Pharmacotechnical Development of a Nasal Drug Delivery Composite Nanosystem Intended for Alzheimer’s Disease Treatment

Direct nose-to-brain delivery has been raised as a non-invasive powerful strategy to deliver drugs to the brain bypassing the blood-brain barrier (BBB). This study aimed at preparing and characterizing an innovative composite formulation, associating the liposome and hydrogel approaches, suitable for intranasal administration. Thermosensitive gel formulations were obtained based on a mixture of two hydrophilic polymers (Poloxamer 407, P407 and Poloxamer 188, P188) for a controlled delivery through nasal route via liposomes of an active pharmaceutical ingredient (API) of potential interest for Alzheimer’s disease. The osmolarity and the gelation temperature (T° sol-gel) of formulations, defined in a ternary diagram, were investigated by rheometry and visual determination. Regarding the issue of assays, a mixture composed of P407/P188 (15/1%, w/w) was selected for intranasal administration in terms of T° sol-gel and for the compatibility with the olfactory mucosal (280 ± 20 mOsmol, pH 6). Liposomes of API were prepared by the thin film hydration method. Mucoadhesion studies were performed by using mucin disc, and they showed the good natural mucoadhesive characteristics of in situ gel formulations, which increased when liposomes were added. The study demonstrated successful pharmacotechnical development of a promising API-loaded liposomes in a thermosensitive hydrogel intended for nasal Alzheimer’s disease treatment.


Introduction
Alzheimer's disease (AD) is the most frequent cause of dementia among the elderly [1]. This disease, characterized by an insidious decline in cognitive and non-cognitive functions, is devastating for patients, their family and society. Many types of neurotransmitters are affected in this chronic and progressive neurodegenerative disorder, and the relative importance of each in relation to clinical findings has not been fully elucidated. Today, no curative treatment exists. While current clinical therapy

In Vitro Measurement of AChE and BuChE Activity
The API inhibitory capacity on acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) biological activity was evaluated through the use of the spectrometric method of Ellman [29], and realized according to the protocol already reported by our team [30]. Donepezil or tacrine were used as a reference standard. Assays were performed with a blank containing all components except acetyl-or butyrylthiocholine, to account for non-enzymatic reactions. The percent inhibition due to the presence of the test compound was calculated by the following expression: ((v 0 -v i )/v 0 ) × 100, where v i is the rate calculated in the presence of inhibitor and v 0 is the enzyme activity. IC 50 values were determined graphically by plotting the % inhibition versus the logarithm of six inhibitor concentrations in the assay solution, using the GraphPad Prism software (version 6.01, GraphPad Software, La Jolla, CA, USA). All experiments were performed in n = 3.

Parallel Artificial Membrane Permeability Assay (PAMPA)
The PAMPA-GIT experiments were conducted using the Pampa Explorer Kit (Pion Inc., Billerica, MA, USA) according to manufacturer's protocol, and realized according to the protocol already reported by our team [31]. Quality control standards with known Pe were used as references: the low/moderately permeable corticosterone (Pe = 202.0 nm/s at pH = 7.4) and theophylline (Pe = 5.0 nm/s at pH = 7.4).

Formulation of Liposomes
Liposomes were formulated according to the adapted method of the thin lipid film hydration [32]. A lipid blend of soybean phosphatidylcholine (SPC) and cholesterol (CHOL) was used at a molar ratio 8:1. Lipid solutions in chloroform/methanol (4:1) were evaporated under nitrogen flow, and left under vacuum for 3-4 h to form a lipid film. This thin lipid film was then hydrated in water or API solution, and vortexed. The yielded multilamellar vesicles (MLVs) were then extruded 13 times with a mini-extruder (Avanti Polar Lipids, Inc., Alabaster, AL, USA) through polycarbonate membranes with a pore diameter of 100 nm (Avanti Polar Lipids, Inc., Alabaster, AL, USA) to form large unilamellar vesicle (LUVs).

Characterization of Liposomes by Dynamic Light Scattering
The average hydrodynamic diameter associated with the polydispersity index (PDI) of the formulated LUVs and the formulated LUVs dispersed in polymer solution were measured by dynamic light scattering (DLS) using a NanoZS ® apparatus (Malvern Instruments SA, Worcestershire, UK). The zeta potential was calculated from the electrophoretic mobility using the Smoluchowski equation, also using a NanoZS ® apparatus. The measurements were performed in triplicate at 25 • C, after a 1:100 dilution in water.

Entrapment Efficiency
Entrapment of the active pharmaceutical ingredient (API) into the liposome was determined. Liposomes were separated by Sepharose ® CL-4B column. The API content in the liposome fraction was analyzed by HPLC and the encapsulation efficiency (% EE) was calculated by considering the initial amount of drug added in the formulation using Equation (1): The amount present in liposomes fraction Total amount of API added × 100 Drug loading (DL) was calculated using Equation (2): % DL = Weight of API entrapped within liposomes Total weight of oil phase of the liposomes × 100 (2) Total weight of oil phase of the liposome corresponds to the total amount of excipients (excluding the water phase) and API used for the preparation of the liposomes.

UHPLC
API concentrations were measured by high-performance liquid chromatography (UHPLC). The apparatus was composed by an Agilent binary pump 1290, an autosampler 1290 and a diode array UV detector 1260 (Agilent technologies, Santa Clara, CA, USA). The column used was a reversed phase column C18 (Restek ® Ultra, 5 µm, 2.1 × 50 mm, Lisses, France). The injection volume and the run-to-run time were 4 µL and 1.53 min, respectively. The flow rate was 0.8 mL/min and the detection wavelength was 214 nm. The mobile phase was initially composed of a mixture of 3% water containing 0.1% (v/v) formic acid (A) and 97% acetonitrile containing 0.1% (v/v) formic acid (B). Then, the composition reached 90% of B after 0.8 min, then 60% of B after 1.5 min by applying a linear gradient, maintained for 1 min. 5% of B was then reached at 2.51 min, maintained for 1.5 min and returned to the initial conditions. Linearity was observed in the range from 2.5 to 100 µM with a determination coefficient greater than 0.999. The column was maintained at 25 • C. It was checked that in presence of liposome and gel, no interaction with API is present.

