Pimozide-loaded nanostructured lipid carriers: Repurposing strategy against lung cancer

Abstract

Objective

This study aimed to repurpose pimozide (PMZ) by incorporating it into nanostructured lipid carriers (NLC) using a modified melting emulsion ultrasonication method.

Methods

We employed stearic and oleic acids in a 1:1 ratio as lipids, with Tween 80 and PEG 4000 as surfactants. The formulation was analyzed for particle size, zeta potential, and encapsulation efficiency. Transmission electron microscopy (TEM) was used to confirm the spherical shape of the particles. The release profile of PMZ-NLC was evaluated under different pH conditions, and anticancer activity was tested on A549 cell lines.

Results

The PMZ-NLC exhibited an average particle size of 136 ± 2.9 nm, a zeta potential of −25.1 ± 0.9 mV, and an encapsulation efficiency of 86% ± 11. TEM confirmed the spherical shape of the NLCs. PMZ release from PMZ-NLC was pH-sensitive, enhancing tumor targeting. IC₅₀ values were 16.5 μM for free PMZ and 12.9 μM for PMZ-NLC after 72 h.

Discussion

PMZ-NLC demonstrated improved anticancer activity compared to free PMZ, suggesting that encapsulation enhances the drug's effectiveness. The pH-sensitive release profile supports its potential for targeted therapy in lung cancer.

Conclusions

PMZ-NLC showed potential as a safe and effective strategy for lung cancer treatment. Further investigation is warranted to evaluate its in vivo efficacy, long-term safety, and clinical application.

Introduction

As the second largest cause of mortality globally, cancer is a major public health problem. The World Health Organization (WHO) predicts that by 2040, there will be 47% more cancer patients than there were in 2020, reaching 16 million fatalities and over 28 million cases. ¹ Lung cancer was the most frequently diagnosed cancer in 2022, responsible for almost 2.5 million new cases, or one in eight cancers worldwide. ² Lung cancers are categorized based on microscopic cell structures into small cell lung cancer (SCLC) and non-SCLC (NSCLC). ³ These types exhibit distinct development patterns and treatment approaches. According to Rinaldi et al. (2006), NSCLC, which makes up (80%–85%) of lung cancer cases, is generally less aggressive than SCLC, which spreads rapidly, and it is typically brought on by tobacco use.^4,5 Cancer development and treatment response are heavily influenced by the tumor microenvironment (TME). One of the most distinguishing aspects of the TME is its acidic nature, which is frequently caused by excessive glycolysis and limited blood supply in tumors. ⁶ This acidic environment, with a pH ranging from 6.5 to 7.0, has a considerable influence on medication efficacy and cellular activity. Researchers can better understand how therapies operate under different environmental pressures by simulating physiological and tumor-specific circumstances at pH values of 7.4 and 4.6. This method aids in improving medication delivery systems and therapy techniques to improve their efficacy in the acidic tumor environment. ⁷

Current treatments for lung cancer, such as chemotherapy and radiotherapy, face significant limitations, including frequent relapses, development of resistance, and severe side effects. ⁸ These treatments often lack precision, targeting not only cancer cells but also healthy cells, leading to detrimental consequences for patients. Additionally, SCLC, known for its rapid growth and spread, often shows limited response to conventional therapies, highlighting the urgent need for novel approaches. ⁹ Drug repurposing offers a viable solution by leveraging existing medications with known safety profiles, potentially reducing the time and cost associated with bringing new cancer treatments to market while providing more effective and targeted therapeutic options.^10,11

A549 is a well-established human lung adenocarcinoma cell line, derived from a patient with NSCLC, specifically adenocarcinoma. ¹² Although A549 cells, which were obtained from a patient with NSCLC adenocarcinoma, are not directly indicative of SCLC, they are frequently utilized in research to examine lung cancer biology, drug resistance, and possible therapies. The findings from research utilizing A549 cells might guide tactics that may assist SCLC patients, particularly in understanding how different medicines affect lung cancer cells and generating innovative therapeutic techniques. ¹³

