Improved Bioavailability of Curcumin in Gliadin-Protected Gold Quantum Cluster for Targeted Delivery.

This study deals with the synthesis of a gliadin-stabilized gold quantum cluster (AuQC) for the encapsulation of curcumin (CUR) and its targeted delivery to the cancer cell. CUR is an anticancer drug containing a hydrophobic polyphenol derived from the rhizome of Curcuma longa. The utilization of CUR in cancer treatment is limited because of suboptimal pharmacokinetics and poor bioavailability at the tumor site. In order to improve the bioavailability of CUR, we have encapsulated it into AuQCs stabilized by a proline-rich protein gliadin because proline-rich protein has the ability to bind a hydrophobic drug CUR. The encapsulation of CUR into the hydrophobic cavity of the protein was confirmed by various spectroscopic techniques. Compared to CUR alone, the encapsulated CUR was stable against degradation and showed higher pH stability up to pH 8.5. The encapsulation efficiency of CUR in AuQCs was calculated as 98%, which was much higher than the other reported methods. In vitro drug release experiment exhibited a controlled and pH-dependent CUR release over a period of 60 h. The encapsulated CUR-QCs exhibited less toxicity in the normal cell line (L929) and high toxicity in breast cancer (MDA-MB239). Thus, it can be used as a potential material for anticancer therapy and bioimaging.

Introduction

Cancer is known as an uncontrolled growth of abnormal cells, and it is considered as the second leading cause of death globally. The conventional treatments for cancer diseases are surgery, chemotherapy, radiotherapy, and hormone therapy.[1] The major challenge of chemotherapy is the nonspecificity of cancer cells, where normal cells are also drastically affected. The common chemotherapeutic agents used for the treatment are platinum derivatives, topoisomerase inhibitors, nucleoside analogues, vinca alkaloids, and taxanes.[2,3] These chemotherapeutic agents, though effective in treating cancer, exhibit severe toxicity in noncancer cells as well.[4] Also, most cancer types show initial susceptibility to chemotherapeutic agents and gain drug resistance through methods such as drug efflux and DNA mutation, thus increasing the chances of reoccurrence of the disease and thereby questioning the continued administration of these drugs.[5] Thus, the discovery of natural phytochemicals with effective growth inhibitory activity in cancer cells and zero toxicity for normal cell lines has been the Holy Grail in the research community.

Among the phytochemicals, curcumin (CUR) [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] is a worthy candidate, which is being explored thoroughly. CUR is a natural hydrophobic polyphenol, isolated from the rhizomes of the perennial herb Curcuma longa.[6] It has drawn significant interest in the recent past owing to its numerous biological and medicinal activities such as antimicrobial, antioxidant, anti-inflammatory, anticarcinogenic, and neuroprotective properties and is also an inhibitor of angiogenesis.[7−9] However, its poor water solubility, high rate of degradation at the physiological condition, and low oral bioavailability, which is essentially due to its hydrophobic nature, limit its use in food and medicine.[9] The poor bioavailability of CUR leads to its low absorption and high rate of metabolism within the living system and rapid elimination from the biological system.[7,10]

With this insight, various carrier systems, such as nanoparticles, liposomes, proteins, polymers, and so forth, have been devised for the encapsulation of CUR.[10] In recent times, several research groups have used biocompatible nanomaterials as drug carriers for targeted therapy. One of the important nanomaterials used as carriers is metal nanoparticles, and among them, gold quantum clusters have specifically raised interest among scientists.[11,12] They are a subnanometer core-sized particle made up of several tens of atoms, which shows molecule-like optical properties.[13] Recently, Govindaraju and co-workers developed CUR-conjugated fluorescent gold nanoclusters for anticancer therapy.[14] The excellent properties of gliadin-stabilized gold quantum clusters (AuQCs), such as easy one-pot synthesis, intensive fluorescence, good aqueous solubility, excellent biocompatibility, extraordinary photostability, extremely small size, and low cytotoxicity, make them a suitable candidate for bioimaging and drug delivery.[15]

Herein, we selected an abundant, low-cost, and sustainable plant protein—wheat gliadin—employed as a reducing as well as stabilizing agent to facilely produce AuQC and exhibit a strong red fluorescence. Gliadin is a proline and glutamine-rich monomeric protein component of wheat gluten and has a high amount of nonpolar amino acids in its primary structure.[16] It is well known that the proline-rich proteins have the ability to bind hydrophobic drugs, which allow mediated and controlled drug release.[17,18] Thus, herein, we have used a gliadin-protected gold quantum cluster for enhanced solubilization of CUR in an aqueous medium, which exhibits excellent stability at the physiological condition. The AuQC–CUR shows significant results in both bioimaging and anticancer therapy, which suggest its great potential for anticancer treatment.

