INTRODUCTION:

Solid lipid nanoparticles (SLNs) are colloidal transporter systems extending from 50 to 1000nm. It is comprised of a lipid having high melting point as a solid center covered by a surface active agent. In this submicron measured system, solid lipid are been utilized rather than fluid lipid (for example oil) to shape SLN. Solid lipid nanoparticles are nanosystems made out of 0.1% (w/w) -30% (w/w) solid lipid scattered in a fluid and settled by the 0.5% (w/w) -5% (w/w) surface active agent.

SLN usually incorporates a lipid core and a surface-active agent that stabilize the particle in a liquid atmosphere. Physiological and non-toxic lipids are used primarily to prepare SLN, making them commonly acceptable for the delivery of topical drugs. SLNs are created either by dispersing or dissolving the hydrophobic substance into the lipid matrix or by dispersing it into the external layer around the lipid nucleus¹.

Artesunate is a butanedioic acid with anti-infective, antimalarial, antiparasitic, repellents properties. It is on the essential medicines list of the WHO. It is administered in few dosage forms such as tablet, injectable and rectal.

The major limitation of Artesunate is its poor solubility leading to low bioavailability, low permeability and short half-life of <43 minutes. This leads to the requirement of high drug dose, increased dosing frequency and patient compliance. After dosing ART is quickly removed from the blood. To overcome this, controlled-release dosage form can be formulated to treat malaria with dosing reduced to once daily. Thus ART SLN can be formulated to resolve low bioavailability and frequent dosing problems².

MATERIAL AND METHOD:

Materials:

Artesunate, an antimalarial drug, was obtained from IPCA laboratories. All the excipients like cetyl palmitate, tween 80, span 60, percirol ATO 5, and glyceryl monostearate used were of analytical grade.

Method:

Artesunate was identified by its color, texture and melting point. Further, a UV spectroscopic method was developed for its characterization^3–5. Standard calibration of the drug was performed in methanol, distilled water and phosphate buffer 6.8 (PBS). The assay technique was validated for linearity, ruggedness and robustness.

The formulation of Artesunate-loaded SLNs dispersions encompassed the utilization of the solvent injection method^6,7. The lipid phase was formed by combining a lipophilic surfactant (Span 60) and lipid, followed by the introduction of ethanol. This lipid phase was heated to 60°C to ensure lipid melting. Subsequently, a measured quantity of Artesunate was incorporated into the molten solution. Subsequently, the resultant solution was drawn into a syringe and swiftly injected into an aqueous phase containing a polysorbate 80 aqueous solution, kept at the identical temperature as the lipid phase. The amalgam was then sonicated for 5-10 minutes utilizing a Probe Sonicator (Sonics-Model VCX 750). Following sonication, the resulting dispersion underwent stirring on a magnetic stirrer (REMI) at 600 revolutions per minute (rpm) for 30minutes to attain nanoparticulate dispersion. The supernatant, containing SLN, was harvested and subjected to drying in a petri dish to obtain SLN. The dispersion was then poured into a petri dish and air-dried for 5-7 days to yield dry particles. The composition of different batches, varied by altering different parameters, is presented in table 1. This method was conducted in triplicate for consistency and reliability of results.

Initial batches of Artesunate SLNs were synthesized through the solvent injection method, employing diverse lipids such as cetyl palmitate, percirol, and GMS. These batches underwent assessment for parameters including entrapment efficiency (EE), physical appearance, and drug loading capacity. Cetyl palpitate exhibited high EE as compared to other lipids. Therefore, it was chosen as the optimal lipid for the formulation of SLNs.

The formulation of Artesunate-loaded SLNs utilized cetyl palmitate as the lipid matrix, Span 60 as the lipophilic surfactant, 1% polysorbate 80 as the co-surfactant, and ethyl alcohol as the organic solvent for drug solubilization. It was observed that the drug and lipid ratio of 1:10 demonstrated high EE. Thus the trial batches were designed in the ratio of 1:5, 1:10 and 1:20.

