Silibinin-Loaded Nanostructured Lipid Carriers for Growth Inhibition of Cisplatin-Resistant Ovarian Cancer Cells

Abstract

Cisplatin is the most often used chemotherapy in the treatment of ovarian cancer (OC), however long-term usage leads to drug resistance and treatment failure. Silibinin is a sparingly water-soluble natural compound with well-known anticancer effects. The use of lipid-based delivery systems is a potential approach for enhancing silibinin's water solubility. In this study, nanostructured lipid carriers (NLCs) containing silibinin were prepared and their inhibitory effects were tested in combination with cisplatin against sensitive/resistant A2780 OC cells. Silibinin-loaded NLCs (silibinin-NLCs) were prepared by the hot homogenization method, and their size, shape, zeta potential (ZP), and encapsulation efficiency (EE), as well as their inhibitory effects, were examined in combination with cisplatin against sensitive/resistant A2780 OC cells. Formulation of silibinin-NLCs using cocoa butter led to spherical-shaped NLCs with a size of 95 nm and EE of 98%. The ZP and the dispersion index of the silibinin-NLCs were −27.12 ± 0.13 mv and 0.12 ± 0.04, respectively. The release kinetics of silibinin-NLCs was best fitted with the zero-order model. The combination of cisplatin and silibinin-NLCs sensitized the cisplatin-resistant A2780 OC cells and exhibited a more synergistic inhibitory effect on A2780 cells as compared with the combination of cisplatin and plain silibinin. The optimized silibinin-NLCs can be considered a suitable drug delivery system for the inhibition of cisplatin-resistant OC cells.

INTRODUCTION

Cancer, a great health concern worldwide is the name given to a collection of diseases characterized by uncontrolled growth and proliferation of healthy cells. Cancer mostly occurs due to the deficiency in cell apoptosis and division process, which is related to alterations in the genetic, epigenetic, and transcription factors.¹

Ovarian cancer (OC) is one of the most common lethal malignancies and has the highest incidence in women worldwide. According to World Health Organization, about 225,500 women are diagnosed annually with OC in the world and 140,200 patients go through cancer-related deaths.² Despite the development of novel therapeutic approaches for OC therapy, chemotherapy is still considered the main strategy for OC treatment.³

Cisplatin is therapeutically used to treat OC; nevertheless, long-term cisplatin therapy might cause chemoresistance in individuals.^4,5 As a result, in the majority of instances, chemotherapy with cisplatin leads to treatment failure owing to drug resistance in OC patients. To overcome drug resistance in OC, innovative therapeutic approaches are urgently needed.⁶ Combination therapy is one of the most efficient strategies for overcoming drug resistance in cancer cells, which targets different signaling pathways involved in the growth and survival of cancer cells.^7,8

Silibinin is a natural flavonoid molecule derived mostly from milk thistle seed that has received widespread attention for its hepatoprotective, antioxidant, and anticancer properties. Several studies have been conducted to investigate the anticancer effects of silibinin in various human malignancies, both in vitro and in vivo. ⁹ The identified mechanisms for silibinin's anticancer actions include induction of apoptosis, suppression of metastasis, and decrease of chemo-induced drug resistance in cancer cells.¹⁰ Moreover, an increased number of studies have demonstrated that silibinin causes growth inhibition and cell cycle arrests in different cancer cells such as OC, which can be related to both cytotoxic and cytostatic effects of silibinin on cancer cells.^10

–13

Silibinin is an efficient chemotherapeutic and chemopreventive drug in a variety of malignancies with few side effects.¹⁴ Furthermore, earlier research has shown that silibinin can make cancer cells more sensitive to chemotherapy drugs like doxorubicin and cisplatin.¹⁵ According to previous reports, silibinin restores cisplatin sensitivity in A2780 cancer cell lines.¹⁶ As a result, silibinin may have interesting clinical uses in combination with chemotherapeutic drugs to overcome drug resistance.

Despite its anticancer properties, silibinin has not yet been used in cancer treatment due to its limited water solubility and low bioavailability. One possible strategy for improving drug solubility is the formulation of weakly water-soluble drugs in organic nanocarriers.^17

–22 Nanostructured lipid carriers (NLCs) are a new type of lipid-based nanoparticles (NPs) made from a solid lipid matrix with a liquid lipid content that overcomes the constraints of solid lipid NPs (e.g., low drug storage capacity and drug explosion during storage). NLCs also possess other exclusive properties such as high biocompatibility and biodegradability, large surface area, small size, and interaction of phases at the interface, making them suitable candidates for drug delivery.²³ The use of NLCs to deliver lipophilic compounds to cancer cells, not only increases their bioavailability but also improves the pharmacokinetic properties through passive targeting due to the enhanced permeability and retention (EPR) effect.

