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Future Journal of Pharmaceutical Sciences
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Dual drug-loaded cubosome nanoparticles for hepatocellular carcinoma: a design of experiment approach for optimization and in vitro evaluation

Future Journal of Pharmaceutical Sciences volume 10, Article number: 38 (2024) Cite this article

Abstract

Background

Liver cancer, a formidable and complex disease, poses a significant global health threat, stemming from various causes, including chronic infections like hepatitis B and C, cirrhosis, and lifestyle factors. In liver cancer treatment, targeted delivery revolutionizes precision therapy, minimizing side effects by directing drugs specifically to cancer cells. This study aims to develop and statistically optimize cubosomal formulations containing piperine and quercetin with the goal of augmenting their activity against hepatocellular carcinoma.

Results

Employing a central-composite design, we utilized Design-Expert® software to guide the experiment. The key formulation variables were the concentration of glyceryl monooleate (GMO) and Poloxamer-407, while the dependent responses were particle size (PS) and entrapment efficiency (EE%). The optimized cubosomal formulation was validated through the utilization of high-resolution transmission electron microscopy (HR-TEM), in vitro release studies, and an in vitro cell proliferation assay conducted on the HepG2 cell line. High-performance liquid chromatography was employed for the determination of piperine and quercetin in the optimized cubosomal nanoparticle. The optimized formulation had a composition of 2.5 (w/w%) GMO and 0.5 (w/w%) Poloxamer 407. The predicted values for PS and EE% were 102.34 and 75.11%, respectively. The cytotoxicity of the optimized cubosomal formulation exhibited enhanced efficacy on the HepG2 cancer cell line, even at lower concentrations, when compared to the standard. Notably, it demonstrated a superior cytotoxic effect on the liver cancer cell line.

Conclusion

The findings of the study indicated that cubosomes exhibit promise as an effective carrier for delivering piperine and quercetin, addressing hepatocellular carcinoma effectively.

Graphical abstract

Background

In the realm of cancer incidence, primary liver cancer holds the fifth position in terms of frequency, solidifying its status as the second most widespread cancer type [1, 2]. Over the course of several decades, the frequency of cancer cases has consistently risen. This underscores the ongoing significance of cancer as a major public health concern, remaining among the primary contributors to global mortality [3,4,5]. Despite the emergence of innovative methods like vaccines, immunotherapy, and gene therapy, which exhibit encouraging treatment outcomes for diverse cancer forms, chemotherapy retains its position as a fundamental strategy in the medical management of cancer [6,7,8].

Natural phytoconstituents play a vital role in impeding diverse mechanisms linked to cancer, including cellular division, cell proliferation, angiogenesis, programmed cell death (apoptosis), and the spread of cancer cells (metastasis) [9, 10]. Numerous herbs and phytoconstituents are utilized for the treatment of chronic liver ailments due to their cost-effectiveness, high safety thresholds, long-lasting positive effects, and minimal adverse reactions [11, 12]. Previous studies suggest that these natural compounds can protect liver cells through diverse mechanisms, including virus elimination, suppression of fibrogenesis, inhibition of oxidative damage, and reduction of carcinogenesis [13,14,15]. These herbal remedies effectively inhibit cell growth at different stages of the cell cycle, disrupt signaling pathways, and suppress gene expression, leading to the prevention of multiple types of cancer [16,17,18].

Quercetin, a member of the flavonoid family, possesses notable antioxidant, anti-inflammatory, and immunoregulatory properties, which contribute to its potential in preventing and treating conditions such as cardiovascular diseases, diabetes, neurodegenerative disorders, and cerebrovascular diseases [19,20,21]. Moreover, studies have reported the inhibitory effects of quercetin on hepatocellular carcinoma (HCC), showing that it can hinder HCC growth by suppressing proliferation and/or promoting apoptosis [22,23,24,25]. Numerous pharmacological investigations have indicated the promising capacity of piperine as a viable medication against cancer [26,27,28]. Its mechanism involves the suppression of pro-survival pathways and the initiation of apoptosis across diverse cancer cell variations. Piperine, identified as a potential anticancer compound capable of regulating autophagy, has demonstrated impressive success in combatting cancer within living organisms [29,30,31]. Recent research indicates that piperine has been utilized both as a standalone treatment and in combination with other drugs to enhance their anticancer effects and effectively manage the process of carcinogenesis [32].

Regrettably, these two medications exhibit limited solubility in water and lead to significant problems linked to sensitivity to light. Furthermore, quercetin has a tendency to experience swift chemical deterioration under conditions of acidic gastric pH. Hence, the integration of these two pharmaceutical agents into a well-suited, singularly lipid-mediated nanostructured arrangement, particularly through cubosomes, proves advantageous in surmounting crucial challenges. Cubosomes emerge as nanoparticles containing lipids and surfactants, showcasing an exceptional cubic liquid crystalline configuration [33, 34]. These minute entities, smaller than a micron, display a dual continuous character, boasting a substantial expanse of surface. Comprising amphiphilic components like polar and nonpolar polymers, lipids, and surfactants, cubosomes possess the innate ability to autonomously structure themselves. The genesis of cubosomes hinges on the self-organization of particular surfactant entities [35, 36].

The clinical effectiveness of anticancer drugs is limited because they tend to spread throughout the body without precision, resulting in unintended harm to healthy tissues and consequent side effects [37]. To address this challenge, researchers have introduced diverse drug transporters designed to transport therapeutic substances with precision to particular locations. One of the most successful approaches involves the utilization of nanocarriers, which capitalize on the enhanced permeation and retention phenomenon, allowing them to accurately target the afflicted areas while minimizing impact on unaffected tissues [38, 39].

