Curcumin intercalated layered double hydroxide nanohybrid as a potential drug delivery system for effective photodynamic therapy in human breast cancer cells

Abstract

Curcumin, a naturally occurring phenolic compound, is a highly potent anticancer agent against many different types of cancers. Recent studies show that curcumin can be used as a photosensitizer in photodynamic therapy for cancer treatment. However, the major disadvantage of curcumin is its poor aqueous solubility. To improve its applicability in cancer therapy, we intercalated curcumin into layered double hydroxide (LDH) with the co-precipitation method and used as a nanohybrid photosensitizer in photodynamic therapy of human breast cancer cells. Powder X-ray diffraction (XRD), TEM and SEM microscopy analyses indicate that curcumin is stabilized in the host interlayer. According to the spectroscopy results, the water solubility and dispersity of intercalated curcumin increased and loading amount of curcumin in LDH is about 50%. The photodynamic effect of curcumin and the curcumin–LDH nanohybrid was studied on the MDA-MB-123 human breast cancer cell line. Optimization of incubation time with free curcumin and curcumin–LDH nanohybrid as the most effective parameter was investigated. The optimum irradiation time of blue LED on photodynamic therapy was determined for both free curcumin and curcumin–LDH nanohybrid. Cell viability studies revealed that the nanohybrid curcumin–LDH were able to show more effective photodynamic effects on the cancer cells as compared to free curcumin. These results suggest that the biocompatible layered double hydroxide can be used as the basis of a tunable curcumin delivery carrier for photodynamic therapy in breast cancer treatment.

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Introduction

Breast cancer is known as the most common cancer for women in the world. Developed countries have a higher population (approximately 25% of all female malignancies) of women with this cancer. Breast cancer is the second leading cause of cancer-related death among females in the world.¹ Recently, various researches have been focused on achieving an effective therapy for breast cancer.^2,3 Photodynamic therapy (PDT) is a novel and useful technique for the treatment of a variety of diseases and cancers. The principle of PDT is to illuminate a photosensitizer with light at specific wavelengths to produce reactive oxygen species (ROS), especially singlet oxygen, in tumor cells. The tumor cells would be destroyed by apoptosis and/or necrosis induced by ROS, achieving, ultimately, the goal of local treatment with minimum invasion.^4,5 The photosensitizer plays a crucial performance in an efficient PDT.^6,7 Curcumin, (1,7-bis(4-hydroxy 3-methoxy phenyl)-1,6-heptadiene-3,5-dione), a natural polyphenolic pigment from Curcuma longa (turmeric) rhizomes, is a well-known anti-carcinogenic, wound-healing and anti-inflammatory agent.^8–10 Poor solubility and bioavailability of curcumin in aqueous solutions have limited the applications of curcumin in medical and clinical applications.¹¹ However, the effective delivery of curcumin for cancer treatment is very interesting for various scientists and needs to be studied. Numerous studies have been reported on modified curcumin with different functionalities for evaluating its anticancer activities.¹² Among the published researches, the encapsulation of curcumin in liposomes, polymeric micelles, and intercalation in cyclodextrins are reported and used for increasing the curcumin solubility and bioavailability.^13,14

Layered double hydroxides (LDHs), also called anionic nano-clays, are an important class of layered inorganic materials that make a network of divalent and trivalent metal cations cross-linked with hydroxide anions and contain charge balancing interlayer anions.¹⁵ In recent studies, LDHs have been used as drug delivery systems due to its good biocompatibility, low cytotoxicity, unique anionic exchange property and pH-controlled release property in acidic environments.^16,17 In some studies, the role of curcumin as a photosensitizer in antimicrobial photodynamic therapy was investigated.^18,19 To date, there are few studies for intercalating curcumin in nanoparticles as a photosensitizer. Furthermore, using LDHs as a nano-drug delivery system for PDT has recently been developed^20,21 but there is no report on curcumin intercalation in LDH as a photosensitizer in PDT.

In this study the curcumin intercalated LDH materials were prepared, characterized and evaluated for curcumin delivery into MDA-MB-231 cell lines as a photosensitizer in PDT. The results show that curcumin intercalated LDH nanohybrids have more photodynamic effect in comparison to free curcumin. It can be suggested that curcumin–LDH nanohybrids can work as a potential drug delivery system for PDT on the MDA-MB-231 breast cancer cell line.

