Mesoporous SBA-15 Silica–Loaded Nano-formulation of Quercetin: A Probable Radio-Sensitizer for Lung Carcinoma

Abstract

Lung cancer is considered as one of the most serious disease worldwide. The progress of drug carriers based on nonmaterial, which selectively hold chemotherapeutic agents to cancer cells, has become a major focus in biomedical research. This study aimed to evaluate the growth inhibition and apoptosis induction of the human lung cancer cells (A-549) by Q-loaded SBA-15 conjugate system. Mesoporous silica nanoparticles (SBA-15) as host materials for transporting therapeutics medicaments were fabricated for targeted drug delivery toward lung cancer. With the objective of increasing bioavailability and aqueous solubility of flavonoids, SBA-15 was successfully loaded with the quercetin (Q)—a major flavonoid and characterized with the help of Fourier-transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The biological investigation on A549 cell line confirmed that the efficacy of Q-SBA-15 is much higher than only Q. Moreover, the apoptotic pathway of synthesized Q-SBA-15 NPs examined that the Q-SBA-15–mediated apoptosis via PI3K/AKT/mTOR signaling pathway. Thus, the newly conjugated Q-SBA-15 system improved the apoptotic fate through caspase-mediated apoptosis via PI3K/AKT/mTOR signaling pathway and hence, it can be potentially employed as an anticancer agent for lung cancer.

Introduction

Lung cancer has emerged as major cause of oncological death worldwide, with neoplasia being generally avoidable by weight control, intake of vegetable food, and reduction of alcohol and tobacco. Owing to the complex molecular biology associated with lung metastases, various therapeutics approaches have been assumed such as chemotherapy, radiotherapy, and immunotherapy capable of defeating tumor growth to some extent.^1-4 Among them, majority of lung cancer treatment requires ionizing radiation (IR) to destroy proliferating cells and exhibits a crucial role in native control after surgery.^5,6 In the course of radiotherapy, ionizing radiation damages the cancerous DNA, thereby facilitating tumor cell apoptosis and necrosis in cancerous tissues.^7,8 Regardless of current improvements in radiation therapy (RT), there are still certain limitations to its use viz.: (a) failed to differentiate healthy and infectious tissue, (b) radiation dosage and hence its therapeutic effects, and (c) intrinsic radiation resistance arises due to incomplete tumor response or recurrence.^9-11 Therefore, a new strategy is required to lower these issue laid by association between IR and photosensitizers that potentiate the radiotherapy outcomes.^12-14 One probable area of development is the use of biomaterial-based radioprotective agent to combine the effects of RT with other therapeutic modalities.^3,13,15,16 Literature reports presented that the antioxidants derived either from exogenous/endogenous sources provide defense system to the body and neutralize excess reactive oxygen species (ROS) to diminish the injury governed by IR-induced.^17,18

In this context, natural occurring bioflavonoids have great therapeutic potential against various cancers as they are involved in a different signaling pathway related to apoptosis via modulating gene expression. ¹⁹ In particular, Q (3,3,4,5,7-pentahydroxyflavone) is of the special interest for its diverse biological effects, inhibiting multiple enzymes involved in cell proliferation, including antiproliferative and apoptotic pathways.^20-22 It can also act as chemoprotective and radioprotective agent, protecting normal cells from the side effects, which offers notable benefits in anticancer treatment.^16,23 Moreover, it induces mitochondrial mediated both up- and down-regulation of pro-apoptotic proteins with the release of cytochrome c.^21-25 As an effective ROS scavenger, Q exerts an antioxidant property which helps in the free radical-scavenging, reducing the level of ROS and inhibiting lipid peroxidation.^26,27 However, its clinical efficacy is restricted mainly due to low polar solubility and chemical instability that eventually lead to insufficient accumulation around cancer cell.^23,28 Thus, alternative schemes based on multifunctional nanomaterials have been carried out to improve the aqueous solubility of Q and radiation responses and thereby achieving enhanced RT with reduced radio-toxicity.^29,30