Pimozide (PMZ) is traditionally used as an antipsychotic medication to manage symptoms such as abnormal brain excitation and tics by blocking type 2 dopamine receptors in the central nervous system. ¹⁴ Recent studies, however, have highlighted its potential anticancer properties, demonstrating efficacy against various cancer cell lines, including liver, leukemia, breast, prostate, brain, and lung cancer cells. The interest in repurposing PMZ for cancer treatment is driven by its established safety profile, low incidence of adverse effects, and affordability. Its extensive research background makes it a promising candidate for drug repurposing efforts aimed at developing new therapeutic strategies for cancer, particularly lung cancer.^15,16

Treatment plans are optimized by considering factors such as the disease's stage, the tumor's origin, genetic mutations, and family history. SCLC, known for its rapid growth and spread, is commonly addressed using chemotherapy and radiotherapy. ¹⁷ Nevertheless, relapses are frequently seen in SCLC cases, and the available treatment methods are limited by the frequent emergence of resistance, lack of precision, and the severe side effects of radiation and chemotherapy. Consequently, research efforts are directed toward drug repurposing. ¹⁸

Drug repurposing, or drug repositioning, is to find new innovative application derived from currently available FDA-approved and clinically utilized medicinal compounds.^19,20 This is frequently accomplished by gathering scientific evidence regarding the drug's impact on a condition that it is not approved to treat.^19,21 Drug repurposing offers several advantages compared to the traditional approach of developing new drugs. Firstly, it carries a lower risk of failure as successful preclinical experiments have already been conducted. Secondly, it reduces the costs associated with preparing a drug for human testing. Thirdly, it accelerates the process of assessing a drug's efficacy in humans. ²² In this study, PMZ will be tested as an anticancer medication with an alternative effect beyond its primary use.

PMZ is a conventional antipsychotic drug that reduces abnormal brain excitation and controls tics by blocking type 2 dopamine receptors in the central nervous system. ²³ PMZ has demonstrated anticancer effects in various cell lines, including liver, ²⁴ leukemia, ²⁵ breast, ²⁶ prostate, ²⁷ brain, ²³ and lung cancer cells. ²⁰ The literature has investigated PMZ's effects on many cancer types, suggesting its promise as an anticancer agent. According to studies, PMZ has cytotoxic effects on numerous cancer cell lines, including breast, prostate, and lung malignancies. Its mode of action is hypothesized to entail inducing apoptosis and inhibiting cell growth. ²⁶ PMZ's anticancer properties are due to its capacity to affect several cellular pathways. It inhibits the Wnt/β-catenin signaling system, which is crucial for cancer cell proliferation and metastasis. PMZ may also influence other pathways involved in cell survival and stress responses. ²⁸ Its extensive research is driven by its safety, low adverse effects, and affordability. ²⁹ However, PMZ's classification as a Biopharmaceutical Classification System (BCS) Class Ⅱ compound with low solubility and high permeability presents challenges for its pharmacokinetic and pharmacodynamic optimization. ³⁰ Fortunately, these difficulties can be solved by encapsulating the medication into a suitable nanocarrier.

Researchers have investigated numerous formulation options to improve PMZ potency and delivery, such as encapsulation in nanostructured lipid carriers (NLCs). NLCs provide benefits such as increased drug solubility, controlled release, and targeted delivery to cancer cells. Studies have shown that encapsulating PMZ in NLCs improves its therapeutic efficacy and reduces negative effects when compared to free PMZ. ³¹

NLC, a second generation of solid lipid nanocarriers (SLNs) that derived from oil-in-water nanoemulsions, are emerging as a promising drug delivery technique.^32–34 These carriers consist of liquid lipid, solid lipid, and surfactants. NLCs are novel drug delivery systems with biodegradable and biocompatible properties, offering controlled release patterns and various dosage forms. ³⁵ NLCs exhibit advantages such as improved drug storage stability, controlled release profiles, and high drug-loading capacity. ³⁵

The current study aimed to repurpose and formulate PMZ into NLC (PMZ-NLC) utilizing a modified melting emulsion ultrasonication method and evaluating the in vitro anticancer activity of the fully characterized PMZ-NLC formula compared to free PMZ.

Materials and methods

Materials

PMZ was sourced from Sigma Aldrich, USA. Oleic acid (OA), stearic acid (SA), PEG-4000, and polysorbate 80 (Tween 80) were obtained from TCI, Japan. MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide) was purchased from Promega, USA. HPLC-grade organic solvents were acquired from Schar Lab and were used without further purification.