Consequently, within this current investigation, our contemplation revolves around crafting a cubosome-based nanoformulation that encapsulates quercetin and piperine. The attainment of desired therapeutic effects from any medicinal agent hinges on precise assessments of the entrapped substances within the nanoformulation. Hence, our objective resides in the development of a reverse phase-high-performance liquid chromatography (RP-HPLC) technique, enabling the simultaneous quantification of both quercetin and piperine loaded within the cubosomes.

Methods

Materials

Quercetin (≥ 95%) was procured from Ausen Chemical Co., Ltd. in China, while piperine (≥ 97%) was acquired from Sigma-Aldrich India. Glyceryl monooleate (GMO) was kindly provided by Mohini Organics India. Poloxamer was obtained from Sigma-Aldrich in India, while potassium dihydrogen phosphate and sodium hydroxide were sourced from Merck Life Science Pvt. Ltd., India. Fetal bovine serum (FBS) and Dulbecco's Modified Eagle Medium (DMEM) solutions were acquired from Sigma-Aldrich in India. High-performance liquid chromatography (HPLC) grade methanol and various other essential chemicals were obtained from Merck Life Science Pvt. Ltd.

Compatibility studies

To evaluate the compatibility of piperine and quercetin and search for any possible interactions between the two compounds, Fourier transform infrared spectroscopy (FT-IR) was performed [40]. In a nutshell, piperine and quercetin were mixed together in a 1:1 ratio (2 mg), and the mixture was kept at 25 ± 2 °C and 60 ± 5% relative humidity (%RH) for 28 days. Piperine, quercetin, and the mixture underwent FT-IR analysis. The effects of piperine, quercetin, and their combination were studied using FT-IR.

Experimental design

Design-Expert software version 13.0 was used to create the experiments (Stat-Ease Inc., Minneapolis, MN, USA). The program was used to design and produce a total of 11 formulations [41,42,43]. To optimize the prepared cubosome formulations, a two-factor three-level (32) experimental design was employed. The independent variables considered were the amount of glyceryl monooleate (X1) and Poloxamer 407 (X2). Meanwhile, the dependent variables assessed were particle size (Y1) and entrapment efficiency (Y2). The statistical plan included three different concentrations of Poloxamer 407 (0.5, 1.25, and 2%) and three different concentrations of glyceryl monooleate (2.5, 3.75, and 5%) (Table 1).

Table 1 Coded values of 32 full factorial design for formulation of cubosomes
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Preparation of cubosomes

Piperine- and quercetin-loaded cubosomes were prepared using the top-down method with the aid of a magnetic stirrer and homogenizer. The process began by melting the lipid (2.5 to 5%), followed by dissolving a precisely weighed quantity of the drug (0.1%) in the melted glyceryl monooleate (GMO). This mixture was then transferred into the melted Poloxamer 407 (0.5 to 2%), resulting in the formation of a cubic phase [44,45,46,47]. To reach the desired volume (30 mL), a measured amount of water was added dropwise using a syringe. The mixture was homogenized using an Ultraturaxx homogenizer at 12,000 rpm for 15 min and subsequently sonicated for 15 min to obtain cubosomes. The prepared cubosomes were then stored in a cool environment. Various formulations of PQC (piperine–quercetin cubosomes) were meticulously prepared, and their respective compositions are elucidated in Table 2.

Table 2 CCD experimental runs with obtained response
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Particle size, PDI and entrapment efficiency

The mean vesicle size and polydispersity index (PDI) of cubosomes loaded with piperine and quercetin were analyzed using a zetasizer. For the analysis, 1 mL of the formulation was diluted with 20 mL of milli-Q water. To separate the untrapped drug, samples from each formulation were subjected to centrifugation at 15,000 rpm and 4 °C for a duration of 1 h. The resulting supernatant was collected and then appropriately diluted with methanol. The estimation of the unbound drug was performed using UV–Vis spectroscopy at a wavelength of 273 nm. The percentage of drug entrapment was calculated using the relevant formula [48, 49].

In vitro release study of cubosomes

The in vitro release study of cubosomes was conducted using the dynamic dialysis method (USP II method). The investigation into drug release employed a tablet dissolution testing apparatus (Electrolab, EDT-08LX) featuring a low-volume conversion kit (EDT-08L/08L × 150 ml). This specialized setup was specifically chosen for assessing the release of the drug from the nanoformulation. Prior to the study, the dialysis membrane was soaked overnight [50,51,52]. Briefly, PQC nanoformulation (5 ml) was transferred to a dialysis bag, which was immersed into a 100 ml of PBS (pH 5.5) containing tween 80 (0.1%). The temperature of dissolution medium was kept at 37 ± 0.5 °C with a stirring speed of 50 rpm. At predetermined time intervals (0.5, 1, 2, 3, 4, 6, 12 and 24 h), aliquots of 1 mL were withdrawn for analysis. The withdrawn aliquots of the drug release medium were replaced with same volume of fresh buffer to maintain the sink condition. The drug concentrations in the samples were analyzed using spectrophotometry at the maximum wavelength (λ max) of 273 nm.

Transmission electron microscopy (TEM)

The surface morphology and structure of optimized cubosomes were examined using a high-resolution transmission electron microscope (JEM-2100, Jeol, Tokyo, Japan). A small volume of the optimized cubosomes was applied onto a carbon-coated grid, allowing the sample to adhere. Subsequently, the specimen was stained with uranyl acetate for a duration of 5 min, followed by staining with lead citrate for 2 min. The grid containing the stained sample was then observed using a transmission electron microscope, with an appropriate level of magnification (20 nm and 100 nm) chosen to capture images [53, 54].

