-poloxamer synthesis. NP-PLA-poloxamer (either P188 or P407, BASF, Germany) were synthesized using two different techniques: a one-step synthesis and a two-step one. First, for the one-step synthesis resulting in NP-PLA-P188 and NP-PLA-P407, the same nanoprecipitation technique described above was used except that poloxamer (5 mg mL⁻¹) was added to the 5 mM sodium carbonate buffer. Then, using the two-step method, NP-PLA were first synthesized exactly as mentioned above and added dropwise into a poloxamer solution in 5 mM sodium carbonate buffer. These solutions were then placed on an agitation wheel for 2 h at 18 rpm. These particles were denoted as NP-PLA-cP188 and NP-PLA-cP407, for referring to the additional poloxamer coating. Similarly to NP-PLA, NP-PLA-poloxamer solutions were resuspended in water before use.

Fluorescent-NP synthesis. Fluorescent NPs were prepared using either DY650 (λ_ex: 653 nm, λ_em: 674 nm, Dyomics, Germany) or DiR (λ_ex: 748 nm, λ_em: 780 nm, Revvity, USA) fluorophores. Using similar processes as described above, fluorophores were added in the organic phase before the nanoprecipitation step at the loading rate of 0.02%. Formulations were resuspended in water and fluorophores content was checked after centrifugation at 16 000 rpm for 20 min. Fluorescence was measured on the total fraction and on the supernatant using a Spark® multimode microplate reader (Tecan, Switzerland).

Nanoparticle characterisation

Physicochemical characteristics. The average hydrodynamic diameter and particle size distribution (polydispersity index, PDI) of nanoformulations were determined by measuring the dynamic light scattering (Zetasizer Nano ZS, Malvern Panalytical, UK). The electrophoretic mobilities (zeta potential) were measured by Laser Doppler velocimetry using the same instrument. Stability of each parameter was also measured over time. The final NPs concentration was determined by multi-angle dynamic light scattering using a Zetasizer Ultra (Malvern Panalytical, UK). All parameters were determined using three replicates and three independent experiments.

This technique was used weekly for one month, as described above, to determine stability at 4 °C. Parameters were determined using three replicates and three independent experiments.

Molecular modelling. Dissipative particle dynamics (DPD) simulations were performed using the Materials Studio 2017 software (Biovia, France). For the water molecules, a coarse graining approach was used where one bead represents 3 molecules of water. The radius of the water bead was set to 2.78 Å and its molecular mass to 54 Da. All the chain lengths of the PLA and poloxamer molecules were scaled to a 1/5 ratio in order to match the size of the simulated NPs (about 20 nm for the final diameter in our simulations, compared to about 100 nm for the experimentally produced NPs). Accordingly, PLA molecules were constructed as linear repetitions of 140 units of lactic acid monomers (LA)_n. Special beads were created for each extremity of the PLA chain, with a start and end bead in order to take into account the hydroxy function at the start of the chain and the carboxylic acid function at the end of the chain.

For the P188, a mesoscale molecule was obtained with the following organization: 16 polyethylene oxide (PEO-5 polypropylene oxide (PPO)-16 PEO. Accordingly, the P407 was simulated with a mesoscale molecule spanning 20 PEO-12 PPO-20 PEO. A supplementary special bead was created for the extremities of the poloxamers to take into account the hydroxy function at the start and the end of each poloxamer chain. All the constructions are displayed in Fig. 2.

For the calculations, all the solubility parameters δ_i were calculated with the software, using either the Synthia module for polymers, or models constructed using the amorphous cell module, or a combination of both methods. For the models constructed as amorphous cells, all the calculations were performed with the Forcite module using the COMPASS II force field for atom parameters and partial charges.

The corresponding solubility parameters were then used to calculate the Flory–Huggins interaction parameters δ_ij for the corresponding binary mixtures, which were calculated using the relation χ_ij = (v/RT)(δ_i − δ_j)² where R is the gas constant, T the absolute temperature and v the mean volume per mole of the two corresponding components.²⁰ The molar volumes for each component were determined using the MOE software. The Flory–Huggins interaction parameters were then converted into DPD repulsion parameters a_ij, which were obtained using the relation a_ij = 25 + 3.50χ_ij.²¹

Our calculations were performed using 300 × 300 × 300 Å cubic periodic boxes of water as starting points for all the dynamics. For the NP-PLA-P188 and NP-PLA-P407 runs, droplets with a radius of 140 Å containing a disordered mixture of water, PLA and poloxamer molecules (respectively P188 and P407) were placed at their center. In order to mimic the experimental ratio of PLA and poloxamer molecules during the nanoprecipitation process, we used the following mass ratio in the Materials Studio: water 99, PLA 66 and poloxamer 33, leading to a PLA/poloxamer mass ratio of 2 : 1 and a water/polymer mass ratio of 1 : 1. In both cases (NP-PLA-P188 and NP-PLA-P407), the rest of the simulation box was filled with water.