Cells

Human adenocarcinoma alveolar basal epithelial cell lines (A549) were obtained from the ECACC, catalog number 86012804, passage number 18. The following materials and kits were utilized in this study: the MTT assay kit from Promega (USA), dimethyl sulfoxide (DMSO) from GCC-UK, 70% alcohol, fetal bovine serum (FBS) from Euroclone (Italy), L-glutamine from Euroclone (Italy), penicillin/streptomycin from Euroclone (Italy), and Dulbecco's Modified Eagle Medium (DMEM) with high glucose. Our study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2013.

Methods

Preparation of PMZ-NLCs

Eight different formulas of PMZ-NLCs were prepared using a modified melting emulsion sonication procedure ³⁶ (Table 1). Briefly, two phases were prepared and heated separately: the aqueous phase (composed of Tween 80 and PEG 4000 dissolved in 1 mL deionized water) and the lipidic phase (composed of SA, OA, and PMZ) were heated to 5–10°C above the melting point of the lipids. The aqueous phase was then gradually added to the lipidic phase under gentle stirring. A 10-min incubation at 80°C was required to form the nanoemulsion. Finally, the nanoemulsion was probe sonicated for 2 min using a 100-watt probe sonicator at 40% amplitude and then quickly frozen for 1 min to solidify the prepared NLCs.

Table 1.

Optimization of PMZ-NLC depending on OA, SA, Tween 80, and PEG4000 different ratios, final volumes of PMZ-NLC were adjusted with deionized water up to 1.0 mL.

Formula number	Pimozide (mg)	OA (mg)	SA (mg)	Tween 80 (μL)	PEG 4000 (μL)
F1	0.5	20	20	30	30
F2	0.5	10	10	30	30
F3	0.5	6	2	30	30
F4	0.5	6	2	20	30
F5	0.5	6	2	10	30
F6	0.5	6	2	10	–
F7	0.5	6	4	10	–
F8	0.5	5	5	30	30

NLC: nanostructured lipid carriers; PMZ: pimozide; OA: oleic acid; SA: stearic acid.

Characterization of NLCs

The NLC sample underwent a comprehensive characterization process using dynamic light scattering, enabling the determination of critical parameters such as average size, polydispersity index (PDI), and zeta potential to assess stability. ³⁷ To ensure uniform sample distribution, meticulous preparation was carried out within an Eppendorf tube, followed by a 60-s vortexing step to achieve homogeneity. To perform these measurements, 5 μL of each sample was mixed with 995 μL of distilled water, creating an opalescent dispersion, dispersant viscosity (0.8872 cP), refractive index (1.332), and a temperature of 25°C. The analysis of particle size and zeta potential was conducted using the Zeta-sizer software from Malvern Instruments. This entire procedure was performed three times (n = 3) to ensure precision and reliability, resulting in a comprehensive and accurate characterization of the NLC sample's properties.

PMZ encapsulation efficiency (EE%)

PMZ quantification within NLCs was performed using an HPLC system composed of a Shimadzu LC-2030 equipped with a UV Detector (Shimadzu, Kyoto, Japan). Separation was achieved on Thermo Scientific™ Hypersil™ BDS C18 HPLC Column, 100 ° A, 5 μm, 4.6 × 150 mm stationary phase ³⁸ using an isocratic mobile phase of 75% methanol with 25% of a 0.1% v/v phosphoric acid solution. The pH of the mobile phase was adjusted to a range of 2.0 to 3.0 using phosphoric acid solution. Following sonication and heating, the mobile phase underwent vacuum filtration. Samples were prepared by disrupting 200 μL of PMZ-NLC with 800 μL methanol via 5-min bath sonication, followed by 1200 rpm centrifugation for 10 min. 20 μL of the supernatant then injected into HPLC to quantify PMZ at 280 nm. ³⁹ The calibration curve of PMZ was linear. The mean % assay of was found to be 101.02% and % recovery was observed in the range of 99.23–101.91%. Relative standard deviation for precision study was found less than 2%. The LOD and LOQ values were found to be 0.553 µg/ml and 1.678 µg/ml respectively. ³⁹ Encapsulation efficiency (EE%) was determined using equation (1) to ascertain the drug content in the NLCs ⁴⁰ :

$$EE(\text{\%})={\displaystyle \frac{(\mathrm{Amount}\mathrm{of}\mathrm{drug}\mathrm{encapsulated}\mathrm{in}\mathrm{NLCs})}{(\mathrm{Amount}\mathrm{of}\mathrm{total}\mathrm{drug}\mathrm{added}\mathrm{in}\mathrm{the}\mathrm{preparation})}\times 100\text{\%}}$$ (1)

