Formulation and In-Vitro Comparison Study between Lipid-Based and Polymeric-Based Nanoparticles for Nose-to-Brain Deliery of a Model Drug for Alzheimer Disease

Certain challenges like the presence of highly complex structure (blood-brain barrier (BBB)), P-glycoprotein efflux, and the particular enzymatic activity stand in the way of the successful delivery of the drug moieties to the brain and make them fruitless. Many efforts have been conducted to overcome the previous. Direct delivery of drugs to the brain after the intranasal application is one of those strategies since it holds a great hope to raise the chances of drug moieties to the brain. Nanoparticles could be the potential to improve nose-to-brain drug delivery since they are able to protect the encapsulated drugs from biological and/or chemical degradation and increase their penetration across biological barriers. Based on the fact that neuroinflammation is associated with neuron death and neurodegenerative diseases like Alzheimer’s, nonsteroidal antiinflammatory drugs (NSAIDs) might play a positive role in the disease. The present study aimed to employ the QbD approach for the first time in optimizing polymeric and lipid-based nanoparticles for the nose-to-brain delivery of Meloxicam (MEL), and to perform a comparison between the pure drug and the formulated nanosystems regarding dissolution profiles, permeability, and mucoadhesiveness.


Introduction
The precense of the blood-brain barrier (BBB) forms the major drawback to the successful delivery of the brain targeting moieties to their active site. On the other hand, the P-glycoprotein efflux supports the preventing role of the BBB by transporting the particles out of the CNS. However, this physiological protective barrier is considered s a key challenge to the pharmaceutical fraternity, with a need to circumvent it so as to deliver drugs to the brain in various CNS disorders [1][2][3].
Nose to Brain delivery route holds a great promise to overcome the BBB, since it transports the drug directly to the brain along the olfactory and trigeminal nerve pathways. These nerve pathways initiate in the nasal cavity at olfactory neuroepithelium and terminate in the brain [4].
Several clinical studies concluded that long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) may protect against the onset of Alzheimer's disease (AD) [5]. Among those, meloxicam exerts its pharmacological properties by inhibiting the enzymatic activity of cyclooxygenase-2 (COX-2) [6], which linked with a neuroprotective action in Alzheimer's disease [7][8][9][10]. Unfortunately meloxicam is a lipophilic drug with a high potency and poor water solubility [11] which decreases its bioavailability when it is applied following the routine routs of administration.
The strategy of designing drugs in an encapsulated form of nanoparticles (NPs) to target the olfactory epithelium could potentially improve the direct CNS delivery of drugs-including biologics [2]. Nanostructured drug delivery systems have been accomplished with an increase in nasal permeability and control drug release [12]. Lipid-based and polymeric nanoparticles have recently attrack the attention for this purpose, Solid lipid NPs (SLNs), as a lipid-based formulation, offers an improvement to the nose-to-brain drug delivery since they are able to protect the encapsulated drug from biological and/or chemical degradation and enhance its characters regarding nasal retention time, good application properties, and adhesion of the SLNs to mucous membranes [13]. On the other hand, polymeric nanoparticles show higher stability, variety of the preparation methods, controlled release of the drug [14].
Since there are many factors can affect the characteristics and properties of the previously mentioned nanosystems, the application of QbD approach presents a logic strategy that saves time, cost, and efforts when developing complex systems such as the nanosystems [14]. Starting by the definition of the quality target product profile (QTPP), then the critical process parameters (CPPs) and critical material attributes (CMAs) that can highly affect the critical quality attributes (CQAs) of the product [15].
In the present research, two types of nanoparticles were prepared following a QbD approach. Physical, chemical, and morphological characterization were conducted. The following step was to evaluate their in vitro behavior regarding release profile, permeation and mucoadhesion properties. Correspondingly, a profound comparison was performed followed by the selection of the optimized nanocarrier system to be a successful candidate for nose-to-brain delivery of anti AD drug formulation.

MEL Loaded SLNs
MEL loaded SLNs were prepared following a modified double emulsion (W1/O/W2) solvent evaporation (DESE) technique [45]. MEL was dissolved in 0.1 M NaOH solution formulating the W1 Phase. The oily phase was prepared by dissolving phosphatidylcholine in cyclohexane. The primary emulsion was formed by adding the W1 phase dropwise into the organic phase using a homogenizing mixer (Hielscher, Germany) (0.5 cycles& 75% amplitude) for 1 min. The resultant nanoemulsion was then added dropwise into the surfactant aqueous solution using the homogenizing mixer (0.5 cycles& 75% amplitude) for 1 min. The final mixture was left then to stir over the night using a magnetic stirrer to allow the evaporation of the organic solvent and thus formulation of the SLNs.