Additionally, a 3x2 factorial design approach was implemented to optimize two independent variables, with each variable being tested at three different levels. The two variables subjected to variation were the quantities of Span 60 and Tween 80, manipulated at low, medium, and high levels. The response variables under consideration encompassed particle size, drug release across a dialysis membrane, and the percentage entrapment efficiency.

Table 1: Composition of different batches of SLN

Batch	Artesunate (mg)	Cetyl palmitate (mg)	Span 60 (mg)	Ethanol (ml)	Tween 80 (%)
F1	10	50	25	5	0.5
F2	10	100	25	5	1
F3	10	200	25	5	1.5
F4	10	50	50	5	0.5
F5	10	100	50	5	1
F6	10	200	50	5	1.5
F7	10	50	75	5	0.5
F8	10	100	75	5	1
F9	10	200	75	5	1.5

All batches underwent thorough evaluation, including assessments of appearance, entrapment efficiency (EE), drug loading efficiency, particle size, Fourier-transform infrared spectroscopy (FT-IR), polydispersibility index, scanning electron microscopy (SEM), zeta potential, Differential Scanning Calorimetry (DSC)⁸, dissolution rate, in-vitro drug release over 8 hours using the dialysis method, ex-vivo release studies, and stability.

The entrapment efficiency of Artesunate in the SLN dispersion was evaluated using meticulous centrifugation at 6000 revolutions per minute (RPM) for 45 minutes, facilitating the sedimentation of the nanoparticles. Subsequently, the supernatant was meticulously decanted without interrupting the Artesunate pellet. The dispersion of SLN and the Artesunate pellet were then utilized for determining the entrapped and un-entrapped Artesunate content, respectively. A fixed volume of both the SLN dispersion and the Artesunate pellet was separately suspended in methanol and analyzed for entrapped and un-entrapped Artesunate content by UV spectrophotometry at 240 nm ^9,10.

Formula:

% EE = [(W _{initial Artesunate} – W _{free
Artesunate}) / (W _{initial Artesunate})] x 100 … (Eq 1)

Where, W is quantity of Artesunate.

The drug loading efficiency was determined by dissolving 10mg of dried SLN in methanol and analyzing it at 240nm spectrophotometrically. The quantity of entrapped Artesunate in the 10mg sample was ascertained and then extrapolated to calculate the total amount present in the entire powder.

Formula:

% Drug Loading = [Artesunate entrapped in SLN/ (Amount of Artesunate added + Amount of lipid added)] x 100 … (Eq 2)

Inverted microscope was used to evaluate average particle size of the formulation. Additionally, the Malvern zeta sizer was employed to conclude both the zeta potential and particle size of all batches.

The surface morphology of the SLNs was studied with the help of SEM at various magnifications.

An FTIR spectrophotometer was employed to assess the compatibility between the bulk material and the resulting SLN. KBr pellets were created and their spectra were subsequently recorded using an IR spectrophotometer (Varian 4640).

DSC thermographs were generated utilizing a Hitachi 7020 differential scanning calorimeter. Samples weighing approximately 3-5mg and were heated at a rate of 10ºC/min under a nitrogen flow of 30ml/min in an aluminum pan. Thermal data analysis was conducted based on the DSC thermograph, followed by the calculation of crystallinity indexes (CI %) for SLNs by the following equation.

CI% = [Melting enthalpy of SLN (J/g)/(Melting enthalpy of bulk material without Artesunate (J/g) x Concentration of lipid)] x 100 … (Eq 3)

The investigation of drug release was conducted utilizing the dialysis bag technique in conjunction with phosphate-buffered saline (PBS) of pH 6.8 as the dissolution medium. Dialysis membranes (specifically Dialysis membrane 50) were preconditioned in PBS of pH 6.8 for 12hours prior to their application. Two milliliters of SLN dispersion, with a concentration equivalent to 1-2mg/ml based on entrapment efficiency, were introduced into the dialysis bag. The dialysis bag was then submerged in a beaker with 150ml dissolution medium and agitated at 100rpm, while maintaining the temperature at 37±0.5ºC. At specific time intervals (15, 30, 45, 60, 120, 240, 360, and 480 minutes), 5 ml samples of the dissolution medium were extracted. To sustain sink conditions, equal volume of freshly prepared dissolution medium was added after each withdrawal. The collected samples were subsequently analyzed using spectrophotometer at 240nm for determination of Artesunate concentration¹¹.