Transmission electron microscope

Transmission electron microscopy (TEM) was employed to investigate the morphology of the PMZ-NLC colloidal solution. Samples were prepared by diluting the PMZ-NLC solution 200-fold with double-distilled water. Negative staining method was then performed using a 1% phosphotungstic acid solution.^41,42

In vitro release assay

In vitro release assay was performed utilizing dialysis method at two different pHs. The experiment was performed by placing 1 mL of free PMZ or PMZ-NLC, diluted in PBS (pH = 7.4), into a cellulose membrane bag (cut off at 50 KD), soaking for 30 mL PBS and 3% tween 80 with pH 7.4 or 4.6. The release medium was shaken at 37°C at 100 rpm. Then, 1 mL of dialysate was removed at specified time intervals (2, 4, 24, 48, and 72 h) and replaced with the same amount of dialysis solution to keep sink condition. HPLC quantification was used to evaluate the amount of PMZ released from the dialysis samples.^43,44

Cell viability assay

In vitro cytotoxic activities of PMZ and PMZ-NLZ were investigated against A549 lung cancer cell lines. A549 cells were cultured in DMEM growth medium (Euroclone^®, Italy), which was supplemented with 10% (v/v) FBS and antibiotics (5 ml / 500 ml penicillin / streptomycin). Cells were cultured at a confluence of 75–95% in a humidified 5% CO₂ incubator at 37 °C. 10 × 10³ cells per well were planted in 96-well plates. Pre-seeded cells were prepared for treatment after a 24-h incubation period. Three different stock solutions (free PMZ, PMZ-NLC, and blank NLC) were prepared at various concentrations through serial dilution ranging from 100 μM to 0.00000625 μM. Each concentration was assessed n = 3. After 72 h of treatment, the culture media was removed, and MTT dye (100 μL) was added to each well. The plate was then incubated for an additional 3 h, and the dye was replaced with 150 μL of stop dye. After 30 min on an orbital shaker, the absorbance of each well was measured using an ELISA reader at wavelengths of 590 and 630 nanometers. This process allowed for the evaluation of cell viability and treatment efficacy.^45,46 IC₅₀ values were calculated using GraphPad Prism 8 software through concentration-viability curve analysis. ⁴⁷ Cell viability was determined using equation (2):

$$\mathrm{Cell}\mathrm{viability}\text{\%}={\displaystyle \frac{\mathrm{Sample}\mathrm{absorbance}-\mathrm{Blank}\mathrm{absorbance}}{\mathrm{Control}\mathrm{absorbance}-\mathrm{Blank}\mathrm{absorbance}}\times 100\text{\%}}$$ (2)

In vitro wound healing assay

The cell migration experiment (wound healing) is a crucial step in studying tumor progression under PMZ treatment and understanding the in vivo behavior. A monolayer of A549 adherent cells was cultured in a 12-well plate (2.0 × 10⁵ cells per well) for 24 h and then subjected to scratching using a pipette tip. ²⁹ After removing the old media, fresh media containing the treatment was added. The scratched areas were assessed using Motic Images Plus 2.0 software, and images were captured with a Nikon P. MICRO-001 phase contrast microscope at time 0 and 72 h, measuring in square micrometers. The percentage of scratch opening was calculated using equation (3). This provided insights to how PMZ treatment affected cell migration over time.

$$\mathrm{Scratch}\mathrm{opening}\text{\%}={\displaystyle \frac{\mathrm{Area}\mathrm{on}\mathrm{Day}2}{\mathrm{Area}\mathrm{on}\mathrm{Day}1}\times 100\text{\%}}$$ (3)

In vitro colony formation assay

The colony formation assay was used to assess the ability of cancer cells to divide and form colonies. A small number of cancer cells (300/well) were initially incubated for 24 h at 37°C in a CO₂ incubator. Afterward, the old media was replaced with fresh media containing the intended treatment and incubated for an additional 72 h. Subsequently, the old media was discarded, and each well was rinsed with PBS to remove any residual treatment.