Gel Formulation
The gel was made on a weight basis using the "cold method" [33]. Poloxamer P407 was mixed with Poloxamer P188, the API, chitosan, gellan gum, HPMC or liposomes and demineralized water (pH 6-7) under magnetic stirring at 500 rpm during at least 30 min at 4 • C, until a homogenous solution was obtained. After dissolution, solutions were stored at 4 • C for 2 h before characterization. Concentrations of Poloxamer P407 (14-25%, w/v) and Poloxamer P188 (0-10%, w/v) were optimized to obtain an instantly gelling system at physiological conditions. API concentration of 1 mg/mL was used.

Physicochemical Properties
The pH of formulation was measured using a pH-meter (Eutech instrument, Landsmeer, The Netherlands). To determine osmolarity, a micro-osmometer autocal type 13 based on the freezing-point method (Roebling, Berlin, Germany) was used. 100 µL of each formulation were introduced in a microtube and measurements were performed. Gelation temperature (T gel ) of formulations was investigated by visual determination. Briefly, formulation was placed in transparent vials under magnetic stirring s in a water bath heated from temperature of 15 • C to 50 • C (at the rate of 2 • C/min). The gelation temperature was recorded when the magnetic bar stopped moving because an increase in viscosity. The visual determinsation of gelation temperature was confirmed by rheometry method.

Rheological Evaluation
All the rheological characterizations were carried out on a rheometer AR1000 (TA Instruments, Guyancourt, France) equipped with a cone-plate geometry (4 cm, 1.59 • ).
Gelation temperature determinations were performed in oscillatory mode. The storage and loss moduli were measured as a function of temperature (from 15 to 50 • C at 5 • C/min) with an oscillatory stress value maintained at 25 Pa in order to ensure being in the linear viscoelastic region in both the liquid and solid state. The frequency was set at 1.0 Hz during the measurements.
The viscosity of the gel forming solutions was measured as a function of shear rate (from 0.1-100 s −1 then from 100-0.1 s −1 ) in steady state at 34 • C to evaluate the interactions between the formulation components.
In order to evaluate the gel strength, storage modulus evolution of hydrogels was recorded during stress sweeps performed above T gel at 50 • C from 0 to 600 Pa in oscillatory mode.

In Vitro Evaluation of Mucoadhesion
The mucoadhesive strength of empty gels (blank) and those containing liposomes was determined using a Texture Analyzer TA-XT Plus (Stable Micro Systems, Cardiff, UK) and a mucin disc test. Porcine Pharmaceutics 2020, 12, 251 6 of 17 mucin discs (250 mg, 13 mm diameter), prepared by direct compression (10 t for 30 s of pig gastric mucin), were horizontally attached onto the lower face of an inert horizontal polycarbonate probe. A force of 5 g was applied for 120 s to ensure contact between formulations and the mucin disc. Then, the probe was raised at a speed of 0.5 mm/s. The mucoadhesive strength was determined from the force of detachment. Measurements were run at 37 • C. A cylindrical probe P/35 (13 mm diameter, aluminum) was used, and an amount of 5 mL of product was delivered on the base.

In Vitro Release Profile
Kinetic release studies were performed in nasal simulated fluid at 34 • C using Franz cells. 200 µL of formulations was placed into a cellulose ester dialysis tube with 100 kDa pore size (Spectrum lab, Gardena, CA, USA), and incubated in simulated nasal fluid (SNF) pH 6.5. at 34 • C under magnetic stirring. SNF was an aqueous solution containing 8,77 mg/mL of NaCl, 2.98 mg/mL KCl and 0.59 mg/mL CaCl 2 . The receptor solution was withdrawn (samples of 100 µL), was replaced at various time points by fresh SNF and the amount of API released from the formulations was determined by HPLC after a 10-fold dilution in methanol. All measurements were performed in triplicate.

Statistical Analysis
Results were expressed as mean values ± standard error of the mean (SEM) or as mean values ± standard deviation. A Student's t-test was used for statistical comparison/analysis, and P <0.05 was considered statistically significant.

Characterization of the Active Pharmaceutical Ingredient (API)
API inhibitor capacity of human AChE and equine BuChE was assessed using the Ellman assay [29]. In this test, Donepezil and Tacrine were used as reference AChE and BuChE inhibitors, respectively. The results are depicted in Table 1. API appeared able to inhibit BuChE with IC 50 value of 573 ± 40 nM, whereas its displays a negligible AChE inhibitor activity with IC 50 value of 14,520 ± 40 nM. Thus, API shows a specific submicromolar inhibition of BuChE.
Both cholinesterase enzymes AChE and BuChE are involved in the hydrolysis of acetylcholine (ACh). In the healthy brain, AChE predominates on BuChE, that is considered to play a minor role in regulating brain levels of ACh. However, during progression of AD in certain brain regions including cortical areas, AChE activity declines while BuChE activity remains unaffected or progressively increases [34]. Finally, in advanced stages of AD, BuChE is considered to play a prominent role in regulating brain ACh levels [35]. Thus, BuChE appears as a valuable target to improve the cholinergic deficit, due to its increasing role predicted in regulating brain ACh levels as the disease progresses. Furukawa-Hibi et al. demonstrated that BuChE inhibition, as well as AChE inhibition, is a viable therapeutic strategy for cognitive dysfunction in AD. Indeed, a selective BuChE inhibitor provides beneficial effects on the cognitive dysfunction induced by amyloid-β peptide in mice [36]. From our experimental results, our API shows a specific submicromolar inhibition of BuChE without affecting AChE (i.e., 25-fold BuChE selectivity). This activity is very promising, especially if compared with reported equine BuChE inhibitory activity of other compounds, e.g., a new series of pyrazinamides, whose showed IC 50 micromolar values [37].
The cholinesterase inhibitory activity of new compounds is classically evaluated using equine serum BuChE due to the economic viability of this enzyme. Purified human BuChE has limited availability from commercial sources, making equine BuChE a reasonable alternative [38]. In the study of Wu et al., synthetic compounds showed similar effectiveness in inhibiting BuChE from equine and human plasma, but with a higher potency over the human enzyme [39]. Similarly, Dighe et al. have shown that their lead was 2-fold more potent in inhibiting human BuChE than equine BuChE [40]. In the same way, we could hope for a better activity in human BuChE.
Having a selective inhibitor of BuChE is innovative because there is currently no equivalent drug available on the market. The API could have a positive role in the attention, executive function, emotional memory and behavior of patients at advanced stages AD by inhibiting the BuChE present in CNS neurons.
To determine the capacity to reach the CNS, the permeability must be evaluated. To analyze the passive permeability characteristics of API, we measured BBB permeability by a PAMPA model, well adapted to High Throughput Screening (HTS) [41]. Theophylline (Pe = 5.0 ± 0.0 nm/s), and corticosterone (Pe = 202.0 ± 3.0 nm/s) were used as low and high permeability standards, respectively. The API poorly diffused through the lipid bilayer, since Pe values (≤20.4 ± 3.0 nm/s), significantly lower than the positive control, indicated a potential difficulty in crossing the BBB. Thus, because of this limited permeability and its good water solubility (>10 mg/mL), the API can be considered as a class III BCS drug [16]. This poor BBB permeability, probably related to the hydrophilic character of the API, would lead to a poor brain bioavailability after intravenous administration.
However, whereas intravenous administration of API is unlikely to reach CNS, BuChE inhibition must occur in the CNS to be a viable therapeutic strategy in advanced AD [3]. Moreover, the API peripheral biodistribution could lead to important side effects, if it is not controlled and limited. Indeed, the heart is naturally rich in cholinesterase (ChE). Its inhibition may affect the cardiac function, especially in elderly patients, many of them having concomitant cardiovascular diseases. Peripheral ChE inhibition potentiates the cardio-inhibitory effect with retarding ACh degradation and leads to negative chronotropic and dromotropic effects through muscarinic receptors [42]. As a consequence, a CNS selectivity is highly desired to avoid any dramatic side effect and improve API efficiency.
Nose-to brain delivery is a powerful strategy to increase this expected CNS selectivity. Drug nasal delivery enables to bypass BBB and permits the API brain delivery. Nevertheless, this route of administration may be limited by enzyme degradation and quick mucociliary clearance [43] if formulation is not optimized. To increase drug transport efficiency into CNS via intranasal delivery, we have chosen to develop an innovative composite formulation based on liposomes included in gels.

