Tailoring Nanostructured Lipid Carriers for Efficient Delivery of Asenapine Maleate in Antipsychotic Treatment

doi:10.21203/rs.3.rs-4680275/v1

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Research Article

Tailoring Nanostructured Lipid Carriers for Efficient Delivery of Asenapine Maleate in Antipsychotic Treatment

https://doi.org/10.21203/rs.3.rs-4680275/v1

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Asenapine Maleate (ASPM), is the atypical antipsychotic agent used for treating various psychological disorders such as schizophrenia and acute mania associated with bipolar disorder. However, the drug's therapeutic potential is intricately linked to its solubility, bioavailability, stability, and ability to be delivered specifically to the central nervous system. The aim of the study was to design, develop, and optimize Nanostructured Lipid Carriers (NLCs) encapsulating Asenapine maleate to improve the therapeutic efficacy. The development utilized a systematic approach; various formulation parameters such as lipid composition, surfactant concentration, and entrapment efficiency were meticulously optimized by employing quality-by-design principles. The optimized ASPM-loaded NLCs demonstrated a particle size of 97.6 ± 2.48 nm, zeta potential of −20mV, and entrapment efficiency of 96.74%, respectively. ASPM-loaded NLC showed initially rapid release (20%) accompanied by sustained release (75%) over 24 hr with 22-fold improvement in oral bioavailability with significantly reduced locomotor count induced by L-DOPA-carbidopa in comparison with the pure drug in Sprague-Dawley rats. Furthermore, in-vitro and in-vivo results underscored the enhanced permeability through the blood-brain barrier with superior pharmacokinetics for the NLC. Collectively, this research elucidates the potential of optimized NLCs as robust drug delivery system for asenapine maleate, offering a promising avenue for advancing therapeutic results in the management of psychosis.

Schizophrenia

Asenapine Maleate

Nanostructured lipid carriers

Central Composite design

Sustained release

Intestinal Lymphatic System

Psychosis, characterized by a disconnection from reality, represents a significant challenge in the realm of psychiatric disorders due to its debilitating impact on affected individuals and their communities [1]. Among the pharmacological interventions employed to manage psychotic symptoms, Asenapine Maleate (ASPM) (Fig. 1) emerges as promising atypical antipsychotic agent with potential benefits in terms of efficacy and tolerability, commonly utilized in the management of Schizophrenia, mania, acute depression and bipolar disorders. However, the therapeutic potential of ASPM is intricately linked to its solubility, bioavailability, stability, and drug delivery directly in central nervous system (CNS). Currently, ASPM is available in the market in the form of sublingual tablets. However, owing to severe drawbacks associated with sublingual tablets highlight the necessity for a development of formulation with improved bioavailability [2]. Despite its therapeutic potential, ASPM's effectiveness is often hindered by obstacles linked with its formulation, such as limited solubility and poor bioavailability. The successful management of psychosis requires the implementation of innovative drug delivery strategies that can ensure both enhanced therapeutic efficacy and patient compliance [3, 4].

Over the years, several methods have been explored to enhance ASPM's pharmaceutical properties, bioavailability, and therapeutic efficacy. Gambhire et al. (2018) developed Solid Lipid Nanoparticles to improve ASPM's bioavailability by bypassing first-pass metabolism [5]. Researchers have conducted numerous studies to devise diverse formulations of Asenapine Maleate, such as sublingual film [6], Thermo-reversible Mucoadhesive in situ Nasal Gel of Asenapine Maleate [7], nanostructured lipid carriers (NLCs) for intranasal Asenapine Maleate delivery to brain [8], and surface modified solid lipid nanoparticles loaded with Asenapine Maleate (SLN) [9, 10]. Moreover, several patents exist for intranasal, transdermal, and injectable ASPM formulations. However, some formulations, such as in-situ gel, have drug degradation and stability issues [7, 11], sublingual tablets can be ineffective if swallowed, leading to a loss of drug in saliva [12], and Solid lipid nanoparticles have crystalline structure which is responsible for poor drug entrapment and loading, with drug expulsion potentially occurring during the crystallization process [13].

Nanostructured lipid carriers (NLCs) formulations have evolved as the most promising area in the pharmaceutical field to address the issues related to poorly soluble and bioavailable drugs. NLCs represent a category of lipid-based nanocarrier systems that amalgamate the benefits of liposomes and solid lipid nanoparticles [14]. The distinctive characteristics of NLCs, including their capacity to enclose hydrophobic drugs within their lipid matrix, effectively enhance solubility and lead to a more consistent and predictable drug release profile, thus tackling the problem of variable bioavailability [15]. Moreover, upon oral administration of NLCs, they gain access to intestinal lymphatic system (ILS) augmenting the bioavailability of Asenapine Maleate. Despite the incomplete understanding of the exact mechanism involved in the lymphatic transport of NLCs, it has been proposed that lipid-based formulation when taken orally stimulates the synthesis of Chylomicrons [16]. Chylomicron acts as a carrier for binding lipid-based drugs, as a result the medication attaches to the chylomicrons, aiding in its uptake in the Intestinal lymphatic system. The intestinal lymphatic system (ILS) is essential in evading first-pass metabolism by allowing lipid-based drugs to enter into the lymphatic system, and reaching systemic circulation more directly through the thoracic duct, which drains into the bloodstream. [17]. NLCs provide a sustained and control drug release mechanism that can maintain therapeutic drug levels in the body, potentially reducing the number of administrations and enhancing patient compliance. Furthermore, owing to their nanoscale size, NLCs offer improved penetration into the central nervous system, which is crucial for targeting the fundamental neurological mechanisms of Schizophrenia [18].