After 14 days, the old media was removed, and paraformaldehyde (PFA) was added to fix the existing cells. The cells were then incubated for an extra hour at 37°C. Following this, the PFA was aspirated, and crystal violet stain was applied to color the preexisting colonies for 30 min. Finally, the plate was rinsed multiple times with deionized water and allowed to dry completely. Colony counting was performed visually to assess the colony-forming ability. Colonies consisting of at least 50 cells were considered valid. ⁴⁸

Statistical analysis

Statistical analysis was performed to ensure the reproducibility and reliability of the results. All experiments were conducted by n = 3, and the data were expressed as mean ± standard deviation. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons. The IC₅₀ values were calculated using nonlinear regression analysis with GraphPad Prism 8 software program. A p-value of less than 0.05 was considered statistically significant.

Control groups

Control groups were essential for the comparative analysis of the experiments. In the cell viability assay, three different stock solutions were prepared: free PMZ, PMZ-NLC, and blank NLC, with each concentration tested by n = 3. Untreated cells (DMSO + Media) served as negative controls to evaluate the baseline cell viability. For the wound healing assay, A549 cells were treated with two concentrations of free PMZ and PMZ-NLC (8 and 16 μM), with untreated cells as controls. In the colony formation assay, media-treated cells were used as controls to assess the natural proliferation capacity of A549 cells. These control groups allowed for a thorough comparative analysis, ensuring the observed effects were due to the treatments administered.

Results and discussions

Dynamic light scattering measurements

In this study, PMZ-NLC formulations were prepared by combining OA and SA at different ratios. OA was chosen for its favorable interaction with the hydrophobic oleate group of Tween 80 (polyoxyethylene sorbitan monooleate), which likely enhanced the stability and release profile of the NLCs. Additionally, we selected OA and SA for their biocompatibility and biodegradability, with OA serving as the liquid lipid and SA as the solid lipid.^49,50 To obtain suitable NLC formulation, many experiments have been conducted to get PMZ-NLC with the best traits and qualities; each experiment involved changing one of the preset factors. These variables are divided into two categories: variables relating to the formula's components (Table 1) and factors related to probe sonication time and power (Figure 1).

Figure 1.

The effect of probe sonication variables on PMZ-NLC particle size for formula F1 (A) effect of sonication time (min) at 40% sonication power of 100 Watt, (B) effect of % sonication power of 100 Watt at 2 min sonication time.

NLC: nanostructured lipid carriers; PMZ: pimozide.

According to Figure 2, NLCs were subjected to probe sonication for 2 min. using 40% of 100-Watt. Moreover, Table 2 showed the average particle size, zeta potential, and PDI for the prepared formulas. The result illustrated that the lipid content plays a significant role in determining the particle size and zeta potential of the nanoparticles. This influence is attributed to the lipid's impact on reorienting crystalline planes, leading to alterations in the particle surface and, consequently, changes in the charge of the produced nanoparticles. ⁵¹ The initial two formulas (F1 and F2) exhibit a nearly neutral charge and large particle diameter, primarily due to their high lipid content. To address this issue, new formulations were developed with SA at one-third the amount of OA (at a 1 : 3 ratio) (F3 to F6). This adjustment reduced particle size due to the higher proportion of liquid lipid, which decreased the viscosity of the solid matrix. ⁵² However, these formulations demonstrated lower stability (as shown in Figure S1). To improve stability, the SA content was increased to 50% w/w of the total lipid mixture (F8). This modification not only enhanced stability (Figure S1E) but also reduced particle size and PDI (Figure 2). High concentrations of PEG (3%) and Tween 80 (3%) were used to produce smaller particles with a narrower size distribution. ⁵³ Increasing stearic acid to 50% improves PMZ-NLC stability by strengthening the solid matrix, minimizing particle aggregation, and increasing encapsulation efficiency. This solid lipid content promotes a more cohesive structure, resulting in a more stable and homogeneous nanoparticle formulation. ⁵⁴ Based on PMZ-NLC stability, the optimal ratio of liquid to solid lipids is 1 : 1 (50 : 50). ⁵⁵

Figure 2.

DLS measurements of blank NLC F8 formula (A) zeta potential, (B) particle size, and DLS measurements of PMZ-NLC F8 formula (C) zeta potential, (D) particle size.

NLC: nanostructured lipid carriers; PMZ: pimozide; DLS: dynamic light scattering.

Table 2.