Experimental Section

Materials

The chemicals, HAuCl4·3H2O, gliadin, folic acid (FA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxy succinimide (NHS), and the membrane dialysis bag (molecular weight cutoff 14 kDa), were purchased from Sigma-Aldrich. Sodium dihydrogen phosphate, sodium hydroxide, and disodium hydrogen phosphate were purchased from Merck. CUR was gifted by Synthite Industries, Kerala, India. All solutions were prepared using Millipore water.

Instrumentation

The absorption spectra were taken using a Varian model Cary wins Bio 100 spectrometer in the range of 200–800 nm. The fluorescence emission spectra were recorded using the Fluoro Max-4C spectrofluorometer (Horiba Instruments, USA). The slits for excitation and emission were set at 5 nm. Lifetime analysis was measured using time-correlated single photon counting with a pulse width of 1.3 ns. High-resolution transmission electron microscopy (HRTEM) images were recorded on a JEOL JEM 2100 instrument with an acceleration voltage of 200 kV. An Omicron ESCA Probe spectrometer with unmonochromatized Mg Kα X-rays was used for X-ray photoelectron spectroscopy (XPS) analysis. Fourier-transform infrared (FTIR) spectra were recorded using a PerkinElmer FTIR spectrometer. Thermal transition measurements were done using a TA Q100-DSC thermal analyzer (TA Instruments, New Castle, Delaware 19720 USA).

Synthesis of AuQC@Gliadin

Synthesis of AuQC@gliadin is briefly explained herein. Gliadin (25 mg/mL) protein powder was dissolved in 0.25 M NaOH solution and heated at 40 °C for 15 min. The above prepared gliadin solution (5 mL) was treated with 5 mL (5 mM) of AuCl4 and vigorously stirred at 55 °C for 3 h. Purification of AuQC@gliadin was performed by dialysis against distilled water for 24 h in a dialysis membrane with a molecular weight cutoff of 14 kDa. The cleaned gold clusters were then freeze-dried and stored at room temperature for further use.

Loading of CUR

The AuQC@gliadin stock solution (5%, w/v) was prepared by dispersing the freeze-dried AuQC@gliadin sample in water. CUR stock solution (4 mg/mL) was prepared in distilled ethanol, and 14 μL of CUR solution was added dropwise per milliliter of AuQC@gliadin under stirring at room temperature. The CUR solution was immediately solubilized in AuQC@gliadin solution, resulting in a bright yellow solution, which is distinct from the golden-yellow solution of AuQC@gliadin. The stirring was continued for 30 min to stabilize the formulation. The unbound CUR was removed by centrifugation at 10 000 rpm for 10 min. The resultant supernatants were then collected, freeze-dried, and stored at room temperature for further use.

Preparation of the FA-Functionalized AuQC@Gliadin–CUR Conjugate

Conjugation of CUR with protein-stabilized AuQC was carried out by the EDC/NHS coupling reaction.[19] An aqueous solution (0.05 M, 0.25 mL) of EDC was treated with 5 mL (1 mg/mL) of AuQC@gliadin and kept stirring for 2 h. To this, 0.25 mL of 0.05 M aqueous solution of NHS was added, followed by the addition of 150 μL of 4 mM FA. After 12 h of continuous reaction, the reaction mixture was centrifuged at 15 000 rpm for 15 min in a 1:1 methanol/2-propanol mixture, followed by washing with DI water twice to obtain FL-AuQC@gliadin. To this solution, 200 μL of CUR stock solution was added, followed by continuous stirring for 30 min to get the FA-AuQC@gliadin–CUR conjugate.

