Formulation, optimization, in vitro and in vivo evaluation of Nateglinide-loaded nanostructured lipid carriers for enhanced bioavailability.

doi:10.21203/rs.3.rs-4328639/v1

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Formulation, optimization, in vitro and in vivo evaluation of Nateglinide-loaded nanostructured lipid carriers for enhanced bioavailability.

https://doi.org/10.21203/rs.3.rs-4328639/v1

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Purpose

Nateglinide belongs to the meglitinide class, oral hypoglycemic drug used in the treatment of insulin-resistant (Type II) diabetes mellitus. Potential constraints associated with NTG delivery include poor aqueous solubility, short action time, and quick elimination, which causes variable bioavailability. Therefore, the aim of the present study was to develop and optimize NLCs formulations to improve the oral bioavailability and efficient delivery of NTG.

Method

NLCs were prepared by a modified HPH method using a box Behenken design approach. Glyceryl Monostearate and Miglyol 812, Acrysol EL 135 were chosen as solid lipid, liquid lipids, and surfactant respectively. Obtained NLCs were characterized for physicochemical properties, in-vitro drug release studies and pharmacokinetic parameters.

Result

NTG-NLCs exhibited small particle size ranging from 142.8 ± 1.67 to 252.7 ± 2.17 nm zeta potential in the ranging from 13.53 mV to 30.93 mV, Polydispersibility index of 0.343±0.071 to 0.417 ±0.058. The average encapsulation efficiency for the NLCs was 89.99%. Optimized NTG-NLC showed particle size 142.8 nm, zeta Potential, 30.93 mV, drug loading 16.04%, and entrapment efficiency 93.48 %. In a pharmacokinetic study, the relative oral bioavailability of nateglinide-NLC was increased by 3.77 times than that of pure nateglinide and 1.54 times than Glinate 60 marketed nateglinide formulation. The half-life of the drug was prolonged by 1.6 times. The solubility and bioavailability of nateglinide is enhanced, coupled with its prolonged release.

Conclusion: NTG-NLC prepared by the modified HPH method is a promising technique to enhance in vitro drug release, bioavailability, and pharmacokinetics.

Box-Behnken design

Nateglinide

bioavailability

dissolution

nanostructured lipid carriers

poorly water-soluble drug

Nateglinide (NTG) is often employed as a medication for oral administration to treat insulin-resistant diabetes, specifically Type II diabetes mellitus. It is a commonly prescribed drug for this purpose. [1]. NTG is a BCS II drug that exhibits relatively poor water solubility and variable bioavailability [2]. As a non-sulfonylurea antidiabetic drug, it rapidly decreases blood glucose levels compared with sulfonylurea [3, 4]. NTG can be utilized as a standalone therapy or in combination with other hypoglycemic agents, presenting an elegant, adaptable, and effective treatment solution for lowering postprandial glucose levels in individuals with type II diabetes. [5, 6].

Drug solubility is a crucial factor in enhancing the oral bioavailability of practically insoluble drugs such as NTG [7]. NTG is an amino acid derivative that triggers insulin secretion from pancreatic β-cells by obstructing potassium ATP channels, which in turn decreases blood glucose levels. This action is dependent on the function of beta cells in the pancreatic islets. NTG is quickly absorbed by the gastrointestinal tract and undergoes rapid clearance, with a half-life of approximately 1.5 hours. [7, 8].

Nateglinide is one of the most efficient antidiabetic drugs; it is lipophilic, that belongs to the meglitinide family, and is recommended at a dose of 60 mg or 120 mg [9]. However, it has low bioavailability, extremely low water solubility (around 0.061 mg/ml), a pKa of 3.1 and a log P value of 4.2 these are significant obstacles to preparing an oral dosage form of nateglinide. Previous research have indicated that NTG exhibits variable bioavailability and pH-dependent solubility.[10]. Literature suggests that its solubility is lower at acidic pH levels and higher at alkaline pH levels. Complete dissolution of the drug in the acidic pH of the stomach environment is necessary to achieve quick oral absorption and rapid achievement of a bioavailable dose of NTG. Addressing pH-dependent solubility is a crucial consideration when designing dosage forms for NTG [11–12]. Therefore, the major obstacle to formulating a drug delivery system that is capable of addressing these issues comprehensively and for enhancing the bioavailability, retention of the drug within the body, and pharmacological potential.

Numerous technologies and polymers can be used to augment the dissolution characteristics and bioavailability of drugs with limited solubility. A lipid-based formulation is a other potential strategy for developing a product from a laboratory scale to a market level. Lipid-based scaffold are impending drug carriers because of their ability to increase lipophilic drug solubility and eventually, oral bioavailability, thereby achieving sustained and controlled drug delivery. [13]

Nanoparticle drug delivery systems have emerged as increasingly prominent technologies. NLCs represent the next generation of lipid nanoparticles. NLCs create unstructured matrices, which improve drug loading and minimize drug exclusion during storage. NLCs have been introduced as cutting-edge pharmaceutical delivery systems as lipid-based nanoparticles. Their exceptional solubilization and dispersal capabilities have made them a promising nano carrier, leading to potential oral drug delivery.[14–16]

Lipids incorporated into NLC offer numerous benefits, including excellent solubilization potential, straightforward and easy manufacturing processes, biodegradable, biocompatibility, the capability to improve the oral bioavailability of hydrophobic drugs, and controlled-drug release. These lipids improve the bioavailability of drugs by overcoming a number of physiological barriers, including enzymatic degradation, P-glycoprotein (P-gp) mediated efflux, drug metabolism by enzyme cytochrome P450, hepatic metabolism, gastrointestinal drug degradation, and permeability problems. They also help to deliver the drug to the intestinal lymphatic transport thereby prevent first pass metabolism. [17–19]

As NLC show excellent solubilization and dispersing capabilities,, protecting the drug from degradation, NLC have emerged as a promising nanocarrier widely employed in oral drug delivery. Following oral administration, NLCs showed superior controlled release within the gastrointestinal tract compared to other lipid-based formulations. NLCs maintain adequate solubility at the site of intestinal absorption for poorly water-soluble drugs, which is attributed to their small particle size and lipid solubilization. [20] The nanosized nature of NLCs contributes to an augmented surface area, improving drug solubility, protecting the drug from degradation, facilitating enhanced interactions with biological systems, and potentially enhancing absorption. NLCs are nontoxic, biodegradable, and biocompatible drug delivery systems. NLCs have potential advantages, including controlled release, increased drug stability, improved bioavailability, and potential for targeted delivery to specific tissues. [21–22]

Hence, the current investigation was aimed at exploring the development and optimization of NTG-NLCs using a modified high-pressure homogenization method employing the Box-Behnken design. The prepared NLCs were characterized for various parameters including entrapment efficiency, drug loading, zeta potential, PDI, and in vitro drug release study. Subsequently, NTG-NLCs were evaluated in Wistar rats to confirm their enhanced oral bioavailability. NLCs can potentially enhance drug solubility and bioavailability.