Preparation of Liposomes
Unloaded (i.e., blank) and API-loaded liposomes (API-Lip) were formulated by the thin lipid film hydration method. Unloaded and API-lip were characterized in terms of granulometric properties, zeta-potential, and encapsulation efficiency ( Table 2). Various SPC and cholesterol concentrations, and molar ratios were assayed, without any significant influence on the liposomes physico-chemical properties (results not shown).
For the retained liposomal formulation, based on a 120 mM SPC and cholesterol mixture, used in a molar ratio of 8:1, the mean hydrodynamic diameter was 119.0 ± 0.7 nm for blank Lip, and 114.9 ± 2.4 nm for API-Lip. For all the formulations, the polydispersity index (PDI) values were lower than 0.1, indicating monomodal size distributions. Encapsulation efficiency of API in Lip was 11%. This corresponds to an API concentration of 1.2 ± 0.1 mg/mL, and a drug loading of 1.4 ± 0.1%. SPC/cholesterol liposome formulations are widely found in the literature, and the particle size properties obtained are comparable to ours results [44,45].
The nose-to-brain passage of nanoemulsions has been studied through live imaging or ex vivo histological examination in rats after nasal administration by Ahmad et al. [46]. This study demonstrated that nanoparticle size plays a determinant role, by directly influencing their in vivo fate. NEs with a size of about 100 nm have longer retention time in nostrils and slower mucociliary clearance than larger ones (i.e., >200 nm). NEs of 100 nm can be transported through the trigeminal and the olfactory nerves to the olfactory bulb. Subsequent uptake and translocation of nanoparticles along axons of the olfactory nerve have been shown in non-human primates and rodents [47]. Morrison and Costanzo established that the diameter of a human olfactory neuron axon ranges from 100 to 700 nm [48]. Therefore, theoretically, axonal transport of up to~500 nm diameter particles is possible in humans. Al Asmari et al. showed that liposomal formulation of donepezil with a size of 120 nm, significantly increased brain concentration after intranasal administration [12]. Similarly, pharmacokinetic and brain targeting studies in rats revealed a significantly high concentration of drug in brain following intranasal administration of solid lipid nanoparticles of donepezil in comparison with a donepezil solution [49]. This demonstrates brain targeting efficiency of solid lipid nanoparticles of 120 nm. As a consequence, liposomes of 120 nm appear really suitable for brain targeting through nasal route and could reach the olfactory bulb through axons.
Furthermore, in ours assays, zeta potential values were only very slightly negative, and broadly neutral for both blank Lip and API-Lip. Surface charge of the nanoparticles could influence the mechanisms involved in their transport from the nasal cavity to the CNS. Indeed, the olfactory pathway is mainly responsible for the translocation of negatively charged nanoparticles, whereas the positively charged nanoparticles reach the brain more slowly, by involving the trigeminal pathway [50]. Intranasally applied nanoparticles have to penetrate the mucus layer covering nasal mucosa to reach the olfactory mucosa, and thus the CNS [51]. Interactions between particles and mucus could lead to trapping and wrapping of nanoparticles in mucus, making them less suitable as efficient vehicles for nose-to-brain delivery [27]. Uncharged nanoparticles, which do not interact with mucus are able to diffuse through mucus more efficiently [52].
Thus, nanoparticles appear well designed to permeate mucus by being small enough, neutral in net surface charge and coated densely with charged groups that prevent hydrophobic bonding to mucins [53].
The liposomes, that we have formulated, present surface charges close to neutrality and very slightly negative, as well in the absence (potential ζ = -5.5 ± 0.3 mV) as in the presence of the encapsulated API (potential ζ = -11.2 ± 1.4 mV), and although in gels (potential ζ = -6.6 ± 0.5 mV) (see after). In view of these particle size and surface charge properties of the formulated liposomes, the prerequisites for allowing the addressing and diffusion of the API towards the olfactory mucosa appear to be satisfied.