Results of PMZ-NLC optimization prepared from different ratios of SA, OA, Tween 80, and PEG 4000 according to DLS measurements of average particle size, zeta potential, and PDI.

Formula number	Z-average (nm) ± SD	Zeta potential (mV)± SD	PDI ± SD
F1	181 ± 12.3	−0.03 ± 0.2	0.227 ± 0.01
F2	257 ± 8.8	−1.0 ± 0.5	0.230 ± 0.01
F3	137 ± 0.5	−18.0 ± 3.3	0.202 ± 0.01
F4	120 ± 2.3	−20.3 ± 0.6	0.251 ± 0.01
F5	101 ± 1.0	−12.7 ± 3.2	0.225 ± 0.01
F6	104 ± 0.2	−20.6 ± 1.1	0.590 ± 0.01
F7	360 ± 2.8	−12.5 ± 4.2	0.366 ± 0.06
F8	136 ± 2.9	−25.1 ± 0.9	0.243 ± 0.02

NLC: nanostructured lipid carriers; PMZ: pimozide; DLS: dynamic light scattering; OA: oleic acid; SA: stearic acid.

As we moved from F3 to F6, the nanoparticle size decreased because the amount of Tween 80 was reduced from 30 to 10 μl. Lower Tween 80 concentrations may reduce nanoparticle size by minimizing surfactant-induced aggregation. This reduction in Tween 80 enhances dispersion during formulation, resulting in smaller and more uniform nanoparticles due to the decreased stabilizing effect of the surfactant. ⁵⁶

We observed that increasing the surfactant concentration significantly affected the particle size and distribution. In our study, the optimal surfactant concentration was 3% v/v for each surfactant, which produced small particles with a narrow size distribution. ⁵³ We also found that sonication duration and amplitude influenced particle size, zeta potential, and PDI. Specifically, longer sonication times and higher amplitudes increased particle size and PDI, likely due to particle aggregation from the added energy. To achieve the best characteristics for PMZ-NLC, we optimized the process with a sonication time of 2 min and an amplitude of 40%.

PMZ entrapment efficiency (EE%)

Encapsulation efficiency of PMZ incorporated into PMZ-NLC was determined using equation (1) utilizing the calibration curves as indicated in the material and method section. Due to the highest stability of F8 compared to other formulations, the percentage of encapsulation efficiency (EE%) for F8 was determined. EE% was found to be 86.6% ± 11, meaning the drug was almost fully loaded within PMZ-NLC compared to previous studies. For example, Uddin et al. reported a 50% EE of PMZ loaded into PLGA nanoparticles, ⁵⁷ while it was improved by Barada et al. to be 76.7% by adding PEG to PLGA nanoparticles. ⁵⁸ NLCs exhibit high %EE due to their solid-liquid lipid matrix, which efficiently stabilizes and maintains the medication. The combination of solid and liquid lipids reduces drug leakage and improves drug-lipid compatibility, resulting in a higher %EE compared to other nanoparticle systems.^34,54

Transmission electron microscopy

TEM has been the most used imaging technique for assessing the structure of nano systems. The size and structural properties of F8 were confirmed using TEM, as shown in Figure 3A. TEM images confirmed NLC spherical shape with proper dispersion and particle size. The size differences in the NLC particles observed in TEM images could be due to variations in the preparation process or aggregation of particles. Factors such as inconsistent mixing or slight changes in conditions can lead to these size discrepancies.^59,60

Figure 3.

(A) TEM images of PMZ-NLC, (B) cumulative release test pattern of PMZ and PMZ-NL at (37 °C) and different pH media.

TEM: transmission electron microscopy; NLC: nanostructured lipid carriers; PMZ: pimozide.

In vitro release assay

An in vitro release assay was conducted to assess the differences in release patterns between PMZ-NLC and free PMZ. This test involved using PBS and 3% Tween 80 media with both acidic pH (4.6), to simulate the environmental conditions of cancer cells, and neutral pH (7.4). The release of both the free drug and the drug loaded in lipid particles was studied over a period of 72 h.