1.1. Materials

NTG was received as a gift from Cipla Ltd. Goa, India. Labrasol and Transcutol HP were purchased from Gattefosse India Pvt. Ltd., Mumbai, Maharashtra, India. Acrysol EL 135 was provided by Corel Pharma (Ahmedabad, Gujrat, India) as a gift. Glyceryl monostearate (GMS), Miglyol 812, Tween 80 were purchased from Sigma Aldrich, Mumbai. Ethanol/methanol was purchased from Himedia Laboratories, Mumbai, India. All other solvents and materials used were of analytical grade.

1.2. Optimization of NTG-NLC

1. Experimental design

To examine the effect of different independent variables on the dependent characteristics of (NLCs), the NTG loaded NLCs were developed using the Box-Behnken design. In this approach, three factors and four levels produced a total of 17 batches, the details of which are presented in Table 1. The compositions are listed in Table 2. The design of experiments was conducted using Design Expert® 12.1 software (Stat-Ease Inc., Minneapolis, MN, USA). [23]

Table 1

Factors and responses used in Box–Behenken design
Factors ( Independent variable )	Level Used
Factors ( Independent variable )	Low (− 1)	Medium (0)	High (+ 1)
X1: Percentage of total lipid	1%	1.5%	2%
X2: Ratio of the liquid lipid to the solid lipid	15	30	45
X3: Concentration of surfactant	1%	1.5%	2%
Responses ( Dependent variable )	Goal
Y1: Particle Size	Minimize
Y2: Zeta Potential	Maximize
Y3:Entrapment Efficiency %	Maximize
Y4: Drug Loading %	Maximize

Table 2

Formulation composition of NTG- loaded NLC (N1 to N17)
Formulation	NTG (% w/v)	Total Lipid (% w/v)	Liquid lipid: Solid lipid (weight ratio)	Acrysol EL 135 (% v/v)	Water ml
N1	0.05	1.5	15:85	1	50
N2	0.05	1.5	30:70	1.5	50
N3	0.05	1.5	15:85	2	50
N4	0.05	2	30:70	2	50
N5	0.05	1	30:70	1	50
N6	0.05	2	15:85	1.5	50
N7	0.05	1.5	30:70	1.5	50
N8	0.05	1.5	45:55	1	50
F9	0.05	1.5	30:70	1.5	50
N10	0.05	2	45:55	1.5	50
N11	0.05	1	45:55	1.5	50
N12	0.05	1	30:70	2	50
N13	0.05	1.5	45:55	2	50
N14	0.05	2	30:70	1	50
N15	0.05	1.5	30:70	1.5	50
N16	0.05	1	15:85	1.5	50
N17	0.05	1.5	30:70	1.5	50

Preparation of NTG-loaded Nanostructured Lipid Carriers

The preparation of NTG-NLCs was achieved through a modified high-pressure homogenization technique [24]. GMS served as the solid lipid, while miglyol 812 and Acrysol EL 135 were used as the liquid lipid and surfactant, respectively. The liquid lipid phase consisted of NTG (0.05% w/v) dissolved in miglyol 812, which was thoroughly mixed with the melted GMS to form the lipid phase. The aqueous phase was created by dissolving Acrysol EL 135 in water, and the aqueous phase was then combined with the lipid phase. The mixture was homogenized using a mechanical stirrer ((Model: 1MLH, REMI Labworld, Mumbai, India) at 1500 RPM for 30 minutes, resulting in the formation of a pre-emulsion. The pre-emulsion was then run through a high-pressure homogenizer (Nitro, GEA Panda plus 2000, USA) at 800–900 bar for 5–6 cycles to create optically clear and homogeneous NLC suspensions. Finally, the resultant NLC suspension was lyophilized using freeze dryer (C-Gen Biotech) and used for characterization. The formulation batch details are presented in Table 2. [25]

Characterization of NTG-NLCs

Particle size, Zeta potential and Polydispersity index (PDI)

The particle size, zeta potential, and polydispersity index (PDI) of NTG-NLCs were determined using the dynamic light scattering technique with a Zetasizer Nano Plus (Malvern Instruments, Worcestershire, UK). The particle size and zeta potential were measured after diluting the NLCs dispersion ten times with distilled water at 25°C. The measurement was performed with a standard laser (4 mW He–Ne, 633 nm) at a fixed angle of 90°. The polydispersity index was calculated to accurately estimate the distribution of particle size for the NLC formulations. [26, 27]

Percent Entrapment efficiency (% EE) and Drug loading (DL)

The evaluation of EE and DL of NLCs was carried out by determining the concentration of free NTG and total NTG in the NLCs dispersion. About 2 ml of NTG-loaded NLCs dispersion underwent centrifugation at 10,000 RPM for 45 minutes to separate the aqueous and lipid phases. To determine the free drug concentration in the aqueous phase, 1 ml of supernatant was diluted with methanol, filtered through 0.45-µm filter, and the drug concentration was estimated by UV spectrophotometer at 210 nm. To determine the concentration of total drug nateglinide in NLCs, 1 ml of NLCs dispersion was lyophilized using a freeze dryer. The lyophilised NLC was then re-suspended in methanol, and the concentration of NTG was determined in NLCs after dilution at 210 nm. The percentage entrapment efficiency (EE %) and drug loading (% DL) were determined using the following equations [28].