For the NP-PLA-cP188 and NP-PLA-cP407 runs, we started our simulation with a central condensed droplet of PLA (with a radius of 99 Å) containing only 5% water (in mass) by using the following mass ratio in Materials Studio: water 5, PLA 95. A shell of poloxamer (P188 or P407) with 5% water in mass was added around the PLA droplet (with a thickness of 14 Å). The diameter of both the PLA core droplet and the poloxamer shell were calculated in order to keep a 2 : 1 mass ratio of PLA in regards to poloxamer, while also ensuring that we kept a total mass of PLA and poloxamer identical to those used in the two first models NP-PLA-P188 and NP-PLA-P407. In both cases (NP-PLA-cP188 and NP-PLA-cP407), the rest of the simulation box was filled with water.

All the DPD calculations included an initial geometry optimization with 250 steps of conjugate gradient. DPD were then conducted using a DPD time scale of 2.6 ps. For each DPD run, we used a total of 230 000 steps with a time step of 0.05, leading to a DPD simulation time of 11 500 DPD units, which is equivalent to a total simulation time of about 30 ns (when using a DPD time scale of 2.6 ps). A final geometry optimization (250 steps of conjugate gradient) was finally performed prior to the analysis of the structures.

Stability in biological fluids. To evaluate NPs stability in biological conditions, artificial mucus was prepared as previously described.²² Briefly, 8% (w/v) of type II mucin from porcine stomach (Sigma Aldrich, USA) was dissolved in a solution of NaCl, KCl, and CaCl₂, 2H₂O. pH was then adjusted to 5.7. NPs (0.5 × 10¹¹ NPs per mL) were added to this artificial mucus, incubated at 37 °C, and characterized as described above. Diameter, homogeneity and charge were determined using three replicates and three independent experiments.

Similarly, artificial cerebrospinal fluid (CSF) was prepared using 0.6% (w/v) of bovine serum albumin (Euromedex, France) in NaCl, KCl, MgCl₂, CaCl₂, Na₂CO₃, Na₂HPO₄, D-glucose, and L-ascorbic acid, and pH was adjusted to 7.35.²³ NPs (0.5 × 10¹¹ NPs per mL) were added to this artificial CSF, incubated at 37 °C, and characterized as described above. Diameter, homogeneity and charge were determined using three replicates and three independent experiments.

In vitro evaluation

Cell cultures. RPMI 2650, which are human nasal epithelial cells, were purchased from ATCC (USA). They were cultured in MEM medium (Sigma Aldrich, USA), supplemented with 10% foetal bovine serum as well as 1% penicillin/streptavidine. They were conserved in a 37 °C incubator (ThermoScientific, USA) under 5% CO₂ and 95% humidity. Cells were passed when 90% confluence was reached.

PC12, which are rat neuronal cells, were also obtained from ATCC (USA) and were cultured in DMEM medium with 10% horse serum (ThermoScientific, USA), 5% foetal bovine serum and 1% penicillin/streptavidine. Cells were kept in a 37 °C incubator (ThermoScientific, USA) under 5% CO₂ and 95% humidity. Cells were passed when 90% confluence was reached.

PC12 cells were also differentiated into neuronal cells. To do so, they were seeded in plates coated with type I collagen (ThermoScientific, USA) at a low density in the cell medium described above. At 2 h after plating, cell medium was replaced by OptiMEM medium (ThermoScientific, USA), supplemented with GlutaMax 4 mM, 0.5% foetal bovine serum and 50 ng mL⁻¹ NGF-β (R&D Systems, USA). Cell medium was changed every other day for a week to obtain differentiated cells with axon-like projections.

Cell viability. Prior to viability assays, PC12 cells were seeded in 96-well plates at 85 000 cells per well and differentiated for 7 days. RPMI cells were seeded in 96-well plates at 85 000 cells per well for 24 h. Cells were counted and formulations were placed onto the cell culture at a concentration of 10⁵ NPs per cell. After 24 h of incubation at 37 °C, 10 μL of PrestoBlue™ Cell Viability Reagent (ThermoScientific, USA) was added in each well for 30 min at 37 °C, for a total volume of 110 μL. Fluorescence was measured using a Spark® multimode microplate reader (λ_ex: 560 nm, λ_em: 590 nm, Tecan, Switzerland). Cell viability values were determined using three replicates and three independent experiments.

Cellular uptake. PC12 cells were seeded in 96-well plates at 85 000 cells per well and differentiated for 7 days. RPMI cells were seeded in 96-well plates at 85 000 cells per well for 48 h. Fluorescent NPs (DY650) were added onto the previously counted cells at a final concentration of 10⁵ NPs per cell. Cells were incubated with NPs for 1 h or 3 h at 37 °C, 5% CO₂. Cells were then rinsed with PBS 1× and detached from the plate using 0.25% trypsin for 5 minutes at 37 °C. Cells were transferred into cytometry tubes (Corning, USA), centrifugated at 400 g, for 5 min, at 4 °C. Supernatant was replaced with 2% foetal bovine serum in PBS 1×. Cells were analysed with a flow cytometer (LSR Fortessa 5L, BD Biosciences, USA). Data were analysed with the OMIQ software (Dotmatics, USA) to isolate fluorescent NPs encapsulating single cells.

In vivo evaluation

Animals. In vivo studies were performed at the animal facility PBES (Plateau de Biologie Expérimentale de la Souris) at the Ecole Normale Supérieure in Lyon, France, and at the animal facility P-PAC (Plateforme du Petit Animal du CRCL) at the Cancer Research Centre of Lyon (CRCL), France. The experiments were performed in accordance with animal welfare regulations for their use for scientific purposes governed by European Directive 2010/63/EU. Protocols were validated by the local Animal Ethics Evaluation Committees (CECCAPP and C2EA-15) and authorized by the French Ministry of Education and Research. To reduce both pain and stress, experimentations were performed after a light anaesthesia using isoflurane. Animals were frequently checked for any degradation of their condition. Animals were divided in groups randomly, without changing cages to reduce stress.

Nasal cavity immunostaining. Female CB6F/1 mice (Charles River Laboratories, France) that were 10-month-old were used to perform nasal cavity immunostaining. These mice have a heterogenous genetic background compared to C57BL/6 or Balb/c mice²⁴ which makes them appropriate for studying the NtBD of NPs. 10-Month-old mice were used as they are considered middle-aged.²⁵

Before administration, DY650-loaded NPs were resuspended and diluted in water to homogenize both the fluorophore concentration and the NPs concentration (10¹⁰ NPs per mL). After gaseous anaesthesia using 3.5% isoflurane, animals were placed in supine position and received 10 μL of formulation in each nostril (n = 2 mice per group). After 1 h or 3 h, mice were anesthetised using 5% isoflurane, and decapitated. As previously described,²⁶ after dissection of the mice head, they were fixed in 4% paraformaldehyde for 3 days at room temperature. Then, decalcification was performed using Osteosoft® reagent (Sigma Aldrich, USA) for 7 days at 4 °C. Cryopreservation was performed using 30% sucrose solution for 3 days at 4 °C. Then, head were places in a mix of 30% sucrose and optimum cutting temperature compound (OCT, Sakura Finetek, The Netherlands) (50/50 v/v) for 1 h and then, only in OCT for 1 h, before freezing the sample using dry ice and placing the sample at −70 °C until processing.

Using a cryostat (Leica Biosystems, Germany), 15-μm thick coronal sections were then performed. As previously described,²⁷ sections were blocked using 1% bovine serum albumin (BSA), and permeabilised with 0.1% Triton 100× in PBS 1×. Sections were incubated with anti-Gap43 primary antibody (Novus Biologicals, USA) (1 : 500 in 0.2% BSA, 0.1% Triton 100× in PBS 1×) overnight at 4 °C. After rinsing the sections with a solution of 0.1% Triton 100× in PBS 1×, a secondary fluorescent anti-rabbit IgG DyLight488 (ThermoScientific, USA) was added (1/500 in the same buffer as the primary antibody) for 2 h at room temperature. Labelling with DAPI (ThermoScientific, USA) was finally performed (1 : 1000 in PBS 1×) for 5 min at room temperature. Images were taken using an inverted fluorescence microscope (Nikon Instruments, USA). Images were then analysed using Fiji software (ImageJ, USA) to homogenise brightness and contrast).

Biodistribution. Biodistribution studies were performed on 6-month-old female SKH1 mice (Charles River Laboratories, France). These mice, that were immunocompetent and hairless, were also mature.^25,28In vivo imaging required the absence of hair as they can attenuate the fluorescence signal. Using an already hairless mice prevented the hair removal process on mice.²⁹

Before administration, DiR-loaded NPs were resuspended in water and diluted to homogenize both the fluorophore concentration and the NPs concentration (10¹⁰ NPs per mL). After gaseous anaesthesia using 3.5% isoflurane, animals were placed in supine position and slowly received 10 μL of formulation in each nostril (n = 4 mice per group). Mice were kept in a supine position for a few seconds before being positioned in a prone position in an open fluorescence in vivo imaging system, the KIS 800 imaging system (Kaer Imaging System, Kaer Labs, France) and images were taken while mice were anesthetised using an exposure time of 500 ms. Filter used was the one corresponding to DiR (λ_ex: 785 nm and λ_em: 800 nm). Images were then taken at 1 h, 3 h, 6 h, and 24 h.

