Thermosensitive Nasal In Situ Gels of Lipid-Based Nanosystems to Improve the Treatment of Alzheimer’s Disease

Thermosensitive in situ gels are promising formulations for the management of Alzheimer’s disease (AD), since they increase the residence time of lipid-based nanosystems in the nasal cavity, improving drug therapeutic efficacy. The purpose of this study is to prepare thermosensitive in situ gels with anticholinesterase inhibitor (RVG)-loaded nanostructured lipid carriers (NLC) and nanoemulsions to improve the residence time of the formulations in the nasal cavity. Different concentrations of thermosensitive polymers were added to the RVG-loaded NLC and to the RVG-loaded nanoemulsion to optimize the gelation temperature of the in situ gels; concentrations of 17% (%, w/w) of Kolliphor® P407 and 0.3% (%, w/w) of MethocelTM K4M were selected. The in situ gels of the RVG-loaded NLC and RVG-loaded nanoemulsion had a particle size, PDI, ZP, and pH of, respectively: 141.70 ± 0.40 nm and 146.10 ± 1.73 nm; 0.45 ± 0.00 and 0.43 ± 0.02; −4.06 ± 1.03 mV and −4.09 ± 0.71 mV, 6.60 ± 0.01 and 7.00 ± 0.02. In addition, these in situ gels showed a non-Newtonian plastic behavior, and the texture parameters presented desirable values for nasal administration. From these results, we concluded that the developed in situ gels can be used to improve the treatment of AD through the nose-to-brain route.


Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder associated with neuronal degeneration and loss of cognitive functions, which lead to severe dementia. Currently, there is no fully effective treatment for AD; drugs are administered in conventional pharmaceutical dosage forms, which reduce the symptoms of the disease while maintaining the patient's quality of life [1,2]. Examples of drugs used in the management of AD include the acetylcholinesterase inhibitors (AChEs), which increase the acetylcholine levels by inhibiting the acetylcholinesterase activity. However, these drugs are generally administered orally and absorbed into the systemic circulation, where they undergo enzymatic and metabolic degradation which decreases their therapeutic effectiveness. In addition, drugs need to cross the blood-brain-barrier (BBB) to reach the central nervous system (CNS), which is challenging [3,4]. Thereby, alternative routes to deliver AChEs inhibitors have been investigated, including nose-to-brain delivery, which allows the direct passage  of the drugs from the nasal cavity to the brain through the olfactory and trigeminal nerves. However, the physiological mechanism of mucociliary clearance is a significant barrier to the absorption of drugs in the nasal cavity, which are quickly eliminated [5][6][7]. To circumvent this limitation, the use of mucoadhesive formulations can improve the drugs' residence time in the nasal cavity through the formation of electrostatic interactions between the formulation and mucin [8]. Thermosensitive polymers, such as poloxamers, and mucoadhesive agents, such as chitosan and hydroxypropyl methylcellulose, have been successfully used to prepare thermosensitive in situ nasal gels [9].
To maximize their mucoadhesive properties, in situ gels have been associated with lipid-based nanosystems. Among these, nanostructured lipid carriers (NLC) and nanoemulsions have proved to be ideal candidates for nasal drug delivery due to their physicochemical properties. The combination of in situ gels with lipid-based nanosystems is useful for the transport of lipophilic drugs, since they are encapsulated in the lipid matrix of the nanosystem within the gel structure, which provides the drugs with high protection and a prolonged release [10,11].
This work aimed to develop thermosensitive in situ nasal gels of NLC and nanoemulsions loaded with an acetylcholinesterase inhibitor (RVG) to improve residence time in the nasal cavity.

Preparation of RVG-Loaded NLC and RVG-Loaded Nanoemulsions
RVG-loaded NLC was previously optimized by Cunha et al. [12] using the high-pressure homogenization method (HPH) and the RVG-loaded nanoemulsions were prepared by the ultrasound technique. Briefly, the lipid phase and the aqueous phase were heated at 70 °C. When both phases were at the same temperature, the aqueous phase was added to the lipid phase. Subsequently, the mixture was emulsified through high-speed stirring with an Ultra-Turrax ® T25 at 13,400 rpm for 5 min. The oil-in-water (O/W) emulsion obtained was sonicated by means of an VCX130 ultrasonic processor, with a power output amplitude of 85% for 15 min. The hot O/W nanoemulsion was transferred to glass vials and cooled to room temperature (20.0 ± 0.5 °C). A drug concentration (RVG) of 0.12% (%, w/w) was added to the lipid phase.