The release of PMZ from NLC particles is enhanced in an acidic pH as shown in (Figure 3B), and this enhances the targeting capability of PMZ-NLC due to the expected accelerated drug release caused by tumors’ acidic extracellular and intracellular environments. This phenomenon is useful for enhancing the delivery and localization of drug PMZ. ⁶¹

Figure 3B showed that free PMZ fully released after 72 h in PBS at 37 °C at both pH levels. In contrast, PMZ-NLC released only 50% of PMZ at pH 7.4 over the same period, and about 75% at pH 4.6. This indicates that PMZ encapsulated in NLCs releases more slowly compared to free PMZ. Additionally, there was minimal difference in PMZ release between neutral and acidic pH levels for the free drug. However, at pH 4.6, PMZ release was 25% higher than at neutral pH, likely due to the neutralization of carboxylate groups in fatty acids, which enhanced PMZ's release. These results suggest that NLCs effectively encapsulate PMZ, allowing for controlled and enhanced release in acidic tumor environments. ⁴³ As PMZ has slightly basic pka of 8.3, the prolonged release at pH 7.4 of PMZ from PMZ-NLC may be explained by the strong electrostatic interaction between fatty acid carboxylate anion and PMZ's cationic charge.

And this validates what Ma et al. (2022) indicated, that Kaempferol was encapsulated in NLC to increase its transport to NSCLC. ⁶² The possible cause of the delay in the release of the loaded drug in both instances is the drug's entrapment in NLC, which has a positive effect on therapeutic outcomes because the modified release pattern may delay the drug's exposure in tumor cells, thereby potentially enhancing the curative effect.

Cell viability assay

In this study, the A549 lung cancer cell line was exposed to different concentrations of PMZ, PMZ-NLC, and blank NLC. The MTT absorbance readings versus treatment concentrations after the experiment were analyzed using GraphPad Prism 8 software, ultimately yielding the half-maximal inhibitory concentration (IC₅₀) values, as depicted in Figure 4A and B.

Figure 4.

Cytotoxicity of PMZ-NLC and free PMZ against A579 cell line.

NLC: nanostructured lipid carriers; PMZ: pimozide.

The IC₅₀ values were 16.52 μM for free PMZ and 12.9 μM for PMZ-NLC (p > .05). This lower IC₅₀ for PMZ-NLC suggests that PMZ-NLC is more effective than free PMZ. This improvement is likely due to the prolonged release of PMZ from the NLCs’ lipid core. The NLCs’ enhanced physicochemical properties and targeting ability are probably due to this prolonged release, especially in acidic environments. ⁶¹ Additionally, the prolonged release of PMZ from NLC may offers stable therapeutic levels, increasing the efficacy against cancer cells by allowing for sustained exposure. This strategy enhances targeting, lowers dose frequency, and reduces adverse effects. A consistent PMZ concentration aids in the treatment of tumor heterogeneity and resistance, resulting in improved therapeutic results and lower systemic toxicity. ⁶³ Moreover, these results indicated that blank NLC is biocompatible and safe as indicated in Figure 4C. Furthermore, the results showed an improved in the cell toxicity when comparing 0.5 × IC₅₀, IC₅₀, and 2 × IC₅₀ of free PMZ compared to PMZ-NLC (Figure 4C). This suggests that PMZ-NLCs were more effective in targeting and killing cancer cells.

In vitro wound healing assay

A monolayer of A549 adherent cells was cultured in a 12-well plate (2.0 × 10⁵ cells per well) for 24 h and then subjected to scratching using a pipette 200 μL tip. Three groups of A549 cells were treated with two concentrations of free PMZ and PMZ-NLC (8 and 16 μM) and one left as control, untreated cells (DMSO + Media). The scratched areas were compared to the untreated cells areas at two different time points: on the day of treatment and 72 h later (Figure 5).

Figure 5.

In vitro wound healing assay for monolayer of A549 adherent cells treated with two concentrations of free PMZ and PMZ-NLC (8 and 16 μM) (A) area in sq. μm at day 1 and day 4 for free PMZ and untreated cells, (B) area in sq. μm at day 1 and day 4 for free PMZ-NLC and untreated cells, (C) percent of wound closure for PMZ and PMZ-NLC at different concentrations.

NLC: nanostructured lipid carriers; PMZ: pimozide.

The inhibition of cell migration was 20% more effective with PMZ-NLC compared to free PMZ at 8 μM (p < .05)(*), while it was less than 10% at 16 μM (p > .05) not significant (ns). These results suggest that PMZ-NLC is more effective at preventing cancer metastasis at lower concentrations of PMZ.