High-performance liquid chromatography (HPLC)

The Agilent Technologies 1220 Infinity II HPLC system was used for quantification of piperine and quercetin in optimized cubosomal formulation. Utilizing a reversed-phase C-18 column (5 µm, 250 × 4.6 mm, ZORBAX), chromatographic separation was accomplished [55,56,57,58]. After conducting numerous trials, the chromatographic conditions were fine-tuned by considering factors such as peak area, retention time, tailing factor, and theoretical plates. The optimal mobile phase was composed of methanol and 0.1% formic acid in water, in the ratio of 80:20. Samples were analyzed at the flow rate of 1 mL/min, and the detection wavelength was set at 273 nm. The prepared cubosomes were centrifuged for 30 min at 15,000 rpm. The supernatant (0.1 mL) was pipetted using a micropipette and made up the volume up to 10 mL of volumetric flask using mobile phase. For each analysis, a 10 μl sample was injected into the column. According to ICH Q2 (R1) criteria, the developed HPLC method's validation was carried out. Linearity, LOD, LOQ, precision, accuracy and robustness were all used to thoroughly evaluate the optimized method.

Cytotoxicity analysis

For this study, the HepG2 cell line, derived from human hepatocellular adenocarcinoma, was procured from NCCS in Pune, India. The cells were cultivated in DMEM-high glucose medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotic–antimycotic solution [59, 60]. Maintaining optimal conditions, the cells were cultured in a controlled environment with 5% CO2, 18–20% O2 and a temperature of 37 °C within a CO2 incubator. Sub-culturing of the cells was carried out every 2 days, and passage number 34 was employed for the experimental procedures. Transfer 200 μl of cell suspension into individual wells of a 96-well plate, targeting a cell density of 20,000 cells per well. Avoid introducing the test agent at this stage. Allow the cells to proliferate for approximately 24 h. Distinct concentrations of cubosomal solutions (50, 60, 70, 80, 90, and 100 µg/ml) were prepared in a solution containing 0.1% DMSO. Subsequently, 50 µl of each concentration's solution was individually introduced into designated wells containing the cells. The plate was then placed in an incubator set at 37 °C, 5% CO2, and 90% humidity for a 24-h duration. A control group was processed without the addition of this formulation. Following the incubation period, 15 µl of MTT solution (5 mg/ml in PBS) was added to the wells, and the plate was further incubated for 4 h at 37 °C. Post-incubation, 100 µl of DMSO was introduced to dissolve the formazan crystals that had formed. The absorbance of the cells was measured at 570 nm utilizing an ELISA plate reader. The mean values for cell viability were compared against the control to assess the impact of cubosomes on the cells. A graphical representation was plotted, correlating cell viability (%) against the concentration levels of the formulation.

Statistical optimization

The experiments conducted in this study were replicated in triplicate, and the outcomes were reported as the mean accompanied by the standard deviation. Both ANOVA and Student’s t test were executed, determining statistical significance for results at a threshold of p < 0.05.

Results

Compatibility studies

The Fourier transform infrared (FTIR) analysis of piperine and quercetin, as depicted in Fig. 1, unveiled the presence of various discernible functional groups. Notably, the spectrum exhibited distinct peaks corresponding to specific vibrational modes, including the aromatic C-H stretch at 2922.61 cm−1, the alcohol O–H stretch at 3393.02 cm−1, the C–O stretch at 1246.50 cm−1, and the aromatic C–C stretch at 1640.32 cm−1. Intriguingly, the physical mixture of the drug and excipients manifested the persistence of these characteristic peaks, underscoring the compatibility of the components. This observation serves to corroborate that the unique chemical identities of both the drug and the excipients remain intact in the composite system.

Fig. 1
figure 1

FTIR spectrum of physical mixture (GMO, Poloxamer 407, piperine, and quercetin)

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Experimental design

Based on the regression coefficient determined in the ANOVA study, it was discovered that independent variables X1 (GMO) and X2 (Poloxamer-407) have a positive sign, indicating a synergistic influence on the outcome Y1 (PS). It was discovered through graphical presentation, namely the response surface plot that raising the level of variable X1 and X2 tends to raise the value of response Y1 (Fig. 2). Both independent variables X1 and X2 had a negative sign, indicating an antagonistic influence on the response Y2. Graphical evidence revealed that as the level of both variables, X1 and X2, is increased, the value of response Y2 was dropped (Fig. 3). Utilizing the Design-Expert software by Stat-Ease, Inc. (version 13.0), it was determined that all responses yielded satisfactory outcomes. The shaded zone with yellow color in the design space (Overlay plot) depicted in Fig. 4 represents the region of successful operating ranges. Design space facilitated the identification of optimal values for the independent parameters: GMO (2.5%) and Poloxamer (0.5%) under these specific concentrations. The model's exceptional ability to forecast values for the response variables was showcased by the remarkably low prediction error (5%) observed between the predicted values and the actual observations.

Fig. 2
figure 2

Response surface plot for response Y1 (Particle size)

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Fig. 3
figure 3

Response surface plot for response Y2 (Entrapment efficiency)

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Fig. 4
figure 4

Design space overlay plot

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Particle size, PDI and entrapment efficiency

The drug-loaded cubosome nanoformulation was prepared through homogenization method, resulting in mean particle sizes of 102.36 nm and 222.4 nm, respectively. The cubosomes exhibited an average PDI in value range 0.1831 to 0.8894. These PDI values are indicative of a narrow and homogeneous particle size distribution, as they are less than 0.9. The zeta potential is a significant variable for evaluating the stability of nanoparticulate systems. Zeta potential was recorded to be between − 13.6 and − 38.9 mV. These zeta potential readings suggest that the cubosome nanoparticles are moderately stable. The presence of free fatty acids within glycerol monooleate's lipid structure, which imparts this negative charge, is the cause of the negative charge that was detected.

Our analysis of the percentage of free and total drug in the cubosome nanoformulation revealed that the encapsulation efficiency (EE) for piperine and quercetin exhibited a range of 53.28% to 81.23%. The optimized concentration of surfactants used is probably responsible for the cubosomes substantially higher EE percentages. Significant amounts of quercetin and piperine were successfully solubilized by these surfactants within the lipid vesicles. The amounts of these substances were thereby markedly increased within the cubosome formulation.