In this research, for the first time, we developed silibinin-loaded NLCs (silibinin-NLCs) composed of cocoa butter as a solid lipid matrix to improve the biological properties of silibinin. Size, surface charge, morphology, drug loading (DL), drug content (DC), encapsulation efficiency (EE), and drug release were all measured in silibinin-NLCs. Furthermore, the cytotoxicity of silibinin-NLCs was assessed in sensitive and cisplatin-resistant A2780 cell lines as a human OC model when used alone and in combination with cisplatin.

MATERIALS AND METHODS

Chemicals and Reagents

N-methyl-2-pyrrolidone (NMP; C₅H₉NO), Poloxamer 407 (C₅₇₂H₁₁₄₆O₂₅₉), Polysorbate 80 (C₆₄H₁₂₄O₂₆), silibinin (C₂₅H₂₂O₁₀), and MTT reagent were purchased from Sigma (St. Louis, MO). Oleic acid (C₁₈H₃₄O₂), ethanol (CH₃CH₂OH), dimethyl sulfoxide (DMSO), and dichloromethane (CH₂Cl₂) were obtained from Merk (Darmstadt, Germany). Deionized distilled water was provided by Shahid Ghazi Pharmaceutical Company (Tabriz, Iran) and used in all steps of the experiments. Nondeodorized cocoa butter was purchased in small disc-shaped chips from Barry Callebaut (Zürich, Switzerland). Fetal bovine serum (FBS) and RPMI-1640 culture media were bought from Gibco (Thermo Fisher Scientific, Waltham, MA). The cell culture experiments were conducted using a class II biological safety cabinet (JAL TAJHIZ, JTLVC2, Karaj, Iran). The study was approved by the local Ethics Committee of Tabriz University of Medical Sciences (code: IR.TBZMED.VCR.REC.1398.463).

Cell Lines

The sensitive A2780 and cisplatin-resistant A2780 cell lines were purchased from the Iranian National Cell Bank (Pasteur Institute, Tehran, Iran). RPMI 1640 medium containing 10% FBS was used to culture the cell lines in a humidified incubator with 5% CO₂ at 37°C.

Formulation of Silibinin-NLCs

Silibinin was loaded in NLC by the hot homogenization method.^24,25 Solid lipid (cocoa butter) and tween 80 as an oil-phase surfactant were heated to 45°C in a hot water bath and placed on a stirrer to form an oil phase. In another vessel, the aqueous surfactant (poloxamer 407) was dissolved in distilled water at the same temperature as the water phase. Silibinin was dissolved in 200 μL NMP and added to the oil phase in a homogenizer (Heidolph Silent Crusher M GmbH & Co., Schwabach, Germany) for 3 min at 5,000 rpm, then the speed was increased to 18,000 rpm to make a water emulsion in oil. Cocoa butter was used to prepare the NLCs and NMP was applied to increase the solubility of silibinin in cocoa butter. Then, the aqueous phase was added dropwise into the oil phase, homogenizing at 18,000 rpm, and the temperature was maintained at 45°C. Finally, the suspension was placed in a fixed location at room temperature (24°C) to form NLCs. Different formulations of silibinin-NLCs (Table 1) were prepared and characterized to select an optimized formulation for the rest of the investigations.

Table 1.

Physicochemical Properties of Silibinin-Loaded Nanostructured Lipid Carrier Formulations