To obtain a more precise localisation of the fluorescence, 3 h after administration of fluorescent-NPs, brains and lungs were harvested and observed using the same KIS 800 imaging system (n = 3 mice per group). Images were taken using an exposure time of 1000 ms.

Images were then analysed using Fiji software (ImageJ, USA) and quantification of the fluorescence was performed. To do so, using a grey scale image of the fluorescence signal, brightness and contrast were homogenised. Threshold was then applied to all the images and integrated density of the signal was measured in the whole image. This number was compared to the integrated density at 0 h to obtain the proportion of the initial fluorescence at different time points.

Statistical analysis

Statistical analyses were performed using GraphPad Prism version 10.1.1 software (USA). When parametric tests are used, normality of the residual was tested using D’Agostino-Pearson omnibus, Anderson-Darling, Shapiro-Wilk and Kolmogorov-Smirnov tests. All of the data, if not otherwise specified, are presented as mean ± SD. Statistical analysis and differences are indicated in the figures.

Results and discussion

Nanoparticles characterisation

In the context of brain disease treatments, the use of NPs for the NtBD is very promising. NP-PLA are considered safe for the nasal mucosa, biodegradable and biocompatible, and can enhance the stability of their cargo and increase penetration, adsorption and bioavailability.³⁰

Physicochemical characteristics. NPs were prepared using the nanoprecipitation method using two different techniques, a one-step synthesis and a two-step synthesis, where the poloxamer was added as a coating onto the NP-PLA. To evaluate the characteristics of the NPs, compare the synthesis method and the type of poloxamer, as well as their relevance for NtBD, dynamic light scattering was measured to determine NPs’ diameter and polydispersity index (PDI). As shown in Fig. 1A, the diameter of synthesized NP-PLA-P188, NP-PLA-P407, NP-PLA-cP188 and NP-PLA-cP407 were around 110 nm and showed no statistical differences compared to NP-PLA. The addition of poloxamers did not impact the NP size. Comparison of the one-step synthesis versus the two-step synthesis showed no statistical differences in terms of diameter.

Fig. 1 NPs characteristics synthetized using the one-step or the two-step synthesis. Diameter and polydispersity index (PDI) of NPs were determined using dynamic light scattering measurements (A); charge was determined by measuring electrophoretic mobility of NPs (B) and concentration was determined using multi-angle dynamic light scattering (C) (n = 3, ordinary one-way ANOVA test followed by Dunnett's multiple comparisons test using PLA-NP as control, ****: p < 0.0001; **: p < 0.01; if no statistical difference is mentioned, the data are not statistically different, p > 0.05).

PDI was below 0.2 for all formulations except NP-PLA-cP188, suggesting homogeneous formulations. No statistical difference was found in terms of homogeneity when poloxamer was added in the synthesis, no matter the type of synthesis or the type of poloxamer. No statistical difference was observed when comparing the two types of synthesis for each poloxamer.

Finally, as shown in Fig. 1B, the zeta potential was evaluated using electrophoretic mobility. The addition of P188 or P407 increased the charge of the particles, with a higher increase when P407 was added. Although both poloxamers share the same triblock copolymer nature, their molecular weights and the ratio of hydrophilic/hydrophilic segments differ. These differences influence how the poloxamers arrange on the NPs surface and, consequently, how charges accumulate at the interface, leading to slight variations in the zeta potential. In every case, NPs remained negatively charged, below −20 mV. When comparing the two synthesis methods with the same materials, no statistical differences were shown, suggesting that the two methods result in a similar NP.

Finally, NPs concentration was evaluated (Fig. 1C) using multi-angle dynamic light scattering and showed a concentration that varied from 1.7 to 4.3 × 10¹² NPs per mL with no statistical difference between formulations.

Other concentrations of poloxamer were tested (from 0.5 mg mL⁻¹ to 20 mg mL⁻¹), and the results of the characterisation are shown in the SI (Fig. S1).