In vitro release study of cubosomes

The in vitro drug release profiles of piperine and quercetin from cubosomes are meticulously illustrated in Fig. 5. The release study, specifically focusing on the optimized batch of the PQC nanoformulation (designated as PQC1), was conducted in a buffer with a pH of 5.5. This investigation was carried out utilizing a tablet dissolution testing apparatus equipped with a low-volume conversion kit. Broadly, the bicontinuous cubosome nanoformulations exhibited a biphasic burst release characterized by a diffusion-based mechanism emanating from the cubic-phase matrix structure. The release of both piperine and quercetin from the cubosome formulation displayed a biphasic pattern. In the initial hour, there was a notable burst release, accounting for approximately 22.43% of piperine and 16.40% of quercetin. Subsequently, after a 24-h duration, the cumulative release escalated to 82.92% for piperine and 61.04% for quercetin.

Fig. 5
figure 5

Cumulative drug release of PQC formulation

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Transmission electron microscopy (TEM)

The surface morphology and structural integrity of the optimized PQC nanoformulation (PQC1) were meticulously scrutinized and substantiated through high-resolution transmission electron microscopy (HR-TEM) imaging. The resulting images revealed that the cubosome particles exhibited a distinctly cubic shape, characterized by a uniform and smooth surface with minimal curvature, as depicted in Fig. 6A, B. Notably, the particles displayed a scattering pattern indicative of their nanoscale dimensions, further accentuating their finely tuned and compact structure.

Fig. 6
figure 6

HR-TEM images of optimized cubosomal nanoformulation (PQC1)

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High-performance liquid chromatography (HPLC)

The optimized method has proven its efficacy in the determination of formulated cubosomes, revealing distinct peaks for both quercetin and piperine. Notably, these peaks were precisely measured at 3.055 and 5.067 min, respectively. The consistent retention times underscore the robustness of the separation method, offering compelling evidence for the successful application of this approach in isolating the two pharmaceuticals within the cubosomes, as illustrated in Figs. 7 and 8. To ensure that the optimized HPLC method is appropriate for the intended use as outlined in ICH Q2 (R1) standards, it has undergone validation. Table 3 provides a summary of the proposed HPLC method's validation parameters, which were determined to be within the ICH Guidelines' standard limits.

Fig. 7
figure 7

HPLC chromatogram depicting standard quercetin and piperine

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Fig. 8
figure 8

HPLC chromatogram depicting quercetin and piperine in cubosomes

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Table 3 Summary of validation parameter
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Cytotoxicity analysis

The statistical findings from the MTT cell cytotoxicity study revealed that the examined compounds exhibited noteworthy cytotoxic attributes against HepG2 cells. Following a 24-h incubation period, the % cell viability of cubosomes at a concentration of 100 μM/mL was determined to be 4.05%. A standard control utilizing 1 μM/mL of doxorubicin was employed, and the cell viability for the standard was established at 32.25% (Fig. 9). The determination of the IC50 value for the cubosomes, based on the percentage drug loading, unequivocally indicates that the designed nanoformulation possesses a superior anticancer effect compared to the standard drug. This superiority may be attributed to its nanosized dimensions, sustained release properties and targeted delivery to cancer cells.

Fig. 9
figure 9

The overlaid bar graph depicting the percentage of cell viability for HepG2 cell lines

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Discussion

This study takes a step toward enhancing liver cancer treatment by focusing on cubosomal formulations containing piperine and quercetin. Piperine and quercetin are compounds known for their potential anticancer properties. By formulating these compounds into cubosomes, the aim is to augment their activity against hepatocellular carcinoma. To assess the compatibility of piperine and quercetin and explore potential interactions between these two compounds, Fourier transform infrared spectroscopy (FT-IR) was conducted. The obtained spectrum revealed clear peaks associated with specific vibrational modes, such as the aromatic C–H stretch observed at 2922.61 cm−1, the alcohol O–H stretch at 3393.02 cm−1, the C–O stretch at 1246.50 cm−1, and the aromatic C–C stretch at 1640.32 cm−1. This observation serves to corroborate that the unique chemical identities of both the drug and the excipients remain intact in the composite system.

The experimental design of this study utilized a central-composite design, guided by the Design-Expert® software. Key formulation variables were the concentration of glyceryl monooleate (GMO) and Poloxamer-407, while particle size (PS) and entrapment efficiency (EE %) were chosen as dependent responses. This approach provides a systematic and statistical method to optimize the formulation for the desired properties. The optimized cubosomal formulation, with a composition of 2.5% (w/w) GMO and 0.5% (w/w) Poloxamer 407, was validated using various techniques. The predicted values for PS and EE% were 102.34 and 76.43%, respectively, indicating successful optimization based on the experimental design. The optimized nanoformulation of piperine and quercetin (PQC1) underwent thorough examination of its surface morphology and structural integrity using high-resolution transmission electron microscopy (HR-TEM) imaging. The images obtained depicted cubosome particles with a well-defined cubic shape, showcasing a consistently smooth and uniform surface with minimal curvature.

The in vitro release study, specifically focusing on the optimized batch of the PQC nanoformulation (designated as PQC1), was conducted in a buffer with a pH of 4.5. After a 24-h duration, the cumulative release escalated to 82.92% for piperine and 61.04% for quercetin. High-performance liquid chromatography was utilized to quantify the concentration of piperine and quercetin within the optimized cubosomal nanoparticles. Significantly, the distinct peaks for piperine and quercetin were precisely identified at 3.055 and 5.067 min, respectively. The consistent retention times emphasize the reliability of the separation method, providing compelling evidence for the effective application of this approach in isolating the two pharmaceuticals within the cubosomes. The cytotoxicity assessment revealed that the optimized cubosomal formulation exhibited enhanced efficacy on the HepG2 cancer cell line, even at lower concentrations compared to the standard treatment. This superior cytotoxic effect specifically on the liver cancer cell line suggests the potential of cubosomes as an effective carrier for delivering piperine and quercetin to address hepatocellular carcinoma.