Screening of Excipients
Kolliphor ® P407 (poloxamer 407) was used as gelling agent, while Methocel TM K4M (hydroxypropyl methylcellulose) was used as mucoadhesive agent. The suitable sol-gel transition temperature for nasal application ranges from 28 to 37 °C [13]. Different concentrations of Kolliphor ® P407 (9, 12, 17, and 20, %, w/w) were added to the NLC dispersion and to the nanoemulsion, and the occurrence of gelation was analyzed after 24 h of storage at 34.4 ± 0.5 °C. After selecting the most suitable concentration of Kolliphor ® P407, 0.3% and 0.5% (%, w/w) of Methocel TM K4M were added to the in situ gels according to a previous work developed by Gadhave et al. [11].

Preparation of Thermosensitive In Situ Gels
In situ gels of RVG-loaded NLC and RVG-loaded nanoemulsions were prepared using the cold technique described by Fatouh et al. and Almeida et al. [14,15]. Briefly, the Kolliphor ® P407 was added to the RVG-loaded NLC dispersion and to the RVG-loaded nanoemulsion and stirred at 743 rpm, at 4.0 ± 0.5 °C. The formed gels were further stored at 4.0 ± 0.5 °C to eliminate the air incorporated during the preparation. Afterwards, Methocel TM K4M was added to the formulations with a mechanical stirrer at 743 rpm. The final formulations were stored at 4.0 ± 0.5 °C for 24 h and then incubated at 34.4 ± 0.5 °C for 24 h.
An in situ control gel was also prepared according to the cold method. Briefly, 0.12% (%, w/w) of RVG, 0.02% (%, w/w) of benzalkonium chloride, 1.70% (%, w/w) of glycerin, and 17% (%, w/w) of Kolliphor ® P407 were dispersed in 100 mL of purified water at 4.0 ± 0.5 °C under magnetic stirring. Then, 0.3% (%, w/w) of Methocel TM K4M was added to the previously prepared gel and mixed with the magnetic stirrer. The prepared formulation was stored in a refrigerator at 4.0 ± 0.5 °C for 48 h and then incubated at 34.4 ± 0.5 °C for 24 h.

Particle Size, Polydispersity Index (PDI), and Zeta Potential (ZP)
For the determination of particle/droplet size and PDI, the RVG-loaded NLC and RVG-loaded nanoemulsion and their respective in situ gels were previously diluted in ultrapure water in a proportion of 1:100. The measurements were performed by a dynamic light scattering technique (DLS) using a Malvern Nano-Zetasizer (Malvern, UK). ZP measurements were performed by laser doppler electrophoresis using the same apparatus. The temperature was set at 25.0 ± 0.5 °C. Each sample was analyzed in triplicate (n = 3), and the results were reported as the mean ± standard deviation (SD).

Organoleptic Analysis
The general macroscopic appearance of the in situ gels was analyzed 48 h after the production, at 20.0 ± 0.5 °C.

pH and Osmolarity
The pH was measured using a BASIS 20 calibrated digital pH meter (Crison Instruments, s.a., Barcelona, Spain), and the osmolarity with a Type 6 osmometer (Löser Messtechnik, Berlin-Spandau, Germany). The values were adjusted to the nasal physiological values, using sodium hydroxide for pH (4.5-6.5) and glycerin for osmolarity (290 mOsm/kg).

Rheological and Texture Analysis
Rheological experiments were conducted in the in situ gels of the RVG-loaded NLC, RVG-loaded nanoemulsion, and in situ control gel with RVG, using a rotational viscometer (Haake VT-550 type Searle (Haake Viscotester™ 550; Thermo Fisher Scientific, Waltham, MA, USA) with a coaxial cylinder sensor (Rotor SV DIN 53019/ISO 3219), at 20.5 ± 0.5 °C and 34.4 ± 0.5 °C. The measurements were performed in triplicate (n = 3), and the results were reported as the mean ± SD. The formulations' mechanical properties of adhesiveness and firmness were evaluated through a texture analysis. A texture analyzer (TA-XT2i; Stable Micro Systems, Godalming, UK) with a load cell of 5 kg, a trigger force of 0.049 N, a 0.5 mm diameter cylindrical probe, a penetration depth of 5 mm, and a test speed of 3 mm⋅s −1 was used. The experiments were performed in triplicate (n = 3), and the results were reported as the mean ± SD, at 20.5 ± 0.5 °C.