% Entrapment Efficiency = \(\frac{\mathbf{W}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t} \mathbf{o}\mathbf{f} \mathbf{t}\mathbf{o}\mathbf{t}\mathbf{a}\mathbf{l} \mathbf{d}\mathbf{r}\mathbf{u}\mathbf{g} – \mathbf{W}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t} \mathbf{o}\mathbf{f} \mathbf{F}\mathbf{r}\mathbf{e}\mathbf{e} \mathbf{d}\mathbf{r}\mathbf{u}\mathbf{g} }{\mathbf{W}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t} \mathbf{o}\mathbf{f} \mathbf{t}\mathbf{o}\mathbf{t}\mathbf{a}\mathbf{l} \mathbf{d}\mathbf{r}\mathbf{u}\mathbf{g}}\) X 100%

% Drug Loading = \(\frac{\mathbf{A}\mathbf{m}\mathbf{o}\mathbf{u}\mathbf{n}\mathbf{t} \mathbf{o}\mathbf{f} \mathbf{e}\mathbf{n}\mathbf{t}\mathbf{r}\mathbf{a}\mathbf{p}\mathbf{p}\mathbf{e}\mathbf{d} \mathbf{d}\mathbf{r}\mathbf{u}\mathbf{g}}{\mathbf{W}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t} \mathbf{o}\mathbf{f} \mathbf{N}\mathbf{L}\mathbf{C} \mathbf{f}\mathbf{o}\mathbf{r}\mathbf{m}\mathbf{u}\mathbf{l}\mathbf{a}\mathbf{t}\mathbf{i}\mathbf{o}\mathbf{n}}\) X 100%

Solid State Characterization of Freeze-Dried NTG-NLCs

I. Fourier Transform Infrared Spectroscopy

The interaction between the drug and excipients in the optimized freeze-dried NTG-NLCs was analyzed by recording their FTIR spectra on the FTIR spectrophotometer (Shimadzu 8400S, Japan), KBr used as the reference standard. The spectral range was set from 4000–400 cm − 1 to examine the effects of NLC formulation on the pure NTG structure. [29]

II. Differential Scanning Calorimetry

To evaluate the crystal structure of NTG and the optimized freeze-dried NTG-NLCs, a DSC analysis was conducted using the DSC instrument Mettler Toledo, Japan, and STAR-SW10 software. The instrument was calibrated for heat flow and temperature. About 2–3 mg of each NTG and the optimized lyophilized NTG-NLCs were separately placed in an aluminum pan and equilibrated at 25°C. The DSC run was performed at a heating rate of 10°C/min over a temperature range of 10° to 400°C, with a scanning rate of 10°/min. [30]

III. Powder X-ray diffraction analysis (PXRD)

PXRD was employed to analyze the crystallinity of NTG and NTG-NLCs. This study was conducted using an X-ray diffractometer, the Miniflex II (Regaku, USA), equipped with a PWR 30 X-ray generator. Operating parameters included a voltage of 40 kV and a current of 25 mA, utilizing the Cu K α line at 1.54056 A° as the radiation source with nickel filter. [31]

IV. Scanning Electron Microscopy (SEM)

SEM was performed to study and analyze the surface morphology of the NTG- NLCs. ZEISS Ultra 55 scanning electron microscope from Carl Zeiss SMT GmbH, Germany was used to examine the NLC. The system was attached with an energy dispersive X-ray (EDX) Oxford ISIS 300 micro-analytical system. Initially samples were diluted with Milli Q water, placed on carbon grid, air-dried at room temperature and coated with chrome. The samples were covered with carbon coating to enhance the electron beam conductivity. Probe current of 45 nA, accelerating voltage of 20 kV, and a counting time of 60 seconds was used during the scan. This technique was employed to observe the external macroscopic structures of the NTG-NLC.[32]

In vitro drug release

The in vitro drug release assessments of NTG from pure NTG suspension, marketed formulation Glinate 60 suspension and optimised NTG‑NLCs suspension was conducted employing the dialysis bag technique. Phosphate buffer pH 7.4 was used as release media for in vitro drug release study. The dialysis bags having a molecular weight 12,000–14,000 Daltons (Himedia-Dialysis membrane 135, Mumbai, India) were preconditioned in the release media (Phosphate buffer pH 7.4) for 24 hours to activate it prior to the study.

To examine the in vitro release profile of NTG, 10 ml of freshly prepared NTG suspension, Glinate 60 suspension and NTG‑NLCs suspension equivalent to 5 mg NTG were added into the separate dialysis bags and tightly sealed with tread from both sides. The dialysis bags were suspended in the separate beaker containing 100 ml of phosphate buffer pH 7.4. These beakers were placed on a magnetic stirrer at 150 rpm, maintaining a temperature of 37°C.

1ml of release media was withdrawn at a predetermined time intervals including 0, 0.5, 1, 1.5, 2, 4, 6, 8, 12 h and an equal amount of fresh buffer was replaced in the beaker to maintain a consistent sink condition. Meanwhile, the release of free NTG from NTG suspension (0.5 mg/ml) was performed in the similar manner. The release studies were performed in triplicate. The samples were subsequently diluted and drug release was quantified by using UV Spectrophotometer at 210 nm. [33–35]

In vivo pharmacokinetic study for optimised NTG-NLC

In vivo pharmacokinetic assessment were performed to evaluate the bioavailability of nateglinide. The study compared the plasma profiles of pure drug NTG suspension, Glinate 60 suspension, and the optimized NTG‑NLC suspension. [36, 37]

The experimental methodology included oral administration of these formulations to the wistar rats of both sex weighing 200 to 250 gm. All animal-related experiments, procedures adhered to the approved guidelines and were conducted with the approval of the committee overseeing experiments on animals (CPCSEA). The approval number for these protocols was 1138 /PO/RCS/RCNRCL/S/08/CPCSEA.

Animals were separated into four groups, each group comprises of six wistar rats. These groups received a streptozotocin single intraperitoneal injection at a dose of 65 mg/kg dissolved in phosphate buffer at pH 7.4. This injection was intended to induce type II diabetes in the rats.

NTG pure drug as well as formulations were orally administered to the wistar rats at a dose of 10.6 mg/ kg of samples. The animals underwent an overnight fasting with continuous access to water throughout the study. [37–38]

The first group received NTG suspension prepared by dissolving the NTG pure drug in a 0.5% sodium carboxymethylcellulose (CMC-Na) solution. The second group was administered the optimized NTG‑NLC dispersion, while the third group received the Glinate-60 suspension (a marketed formulation of nateglinide) via oral gavage. The fourth group served as the control group without any treatment.