The findings of this study highlight the promise of cubosomes in liver cancer therapy. The targeted delivery system not only optimizes drug efficacy but also minimizes side effects, addressing a critical need in the complex landscape of liver cancer treatment. The use of advanced techniques such as HR-TEM and high-performance liquid chromatography adds credibility to the study, ensuring a thorough validation of the optimized cubosomal formulation. In conclusion, this research contributes to the growing body of knowledge seeking innovative solutions for liver cancer treatment. The effectiveness of cubosomal formulations containing piperine and quercetin against hepatocellular carcinoma suggests a potential breakthrough in precision therapy. As further research and clinical trials unfold, these findings may pave the way for a more targeted and efficient approach to combat liver cancer, ultimately improving patient outcomes on a global scale.

Conclusion

The objective of this research was to develop precision-targeted cubosomes containing piperine and quercetin to address Hepatocellular carcinoma. Effective formulation and fine-tuning of cubosomes loaded with piperine and quercetin were accomplished employing central composite factorial design methodology in conjunction with the Design-expert software. The prepared cubosomes demonstrated well-suited nanoscale vesicles, with particle dimensions falling within the nanoscale and achieving peak entrapment efficiency levels. Following the design of experiments (DoE) and the desirability criteria, it was determined that formulation (C1) containing GMO (2.5%) and Poloxamer 407 (0.5%) stood out as the most favorable selection, exhibiting the most compact particle size (102.3 nm) and greatest entrapment efficiency (76.43%). An analytical technique was established for piperine and quercetin using HPLC under standard environmental conditions. The solvent system utilized consisted of HPLC-grade methanol and 0.1% formic acid in water an 80:20 ratio. The quantitative analysis demonstrated well characteristics peaks for both drugs in the optimized cubosomal formulation, with a retention time of 3.0 min for quercetin and 5.0 min for piperine. The investigation on the HepG2 cell line using the cubosomes loaded with piperine and quercetin, carried out through the MTT assay. Following a 24-h incubation period, the % cell viability of cubosomes at a concentration of 100 μM/mL was determined to be 4.05%. A standard control utilizing 1 μM/mL of doxorubicin was employed, and the cell viability for the standard was established at 32.25%. This outcome serves as confirmation of the formulation's effectiveness in treating hepatocellular carcinoma; this formulation stands as a promising and preferred dosage form poised to be a leading option in the near future.

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References

  1. Bolondi L, Cillo U, Colombo M, Craxì A, Farinati F, Giannini EG, Golfieri R, Levrero M, Pinna AD, Piscaglia F, Raimondo G (2013) Position paper of the Italian Association for the Study of the Liver (AISF): the multidisciplinary clinical approach to hepatocellular carcinoma. Dig Liver Dis 45(9):712–723. https://doi.org/10.1016/j.dld.2013.01.012

    Article  PubMed  Google Scholar 

  2. Craig ER, Londoño AI, Norian LA, Arend RC (2016) Metabolic risk factors and mechanisms of disease in epithelial ovarian cancer: a review. Gynecol Oncol 143(3):674–683. https://doi.org/10.1016/j.ygyno.2016.10.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Cuccurullo V, Di Stasio GD, Mazzarella G, Cascini GL (2018) Microvascular invasion in HCC: the molecular imaging perspective. Contrast Media Mol Imaging. https://doi.org/10.1155/2018/9487938

    Article  PubMed  PubMed Central  Google Scholar 

  4. Mariotto AB, Robin Yabroff K, Shao Y, Feuer EJ, Brown ML (2011) Projections of the cost of cancer care in the United States: 2010–2020. J Natl Cancer Inst 103(2):117–128. https://doi.org/10.1093/jnci/djq495

    Article  PubMed  PubMed Central  Google Scholar 

  5. Chaturvedi AK, Anderson WF, Lortet-Tieulent J, Curado MP, Ferlay J, Franceschi S, Rosenberg PS, Bray F, Gillison ML (2013) Worldwide trends in incidence rates for oral cavity and oropharyngeal cancers. J Clin Oncol 31(36):4550. https://doi.org/10.1200/JCO.2013.50.3870

    Article  PubMed  PubMed Central  Google Scholar 

  6. Lacoste B, Raymond VA, Cassim S, Lapierre P, Bilodeau M (2017) Highly tumorigenic hepatocellular carcinoma cell line with cancer stem cell-like properties. PLoS ONE 12(2):e0171215. https://doi.org/10.1371/journal.pone.0171215

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chu E, Sartorelli AC (2018) Cancer chemotherapy. Lange’s Basic and Clinical Pharmacology 948–76

  8. Gudasi S, Gharge S, Koli R, Kagawad P (2024) exploring in-silico, in-vitro antioxidant, and cytotoxic potential of valerian Wallichii by on cervical epithelial carcinoma (HeLa) cell lines. Chem Biodivers. https://doi.org/10.1002/cbdv.202302072

    Article  PubMed  Google Scholar 

  9. George BP, Chandran R, Abrahamse H (2021) Role of phytochemicals in cancer chemoprevention: insights. Antioxidants 10(9):1455. https://doi.org/10.3390/antiox10091455

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ranjan A, Ramachandran S, Gupta N, Kaushik I, Wright S, Srivastava S, Das H, Srivastava S, Prasad S, Srivastava SK (2019) Role of phytochemicals in cancer prevention. Int J Mol Sci 20(20):4981. https://doi.org/10.3390/ijms20204981