Overall, homogenous and negatively charged 110 nm were produced using either synthesis method and either poloxamer. Although NPs ranging from 90 nm³¹ to 360 nm³² have been used for NtBD and have been found in the brain after nasal administration, Cruz et al. demonstrated that 100 nm polymeric NPs had a better brain penetration than 200 nm and 800 nm NPs after intranasal (IN) administration.¹³ Similarly, both negative and positive NPs have been used for NtBD^33,34 and it has been suggested that negative NPs used the olfactory pathway and positively charged NPs used the trigeminal pathway.³⁵ Since transport via the trigeminal pathway was described to be longer (17 to 56 h) compared to the olfactory pathway (1.5 to 6 h),³⁶ our research focused on obtaining negative NPs. From the results of Fig. 1, all poloxamer-based NPs were selected as compatible with NtBD in terms of size and charge.

Molecular modelling. The absence of significant differences between formulations prompts us to further investigate the NP behaviour. The one-step synthesis was expected to lead to a homogenous repartition of the two polymers in the NP whereas the two-step synthesis could result in a core composed of PLA surrounded by a corona of poloxamer. Molecular modelling was performed to investigate these hypotheses.

Over the last few years, DPD simulations have been used to investigate the behaviour of NP-PLA with a variety of biologically relevant small molecules.^37–39 This non-atomistic simulation is particularly well-adapted to NP modelling. These simulations use beads, which represent a functional group of a molecule or a group of several molecules like water.

In order to keep the simulation times within reasonable limits, our systems were set up to match the nanoprecipitation process of NPs with a final diameter of about 20 nm, which roughly corresponds to a 5 times scaled-down model, as the experimentally obtained NPs have a measured diameter of about 100 nm. PLA, water, and poloxamer molecules were set up with different types of beads (Fig. 2).

Fig. 2 (A) Schematic representation of the 7 different kinds of beads used for the dissipative particle dynamics (DPD) calculations. Linear chains of lactic acid monomers: (LA)_n for the PLA chains, LA_start for the hydroxy extremity of the chain, and LA_end for the carboxylic extremity of the chain; water (W), where one bead represents 3 molecules of water; linear chains of poloxamer: PPO for the central polypropylene oxide units, PEO for the external polyethylene oxide units, and PEO_ext for both the hydroxy extremities of the chain. (B) Representations of the PLA and poloxamer molecules. The groups of atoms corresponding to the different beads are evidenced using colored backgrounds. For the PLA chains, n = 138 for a total length of 140 units including both extremities. For P188, a = 16 and b = 5. For P407, a = 20 and b = 12.

The systems were formed using two very different starting points. First, in order to simulate the formation of NP-PLA-P188 and NP-PLA-P407, the modelling started with completely disordered mixtures of water, PLA and poloxamer to mimic the experimental conditions (Fig. 3A and B). Then, in order to simulate the two-step synthesis, resulting in NP-PLA-cP188 and NP-PLA-cP407, we started our simulation with a central droplet of PLA containing only 5% of water surrounded by a thin shell of poloxamer also containing 5% of water (Fig. 3C and D).

Fig. 3 Comparison of DPD simulations for the NP formation. Cross sections of the starting point of the DPD simulations (A), (D), (G) and (J). Cross section of the end state of the simulations (B), (E), (H) and (K). Surface of the complete NP surface, the water molecules are not displayed for clarity (C), (F), (I) and (L).

Simulations and analysis of the DPD simulations were performed with the software mentioned above. The four simulation runs reached an equilibrium within the first few ns of the simulation and the total simulation time was 30 ns.

For NP-PLA-P188 and NP-PLA-P407 (Fig. 3A and B), during the first nanoseconds of the simulations, the PLA and poloxamer chains undergo a volume contraction and most of the water molecules are expelled outside of the forming NP. At the end of the simulation, few water molecules remains trapped in small clusters inside the newly formed smooth spherical particle. Those clusters are randomly distributed inside the particle and most of them interact with the negatively charged extremity of PLA chains (green bead, Fig. 3E and F). The inside of the NP is quite hydrophobic with lots of non-charged LA monomers, PEO and PPO monomers. The surface of the NP is covered in comparable proportions of PLA monomers and PEO monomers from the poloxamer chains. A close analysis of the surface (Fig. 3K and L) reveals that most of the hydroxy and negatively charged acidic extremities of the PLA chains (orange and green beads, respectively) are concentrated onto the surface of the NPs in direct interaction with the surrounding water. For the poloxamer chains, PEO monomers (blue beads), which are slightly more hydrophilic than their PPO (purple beads) counterparts, are by far more present at the surface, while the PPO monomers are more deeply located inside the NPs and very few of them are actually visible on the surface (Fig. 3I and J). This is especially relevant with the P188 which seems to hide its shorter chains of hydrophobic PPO units more deeply than the longer P407. In both cases, the more hydrophilic hydroxy extremities of the poloxamer chains (dark blue beads) also end up highly concentrated on the NP surface (Fig. 3I and J).