Given the aforementioned potential of NLCs, the application of NLCs as a delivery platform for ASPM shows significant promise. The main objective of the study was to formulate ASPM-loaded NLCs to enhance its solubility, bioavailability, and therapeutic effectiveness. Employing a systematic methodology, various formulation parameters such as lipid composition, surfactant concentration, and ASPM loading were methodically optimized utilizing quality-by-design principles [19]. High shear homogenization was adapted to prepare ASPM loaded NLCs and optimization was carried out using a Central Composite Design. The optimized NLCs underwent evaluation based on particle size, zeta potential, entrapment efficiency as well as FTIR, DSC, PXRD, and in-vitro drug release studies. Subsequently, in-vivo pharmacokinetic and pharmacodynamic investigations were performed on Sprague-Dawley rats, comparing the results with pure ASPM.

2.1. Materials

Asenapine Maleate (ASPM) was obtained as gift sample by MSN Organics Pvt. Ltd (Hyderabad, Telangana State, India), Glyceryl Monostearate obtained from Mohini Organics Pvt. Ltd. (Mumbai, India), Gelucire 44/14 was gifted by Gattefosse (St-Priest, Cedex, France). Capmul MCM was gifted by ABITEC Corporation, Columbus, USA. HPLC grade Acetonitrile was procured from Merck India Ltd

2.2. Selection of solid lipids, liquid lipids and surfactants

Determination of solubility of the ASPM in solid lipids is an important factor in achieving high entrapment in a lipid matrix. To assess the solubility of ASPM in different solid lipids, 1 gram of each solid lipid was melted in a test tube above its respective melting point using a water bath [20]. The ASPM was added into molten lipid by increasing ASPM concentration and mixed for 5 min by using a cyclo-mixer (CM 101, REMI, Mumbai, India), the test tubes were then visually inspected for solubility, miscibility, and clarity.

A variety of liquid lipids were chosen to assess the solubility of ASPM. An excess amount of the drug was added to 1 ml of selected liquid lipids contained in single cap tube. Rotatest shaker was used to stir the cap tubes for 24 hours at a temperature of 25 ℃. Upon reaching equilibrium, samples were centrifuged at 10,000 rpm to remove any undissolved drug particles for 10 minutes [21]. The supernatant from each cap tube was filtered in order to provide clear sample for analysis [22]. The concentration of dissolved ASPM in the liquid lipids was determined using High-Performance Liquid Chromatography (HPLC). The solubility study was conducted three times to ensure accuracy and reliability. Gelucire 44/14 was employed as a surfactant for the formulation of Asenapine maleate loaded NLCs.

The concentration of ASPM was determined by using a reverse-phase high-performance liquid chromatography (RP-HPLC) system (Agilent Technologies 1200 series) equipped with a UV detector, an autosampler and column manager. The mobile phase comprised of methanol, water, and triethylamine in the ratio of (800:200:0.75 v/v) was used as the mobile phase, and the flow rate was adjusted to 1.0 mL/min. At 257 nm, the elution was conducted on a C-8 column Xterra RP (85 µm, 250 X 4.66 mm). The Open Lab CDS program was used for analyzing the data and chromatograms.

2.2.1. Screening of Solid lipid to liquid lipid ratio

To determine optimal ratio of solid lipid (Glyceryl Monostearate) to liquid lipid (Capmul MCM), the surfactant Gelucire 44/14, the concentration of ASPM, and the total lipids were maintained at 2.5%w/v, 100 mg and 400 mg, respectively [23]. Using high Shear homogenization technique with 10,000 rpm for 10 minutes, the 20 ml of ASPM-loaded NLCs were prepared by altering the solid-to-liquid lipid ratio as specified in Table 1. Entrapment efficiency was taken into consideration while optimizing the solid-to-liquid lipid ratio.

Table 1

Screening solid lipid and liquid lipid ratio.
Batch No	Solid Lipid (Glyceryl Monostearate)	Liquid Lipid (Capmul MCM)	Entrapment efficiency (%)
PF1	360 (90%)	40 (10%)	35.10 ± 0.89
PF2	320 (80%)	80 (20%)	40.29 ± 1.07
PF3	280 (70%)	120 (30%)	54.10 ± 1.45
PF4	240 (60%)	160 (40%)	43.25 ± 1.56

2.2.2. Screening of NLC preparation parameters

The NLC preparation parameters, such as homogenization time and rotation per minute, were optimized by keeping a constant amount of total lipid, ASPM concentration, and surfactant Gelucire 44/14 as 400 mg a ratio of 7:3 of solid lipid (Glyceryl Monostearate) to liquid lipid (Capmul MCM), 100 mg and 2.5%w/v, respectively and by varying homogenization time and rotation per minute in order to get optimum particle size and entrapment efficiency is illustrated in Table 2.