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zheng Z, Zhang L, Hou X (2022) Potential roles and molecular mechanisms of phytochemicals against cancer. Food Funct 13(18):9208–9225. https://doi.org/10.1039/D2FO01663J

    Article  CAS  PubMed  Google Scholar 

  12. Bhoi A, Dwivedi SD, Singh D, Singh MR, Keshavkant S (2023) Worldwide health scenario from the perspective of herbal medicine research. Phytopharmaceuticals and herbal drugs. Academic Press, New York, pp 13–32. https://doi.org/10.1016/B978-0-323-99125-4.00014-7

    Book  Google Scholar 

  13. Yadav R, Das J, Lalhlenmawia H, Tonk RK, Singh L, Kumar D (2021) Targeting cancer using phytoconstituents-based drug delivery. Advanced drug delivery systems in the management of cancer. Academic Press, New York, pp 499–508. https://doi.org/10.1016/B978-0-323-85503-7.00033-X

    Book  Google Scholar 

  14. Domitrović R, Potočnjak I (2016) A comprehensive overview of hepatoprotective natural compounds: mechanism of action and clinical perspectives. Arch Toxicol 90:39–79. https://doi.org/10.1007/s00204-015-1580-z

    Article  CAS  PubMed  Google Scholar 

  15. Li S, Tan HY, Wang N, Cheung F, Hong M, Feng Y (2018) The potential and action mechanism of polyphenols in the treatment of liver diseases. Oxid Med Cell Longev. https://doi.org/10.1155/2018/8394818

    Article  PubMed  PubMed Central  Google Scholar 

  16. Singh AK, Sharma N, Ghosh M, Park YH, Jeong DK (2017) Emerging importance of dietary phytochemicals in fight against cancer: role in targeting cancer stem cells. Crit Rev Food Sci Nutr 57(16):3449–3463. https://doi.org/10.1080/10408398.2015.1129310

    Article  CAS  PubMed  Google Scholar 

  17. Anwanwan D, Singh SK, Singh S, Saikam V, Singh R (2020) Challenges in liver cancer and possible treatment approaches. Biochim Biophys Acta Rev Cancer 1873(1):188314. https://doi.org/10.1016/j.bbcan.2019.188314

    Article  CAS  PubMed  Google Scholar 

  18. Liu CY, Chen KF, Chen PJ (2015) Treatment of liver cancer. Cold Spring Harb Perspect Med. https://doi.org/10.1101/cshperspect.a021535

    Article  PubMed  PubMed Central  Google Scholar 

  19. Li Y, Yao J, Han C, Yang J, Chaudhry MT, Wang S, Liu H, Yin Y (2016) Quercetin, inflammation and immunity. Nutrients 8(3):167. https://doi.org/10.3390/nu8030167

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Al-Khayri JM, Sahana GR, Nagella P, Joseph BV, Alessa FM, Al-Mssallem MQ (2020) Flavonoids as potential anti-inflammatory molecules: a review. Molecules 27(9):2901. https://doi.org/10.3390/molecules27092901

    Article  CAS  Google Scholar 

  21. Deepika MPK (2022) Health benefits of quercetin in age-related diseases. Molecules 27(8):2498. https://doi.org/10.3390/molecules27082498

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fernández-Palanca P, Fondevila F, Méndez-Blanco C, Tuñón MJ, González-Gallego J, Mauriz JL (2019) Antitumor effects of quercetin in hepatocarcinoma in vitro and in vivo models: a systematic review. Nutrients 11(12):2875. https://doi.org/10.3390/nu11122875

    Article  PubMed  PubMed Central  Google Scholar 

  23. Ji YI, Li LI, Ma YX, Li WT, Li L, Zhu HZ, Wu MH, Zhou JR (2019) Quercetin inhibits growth of hepatocellular carcinoma by apoptosis induction in part via autophagy stimulation in mice. J Nutr Biochem 69:108–119. https://doi.org/10.1016/j.jnutbio.2019.03.018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Wu L, Li J, Liu T, Li S, Feng J, Yu Q, Zhang J, Chen J, Zhou Y, Ji J, Chen K (2019) Quercetin shows anti-tumor effect in hepatocellular carcinoma LM3 cells by abrogating JAK2/STAT3 signaling pathway. Cancer Med 8(10):4806–4820. https://doi.org/10.1002/cam4.2388

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Abdu S, Juaid N, Amin A, Moulay M, Miled N (2022) Effects of sorafenib and quercetin alone or in combination in treating hepatocellular carcinoma: in vitro and in vivo approaches. Molecules. https://doi.org/10.3390/molecules27228082

    Article  PubMed  PubMed Central  Google Scholar 

  26. Mitra S, Anand U, Jha NK, Shekhawat MS, Saha SC, Nongdam P, Rengasamy KR, Proćków J, Dey A (2022) Anticancer applications and pharmacological properties of piperidine and piperine: a comprehensive review on molecular mechanisms and therapeutic perspectives. Front Pharmacol 12:772418. https://doi.org/10.3389/fphar.2021.772418

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Quijia CR, Chorilli M (2022) Piperine for treating breast cancer: a review of molecular mechanisms, combination with anticancer drugs, and nanosystems. Phytother Res 36(1):147–163. https://doi.org/10.1002/ptr.7291

    Article  CAS  PubMed  Google Scholar 

  28. Zadorozhna M, Tataranni T, Mangieri D (2019) Piperine: role in prevention and progression of cancer. Mol Biol Rep 46(5):5617–5629. https://doi.org/10.1007/s11033-019-04927-z

    Article  CAS  PubMed  Google Scholar 

  29. Rather RA, Bhagat M (2018) Cancer chemoprevention and piperine: molecular mechanisms and therapeutic opportunities. Front Cell Dev Boil 6:10. https://doi.org/10.3389/fcell.2018.00010