Formulation	Cocoa butter (mg)	Silibinin (mg)	Tween 80 (mg)	Poloxamer 407 (mg)	Size (nM)	PDI	Zeta-potential (mv)	EE ± SD (%)	DL ± SD (mg/g)	DC (%)
F1	504	20	10	450	26.41 ± 1.64	0.12 ± 0.2	−29.2 ± 0.3	98.70 ± 0.002	18.45	1.82
F2	500	20	20	500	127.9 ± 8.54	0.23 ± 0.01	−21.9 ± 0.23	98.11 ± 0.0023	17.54	0.37
F3	500	20	30	550	103.11 ± 17.18	0.35 ± 0.02	−25.9 ± 0.19	97.61 ± 0.0017	16.66	0.47
F4	500	20	40	600	173.07 ± 43.77	0.42 ± 0.03	−21.2 ± 0.21	99.17 ± 0.0021	15.87	0.16
F5	300	20	50	350	129.8 ± 2.28	0.18 ± 0.02	−20.9 ± 0.13	94.05 ± 0.0014	40	3.76
F6	300	30	20	350	108.9 ± 29.05	0.15 ± 0.03	−30.1 ± 0.43	98.30 ± 0.0023	40	3.93
F7	300	30	20	350	95.3 ± 2.29	0.12 ± 0.04	−27.12 ± 0.13	98.75 ± 0.0026	40	3.95
Blank	300	—	20	350	116 ± 21.22	0.43 ± 0.1	−27.13 ± 0.14	—	—	—

DC, drug content; DL, drug loading; EE, encapsulation efficiency; PDI, polydispersity index; SD, standard deviation.

Characterization of Silibinin-NLCs

The size, polydispersity index (PDI), and zeta potential (ZP) of lipid nanocarriers were measured by the dynamic light scattering (DLS) technique using Microtrac (Pennsylvania, FL) size/zeta analyzer. To measure ZP air bubbles were removed before using the device. All experiments were repeated three times.

Determination of DL and EE

The EE of NLCs was determined using the filtration method by Amicon filter with a 10 kDa molecular weight cutoff membrane (Amicon Ultra-4 10k; Millipore).²⁶ Briefly, the Amicon filter was filled with silibinin-NLC suspension and centrifuged ( × 3) at 1,500 × g for 3 min. The filtrated solution was analyzed to determine the amount of plain silibinin, DL (mg/g), and EE (%) by ultraviolet-visible spectroscopy (UV-1800; Shimadzu, Kyoto, Japan) at 254.5 nm. To prepare standard calibration curve, silibinin was dissolved in NMP and standard concentrations were prepared (5, 10, 20, 40, 60 μg/mL). Before using standard solutions, the cuvette was washed with deionized distilled water and the system was calibrated by reading the absorbance of the stock solution prepared from NMP and tween 80 as the blank sample. The following equations were used to calculate silibinin EE and DL and DC: $E E % = \frac{(T o t a l d r u g - U n l o a d e d d r u g)}{t o t a l d r u g} \times 100$ (1) $D L m g ∕ g = \frac{W_{d l}}{W_{N L C}}$ (2)

D C % = \frac{T o t a l d r u g - U n l o a d e d d r u g}{W_{N L C}} \times 100

(3)

In Equations (2) and (3), W_dl and W_NLC represent the amount of loaded silibinin and the weight of the solid mass of NLCs, respectively.

In Vitro Release

The drug release profile of NLCs was investigated using a dialysis bag (molecular weight cutoff = 12,000–14,000) at physiological pH (7.4) and temperature of 37°C. To study drug release, 2 mL of silibinin-NLCs was placed in a dialysis bag. The dialysis bag was soaked in 100 mL of prepared phosphate-buffered saline (PBS) sink solution (1 mL NMP, 1 μL tween 80, and 100 mL PBS), then the whole system was placed on a stirrer. One milliliter of the sample was withdrawn and replaced with 1 mL of fresh PBS sink solution at various time intervals. The amount of drug released by NLCs was measured by the ultraviolet (UV) spectrophotometer at 254.5 nm.

Cell Culture and Cell Viability Assay

The anticancer properties of plain silibinin and silibinin-NLCs were assessed using an MTT assay in vitro.^27,28 A2789-sensitive and A2780-resistant (resistant to 20 μM cisplatin) cells were seeded in 96-well plates (3,000 × 10³ cells per/well) and incubated overnight. Then, the cells were exposed to the treatments, including plain silibinin, cisplatin, a combination of plain silibinin and cisplatin, blank NLCs, silibinin-NLCs, a combination of silibinin-NLCs and cisplatin. Untreated cells and the DMSO-treated cells were assumed as negative and positive controls, respectively. After 48-h treatment, the medium was removed and MTT solution (0.5 mg/mL) was added to the wells and incubated at 37°C for 4 h. After that, the MTT solution was removed, and DMSO was added to solubilize formazan crystals. Finally, absorbance was measured by an enzyme-linked immunosorbent assay plate reader at 570 nm. To calculate the IC₅₀ value of compounds GraphPad Prism 6 (GraphPad Software, Inc.) was used.