Table 2

Screening of NLC preparation parameters.
Batch Nos	NLC preparation parameters		Size (nm)	Entrapment efficiency (%)
Batch Nos	Rotation per minute	Homogenization time (min)	Size (nm)	Entrapment efficiency (%)
PF3	10000	10	206.37 ± 3.54	54.10 ± 1.45
PF5	10000	15	242.10 ± 4.25	42.57 ± 1.74
PF6	15000	10	229.44 ± 2.89	38.26 ± 1.32
PF7	15000	15	384.26 ± 5.96	31.77 ± 2.59

2.3. Preparation of ASPM loaded NLCs

NLCs were formulated by adapting high-shear homogenization technique followed by probe sonication. The procedure for formulation was executed in two distinct phases, where one segment comprised a binary blend of lipids and drugs, meanwhile the other segment involved an aqueous solution containing surfactant [24]. The drug and binary lipid amalgamation were melted at a temperature 5℃ above the melting point of the solid lipid. The aqueous phase was heated to the same temperature. Once the aqueous phase and the molten lipid phase are both at the same temperature, combine them. This is usually accomplished by gradually mixing the aqueous phase into the lipid phase while utilizing a high-shear homogenizer (HSH, Kinematica AG, Polytron, Switzerland) to apply high-shear pressures to the mixture. The lipid phase is broken down by the homogenizer into smaller droplets scattered throughout the aqueous phase and further subjected to probe sonication. The temperature was meticulously sustained at a constant level during the emulsification procedure. The resultant lipidic dispersion was cooled to ambient temperature for a duration of 10 minutes to yield ASPM NLCs [25].

2.4. Optimization of ASPM-loaded NLCs using the Central Composite Design

Preliminary experiments were conducted based on prior knowledge, process assessment, and examination of process variables [26]. The evaluation from the initial study showcased that the concentrations of surfactant and drug lipid ratio significantly affected the formulation of NLCs. The Central Composite design comprises a thorough factorial layout involving central points and a sequence of axial points, facilitating the computation of curvature. The impact of independent factors such as Drug lipid ratio (A) and Gelucire 44/14 concentration (B) on dependent factors like particle size (Y1) and entrapment efficiency (Y2) was assessed [27]. Thirteen formulations were prepared, and their effects on the responses were evaluated. The data acquired from the 13 test runs was subjected to analysis utilizing the Design Expert software (Stat-Ease, version 13.0, USA). To impact of independent factors on responses was examined by constructing contour plots and 3-D surface response plots. Multiple linear regression analysis was employed to ascertain the relationship between independent variables and the responses.

The equation fitted was

Y = β0 + β1A + β2B + β12AB + β1²A² + β2²B²

The model's significance was determined through ANOVA, accompanied by a student t-test. The variables' impact on the responses was deemed substantial if the p-value was below the predetermined significance level (p < 0.050), as indicated by the ANOVA findings.

2.5. Evaluation of ASPM loaded NLCs

2.5.1. Particle size and zeta potential

The particle size of ASPM loaded NLCs was determined by Dynamic Light Scattering (DLS) technique using Malvern zeta sizer Zetasizer (Nano-ZS90, Malvern, Worcestershire, UK). The Zeta potential was assessed based on electrophoretic light scattering (ELS)under an applied electric field. Before measurements, ASPM loaded NLCs was diluted ten times with distilled water [28]. Particle size and zeta potential were executed by introducing 1 ml of the sample in a polystyrene cuvette for particle size evaluation, while zeta potential was determined using a disposable folded capillary cell at a temperature of 25 ℃

2.5.2. Entrapment efficiency

The percentage Entrapment Efficiency (EE) of Asenapine Maleate loaded NLCs was determined by employing the centrifugation methodology [29]. The 5 ml of optimized ASPM-loaded NLCs was centrifuged at 15000 revolution per minute at a cold temperature for 15 minutes. After further diluting the resulting supernatant with methanol, the validated HPLC technique was used to quantify the free ASPM. The entrapment efficiency was calculated as EE (%) = [(Total ASPM - Free ASPM) / Total ASPM] x 100, Where the total ASPM is the initial amount of ASPM added formulation and free ASPM is the amount of unencapsulated ASPM.

2.5.3. Transmission electron microscopy

The structural morphology of ASPM-loaded NLCs was carried out utilizing a transmission electron microscope (TEM). After being diluted tenfold with distilled water, the optimized NLCs were placed on a copper coated TEM grid and then stained with suitable electron-dense material, such as phosphotungstic acid (one percent) [30]. Subsequently, the sample was air dried at room temperature and focused onto a photographic film grid for imaging under a transmission electron microscope (Philips, model Tecnai 20, Holland). The study was carried out at a two hundred kV voltage of acceleration and 40,000x resolution. Soft Imaging Systems, located in Reutlingen, Germany, developed the AnalySIS® program, which was used to estimate the dimensions of the ASPM-loaded NLCs.

2.5.4 Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy was utilised for analysing both pure ASPM and the optimized ASPM-loaded NLCs. An IR Affinity 1S FTIR instrument (Perkin Elmer) was used for analyzing a 1 mg sample across a wavenumber range of 4000 to 400 cm-1. The resolution was set to 4 cm-1 while the instrument was in the transmittance mode of operation. The functional groups of optimized ASPM-loaded NLCs were compared with pure ASPM groups.