The Artesunate release from all SLN batches was investigated to determine the most appropriate kinetic model. Different models consisting Korsmeyer-Peppas, first-order, Higuchi, zero-order, and Hixson-Crowell were evaluated. The correlation coefficient (R²) was evaluated for every model, and the mechanism of Artesunate release was inferred based on these R² values. The analysis was performed using PCP Dissolution Version 3 software for this release kinetic study¹².

Ex-vivo release studies were performed to compare the release of Artesunate from plain Artesunate solution and from SLN dispersion using freshly obtained 3 cm sections of chick ileum. One end of each ileum section was securely sealed with a thread, and 2 ml of plain Artesunate suspension was introduced through the other open end. Similarly, another ileum section was filled with 2ml of SLN dispersion, and both ends of the ileum segments were sealed tightly. These sealed portions were individually placed in two jars of a USP dissolution apparatus (Type 2) containing 900ml of dissolution media (distilled water) and run for 2 hours at 50rpm. Continuous aeration at a rate of 1-2 bubbles per second was maintained, while the temperature was controlled at 37±0.5ºC. Sample (5ml) was removed at scheduled time periods, and the amount of released Artesunate was quantified spectrophotometrically at 240 nm. This experimental procedure was conducted in triplicate to ensure reliability and consistency of results ¹³.

Stability study was executed to assess the stability of the SLN formulation. Short-ranged accelerated stability testing was performed subsequently after 1 month, while long-term stability testing was conducted after 3 months under controlled conditions of (30°C±2°C, 65% RH± 5% RH) and accelerated environments of (40°C±2°C, 75%±5% RH). Following storage, samples were examined for changes in any physical appearance, consistency, entrapment efficiency, and Artesunate loading¹⁴.

RESULTS AND DISCUSSION:

Artesunate was white crystalline powder with melting point of 132-134°C. The calibration curve in methanol, water and PBS 6.8 were found to be linear with good ruggedness and robustness at the lambda max of 240 nm. The detection limit and quantitation limit for Artesunate were observed at 0.0242µg/ml and 0.0733 µg/ml respectively.

The batches F1 to F9 were evaluated for its particle size, appearance, drug loading capacity, and EE. The outcome obtained are stated in table 2.

Table 2: Evaluation results for batches F1 to F9

Batch	Appearance	% EE	Drug loading capacity	Mean particle size (µm)
F1	Colloidal dispersion	78.3±1.44	4.89	2.655
F2	Colloidal dispersion	82.4±2.45	5.08	1.61
F3	Colloidal dispersion	91.1±2.44	5.79	1.499
F4	Colloidal dispersion	78±0.74	5.30	1.695
F5	Colloidal dispersion	83.4±1.33	5.38	1.38
F6	Colloidal dispersion	89.1±1.11	5.53	1.39
F7	Colloidal dispersion	78.7±1.89	6.29	1.88
F8	Colloidal dispersion	83.3±2.75	6.31	1.514
F9	Colloidal dispersion	88.1±3.45	6.93	1.53

Table 3: Release kinetics data

Batch	Zero Order	First Order	Matrix	Korsmeyer Peppas			Hixon Crowell	Best fit model
Batch	Zero Order	First Order	Matrix	R2	n	K	Hixon Crowell	Best fit model
F1	0.6917	0.6917	0.9771	0.9768	0.3943	0.0000	0.6917	Matrix
F2	0.7687	0.7687	0.9903	0.9923	0.4196	0.0000	0.7687	Peppas
F3	0.7848	0.7848	0.7848	0.9978	0.4194	0.0000	0.7848	Peppas
F4	0.7486	0.7486	0.9888	0.9966	0.4045	0.0000	0.7486	Peppas
F5	0.7850	0.7850	0.9930	0.9958	0.4260	0.0000	0.7850	Peppas
F6	0.6839	0.6839	0.9749	0.9726	0.3988	0.0000	0.6839	Matrix
F7	0.7125	0.7125	0.9803	0.9858	0.3777	0.0000	0.7125	Peppas
F8	0.6454	0.6454	0.9677	0.9690	0.3765	0.0000	0.6454	Peppas
F9	0.7496	0.7496	0.9878	0.9936	0.3995	0.0000	0.7496	Peppas

R2 is coefficient of correlation, n is exponent of diffusion and k is rate constant.