CompuSyn software (CompuSyn v1.0; CompuSyn, Inc.) was used to calculate combination index (CI) values. Fraction affected (Fa) is the fraction of cells inhibited after the drug exposure and defined between 0 and 1. For example, Fa = 0.5 shows that 50% of cells were inhibited after drug exposure.²⁹ According to the Chou-Talalay method, antagonistic, additive, and synergistic effects were indicated, respectively, by CI >1.1, CI = 0.9–1.1, and CI <0.9.³⁰

Statistical Analysis

In the present study, each experiment was carried out in triplicate and all data were represented by the mean ± standard deviation. Two-way analysis of variance was used for statistical analysis followed by Tukey's post hoc tests for multiple comparisons. The p-values lower than 0.05 (p < 0.05) were considered significant.

RESULTS

Preparation and Physicochemical Properties of Silibinin-NLCs

The hot homogenization technique was used to create silibinin-NLC with varying amounts of drug, lipid, and surfactants. Size, ZP, PDI, EE, DL, and DC of the prepared silibinin-NLCs and blank NLC have been summarized in Table 1. For the remaining experiments, the optimum formulation was chosen based on acceptable DL and DC as well as ideal size and EE.

EE, DL, and DC

As shown in Figure 1 and Table 1, DLS analysis revealed that the formulations ranged in size from 80 to 120 nm, and the optimized formulation (F7) had an average size of 95.3 ± 2.29. The PDI index reveals the dispersion quality of NPs. PDI index ≤0.1 demonstrates the highest quality of dispersion and monodispersity, and PDI ≤0.5 is defined as acceptable dispersity.³¹ The F7 formulation had the best dispersion quality, with a PDI score of 0.12 ± 0.04, suggesting monodispersity of silibinin-NLCs. Furthermore, the F7 formulation presented the highest amount of EE (98.75% ± 0.0026%) and DL (40 mg/g). Figure 1A and B shows the morphology of silibinin-NLCs and blank NLCs obtained by scanning electron microscope in the F7 formulation. The results revealed that silibinin-NLCs and blank NLCs were spherical. The optimized silibinin-NLC (F7) was used for the rest of the analyses, including in vitro release study and cytotoxic analysis.

Fig. 1.

SEM image of (A) silibinin-NLCs and (B) Blank NLCs, and particle size of (C) silibinin-NLCs and (D) Blank NLCs. NLCs, nanostructured lipid carriers; silibinin-NLCs, silibinin-loaded nanostructured lipid carriers. SEM, scanning electron microscope.

Drug Release and Kinetics of Release of Silibinin-NLCs

To evaluate the drug release at different time intervals, the released samples were analyzed by UV spectroscopy. The drug release profile of silibinin-NLCs and plain silibinin was observed for 96 h and data were presented in Figure 2.

Fig. 2.

The cumulative release profile of plain silibinin and silibinin-NLCs in vitro after (A) 72 h and (B) first 4 h. Data are representative of three independent replicates and the values are the mean of triplicates ± SD. SD, standard deviation.

It was observed that the release of silibinin from NLCs consists of two phases. The initial phase (first 24 h) exhibited a burst release of the drug (45% ± 0.93%) mainly due to the presence of the drug in the outer membrane of NPs. In the second phase, the drug release slope decreased over time and this condition continued up to 96 h. Moreover, the release profile of plain silibinin demonstrated the permeability of the dialysis bag against the drug.

Table 2 shows silibinin release kinetics fitted on different models including zero-order models, Korsmeyer–Peppas, Higuchi, Hixon Crowell, and first order. The results demonstrated that silibinin cumulative release follows the zero-order kinetic model, with a regression coefficient of 0.9. In the zero-order model, the dissolution of drug molecules is only affected by time. Zero-order model is true for slow-release drugs and follows the Equation (4):

Table 2.