2.5.6 Differential scanning calorimetry

The thermal characteristics of both pure ASPM and ASPM-loaded NLCs were assessed through the use of differential scanning calorimetry. Each sample was carefully weighed and then heated in a sealed aluminium pan with perforations, at a controlled scanning rate of 20 ℃/min, spanning temperatures from 30 to 220 ℃. Throughout the analysis, nitrogen gas was purged at a flow rate of 50 ml/min to create an inert atmosphere, and the resulting thermogram was recorded for further examination.

2.5.7 Powder X-ray diffraction

The crystallinity of ASPM in NLCs was determined by conducting powder X-ray diffraction on pure ASPM and optimized ASPM-loaded NLC using a Bruker D8 Advance diffractometer. The X-ray pattern was examined using dermic X-ray tube, featuring 2.2 KW copper anode along with a Nickel filter, and a Lynx eye detector. Samples were assessed between 10 and 80 ℃ at a 2θ angle at rate of scanning as 0.30℃/min using X-powder 12 Version-2014.04.37 software.

2.5.8 In-vitro ASPMs release study

The release of ASPM from ASPM-loaded NLCs were investigated through in-vitro experiments. This involved studying the release profile in 1.2 pH 0.1N hydrochloric acid (HCl) for two hours and 7.4 pH phosphate buffer for twenty-four hours. The dialysis technique was employed to evaluate the release kinetics. A volume of 2 ml of ASPMs loaded NLCs corresponding to 10 mg of ASPM equivalent was introduced in a dialysis bag (MWCO 12000Da) immersed in 100 ml of media and positioned on a magnetic stirrer set at a speed of 100 rpm. The temperature was carefully maintained at 37 ± 5 ºC throughout the studies. At predefined time intervals (0.5, 1, 2, 4, 8, 12, 22, 24), 5 ml aliquots were withdrawn and replaced with a equal volume of buffer solution to ensure constant sink condition [33]. The withdrawn aliquots were filtered using membrane filter and analyzed by validated HPLC method. Triplicate measurements were made and the cumulative percentage of ASPM release was calculated.

2.5.9 In-vivo pharmacokinetic study

In-vivo pharmacokinetic study was carried out on healthy male Sprague-Dawley rats with weight ranging from 200 to 250 g, that were kept at the Central Animal Research Facility, Manipal Academy of Higher Education, Manipal. The experimental protocol in the present study was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the Institutional Animal Ethics Committee (IAEC) of Kasturba Medical College, Manipal, under Approval No: IAEC/KMC/06/2044. The rats were accommodated in polypropylene cages under controlled conditions, maintaining a temperature of 25 ± 2°C, and subjected to a 12-hour light-dark cycle. They had ad libitum access to both food and water throughout the study period. After an overnight fast, the rats were divided into two groups containing six animals each. Group I (control group) received solution of pure ASPM (p.o; n = 6) and the second group, Group II (Test group) received optimized formulation containing ASPM-loaded NLCs (p.o.; n = 6), both at a dosage of 5 mg/kg body weight. Serial blood samples were collected from the retro-orbital sinus at various time intervals ranging from 0 to 48 hours in tubes containing anticoagulant (Disodium EDTA). After ten minutes of centrifugation at 5000 rpm, the clear plasma was kept at a temperature of -80 ºC for subsequent investigation [34]. The quantification of Asenapine Maleate in the samples was carried out using an RP-HPLC-based bioanalytical method, and the pharmacokinetic parameters were determined utilizing PK Solution 2.0 software.

2.5.10 Pharmacodynamics studies

The evaluation of pharmacodynamic effects for the optimized formulation was conducted in terms of locomotor activity (LMA) using a psychostimulant-induced hyperactivity model in comparison with plain ASPM drug. Psychostimulant-induced hyperactivity was induced in SD rats by administration of Levodopa and carbidopa, leading to increased locomotor activity (LMA). The impact of the formulations on reverting these conditions was observed. The SD rats were divided into several groups for treatment.

Group 1: Normal control group with no intervention. Group 2: Control group (Positive) receiving L-Dopa (10 mg/kg) and carbidopa (2.5 mg/Kg) intraperitoneally. Group 3: L-Dopa-carbidopa induced rats treated with pure Asenapine solution which was given orally (5 mg/kg) Group 4: L-Dopa-carbidopa induced rats treated with optimised Asenapine loaded NLCs received orally (5 mg/kg).

Locomotor activity was checked at five-minute intervals at one, two, and three hours using a Digital Actophotometer (Ikon Instruments, New Delhi, India). The LMA assessment was conducted one hour after receiving L-dopa-carbidopa in group 2. In groups 3 and 4, pure ASPM was given 30 minutes after L-dopa-carbidopa was administered, and LMA was assessed 30 minutes after Asenapine loaded NLCs were given.

2.6 Statistical analysis

The results are expressed as the mean ± SEM for each group. Statistical differences were evaluated using a one-way analysis of variance (ANOVA). Results were statistically significant at p < 0.05.

3.1. Selection of solid lipids, liquid lipids and surfactants

The encapsulation of ASPM in NLCs was greatly aided by its solubility in lipids (solid and liquid) and surfactants. Figure 2 illustrates the solubility of ASPM in different solid lipids, liquid lipids, and surfactants. It demonstrates that Glyceryl Monostearate has a greater solubility of ASPM than other solid lipids. Similarly, the solubility of ASPM was higher in Capmul MCM as compared with other liquid lipids selected as liquid lipids for NLC preparation. Also, in the solid lipid, Gelucire 44/14 showed ASPMs solubility of 14mg/gram, selected as surfactant for formulation ASPM loaded NLCs.

3.2. Screening of Solid lipid to liquid lipid ratio

In the preliminary screening of solid lipid (Glyceryl Monostearate) to liquid lipid (Capmul MCM) ratio, as shown in Table 1, The entrapment efficiency was found to increase with the rise in liquid lipid concentration up to 30% of the total lipid, possibly due to enhanced miscibility between solid and liquid lipids [36]. The addition of liquid lipids can change the fluidity and crystallinity of lipid matrix thus modifying its physical properties. These modifications can influence the structure and morphology of NLC particles, potentially drug entrapment efficiency, by altering the amount of the drug entrapped within the NLC matrix. Further, increasing the liquid lipid to 40% showed reduced entrapment efficiency, which may be because of both lipids mixing at a specific ratio after that, Capmul MCM remaining as such and not forming the NLC [37]. Considering entrapment efficiency, the solid lipid (Glyceryl Monostearate): Liquid lipid (Capmul MCM) (7:3) ratio was undertaken for further study.

3.3 Screening of NLC preparation parameters

The NLC preparation parameters, such as homogenization time and revolution per minute, were optimized in order to get optimum particle size and entrapment efficiency (Table 2). An increase in High shear homogenization RPM from 10000 to 15000 resulted in a rise in particle size from 229.44 ± 2.89 to 384.26 ± 5.96 since higher shear rates can break down larger particles into smaller fragments. However, these fragments might quickly reaggregate due to the increased surface energy and collisions induced by the high shear environment. The increase in particle size at 15,000 RPM compared to 10,000 RPM is primarily due to enhanced collision frequency and energy, leading to aggregation and coalescence, especially if stabilization mechanisms are insufficient. In comparison to 10000 rpm, the particle size was higher at 15000 rpm, while entrapment efficiency decreased. High shear rates can negatively impact the entrapment efficiency of NLCs primarily due to surfactant desorption, lipid disruption, increased collision frequency, and enhanced diffusion of the drug into the external phase. At fifteen minutes, similar observations were recorded. Thus, for the subsequent optimization of ASPM-loaded NLC using Central Composite Design, fixed levels were defined such as HSH RPM of 10000 was set for 10 minutes and Gelucire 44/14 as a surfactant.

3.4. Optimization by Central Composite Design

A total of thirteen formulations were prepared, and their result data was evaluated using statistical software Design Expert® (Version 13, Minneapolis, MN) Table 3 The purpose of the contour plots and 3-D surface response plots was to examine the impact of independent factors on response patterns. To determine the relationship between the responses and the independent variables, regression analysis was done. The model's significance was examined using an ANOVA followed by student t-test [38].

Table 3

Evaluation of Central Composite design batches
Formulation	Drug Lipid ratio	Surfactant Concentration (%w/v)	Size (nm)	Entrapment Efficiency (%)
F1	4	1	253.2 ± 2.45	37.44 ± 1.65
F2	6	1	199.5 ± 1.65	49.56 ± 2.10
F3	8	1	135.1 ± 4.56	62.54 ± 1.95
F4	4	2.5	187.9 ± 3.54	54.10 ± 1.45
F5	6	2.5	132.1 ± 3.41	66.56 ± 2.23
F6	8	2.5	96.6 ± 1.74	80.73 ± 1.29
F7	4	4	192.0 ± 3.10	42.76 ± 1.06
F8	6	4	174.7 ± 2.03	54.69 ± 2.09
F9	8	4	157.2 ± 2.89	69.53 ± 1.95
F10	6	2.5	128.5 ± 1.15	65.98 ± 2.10
F11	6	2.5	129.4 ± 1.65	66.02 ± 1.76
F12	6	2.5	136.9 ± 1.89	90.11 ± 3.93
F13	6	2.5	130.2 ± 1.36	89.53 ± 4.82

3.4.1. Effect of independent variables on particle size (Y1)

The variation in particle size ranged from 96.6 ± 1.74 to 253.2 ± 2.45, indicating a considerable influence of the selected independent factors on the NLCs' size. The Model F-value of 5.12, with a p-value of less than 0.05 (0.0294), indicates that the model holds significance. The fact that the value of adequate precision is greater than 4 (7.5) suggests a sufficient signal-to-noise ratio. The ANOVA data reveals a notable effect of drug lipid ratio on particle size (p 0.0294). Conversely, the higher p-value (p > 0.1) indicates an insignificant impact of Gelucire 44/14 concentration on particle size. The quadratic terms in the equation demonstrate the presence of non-linearity in the response, further supported by the surface plots. The regression equations of the responses for particle size for factors A and B are given below:

Particle size (Y1) = + 203.7 + 164.4*A + 0.64*B – 0.97*AB -39.72*A2–0.07*B2

As illustrated from equation the surfactant concentration has a generally positive effect on particle size, meaning that increasing the surfactant concentration tends to increase the particle size. However, there is a point at which further increases in surfactant concentration will start to decrease the particle size. The drug-to-lipid ratio also has a positive effect on particle size, though the increase is more gradual. Similar to surfactant concentration, there is a point where higher drug-to-lipid ratios will begin to decrease the particle size. When both surfactant concentration and drug-to-lipid ratio are increased together, their combined effect slightly reduces the particle size compared to their individual effects. To achieve the optimal particle size, both factors must be balanced, considering their individual and combined influences.