Preparation of in Situ-Forming Gel
We attempted to develop an aqueous formulation of API that allows an increased residence time in the nasal cavity. Hydrogels are a particularly attractive type for intranasal API administration.
Widely used in various medical fields [54][55][56], the hydrogels are devoid of potentially toxic organic solvents since they are composed of a large amount of water and a network of cross-linked polymers.
Poloxamers are biodegradable by erosion [57] and biocompatible polymers [58]. Based on these safety data and the information that the manufacturing process can be controlled to limit unwanted impurities, the Cosmetic Ingredient Review Expert Panel concluded that poloxamers are safe as used [59].
Poloxamers are composed of a hydrophobic POP block, sandwiched between two hydrophilic POE blocks [60]. Among them, P407 is a non-ionic surfactant, endowed with gelation properties above a defined sol-gel transition temperature (T gel ). Below this temperature, the sample remains fluid, and above, the solution becomes semi-solid. This phenomenon of thermogelling is perfectly reversible. When the temperature increases, copolymer macromolecules aggregate into micelles. This micellization is due to the dehydration of hydrophobic POP blocks, which represents the very first step towards gelation. When the polymer concentration is high enough, the micellization is followed by gelation.
Surprisingly and unlike some empirical data from the literature [61], we experimentally observed that hydrogel formulations based on the sole P407 could not lead to a T gel compatible with our specifications (data not shown). According to information provided by the supplier, the production mode of P407 has recently evolved, thus modifying the average molecular weight of the poloxamers produced, and resulting in a direct modification of the gelation temperatures. Hence, to further modulate sol-gel temperature of P407 hydrogel, P188 was added in defined proportions.
Various ternary mixtures of P407, P188, and water were tested. The gelation temperatures and osmolarity of theses formulations were plotted on ternary diagrams ( Figure 1). For all the formulations, pH is acceptable (optimum pH for nasal rote is 6 ± 3). Increase of P407 relative content leads to a gelation temperature decrease, and to higher osmolarity values. P188 relative content increases both the gelation temperature and the osmolarity, as previously reported in the literature [62]. Poloxamers are composed of a hydrophobic POP block, sandwiched between two hydrophilic POE blocks [60]. Among them, P407 is a non-ionic surfactant, endowed with gelation properties above a defined sol-gel transition temperature (Tgel). Below this temperature, the sample remains fluid, and above, the solution becomes semi-solid. This phenomenon of thermogelling is perfectly reversible. When the temperature increases, copolymer macromolecules aggregate into micelles. This micellization is due to the dehydration of hydrophobic POP blocks, which represents the very first step towards gelation. When the polymer concentration is high enough, the micellization is followed by gelation.
Surprisingly and unlike some empirical data from the literature [61], we experimentally observed that hydrogel formulations based on the sole P407 could not lead to a Tgel compatible with our specifications (data not shown). According to information provided by the supplier, the production mode of P407 has recently evolved, thus modifying the average molecular weight of the poloxamers produced, and resulting in a direct modification of the gelation temperatures. Hence, to further modulate sol-gel temperature of P407 hydrogel, P188 was added in defined proportions.
Various ternary mixtures of P407, P188, and water were tested. The gelation temperatures and osmolarity of theses formulations were plotted on ternary diagrams ( Figure 1). For all the formulations, pH is acceptable (optimum pH for nasal rote is 6 ± 3). Increase of P407 relative content leads to a gelation temperature decrease, and to higher osmolarity values. P188 relative content increases both the gelation temperature and the osmolarity, as previously reported in the literature [62]. By using 15 wt% of P407 and 1 wt% of P188, an optimal formulation is obtained, characterized by a gelation temperature of 34.5 ± 0.3 °C, an osmolarity of 277 ± 4 mOsm, and a pH of 6.5. Gelation temperature of this P407/P188 mixture was confirmed by rheological determination, illustrated by the sudden increase of the storage modulus as the gel forms ( Figure 2). When the storage modulus value overcomes the loss modulus value, the elastic behavior prevails on the viscous behavior. The physicochemical properties of this formulation are therefore suitable for intranasal administration according to the European pharmacopeia specifications [28], since the use of this isotonic formulation, with a stabilized pH value, would not adversely affect the functions of the nasal mucosa and its cilia. Poloxamers are composed of a hydrophobic POP block, sandwiched between two hydrophilic POE blocks [60]. Among them, P407 is a non-ionic surfactant, endowed with gelation properties above a defined sol-gel transition temperature (Tgel). Below this temperature, the sample remains fluid, and above, the solution becomes semi-solid. This phenomenon of thermogelling is perfectly reversible. When the temperature increases, copolymer macromolecules aggregate into micelles. This micellization is due to the dehydration of hydrophobic POP blocks, which represents the very first step towards gelation. When the polymer concentration is high enough, the micellization is followed by gelation.
Surprisingly and unlike some empirical data from the literature [61], we experimentally observed that hydrogel formulations based on the sole P407 could not lead to a Tgel compatible with our specifications (data not shown). According to information provided by the supplier, the production mode of P407 has recently evolved, thus modifying the average molecular weight of the poloxamers produced, and resulting in a direct modification of the gelation temperatures. Hence, to further modulate sol-gel temperature of P407 hydrogel, P188 was added in defined proportions.