The Kinetic Mathematical Models Were Used to Fit the Release Data

	Zero-order (R ²)	First-order (R ²)	Higuchi (R ²)	Hixson–Crowell (R ²)	Peppas Korsmeyer (R ²)
Equation	$F = k_{0} t$	$L n (1 - F) = - k_{f} t$	$F = k_{H} \sqrt{t}$	$1 - \sqrt[3]{1 - F} = k_{1 ∕ 3} t$	$F = k_{k p} t^{n}$
Silibinin-NLCs	0.9	0.77	0.86	0.82	0.84

Parameters of models were obtained by linear regression (R ²). “F” represents a fraction of the drug released up to time t. All ks are constant of the mathematical models. “n” is the release exponent of the Peppas–Korsmeyer model.

Ln, natural logarithm; silibinin-NLCs, silibinin-loaded nanostructured lipid carriers.

M_{0} - M_{t} = K_{o} t

(4)

where M ₀ is the initial concentration of the drug released in time 0 and usually is considered zero, M_t is the concentration of the drug released in time t, and K ₀ is the zero-order release constant.

Antiproliferative Properties of Silibinin-NLCs in A2780 Cancer Cells

To evaluate the functional delivery of silibinin-NLCs, an MTT assay was conducted. Sensitive and cisplatin-resistant A2780 cancer cells were treated with plain silibinin, silibinin-NLCs, and the combination of plain silibinin and/or silibinin-NLCs with cisplatin. A2780 cell was selected as an OC model, for which cisplatin is one of the first-line treatments. Table 3 represents the IC₅₀ values obtained by MTT assay for each compound. As demonstrated in Table 3, silibinin-NLCs dramatically lowered the IC₅₀ values in both types of cell lines when compared with plain silibinin (p < 0.001), indicating that silibinin encapsulated in NLCs is functional and can effectively inhibit cancer cell growth.

Table 3.

The IC₅₀ Values of Different Compounds Toward A2780 Cells

	A2780-sensitive cells	A2780-resistant cells
Compounds	IC₅₀ (μM) ± SD
Plain silibinin	93.68 ± 4.45	123.9 ± 3.95
Silibinin-NLCs	68.04 ± 1.99	86.105 ± 1.69
Cisplatin	12.69 ± 1.79	60.41 ± 4.3

Figure 3 exhibits the viability of A2780 cells, which were treated with various concentrations of plain silibinin, silibinin-NLCs, the combination of plain silibinin with cisplatin, and the combination of silibinin-NLCs with cisplatin. As shown in Figure 3, the blank NLCs had no significant cytotoxic effect in comparison to untreated groups in both cell lines (p > 0.05), demonstrating the nontoxicity of the NLCs. Figure 3 further shows that at concentrations ranging from 40 to 120 M, the cell viability of both sensitive and resistant A2780 cells treated with silibinin-NLCs was much lower compared with cells treated with plain silibinin (p < 0.001). Furthermore, all of the test groups that received cisplatin and silibinin-NLCs at varied doses showed a substantial reduction in cell viability when compared with those treated with cisplatin and plain silibinin. Overall, our findings demonstrated the efficacy of the formulated silibinin-NLCs as a drug delivery system. Tables 4 and 5 represent Fa and CI data obtained by Compusyn software for actual experimental combinations of cisplatin with plain silibinin and/or silibinin-NLCs, respectively.

Fig. 3.

Cytotoxicity of cisplatin, plain silibinin, silibinin-NLCs, and combination of plain silibinin and silibinin-NLCs with cisplatin in (A) sensitive A2780 cells and (B) cisplatin-resistant A2780 cells. All experiments were conducted in triplicate and data are shown as mean ± SD (***p < 0.001). ns, not significant.

Table 4.

Fraction Affected and Combination Index Values for the Combination of Plain Silibinin and Cisplatin in A2780 Cell Lines

Combination		A2780-sensitive cells		A2780-resistant cells
Cisplatin (μM)	Plain Silibinin (μM)	Fa	CI	Fa	CI
5	20	0.37	0.90847	0.07	0.93984
10	40	0.57	0.90332	0.28	0.77662
20	80	0.76	0.86646	0.59	0.77799
30	120	0.84	0.85375	0.81	0.66282
40	160	0.91	0.6649	0.89	0.63508

CI, combination index; Fa, fraction of inhibited cells.

Table 5.