Materials and methods

Materials

Curcumin (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (C₂₁H₂₀O₆), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), trypan blue solution 0.4% and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO, USA). Fetal bovine serum (FBS), phosphate buffered saline (PBS) and antibiotics were purchased from Gibco (Gibco BRL). Dulbecco's Modified Eagle Medium (DMEM) was purchased from Invitrogen (Invitrogen, Carlsbad, California, US). All the other reagents were obtained from Merck. Deionized (D.I.) water was used for the entire experiment.

Synthesis of LDH

An aqueous solution (100 ml) containing NaOH (0.2 mol) was added dropwise to a solution (160 ml) containing Mg (NO₃)₂·6H₂O (0.006 mol) and Al(NO₃)₃·9H₂O (0.002 mol) under a nitrogen atmosphere with vigorous stirring until the final pH 10 was achieved. The resulting slurry was transferred in a Teflon lined autoclave for hydrothermal calcination at 400 °C for 2 h and then was cooled, filtered, washed with fresh and CO₂ free de-ionized water until pH 7 was achieved and finally dried in vacuum at room temperature for 12 h giving the product.

Intercalation curcumin into LDHs

An aqueous solution of (100 ml) NaOH and curcumin (0.0003 M) was added dropwise to a solution (250 ml) containing Mg (NO₃)₂·6H₂O (0.006 M) and Al(NO₃)₃·9H₂O (0.002 M) (molar ratio Mg/Al = 3.0), under a nitrogen atmosphere, with vigorous stirring until a final pH of 10 was achieved. The resulting slurry was transferred in a Teflon lined autoclave for hydrothermal calcination at 400 °C for 2 h and was then cooled, filtered, washed with fresh and CO₂ free de-ionized water until pH 7 was achieved and finally dried in vacuum at room temperature for 12 h giving the nanohybrid product (curcumin intercalated LDH nanohybrid).

Loading efficiency and solubility study of curcumin

To determine the loading efficiency of curcumin (%) in curcumin–LDH-NPs, UV-vis spectrophotometric studies (using a Cary 100 spectrophotometer) at 430 nm were carried out. A standard curve in the range of 0–20 μg ml⁻¹ curcumin was plotted. The curcumin content present in curcumin–LDH-NPs was calculated by loading efficiency using eqn (1).

Aqueous solubility of curcumin and curcumin–LDH nanohybrid was determined by dissolving each separately in 0.01 M of PBS at pH 7.4. The nanohybrid increased the aqueous solubility of curcumin up to 6.15. For this purpose, a certain amount of both curcumin and curcumin nanohybrid was used (100 μg ml⁻¹). The sample solution was stirred for 5 min. Then, a certain amount of clear solution for each sample was transferred in a quartz cell for measuring maximum absorbance of solution by considering A_max. Furthermore, we determined the baseline for each graph (curcumin and curcumin–LDH nanohybrid), and then we determined the difference between baseline and maximum absorption in each graph to eliminate baseline effects. The enhancement of solubility of curcumin in the nanohybrid could be calculated as A_{max-curcumin–nanohybrid}/A_max-curcumin.

Characterization of curcumin–LDH nanohybrid

The absorption spectra were obtained using a Cary 100 UV-vis spectrophotometer, equipped with quartz cuvettes. Fluorescence spectra were obtained on a Cary Eclipse fluorescence spectrophotometer equipped with a thermostatically controlled cell holder at ambient temperature. The monochromatic slits were set at 5 nm for excitation and emission to reduce the intensity of the signal depending on the experiment. Powder X-ray diffraction (XRD) patterns were obtained by a Rigaku Miniflex X-ray diffractometer using CuKα radiation (λ = 0.154 nm). The morphology of samples was examined using a scanning electron microscope (SEM). The surface morphology of the samples was analyzed using a transmission electron microscope (TEM).

Cell line and culture

Human breast cancer cell line, MDA-MB-231, was obtained from the Institute of Pasture, Tehran, Iran. These cells were grown in DMEM medium supplemented with 10% FBS, 100 IU ml⁻¹ penicillin, and 100 μg ml⁻¹ of streptomycin and then incubated in a humidified incubator containing 5% CO₂ at 37 °C. For the experiments, the cells were removed by trypsinizing (trypsin 0.025%, EDTA 0.02%) and washed with phosphate-buffered saline (PBS).