The drug release up to 8 hours by dialysis method was studied for all the batches. The following figure 1 show cases the obtained results.

Figure 1: Percentage drug release by dialysis membrane for batches F1 to F9

According to Table 3, Batch F1 and F6 demonstrate a matrix-type release pattern, whereas the remaining (F2, F3, F4, F5, F7, F8, and F9) adhere to the Korsmeyer-Peppas model. In this, the mechanism of release of drug is signified by the release exponent (n), where a higher 'n' value indicates a stronger influence of the polymer matrix on drug release. Thus, when 'n' is the highest among all batches, the formulation follows the Korsmeyer-Peppas model. Batches F3, F5, F6, and F9 display favorable characteristics, such as desirable entrapment efficiency and particle size.

The criteria for release pattern and duration were carefully considered in the evaluation. Conversely, batches F1, F4, and F7 displayed the lowest entrapment efficiency and drug release below 80%, rendering them unsuitable when compared to other batches. These batches exhibited low entrapment efficiency and limited drug loading capacity, potentially attributed to higher concentrations of lipophilic surfactant. Batches F1 and F2 presented larger particle sizes and slight turbidity, with F1 exceeding a release of 110%. Consequently, batches F5 and F6 showed smaller particle size, uniform distribution, reproducibility, long-term stability, and requisite characteristics for a stable SLN formulation.

Upon comprehensive evaluation of all pertinent data, Batch F5 underwent characterization through techniques such as Zeta potential analysis, SEM, DSC.

The SLN displayed 278.1nm particle size and 0.244 of PDI value, both of which are within acceptable limits, indicating a narrow distribution of particle size. Furthermore, the zeta potential value of the SLN was found to be -23.9, indicating good physical stability of the SLN formulation.

The FE-SEM image (figure 2) of dried SLN batch F5 revealed a particle size of 246nm with uniform distribution.

Figure 2: FE-SEM of F5 batch

The infrared spectra of Artesunate, lipid, and SLN were examined to identify and characterize the bands associated with the functional groups present in each component.

Scan A of the pure drug Artesunate exhibited characteristic bands at 3277 cm^-1, 1004 cm^-1, 1150 cm^-1, and 1373 cm^-1. Scan B of bulk cetyl palmitate displayed characteristic bands at 2950 cm^-1, 1640 cm^-1, and 1000 cm^-1. Scan C of the SLN exhibited bands at 3441 cm^-1, 2918 cm^-1, 1108 cm^-1, and 955 cm^-1.

Observations derived from the IR spectra of the SLN revealed a decrease in intensity for some bands of the drug and lipid, and certain bands vanished entirely, suggesting the integration of Artesunate into the lipid matrix, leading to SLN formation. Notably, several bands within the 1000-1500 cm-1 range, present in the pure Artesunate spectra, exhibited diminished intensity in the SLN spectra. This reduction in the distinctive bands of the drug within the SLN spectra suggests that the drug molecules are enveloped by the lipid matrix, indicating a physical interaction among the SLN and the drug. Consequently, the FTIR analysis confirms that during the encapsulation process of Artesunate, the characteristic peaks disappear due to interactions among the reactive groups of Artesunate with lipid matrix. This underscores the physical interaction among the excipients (lipid and surfactant) and Artesunate within the SLN formulation, rather than a chemical one.

DSC was employed to investigate the SLN for polymorphism and the degree of crystallinity. The table 4 briefs data derived from these profiles.