Fraction Affected and Combination Index Values for the Combination of Silibinin-Loaded Nanostructured Lipid Carriers and Cisplatin in A2780 Cell Lines

Combination		A2780-sensitive cells		A2780-resistant cells
Cisplatin (μM)	Silibinin-NLCs (μM)	Fa	CI	Fa	CI
5	20	0.40	0.96886	0.19	0.80178
10	40	0.65	0.77892	0.45	0.70747
20	80	0.82	0.70405	0.72	0.65062
30	120	0.90	0.57951	0.82	0.63197
40	160	0.95	0.40092	0.93	0.42783

The combination of cisplatin with both forms of the plain silibinin and silibinin-NLCs resulted in a synergistic growth-inhibitory effect, which was indicated with CI values lower than 0.9 in both sensitive and resistant A2780 cancer cells. The CI values for the combination of plain silibinin and cisplatin at Fa = 0.5 were 0.90 in sensitive and 0.74 in resistant cancer cells. The CI values for silibinin-NLCs and cisplatin at Fa = 0.5 were 0.91 in sensitive and 0.69 in resistant cancer cells. Furthermore, a comparison of the obtained CI values and CI-Fa plots in sensitive and resistant cancer cells at Fa = 0.5 (Fig. 4) reveals that the resistant A2780 cells were more affected by combination therapy of the plain or formulated silibinin with cisplatin. Importantly, the combination therapy with silibinin-NLCs and cisplatin resulted in a stronger synergistic inhibitory effect than treatment with plain silibinin and cisplatin (CI = 69 vs. CI = 0.74) in resistant cells. Details are presented in Supplementary Tables S1 and S2.

Fig. 4.

CI (Fa-CI) plot indicating effect levels of plain silibinin and silibinin-NLC in combination with cisplatin (A) A2780-sensitive cells and (B) A2780-resistant cells. CompuSyn software was used for generating Fa-CI plots. CI, combination index; Fa, fraction affected.

DISCUSSION

OC is one of the leading causes of mortality in females globally. The main chemotherapies for the initial treatment of OC are platinum and cisplatin analogs.⁸ Nonetheless, most OC patients eventually relapse or die, in large part due to cancer cells' resistance to the commercial treatments.

Combination therapy is one of the crucial approaches to reducing OC cell drug resistance. Silibinin is a naturally occurring anticancer drug that has mostly been used in cancer therapy and combination therapy. Despite silibinin's notable antiproliferative capabilities against cancer cells, its limited water solubility and bioavailability are the key barriers limiting clinical application. Drugs' ability to penetrate the tumor environment is hampered by their poor water solubility. The development of lipid-based nanocarriers is a promising strategy for increasing the bioavailability of weakly water-soluble drugs, such as silibinin.³² Furthermore, drug delivery by nanocarriers overcomes multidrug resistance in cancer cells, which may lead to an increase in drug concentration in the tumor microenvironment. Our findings show that encapsulating silibinin in NLCs considerably reduces the disadvantage of silibinin's poor solubility and overcomes A2780 cancer cell resistance when used in conjunction with cisplatin.

Silibinin-NLCs were prepared using the hot homogenization technique with cocoa butter. The size and ZP of the NLCs were in the range of 26–173 nm and −20 to −30 mV, respectively. The low PDI revealed the monodispersity of the prepared NLCs. In a similar study, silibinin polymeric NPs, prepared by an evaporation method, was measured to be 219.2 nm in size and −12.5 nm in ZP. The EE of the polymeric NPs was determined to be 94%.³³ Another study employed the thin-layer film hydration approach to generate nanoliposomes containing silibinin. The NP size was 46.3 nm and the EE was 24.37, suggesting that the tiny size of the NPs caused low DL on the nanoliposomes.³⁴ Several lines of studies have shown that size is an important factor in the EPR effect and passive drug targeting.

When anticancer drug-loaded NPs in the 20–200 nm size range are administered intravenously, they can avoid renal clearance and cannot cross normal blood vessels, but they can spread to tumor vasculature and aggregate around the tumor due to abnormalities in the tumor lymphatic system.³⁵ Particles having a molecular weight larger than 40 kDa generally avoid renal clearance, have a prolonged biological half-life, and hence accumulate more drugs in the tumor. The EPR effect causes the accumulation of a high concentration of drugs in tumor cells.³⁶

Another aspect influencing the EPR effect is the drug's release rate. Too slow or too fast drug releases result in inadequate concentration and poor accumulation of drugs inside the tumor, resulting in unwanted systemic toxicity and limited therapeutic impact.³⁷ According to our findings, the release rate of silibinin-NLCs was around 45% in the first 24 h, whereas in Ochi et al's work, the release rate of silibinin from nanoliposomes was 14% (w/w) in the first 48 h.³⁴ Different compositions of lipid-based NPs can be one of the main reasons for the different release profiles of silibinin. According to Patel et al, the silibinin release percentage from several formulations of solid lipid nanoparticles (SLNs) was in the range of 50.21 − 81.40 after 12 h.³² The authors revealed that the type of surfactant and lipid are affecting parameters of in vitro drug release of silibinin from SLNs. It was shown that higher drug releases were related to Tween-80 and Poloxamer 407, respectively, which can be due to their lower hydrophilic−lipophilic balance value among other surfactants.