Blood samples, 0.5 ml each, were collected through the retro-orbital plexus at predermined intervals (0, 0.5, 1, 1.5, 2, 4, 6, 8, and 12 hours) and placed into microcentrifuge tubes containing 2.6 mol sodium edetate in order to prevent coagulation. These collected blood samples were then centrifuged at for 15 minutes at 5000 RPM to separate the plasma. The obtained plasma samples were kept in a deep freezer until NTG content could be ascertained through further analysis. The objective of this study was to assess the plasma profiles and concentrations of NTG and its formulations over time in the rat model. [37, 39]

Bioanalytical method:

In this investigation, chromatographic separation was carried out using a formerly validated chromatographic method. NTG was extracted from plasma samples by mixing 1 ml of acetonitrile, followed by centrifugation. The NTG concentration in the resultant supernatant plasma was measured with high-performance liquid chromatography (HPLC), Agilent 1260, Santa Clara, USA.

For the chromatographic separation BDS Hypersil, C18 column (250 mm× 4.6mm with a particle size of 5µm, Thermo scientific, Waltham, MA, USA) was utilised. Mobile phase encompass of 65:35 v/v mixture of acetonitrile and phosphate buffer pH 3.0 (pH adjusted with diluted ortho-phosphoric acid), and the flow rate was adjusted at1 ml/min. The effluents were analysed at wavelength 210 nm using Photodiode array detector.

This method facilitated the quantification and analysis of NTG concentrations in the plasma samples, enabling precise measurement and assessment of the drug in the biological samples. [38–41]

Pharmacokinetic analysis:

Data acquisition was conducted using PKSolver 2.0 software. Various pharmacokinetic parameters including peak plasma concentration time (Tmax), highest plasma concentration (Cmax), and area under the curve (AUC 0-t) were derived from the plasma drug concentration-time graph from 0 to 24 hours. Linear trapezoidal rule was used for determination of the area under the curve (AUC).

The plasma drug concentration–time plot was used to determine the maximum concentration of nateglinide in plasma (Cmax), time required to achieve the peak plasma concentration (Tmax), volume of distribution and half-life. Furthermore the relative bioavailability (F) of NTG-NLC was assessed by establishing the ratio between the AUC of NTG-NLC and AUC of NTG suspension.[41, 42]

Relative Bioavailability (F)

Relative bioavailability is a measure of the bioavailability, often estimated as the area under the curve (AUC), of a test formulation of a specific drug in comparison to a reference formulation of the same drug. This comparison provides insights into how well the test formulation performs concerning absorption and availability in the body relative to a known or standard formulation of the drug.

Relative Bioavailability (F) = \(\frac{\mathbf{A}\mathbf{U}\mathbf{C} \mathbf{S}\mathbf{t}\mathbf{a}\mathbf{n}\mathbf{d}\mathbf{a}\mathbf{r}\mathbf{d}}{\mathbf{A}\mathbf{U}\mathbf{C} \mathbf{T}\mathbf{e}\mathbf{s}\mathbf{t}}\) × 100%

I. Solubility of Nateglinide in liquid lipids

The process of selecting suitable lipids and other excipients holds significant importance while developing NLCs, particularly for poorly soluble drug like nateglinide.

NTG solubility in lipids is significant for achieving elevated drug loading, ensuring its efficient integration within the lipid matrix.

For nateglinide, an assessment of its saturation solubility was conducted across various lipids. Lipids showing good saturation solubility were selected for the formulation of NLC. Solubility of pure NTG was found highest in transcutol HP, miglyol 812 and acrysol EL 135. NTG saturation solubility in various liquid lipids is depicted in Table 3.

Table 3

Saturation solubility of Nateglinide in different liquid lipids
Lipid excipients	Solubility (mg/g)
Transcutol HP	202.34 ± 1.78
Miglyol 812	168.65 ± 1.02
Acrysol EL 135	176.18 ± 1.14
Labrasol	78.86 ± 2.05
Tween 80	99.96 ± 2.26
PEG 400	86.72 ± 2.26

II. Solubility of Nateglinide in Solid Lipids

Solubility of drug within the lipid matrix significantly impacts the entrapment of NTG in NLC. Additionally, the composition of surfactants, lipid in NLC has significant impact on particles in NLC. To ascertain these factors, extensive solubility studies were conducted to evaluate NTG solubility in various solid lipids and liquid lipids before formulating NTG–NLC.

Saturation solubility of NTG was found notably higher in Glycerol mono stearate, therefore it is selected as solid lipid to formulate NTG-NLC. NTG solubility in different solid lipids is depicted in Table 4.

Table 4

The solubility of nateglinide in various solid lipids (Mean ± SD, n = 3)
Solid Lipid excipients	Solubility (mg/g)
Glycerol mono stearate	68.76 ± 3.24
Hydrogenated vegetable oil.	37.14 ± 3.46

Evaluation of Nateglinide Loaded Nanostructured Lipid Carriers

I. Entrapment Efficiency (EE %) and Drug loading (DL %)

NLC formulations exhibited excellent entrapment efficiency ranging from 85.48 to 92.14%. The average entrapment efficiency of all the NTG-NLC was found 89.39%. Optimised NLC is having 85.48% entrapment efficiency. There was remarkable increase in entrapment efficiency at 1% total lipid concentration. Moreover, as quantity of liquid lipid increased it corresponded to augmentation in entrapment efficiency. The solid lipid to liquid lipid ratio demonstrated a substantial effect on the extent of entrapment efficiency. The greater entrapment efficiency could potentially be attributed to the lipophilic nature of NTG and its increased solubility in certain lipids.[43] High entrapment efficiency shows that NLC has a large capacity to encapsulate NTG. Effect of percentage of total lipid and liquid lipid to total lipid concentration ratio was observed on the EE%. It was observed that higher the liquid lipid concentration, more was the EE%, while less the percentage of total lipid, greater was the EE%. [44]

The surface response graph (J) and linear graph (E) are illustrated in Fig. 4, demonstrating that independent variables affects entrapment efficiency. ANOVA study shows F-value of 6.61 and P-value of 0.0188, this indicate significant model. Mathematical equation for entrapment efficiency is as follows.

Y3 = + 11.89-0.6663A + 0.5175 B + 0.0463C-1.71AB + 0.9800AC-0.1375BC + 0.9225A²-1.05B²

Where, Y3 is EE %, A is Percentage of total lipid, B is Ratio of the liquid lipid to the solid lipid, C is Concentration of surfactant. [45]

Drug loading for all NLC formulations was good. Percentage Drug loading was found in between 8.46 to 16.04%. From the optimisation graphs, it can be concluded that drug loading of the NLC formulations was increased when liquid lipid to solid lipid ratio was increased. Among all the above batches, N11- NLC has shown maximum drug loading of 16.04%. For DL% F-value of 7.57 and P-value of 0.0062, this indicate significant model. Mathematical equation for entrapment efficiency is as follows.