Calculation of Encapsulation Efficiency

Encapsulation efficiency (EE) of CUR bound to AuQC@gliadin was determined by adding 200 μL of CUR stock solution in 10 mL of solution of AuQC@gliadin and kept stirring for 30 min at room temperature. The solution was centrifuged for 10 min at 10 000 rpm to pelletize the undissolved CUR. The pellet was carefully dissolved in a known amount of ethanol, and CUR was quantified spectrophotometrically at 428 nm. The EE was calculated based on eq .[20]

In Vitro Release of CUR

Direct dispersion method was employed to study the in vitro drug release of CUR from AuQC@gliadin–CUR.[21−23] A known quantity of CUR-loaded AuQCs was taken in 3 mL of 0.1 M phosphate-buffered saline (PBS) at pH 5 and 7.4 and incubated in a water bath shaker at 37 °C. This study was carried out for a time period of 60 h where the tubes were taken out at definite time intervals and centrifuged at 10 000 rpm for 10 min. This was done in order to pelletize the released drug, while the entrapped drug within the gold quantum cluster remained in the supernatant. Subsequently, the pellets were dissolved in 3 mL of ethanol, and the amount of CUR released was quantified using a spectrometer at a wavelength of 428 nm.

Cell Culture

C6 cells were cultured in the F-12K basal medium and MDA-MB231 and L929 cells in Dulbecco’s modified Eagle’s medium, which were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were incubated at 37 °C for 24 h with 5% CO2 and were observed for healthy growth after incubation. On reaching 80–90% confluency, they were trypsinized using trypsin–EDTA solution (0.25% w/v trypsin, 0.54 mM EDTA) to detach them from the flask and centrifuged at 3000 rpm for 3 min. The cells were then resuspended in the medium for further studies.

Cytotoxicity Assay

To evaluate the cytotoxicity of the prepared formulation, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium (MTT) assay was performed. It is a colorimetric test based on the selective ability of viable cells to reduce the tetrazolium component of MTT into formazan crystals, which is purple in color.[24] For cytotoxicity experiments, MDA-MB 231 and L929 cells were seeded on a 96-well plate and incubated at 37 °C for 24 h. Subsequently, both the cell lines were added with four different concentrations (25, 50, 100, 200 μg) of FA-conjugated AuQC@gliadin–CUR. After the incubation of FA-AuQC@gliadin–CUR in serum-free media for 24 h, the media were replaced with 90 μL of fresh media, followed by the addition of 10 μL (5 mg/mL) MTT reagent. The media were removed after 4 h incubation at 37 °C. Dimethyl sulfoxide (100 μL) was then added to the wells and incubated for 30 min. The optical density of the solutions was then determined at 570 nm using a microplate reader. The data were plotted against the concentration of FA-AuQC@gliadin–CUR versus relative cell viability (%).

Cellular Uptake Studies

Cellular uptake studies were done to monitor the relative uptake of FA-AuQC@gliadin–CUR in different cell lines and also to standardize the time required for the maximum uptake of the formulation by the cells. The study was done in L929 (normal cell line), C6 glioma (brain cancer cell), and MDA-MB231 (breast cancer) cells. These cells were cultured as described above and were seeded into four-well dishes. The cells were incubated at 37 °C for 24 h so that they can attach to the well. Followed by incubation, the cell lines were treated with 100 μg of FA-AuQC@gliadin–CUR at three different time point incubations (1, 2, and 4 h). After each time point of incubation, the cells were fixed using 4% paraformaldehyde solution and were mounded on glass slides. The cellular uptake and the fluorescence property of the FA-conjugated AuQC@gliadin–CUR were observed with HcRed and fluorescein isothiocyanate using a confocal microscope.

Stability Measurements

Stability of the prepared formulation in various pH (5, 6, 7, 7.4, 8.6, and 9) buffers was studied spectrophotometrically. In order to study the stability of the conjugate at pH 7.4 with time, CUR and AuQC@gliadin–CUR were dispersed in phosphate buffer and incubated at room temperature. The concentration of CUR at different time intervals was calculated by measuring the absorbance at 428 nm.

Results and Discussion

Characterization of AuQC@gliadin

The method for the synthesis of AuQC@gliadin is similar to our previous method for the synthesis of gluten-protected AuQC.[25] Gliadin-protected AuQC was synthesized by in situ reduction of chloroauric acid. Spectroscopic and microscopic techniques were used to confirm the successful formation of AuQC@gliadin. The UV–visible absorption and emission spectrum of AuQC@gliadin are shown in Figure .