The release profile of silibinin-NLCs, according to our observations, obeyed multiple mathematical models, however, the obtained formulation was best matched to the zero-order model, suggesting that the developed formulation may offer sustained regulated release of drug over extended hours. Previous studies on silibinin-loaded SLNs reported the Higuchi model as the best-fitted model for silibinin release.³² In the studies done on silibinin-loaded micelles, silibinin release kinetics from polymeric micelles followed the Peppas model.³⁸ Peppas model mostly describes drug release kinetics from polymeric delivery systems.³⁹

We adopted a combined treatment method in this study to overcome cisplatin resistance in ovarian A2780 cancer cells. We found that the combination of cisplatin and plain silibinin synergistically inhibited normal and resistant A2780 cells' growth rate. This finding was consistent with a prior study that found silibinin restored cisplatin sensitivity in A2780-resistant cells.¹⁶ Several studies have proven that when silibinin is combined with chemotherapeutics such as cisplatin, metformin, paclitaxel, and doxorubicin, silibinin synergistically improves growth inhibitory effects in several types of cancer cells.^13,40 The suppression of signal transducer and activator of transcription 3 (STAT3) may be one of the mechanisms through which silibinin sensitizes cancer cells to chemotherapy.⁴¹ Previous report has shown that an increase in activated STAT3 enhances chemoresistance in OC cells.⁴²

Furthermore, our findings revealed that combination therapy with plain silibinin and silibinin-NLC with cisplatin resulted in a stronger synergistic inhibitory impact on resistant cancer cells than on sensitive cells. Ji et al reported that cisplatin-resistant OC cells exhibit a greater amount of activated STAT3.⁴³ Previous research has shown that silibinin inhibits STAT3 activity,^11,41 therefore, cisplatin-resistant OC cells should be more impacted by the combination of silibinin and cisplatin than sensitive cancer cells.

In comparison to the combination treatment with plain silibinin and cisplatin, silibinin-NLCs had a higher synergistic inhibitory effect with cisplatin. This finding implies that the new formulation of silibinin-NLC successfully delivers functional silibinin to the cells and likely provides a larger concentration of silibinin inside cancer cells than the plain drug.

CONCLUSION

One of the primary challenges in the chemotherapeutic treatment of many forms of cancer is drug resistance. Silibinin is a naturally occurring molecule that improves the therapeutic efficacy of chemotherapy drugs in a variety of malignancies. We explored the effect of silibinin-NLCs on overcoming cisplatin resistance in A2780 cells in this work. In both cisplatin-sensitive/resistant A2780 cancer cells, the growth inhibitory impact of formulated silibinin-NLCs was shown to be substantially greater than that of free silibinin. Furthermore, as compared with the combination therapy with free silibinin and cisplatin, the combination therapy with silibinin-NLCs and cisplatin had a more powerful synergistic growth inhibitory impact on sensitive and resistant A2780 cells. Overall, our findings suggest that the delivery of silibinin by NLCs might be an effective and promising approach for the treatment of chemotherapy-resistant OC.

Footnotes

ACKNOWLEDGMENTS

The authors would like to express their gratitude to the Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran for the technical support for this study.

AUTHORs' CONTRIBUTIONS

S.J.: Investigation, formal analysis, and writing—original draft preparation. A.B.: Investigation and writing—original draft preparation. M.E.: Conceptualization, supervision and writing—review and editing. O.M.: Conceptualization, supervision, and writing—review and editing.

DISCLOSURE STATEMENT

The authors declare that they have no competing interests.

FUNDING INFORMATION

This project has been supported by the Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran (Grant No: 63131).

SUPPLEMENTARY MATERIAL

Supplementary Table S1

Supplementary Table S2

Abbreviations Used

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