For the two second models with a condensed PLA core coated with a shell of poloxamer (NP-PLA-cP188 and NP-PLA-cP407, Fig. 3C and D), the volume contraction at the start of the simulations is extremely limited as these constructions contain only 5% of water (in mass). However, most of the water molecules are still expelled outside of the forming NP (Fig. 3G and H). A strong mixing of the PLA and poloxamer chains is evidenced from the start of the simulations, and the end results are extremely similar to what is observed in the two first simulations with the random starting points for NP-PLA-P188 and NP-PLA-P407 (Fig. 3K and L). This observation highlights the high compatibility between these two polymers whose chains end up deeply intricated at the end of the simulations. All the previous observations about the location of the extremities of the chains and their repartition are still valid and the final results at the end of the simulations are extremely comparable.

Coherent results were obtained, compared to what was found in vitro. The simulation showed no strong difference between the two types of synthesis, which correlates with the first in vitro results, confirming the potential similarity of the two formulations. Furthermore, the in silico model explained the negatively charged surface of the NPs and a more hydrophilic surface and hydrophobic core.

Stability of the formulations. The stability of all formulations was evaluated by characterizing formulations over time when they were stored at 4 °C in 5 mM sodium carbonate buffer (Fig. 4A). Determination of the diameter over time showed that diameters remained below 150 nm, suggesting stability of all formulations at least for 35 days. Similarly, homogeneity and charge, respectively under 0.2 and −20 mV, were stable for at least the period of time (see Fig. S2A and S3A). These results suggest a stability of conservation of all formulations for at least 35 days.

Fig. 4 NPs’ diameter stability of conservation at 4 °C and in physiological conditions. NPs’ diameter was determined over time at 4 °C in 5 mM sodium carbonate buffer (A). NPs, resuspended in water, were also diluted in artificial mucus (B) or in artificial cerebrospinal fluid (C) and the diameter was determined over time, CSF: cerebrospinal fluid (n = 3, Kruskal–Wallis test followed by Dunn's multiple comparisons test using the parameters before dilution as control, *: p < 0.05; **: p < 0.01; if no statistical difference is mentioned, the data are not statistically different, p > 0.05).

To further evaluate the relevance of our formulations for the NtBD, we evaluated their stability in two different biological media that can be encountered during NtBD, mucus and cerebrospinal fluid.

NPs were first incubated in artificial mucus at 37 °C and their physicochemical characteristics (diameter, PDI, and charge) were then evaluated. As shown in Fig. 4B, just after the incubation in artificial mucus started, NP-PLA diameter drastically increased (above 2000 nm) and remained above 500 nm for at least 24 h suggesting their aggregation and instability. Formulations with poloxamer remained the same in terms of diameter, proving the interest of using poloxamer in these conditions. PDI remained belon 0.5 except for NP-PLA. PDI was also increased for NPs coated with poloxamer upon dilution but decreased below 0.5 after 6 h (Fig. S2B) and zeta potential evaluation showed that the charge increased especially upon dilution but remained negative (Fig. S3B).

After incubation in artificial cerebrospinal fluid at 37 °C and evaluation of the physicochemical characteristics, similar conclusions were obtained (Fig. 4C). The diameter of NP-PLA first increased to 160 nm right after dilution but then was of 1000 nm 6 h after dilution and remained above 400 nm after 24 h of incubation. Diameters of formulations with poloxamers were also stable in artificial cerebrospinal fluid. PDI increased only for NP-PLA and was above 0.5 6 h after dilution (Fig. S2C). The NPs’ charge increased after dilution in cerebrospinal fluid but remained negative (Fig. S3C).

These results using mucus or cerebrospinal fluid showed that NPs composed of PLA and poloxamer are more stable in biological conditions. The increase in surface hydrophilicity confirmed by molecular modelling can explain the improved stability of our NPs when in contact with simulated biological fluids. We observed that poloxamers increased the colloidal stability after contact with either artificial nasal mucus or cerebrospinal fluid, showing the interest of using poloxamer in the synthesis to obtain suitable NPs for in vivo use. Plus, the surface hydrophobicity can impact the NPs’ bioavailability, as it can trigger the opsonisation and the elimination of the NPs from the body. NPs containing poloxamer are more stealth and could better avoid being eliminated by the immune system as it has already been demonstrated in a previous study.⁴⁰

In vitro evaluation

Cell viability. After physicochemical characterisation, evaluation of cell viability was performed using two cell models of the major cell types that compose the olfactory mucosa,⁴¹ human nasal epithelial cells, RPMI 2650, and rat neuronal cells, PC12. PC12 is a model of choice for in vitro experimentation due to its availability and its ability to differentiate into cells manifesting morphological and functional neuronal characteristics.⁴²

Viability was measured after 24 h of contact with the different formulations (Fig. 5A and B) and remained above 80% for every formulation and did not show a significant difference with the viability of cells alone. Plus, no strong difference between formulation suggested that the formulations were not toxic for both cell types.

