*Article* **Development of In Situ Gelling Meloxicam-Human Serum Albumin Nanoparticle Formulation for Nose-to-Brain Application**

**Gábor Katona 1,\* , Bence Sipos <sup>1</sup> , Mária Budai-Sz ˝ucs <sup>1</sup> , György Tibor Balogh 2,3, Szilvia Veszelka <sup>4</sup> , Ilona Gróf 4 , Mária A. Deli <sup>4</sup> , Balázs Volk <sup>5</sup> , Piroska Szabó-Révész <sup>1</sup> and Ildikó Csóka <sup>1</sup>**


**Abstract:** The aim of this study was to develop an intranasal in situ thermo-gelling meloxicam-human serum albumin (MEL-HSA) nanoparticulate formulation applying poloxamer 407 (P407), which can be administered in liquid state into the nostril, and to increase the resistance of the formulation against mucociliary clearance by sol-gel transition on the nasal mucosa, as well as to improve drug absorption. Nanoparticle characterization showed that formulations containing 12–15% *w*/*w* P407 met the requirements of intranasal administration. The Z-average (in the range of 180–304 nm), the narrow polydispersity index (PdI, from 0.193 to 0.328), the zeta potential (between −9.4 and −7.0 mV) and the hypotonic osmolality (200–278 mOsmol/L) of MEL-HSA nanoparticles predict enhanced drug absorption through the nasal mucosa. Based on the rheological, muco-adhesion, drug release and permeability studies, the 14% *w*/*w* P407 containing formulation (MEL-HSA-P14%) was considered as the optimized formulation, which allows enhanced permeability of MEL through blood– brain barrier-specific lipid fraction. Cell line studies showed no cell damage after 1-h treatment with MEL-HSA-P14% on RPMI 2650 human endothelial cells' moreover, enhanced permeation (four-fold) of MEL from MEL-HSA-P14% was observed in comparison to pure MEL. Overall, MEL-HSA-P14% can be promising for overcoming the challenges of nasal drug delivery.

**Keywords:** quality by design; rapid equilibrium dialysis; muco-adhesion; brain PAMPA; RPMI 2650 nasal epithelial cell

## **1. Introduction**

Albumin is a versatile, biodegradable drug carrier for numerous therapeutic agents that have poor water solubility, unsatisfying pharmacokinetics with low circulation halflife, inefficient targetability and even instability in vivo. Strategies for applying albumin for drug delivery can be classified broadly into exogenous and in situ binding formulations that utilize covalent attachment, non-covalent association, or encapsulation of the drug in the form of albumin-based nanoparticles [1].

Neurodegenerative diseases are associated with neuroinflammation. The combination of albumin with non-steroid anti-inflammatory drugs (NSAID) can be promising in therapy, which depends on passing the blood–brain barrier (BBB). NSAIDs can have a protective effect in neurodegenerative diseases through different mechanisms. They can depolarize

**Citation:** Katona, G.; Sipos, B.; Budai-Sz˝ucs, M.; Balogh, G.T.; Veszelka, S.; Gróf, I.; Deli, M.A.; Volk, B.; Szabó-Révész, P.; Csóka, I. Development of In Situ Gelling Meloxicam-Human Serum Albumin Nanoparticle Formulation for Nose-to-Brain Application. *Pharmaceutics* **2021**, *13*, 646. https://doi.org/10.3390/ pharmaceutics13050646

Academic Editor: Juan José Torrado

Received: 25 March 2021 Accepted: 28 April 2021 Published: 1 May 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the mitochondria, therefore inhibiting calcium ion uptake, due to the ionizable carboxylic group [2,3]. Moreover, inhibition of cyclooxygenase (COX) enzymes can suppress glia activity and reduce amyloidosis [4]. COX-2 inhibitor NSAIDs such as meloxicam (MEL) can be advantageous in the treatment of neurodegenerative disorders as they have improved anti-amnesic activity through inhibiting lipid peroxidation and acetylcholinesterase activity in the brain [2] supplemented by additional antioxidant effect [3], but the therapeutic application is limited due to the poor BBB transport [5]. To overcome this obstacle, choosing the appropriate carrier system and route of administration has a prominent role [6].

