Preparation and Characterization of Liposomal N-Acetylcysteine

N-acetylcysteine (NAC) is a thiol-containing compound widely recognized for its antioxidant activity and its role as a precursor for glutathione synthesis. Beyond its established clinical use as an antidote for acetaminophen toxicity, NAC has been investigated for potential applications in neurological disorders because of its antioxidant, anti-inflammatory and redox-modulating properties¹⁻³. Reviews of NAC in neurological disease have described experimental evidence relevant to multiple sclerosis and other disorders in which oxidative stress and inflammatory signaling contribute to neuronal injury⁴.

Liposomal drug-delivery systems consist of phospholipid bilayer vesicles capable of incorporating hydrophilic compounds into the aqueous compartment and interacting with hydrophobic compounds within the lipid bilayer. Their physicochemical properties can be adjusted by changing lipid composition, cholesterol content, vesicle size, surface charge and preparation conditions⁵⁻⁷. Such characteristics make liposomes attractive carriers for improving drug protection and controlling drug disposition.

Among the different preparation approaches, thin-film hydration is a widely used laboratory method in which lipids are dissolved in an organic solvent, the solvent is removed to form a dry lipid film, and the film is hydrated with an aqueous phase containing the drug or drug solution⁵'⁸. The resulting vesicles may subsequently be reduced in size by sonication or other size-reduction techniques. Because formulation variables can simultaneously affect several quality attributes, design-of-experiments approaches are useful for systematic optimization.

The Box-Behnken design is a response-surface methodology that allows simultaneous evaluation of multiple factors and responses while reducing the number of experimental runs compared with some full-factorial approaches⁹. Desirability-based optimization can then be used to identify a compromise formulation that meets predefined goals for multiple responses.

The present study therefore aimed to formulate Lipo-NAC using soy lecithin and cholesterol, optimize the formulation using a three-factor Box-Behnken design, and characterize the optimized formulation with respect to particle size, PDI, zeta potential, morphology and drug entrapment. Short-term stability was also examined under refrigerated and room-temperature conditions.

MATERIALS AND METHODS

N-acetylcysteine, soy lecithin and cholesterol were used for formulation development. Chloroform was used as the organic solvent during lipid-film formation, and distilled water was used for hydration. The instrumentation utilized included a UV spectrophotometer, FTIR spectrophotometer, dynamic light-scattering/zeta-potential analyzer, probe sonicator, rotary evaporator and transmission electron microscope.

For preformulation characterization, FTIR data for pure NAC were obtained using the potassium bromide disc method. FTIR spectra of cholesterol, soy lecithin and the combined formulation components were also recorded for physicochemical characterization and assessment of chemical compatibility¹⁰⁻¹².

A three-factor Box-Behnken design was generated using Design-Expert software (version 11). The independent variables were phospholipid concentration (A), cholesterol concentration (B) and NAC concentration (C). The dependent responses were particle size (PS) and entrapment efficiency (EE). Seventeen experimental runs were generated. Analysis of variance (ANOVA) was used to assess model significance and the contribution of formulation factors. Numerical optimization was performed using desirability-based constraints, with the goal of minimizing particle size while maximizing EE⁹.

Lipo-NAC was prepared by the thin-film hydration method. Soy lecithin and cholesterol were accurately weighed and dissolved in chloroform in a round-bottom flask. The organic phase was evaporated using a rotary evaporator at approximately 45-46°C for 15 min at 70 rpm under vacuum, producing a thin lipid film on the inner wall of the flask. NAC was dissolved in 5 mL distilled water, and the lipid film was hydrated with 20 mL distilled water with rotation/stirring at room temperature. The resulting suspension was probe-sonicated for 30 min. The formulation was centrifuged at 36,000 rpm for 30 min to separate unencapsulated drug. The resulting liposomal preparation was lyophilized and stored at 2-8°C until analysis⁸'¹³.