Figure 2

(A) Optical absorption (black trace) and emission (blue trace, λex = 380 nm) spectra of AuQC@gliadin. The inset shows the photographs of aqueous solution of AuQC@gliadin under (i) UV light and (ii) visible light. (B) TEM images of AuQC@gliadin. The inset shows the HRTEM image of AuQC@gliadin in the scale of 2 nm; (C) SAED pattern of AuQC@gliadin; and (D) binding energy of AuQC@gliadin determined from XPS.

AuQC@gliadin showed a characteristic broad featureless absorption peak with a small hump at 280 nm, which is due to the presence of aromatic amino acid present in gliadin as AuQC@gluten (Figure A).[25−27] The possibility of Au nanoparticle formation was eliminated as the surface plasmon resonance peak was not visible at around 520 nm. At an excitation of 380 nm, the fluorescence spectra of the formed cluster showed an emission maximum centered at 685 nm along with a weak emission peak around 460 nm, which is due to the aromatic amino acid present in gliadin (Figure A). The formed cluster showed an intense red emission under UV light (365 nm) [inset of Figure A(i)] and light brown color under visible light [inset of Figure B(ii)]. TEM image and XPS analysis further confirmed the formation of AuQC. The TEM image of AuQC@gliadin (Figure B) demonstrated that the as-prepared AuQC has good monodispersity with a particle size of ∼2 nm. The hydrodynamic volume of AuQC@gliadin was analyzed using the dynamic light scattering (DLS) technique. The average size of AuQC@gliadin was found to be 60 ± 2 nm (Figure S1).

HRTEM and selected area electron diffraction (SAED) pattern demonstrated the crystallinity of the as-formed QCs. XPS determined the binding energy for Au 4f7/2 and 4f5/2 of AuQC@gliadin to be 84.5 and 88.1 eV, respectively, which corresponds to Au(0). This confirmed the reduction of Au(III) to Au(0) after the formation of clusters (Figure B).

Characterization of AuQC@Gliadin–CUR

Simple one-step mixing method was used for the loading of CUR into AuQC@gliadin to form the AuQC@gliadin–CUR hybrid. The schematic representation for the encapsulation of CUR into AuQC@gliadin is illustrated in Figure . The EE of CUR in AuQC@gliadin was calculated and found to be 98.17 ± 0.25%. The formed AuQC@gliadin–CUR hybrid was then lyophilized and resuspended in water. It was found that the lyophilized powder of the AuQC@gliadin–CUR hybrid showed a complete dispersion in an aqueous medium (Figure b, inset), whereas CUR alone remained completely insoluble in water with undissolved flakes clearly visible in the suspension (Figure b inset). The photophysical studies of AuQC@gliadin and the hybrid have been carried out to understand the interaction of CUR with AuQC@gliadin. Noticeable changes in the absorption and fluorescence spectra of CUR and AuQC@gliadin were observed after interaction of these two entities. Figure A,B shows the UV–vis absorption and emission spectra of AuQC@gliadin with different concentrations of CUR. CUR alone showed an absorption peak at 428 nm in the aqueous buffer, which is the signature of its basic diaryl heptanoic chromophore group.[28] The inclusion of CUR in AuQC@gliadin showed the broadening with a slight blue shift in the absorption peak and a noticeable increase in the absorption intensity (Figure A). The blue shift in the absorption maxima suggests the nonpolar vicinity of the CUR molecules.[29,30] Upon addition of CUR to AuQC@gliadin, noticeable changes in the fluorescence intensity and peak position at 480 nm of QC were observed. On addition of increasing concentration of CUR to QC, the broadening of the emission peak at 480 nm and its complete shifting to 505 nm were noticed, which corresponds to CUR emission (Figure B).

Figure 1

Schematic representation for the targeted delivery of CUR by FA-conjugated AuQC.

Figure 3

(A,B) Respective absorption and emission spectra of AuQC@gliadin with different concentrations of CUR (4.4 μM to 36.4 μM). The inset of (A) shows (a) CUR in AuQC@gliadin and (b) CUR in water; (C,D) respective absorption and emission spectra of CUR with different volumes of AuQC@gliadin (100 μL to 800 μL).