MEL Loaded PLGA NPs
MEL loaded PLGA NPs were prepared using a double emulsion (W1/O/W2) solvent evaporation (DESE) technique [16]. First, formation of a primary W1/O emulsion, where the aqueous solution of the MEL was added to the PLGA solution in ethyl acetate upon sonication in ice bath. This was followed by the formation of a double emulsion (W1/O/W2) by dispersing the primary emulsion in an external aqueous phase containing poly vinyl alcohol (PVA) as a stabilizer, with the use of sonication in ice bath. Finally, organic solvent evaporation over the night resulted in the formation of MEL loaded NPs.
Both types of NPs were harvested by centrifugation at 16000× g for 1 h at 10 °C (Sigma, Germany) and washed 3 times with deionized water to remove unentrapped drug, surfactants and remaining organic solvent. The NPs were then resuspended in 2 mL of 10% (w/v) trehalose aqueous solution, frozen at −20 °C, and were finally freeze-dried (Christ, Germany) at −40 °C for 72 h.

Mean Particle Diameter, Size Distribution and Zeta Potential
The average hydrodynamic diameter (Z-average), polydispersity index (PDI), and surface charge (zeta potential) of the NPs were analyzed in folded capillary cells, using Malvern nano ZS instrument (Malvern Instruments, Worcestershire, UK).

Encapsulation Efficacy and Drug Load
The obtained NPs were separated from the preparation medium by centrifugation and washed 3 times, each time was followed by a centrifugation to obtain NPs pellets. Meloxical then was extracted using chloroform. The mixture was moved to a separatory funnel and the aqueous phase was withdrawn to determine its drug content by HPLC, and the EE and DL were calculated according to the following equations:

Scanning Electron Microscopy (SEM)
The morphological appearance of NPs was investigated using scanning electron microscopy (SEM) (Hitachi S4700, Hitachi Scientific Ltd., Tokyo, Japan) at 10 kV.

Fourier-Transform Infrared Spectroscopy (FTIR)
The chemical interactions between the drug and excipients were analyzed by a Thermo Nicolet AVATAR FTIR spectrometer (Thermo-Fisher, Waltham, USA.

X-ray Powder Diffraction XRPD
The X-ray powder diffractograms of citral SLNs, GMS, and the physical mixture of citral and GMS were obtained in the angular range of 3-40° 2θ at a step time of 0.1 s and a step size of 0.007°at ambient temperature. Monochromatic CuΚα1 radiation (with λ = 1.5406 Å) at 40 kV and 40 mA was used as the X-ray source. The same was repeated with the polymeric NPs.

Dissolution Test
Dissolution of meloxicam and the drug release from MEL-NPs was determined using a dialysis bag diffusion technique using a dialysis membrane [17][18][19]. The samples were analyzed spectrophotometrically at λ max of 346 nm (Jasco V730 UV-VIS spectrophotometer (ABL&E-JASCO Ltd., Budapest, Hungary).

Permeation Test
In vitro permeation of the prepared NPs was investigated using Side-by-side type apparatus. An accurate weights of MEL and MEL NPs equivalent to 1 mg of MEL were suspended in 9 mL of simulated nasal electrolyte solution (SNES), then placed in the donor chamber. On the other hand, 9 mL of pH 7.40 phosphate buffer was placed in the acceptor chamber. A semi-permeable cellulose membrane, previously impregnated in isopropyl myristate for 1 h, was placed between the two chambers as membrane to mimic the nasal mucosa.
Diffusion was investigated for 1 h comparing pure MEL, optimized SLNs formulation, and optimized PLGA NPs.

Mucoadhesiveness Test
Mucoadhesion was determined following the direct method (turbidimetric method) as following: briefly, the mucin was in PBS 6.4 (0.5 mg mL−1) and the NPs were mixed and incubated at 37 °C with continuous stirring with predetermined times of 1, 2, 3 and 4 h [20,21].

Risk Assessment
Risk assessment was conducted to rank and prioritize the factors with the highest impact on product quality. The first step of QbD-based risk assessment study was to set the QTPP encompassing the desired quality attributes in MEL-NPs, followed by the selection of CPPs and CMAs. The previous is summarized and ranked as in Figure 1

Morphology, Size, ZP and EE
The resulted NPs were smooth and spherical in shape as shown in Figure 6.

Compatibility Study
The FT-IR spectra, XRD patterns of MEL, both loaded NPs formulations, and the used excipients are presented in Figure 8.

In vitro Release
The in vitro dissolution profiles of pure MEL and MEL loaded NPs were investigated in intranasal-simulated conditions, using simulated nasal electrolytic solution (SNES) medium (pH of 5.6) and the results are shown in Figure 4.

In Vito Permeation
Permeation test has been performed in vitro for pure MEL solution and MEL-NPs, following similar conditions for nose-to-brain delivery route ( Figure 5).

In vito Mucoadhesion
The mucoadhesion was determined by turbidity analysis method to understand how the NPs will be retained, since a strong mucoadhesion suggests a close contact with absorption site, thus ensuring the effective absorption following the nasal administration ( Figure 6).