The response surface and contour plot further substantiate this effect. The contour plot indicates particle sizes below 130 nm for all concentrations of Gelucire 44/14, as well as at medium and lower levels of surfactant concentration. The smallest particle size is achieved at lower levels of both factors. This conclusion can be inferred from the data.

3.4.2. Effect of independent variables on entrapment efficiency (Y2)

The entrapment efficacy (Y2) of ASPM-loaded NLCs ranged from 42.76%±1.06 to 80.73%±1.29. The findings validate the noteworthy influence of the chosen independent factors (A and B) on the entrapment effectiveness of NLCs loaded with ASPM. The Model F-value of 5.58 with a p-value less than 0.05 (0.0235) indicates that the model holds significance. The analysis of variance (ANOVA) data further demonstrates a substantial effect of sonication time on the entrapment efficacy (p < 0.0200). Conversely, the higher p-value (p > 0.1) suggests a lack of significant effect of Gelucire 44/14 concentration on the entrapment efficacy [39].

The regression equations of the responses for particle size and entrapment efficiency for factors A and B are given below:

Entrapment efficiency (Y2) = + 16.793 + 39.54*A − 0.074*B − 0.027*AB – 7.09*A2 + 0.13*B2

According to equation, it is evident that factor A has positive influence and B independent variables has negative influences on entrapment efficiency. However, the Gelucire 44/14 showed a stronger positive effect on entrapment efficiency as compared to the drug lipid ratio. As the drug Gelucire 44/14 concentration increases, the entrapment efficiency increases. At lower Gelucire 44/14 concentration and low drug lipid ratio showed a decrease in entrapment efficiency; further improvement in the concentration of A and time of B showed a increase in entrapment efficiency, which can be because to higher cohesive forces between Gelucire 44/14 molecules at high concentration leads to the exclusion of ASPM from NLCs. At low drug-lipid ratios, entrapment efficiency might be suboptimal due to insufficient drug loading, resulting in excess lipids without effectively encapsulating the drug. Conversely, at high drug-lipid ratios, entrapment efficiency might decrease due to saturation of lipid carriers, leading to drug expulsion or poor encapsulation efficiency. This effect was further supported by the contour plots. Model selection for response analysis was guided by sequential model sum of squares, lack of fit, and model summary statistics. The quadratic model was favoured for both responses due to a p-value of < 0.0001, low standard deviation, high R-squared value, and a lower Predicted Residual Error Sum of Squares (PRESS). With a Model Prob > F value of less than 0.05, the experimental data's analysis of variance (ANOVA) further supported the model's significance.

The influence of the selected independent variables on the dependent variable was elucidated using contour plots. These plots indicated that maintaining Gelucire 44/14 (A) within the 0 to 1 level range and Drug to lipid ratio (B) within the − 1 to 1 level range (with a preference for higher levels) could achieve the desired particle size (< 200 nm). To achieve a target drug release percentage of over 50% after 24 hours, it was recommended to select Gelucire 44/14 (A) levels ranging from − 0.5 to + 0.5. The contour plots highlighted that the surfactant concentration (Gelucire 44/14) has a more significant impact on the size and entrapment efficiency of NLCs compared to the lipid mixture.

3.5. Particle size, zeta potential, and entrapment efficiency

The optimized ASPM-loaded NLC exhibited a particle size of 97.6 ± 2.48 nm (Fig. 4a). It exhibited a single narrow peak indicating the NLCs possess particle size within the nanometer range, ensuring stability and preventing aggregation. Additionally, the polydispersity index (PDI) was 0.20, indicating monodispersity. A lower PDI value signifies a higher degree of homogeneity. Moreover, the zeta potential of NLCs was − 20mV (Fig. 4b); numerous studies have demonstrated the influence of zeta potential on the stability of NLCs, with a higher value indicating electrostatic repulsion between particles. The theory of DLVO states that the repulsion caused by the electric double-layer serves to stabilize the NLC, thereby preventing aggregation from occurring as a result of the particles' notably negative charge [40]. The entrapment efficiency was 90.1%, which was influenced by solid lipid to liquid lipid ratio.

3.6. Transmission electron microscopy

A transmission electron microscope (TEM) was used to examine the morphology of optimized ASPM-loaded NLCs. NLCs were uniformly distributed and were spherical in shape (Fig. 5). The values obtained for the particle size were in agreement with the range of particle sizes shown in the TEM image.

3.7. Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy was employed to examine both pure ASPM and the optimized ASPM-loaded NLC to identify any chemical interactions between the drug and lipid. As Illustrated in Fig. 6, FTIR spectra of Asenapine Maleate showed distinct peaks at particular wavenumbers: 2047 cm⁻¹ for the –C–O bond, 1614 cm⁻¹ for the aryl C = C bond, 1572 cm⁻¹ and 1483 cm⁻¹ for the = C–H functional groups of the aryl ring, and 2960 cm⁻¹ for the sp³ C–H stretch. Furthermore, the ASPM-loaded NLC exhibited identical peaks to ASPM, indicating no chemical interaction between Asenapine Maleate and the lipids.

3.8. Differential scanning calorimetry (DSC)

The thermal properties of pure ASPM and ASPM-loaded NLCs were investigated using differential scanning calorimetry. Shown in Fig. 7, ASPM displayed a distinct endothermic peak at 146℃ with an enthalpy fusion (∆H) of 105 J/g, signifying its crystalline nature. However, the disappearance of ASPM peaks in the NLC formulation indicated the transformation of the drug from crystalline to amorphous states, with ASPM being distributed at a molecular level within the lipid matrix in NLC.

3.9. X-ray diffraction (X-RD)

X-ray diffraction analysis was conducted on pure ASPM and the optimized ASPM-loaded NLC using a Bruker D8 Advance diffractometer to assess the crystallinity of ASPM within the NLCs. The XRD pattern, depicted in Fig. 8, revealed sharp peaks at diffraction angles 2θ (9.50, 20.40, 21.90) corresponding to Asenapine Maleate. The XRD pattern of Asenapine Maleate-loaded NLCs exhibited distinct characteristics. Importantly, no prominent diffraction peaks were observed in the examined 2θ range, indicating the absence of well-defined crystalline structures within the NLCs.

3.10. In-vitro ASPM release

The in-vitro ASPM release studies were carried out for pure ASPM and optimized ASPM-loaded NLC in Phosphate buffer having a pH 7.4 using the dialysis technique. The data depicted in Fig. 9, illustrates that pure ASPM exhibited a 15% release within a 24-hour timeframe, a phenomenon potentially attributed to its limited aqueous solubility. Conversely, the optimized ASPM loaded NLC displayed a biphasic release profile, characterized by an initial burst followed by sustained release over 24 hours. The 20% initial burst release in 8 hrs was due to the release of ASPM molecules situated or close to the surface of the NLCs. After burst release, it demonstrated a sustained drug release pattern for up to 24 hrs (75% release), indicating the diffusion of encapsulated ASPM from the NLC lipid matrix. The sustained release phase demonstrates the potential of the NLCs to provide prolonged ASPM release, which is desirable for maintaining therapeutic drug levels over an extended period and has significant clinical implications, particularly in the treatment of Schizophrenia [41]. This may result in improved patient compliance and reduced side effects associated with fluctuating drug concentrations.

3.11 In vivo Pharmacokinetic Studies

The mean plasma concentration-time data for the pure drug and ASPM loaded NLCs in SD rats on oral administration of ASPM at a dose of 5 mg/kg is depicted in Fig. 11, along with the corresponding pharmacokinetic parameters outlined in Table 4. The marketed sublingual tablet of ASPM demonstrates an elimination half-life (t_1/2) of around 24 hours; in contrast, the t_1/2 values were recorded at 33.6 ± 6.9 hours for the pure drug and 41.06 ± 11.8 hours for NLCs after oral administration in rats. The average residence time of ASPM was calculated to be 47.53 ± 7.99 hours for the pure drug and 55.035 ± 7.06 hours for ASPM-NLCs. ASPM-NLCs exhibited a significant increase in AUC_0 − t in comparison to pure drug (81169.693 ± 4195.6 vs 3353.09 ± 246.6 h*ng/ml; p < 0.001). The time required by the drug to reach peak plasma drug concentration was notably extended in ASPM-NLCs (8 hours) versus the pure drug (1 hour). The pharmacokinetic profile of Asenapine loaded NLCs show two distinct peaks at one hour and eight hours; but the peak concentration (C max) of 6426.0 ± 1947.1 ng/ml was seen at eight hours (Fig. 10). ASPM-NLCs showed a considerable rise in AUC_0 − t in comparison to the pure drug, possibly because of its nano size and evasion of first-pass metabolism by absorption via the lymphatic route. The initial peak at 1 hour in ASPM-NLCs may be due to release of surface adsorbed drug and unentrapped-free drugs and followed rapid absorption. The gradual release of a drug from Asenapine loaded NLCs, leading to a peak concentration at around 8 hours, is a result of increase in permeability by the use of surfactants, leading to enhanced absorption. This mechanism enhances the pharmacokinetic profile by improving solubility, bioavailability, maintaining therapeutic levels, reducing dosing frequency, and minimizing side effects.