In vitro colony formation assay

This test was performed to evaluate the effectiveness of PMZ-NLC in limiting the capacity for sustained proliferation comparing to free PMZ, in order to determine whether the new formulation improved the drug's cytotoxic effect. ⁴⁸ In vitro colony formation was evaluated utilizing three different concentrations of PMZ in either free drug or in PMZ-NLC that compromising the IC₅₀, 0.5 × IC₅₀, and 2 × IC₅₀. Following the assay procedure adapted in the methodology section and after 14 days of cell treatments with PMZ and PMZ-NLC, colony counting was performed visually to assess the colony-forming ability (Figure 6). Colonies consisting of at least 50 cells were considered valid.

Figure 6.

In vitro colony formation assay for A549 cells treated with different concentrations of either (A) free PMZ, (B) PMZ-NLC. For free PMZ, the number of colonies were 30 and 35 clones when cell treated with 0.5 × IC₅₀, while no colonies were observed after treating cells with IC₅₀ and 2 × IC₅₀ of free PMZ. Media treated cells showed 35 clones. For PMZ-NLC treatment; the number of colonies was two when cell treated with 0.5 × IC₅₀, while no colonies were observed after treating cells with IC₅₀ and 2 × IC₅₀ of free PMZ. Media treated cells showed 34 clones.

NLC: nanostructured lipid carriers; PMZ: pimozide.

The obtained results from the colony formation and wound healing assays support the release assay findings; wherein the migration of A549 cells was 10% more inhibited by the PMZ-NLC, which has an IC₅₀ of 12 µM, compared to the PMZ-treated cells, and this supports the research conducted by Dakir et al. in 2018 on how PMZ can prevent lung cancer cells from metastasizing. ²⁶ Additionally, PMZ-NLC treated cells inhibited colony formation at the employed concentration, but PMZ treated cells inhibited colony formation only at IC₅₀ and 2 × IC₅₀.

The enhanced anticancer activity of PMZ-NLC compared to free PMZ can be attributed to several mechanistic factors. Firstly, the NLCs provide a sustained and controlled release of PMZ, leading to prolonged exposure of cancer cells to the drug. This slow-release mechanism ensures that a higher concentration of PMZ remains in the vicinity of the tumor for an extended period, increasing its therapeutic efficacy. Additionally, the pH-sensitive release pattern of PMZ-NLC targets the acidic microenvironment of tumors, ensuring more efficient drug delivery specifically to cancer cells while sparing healthy tissues. The encapsulation within NLCs also improves the solubility and bioavailability of PMZ, overcoming the limitations posed by its low solubility as a BCS Class II compound.

The findings of this study have significant clinical implications. PMZ-NLC demonstrates a higher anticancer activity with potentially reduced side effects compared to free PMZ, due to its targeted delivery and controlled release properties. This targeted approach can minimize the systemic toxicity often associated with conventional chemotherapy. Dosage optimization of PMZ-NLC will be crucial to maximize its therapeutic benefits while minimizing adverse effects. The promising results from the in vitro studies suggest that PMZ-NLC could be a valuable addition to the current lung cancer treatment regimens, offering a novel therapeutic strategy with the potential for better patient outcomes.

Future research directions

Future study should focus on testing F8's rapid and long-term stability, including physical, chemical, and microbiological stability, because the stated four-day stability does not guarantee long-term efficacy. Prior to in vivo research, in vitro oral absorption should be assessed using models such as CaCO-2 cells to ensure sufficient bioavailability. Subsequent in vivo investigations in animal lung cancer models will be critical in determining PMZ-NLC's effectiveness and safety. Furthermore, investigating combination therapy with additional chemotherapeutic drugs or targeted therapies may improve anticancer efficacy and address resistance mechanisms. Clinical studies will be required to evaluate the appropriate dose, treatment regimens, and overall efficacy in lung cancer patients.

Conclusion

In conclusion, this study demonstrated that PMZ could be effectively formulated into NLC using a modified melting emulsion ultrasonication method. The PMZ-NLC showed improved physicochemical properties, including smaller particle size, high encapsulation efficiency, and pH-sensitive release. In vitro tests revealed that PMZ-NLC had superior anticancer activity against A549 lung cancer cells compared to free PMZ, as indicated by IC₅₀ values. These results underscore the potential of PMZ-NLC as a more effective, targeted therapy for lung cancer, with promising implications for drug repurposing and nanomedicine.