Fig. 5 In vitro evaluation of the different NPs. Cell viability when in contact with NPs for 24 h. Viability was measured using the PrestoBlue assay on (A) RMPI 2650 epithelial cells and on (B) neuron-like PC12 cells (n = 3, one-way ANOVA and Dunnett's multiple comparisons test, the data are not statistically different, p > 0.05). Then, cellular uptake was evaluated after 1 h or 3 h of contact between fluorescent (DY650) NPs and either (C) RPMI 2650 cells or (D) neuron-like PC12 cells (n = 3, data presented as mean ± SEM, formulations were compared with each other using Kruskal–Wallis’ test followed by Dunn's multiple comparisons test, times were compared with Mann–Whitney test, ns: p > 0.05).

Overall, formulations had no impact on either neuronal-like PC12 cells or epithelial RPMI 2650 cells, suggesting that they will not impact cell viability of the olfactory mucosa after IN administration.

Cellular uptake. Evaluation of cellular uptake was performed using flow cytometry after 1 h or 3 h of contact between fluorescent NPs and either RPMI 2650 cells or differentiated PC12 cells. RPMI 2650 cellular uptake (Fig. 5C) was around 1.2% for all formulations after 1 h of contact and 2.4% after 3 h of contact, no differences were found between formulations, and between durations of contact. PC12 cellular uptake was then evaluated (Fig. 5D), and the values were much higher, of 22.7% after 1 h and 44.3% after 3 h of contact. Similarly, no differences were evidenced either between formulations or durations. No differences were also found between the two cell types.

These results evidenced the uptake of NPs by epithelial and neuronal cells, suggesting the use of several pathways for the NtBD, both through the epithelial cells, and though neuronal cells to reach the olfactory bulbs and the brain.

In vivo distribution

To visualise NPs in vivo in mice models, fluorescent-NPs (DY650 and DiR) were synthesised and exhibit similar characteristics as non-fluorescent-NPs (Table S4). Encapsulation was above 95% for every formulation and both fluorophores.

Nasal cavity slices. Immunostaining of nasal cavity slices was performed to locate the NPs in the structures of the mice head 3 h after IN administration of fluorescent NPs (DY650). Fig. 6A shows the nasal cavity and the olfactory bulbs. Olfactory neurons are visible in the mucosa (in green), along with axon bundles in the lamina propria. It was clear that a large proportion of the fluorescent NPs (in red) are located in the mucosa and the lamina propria 3 h after IN administration. Plus, when looking more specifically at the olfactory bulb, nanoparticles were visible 3 h after IN administration. Similar observations were performed 1 h after IN administration, showing fluorescent NPs in the epithelium and in the olfactory bulb (see Fig. S4). After IN administration, NPs were able to reach the olfactory bulb, no matter the type of synthesis or the type of poloxamer.

Fig. 6 (A) Nasal cavity immunostaining of nucleus (in blue), immature olfactory neurons (Gap43, in green) after intranasal administration of fluorescent-NPs containing DY650 (in red). Slices were obtained at the beginning of the olfactory bulb and show either the nasal cavity and the olfactory bulbs 3 h after administration. Images only showing DY650-loaded NPs in the olfactory bulb were also taken. Scale bar: 100 μm (n = 2). (B) Fluorescent tomography of mice after intranasal administration of fluorescent NPs. Dorsal views were taken at different time points, and quantification was performed of these images using ImageJ (C). Brain and lungs were harvested and observed using the same device 3 h after administration. Fluorescence was also quantified (D) (n = 4 (B) and (C), n = 3 (D), data are presented as mean ± SEM, Kruskal–Wallis test followed by Dunn's multiple comparisons test using the NP-PLA group as control, no statistical difference were shown, p > 0,05; the most relevant mouse was chosen for the images).

The lack of differences between formulations, especially formulations with poloxamers compared to NP-PLA, was not expected as it was observed that poloxamer was essential for colloidal stability in the mucus and in the cerebrospinal fluid. These results showed that the in vitro evaluation of the stability of NPs in biological fluids is not a predictive method to evaluate in vivo crossing of the nasal mucosa. Further models could be used in order to select formulations adapted to NtBD, such as air–liquid interface culture or permeability evaluation using Franz diffusion cell for example.