Effect of different incubation time with curcumin and curcumin–LDH nanohybrid on human breast cancer cells

We designed the experiment for determining the optimum incubation time of curcumin and curcumin–LDH nanohybrid on cells via counting live and dead cells after specific times. About 1 × 10⁴ MDA-MB-231 cells in culture medium were seeded in petri dishes and incubated overnight at 37 °C with 5% CO₂. After 24 h, cells were incubated in a fresh growth medium containing different concentrations of curcumin and curcumin–LDH nanohybrid (0, 10, and 25 μg ml⁻¹). After a specified period of incubation (1, 4, 24, and 48 h) the growth medium containing curcumin and curcumin–LDH nanohybrid was removed and cells were washed with PBS. One plate of treated cells was irradiated with a blue LED source (465 nm; power density: 34 mW cm⁻²) and another was kept in the dark, outside the incubator, for 30 min. Cell viability was determined by the trypan blue exclusion method. All experiments were repeated three times.

Effect of different irradiation time on human breast cancer cells

For determining the effect of different irradiation time, MDA-MB-231 cells at a density of 1 × 10⁴ cells per well were seeded in 96-well flat-bottomed micro titer plates (Jet Biofil cat. no. TCP011096). After 24 h, the cells were incubated in a fresh growth medium containing different concentrations of curcumin and curcumin–LDH nanohybrid (0, 10, and 25 μg ml⁻¹). After a further incubation of 24 h, the cells were washed with PBS. One plate of treated cells was irradiated with a blue LED source (465 nm; power density: 34 mW cm⁻²) and another was kept in the dark, outside the incubator, for specified time periods (5, 10, 15, 20 and 30 min). All irradiations were performed at room temperature (25 °C). The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell viability. All experiments were repeated three times.

In vitro photodynamic assay

MDA-MB-231 cells were grown in medium culture cell and after reaching 80–90% confluence; MDA-MB-231 cells were washed with PBS, afterwards detached from the flask by addition of 1.0 ml of 0.25% trypsin for 1−3 min at 37 °C. MDA-MB-231 cells (1 × 10 ⁴ cells per well) were seeded into two 96-well plates. The cells were then treated with curcumin and curcumin–LDH nanohybrid at different concentrations. After a further incubation of 24 h, one plate was irradiated with a blue LED source (465 nm; power density: 34 mW cm⁻²) and another was kept in the dark for 30 min.

To determine cytotoxicity of void LDH (drug free), additional groups of cells were incubated with different concentrations of LDH (0–100 μg ml⁻¹) and cell viability was measured in the presence and absence of LED irradiation. All irradiations were performed at room temperature (25 °C). The MTT assay was used to determine the cell viability. All experiments were repeated three times.

Cell viability

Cell survival was determined by the MTT colorimetric assay. In brief, culture medium was removed and cells were incubated in medium with 0.5 mg ml⁻¹ of 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT) for 4 h at 37 °C. The resulting formazan crystals were dissolved with 100 μL dimethyl sulphoxide (DMSO) and shaken for 15 min. The absorbance was measured at 540 nm using an ELISA reader (Hyperion, Inc., FL, U.S.A.).

Statistical analysis

Statistical analysis was performed with a Student's t-test (two tailed). All values are expressed as means ± SD. Results are expressed as with n denoting the number of experiments. P < 0.05 was considered as statistically significant.

Results

Physicochemical characterization of curcumin–LDH complex

Curcumin loading efficiency was found to be about 50%. Solubility of curcumin and curcumin–LDH nanohybrid was also determined by dissolving both curcumin and curcumin–LDH nanohybrid into aqueous solutions and compared (Fig. 1A). It was revealed that curcumin–LDH nanohybrid completely dissolved, and a clear orange well-dispersed liquid could be observed. The solubility of curcumin in nanohybrid was increased 6.15 times in comparison to neat curcumin in solution.

Fig. 1 (A) The comparative solubility of curcumin and curcumin–LDH nanohybrid in PBS [(left) curcumin & (right) curcumin–LDH nanohybrid in PBS (0.01 M, pH 7.4)]; (B) absorbance spectra and (C) fluorescence emission spectra of free curcumin (solid line), curcumin–LDH complex (dashed line) and LDH (dotted line) in an aqueous solution.

