1. INTRODUCTION:

Approximately 60-70% of drugs are classified as BCS Class II (low solubility/high permeability) or Class IV (poor solubility/low permeability). These classifications present challenges related to stability, solubility, dissolution, and therapeutic efficacy.

Nanostructured lipid carriers (NLCs) are a new generation of colloidal carrier systems made using combinations of solid and liquid lipids. These carriers offer various advantages over conventional nanocarriers, including higher permeability, enhanced bioavailability, fewer adverse effects, prolonged half-life, higher drug loading capacity, reduced drug leakage during storage, and improved physical stability. Solid lipid nanoparticles (SLNs), in contrast, face challenges such as poor drug loading capacity, drug leakage, and ineffective entrapment. NLCs were developed to address these issues. The characterization of NLCs was conducted using techniques such as transmission electron microscopy (TEM), zeta potential analysis, in-vitro diffusion studies, in-vitro dissolution methods, and ex-vivo permeation studies.^1-2

Salbutamol sulphate is a short-acting, selective β1-adrenergic receptor agonist classified under BCS Class I. It is utilized to manage asthma, bronchospasm, and reversible airway blockage by dilating the airways in the lungs. The half-life of inhaled or oral Salbutamol sulphate ranges from 2.7 to 5hours, with a bioavailability of approximately 50%. It is metabolized in the gastrointestinal tract and liver.

In recent years, researchers have mainly focused on lipid nanoparticles, such as SLNs, NLCs, niosomes, and liposomes, for antiasthmatic treatments to reduce side effects and improve efficacy. To address the challenges associated with Salbutamol, this study developed a Nanostructured Lipid Carriers (NLC) system.

Pharmaceutical NLCs can enhance bioavailability and bypass first-pass metabolism. Therefore, the developed Salbutamol sulphate-loaded NLC in-situ gel can be considered a suitable nasal delivery system for asthma treatment.^3-4

2. MATERIAL AND METHOD:

2.1 Chemicals:

Salbutamol sulphate was acquired as a complimentary sample from Raptakos Brett & Co. Ltd. (Mumbai, India). Glyceryl monostearate and Capmul PG-8-70 NF were received as complimentary samples from Loba Chemicals Pvt. Ltd. (Mumbai, India). Tween 80 obtained as gift samples from Loba chemicals Pvt. Ltd. (Mumbai, India). Methanol and HPMC E15 were received as complimentary samples from Research Lab Fine Chem. All remaining reagents and compounds were of analytical grade.⁵

Selection of lipids, surfactants and method for the preparation of NLCs

To begin with, it is essential to screen the components before formulating Nanostructured Lipid Carriers (NLCs). This phase facilitates the identification of the most appropriate solid and liquid lipids for the manufacture of nanostructured lipid carriers (NLC), hence assuring optimal drug entrapment and maximal drug loading inside the lipid matrix. The solubility of the medication in different solid and liquid lipids must be assessed. For solid lipids, such as Glycerol Monostearate and Capmul PG-8-70, a weighed amount of the drug is added to a glass vial, which is then heated at 10°C. The drug is continuously added until saturation is achieved.^6-7

To evaluate the solubility of the medicine in various liquid lipids (such as oleic acid, castor oil, and olive oil), the drug is incrementally introduced into micro-centrifuge tubes until dissolution ceases. The quantity of drug absorption is ultimately determined using 1 gram of liquid lipid.

Based on the size and stability of the formulation, various surfactants are selected according to their Hydrophilic-Lipophilic Balance (HLB) values (e.g., Tween 80, Tween 20, Span 80-LQ-(TH), and Span 83-LQ-(SG)). The impact of surfactants on the stability of NLCs is measured, and the stability of blank NLCs is observed by examining phase separation in the formulation.⁸

Table no.1: optimization of salbutamol sulphate loaded nanostructured lipid carriers using design of expert (DoE) software

Variable		Optimization levels
Independent variables (Factors)	Dependent variables (Responses)	Low (-1)	Medium (0)	High (+1)
Solid lipid and liquid lipid (%)	Particle size (nm)	70:30 (-1)	80:20 (0)	90:10 (+1)
Surfactant concentration (%)	Entrapment efficiency (%)	0.5 (-1)	1.25 (0)	2.0 ((+1)