    Article  Google Scholar 

  30. Thawabteh A, Juma S, Bader M, Karaman D, Scrano L, Bufo SA, Karaman R (2019) The biological activity of natural alkaloids against herbivores, cancerous cells and pathogens. Toxins 11(11):656. https://doi.org/10.3390/toxins11110656

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Swetha M, Keerthana CK, Rayginia TP, Anto RJ (2022) Cancer chemoprevention: a strategic approach using phytochemicals. Front Pharmacol 12:809308. https://doi.org/10.3389/fphar.2021.809308

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Bolat ZB, Islek Z, Demir BN, Yilmaz EN, Sahin F, Ucisik MH (2020) Curcumin-and piperine-loaded emulsomes as combinational treatment approach enhance the anticancer activity of curcumin on HCT116 colorectal cancer model. Front Bioeng Biotechnol 8:50. https://doi.org/10.3389/fbioe.2020.00050

    Article  PubMed  PubMed Central  Google Scholar 

  33. Zhai J, Fong C, Tran N, Drummond CJ (2019) Non-lamellar lyotropic liquid crystalline lipid nanoparticles for the next generation of nanomedicine. ACS Nano 13(6):6178–6206. https://doi.org/10.1021/acsnano.8b07961

    Article  CAS  PubMed  Google Scholar 

  34. Hartnett TE, Ladewig K, O’Connor AJ, Hartley PG, McLean KM (2014) Size and phase control of cubic lyotropic liquid crystal nanoparticles. J Phys Chem B 118(26):7430–7439. https://doi.org/10.1021/jp502898a

    Article  CAS  PubMed  Google Scholar 

  35. Abourehab MA, Ansari MJ, Singh A, Hassan A, Abdelgawad MA, Shrivastav P, Abualsoud BM, Amaral LS, Pramanik S (2022) Cubosomes as an emerging platform for drug delivery: a review of the state of the art. J Mater Chem 10(15):2781–2819. https://doi.org/10.1039/D2TB00031H

    Article  CAS  Google Scholar 

  36. Karami Z, Hamidi M (2016) Cubosomes: remarkable drug delivery potential. Drug Discov Today 21(5):789–801. https://doi.org/10.1016/j.drudis.2016.01.004

    Article  CAS  PubMed  Google Scholar 

  37. Manzari MT, Shamay Y, Kiguchi H, Rosen N, Scaltriti M, Heller DA (2021) Targeted drug delivery strategies for precision medicines. Nat Rev Mater 6(4):351–370. https://doi.org/10.1038/s41578-020-00269-6

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  38. Tian H, Zhang T, Qin S, Huang Z, Zhou L, Shi J, Nice EC, Xie N, Huang C, Shen Z (2022) Enhancing the therapeutic efficacy of nanoparticles for cancer treatment using versatile targeted strategies. J Hematol Oncol 15(1):1–40. https://doi.org/10.1186/s13045-022-01320-5

    Article  Google Scholar 

  39. Urandur S, Marwaha D, Gautam S, Banala VT, Sharma M, Mishra PR (2018) Nonlamellar liquid crystals: a new paradigm for the delivery of small molecules and bio-macromolecules. Ther Deliv 9(9):667–689. https://doi.org/10.4155/tde-2018-0038

    Article  CAS  PubMed  Google Scholar 

  40. Reddy DV, Navaneetha K (2015) Comparative assessment of potential of various polymers in the formulation development and evaluation of mucoadhesive microspheres of glipizide. J Glob Trends Pharm Sci 6(1):2379–2387

    CAS  Google Scholar 

  41. Koli R, Mannur VS, Gudasi S, Singadi R, Nashipudi A (2023) Development of directly compressible polyherbal tablets by using QbD approach a novel immunomodulatory material. J Med Pharm Allied Sci 11(16):5476–5484. https://doi.org/10.55522/jmpas.V11I6.4520

    Article  Google Scholar 

  42. Liu WY, Lin CC, Hsieh YS, Wu YT (2021) Nanoformulation development to improve the biopharmaceutical properties of fisetin using design of experiment approach. Molecules 26(10):3031. https://doi.org/10.3390/molecules26103031

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Rapalli VK, Banerjee S, Khan S, Jha PN, Gupta G, Dua K, Hasnain MS, Nayak AK, Dubey SK, Singhvi G (2021) QbD-driven formulation development and evaluation of topical hydrogel containing ketoconazole loaded cubosomes. Mater Sci Eng C 119:111548. https://doi.org/10.1016/j.msec.2020.111548

    Article  CAS  Google Scholar 

  44. Gaballa SA, El Garhy OH, Abdelkader H (2020) Cubosomes: composition, preparation, and drug delivery applications. J Adv Biomed Pharm Sci 3(1):1–9. https://doi.org/10.21608/JABPS.2019.16887.1057

    Article  Google Scholar 

  45. Alavi M, Nokhodchi A (2020) Micro-and nanoformulations of paclitaxel based on micelles, liposomes, cubosomes, and lipid nanoparticles: Recent advances and challenges. Drug Discov Today 27(2):576–584. https://doi.org/10.1016/j.drudis.2021.10.007

    Article  CAS  Google Scholar 

  46. Chettupalli AK, Ananthula M, Amarachinta PR, Bakshi V, Yata VK (2021) Design, formulation, in-vitro and ex-vivo evaluation of atazanavir loaded cubosomal gel. Biointerface Res Appl Chem 11:12037–12054. https://doi.org/10.33263/BRIAC114.1203712054

    Article  Google Scholar 

  47. Kurangi B, Jalalpure S, Jagwani S (2021) Formulation and evaluation of resveratrol loaded cubosomal nanoformulation for topical delivery. Curr Drug Deliv 18(5):607–619. https://doi.org/10.2174/1567201817666200902150646