Discussion
Analyzing the results of the risk assessment as a part of QbD and based on the risk priority number RPN demonstrated the most highly influential CPP was sonication time, while the most highly influential CMAs were lipid/polymer type, lipid/ polymer concentration, surfactant type and surfactant concentration as shown in Figure 7.  Both types of NPs comply with the size requirement of the administration via the nose-to-brain route which preferred to be up to 200 nm [22,23] The zeta potential values obtained for PLGA NPs and SLNs were −16.2 ± 1.81, −43.65 ± 1.47 mV, respectively, which is logical due to the negative charge of phosphatidylcholine estimated at between −10 mV and −30 mV at neutral pH [24] due to the presence of phosphate and carboxyl groups, while the negatively charged carboxyl groups on PLGA only are the cause behind the negative charge.
The FTIR spectra of the NPs showed that no changes occurred in the MEL chemical structure and did not present a significant difference in the main functional groups of MEL. The absorption band at 3290 cm −1 correspondings to -NH stretch appears to overlap with -OH group of phosphatidylcholines, which is represented at 3200-3400 cm −1 . Likewise, the absorption band at 1650 cm −1 match a slight shift of C=O. Hence, there is no interaction between MEL and the other SLNs excipients, and they are compatible with each other.
Similarly, absorption peaks of the materials used for polymeric NPs preparation (PLGA, Poloxamer) exhibited compatibility with MEL since the previously mentioned two chemical groups of MEL were maintained the same after PLGA NPs formulations The XRD of MEL gave unique fingerprint patterns owing to its crystalline structure. However, both SLNs and PLGA NPs did not show the characteristic fingerprints for the drug in their XRD pattern. This confirms that the drugs are present in a nanocrystalline state in the NPs [25]. The previous results are in agreement with the results of FTIR.
Based on Figure 4, it is evident that pure MEL demonstrates a poor solubility (5.10 ± 0. 9 µg/mL, over 360 min, at 35 °C) due to the chemical structure it has and the weak acidic character resulted in this medium (pKa = 3.43) [26]; fabrication of MEL in nanoformulations showed a significant increase in the dissolution rate than that of pure MEL (approximately 4-5 times higher), this goes in line with the results previously reported by Katona et al. and might be due to the nanosize and the increased specific surface area that the NPs have [27].
The release behavior from the NPs showed a sustained release pattern starting by a mild initial burst release during the first hour, where 12.94 ± 0.86%, 11.79 ± 0.74 of MEL was released from the PLGA NPs and SLNs, respectively, which has been frequently reported for polymeric NPs [28,29] and SLNs [13,17]. This could be explained by the presence of the surface-adsorbed drug on the NPs, in addition to the drug molecules that exist close to the surface having weak interactions with the NPs system. This was followed by a slow-release profile until 6 h, where only 25.26 ± 2.39%, 21.37 ± 1.47 of cumulative MEL release was observed for PLGA NPs and SLNs, respectively as the encapsulated drug slowly diffused through the NPs core [17]. The previous results point out that the majority of the drug remained in the NPs after their contact with the nasal mimetic conditions, and able to be released inside the targeted position.
The mucoadhesive strength was detected by calculating binding efficiency of mucin to PLGA NPs, and SLNs, which were 36.55%, 57.59%, respectively by the end of the experiment as Figure 6 represents. Since mucin is a highly glycosylated and negatively charged protein, the negatively charged SLNs showed a modest affinity driving especially by electrostatic interactions between mucin and SLNs and since SLNs are higher negatively charged, the electrostatic interactions between them and mucin will be higher than those with PLGA NPs. This observation is in close agreement with previous studies [30,31] A significant enhancement of MEL permeability through the semipermeable membrane was achieved when MEL was formulated in NPs in comparison with the pure MEL solution. This could be due to the nanoscale size of the prepared nanosystems which have the best nasal permeation properties as previously reported by Gänger et al. [22]. Moreover, the spherical and smooth surface of both NPs, as confirmed by SEM images, leads to the least friction with the membrane surface in comparison with the needle-shape particles [32]. Stabilizing these NPs using poloxamer, a permeation enhancer, further improve their permeation properties [33], by inhibiting the efflux pumps in addition to lowering the membrane fluidity when it is used in vivo [34].
Interestingly, SLNs showed superior permeability over PLGA NPs, which might be explained by the lipophilic properties of these lipid-based nanosystems [35], which exceed those of PLGA NPs [32].

Conclusions
The present research work put forward solid lipid nanoparticulate (SLNs) and polymeric based nanoparticles PBNs for the nose to brain delivery of meloxicam. QbD concept was employed for the first time to analyze the previously research efforts so we could define the critical process parameters and the critical material attribute.
All the measurements were found to be in an acceptable range. Spherical nanoparticles were obtained for both SLNs and PBNs with a diameter of 142.06, 94.76 nm, and a ZP of −16.2, −43.65, respectively. The resulted nanoparticle showed good compatibility with used materials based on FTIR and XRD measurements. Higher entrapment efficacy and drug load were noticed with SLNs (87.26%, 2.64%, respectively). Better in vitro drug release, permeation and mucoadhesion accomplished the formulation of meloxicam in SLNs more than PBNs. However, ex-vivo data is still needed to investigate cell viability, permeability, and cytotoxicity. Then in vivo measurements are critical to detect brain concentration and distribution in between brain deferent parts evaluate the risk/benefit ratio.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript: