Improving Anticancer Activity of Chrysin using Tumor Microenvironment pH-Responsive and Self-Assembled Nanoparticles.

Chrysin is a natural bioactive compound with potential biological activities. However, unfavorable physicochemical properties of native chrysin make it difficult to achieve good therapeutic efficacies. In this study, poly(ethylene) glycol (PEG4000)-conjugated chrysin nanoparticles were prepared. The PEG4000 was conjugated to chrysin through cis-aconityl and succinoyl linkers to achieve tumor microenvironment-specific drug release from PEGylated nanoparticles. The conjugation of PEG and chrysin via succinoyl (PCNP-1) and cis-aconityl (PCNP-2) linkers was confirmed by the 1H NMR and FTIR analysis. The nanoparticles were characterized by DLS, TEM, XRD, and DSC analysis. Comparatively, PCNP-2 showed a better drug release profile and higher anticancer activity against human breast cancer cells than chrysin or PCNP-1. The apoptosis studies and colony formation inhibition assay revealed that the PCNP-2 induced more apoptosis and more greatly controlled the growth of human breast cancer cells than pure chrysin. Thus, the use of PCNPs may help to overcome the issues of chrysin and could be a better therapeutic approach.

Introduction

In the 21st century, phytoconstituents have emerged as the most prominent source of many anticancer agents and represent an alternative to synthetic chemotherapeutic agents for cancer treatment.[1] A wide range of phytoconstituents, particularly bioflavonoids, have shown potential anticancer activities against various cancers. Chrysin (5,7-dihydroxy-2-phenyl-4H-chromen-4-one) is one such natural, plant-based bioflavonoid with broad-spectrum anticancer activity.[2] This molecule is extracted from Passiflora caerulae, Passiflora incarnata, honey, propolis, and honeycombs.[3] The preclinical studies of chrysin have shown its potential anticancer effects through selective cell signaling mechanisms, which are connected to cell proliferation, inflammation, angiogenesis, and cancer metathesis.[4−6] Chrysin was found to be effective against various cancers including breast, lung, bladder, thyroid, prostate, colon, etc.[2,5,7−9]

Despite the potential efficacy of chrysin in cancer treatment, some major challenges hinder the clinical use of chrysin. These challenges are low aqueous solubility (3 μg/mL), poor cellular uptake, physiochemical instability, rapid metabolism, and low oral bioavailability.[10,11] Therefore, in the past few years, many attempts have been made to improve the physicochemical properties and delivery of chrysin. Researchers have used micronization,[12] nanoemulsion, nanomicelles, nanosuspension, host–guest complexes, different derivatization, and salt formation approaches.[13−17] However, these systems still have a few drawbacks and cannot deliver the drug site-specifically. Therefore, inspired by the specific tumor microenvironment, we have developed self-assembled nanoparticles of chrysin by conjugating it with PEG4000 via acid-sensitive linkers. Further, it is reported that the drug–polymer conjugated system shows high drug loading and enhanced therapeutic efficacy in comparison to the physical encapsulation of the drug-loaded system.[18] In a study, chrysin-loaded PLGA–PEG nanoparticles were also prepared and used against T47D (IC50 = 31.28 μM) and MCF7 (IC50 = 52.54 μM) breast cancer cells[19] and gastric cancer AGS cell (IC50 = 36.8 μM) lines.[20] In another interesting study, PEG1-chrysin conjugate was developed for the delivery of doxorubicin where the directly conjugated chrysin did not show any sign of toxicity against the HepG2 cells.[21]

PEG is an FDA-approved, hydrophilic polymer and is generally used as an excipient in different pharmaceutical formulations.[22−25] High biocompatibility and hydrophilic nature make PEG an attractive polymer for formulation development.[26] PEGylation of hydrophobic drugs improves their aqueous solubility, improves cellular uptake, prolongs circulation in the blood and therefore in vivo performance, and improves overall therapeutic efficacy.[27−31] The development of a pH-responsive system helps in the site-specific delivery of therapeutic agents, particularly in cancer chemotherapy. The pH of the tumor microenvironment (pH 6.5–6.8) is slightly acidic as compared with physiological pH 7.4. This pH difference provides an opportunity for selective delivery of drugs using pH-sensitive drug carriers.[32−34] Herein, we have conjugated chrysin to PEG using two pH-sensitive linkers, i.e., succinoyl and cis-aconityl, and evaluated its anticancer activity. The synthesized system was able to self-assemble at the nanoscale, which was characterized by different analytical techniques.