Table 4

The pharmacokinetic parameters of asenapine maleate and asenapine maleate- nanostructured lipid carriers when administered orally in rats.
Parameters	ASPM	ASPM NLCS
C max (ng/mL)	313.82 ± 70.5	6426.0 ± 1947.14***
T max (h)	1 ± 0	8 ± 0***
AUC_0 − t (h*ng/ml)	3353.09 ± 246.60	81169.693 ± 4195.6***
AUC_0−∞ (h*ng/ml)	6248.11 ± 813.42	136746.861 ± 3137.49***
t1/2 (h)	33.67 ± 6.98	41.065 ± 11.82***
MRT _0−∞	47.53 ± 7.99	55.035 ± 7.06***
All values are presented as mean ± SD, n = 6; ***p < 0.001. ASPM: Asenapine maleate; AUC: Area under the curve; MRT: Mean residential time; NLC: Nanostructured lipid carrier; t1/2: Elimination half-life;

3.12. Pharmacodynamics studies

The locomotor activity was found to be normal in Control group of animals when measured by using digital Actophotometer, with locomotor counts of 378 ± 32 after one hour, 380 ± 20 after two hours, and 404 ± 29 after three hours. Baseline LMA values were recorded in untreated animals (control) which provides a reference point for comparison with other test groups. Positive control group received intraperitoneal injection of 2.5mg/kg and 10mg/kg of carbidopa and L-DOPA. It was observed that the LMA in positive control animal group was significantly elevated (p < 0.001), with peak locomotor counts of 572 ± 48 after one hour, 685 ± 38 after two hours, and 705 ± 50 after three hours, in contrast to the baseline LMA levels (Fig. 12). Reduction in LMA after administration of the pure drug, at the identical dose of 5 mg/kg as asenapine, resulted in a noteworthy decrease in locomotor count (1 hour: 122 ± 41, 2 hours: 134 ± 32, 3 hours: 100 ± 30) in comparison to the positive control animal group and normal control animal groups, which was found to decrease further with Asenapine maleate loaded NLCs (1 hour: 50 ± 10, 2 hours: 31 ± 20, 3 hours: 35 ± 09). The marked decrease in induced LMA was seen with Asenapine maleate loaded NLCs implies enhanced pharmacodynamic efficacy of NLCs containing ASPM when compared with the pure drug.

The development and optimization of ASPM-loaded NLCs were effectively executed in the current study, employing a Central composite design with Glyceryl Monostearate as the solid lipid, Capmul MCM as liquid lipid and Gelucire 44/14 as the surfactant. The optimized ASPM-loaded NLCs demonstrated favourable characteristics in terms of particle size (97.6 ± 2.48 nm), zeta potential (-20mV), and entrapment efficiency (96.74%). Notably, Asenapine maleate loaded NLC exhibited an initial burst release of 20%, succeeded by sustained release of 75% over a 24-hour period. Furthermore, pharmacokinetic assessments conducted in vivo indicated a substantial 22-fold enhancement in bioavailability with the developed ASPM-loaded NLCs, alongside a significant reduction in L-DOPA-carbidopa-induced locomotor count in comparison to the pure drug in Sprague Dawley rats. Thus, the successful development of Asenapine maleate loaded NLCs exhibited improved performance both in in vitro and in vivo.

Acknowledgements:

The authors are grateful to Sanofi India Ltd for providing facilities to characterize the formulation.

Funding statement:

This work does not involve any funding sources

Informed consent. All authors have seen and approved the manuscript for submission

Research involving Human and Animal statement: Yes

Ethics approval consent to participate:

Animal studies were carried out on healthy male Sprague-Dawley rats with weight ranging from 200 to 250 g, that were kept at the Central Animal Research Facility, Manipal Academy of Higher Education, Manipal. The experimental protocol in the present study was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the Institutional Animal Ethics Committee (IAEC) of Kasturba Medical College, Manipal, under Approval No: IAEC/KMC/06/2044.

Conflict of Interest:

The author declares no conflict of interest.

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Tailoring Nanostructured Lipid Carriers for Efficient Delivery of Asenapine Maleate in Antipsychotic Treatment

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Selection of solid lipids, liquid lipids and surfactants

2.2.1. Screening of Solid lipid to liquid lipid ratio

2.2.2. Screening of NLC preparation parameters

2.3. Preparation of ASPM loaded NLCs

2.4. Optimization of ASPM-loaded NLCs using the Central Composite Design

2.5. Evaluation of ASPM loaded NLCs

2.5.1. Particle size and zeta potential

2.5.2. Entrapment efficiency

2.5.3. Transmission electron microscopy

2.5.4 Fourier transform infrared spectroscopy

2.5.6 Differential scanning calorimetry

2.5.7 Powder X-ray diffraction

2.5.8 In-vitro ASPMs release study

2.5.9 In-vivo pharmacokinetic study

2.5.10 Pharmacodynamics studies

2.6 Statistical analysis

3. Results and discussion

3.1. Selection of solid lipids, liquid lipids and surfactants

3.2. Screening of Solid lipid to liquid lipid ratio

3.3 Screening of NLC preparation parameters

3.4. Optimization by Central Composite Design

3.4.1. Effect of independent variables on particle size (Y1)

3.4.2. Effect of independent variables on entrapment efficiency (Y2)

3.5. Particle size, zeta potential, and entrapment efficiency

3.6. Transmission electron microscopy

3.7. Fourier transform infrared spectroscopy

3.8. Differential scanning calorimetry (DSC)

3.9. X-ray diffraction (X-RD)

3.10. In-vitro ASPM release

3.11 In vivo Pharmacokinetic Studies

3.12. Pharmacodynamics studies

4. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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