Screening of Excipients
The concentrations of Kolliphor ® P407 that allowed the sol-gel transition at 34.4 ± 0.5 °C was 17% (w/w). According to a previous work developed by Gadhave et al., the Methocel TM K4M concentration selected was 0.3% (%, w/w) [11]. Table 1 shows the results of the particle size, PDI, and ZP of the RVG-loaded NLC and RVG-loaded nanoemulsions and their respective in situ gels.  Table 1, it can be seen that the particle/droplet size of the RVG-loaded NLC and RVG-loaded nanoemulsion increased slightly after incorporation in the in situ gels. Similar results have been reported in other studies. For example, Fatouh et al. [14] observed a slight increase in the particle size of agomelatine-loaded SLN (167.7 ± 0.42 nm) after incorporation in an in situ gel (175.75 ± 1.10 nm), and Pires et al. [16] verified an increase in the droplet size of a fosphenytoin-loaded nanoemulsion (216.4 ± 10.5 nm) after incorporation in a thermosensitive gel (219.7 ± 26.8 nm).

Organoleptic Analysis
The organoleptic analysis of the formulations showed that the in situ gels were opaque and had a homogeneous appearance without visibly notable phase separation, before and after gelation.

pH and Osmolarity Measurements
The mean pH and osmolarity values obtained for the in situ gels of RVG-loaded NLC and the RVG-loaded nanoemulsion were 6.60 ± 0.01 and 275 ± 0.02 and 7.00 ± 0.02 and 280 ± 0.00 mOsm/kg, respectively. Thus, the prepared formulations can be used for nasal application without damaging the nasal mucosa [17]. Figures 1A,B show that the in situ gels of RVG-loaded NLC and RVG-loaded nanoemulsions exhibited a non-Newtonian plastic behavior, which means that viscosity decreases with increasing shear rate. Moreover, a decrease in the formulations' apparent viscosity with time was not observed, which indicates the absence of thixotropy (the upper and lower curves of the rheograms overlap). Other authors have reported the same behavior as desirable for nasal formulations [18][19][20]. Figure 1B also shows the effect of lipid-based nanosystems on the flow behavior. As expected, in situ gels of RVG-loaded NLC and RVG-loaded nanoemulsions had a lower viscosity than the in situ control gel, since lipid-based systems interfere with the consistency of the gels, decreasing their apparent viscosity.

Rheological and Texture Analysis
Regarding the low viscosity of the in situ control gel with RVG before its gelation at 34.4 ± 0.5 °C, it is impossible to evaluate its flow behavior at 20.5 ± 0.5 °C.  (Figure 2A), it was observed that the in situ gel of RVG-loaded NLC exhibited a higher firmness than the in situ gels of RVG-loaded nanoemulsions. This could be related to the lower viscosity of the nanoemulsions at 20.5 ± 0.5 °C than NLC. However, all formulations had higher values of firmness than the in situ control gel, meaning that the presence of the NLC and of the nanoemulsions increases the firmness of the in situ gels. Concerning the adhesiveness (Figure 2B), the in situ gel of RVG-loaded NLC also exhibits higher values of adhesion (−0.945 N.mm and −1.041 N.mm, respectively) than the in situ gel of RVG-loaded nanoemulsions (−0.625 N.mm). In addition, both formulations presented a higher adhesion than the in situ control gel (−0.4863 N.mm). These results suggest that the in situ gels of lipid-based nanosystems increase the formulations' retention time in the nasal cavity [21].

Conclusions
The developed thermosensitive in situ gels of RVG-loaded NLC and of RVG-loaded nanoemulsions had adequate values of particle/droplet size, PDI, and ZP for nasal administration. They also presented a homogeneous appearance and values of pH and osmolarity compatible with the physiological values of the nasal cavity. Rheological studies demonstrated that both of the thermosensitive in situ gels exhibited a plastic non-Newtonian behavior without thixotropy. Regarding the texture analysis, it was observed that the addition of thermosensitive and mucoadhesive polymers to the RVG-loaded NLC and RVG-loaded nanoemulsions increased their firmness and adhesiveness. From these results, we conclude that the developed thermosensitive in situ gels can be used to improve the treatment of AD through the nose-to-brain route. However, this study is under development, and further in vitro and ex vivo studies should be performed to confirm this application.