CUR alone in buffer showed a weak broad fluorescence peak at 570 nm when excited at 430 nm (black trace of Figure D).The blue shift in the emission spectrum of CUR from 570 to 505 nm is due to the entrapment of CUR in AuQC@gliadin. Previous reports for the interaction of CUR with proteins support this observation.[30−32] The binding of CUR to bovine serum albumin, human serum albumin ,and soy protein isolate showed fluorescence maxima at 510, 515, and 500 nm, respectively.[30−32] CUR is known to bind to the hydrophobic domain of the protein molecules and this nonpolar environment of CUR in AuQC@gliadin is held responsible for the blue shift in its emission maxima.[30,31,33,34] Moreover, the interaction of CUR with AuQC@gliadin can be confirmed by the progressive reduction in fluorescence intensity at 680 nm on addition of increasing concentrations of CUR (Figure B).

Similarly, the enhanced solubilization of CUR in an aqueous solution of AuQC@gliadin was demonstrated by monitoring the absorption and emission spectra of CUR with various concentrations of AuQC@gliadin. The absorption and emission spectra of CUR at different volumes of AuQC@gliadin are shown in Figure C,D. On addition of increasing volume of AuQC@gliadin to CUR in the buffer, remarkable changes were observed in the absorption and fluorescence spectra of CUR. The increase in absorption intensity at 428 nm along with the broadening in the absorption peak confirmed the improved solubility of CUR in the gold quantum cluster solution. Moreover, the addition of AuQC@gliadin greatly improved the fluorescence intensity of CUR at 505 nm, compared to that in an aqueous medium, together with the blue shift from 575 to 505 nm in the fluorescence peak, which suggests that CUR in AuQC@gliadin binds to the hydrophobic pockets of the protein and thus experiences a nonpolar environment. The fluorescence lifetime analysis further supported the encapsulation of CUR in protein-stabilized AuQCs. The fluorescence decays of CUR, AuQC@gliadin, and AuQC@gliadin–CUR were recorded with an excitation wavelength of 405 nm, and the fluorescence decays were monitored to their emission maxima (Figure ).

Figure 4

(A) Lifetime of AuQC@gliadin and AuQC@gliadin–CUR at 680 nm emission and (B) lifetime of CUR and AuQC@gliadin–CUR at 550 nm emission.

All the lifetime values were obtained by fitting the fluorescence decay curves biexponentially, as tabulated in Table . When comparing the average lifetime of CUR and encapsulated CUR, the encapsulated CUR shows increased average lifetime value.

Table 1

Lifetime Data of AuQC@Gliadin, AuQC@Gliadin–CUR, and CUR

sample name	A1	τ1 (ns)	A2	τ2 (ns)	χ2	τav (ns)
CUR (550 nm)	14.96	6.84	85.04	1.48	1.37	3.88
AuQC@gliadin–CUR (550 nm)	43.74	2.16	56.26	8.2	1.37	7.17
AuQC@gliadin (680 nm)	84.74	60.25	59.69	3.29	1.39	59.69
AuQC@gliadin–CUR (680 nm)	78.78	44.5	21.22	2.68	1.1	43.83

The increased value of the average lifetime is due to the entrapment of CUR in the hydrophobic pockets of the protein, which disrupt the excited-state intramolecular proton-transfer process, whereas the average lifetime of AuQC@gliadin decreased when CUR was introduced, which is due to the interaction of CUR with QCs.

The FTIR analysis was carried out to understand the conformation changes of the protein after incorporation of CUR. Figure A shows the FTIR spectra of (i) AuQC@gliadin (black), (ii) AuQC@gliadin–CUR (blue), and (iii) CUR (red). The secondary structure of gliadin was described by the protein amide I band at 1600–1690 cm–1 (due to C–O stretching) and amide II band at 1480–1575 cm–1 (C–N stretch coupled with the N–H bending mode).[35,36] Noncovalent interactions such as van der Waals interactions, hydrophobic interactions, and hydrogen bonds are generally known to lower the energy of the corresponding part of the molecule, reduce the force constants of the bonds, and therefore decrease its absorption frequency.[37]

Figure 5

(A) FTIR spectra, (B) TGA, and (C) DSC analysis of AuQC@gliadin, CUR, and AuQC@gliadin–CUR, respectively.