Fig. 1B shows the absorption spectra of curcumin loaded in LDH (curcumin–LDH complex), free curcumin and LDH in an aqueous solution. The absorption spectra of curcumin–LDH showed the higher absorbance peak (512 nm) with a red shift as compared to free curcumin (430 nm). As observed in Fig. 1B, fluorescence spectra of the curcumin–LDH nanohybrid showed the same pattern in the fluorescence peak with reduction in intensity as compared to free curcumin. The fluorescence intensity of intercalated curcumin quenched in comparison with free curcumin fluorescence spectra. The XRD, SEM and TEM results proved the spectrophotometry results regarding intercalation of curcumin in the layers of the LDH structure.

Structural and morphological characterization

Curcumin intercalation into the interlayer region of LDH was confirmed by X-ray diffraction (XRD) analysis. The XRD patterns of curcumin-Mg₂Al-LDH samples are shown in Fig. 2A. In each case, a typical LDH XRD pattern exhibits the characteristic reflections of the LDH layered structure with a series of peaks related to the stacked layers; strong lines at low angle (marked by *), indicating the guests have been successfully intercalated into the LDH gallery to produce a supramolecular structure.²² Furthermore, the left shift of these peaks in the curcumin–LDH XRD pattern is due to intercalation of curcumin in expanded layers.

Fig. 2 (A) The XRD patterns of curcumin–LDH and free LDH ((*) indicates strong lines at low angle) (B) SEM image of the curcumin–LDH sample (C) TEM image of the curcumin–LDH nanohybrid.

SEM (Fig. 2B) and TEM (Fig. 2C) images show that the sample of curcumin–LDH nanohybrid possesses uniform plate-like morphology with particle sizes ranging from 70 to 90 nm.

Cytotoxicity of void LDH nanoparticle

To determine the toxicity of LDH used for curcumin loading, the effect of LDH (void) on cell viability was studied. Viability of cells treated with different concentrations of LDH (up to 100 μg ml⁻¹; for 24 h) did not change significantly (Fig. 3). The sample of LDH does not show PDT effect as well as cytotoxicity, demonstrating its biocompatibility as reported previously.²³

Fig. 3 Viability of MDA-MB-231 breast cancer cells treated with indicated concentrations of LDH for 24 h and then one group kept in dark and another exposed to blue LED for 30 min. The results are expressed as the mean ± SD (n = 3).

Pre-incubation time dependent of curcumin–LDH nanohybrid phototoxicity on human breast cancer cells

The effect of various pre-incubation times with curcumin and curcumin–LDH nanohybrid on viability of cells was investigated with and without exposure to light. As observed in Fig. 4, no significant change in viability was observed for 1 and 4 h of incubation. Dark toxicity of the curcumin–LDH nanohybrid slightly increased with increasing incubation times (24 and 48 h). 98% viability was observed in cells pretreated with 25 μg ml⁻¹ curcumin–LDH nanohybrid for 24 h. Exposure of these cells to blue LED light (∼34 mW cm⁻²) decreased the viability to 61%. On the other hand, increasing incubation time did not have a significant influence on the viability of cells pretreated with a similar concentration of free curcumin.

Fig. 4 Viability of cells treated with 0, 10 and 25 μg ml⁻¹ of free curcumin and equivalent concentration of curcumin–LDH complex for 1, 4, 24 and 48 h and then kept in dark (A) and or exposed to blue LED (B) for 30 min. Viability of cells after different treatments was compared with untreated controls. The results are expressed as the mean ± SD (n = 3, *P < 0.05 compared with control group).

The results of 48 h incubation are similar to 24 h trials. The viability of treated cells with 25 μg ml⁻¹ curcumin–LDH nanohybrid after 48 h was 97%. Exposure of these cells to light (∼34 mW cm⁻²) reduces the cell viability to 54%. Blank test shows that the irradiation poses no influence on cell viability.

Effect of different irradiation time on human breast cancer cells

The effect of light dose on cell death pretreated with either free or curcumin–LDH monohybrid was determined. The results presented in Fig. 5 show that there is no significant difference in the viability of the cells treated with free curcumin and curcumin–LDH nanohybrid kept dark. However, the viability of treated cells with 25 μg ml⁻¹ curcumin–LDH nanohybrid and irradiated decreased gradually in a light dose dependent manner. The viability of the cells (curcumin–LDH nanohybrid treatment) exposed to 10, 15, 20 and 30 min irradiation were 90%, 83%, 70% and 61%, respectively. The viability of free curcumin treated cells that were exposed to blue LED light did not change significantly (Fig. 5). The blank test shows that the various irradiation times pose no influence on the cell viability.