Nasal administration can be a suitable means of transport route for that purpose; moreover, it has been reported that the initial formation of Alzheimer's disease begins in the entorhinal cortex, a region innervated by the olfactory nerves, then progresses according to the corresponding pattern [7]. Due to the high surface area and rich vascularization, drugs or drug-delivery systems can be easily absorbed from the nasal cavity; moreover, first-pass metabolism is negligible through this administration route, which can be advantageous in terms of preserving pharmacological activity [8–10]. Nano drug delivery systems (nanoDDSs) are able to transport drugs as cargo, bypassing the BBB through the trigeminal and olfactory nerves directly into the brain [11]. Moreover, they support intranasal NSAID administration, due to their poor water solubility at nasal pH (5.3–5.6) and low residence time (10–15 min) due to mucociliary clearance [12]. In our previous study was demonstrated that MEL-human serum albumin (HSA) nanoparticles could be successfully applied for nose-to-brain delivery, with improved in vivo brain targeting efficacy [13].

As mucociliary clearance is a limiting factor in nose-to-brain delivery, the application of viscosity enhancers or mucoadhesive polymers can be advantageous by increasing the residence time on the nasal mucosa, which supports drug absorption [14–16]. To satisfy these requirements formulation of in situ thermo-gelling systems can be efficacious. Poloxamer 407 (P407), a triblock copolymer consisting of a hydrophobic residue of polyoxy-propylene (POP) between the two hydrophilic units of poly-oxyethylene (POE), can be applied for development of in situ thermo-reversible gelling systems, through temperaturecontrolled micelle forming [17–19]. Thermo-gelling occurs due to hydrophobic interactions between the P407 copolymer chains [20]. By optimization of P407 concentration in the formulation, a sol–gel transition can be reached at the temperature of the nasal cavity, while it remains in a liquid state below that temperature during storage and administration.

As continuous improvement is part of industrial manufacturing and research processes, the quality management of nanocarriers having higher potential, in the case of beneficial therapeutic applications and effects, should be of paramount importance. Quality by Design (QbD) offers a proper methodology based on knowledge and risk assessment, ensuring the quality, safety and efficacy of the desirable nanocarrier [21]. In the case of noseto-brain applicable nanocarriers, many physiological and pharmaceutical aspects must be taken into account, which are adapted to the versatile biological and chemical aspects of this route. As part of the QbD assessment, a comparison study was performed between MEL-HSA and MEL-HSA-P407 formulations to evaluate the change of risk severity during a continuous development process [22].

Our aim was to optimize an in situ thermo-gelling MEL-HSA-P407 formulation, which can be administered in liquid state into the nostril and to increase resistance of formulation against mucociliarly clearance by sol–gel transition on the nasal mucosa, as well as to improve drug absorption.

#### **2. Materials and Methods**

#### *2.1. Materials*

Meloxicam (MEL, 4-hydroxy-2-methyl-*N*-(5-methyl-2-thiazolyl)-2*H*-benzothiazine-3 carboxamide-1,1-dioxide) was donated by EGIS Pharmaceuticals Plc. (Budapest, Hungary) for research work. Human serum albumin (HSA, lyophilized powder, purity > 97%), fluorescein isothiocyanate-labelled HSA (FITC-HSA), Tween 80 (Tween), P407, disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), polar brain

lipid extract, cholesterol, mucin from porcine stomach (Type III), and all reagents for cell line studies were purchased from Sigma Aldrich Co. Ltd. (Budapest, Hungary) if not indicated otherwise. Analytical grade solvents such as methanol, dimethyl sulfoxide (DMSO) and dodecane were purchased from Molar Chemicals (Budapest, Hungary). Sodium hyaluronate (NaHA, Mw = 1400 kDa) was obtained from Gedeon Richter Plc. (Budapest, Hungary). In all experiments, water was purified by the Millipore Milli-Q® 140 Gradient Water Purification System. lipid extract, cholesterol, mucin from porcine stomach (Type III), and all reagents for cell line studies were purchased from Sigma Aldrich Co. Ltd. (Budapest, Hungary) if not indicated otherwise. Analytical grade solvents such as methanol, dimethyl sulfoxide (DMSO) and dodecane were purchased from Molar Chemicals (Budapest, Hungary). Sodium hyaluronate (NaHA, Mw = 1400 kDa) was obtained from Gedeon Richter Plc. (Budapest, Hungary). In all experiments, water was purified by the Millipore Milli-Q® 140 Gradient Water Purification System.