After interaction with CUR, the peak corresponding to amide I shifted to lower wavenumber from 1633 to 1630 cm–1 and the amide II band shifted from 1536 to 1528 cm–1. This shift in wavenumber resulted from the binding of CUR to the protein C=O, C–N, and N–H groups. The N–H stretching frequency of gliadin is decreased from 3280 to 3278 cm–1 because of the interaction of CUR with the N–H groups of the protein.[38] Moreover, the decrease in signal intensity of the amide bands of the protein furthermore suggested the changes in the protein conformational states upon interaction with CUR.[38] CUR has its phenolic −OH stretching peak at 3504 cm–1 and its other characteristic bands at 1027/857 cm–1 (C–O–C stretching), 1275 cm–1(aromatic C–O stretching), 1428 cm–1 (olefinic C–H bending), 1502 cm–1 (C=O and C=C vibrations) and at 1602 cm–1 stretching vibrations of the benzene ring. These peaks were attenuated in the AuQC@gliadin–CUR spectrum because of the higher bandwidth of protein bands, which suggests that CUR is located within the macromolecular protein moiety by van der Waals forces and hydrophobic interactions. Thermal analysis of AuQC@gliadin, CUR, and AuQC@gliadin–CUR was carried out to understand the changes in the thermal behavior of AuQC@gliadin after incorporation of CUR. Figure B,C shows the thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) analysis of AuQC@gliadin, CUR, and AuQC@gliadin–CUR.

The thermogravimetric curves of AuQC@gliadin and CUR-loaded AuQC demonstrated the first stage weight loss by the gliadin samples at around 100 °C because of moisture loss at increasing temperature.[39] The second stage weight loss resulted from the decomposition of gliadin at 246 °C in AuQC@gliadin and 249 °C in AuQC@gliadin–CUR. This shift in decomposition temperature is attributed to the incorporation of CUR to AuQC@gliadin and implies the enhanced stability of AuQC@gliadin after CUR incorporation. The TGA curve for pure CUR showed a rapid weight loss of around 260 °C, which may be attributed to the decomposition of substituent groups of CUR.[40]

The DSC thermograms of AuQC@gliadin showed an endothermic peak at 77 °C because of the unfolding and denaturization of the protein.[39] The incorporation of CUR increased the thermal stability of gliadin in AuQC as the denaturization temperature shifted to a higher temperature of 92 °C in the case of AuQC@gliadin–CUR. The DSC of CUR alone shows an endothermic peak at 183 °C, which corresponds to the melting of CUR.[41] The endothermic peak of CUR was absent in the conjugate, which confirms the molecular incorporation of CUR in AuQC.

Stability of CUR

As mentioned earlier, CUR has very little solubility in water and is chemically unstable under physiological conditions, which is a major issue that concerns its bioavailability.[42] It is well known that CUR is highly unstable at physiological pH and undergoes rapid degradation into different products such as bicyclopentadione, vanillin, and ferulic acid.[43] It is reported that the binding of CUR to the hydrophobic pockets of protein can greatly improve solubilization and arrest its degradation.[32,44] In order to investigate the stability/biodegradability of CUR, the changes of the relative intensity of the characteristic absorption maximum of CUR in physiological condition were measured as a function of time (Figure ). The absorption peak at 428 nm of free CUR showed a rapid degradation in PBS solution with only 60% of CUR remaining after 12 h of incubation, whereas in the case of encapsulated CUR, the absorption peak showed remarkable stability under the same condition, with more than 92% of CUR remaining even after 12 h of incubation (Figure A). Similarly, we have analyzed the stability of the encapsulated CUR in different pH conditions. Figure B shows the absorption spectrum of AuQC@gliadin–CUR at different pH ranging from 5 to 9. The inset shows the photographs of the AuQC@gliadin–CUR solution in different buffers. The entrapped CUR is highly stable from pH 5 to 8.6, indicating the protection of CUR from hydrolytic degradation.

Figure 6

(A) Plot showing the stability of AuQC@gliadin–CUR and CUR in aqueous buffer pH 7.4 and (B) absorption spectra indicating the pH stability of AuQC@gliadin–CUR at pH 5, 6, 7, 7.4, 8.6, and 9.