Fig. 5 Viability of cells treated with 25 μg ml⁻¹ of free curcumin and equivalent concentration of curcumin–LDH complex for 24 h and then one group kept in the dark and another exposed to blue LED for 5, 10, 15, 20 and 30 min. The results are expressed as the mean ± SD (n = 3, *P < 0.05 compared with control (blank) group).

In vitro photodynamic activities of curcumin–LDH nanohybrid

The PDT action of curcumin–LDH photosensitizers was further studied by in vitro tests performed with MDA-MB-231 cells. The impact of curcumin–LDH concentration on PDT effectiveness was studied. The MDA-MB-231 cells were incubated in the presence of different concentrations of curcumin–LDH for 24 h, followed by washing with PBS and irradiated with blue LED (465 nm with power density: 34 mW cm⁻²).

The best PDT behavior was demonstrated with the dosage of 100 μg ml⁻¹ of curcumin–LDH nanohybrid (the difference between the dotted green and dashed red bar: 38) (Fig. 6B). For comparison, the PDT performance of free curcumin was also studied by incubating MDA-MB-231 cells with medium containing free curcumin.

Fig. 6 The PDT performance of (A) curcumin and (B) curcumin–LDH with various concentrations after 24 h incubation. MDA-MB-231 breast cancer cells were used in these cases. The results are expressed as the mean ± SD (n = 3, *P < 0.05 compared with the no irradiation group).

After 24 h incubation of cells with 100 μg ml⁻¹ of free curcumin the cell viability was 99% (no irradiation) and 95% (irradiation), indicating some cytotoxicity as well as rather poor PDT effectiveness (Fig. 6A). In the case of curcumin–LDH nanohybrid (Fig. 6B), the cell viability after 24 h of treatment was found to be 90% (no irradiation) and 52% (irradiation), which demonstrates largely-enhanced PDT effectiveness and acceptable cytotoxicity.

Microscopic study

To visualize the phototoxicity effect, images of MDA-MB-231 cells treated with curcumin and curcumin–LDH nanohybrid, without irradiation and under irradiation, were studied under invert microscopy. As observed in Fig. 7, there is a morphological difference between cells treated with free curcumin and curcumin–LDH nanohybrid (C and F panels).

Fig. 7 Invert microscopy images (40×) of MDA-MB-231 cells treated with free curcumin and curcumin–LDH (100 μg ml⁻¹, 24 h incubation) without irradiation: (A) blank, (B) curcumin and (C) curcumin–LDH. MDA-MB-231 cells treated with curcumin and curcumin–LDH (100 μg ml⁻¹, 24 h incubation) under irradiation (30 min irradiation): (D) blank, (E) curcumin and (F) curcumin–LDH.

Discussion

To date, cancer therapy by natural anticancer compounds with low side effects in comparison to chemical anticancer drugs has attracted most scientists in the world. Curcumin is the major constituent of turmeric powder, extracted from the rhizomes of the plant Curcuma longa.²⁴ Various researches have been done on curcumin applications as a natural polyphenolic compound for its anticancer effect against many different types of cancers.^25,26 However, the poor solubility of curcumin leads to low bioavailability and limit the potential effect of curcumin in cancer therapy. Recent studies have shown that the toxic effect of curcumin can be improved by encapsulating it in liposomes, micelles, silica and cyclodextrin nanoparticles.^27,28 There are few studies about the potential role of curcumin as a photosentitizer in photodynamic therapy.²⁹ To date, there is no report on curcumin–LDH nanohybrid applications in photodynamic therapy. The present study describes phototoxicity effect of curcumin–LDH nanohybrid in comparison to free curcumin in photodynamic therapy of breast cancer cells.