Mesoporous silica nanoparticulate (MCM-41, MCM-48, and SBA-15) has been familiarized for the drug delivery purposes and still they are being investigated.^31,32 SBA-15 is one of the common 2D hexagonally arrayed mesoporous materials which ensures the easy access of drug molecules due to its biocompatibility and a low cytotoxicity.^32,33 The kinetic release of the drug molecule can be control by the modulating the pore diameter pore diameter, which directs the expulsion of from SBA-15. ³⁴ It is imperative to analyze the biological effects of Q-loaded SBA-15 mesoporous nanomaterial induce cytotoxicity.

Although several studies regarding the encapsulation of quercetin by other nanoparticulate system have been documented in literature, however cytotoxicity studies of SBA-15 encapsulated Q against lung cancer are scarcely reported. Present investigation aimed to evaluate the growth inhibition and apoptosis induction of the human lung cancer cells (A-549) by Q-loaded SBA-15 conjugate system (Figure 1). Furthermore, we determined that the efficacy of Q-SBA-15 is better than only Q alone. Therefore, we conclude that the coupling of antioxidant to mesoporous silica nanoparticles as a new carrier that generates lower oxidative stress and beneficial in term of ROS reduction, cellular viability, and protective effects mediated apoptosis pathway involved via AKT/PI3K/mTOR axis.

Figure 1.

Diagram showing Q-loaded SBA-15 conjugate system and its mode of action.

Experimental Section

Chemical and Cell Lines

P123 (Poly (ethylene glycol)-block-poly (propylene glycol)-block-poly (ethylene glycol), TEOS (tetraethyl orthosilicate) were procured from Sigma Aldrich, United States.

Human lung carcinoma (A549) cell line of passage four was acquired from ATCC, United States. Cell culture media components: trypsin, DMEM, FBS, PSN antibiotic cocktail, and EDTA were purchased from Gibco (Grand Island, NY, United States). Antibodies were procured from Santa Cruz Biotechnology, Dallas, Texas, United States and eBioscience, San Diego, United States. Dyes were bought from Thermo Fisher Scientific, United States.

Synthesis of Mesoporous SBA-15

In this synthesis, the originator used was tetraethyl orthosilicate (TEOS) and the template used was P123. ³⁵ The procedure is called sol-gel solution, and the solution is in acidic state. ³⁶ First step, 10 mL ethanol was added to 62 g TEOS and stirred for 45 min at 25°C. Second, 10 mL ethanol was mixed with 18 mL HCl. This solution then was mixed with ethanol (20 mL) and 90 mL distilled water and stirred for 45 min. The mixed solution was then refluxed for 3 h at temperature of 60°C. Later, the surfactant solution was added drop wise into TEOS solution and kept on stirring awaiting the solution turned into gel. The obtained gel was dried at 100°C for an hour and calcined for 5 h before subjected for characterizations.

Quercetin Loading

SBA-15 particles were added into Q solution and incubated in a shaker at room temperature overnight. Q molecules could diffuse in solution into the pore channels. The Q-loaded SBA-15 was removed by centrifugation and rinsed with water to get rid of the redundant Q. Finally, the resulting particles were dried in vacuo at 60°C after being washed with water to yield Q-loaded SBA-15. The amount of Q-loaded into SBA-15 was determined by UV–VIS spectroscopy at 369 nm (λ_max = 369 nm) ³³

$Loading Content=\frac{Initial weight of Q-Superna\tan t weight of Q}{Weight of particles}\times 100\%$

Characterizations

For TEM analysis, the dispersed ethanolic solution of material was sonicated for 5 min before placing onto the carbon-coated copper grid. FT-IR spectrum of the Q-SBA-15 prepared as KBr pellets was performed on a Perkin Elmer IR 783 spectrophotometer in the range of 4000–4400 cm⁻¹. ³⁷

Cell Culture and MTT Assay

The A549 cells were maintained in DMEM comprising 10% FBS with 1% antibiotic cocktail as per manufacturer’s protocol. Propagation of the cells takes place in humidified incubator supplied with 5% CO₂ and maintained at 37°C. After confluence (70−75%), cells were collected in PBS medium with EDTA (.52 mM) and trypsin (.25%) and plated at a suitable density to allow them to re-equilibrate before the examination. ³⁸

To measure cell viability and proliferation forms, MTT (4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was done. ³⁹ In this assay, the cells were plated in 96 well plates (4 × 10³ cells/well) and treated in presence and absence of different concentrations of Q and Q-SBA-15 for 24 h. After the incubation period of treatment, the cells were incubated with MTT solution for 4 h. The cells after treatment were incubated with MTT solution for 4 h. The yellow tetrazolium salt (MTT) decreases in metabolically active cells to form insoluble purple formazan crystals, which were solubilized by the addition of acidic isopropanol. The color change was determined spectrophotometrically at 595 nm via an ELISA reader. The same protocol has been followed for HEK 293. Each assay was repeated three times and in triplicate for each type of sample, and the cytotoxicity was evaluated using absorbance values as follows ⁴⁰

$\text{Cell viability}=\frac{\text{OD of Control}-\text{OD of treated}}{\text{OD of Control}}\times 100$

Quantification of Apoptosis/Necrosis

During this process, properly washed control and treated A549 cells were stained with propidium iodide (PI) and Annexin V-FITC in agreement with the seller’s guidelines (Calbiochem, Merck Millipore, Burlington, Massachusetts, United States). ⁴¹ A flow cytometer (BD LSRFortessa, San Jose, CA, United States) was used to detect the percentages of early and/or late live, apoptotic, and necrotic cells. The observed data were investigated through the FlowJo software (version 10.0).

Immunofluorescence

Separately, control/treated A549 cells were washed twice for 10 min in PBS (.01 M) and incubated for 1 h in blocking solution having normal bovine serum (2%) and Triton X-100 (.3%). ⁴² After this, the cells were incubated overnight at 4°C with the corresponding primary antibody (p-AKT, p-PI3K and cytochrome c), followed by washing and incubation with one-to-one fluorophore-conjugated secondary antibodies (antimouse/rabbit FITC and PE) for 2 h. Each slide was counterstained with nuclear dye (DAPI) for 10 min and mounted with the ProLong antifade reagent, which was identified by a confocal microscope. The dilution of primary and secondary was 1:500 and 1:100, respectively. Olympus FV10i microscope was used for the experiment. The data was analyzed by FluoView software.

Measurement of Caspase-9 Activity

Commercially available caspase-9 colorimetric assay kit (BioVision Research Products, Mountain View, CA) was applied to determine the caspase-9 activities as per the manufacturer’s protocols. Absorbance was recorded at 405 nm by an ELISA reader. ⁴³

In Vitro Quercetin Release

The in vitro drug release experiments were conducted at different pHs 5.5 and 7.4 with 10 μg/mL of Q-SBA-15 loaded into a dialysis bag; .01 mg of EDTFP-1 was dispersed in a known amount (1 mL) of PBS of 7.4 and 5.5 solutions, transferred to dialysis bag, and kept at 37°C. The resulting suspension was immersed into 10 mL of PBS and pH 5.5 solutions and incubated at 130 r/min for a definite time period (9 h). Five hundred microliter aliquots were drawn from the above dispersions after 30 min intervals; this was replaced by equal volume of the fresh PBS solution and pH 5.5 buffer for retaining the release medium same. Release of Q was studied through the UV spectrophotometric analysis at 370 nm.

Statistical Analysis

All values were presented as standard error mean (±SEM). Statistical meaning and differences among the set condition was evaluated through one-way analysis of variance (ANOVA) test followed by OriginPro 8.0 software. The critical significance level was set at P < .05.