Various ternary mixtures of P407, P188, and water were tested. The gelation temperatures and osmolarity of theses formulations were plotted on ternary diagrams ( Figure 1). For all the formulations, pH is acceptable (optimum pH for nasal rote is 6 ± 3). Increase of P407 relative content leads to a gelation temperature decrease, and to higher osmolarity values. P188 relative content increases both the gelation temperature and the osmolarity, as previously reported in the literature [62]. By using 15 wt% of P407 and 1 wt% of P188, an optimal formulation is obtained, characterized by a gelation temperature of 34.5 ± 0.3 °C, an osmolarity of 277 ± 4 mOsm, and a pH of 6.5. Gelation temperature of this P407/P188 mixture was confirmed by rheological determination, illustrated by the sudden increase of the storage modulus as the gel forms ( Figure 2). When the storage modulus value overcomes the loss modulus value, the elastic behavior prevails on the viscous behavior. The physicochemical properties of this formulation are therefore suitable for intranasal administration according to the European pharmacopeia specifications [28], since the use of this isotonic formulation, with a stabilized pH value, would not adversely affect the functions of the nasal mucosa and its cilia. Poloxamers are composed of a hydrophobic POP block, sandwiched between two hydrophilic POE blocks [60]. Among them, P407 is a non-ionic surfactant, endowed with gelation properties above a defined sol-gel transition temperature (Tgel). Below this temperature, the sample remains fluid, and above, the solution becomes semi-solid. This phenomenon of thermogelling is perfectly reversible. When the temperature increases, copolymer macromolecules aggregate into micelles. This micellization is due to the dehydration of hydrophobic POP blocks, which represents the very first step towards gelation. When the polymer concentration is high enough, the micellization is followed by gelation.
Surprisingly and unlike some empirical data from the literature [61], we experimentally observed that hydrogel formulations based on the sole P407 could not lead to a Tgel compatible with our specifications (data not shown). According to information provided by the supplier, the production mode of P407 has recently evolved, thus modifying the average molecular weight of the poloxamers produced, and resulting in a direct modification of the gelation temperatures. Hence, to further modulate sol-gel temperature of P407 hydrogel, P188 was added in defined proportions.
Various ternary mixtures of P407, P188, and water were tested. The gelation temperatures and osmolarity of theses formulations were plotted on ternary diagrams (Figure 1). For all the formulations, pH is acceptable (optimum pH for nasal rote is 6 ± 3). Increase of P407 relative content leads to a gelation temperature decrease, and to higher osmolarity values. P188 relative content increases both the gelation temperature and the osmolarity, as previously reported in the literature [62]. By using 15 wt% of P407 and 1 wt% of P188, an optimal formulation is obtained, characterized by a gelation temperature of 34.5 ± 0.3 °C, an osmolarity of 277 ± 4 mOsm, and a pH of 6.5. Gelation temperature of this P407/P188 mixture was confirmed by rheological determination, illustrated by the sudden increase of the storage modulus as the gel forms ( Figure 2). When the storage modulus value overcomes the loss modulus value, the elastic behavior prevails on the viscous behavior. The physicochemical properties of this formulation are therefore suitable for intranasal administration according to the European pharmacopeia specifications [28], since the use of this isotonic formulation, with a stabilized pH value, would not adversely affect the functions of the nasal mucosa and its cilia. Poloxamers are composed of a hydrophobic POP block, sandwiched between two hydrophilic POE blocks [60]. Among them, P407 is a non-ionic surfactant, endowed with gelation properties above a defined sol-gel transition temperature (Tgel). Below this temperature, the sample remains fluid, and above, the solution becomes semi-solid. This phenomenon of thermogelling is perfectly reversible. When the temperature increases, copolymer macromolecules aggregate into micelles. This micellization is due to the dehydration of hydrophobic POP blocks, which represents the very first step towards gelation. When the polymer concentration is high enough, the micellization is followed by gelation.
Surprisingly and unlike some empirical data from the literature [61], we experimentally observed that hydrogel formulations based on the sole P407 could not lead to a Tgel compatible with our specifications (data not shown). According to information provided by the supplier, the production mode of P407 has recently evolved, thus modifying the average molecular weight of the poloxamers produced, and resulting in a direct modification of the gelation temperatures. Hence, to further modulate sol-gel temperature of P407 hydrogel, P188 was added in defined proportions.
Various ternary mixtures of P407, P188, and water were tested. The gelation temperatures and osmolarity of theses formulations were plotted on ternary diagrams (Figure 1). For all the formulations, pH is acceptable (optimum pH for nasal rote is 6 ± 3). Increase of P407 relative content leads to a gelation temperature decrease, and to higher osmolarity values. P188 relative content increases both the gelation temperature and the osmolarity, as previously reported in the literature [62]. By using 15 wt% of P407 and 1 wt% of P188, an optimal formulation is obtained, characterized by a gelation temperature of 34.5 ± 0.3 °C, an osmolarity of 277 ± 4 mOsm, and a pH of 6.5. Gelation temperature of this P407/P188 mixture was confirmed by rheological determination, illustrated by the sudden increase of the storage modulus as the gel forms ( Figure 2). When the storage modulus value overcomes the loss modulus value, the elastic behavior prevails on the viscous behavior. The physicochemical properties of this formulation are therefore suitable for intranasal administration according to the European pharmacopeia specifications [28], since the use of this isotonic formulation, with a stabilized pH value, would not adversely affect the functions of the nasal mucosa and its cilia.