Y4 = + 90.00-0.6463A-0.2825B-0.7462C-2.59AB + 0.1125AC + 0.0150BC

Y4 is Drug Loading % A is Percentage of total lipid, B is Ratio of the liquid lipid to the solid lipid, C is Concentration of surfactant [46]

Particle size, Zeta potential and Polydispersity index (PDI)

NTG-NLC dispersions prepared by high pressure homogenization technique exhibited small particle size ranging from 142.8 ± 1.67 to 252.7 ± 2.17 nm and zeta potential obtained was from 13.53 mV to 30.93 mV. Poly dispersibility index (PDI) was obtained in the range of 0.343 ± 0.071 to 0.417 ± 0.058. Smaller particle size may be due to modified method of HPH. Formulation of pre-emulsion significantly reduced the particle size. Reduced particle size in nano formulations increases surface area thereby solubility and bioavailability.

Positive zeta potential values were observed in all NTG-NLCs.. The greater value of zeta potential, exhibits increased electrostatic repulsion and thereby increased stability. Consequently, greater the value of zeta potential, irrespective of the sign (either negative or positive) indicates good stability of the formulation. From the quadratic model it was observed F Value was 12.40and P-value was 0.0004 this explains the model is significant. A positive sign shows a synergistic effect, and the equations fit to the data. The mathematical equation for particle size is as follows

Y2 = 21.70-2.25A + 6.43B + 1.03C, where Y2 is Zeta Potential, A is Percentage of total lipid, B is Ratio of the liquid lipid to the solid lipid, C is Concentration of surfactant.

Increased total lipid content in NLC led to significant augmentation in the particle size. Conversely, as the liquid lipid content in formulation increased particle size was found reduced. Moreover, particle size of NLC was reduced as liquid lipid to solid lipid concentration ratio increased. At 1% lipid and 1.5% surfactant concentration, NLC with smallest particle size and low PDI were obtained. The optimized Nateglinide NLC, N11 exhibited a particle size of 142.8 ± 1.67 nm, a zeta potential of 30.93 mV, a DL% of 16.04%, EE% of 93.48% and PDI of 0.332.

These results highlight the influence of lipid concentrations, ratios, and surfactant content in tailoring drug loading, particle size, stability, and entrapment efficiency in NLC formulations. Particle size reduction could potentially be accredited to the presence of a surfactant (acrysol el 135 at 1.5%) utilized in formulating NLCs. Furthermore, specific details regarding evaluation parameters DL %, EE %, zeta potential and particle size values of nateglinide NLC formulations are illustrated in Table 5. [47]

Table 5

Evaluation of Nateglinide Nanostructured Lipid Carriers (N1-N17)
Formulations	Factor			Response
	X1	X2	X3	Y1	Y2	Y3	Y4
	Total lipid %	L. lipid/ S. lipid	% of Acrysol	Particle Size (nm)	Zeta Potential (mV)	DL (%)	EE (%)
N1	1.5	15:85	1	245.5 ± 1.74	17.26	9.04	89.40
N2	1.5	30:70	1.5	221.4 ± 2.64	18.99	11.25	89.48
N3	1.5	15:85	2	238.1 ± 2.48	17.45	9.04	89.60
N4	2	30:70	2	214.3 ± 2.22	22.28	12.11	88.88
N5	1	30:70	1	186.5 ± 2.34	26.02	11.56	92.14
N6	2	15:85	1.5	252.7 ± 2.17	13.53	10.91	92.06
N7	1.5	30:70	1.5	224.8 ± 3.14	19.89	11.98	89.95
N8	1.5	45:55	1	189.4 ± 1.94	26.87	9.01	89.36
N9	1.5	30:70	1.5	228.9 ± 2.24	20.28	12.24	90.18
N10	2	45:55	1.5	178.3 ± 1.84	29.79	9.87	85.77
N11	1	45:55	1.5	142.8 ± 1.67	30.93	16.04	93.48
N12	1	30:70	2	170.2 ± 2.90	28.23	10.06	88.70
N13	1.5	45:55	2	165.8 ± 2.24	28.99	8.46	89.62
N14	2	30:70	1	228.7 ± 1.54	18.54	9.69	91.87
N15	1.5	30:70	1.5	223.5 ± 2.12	13.75	11.84	90.14
N16	1	15:85	1.5	212.4 ± 2.28	16.93	10.25	89.43
N17	1.5	30:70	1.5	226.3 ± 2.34	19.15	12.14	89.86

Particle size, polydispersity index and zeta potential values for optimised NLCs N11 are shown in Fig. 1.

From the ANOVA results, quadratic model it was observed F Value was 73.87 and P-value found < 0.0001 this explains the model is significant. R² value 0.9955 shows linearity. A positive sign indicate a synergistic effect, and the equations fits the data. The polynomial mathematical equation for particle size is as follows.[48]

Y1 = 224.40 + 20.25 A- 34.12 B-7.62 C-1.00 AB + 0.5 AC-4.25 BC-19.07A²-9.33 B²-5.83C²

Y1 is particle size, A is Percentage of total lipid, B is Ratio of the liquid lipid to the solid lipid, C is Concentration of surfactant.

Selection of NLC using the Desirability Function

Optimised NLCs was selected on the basis of desirability function. The following criteria, i.e., achieving lowest particle size and highest percentage of DL, EE, Zeta potential was used to select the NLC. Results indicated that among the formulations assessed, N-11 formulation had the greatest desirability of 0.935. Concentration of optimized liquid lipid to the solid lipid ratio, optimized surfactant concentration which integrated the desirability function was decided from response surface methodology.

Solid state characterization of freeze dried NTG-NLC

I. Fourier Transform Infrared Spectroscopy:

Structural changes in drug before and after formulation were monitored with FTIR spectra.

FT-IR analysis of nateglinide (NTG) revealed distinct absorption bands at various wave numbers, each corresponding to specific functional groups present in the compound.