At pH 9, a slight blue shift of the maximum absorption wavelength with a decrease in absorbance was observed, which is due to the degradation of CUR in more alkaline condition. Thus, the hybrid developed in this study shows superior stability over many reported formulations.[9,45−48]

In Vitro Release of CUR from AuQC@Gliadin–CUR

Before checking the anticancer activity of the drug-loaded quantum cluster, in vitro release kinetics was studied using UV–vis spectroscopy. The study was carried out with PBS at pH 5 and 7.4. The percentage of drug released from AuQC@gliadin–CUR at predetermined time intervals was calculated using the standard curve prepared for CUR. Figure shows the in vitro drug release profile of AuQC@gliadin–CUR at pH 5 and 7.4. The in vitro dug release profile shows that CUR release was more at pH 5 compared with that at pH 4. A sustained release of CUR was observed after the initial burst release, and 97.8% of the encapsulated CUR was released at pH 5 within 60 h. The initial burst release could be due to the attached CUR molecules on the surface of the AuQCs and the sustained release from the entrapped CUR.[21] In pH 7.4, the total release was only 35.4% at the same time, and the results indicated that the release of CUR was slower at physiological pH than under acidic condition. Because the cancer cells have an acidic extracellular environment, the release of CUR at acidic (pH ≈ 5) is more suitable for cancer therapy and noncancer cells at physiological pH are likely to be least affected by our formulation. The higher release rate CUR at acidic pH could be the conformational changes of gliadin at this pH, which facilitate enhanced CUR release.

Figure 7

Drug release profile showing the release percentage of CUR from AuQC@gliadin–CUR over 60 h at pH 5 and 7.4.

Characterization of Folate-Conjugated AuQC@Gliadin–CUR

FA has been conjugated to AuQC@gliadin for targeted delivery of CUR to the cancer cell because the membrane-associated FA receptor is overexpressed in cancer cells but remains at a very low level in most normal tissues.[49] FA-AuQC@gliadin–CUR was formed by chemically linking FA to the protein via EDC/NHS coupling. The conjugation of FA was confirmed by FTIR spectroscopy and UV–vis absorption study. Figure A,B shows the respective absorption and FTIR spectra of FA and FA-AuQC@gliadin–CUR, respectively. The absorption spectrum of FA-AuQC@gliadin–CUR shows three characteristic peaks at 282, 361, and 420 nm. The peaks at 282 and 361 nm are accorded with the characteristic absorbance of FA, and the absorbance at 420 nm is originated from CUR. This indicates that FA was successfully conjugated with AuQC@gliadin. Further conjugation of FA toward QC was confirmed by FTIR spectroscopy. The FTIR spectra of FA showed characteristic peaks at 3538 and 3413 cm–1 because of the stretching of −OH and −NH of the glutamic acid and pterinic portion, respectively.[50] Also, the peak at 1690 cm–1 corresponded to the stretching of different −C=O groups and the band at 1604 cm–1 resulted from −NH bending. Moreover, the band at 1482 cm–1 has arisen from the vibration of the pterinic group.[51] On inspecting the spectra of FA-AuQC@gliadin–CUR, the successful conjugation of FA to AuQC@gliadin–CUR was confirmed. As evident from Figure B, the −OH stretching peak of FA at 3538 cm–1 and the NH stretching at 3413 cm–1 with an increase in intensity could be found in the FTIR spectra of FA-AuQC@gliadin–CUR because of the overlapping of these functional groups with those in AuQC@gliadin–CUR. A shift in wavenumber of the amide I group (C–O stretch) from 1630 to 1636 cm–1 was observed on conjugation of FA to AuQC@gliadin–CUR. The amide II band of AuQC@gliadin–CUR also showed a shift from 1528 to 1535 cm–1, which is attributed to the newly formed C–N bond.[50]

Figure 8

(A) Absorption spectra showing FA and FA-conjugated AuQC@gliadin–CUR (AuQC@gliadin-FA–CUR). (B) FTIR spectra of (a) FA and (b) FA-conjugated AuQC–CUR.

In Vitro Cytotoxicity to Normal and Cancer Cells

It is necessary to evaluate the toxicity profile of a nanoprobe for biomedical applications. To evaluate the cytotoxicity of FA-AuQC@gliadin–CUR, we have treated L929-normal cell line and MDA-MB231 breast cancer cells to a series of equivalent concentrations of the nanoprobe for 24 h, and the percentage of viable cells was quantified by the use of the MTT assay (Figure ). The results from the MTT assay showed that AuQC@gliadin-FA–CUR exerts a significant concentration-dependent cytotoxicity to MDA-MB 231 cells and at the same time it is least toxic to the normal cell line—L929. This further supports the targeted delivery of CUR to the cancer cell without affecting normal cells and indicates that CUR remains active even after conjugation with AuQC@gliadin.