high T • sol-gel transition; (b):
Pharmaceutics 2020, 12,251 Poloxamers are composed of a hydrophobic POP blo POE blocks [60]. Among them, P407 is a non-ionic surfa above a defined sol-gel transition temperature (Tgel). Belo fluid, and above, the solution becomes semi-solid. This p reversible. When the temperature increases, copolymer mac micellization is due to the dehydration of hydrophobic PO step towards gelation. When the polymer concentration is h by gelation.
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By using 15 wt% of P407 and 1 wt% of P188, an optimal formulation is obtained, characterized by a gelation temperature of 34.5 ± 0.3 • C, an osmolarity of 277 ± 4 mOsm, and a pH of 6.5. Gelation temperature of this P407/P188 mixture was confirmed by rheological determination, illustrated by the sudden increase of the storage modulus as the gel forms ( Figure 2). When the storage modulus value overcomes the loss modulus value, the elastic behavior prevails on the viscous behavior. The physicochemical properties of this formulation are therefore suitable for intranasal administration according to the European pharmacopeia specifications [28], since the use of this isotonic formulation, with a stabilized pH value, would not adversely affect the functions of the nasal mucosa and its cilia.

Preparation of API Delivery Composite Formulation
In situ forming gel containing liposomes was prepared by the addition of poloxamers to an aqueous solution containing formulated liposomes. In these conditions, liposomes present higher mean hydrodynamic diameter values (140.8 ± 0.5 nm in the poloxamer solution at 25 °C vs. 119.0 ± 0.7 nm just after liposome formulation). The PDI remained lower than 0.1, and the zeta potential values remained broadly neutral ( Table 2). This increase in the average liposome diameter included in the P407/P188 gel formulation may suggest an interaction between the poloxamers and the phospholipids forming the liposomes.
The addition of liposomes in gel formulation did not significantly modify the gelation temperature (Figure 2), nor the osmolarity ( Table 3). The pH value decreased slightly but remained in the optimum value range. Free API also did not interfere with gel properties (Table 3). Thus, neither API nor liposome addition to P407/P188 (15/1 wt%) gel significantly changed the gel formulation properties that respect the nasal physiologic conditions. Note: values are expressed as mean ± standard deviation (n = 3).
The rheological behavior of gel formulations containing liposomes or not were determined during shear rate ramps. In all cases, the shear viscosity progressively decreased (shear thinning) when the shear rate increased. All formulations behaved as a shear-thinning (non-Newtonian) fluids. The presence of liposomes increased the viscosity regardless of the shear rate, and higher content of liposomes led to higher viscosity (Figure 3). Such a viscosity increase with the dispersed phase concentration is classically observed for emulsions or suspensions.

Preparation of API Delivery Composite Formulation
In situ forming gel containing liposomes was prepared by the addition of poloxamers to an aqueous solution containing formulated liposomes. In these conditions, liposomes present higher mean hydrodynamic diameter values (140.8 ± 0.5 nm in the poloxamer solution at 25 • C vs. 119.0 ± 0.7 nm just after liposome formulation). The PDI remained lower than 0.1, and the zeta potential values remained broadly neutral ( Table 2). This increase in the average liposome diameter included in the P407/P188 gel formulation may suggest an interaction between the poloxamers and the phospholipids forming the liposomes.
The addition of liposomes in gel formulation did not significantly modify the gelation temperature (Figure 2), nor the osmolarity ( Table 3). The pH value decreased slightly but remained in the optimum value range. Free API also did not interfere with gel properties (Table 3). Thus, neither API nor liposome addition to P407/P188 (15/1 wt%) gel significantly changed the gel formulation properties that respect the nasal physiologic conditions. Note: values are expressed as mean ± standard deviation (n = 3).
The rheological behavior of gel formulations containing liposomes or not were determined during shear rate ramps. In all cases, the shear viscosity progressively decreased (shear thinning) when the shear rate increased. All formulations behaved as a shear-thinning (non-Newtonian) fluids. The presence of liposomes increased the viscosity regardless of the shear rate, and higher content of liposomes led to higher viscosity (Figure 3). Such a viscosity increase with the dispersed phase concentration is classically observed for emulsions or suspensions.
To evaluate the strength of the gels above T gel , the evolution of storage (G ) and loss (G") moduli of the gel and liposomes-containing gels were measured as a function of the oscillatory stress ( Figure 4). For all gels, G was much higher than G" at low oscillatory stress, which is typical of solid-like materials. The presence of liposomes in the hydrogel significantly increased the storage modulus, and this increase was higher if liposome concentration increased. The stress at which the hydrogel lost 50% of its stiffness also increased significantly with the liposome concentration. Thus, it appears that liposomes simultaneously increase the hydrogel's stiffness and its strength. This leads us to think that liposomes interact with poloxamers, and play a role in the gel structuration. Moreover, the increase of liposomes average diameter in presence of poloxamers could also be related to interactions between poloxamers with the liposomes surface. Indeed, poloxamers exhibit surfactant properties and could interact with the phospholipid bilayer. Bochot et al. reported a destabilization of liposomes by P407 at low concentration [63]. P407 has a different behavior depending on its concentration in the formulation. Within dilute solutions (2%), its chains possess a greater fluidity that could lead to the insertion into phospholipid bilayer. A study of Wu and Lee also reports the existence of a saturation concentration of poloxamers, below which poloxamers can insert themselves in a lipid bilayer without disrupting liposomes and above which they instead disintegrate liposomes [64]. Polymer-liposomes complexes were prepared by Cho et al. The size of these complexes increased as the polymer content increased [65]. Polymer interactions with membranes would be due in particular to the central POP hydrophobic part of the copolymers [66]. There are two possibilities to explain the increase of liposome size: either the inclusion (association) of the polymers within the lipid bilayers or their adsorption on the vesicle surfaces. Based on adsorption method, Tian et al. prepared liposomes surfaces modified with P188 [67]. On the same way, the size of the liposomes gradually increased with increasing P188 content. Anyway, in our present study, whatever the precise interactions between poloxamers and liposomes, they do not affect sol-gel transition temperature but modify gel properties.  To evaluate the strength of the gels above Tgel, the evolution of storage (G′) and loss (G″) moduli of the gel and liposomes-containing gels were measured as a function of the oscillatory stress ( Figure  4). For all gels, G′ was much higher than G″ at low oscillatory stress, which is typical of solid-like materials. The presence of liposomes in the hydrogel significantly increased the storage modulus, and this increase was higher if liposome concentration increased. The stress at which the hydrogel lost 50 % of its stiffness also increased significantly with the liposome concentration. Thus, it appears that liposomes simultaneously increase the hydrogel's stiffness and its strength. This leads us to think that liposomes interact with poloxamers, and play a role in the gel structuration. Moreover, the increase of liposomes average diameter in presence of poloxamers could also be related to interactions between poloxamers with the liposomes surface. Indeed, poloxamers exhibit surfactant properties and could interact with the phospholipid bilayer. Bochot et al. reported a destabilization of liposomes by P407 at low concentration [63]. P407 has a different behavior depending on its concentration in the formulation. Within dilute solutions (2 %), its chains possess a greater fluidity that could lead to the insertion into phospholipid bilayer. A study of Wu and Lee also reports the existence of a saturation concentration of poloxamers, below which poloxamers can insert themselves in a lipid bilayer without disrupting liposomes and above which they instead disintegrate liposomes [64]. Polymer-liposomes complexes were prepared by Cho et al. The size of these complexes increased as the polymer content increased [65]. Polymer interactions with membranes would be due in particular to the central POP hydrophobic part of the copolymers [66]. There are two possibilities to explain the increase of liposome size: either the inclusion (association) of the polymers within the lipid bilayers or their adsorption on the vesicle surfaces. Based on adsorption method, Tian et al. prepared liposomes surfaces modified with P188 [67]. On the same way, the size of the liposomes gradually increased with increasing P188 content. Anyway, in our present study, whatever the precise interactions between poloxamers and liposomes, they do not affect sol-gel transition temperature but modify gel properties.