The absorption band observed at 2927 cm⁻¹ indicates the presence of -OH stretching (asymmetric), while the band at 1741 cm⁻¹ represents C = O stretching. Additionally, the absorption band at 2866 cm⁻¹ corresponds to symmetric aliphatic CH stretching, while the band at 3064 cm⁻¹ represents aromatic C-H stretching. Furthermore, the absorption band observed at 3300 cm⁻¹ indicates aromatic N-H stretching. Peaks in the range of 1200–1350 cm⁻¹ can be attributed to the O-C group. These characteristic peaks obtained from the FT-IR analysis match those reported in previous studies on nateglinide [49].

The FTIR spectrum of optimised NTG- NLC showed new characteristic peaks attributed to the excipients (lipids and surfactant like GMS, Miglyol 812 and Acrysol EL135)) were at positions 2360 cm^− 1, 1743 cm^− 1, 1064.71 cm^− 1 etc, the characteristic intensity of some peak was decreased. All characteristic peak for NTG were observed in lyophilized NTG-NLC spectra, this indicate there are no chemical interactions between NTG and lipids. The entrapment of NTG in NLC depend on solubilization of NTG in lipids.

This analysis confirmed the structural integrity of the chosen excipients within the developed NLC formulation. Additionally, the FTIR spectra indicated the absence of any significant physicochemical interaction between the drug and the excipients in the NLC formulation. Overlay FTIR spectroscopy of NTG and NTG-NLC is depicted in Fig. 2, illustrating the comparison between their respective spectra for further analysis.[50]

II. Differential Scanning Calorimetry

DSC analysis of Nateglinide and NTG-NLC was conducted. The DSC thermogram of NTG-NLC demonstrated no evidence of any chemical reaction occurring between NTG and the excipients in the formulation.

In the case of pure nateglinide, its DSC thermogram exhibited a characteristic peak at 134.56°C, signifying its melting range. Conversely, the DSC thermogram of pure NTG displayed a peak within the range of onset 130.06°C to endset 134.50°C, thermal pattern indicating NTG was in a crystalline form. This analysis suggests that NTG existed in a crystalline state prior to its incorporation into the NLC (NTG-NLC) formulation. [51]

In DSC of lyophilized NTG-NLC, an intense endothermic peak was observed within the range of onset 54.12°C to endset 79.45°C. Widening of peak was observed, is likely attributed to reduced particle size and molecular dispersion of drug in the NLC and lipids present in formulation. Reduction in the melting point was observed in DSC thermogram of NLCs. The comparative DSC of pure drug NTG and optimized NLC formulation shown in Fig. 3. [52]

III. PXRD of Nateglinide pure drug and Optimized NLC:

In the X-ray diffraction (XRPD) pattern of Pure NTG sharp diffraction peak appeared at two-theta values 5.64º, 8.53º, 14.72º, 16.78º, 17.96º, 19.12º and 21.32 º, indicating its crystalline nature. However, in the NTG-NLC formulation, these intense peaks were notably absent. Instead, the peaks observed in the same range exhibited reduced intensity, suggesting a molecular dispersion of the NTG within the NLC. [53]

The peaks were deformed in the same range in the XRD diffractogram of NTG-NLC formulation. This confirmed that NTG is completely in amorphous state in optimized NLCs formulation. An overlapped diffractogram of pure NTG drug and optimized NLC formulation are shown in Fig. 3. [54]

IV. Scanning Electron Microscopy of optimized NLC

SEM examined the optimized lyophilized formulation of NLC-N11 for analysing and investigating the characteristic shape and surface morphology of the formulated nanoparticles. The optimised NLC formulation was having nano size and globular shape with rough surface. Irregular size and variable morphology of the NTG-NLC attributed incorporation of NTG in lipid. NLC suspension during lyophilisation process aggregates, these aggregates appeared in micro size when observed in SEM. SEM for NLC-N11 is shown in Fig. 5. [55]

In vitro NTG release from optimised NTG-NLC formulation

The cumulative percentage drug release through the NTG suspension, NTG-NLC, and NTG-marketed tablets Glinate 60 suspension was determined in Phosphate buffer pH 7.4 release media. About 23.02% of nateglinide was released from pure nateglinide solution at 2 h, while about 80.78% nateglinide was released for NTG marketed formulation Glinate-60 at 2h and around 92% of nateglinide was released from the optimised NTG-NLCs at 1.5 h. Drug release through NLC exhibited sustained pattern for 12 hr.[56–57]

The drug release rate from NTG -NLC was faster than NTG solution and marketed formulation, this accelerated release is likely attributed to the reduced particle size and larger surface area available in the NTG-NLC. Through the optimized NTG-NLC formulation, maximum drug release occurred within 1.5 hours, without any observed burst release, indicating effective encapsulation of NTG within the NLC. Enhanced dissolution in the optimized NLC can be attributed to the nano-sized particles, enhanced surface area, solubility enhancement of NTG in NLC core and conversion of nateglinide into amorphous form. Optimized NLC formulation was showing excellent in vitro drug release performance. Detailed results of the in vitro drug release study are presented in Table 6 and illustrated in Fig. 5, highlighting the prominent differences in drug release profiles between the formulations.[58]

Table 6

*In vitro* drug release study through Nateglinide Pure Drug, Glinate 60 and Nateglinide optimized NLC
Time in hr	Percentage drug release through formulation (%)
Time in hr	Nateglinide Pure Drug	Glinate 60 Marketed Product	Nateglinide optimised Nanostructured Lipid Carriers
0	0	0	0
0.5	5.7 ± 0.23	21.56 ± 1.26	32.29 ± 1.21
01	11.27 ± 1.12	45.45 ± 1.68	67.57 ± 2.24
1.5	16.98 ± 1.46	70.59 ± 2.08	92.38 ± 2.04
02	23.02 ± 1.09	80.78 ± 3.42	86.04 ± 1.12
04	20.69 ± 2.05	66.49 ± 2.14	80.34 ± 1.08
06	18.23 ± 1.04	56.32 ± 2.83	76.59 ± 1.34
08	15.27 ± 1.46	34.67 ± 1.56	65.89 ± 1.18
12	10.88 ± 1.14	22.48 ± 1.12	38.04 ± 1.46

Pharmacokinetic study of Nanostructured Lipid formulation of Nateglinide in Streptazotocin induced diabetes in rat’s in vivo drug release:

NTG-NLC-N11, NTG suspension, and Glinate 60 suspension were administrated through oral route to the wistar rats of either sex. The average plasma concentration (ng/ml) in rats, through NTG suspension, Glinate 60 and NTG-NLC suspension was determined. The study was conducted for 12 hrs.