Preparation of Salbutamol sulphate nanostructured lipid carriers (NLC):

Salbutamol sulphate-loaded NLCs were prepared using the ultrasonication emulsion technique. The process involved preparing the aqueous and lipid phases separately. Glyceryl monostearate, a solid lipid, and Capmul PG-8-70 NF, a liquid lipid, were combined and heated to a temperature of 10°C above their melting point. The melted lipid mixture was then blended with calculated amounts of Salbutamol sulphate to form a homogeneous and transparent lipid mixture. Meanwhile, Tween 80 was solubilized in water and heated to match the temperature of the lipid phase. The aqueous phase was then incorporated into the lipid phase and sonicated with a probe sonicator for 30 minutes. The prepared NLC was subsequently permitted to cool to ambient temperature prior to further characterisation.^9-11

Preparation of salbutamol sulphate loaded in-situ gel by cold method:

The in-situ gel was prepared using the cold method. HPMC E15 was used as mucoadhesive polymer in different ratios along with thermosensitive polymer Poloxamer 407. A measured quantity of HPMC E15, ranging from 0.1% to 0.5% w/v, was dissolved in 10ml of cold NLC dispersion at 4°C with continuous stirring. Poloxamer 407 was added with constant stirring and kept at 4⁰C overnight until to form a clear solution.¹²

Evaluation and characterization (NLC):

Particle size:

The particle size, polydispersity index (PDI), and zeta potential were evaluated using photon correlation spectroscopy. This was carried out with a Malvern Zetasizer (Nano ZS 90, Malvern Ltd., UK).

Morphological study (TEM):

The morphology of the formulation was analyzed using Transmission Electron Microscopy (TEM), specifically with a JEM-1200EX JEOL microscope (Tokyo, Japan). To prepare the samples, a dispersion of Salbutamol sulphate-loaded NLCs was diluted 50-fold with double-distilled water. This dispersion was then placed on a copper grid coated with an amorphous carbon film and negatively stained with 1% phosphotungstic acid. The prepared sample was then examined under the TEM.¹³

Entrapment efficiency (%):

The drug's entrapment percentage in NLC formulations were measured using high-speed cooling centrifugation (Beckman Instruments TLX-120 Optima Ultracentrifuge) at 120000rpm for 2hours at 4°C, the concentration of the unbound drug in the supernatant was measured using a double beam UV-Visible Spectrophotometer. Entrapment efficiency was calculated using the following formula¹⁴

Entrapment Efficiency = (Total amount of drug−Total amount of free drug/Total amount of drug) ×100

In-vitro diffusion studies:

The in-vitro drug diffusion study was performed using a diffusion cell assembly. For the evaluation of the drug-loaded NLC gel, a dialysis membrane with a molecular weight cutoff greater than 12,000 (from Hi Media) was utilized, and a pH 7.4 phosphate buffer solution served as the medium. The evaluation process was conducted at a wavelength of 371nm.¹⁵

Ex-vivo permeation studies

Ex-vivo skin permeation studies were carried out using a Franz diffusion cell, which has a 10ml capacity and an effective diffusion area of 2.5 cm². Sheep nasal mucosa was placed between the donor and receptor compartments of the diffusion cell. The donor chamber was filled with 0.2ml of the sample, while the receptor chamber contained 7ml of phosphate buffer saline at pH 6.4. The temperature of the medium was maintained at 37°C±2°C. Samples of 1ml were taken from the receiver compartment every 30minutes for a duration of up to 6 hours, with an equal volume of fresh medium added to replace each withdrawn sample.¹⁶

Result and discussion

Screening of lipids

Based on the lipid screening results, Glyceryl monostearate and Capmul PG-8-70 NF were chosen as the solid lipids for NLC formulation due to their high drug solubility. Additionally, these lipids possess beneficial properties such as good flowability, non-toxicity, and approved regulatory status.