Mucoadhesion Properties
The thermosensitive properties of P407 have been widely exploited in the development of in situ nasal forming gels [68]. In contrast to P188, P407 is rather described as having limited mucoadhesion properties [26]. For this reason, it appeared interesting to integrate in the formulation a highly mucoadhesive polymer in order to optimize the interactions between the gel and the nasal mucosa. Chitosan and hydroxypropylmethyl cellulose (HPMC) are very widely described as mucoadhesive excipients, able to be incorporated in a gel in order to increase its mucoadhesion properties, and promote the API diffusion to the CNS [62].

Mucoadhesion Properties
The thermosensitive properties of P407 have been widely exploited in the development of in situ nasal forming gels [68]. In contrast to P188, P407 is rather described as having limited mucoadhesion properties [26]. For this reason, it appeared interesting to integrate in the formulation a highly mucoadhesive polymer in order to optimize the interactions between the gel and the nasal mucosa. Chitosan and hydroxypropylmethyl cellulose (HPMC) are very widely described as mucoadhesive excipients, able to be incorporated in a gel in order to increase its mucoadhesion properties, and promote the API diffusion to the CNS [62].
In our studies, it appeared that the addition of excipients (HPMC or chitosan), at the defined classically used concentration of 0.5 wt%, didn't significantly affect the sol-gel temperature (data not shown).
Mucoadhesion performance of these formulations were assessed on a mucin disc ( Figure 5). In the literature, when the concentration of HPMC increased from 0.1% to 1.0% in P407/P188/HPMC gel, an increase in mucoadhesive strength was measured from 4560 dyn/cm 2 to 1 0935 dyn/cm 2 [23]. Surprisingly, in our case, no difference was observed between mucoadhesion properties of the gel formulated either without or with 0.5 wt% HPMC or chitosan. For chitosan, this unexpected result could be due to the low molecular weight of the polysaccharide, that we used. Indeed, after comparing different types of chitosans, some authors previously showed that the increase in the chitosan molecular weight and viscosity could expand the adhesiveness of gels [69].

API In Vitro Release
Drug release from liposomes, hydrogels and composite formulation was performed in nasal simulated fluid, on Franz cells ( Figure 6).
A sustained API release was observed with liposomes or hydrogel, and a maximal delayed release was obtained with the composite formulation (the time to reach a 50% release of API, T50% = 60 min) in comparison with a drug solution (T50% = 20 min). Thus, it appears that, by modulating the nature of galenic form, and/or by using liposomes, the API diffuses according to different release kinetics.

API In Vitro Release
Drug release from liposomes, hydrogels and composite formulation was performed in nasal simulated fluid, on Franz cells ( Figure 6).
A sustained API release was observed with liposomes or hydrogel, and a maximal delayed release was obtained with the composite formulation (the time to reach a 50% release of API, T50% = 60 min) in comparison with a drug solution (T50% = 20 min). Thus, it appears that, by modulating the nature of galenic form, and/or by using liposomes, the API diffuses according to different release kinetics.

API In Vitro Release
Drug release from liposomes, hydrogels and composite formulation was performed in nasal simulated fluid, on Franz cells ( Figure 6).
A sustained API release was observed with liposomes or hydrogel, and a maximal delayed release was obtained with the composite formulation (the time to reach a 50% release of API, T50% = 60 min) in comparison with a drug solution (T50% = 20 min). Thus, it appears that, by modulating the nature of galenic form, and/or by using liposomes, the API diffuses according to different release kinetics.
On the contrary, liposome addition in the gel led to a significant increase of the detachment force and of the adhesion work ( Figure 5). Poloxamers are able to form entanglements or non-covalent bonds with mucus, thus favoring a greater degree of interaction with various biological tissues [21]. In our formulation, interactions between liposomes and poloxamers could impact the strength of the gel and further, positively influence their interactions with the mucin. In these conditions, an increased nasal residence time should be obtained, and so in better API bioavailability in the olfactory mucosa. It can be noted that the granulometric properties of the liposomes were kept unchanged at the end of these experiments (data not shown).