Through the NTG -NLC maximum average plasma concentration 1208.2 ng/ml was observed after 2 hrs of administration. While through Glinate 60 (marketed tablet), maximum average plasma concentration 945.6 ng/ml was observed after 2 hrs of administration. Through pure drug nateglinide suspension, maximum average plasma concentration 482.2 ng/ml was observed after 1.5 hrs of administration. In vivo drug release through optimized NLC was faster and greater than pure drug as well as marketed tablet, Glinate 60 suspension. The concentration of NTG in NLC- N11 was higher than the pure NTG suspension as well as Glinate 60 suspension in the plasma post-administration in wistar rats. The Cmax of NTG-NLC-N11 (1208.2 ng/ml), was appreciably higher than Glinate 60 suspension (945.6 ng/ml) and NTG suspension (482.2 ng/ml). The AUC of (0-12hr) value of NTG-NLC- N11 (8679.23 h.ng /ml) was considerably higher compared to Glinate 60 Suspension (5621.12 h. ng/ml ) and the NTG suspension (2298.04 h.ng/ml ). [58]

Considering the AUC (0-12hr), the bioavailability of NTG-NLC-N11 was determined to be 3.77 times superior than the NTG suspension and 1.54 times superior than the Glinate 60 suspension.C max of NTG-NLC was found 1208 ng /ml, while that of pure NTG was 482 ng/ml. C max of the optimized NTG- NLC formulation exhibited a significantly higher C max (2.5 times) than that of NTG after oral administration to the rats. The enhanced Cmax and AUC (0–12) of the optimized NTG-NLC formulation could be attributed to increased solubilization of NTG within the lipid polymeric matrix of the NLC. Moreover, the Tmax of NTG-NLC-N11 (2 h) was greater than that of pure NTG suspension (1.5 h), which might be due to the presence of solid lipids in the NLCs, which might influence the drug release kinetics.[59]

The t \(\frac{1}{2}\) of NTG-NLC-N11 (3.81 hr) was significantly longer than the NTG-suspension (2.31 hr) and Glinate 60 Suspension (2.30 h). NTG-NLC-N11 displayed the slower elimination rate (Ke) of 0.181/h, as compared to NTG-suspension 0.310 /h., which specify prolonged elimination of NTG through NTG-NLC-N11 formulation. Consequently, the pharmacokinetic findings suggest that NLCs formulations have enhanced the oral bioavailability of nateglinide. The provided pharmacokinetic parameters are detailed in the table. From the in vitro study of the NLC revealed that the incorporating NTG into the lipid based NLC has enormously enhanced drug solubilization, which resulted in extensively augmented drug release.

The in vitro release study of NTG-NLC demonstrated superior drug release in comparison to both the pure drug and Glinate 60 tablet suspension.

Figure 5 depicts the plasma concentration-time profiles after administration of the, Glinate 60, and the NLC-N11 formulation through oral gavage.

Crucial pharmacokinetics parameters are summarized in the Table 7. Significant variations were observed in these important pharmacokinetic factors following oral administration of NTG-NLC to rats.[60]

Table 7

Average plasma concentration (ng/ml) through Nateglinide Pure Drug, Glinate 60 and Nateglinide optimized NLC (*in vivo* study)
Time Point in Hr	Nateglinide Pure Drug	Nateglinide Marketed Product Glinate 60	Nateglinide Optimised Nanostructured Lipid Carriers
0	0	0	0
0.5	165.1 ± 8.08	346.9 ± 18.57	742.1 ± 38.66
1	384.2 ± 10.21	559.4 ± 26.21	864.4 ± 41.54
1.5	482.7 ± 11.15	672.1 ± 21.16	988.6 ± 42.78
2	398.2 ± 10.01	945.6 ± 24.46	1208.2 ± 46.98
4	286.8 ± 9.87	876.5 ± 26.59	1065.9 ± 44.25
6	198.1 ± 9.43	542.4 ± 19.28	924.2 ± 38.46
8	87.3 ± 8.24	216.9 ± 14.36	450.1 ± 31.01
12	28.2 ± 5.41	84.7 ± 10.14	272.2 ± 28.67

Table 8

Pharmacokinetic parameters for through Nateglinide Pure Drug, Glinate 60 and Nateglinide optimized NLC
Parameters	Nateglinide Pure Drug	Nateglinide Marketed Product Glinate 60	Nateglinide optimised Nanostructured Lipid Carriers
Cmax [ng/ml]	482.2 ± 11.15	945.6 ± 24.46	1208.2 ± 46.98
Tmax [hrs]	1.5 ± 0.05	2 ± 0.04	2 ± 0.05
AUC(0–12) [h.ng/ml]	2298.04 ± 168.92	5621.12 ± 89.96	8679.23 ± 96.80
t1/2 [hrs]	2.31 ± 0.12	2.30 ± 0.16	3.81 ± 0.21
MRT (h)	4.36 ± 0.08	4.782 ± 0.06	6.634 ± 0.12
Vd [L/kg]	1.145	0.434	0.376
Cl [ml/min/kg]	0.351	0.136	0.0816
KE	0.31	0.301	0.181

Relative Bioavailability

The relative bioavailability of nateglinide NLC was assessed in comparison to both the marketed formulation Glinate 60 and the pure nateglinide drug. The relative bioavailability of NTG-NLC and NTG marketed drug was compared, determined to be 154.40%. Additionally, the relative bioavailability of NTG-NLC compared to the pure NTG drug was found to be 377.67%. The fold enhancement in relative bioavailability is presented in Table 9.[61–62]

Table 9

Relative Bioavailability of NTG-NLC formulations
Relative Bioavailability of NTG -NLC Vs. NTG Marketed drug	\(\frac{8679}{5621}\) × 100%	154.40%
Relative Bioavailability of NTG -NLC Vs. NTG pure drug	\(\frac{8679}{2298}\) × 100%	377.67%
Fold enhancement in Bioavailability as compared to pure NTG	3.77 fold enhancement in bioavailability.
Fold enhancement in Bioavailability as compared to Glinate 60	1.54 fold enhancement in bioavailability.