    Article  CAS  PubMed  Google Scholar 

  48. Calvo NL, Sreekumar S, Svetaz LA, Lamas MC, Moerschbacher BM, Leonardi D (2019) Design and characterization of chitosan nanoformulations for the delivery of antifungal agents. Int J Mol Sci 20(15):3686. https://doi.org/10.3390/ijms20153686

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ucisik MH, Küpcü S, Schuster B, Sleytr UB (2013) Characterization of curcuemulsomes: nanoformulation for enhanced solubility anddelivery of curcumin. J Nanobiotechnol 11:1–3. https://doi.org/10.1186/1477-3155-11-37

    Article  CAS  Google Scholar 

  50. Ramadan SE, El-Gizawy SA, Osman MA, Arafa MF (2023) Application of design of experiment in the optimization of apixaban-loaded solid lipid nanoparticles: in vitro and in vivo evaluation. AAPS PharmSciTech 24(6):167. https://doi.org/10.1208/s12249-023-02628-2

    Article  CAS  PubMed  Google Scholar 

  51. Pulicharla R, Marques C, Das RK, Rouissi T, Brar SK (2016) Encapsulation and release studies of strawberry polyphenols in biodegradable chitosan nanoformulation. Int J Biol Macromol 88:171–178. https://doi.org/10.1016/j.ijbiomac.2016.03.036

    Article  CAS  PubMed  Google Scholar 

  52. Venkateswarlu V, Manjunath K (2004) Preparation, characterization and in vitro release kinetics of clozapine solid lipid nanoparticles. J Control Release 95(3):627–638. https://doi.org/10.1016/j.jconrel.2004.01.005

    Article  CAS  PubMed  Google Scholar 

  53. Malatesta M (2021) Transmission electron microscopy as a powerful tool to investigate the interaction of nanoparticles with subcellular structures. Int J Mol Sci 22(23):12789. https://doi.org/10.3390/ijms222312789

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Wilson BK, Prud’homme RK (2021) Nanoparticle size distribution quantification from transmission electron microscopy (TEM) of ruthenium tetroxide stained polymeric nanoparticles. J Colloid Interface Sci 604:208–220. https://doi.org/10.1016/j.jcis.2021.04.081

    Article  ADS  CAS  PubMed  Google Scholar 

  55. Carvalho D, Jesus Â, Pinho C, Oliveira RF, Moreira F, Oliveira AI (2023) Validation of an HPLC-DAD Method for quercetin quantification in nanoparticles. Pharmaceuticals 16(12):1736. https://doi.org/10.3390/ph16121736

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Kurangi B, Jalalpure S (2020) A validated stability-indicating RP-HPLC method for piperine estimation in black pepper, marketed formulation and nanoparticles. Indian J Pharm Educ Res 54(3s):s677–s686

    Article  CAS  Google Scholar 

  57. Koli R, Mannur VS (2023) Determination of aflatoxins using chromatographic methods in several foods, feed and herbal medicine products: an analytical review (from 2010 to 2022). J Liq Chromatogr Relat 46(11–15):238–254. https://doi.org/10.1080/10826076.2023.2247484

    Article  CAS  Google Scholar 

  58. Khursheed R, Wadhwa S, Kumar B, Gulati M, Gupta S, Chaitanya MV, Kumar D, Jha NK, Gupta G, Prasher P, Chellappan DK (2022) Development and validation of RP-HPLC based bioanalytical method for simultaneous estimation of curcumin and quercetin in rat’s plasma. S Afr J Bot 149:870–877. https://doi.org/10.1016/j.sajb.2021.12.009

    Article  CAS  Google Scholar 

  59. Gudasi S, Gharge S, Koli R, Patil K (2023) Antioxidant properties and cytotoxic effects of Oxalis corniculata on human Hepatocarcinoma (Hep-G2) cell line: an in vitro and in silico evaluation. Future J Pharm Sci 9(1):25. https://doi.org/10.1186/s43094-023-00476-2

    Article  Google Scholar 

  60. Mohamed EE, Abdel-Moneim A, Ahmed OM, Zoheir KM, Eldin ZE, El-Shahawy A (2022) Anticancer activity of a novel naringin-dextrin nanoformula: preparation, characterization, and in vitro induction of apoptosis in human hepatocellular carcinoma cells by inducing ROS generation, DNA fragmentation, and cell cycle arrest. J Drug Deliv Sci Technol 75:103677. https://doi.org/10.1016/j.jddst.2022.103677

    Article  CAS  Google Scholar 

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Acknowledgements

The authors are very thankful to Principal Dr. S. S. Jalalpure and Vice Principal Dr. M. B. Patil KLE College of Pharmacy, Belagavi, for their support and guidance. The authors are also thankful to the Department of Pharmaceutical Quality Assurance and KLE College of Pharmacy, Belagavi.

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  1. Department of Pharmaceutical Quality Assurance, KLE College of Pharmacy, KLE Academy of Higher Education and Research, Nehru Nagar, Belagavi, Karnataka, 590010, India

    Poorvika Badiger, V. S. Mannur & Rahul Koli

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  1. Poorvika Badiger
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  2. V. S. Mannur
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We affirm that "all authors have read and approved the manuscript." Each contributor played an equal role and actively engaged in this research project. Both PB and RK conscientiously reviewed the manuscript titled "Dual Drug-Loaded Cubosome Nanoparticles for Hepatocellular Carcinoma: A Design of Experiment Approach for Optimization and In Vitro Evaluation." Their efforts were conducted under the supervision of Dr. VSM, who offered guidance and assistance in navigating research complexities.

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Badiger, P., Mannur, V.S. & Koli, R. Dual drug-loaded cubosome nanoparticles for hepatocellular carcinoma: a design of experiment approach for optimization and in vitro evaluation. Futur J Pharm Sci 10, 38 (2024). https://doi.org/10.1186/s43094-024-00607-3

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