Result and Discussion

Characterizations

The FT-IR spectrum of calcined Q-SBA-15 sample was recorded on Perkin Elmer IR 783 spectrophotometer (Figure 2). Q comprises of two benzene rings linked by pyran or pyrone. The spectrum displayed asymmetric and symmetric stretching vibrations bands at 1085 and 801 cm⁻¹, respectively, which belong to the Si–O–Si mesoporous silica in Q-SBA-15–conjugated framework. A weak band around 965 cm⁻¹ represents Si–OH stretching. The band at 1636 cm⁻¹ was credited to the carboxyl group (–C=O) stretching vibration of Q and a broad envelop near 3451 cm⁻¹ ascribed to the characteristic –OH stretching vibration due to presence of phenolic group of Q.

Figure 2.

FTIR spectrum of synthesized Q-SBA-15 in the range of 4000–4400 cm⁻¹.

High-resolution transmission electron microscopy (HRTEM) images and SAED pattern of SBA-15 nanoparticles are shown in Figure 3. It is evident from the Figure 3(A) that hexagonally order pore channels of SBA-15 material were observed under 200 nm scale bar. The pores are observed in Figure 3(B), and the SAED pattern confirmed the mesoporous structure of SBA-15 (Figure 3(C)). After Q loading, the structure remains same (Figure 4) which revealed that there is no alteration in the SBA-15 after loading of Q.

Figure 3.

TEM images of synthesized SBA-15 (A). under 200 nm scale bar (B) under 10 nm scale bar (C). SAED pattern of SBA-15.

Figure 4.

TEM images of synthesized Q-SBA-15 (A). under 500 nm scale bar (B) under 200 nm scale bar (C) under 50 nm scale bar.

MTT Assay

The MTT assay was carried out to evaluate the cytotoxicity of the Q and loaded with SBA-15 (Q-SBA-15) at the varying concentrations (0–20 μg/mL) against A549. The treatment of Q and Q-SBA-15 was for 24 h. In the event of A549 cell line, the colorimetric data (Figure 5) showed that after 15 μg/mL of Q-SBA-15 treatment, almost all the cells were dead, whereas in case of Q, the dose was 25 μg/mL. While in case of Q alone, the data implicated that 30, 50, and 70% cells were dead at 12.35, 20.09, and 26.82 μg/mL, respectively, after 24 h of treatment. The cytotoxicity of only SBA-15 was also evaluated, which displays insignificant effect up to 40 μg/mL of concentration. However, Q-SBA-15 conjugated system exhibited IC30, 50, and 70 doses at 6.29, 9.57, and 16.5 μg/mL, respectively. From this data, we have established that Q-SBA-15 is more potent (85% of cells death) than only Q (65% of cells death) at the same concentration 20 μg/mL. We have also checked the cytotoxicity of Q-SBA-15 on HEK-293 cell line; up to 20 μg/mL, there was no such kind of toxicity. So 9.57 μg/mL dose was selected for further biological experiments.

Figure 5.

(A) MTT assay of Quercetin, Q-SBA-15, (B) only SBA-15 on A549 and (C) Q and Q-SBA-15 on HEK 293 cell line after 24 h of treatment; Each value represents the mean ± SE of three experiments, n = 3, (*P < .05, **P < .01, ***P < .001) compared with the control.

Q-SBA-15 Induced Apoptosis in A549 Cells

It is established that phosphatidylserine (PS), a phospholipid component, translocated in the cell membrane and externalized during apoptosis process. Annexin-V is a specific PS-binding protein that can be used to distinguish the apoptotic cells when conjugated with an exact fluorophore. Interestingly, as shown in Figure 6, the percentage of early and late apoptotic population increased progressively after 10 μg/mL of Q-SBA-15 treatment in time-dependent manner (12 and 24 h). With 12 h of Q-SBA-15 treatment, the viable cells were reduced to 44.8% following the enhancement of early/late apoptotic and necrotic population of 45.7, 9.2, and .3%, respectively. After 24 h of treatment, the viable cells decreased to 17.1% following the early/late apoptotic and necrotic population of 32.8%, 49, and .3%. This data confirmed that Q-SBA-15 induced cell death is due to apoptosis.