Biodistribution. To further understand the fluorescent (DiR) NPs’ fate in the body, biodistribution kinetics were performed using fluorescence tomography. Fluorescence imaging is a very advantageous technique as it is quite sensitive and specific. In contrast, background autofluorescence can occur.⁴³ Therefore, a fluorochrome excited at 750 nm was selected to perform the biodistribution evaluation and avoid any autofluorescence.⁴⁴

Fig. 6B illustrates the most relevant mouse in each group chosen by localisation of the fluorescence. Images at the time of administration were used to evaluate the quality of the administration. In every group, the fluorescence was visible in the nasal cavity at the time of administration, with fluorescence being present from the nostrils upon to below the eyes. Then over time, the fluorescence was less present in the nostrils and seemed to be located deeper in the nasal cavity. At 6 h, fluorescence seemed to be located just in between the eyes, in the olfactory bulbs for every formulation. In the end, the fluorescence decreases for all groups to be almost extinct 24 h after IN administration as shown in Fig. 6B and C. The images were used for quantification of the fluorescence as shown in Fig. 6C, also demonstrating that there is no statistical difference between formulation when compared to each other at any time point.

Finally, brain and lungs were observed 3 h after administration, and fluorescence was quantified (Fig. 6D). The results showed the lack of fluorescence in the lungs that was already visible in whole body images. Even if no strong differences were evidence between formulations, NPs containing poloxamer seemed to be slightly more abundant in the brain, 3 h after IN administration. The larger difference was found between NP-PLA and NP-PLA-P188 suggesting the latter should be the better formulation for NtBD (Fig. 6D). Plus, when comparing the quantity of fluorescence, the larger difference was also found between NP-PLA-P188 and NP-PLA-P407 also suggesting that NP-PLA-P188 seems to be the best candidate for the NtBD (Fig. 6D).

After evaluation of the NPs’ biodistribution after IN administration, no strong differences were detectable, all formulations were localised in the mice's head very early on, similarly to what was found using immunofluorescence staining. Dissection of the mice brain and lungs and evaluation of the fluorescence quantity in the organs showed a slightly stronger brain delivery for the NPs containing poloxamer, especially NP-PLA-P188, as well as a smaller number of NPs in the lungs for the NP-PLA-P188.

Overall, both olfactory bulbs immunostaining and in vivo imaging showed the presence of NPs in the olfactory bulbs suggesting their potential use for the NtBD of drugs. Quantification of the brain fluorescence resulted in the superiority of NP-PLA-P188. Those NPs could be further evaluated for different applications such as Alzheimer's disease treatment or even anti-cancer therapy.⁹ Plus, information on the NPs’ fate could be explored by evaluating metabolization and degradation of our formulations. And finally, the interest of inorganic NPs, such as iron oxide or gold NPs, have been already demonstrated and a combination between these inorganic NPs and our polymeric NPs could also us to visualise our NPs using highly relevant imaging systems for cerebral imaging such as MRI.

Conclusions

Polymeric NPs of PLA and poloxamer were optimized to obtain a suitable delivery system for the NtBD. After in vitro characterization, synthesized NPs containing poloxamer were found to be promising for the NtBD as they were stable in different biological fluids, and non-toxic for the nasal mucosa. In vivo IN administration confirmed that these NPs are localised in the olfactory bulb 3 h after administration. Biodistribution showed a stronger distribution of NP-PLA-P188 in the brain compared to other formulations. These results are encouraging for the use of these NP-PLA-P188 as a platform for the NtBD of brain disease treatments.

Author contributions

Marie Bolon: data curation, formal analysis, investigation, supervision, validation, visualisation, writing – original draft, writing – review & editing. Morgane Marie: data curation, validation, writing – original draft. Simon Megy: investigation, methodology, validation, writing – original draft. Raphael Petrakis: methodology, validation. Raphaël Terreux: resources, supervision. Maxime Fieux: project administration, supervision, writing – review & editing. Claire Monge: conceptualization, investigation, project administration, resources, supervision, visualisation, writing – review & editing. Sophie Richard: conceptualization, funding acquisition, investigation, project administration, resources, supervision, validation, visualisation, writing – review & editing.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the SI. Supplementary characterisation data are presented. Further images of immunofluorescence staining of nasal cavity 1 h after intranasal administration were also provided. See DOI: https://doi.org/10.1039/d5tb00947b

Acknowledgements

We would like to thank C. Garapon and R. Rovera for their help with the in vivo experiments and Dr N. Meunier for his advice on the immunofluorescence staining of the nasal cavity. Finally, we would like to acknowledge the contribution of SFR Biosciences (UAR3444/CNRS, US8/Inserm, ENS de Lyon, UCBL) imaging facility LyMIC/PLATIM and animal facility PBES. We also would like to acknowledge the P-PAC animal facility. This work was financially supported by the Claude Bernard University, Lyon, France, and the National Scientific Research Centre of France.

Notes and references

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