From the results, it is concluded that the red shift in curcumin–LDH UV-vis spectra is related to curcumin intercalation in LDH and the chemical environment around the molecule that decreases the water repulsion and chemical potential for the host molecule. Because of the hydrophobic structure and poor water solubility of curcumin, the absorbance of free curcumin is low. However, when curcumin intercalates in LDH layers, the absorbance peak increases. Increases in the absorbance peak of curcumin–LDH demonstrated that the curcumin in the LDH nanohybrid was well dispersed in solution and a stable dispersion was obtained. The consequent swelling of LDH nanoparticles makes stable dispersions for solutions.³⁰ These findings are consistent with the results of previous studies showing that the curcumin intercalated LDHs are more stable and also have release properties with future potentials for therapeutic applications.³¹ Reduction in the fluorescence spectra of curcumin–LDH demonstrates that by intercalating the fluorescent dye (curcumin) in the layered structure (LDH), some of the exited electrons could not return to lower electron layers; therefore, electron transferring decreases by intercalation and also creates a quenching effect on curcumin fluorescence.³² To the best of our knowledge, oxygen molecules could work as quenchers.³³ It could be suggested that the reduction in fluorescence intensity of exited curcumin is caused by oxygen in the LDH nanohybrid complex (FRET like reaction). The population of excited electrons increases and subsequently it could generate more singlet oxygen using excited electrons in a radical-production route.^34–36 It should be noted that irradiation could also increase the excited electrons and singlet oxygen production in PDT.³⁷ Furthermore, experiments have been performed to prove the intercalation of curcumin in the LDH layers such as XRD, SEM and TEM techniques. According to sharp and symmetric reflections in the LDH XRD patterns, it can be concluded that there is a well-crystallized hydrotalcite-like phase for LDH structure. The left shift in 2θ (2θ = 22.46 (for LDH) and 2θ = 22.26 (for curcumin–LDH)) and sharp lines at low angle clearly demonstrate the intercalation of curcumin in the interlayer region of the LDH complex. The basal interlayer space d₀₀₃ was increased by intercalating of curcumin in LDH layers (d_LDH = 7.98 Å and d_{curcumin–LDH} = 8.52 Å). The obtained results are in agreement with previous studies, which indicates that the curcumin can be intercalated in the interlayer region of LDH.³¹ Hexagonal lamellar structural morphology and a relative uniform size for synthesized LDH and nanohybrid is presented in SEM and TEM images. The narrow size distribution in crystalline beads (70–90 nm) confirms that the hydrophilic properties of LDH chemical structure could improve the curcumin water solubility and stability of the curcumin–LDH nanohybrid dispersion.

The phototoxicity of void LDH results clearly shows that LDH has no significant toxicity on the cell incubated with LDH kept dark or irradiated. These observations suggest that LDH can be used as a biocompatible drug carrier in photosensitizer delivery to cancer cells. Recently, LDHs have been introduced as a pH-responsive controlled release systems.²¹ As described previously, at pH 7.4, the amount of ZnPcPS4 released from LDH–ZnPcPS4 was lower than at pH 6.5 and 5.0, typical of those in tumor stroma or subcellular organelles such as cytoplasm and endosomes.³⁸ Therefore, the LDH nanohybrid could minimize photosentitizer release in the bloodstream and favorably quick-release photosentitizer from LDH once reaches the acidic tumor stroma, which is certainly favorable for targeted cancer treatment.²¹ Pre-incubation time studies reveal that in the dark, the cell viability for LDH, curcumin and curcumin–LDH nanohybrid has the same pattern and does not show significant differences. Irradiation in various times with blue LED clearly shows the phototoxicity of curcumin–LDH nanohybrid. The obtained results from photodynamic studies reveal that curcumin–LDH nanohybrids exhibit excellent photodynamic activity in comparison with free curcumin as a photosensitizer. Therefore, with no doubt, the water dispersion and solubility effect of LDH could improve the photosensitizer penetration and distribution in cells for effective photodynamic therapy performance. Moreover, the deformation and destruction of breast cancer cells due to good penetration and distribution of curcumin–LDH nanohybrid was determined using invert microscopy.

Conclusion

Altogether, our investigation suggests that the curcumin–LDH nanohybrid shows more phototoxic effects on breast cancer cells than compared to free curcumin due to an increase in aqueous solubility and stability of curcumin at physiological pH. In addition, photodynamic activity of curcumin in the nanohybrid is enhanced as indicated by an increase in cell killing. Increased photodynamic activity of curcumin delivered through LDH in breast cancer cells suggests that LDH could be a powerful delivery vehicle for improving photodynamic efficacy of curcumin for breast cancer treatment. Although our experiments on breast cancer cells confirm the phototoxic effect of curcumin–LDH nanohybrid, the precise mechanism is still unknown and remains to be elucidated.

Acknowledgements

The authors gratefully acknowledge the vice chancellor of medical laser research center, ACECR, and Tehran University of medical sciences (TUMS) for providing permissions and facilities to carry out the present study.

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