Meloxicam (MEL, 4-hydroxy-2-methyl-*N*-(5-methyl-2-thiazolyl)-2*H*-benzothiazine-3-carboxamide-1,1-dioxide) was donated by EGIS Pharmaceuticals Plc. (Budapest, Hungary) for research work. Human serum albumin (HSA, lyophilized powder, purity > 97%), fluorescein isothiocyanate-labelled HSA (FITC-HSA), Tween 80 (Tween), P407, disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), polar brain

#### *2.2. Preparation of In Situ Gelling MEL-HSA Nanoparticle Formulations 2.2. Preparation of In Situ Gelling MEL-HSA Nanoparticle Formulations*

*Pharmaceutics* **2021**, *13*, x 3 of 22

MEL-HSA nanoparticles were produced by applying a modified coacervation method (Figure 1) according to the following steps [23]: first, Tween-80 was dispersed in 4 mL of HCl solution (0.1 M), whereas MEL was dissolved in 4 mL of NaOH solution (0.1 M) and HSA was dissolved in 8 mL of purified water. Then, the Tween 80 solution was added dropwise (0.5 mL/min) to the MEL solution at 4 ◦C under constant stirring (800 rpm). Next, this MEL–Tween 80 solution was added dropwise to the HSA solution at 4 ◦C under constant stirring (800 rpm). After complete homogenization, additional HCl was added dropwise to adjust pH to 5.6, and as a result the solution became turbid. Then, the formulation was incubated for 12 h under constant stirring (800 rpm) to obtain nanoparticle dispersion. P407 in various concentrations (based on preliminary experimental results 12, 13, 14, 15 and 16% *w*/*w*) was added to the formulation and kept in a cool place (5 ± 3 ◦C) overnight until complete hydration and dissolution of the polymer, before further investigations. MEL-HSA nanoparticles were produced by applying a modified coacervation method (Figure 1) according to the following steps [23]: first, Tween-80 was dispersed in 4 mL of HCl solution (0.1 M), whereas MEL was dissolved in 4 mL of NaOH solution (0.1 M) and HSA was dissolved in 8 mL of purified water. Then, the Tween 80 solution was added dropwise (0.5 mL/min) to the MEL solution at 4 °C under constant stirring (800 rpm). Next, this MEL–Tween 80 solution was added dropwise to the HSA solution at 4 °C under constant stirring (800 rpm). After complete homogenization, additional HCl was added dropwise to adjust pH to 5.6, and as a result the solution became turbid. Then, the formulation was incubated for 12 h under constant stirring (800 rpm) to obtain nanoparticle dispersion. P407 in various concentrations (based on preliminary experimental results 12, 13, 14,15 and 16% *w*/*w*) was added to the formulation and kept in a cool place (5 ± 3 °C) overnight until complete hydration and dissolution of the polymer, before further investigations.

**Figure 1.** Preparation of MEL-HSA-P407. **Figure 1.** Preparation of MEL-HSA-P407.

#### *2.3. QbD-Based Comparative Risk Assessment 2.3. QbD-Based Comparative Risk Assessment*

As the in situ thermo-gelling carrier system is part of a continuous development process, it needs to be compared to the HSA nanoparticles, validating the product life cycle, and therefore ensuring quality. At first, the quality target product profile (QTPP) was determined based on the mandatory requirements of a nose-to-brain applicable nanocarrier. The critical quality attributes (CQAs) were also determined as they are physicochemical As the in situ thermo-gelling carrier system is part of a continuous development process, it needs to be compared to the HSA nanoparticles, validating the product life cycle, and therefore ensuring quality. At first, the quality target product profile (QTPP) was determined based on the mandatory requirements of a nose-to-brain applicable nanocarrier. The critical quality attributes (CQAs) were also determined as they are physicochemical factors affecting product safety, efficacy and quality. Critical process parameters (CPPs) and critical material attributes (CMAs) were not taken into account during the risk assessment process as an extensive optimization of P407 concentration was performed. For the identified elements, risk levels were assigned for both the MEL-HSA and MEL-HSA-P407. A three-level scale was used to describe the relation between these parameters: each relation was assigned with a "high" (H), "medium" (M) or "low" (L) attributive. To quantify the

risk values, LeanQbD® Software (QbD Works LLC, Fremont, CA, USA) was used. As the output of this comparative risk assessment, severity scores were compared and evaluated to determine the influence of the in situ thermo-gelling carrier system on product quality.