Table 4: DSC results of drug, lipid and SLN

Material	T_onset (°C)	T_max (°C)	H (mJ/mg)
Artesunate	150.7	170.5	25.45
Cetyl Palmitate	51.2	57.3	197.0
Drug loaded SLN	49.60	52.56	22.58

The decrease in melting transition in lipid SLN compared to Artesunate-SLN can be attributed to reduced particle size, increasing surface area (Gibbs-Thomson effect). The shift in melting transition may be due to entrapped drug and surfactant molecules. DSC profiles of SLN didn't show melting at Artesunate's point (132°C), indicating successful incorporation into the lipid phase. Reduced melting enthalpies suggest a chemical interaction. Artesunate exhibited peaks at 150.7°C and 170.5°C, but SLN showed a peak at 49.6°C, suggesting an amorphous form, potentially improving dissolution.

Ex-vivo drug release analysis compared drug release between a plain drug solution and SLN batches F5 and F6. The plain drug solution showed 12.39% drug release after 2 hours, while SLN dispersion via the solvent injection method demonstrated notably higher release: 97.5% for batch F5 and 73.34% for batch F6. This indicates a significant enhancement in drug release from SLN compared to the plain solution, likely due to the reduced particle size of SLN, increasing surface area and promoting dissolution. Additionally, the presence of a surfactant further facilitated drug release from SLN compared to the plain solution. These findings illustrate improved dissolution achieved through SLN formulation, as shown in Figure 3.

Figure 1: Ex-vivo drug release of plain ART and SLN

Batch F5 underwent additional stability testing. Throughout these studies, no notable alterations in the evaluation parameters were observed before and after the study period. The stability assessment revealed that the formulation maintained a clear colloidal dispersion and consistent homogeneity throughout the duration of the study.

Statistical Analysis of Data¹⁵:

Impact of variables on drug release at the 8-hour mark:

To assess the influence of the independent variable on various dependent variables, response surface method (RSM) was used as the experimental design. Specifically, the ratio of surfactant was varied to investigate its effect on both the percent drug release and percent entrapment efficiency. The analysis revealed that both the percent entrapment efficiency and percent drug release were dependent on the variations in both variables.

The impact of the variables on the drug release for 8 hr. was shown below.

Drug release (at 8 hours) = +82.04+12.82A+14.70B … (Eq 4)

Where A: Concentration of Span 60; B: Concentration of Tween 80

Contour plots and response analyses were utilized to generate response plots (Figure 4 and 5). Statistical optimization performed by the software indicated that a quadratic model is followed by cumulative drug release. Statistically significant model terms were identified for drug release at the 8-hour mark, with a high R² value of 0.8793, suggesting a good fit to the quadratic model. The F value of 21.85 indicates that the model was considerable. The "Adequate precision," measuring signal-to-noise ratio, was 13.191, well above the desired value of 4, showing an adequate signal. This suggests, model could effectively guide exploration within the design space.

The P-value presented compelling evidence of the relationship amongst the two independent variables and the responses. A 'Predicted value' < 0.05 signified that all polynomial equations and model terms were statistically substantial, as assessed through ANOVA in the Design Expert software.

Validation of statistical model:

Following statistical assessment of optimized batch F5 using Design Expert 11 software, the actual values for % cumulative drug release at 8 hours and entrapment efficiency closely mirrored the anticipated values, implying effective validation of the model, as shown in Table 5.

ACKNOWLEDGEMENT:

Authors would like to express our deepest gratitude to IPCA Laboratories, Mumbai for providing us with Artesunate as a gift sample for the research work.

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Received on 28.03.2024 Revised on 10.11.2024

Accepted on 18.03.2025 Published on 02.08.2025

Available online from August 08, 2025

Research J. Pharmacy and Technology. 2025;18(8):3622-3628.

DOI: 10.52711/0974-360X.2025.00521

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Polymers	Coded levels	Actual values	Response	% drug release at 8 hours	Entrapment efficiency (%)
			Predicted Value	82.0389	83.6
			Observed value	96.7	83.4
Span 60	0	50 mg	Standard deviation	7.22641	1.0395
Tween 80	0	1 %	Standard error mean	7.6173	1.09573