Particle Size

The particle size, polydispersity index (PDI), and zeta potential were measured using the Malvern Zetasizer NANO ZS90 (Malvern, Worcs, UK). The particle size of the sample was determined to be 85.6nm, with a PDI of 0.270. The zeta potential was recorded at -19.41mV, indicating a negative surface charge. The results are illustrated in the accompanying.¹⁷

Morphological study

Morphological studies performed using TEM for surface morphology. TEM images (1:100 dilutions of Salbutamol sulphate NLC) confirmed that the particles possess a spherical shape, as shown in figure no.1.

Figure 1: TEM image of optimized batch of NLCs (F-III)

Entrapment Efficiency (%):

Nanostructured lipid carriers (NLCs) were formulated using various proportions of drug, Glyceryl Monostearate, Capmul PG-8-70 NF, and Tween 80. The entrapment efficiency ranged from 72.5% to 82.5%. Formulation F3 exhibited the highest entrapment efficiency at 82.5%, while formulation F4 showed the lowest entrapment efficiency at 72.5%.

Evaluation of Salbutamol sulphate loaded in-situ gel

Formulation batch F-I, containing HPMC E15 at 0.1% w/v and Poloxamer at 14% w/v, was used to produce in-situ gels within a 5-minute time frame.¹⁸

In-vitro diffusion studies

The drug release study was conducted using a phosphate buffer solution with a pH of 6.4 for the marketed aqueous formulation. NLC dispersion and nanostructured lipid carriers (NLC) loaded in-situ gel separately using dialysis bag technique. The comparison results showed that the NLC dispersion and NLC-loaded in-situ gel exhibited sustained release action compared to the marketed aqueous solution.

Figure 2: In-vitro release profile

Ex-vivo permeation studies:

An ex-vivo permeation study was conducted using the Franz diffusion cell and sheep nasal mucosa as the biological membrane to evaluate the formulated nanostructured lipid carriers (NLC) dispersion and NLC-loaded in-situ gel. The optimized batches of both formulations were compared.¹⁹

Figure 3: Ex-vivo permeation drug release profile

Drug release kinetics

The results of the in-vitro release studies were plotted using various kinetic models, the highest regression coefficient value of 0.9970 was observed for the zero-order model. This indicates that the release data best fit the zero-order kinetic plot model.

Figure 4: Salbutamol sulphate containing NLCs loaded in- situ gel

NLCs in-situ gel:

For all formulations, the R² values were higher for the zero-order model compared to the Higuchi model, Korsmeyer-Peppas model, Hixson-Crowell model, and first-order model. This suggests that drug release from the formulations followed zero-order kinetics. Specifically, the release data for Salbutamol sulfate from the NLC-loaded in-situ gel was best described by the zero-order model, with an R² value of 0.9970, indicating sustained drug release.

Stability studies²⁰

Table2: Stability study for salbutamol sulphate containing NLCs loaded in- situ gel

Time (Month)

Physical parameters

Gelation properties

Viscosity (cp)

Mucoadhesive strength (Dyne/cm²)

No change

2197

2496.81

CONCLUSION

In this study, Salbutamol sulphate-loaded nanostructured lipid carriers (NLCs) were prepared using the melt emulsification ultra-sonication method, optimized through central composite design (CCD). The resulting NLCs were then developed into an in-situ gel. This Salbutamol sulphate-loaded NLC-based in-situ gel was designed for intranasal administration of Salbutamol sulphate. Administering via the nasal route bypasses first-pass metabolism, making the NLC in-situ gel a promising nasal delivery system for Salbutamol sulphate in the treatment of asthma.

ACKNOWLEDGMENT:

The authors are also thankful toSmt. Kashibai Navale College of Pharmacy, Kondhwa (Bk.), Pune, Maharashtra for availing research facility.

CONFLICT OF INTEREST:

The authors report there are no competing interests to declare.

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Received on 21.02.2025 Revised on 16.04.2025

Accepted on 05.05.2025 Published on 10.07.2025

Available online from July 17, 2025

Asian J. Pharm. Res. 2025; 15(3):269-273.

DOI: 10.52711/2231-5691.2025.00043

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