API In Vitro Release
Drug release from liposomes, hydrogels and composite formulation was performed in nasal simulated fluid, on Franz cells ( Figure 6).
A sustained API release was observed with liposomes or hydrogel, and a maximal delayed release was obtained with the composite formulation (the time to reach a 50% release of API, T 50 % = 60 min) in comparison with a drug solution (T 50 % = 20 min). Thus, it appears that, by modulating the nature of galenic form, and/or by using liposomes, the API diffuses according to different release kinetics. simulated fluid, on Franz cells ( Figure 6).
A sustained API release was observed with liposomes or hydrogel, and a maximal delayed release was obtained with the composite formulation (the time to reach a 50% release of API, T50% = 60 min) in comparison with a drug solution (T50% = 20 min). Thus, it appears that, by modulating the nature of galenic form, and/or by using liposomes, the API diffuses according to different release kinetics. Already, in the literature, it was showed that liposome incorporation into a hydrogel promoted prolonged API release, resulting in increased API residence time at the site of administration. For example, calcein release from liposomal gels was found slower in comparison to control gels, and could be further retarded by using rigid-membrane liposomes, i.e., cholesterol-based liposomes as in our formulation [70]. It is known that drug release from liposomes mainly depends on lipid composition, the size of liposomes, and the extent of lipid packing [71].
To date, the ideal pharmacokinetics profile to reach is not defined for our studied API. Considering all our results, it will be possible to optimize it by using either the drug solution, the API hydrogel, the liposomal or the API composite formulation after nasal administration.
The study of Al Asmari et al. showed that a more rapid and a greater transport of liposomal donepezil into the brain could be obtained, with similar release kinetic [12]. This probably would not be the case with our formulations. In the study of Arumugam et al., a significantly higher level of simulated fluid, on Franz cells ( Figure 6).
A sustained API release was observed with liposomes or hydrogel, and a maximal delayed release was obtained with the composite formulation (the time to reach a 50% release of API, T50% = 60 min) in comparison with a drug solution (T50% = 20 min). Thus, it appears that, by modulating the nature of galenic form, and/or by using liposomes, the API diffuses according to different release kinetics. Already, in the literature, it was showed that liposome incorporation into a hydrogel promoted prolonged API release, resulting in increased API residence time at the site of administration. For example, calcein release from liposomal gels was found slower in comparison to control gels, and could be further retarded by using rigid-membrane liposomes, i.e., cholesterol-based liposomes as in our formulation [70]. It is known that drug release from liposomes mainly depends on lipid composition, the size of liposomes, and the extent of lipid packing [71].
To date, the ideal pharmacokinetics profile to reach is not defined for our studied API. Considering all our results, it will be possible to optimize it by using either the drug solution, the API hydrogel, the liposomal or the API composite formulation after nasal administration.
The study of Al Asmari et al. showed that a more rapid and a greater transport of liposomal donepezil into the brain could be obtained, with similar release kinetic [12]. This probably would not be the case with our formulations. In the study of Arumugam et al., a significantly higher level of Solution API 1 mg/mL, simulated fluid, on Franz cells ( Figure 6).
A sustained API release was observed with liposomes or hydrogel, and a maximal delayed release was obtained with the composite formulation (the time to reach a 50% release of API, T50% = 60 min) in comparison with a drug solution (T50% = 20 min). Thus, it appears that, by modulating the nature of galenic form, and/or by using liposomes, the API diffuses according to different release kinetics. Already, in the literature, it was showed that liposome incorporation into a hydrogel promoted prolonged API release, resulting in increased API residence time at the site of administration. For example, calcein release from liposomal gels was found slower in comparison to control gels, and could be further retarded by using rigid-membrane liposomes, i.e., cholesterol-based liposomes as in our formulation [70]. It is known that drug release from liposomes mainly depends on lipid composition, the size of liposomes, and the extent of lipid packing [71].
To date, the ideal pharmacokinetics profile to reach is not defined for our studied API. Considering all our results, it will be possible to optimize it by using either the drug solution, the API hydrogel, the liposomal or the API composite formulation after nasal administration.
The study of Al Asmari et al. showed that a more rapid and a greater transport of liposomal donepezil into the brain could be obtained, with similar release kinetic [12]. This probably would not be the case with our formulations. In the study of Arumugam et al., a significantly higher level of API-loaded liposomes , A sustained API release was o release was obtained with the comp 60 min) in comparison with a drug s nature of galenic form, and/or by u kinetics. Gel with API 1mg/mL, Already, in the literature, it was prolonged API release, resulting in example, calcein release from liposo could be further retarded by using ri our formulation [70]. It is known composition, the size of liposomes, a To date, the ideal pharmacok Considering all our results, it will be hydrogel, the liposomal or the API c The study of Al Asmari et al. s donepezil into the brain could be ob be the case with our formulations. I Gel with API 1mg/mL, simulated fluid, on Franz cells ( Figure 6).
A sustained API release was observed with liposomes or hydrogel, and a maximal delayed release was obtained with the composite formulation (the time to reach a 50% release of API, T50% = 60 min) in comparison with a drug solution (T50% = 20 min). Thus, it appears that, by modulating the nature of galenic form, and/or by using liposomes, the API diffuses according to different release kinetics. Already, in the literature, it was showed that liposome incorporation into a hydrogel promoted prolonged API release, resulting in increased API residence time at the site of administration. For example, calcein release from liposomal gels was found slower in comparison to control gels, and could be further retarded by using rigid-membrane liposomes, i.e., cholesterol-based liposomes as in our formulation [70]. It is known that drug release from liposomes mainly depends on lipid composition, the size of liposomes, and the extent of lipid packing [71].
To date, the ideal pharmacokinetics profile to reach is not defined for our studied API. Considering all our results, it will be possible to optimize it by using either the drug solution, the API hydrogel, the liposomal or the API composite formulation after nasal administration.
The study of Al Asmari et al. showed that a more rapid and a greater transport of liposomal donepezil into the brain could be obtained, with similar release kinetic [12]. This probably would not be the case with our formulations. In the study of Arumugam et al., a significantly higher level of API-loaded Liposomes in gel.
Already, in the literature, it was showed that liposome incorporation into a hydrogel promoted prolonged API release, resulting in increased API residence time at the site of administration. For example, calcein release from liposomal gels was found slower in comparison to control gels, and could be further retarded by using rigid-membrane liposomes, i.e., cholesterol-based liposomes as in our formulation [70]. It is known that drug release from liposomes mainly depends on lipid composition, the size of liposomes, and the extent of lipid packing [71].
To date, the ideal pharmacokinetics profile to reach is not defined for our studied API. Considering all our results, it will be possible to optimize it by using either the drug solution, the API hydrogel, the liposomal or the API composite formulation after nasal administration.
The study of Al Asmari et al. showed that a more rapid and a greater transport of liposomal donepezil into the brain could be obtained, with similar release kinetic [12]. This probably would not be the case with our formulations. In the study of Arumugam et al., a significantly higher level of drug in the brain was found after intranasal administration of liposomes of rivastigmine compared to the intranasal administration of drug solution [14]. Moreover, these authors demonstrated the longer half-life in the brain after intranasal administration of liposomes than intranasal or oral administration of rivastigmine solution. In these conditions, the efficacy of the API into the CNS can be optimized. The best formulation to use will be defined from further in vivo pharmacokinetics and pharmacodynamics studies.

Conclusions
We based our present study on an API, acting as a promising selective BuChE inhibitor. An innovative composite formulation was designed to enable administration of this API by intranasal route, thus allowing prolonged residence time in the nasal cavity, and a controlled drug release. Such a formulation strategy should permit not only to circumvent the low API BBB-permeability, but also to decrease its systemic exposure and its potential correlated cardiac toxicity effects, and also ultimately, to increase its bioavailability in the CNS.
This formulation consists in a thermosensitive gel, endowed with mucoadhesive properties and carrying liposomes of a promising API for the treatment of AD. The entire formulation is indicated and compatible for intranasal administration, a particularly advantageous route for the outpatient management of patients suffering from this major neurodegenerative disease and having no curative treatment to date. This composite formulation can be used as a whole, or each element (solution, thermosensitive gel, liposomes) can be taken separately, enabling a defined but different API release profile. Further in vivo experiments will contribute to determine ideal in vivo pharmacokinetics and pharmacodynamics of this API. The formulations that we developed will thus permit to optimize API delivery into the CNS in order to obtain a therapeutic effect in AD.