Relative bioavailability (F) = \(\frac{\mathbf{A}\mathbf{U}\mathbf{C} (0-\mathbf{t}) \mathbf{T}\mathbf{e}\mathbf{s}\mathbf{t} }{\mathbf{A}\mathbf{U}\mathbf{C} (0-\mathbf{t}) \mathbf{R}\mathbf{e}\mathbf{f}\mathbf{e}\mathbf{r}\mathbf{e}\mathbf{n}\mathbf{c}\mathbf{e} }\) × 100%

The inherent pH-dependent solubility of the pure nateglinide drug poses a significant challenge in formulating its dosage form. Nateglinide exhibits practical insolubility in aqueous environments, is the major constraint in its bioavailability and therapeutic application. The enhanced Cmax and AUC (0–12) observed in the optimized NTG-NLC formulation can be attributed to increased solubilization of NTG within the gastrointestinal tract. This improved solubilization is likely due to the lipid matrix present in the NLC system along with the inclusion of effective surfactants. The optimized NLC formulation has notably enhanced the bioavailability of nateglinide. Additionally, the surfactant Acrysol EL 135 aids in facilitating the permeability of nateglinide, contributing to this improved bioavailability [62–63]

The NLC formulations include lipids such as Miglyol 812 and the surfactant Acrysol EL 135. These lipids not only augment permeability across the intestinal membrane but also enhance the affinity and interaction between NLC and the intestinal membrane.

Nateglinide nanostructured lipid carriers can be the best method to improve the bioavailability and performance.

NTG-NLC enhances drug solubility, permeability of drug across intestinal mucosa thereby appears to be the most promising approach for significantly enhancing bioavailability and overall performance. Optimised NLC exhibits enhanced in vitro as well as in vivo performance. Formulation of NLCs for nateglinide like hydrophobic drug can represent the most efficient formulation for oral drug delivery.[63]

The enhancement of oral bioavailability for NTG was successfully achieved through the optimization of NTG-NLC using a design of experiments approach. The optimized NTG-NLC formulation exhibited desirable attributes, including reduced particle size, uniform size distribution, and elevated encapsulation efficiency.

Optimized formulation of NTG- NLC obtained through Box- Behenken design shows particle size 142.8 nm, Zeta Potential, 30.93 mV, drug loading 16.04%, and entrapment efficiency 93.48%. NLC formulations exhibited excellent entrapment efficiency ranged from 85.77 to 93.48%. Drug loading was obtained ranging from 8.46–16.04%. The average encapsulation efficiency was 89.99%. The crystallization behavior of NTG within the NLC was examined through PXRD and DSC. It was found that the drug remained in an amorphous state within the NLC matrix.

NTG-NLC dispersions prepared with modified HPH method exhibited small particle size ranging from 142.8 ± 1.67 to 252.7 ± 2.17 nm and zeta potential ranging from 13.53 mV to 30.93 mV. PDI was obtained in the range of 0.343 ± 0.071 to 0.417 ± 0.058. Modified HPH method and formulation of pre-emulsion significantly contributed to the reduction in particle size. Reduced particle size of nano formulations leading to improved solubility and bioavailability of the formulation. All zeta potential values of the NLC formulations were positive, indicating increased electrostatic repulsion and, consequently, enhanced stability. In the in vitro study, NTG–NLC exhibited a C max at 2 hours, prolonged drug release, no significant burst release was observed.

In the oral bioavailability study conducted in Wistar rats, the relative bioavailability of NTG-NLC compared to the NTG marketed drug was determined to be 154.40%. Additionally, the relative bioavailability of NTG-NLC compared to the pure NTG drug was found to be 377.67%. This indicates a 3.77-fold enhancement in bioavailability for NTG-NLC compared to the pure NTG suspension and a 1.54-fold enhancement in comparison with the Glinate 60 marketed nateglinide formulation. The NLC of NTG, prepared using the HPH method, effectively enhanced the bioavailability of the pure drug.

Funding

The authors did not received any financial support, grant or funding from any organization.

Conflict of Interest: All authors (Shradha S Tiwari*^a, Yuvraj D Dange^a, Sandip M Honmane^a, Mahesh G Saralaya^a, Surendra G. Gattani^b, Shailesh J Wadher^b, Rohit R. Sarda^c, Paresh R Mahaparale^d) declare that they have no conflict of interest.

Financial Interest

The authors declare that they have no financial interest.

Ethics approval (include appropriate approvals or waivers)

The submitted work is original and has not been published anywhere else.

Ethical Approval for Animal Study

All animal-related experiments, procedures adhered to the approved guidelines and were conducted with the approval of the committee overseeing experiments on animals (CPCSEA). The approval number for these protocols was 1138 /PO/RCS/RCNRCL/S/08/CPCSEA.

Statement of Human and Animal Rights, and/or Statement of Informed Consent

N/A, human study is not involved in this research.

Consent to participate

N/A

Consent form for publication

N/A

Availability of data and material (data transparency)

Data related to publication is available.

Author statement

This submission is to undertake that:

• All authors of this research paper have directly participated in the planning, execution, or analysis of this study;

• All authors of this paper have read and approved the final version submitted;

• The contents of this manuscript have not been copyrighted or published previously;

• The contents of this manuscript are not now under consideration for publication elsewhere;

• The contents of this manuscript will not be copyrighted, submitted, or published elsewhere, while still under consideration by this journal.

Declaration of competing interest:

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Formulation, optimization, in vitro and in vivo evaluation of Nateglinide-loaded nanostructured lipid carriers for enhanced bioavailability.

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Version 1

Abstract

Figures

Introduction

Materials and methods

1.1. Materials

1. Experimental design

Preparation of NTG-loaded Nanostructured Lipid Carriers

Characterization of NTG-NLCs

Particle size, Zeta potential and Polydispersity index (PDI)

Percent Entrapment efficiency (% EE) and Drug loading (DL)

I. Fourier Transform Infrared Spectroscopy

II. Differential Scanning Calorimetry

III. Powder X-ray diffraction analysis (PXRD)

IV. Scanning Electron Microscopy (SEM)

Bioanalytical method:

Pharmacokinetic analysis:

Relative Bioavailability (F)

Results And Discussion

I. Solubility of Nateglinide in liquid lipids

II. Solubility of Nateglinide in Solid Lipids

I. Entrapment Efficiency (EE %) and Drug loading (DL %)

Particle size, Zeta potential and Polydispersity index (PDI)

Selection of NLC using the Desirability Function

I. Fourier Transform Infrared Spectroscopy:

II. Differential Scanning Calorimetry

III. PXRD of Nateglinide pure drug and Optimized NLC:

IV. Scanning Electron Microscopy of optimized NLC

Relative Bioavailability

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1