Particle size and PDI were measured by dynamic light scattering with a 60-s measurement time and a scattering angle of 165°. Zeta potential was measured at 25°C using a 90° detection angle. Morphology was assessed by TEM. Entrapment efficiency was determined by measuring unentrapped NAC in the post-centrifugation supernatant using UV spectrophotometry and calculating the difference between the total amount of drug initially added (W) and the amount detected in the supernatant (w) via the following equation: $$\% \text {Entrapment efficiency} = \left [\frac {W-w}{W}\right] \times 100$$

Optimized Lipo-NAC samples were sealed in 20-mL glass vials and stored for 30 days at 4°C or at room temperature (25±2°C; 60±5% relative humidity). Samples were examined at predetermined intervals (0, 10, 20 and 30 days) for drug entrapment using UV measurement at 205 nm for the stability assessment.

RESULTS AND DISCUSSION

The Box-Behnken design generated 17 experimental runs, demonstrating substantial variation in particle size and EE across the formulation space. ANOVA evaluation of the model terms showed that phospholipid concentration (factor A) was significant for EE, while cholesterol concentration (factor B) was significant for particle size. The reported lack-of-fit p-values were >0.05 for both responses, indicating non-significant lack of fit. Response-surface and contour plots indicated that increasing phospholipid and cholesterol levels generally increased particle size. For EE, cholesterol exerted a positive influence, and its interaction with phospholipid levels suggested a combined influence on the lipid matrix.

The desirability approach identified an optimized formulation with a predicted particle size of 69.968 nm and a predicted EE of 76.575%. The experimentally observed values showed reasonable agreement with these predictions, as summarized in Table 1.

Table 1: Experimental Validation of the Optimized Lipo-Nac Formulation

The optimized Lipo-NAC showed a mean particle size of 65.8±2.05 nm and a low PDI of 0.291±0.02, reflecting a narrow and uniform particle-size distribution. Nanometer-scale distribution is a critical quality attribute for liposomal delivery systems because it heavily dictates in vivo disposition¹⁴. The measured zeta potential was -47.8 mV, indicating a strongly negative surface charge. While the initial target range was noted as ±30 mV, magnitudes greater than 30 mV imply high physical stability due to substantial electrostatic vesicle-vesicle repulsion, though further long-term testing is needed to completely rule out slow aggregation over time¹⁴'¹⁵. TEM examinations verified that the structural morphology consisted of predominantly spherical vesicular networks.

The primary dataset reports an observed EE of 72.5±0.36%. However, a secondary data entry mentions an unentrapped NAC quantity of 71,308 µg derived from an initial total of 250,000 µg. Applying the standard mathematical equation: $$\% \text {EE} = \left [\frac {250,000 - 71,308}{250,000}\right] \times 100 = 71.48\%$$ This yields 71.48%, revealing a slight internal discrepancy against the alternative 75.47% phrase noted in baseline laboratory reports. Authors must confirm the verified calibration standards and batch assay sheets prior to journal routing.

Stability testing demonstrated that temperature plays an essential role in vesicle retention. Under refrigeration (4°C), the drug entrapment value stayed constant at 52% across the full 30-day timeline. Conversely, storage at room temperature (25±2°C) triggered an incremental leakage of the compound over the tracking intervals, as contextualized in Table 2.

Table 2: Short-Term Stability Performance Evaluation Across 30 Days

The apparent decrease from the freshly optimized experimental EE (72.5%) down to a baseline of 52% at Day 0 of the stability run indicates a potential change in sampling protocols, batch volumes, or tracking conditions (such as the shift from 254 nm to 205 nm analytical detection wavelengths). This structural variance highlights that refrigeration provides superior short-term protection, but complete long-term validation must be established to satisfy international criteria.

CONCLUSION

A liposomal formulation of N-acetylcysteine was successfully developed using soy lecithin and cholesterol and optimized by Box-Behnken response-surface methodology. The optimized formulation exhibited a nanoscale particle size (65.8±2.05 nm), PDI of 0.291±0.02, and zeta potential of -47.8 mV, with predominantly spherical morphology by TEM. Short-term storage at 4°C showed better retention of entrapped drug than room-temperature storage. These findings support further development of Lipo-NAC as a candidate nanocarrier for studies of NAC delivery. However, the minor discrepancies in mathematical entrapment percentages and UV tracking wavelengths must be finalized against the raw laboratory notebooks, and comprehensive evaluations of long-term kinetics, scaling mechanics, and in vivo efficacy remain necessary before clinical translations can progress.

Acknowledgement

The authors acknowledge Nirmala College of Pharmacy, Muvattupuzha, Kerala, India, for providing the necessary facilities and instrumentation to carry out this research work.

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