Figure 9

Cytotoxicity studies by the MTT assay in (A) L929 cell line and (B) MDA-MB-231 cell line.

Herein compared to cancer cells, normal cells are also slightly affected because the immortalized cells cannot sustain continuous treatment for 24 h, and this could also be a governing factor for the unexpected cell death in these cell lines. Moreover, normal cell lines show significant expression of folate receptors and though not overexpressed as these cell lines, there are still distributions of the folate receptors among normal cell lines.[52] The least toxicity of L929 cells with that of MDA-MB231 may be attributed to the presence of these folate receptors in the normal cells which on prolonged exposure (24 h) of FA-AuQC@gliadin–CUR may have facilitated its uptake.

There are several reports on the cytotoxic effects of CUR in several types of cancers and mechanisms by which they act.[53,54] In C6 cells, CUR is known to reduce the cell survival in a p53- and caspase-independent manner, which is an effect correlated with the inhibition of AP-1 and NFB signaling pathways,[55] whereas in MDA-MB-231 cells, apoptosis is induced through the regulation of ROS.[56] Thus, AuQC@gliadin as a CUR carrier is reassuring to the cancer treatment regime because of its ability to administer CUR without the loss of its medicinal efficacy.

Cellular Uptake of FA-AuQC@Gliadin–CUR

The in vitro cellular uptake studies via confocal fluorescent imaging were done after first, second, and fourth hour of incubation, and the images are shown in Figures , 11, and S2. On analyzing the uptake intensities of all the three cell lines, it is confirmed that maximum uptake was observed in the cancer cells after 4 h of incubation. It is long known that nanotechnology-driven drug delivery systems enhance the delivery to targeted cancer cells by benefitting from the unique vasculature characteristics of tumors.[57] L929 cells (normal cell line) showed no fluorescence at 4 h compared to C6 and MDA-MB231 (cancer cell lines) suggesting minimal to zero uptake of the formulation by these cells. This makes AuQC@gliadin a felicitous CUR carrier because the undesirable effects of traditional chemotherapeutic agents may be overcome as it augments its availability at the tumor site alone with least damage on healthy tissues. Also, a gradual increase in the fluorescence intensity was observed with the increase in incubation time (from 1 to 4 h) in cancer cells. The morphological changes in the cells after treatment with FA-AuQC@gliadin–CUR have been inspected by microscopic observation. Compared to L929 cells, both C6 and MDA-MB231 cells have undergone obvious morphological changes as is evident from Figures , 11, and S2. With an increase in incubation time, there was a retraction of cellular processes with appearance of common apoptotic features such as cell shrinkage, membrane blebbing, rounding, and so forth. Also, disintegration of the cancer cells was at its peak at 4 h of incubation, and more cells detached from the substratum leaving only a few attached. This confirms the selective toxicity of our conjugate in cancer cell lines.

Figure 10

Confocal fluorescence images of MDA-MB-231-breast cancer cells treated with AuQC@gliadin-FA–CUR at the first, second, and fourth hour of incubation.

Figure 11

Confocal fluorescence images of L929 cells treated with AuQC@gliadin-FA–CUR at the first, second, and fourth hour of incubation.

Conclusions

We have synthesized a gliadin-protected gold quantum cluster using a simple one-pot synthesis strategy and have effectively conjugated CUR to the cluster through instant mixing. Optical and thermal studies, microscopic analysis, EE, in vitro drug release, cellular uptake, and cytotoxicity of the prepared formulation were performed. The EE of CUR was 98.37 ± 0.25%, and the release study showed 97.8% release of the drug within 60 h in pH 5. The cellular uptake studies also demonstrated maximum uptake by C6 and MDA-MBA-231 cells and minimum uptake by L929 cells. Poor bioavailability upon oral administration and lack of absorption restricts the therapeutic application of hydrophobic and nearly water-insoluble CUR. Despite their phenomenal anticancer activity, many pharmaceutical companies refrain from using them as such owing to the fact that they form drug aggregates in highly localized concentrations at the sites of their deposition. This remains tantalizing to the scientific community knowing how active these compounds are toward their molecular targets. CUR, which falls into this category of compounds, is in dire need for a suitable carrier system to overcome these limitations without compromising its activity which AuQC@gliadin is expected to fulfill.