Figure 6.

Annexin V-FITC/PI data after 12 and 24 h of 10 μg/mL Q-SBA-15 treatment against A549, where Q3, Q4, Q2, and Q1 indicate viable cell, early apoptosis, late apoptosis, and necrosis, respectively. Q-SBA-15 Dephosphorylated AKT/PI3K/mTOR pathway in A549 cells.

Regulating PI3K/AKT/mTOR pathway in cancer cells acts a key aspect to make cancer cell viable for cell death during apoptosis. After treatment with 10 μg/mL of Q-SBA-15, the expression of p-AKT, p-PI3K, and p-mTOR was checked by confocal microscopy. As shown in Figure 7, it has been clearly exposed that the expression of p-AKT and p-PI3K has been decreased over the control one, after treatment of 10 μg/mL of Q-SBA-15 in time-dependent manner, where DAPI is used as nuclear stainer. As p-AKT down-regulates after treatment of Q-SBA-15, the expression of p-mTOR and cytochrome c has been analyzed by confocal microscopy (Figure 8). The data has shown that the enhancement in the expression of p-mTOR and cytochrome c has been observed in a time-dependent manner. Hence, from the microscopic images, it has been indicated that the Q-SBA-15–mediated apoptosis occurs via PI3K/AKT/mTOR signaling pathway.

Figure 7.

Expression of p-AKT and p-PI3K after 12 and 24 h of 10 μg/mL Q-SBA-15 treatment against A549, DAPI used as nuclear stainer.

Figure 8.

Expression of p-mTOR and cytochrome c after 12 and 24 h of 10 μg/mL Q-SBA-15 treatment against A549, DAPI used as nuclear stainer.

Expression of Caspase-9 Activity

In order to corroborate the Q-SBA-15 induced cell death via PI3K/AKT/mTOR signaling pathway, the caspase-9 assay has been performed with different time interval. The expression of cytochrome c has been increased after treatment of Q-SBA-15 with respect to time. The final apoptotic fate was determined by the activity of caspase-9 (Figure 9), which was simultaneously enhanced with the treatment of Q-SBA-15 in a time-dependent manner. Thus, we concluded that Q-SBA-15–mediated apoptosis might be regulated through the PI3K/AKT/mTOR signaling pathway.

Figure 9.

Expression of caspase-9 after 12 and 24 h of 10 and 20 μg/mL Q-SBA-15 and Q, respectively. Each value represents the mean ± SE of three experiments, n = 3, (*P < .05, **P < .01) compared with control.

In Vitro Quercetin Release

In vitro Q release of Q-SBA-15 was evaluated at pH 5.5 and 7.4 as shown in Figure 10. The data shows that at acidic pH, the release of Q was found to be much higher at pH 7.4 as we know the pH of cancer cell is around 5.5. Therefore, we can tell that the apoptosis rate of Q-SBA-15 is much higher than only Q because of the pH-dependent delivery of Q-SBA-15.

Figure 10.

In vitro time-dependent pharmacokinetics of Q release from Q-SBA-15 at pH of 5.5 and 7.4.

Conclusion

In this study, Q-loaded Q-SBA-15 NPs were obtained by a multistep process. This study confirmed that the efficacy of Q-SBA-15 is much higher than only Q. In addition, the obtained data of apoptotic pathway suggested that the Q-SBA-15–mediated cell death is caspase-mediated apoptosis via PI3K/AKT/mTOR signaling pathway. Thus, Q-SBA-15 NPs have a huge prospective to be employed as an anticancer agent to overcome the epidemiology of lung cancer in future. However, further investigations are needed to be conducted on different in vitro and in vivo experimental models.