Experimental Section

Materials

Chrysin (CHRY), 4-dimethylaminopyridine (DMAP), cis-aconitic anhydride (CAA), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), hydroxybenzotriazole (HOBt), fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), phosphate buffer saline (PBS), dialysis tubing (molecular weight cut off 2 kDa), acridine orange, and ethidium bromide were obtained from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-Dimethylthiahiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Minimum Essential Medium (MEM) and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from HiMedia Laboratories (Mumbai, India). Succinic anhydride (SA), triethylamine, diethyl ether, glacial acetic acid, and dichloromethane were purchased from Rankem (Mumbai, India).

Synthesis of pH-Sensitive PEGylated Chrysin Conjugate

The PEGylated chrysin conjugates were synthesized by a two-step reaction. First, the hydroxyl group of PEG4000 was modified into succinoyl and cis-aconityl functionals using succinic anhydride and cis-aconitic anhydride, respectively.[35] In the next step, after 6 h of stirring, a solution of modified PEG4000 (mPEG, 0.500 g, 0.12 mmol), EDC (0.285 g, 0.18 mmol), and NHS (0.07 g, 0.06 mmol) in DMSO was dropwise added to the solution of chrysin (0.30 g, 0.12 mmol) in DMSO. The mixture was stirred for 24 h under nitrogen conditions. The resulting solution was dialyzed (MWCO 2 kDa) against ultrapure Milli-Q water, with the water changed every 6 h cycle, up to 3 days, and the product was collected by lyophilization.

Characterization of PEGylated Chrysin Conjugates

The PEGylated chrysin conjugates were characterized by proton nuclear magnetic resonance (Bruker 500-MHz Ultra shield plus NMR instrument) and Fourier transform infrared (PerkinElmer Spectrum 65 series, USA) spectroscopic techniques. For the NMR analysis, the PEGylated chrysin conjugates were dissolved in CDCl3, and pure chrysin was dissolved in DMSO-d6 solvent. Then both were scanned using a 1H NMR (500 MHz) instrument. For the FTIR analysis, samples were mixed with KBr, pelletized, and scanned from 4000 to 400 cm–1. The critical aggregation concentrations of the conjugates were measured using the pyrene fluorescent method, as previously reported.[36] A 25 μL aliquot of pyrene solution (6 × 107 M) was incubated with 5–500 μg/mL of PEGylated chrysin conjugates in the dark and at room temperature for 3 h. After that, samples were used to measure fluorescence intensities at a 339 nm excitation and 383 and 373 nm emission intensities. The fluorescence intensities were measured using a microplate reader (Synergy H1 Hybrid Reader, Biotek; Winooski, VT, USA). The intensity ratio and concentration of PEGylated chrysin conjugates were plotted to determine the critical micelle concentration (CMC) value.

Preparation of PEGylated Chrysin Nanoparticles (PCNP)

To obtain PEGylated chrysin nanoparticles, i.e., PCNP-1 (PEG4000–chrysin via succinoyl linker) and PCNP-2 (PEG4000–chrysin via cis-aconityl linker), the conjugates were dissolved in acetone at a concentration of 10 mg/mL. Then, 3 mL of this solution was dropwise added to 15 mL of water under stirring at 1000 rpm. After that, the mixtures were kept stirring (1000 rpm, RT) for 3 h to evaporate acetone. The chrysin content in nanoparticles was quantified by a UV–visible spectrophotometer at 266 nm, and % drug content was calculated by the following equation.

Characterizations of PCNP

The size distribution of PCNP-1 and PCNP-2 was determined using DLS technique (Malvern Instruments, UK). TEM instrument (JEOL JEM-2100 TEM, Tokyo, Japan) was used to confirm the morphology of nanoparticles. Powder XRD (D8 Advance, Bruker, Germany) and DSC (DSC 4000, PerkinElmer, USA) instruments were used to confirm the physical state of the prepared nanoparticles.[35]

In Vitro Drug Release Study

The release of chrysin from PCNP-1 and PCNP-2 was performed using the dialysis method.[37] PCNP-1 and PCNP-2 nanoparticles were separately placed in a dialysis bag (MWCO 2 kDa). The formulation-containing dialysis bag was immersed in 100 mL of sodium acetate buffer (pH 5.0), phosphate buffer (pH 6.5), or phosphate buffer saline (pH 7.4). The experiment was performed at 37 °C and with 100 rpm stirring speed. At different intervals, aliquots from the released media were collected for drug analysis and replenished with an equivalent volume of fresh media.

Stability Study

The stability study of prepared PCNPs was performed by measuring particle size (colloidal stability) and % drug content (drug stability) over a period of 96 h at refrigeration temperature. The change in particle size was measured by DLS method, and % chrysin content was measured using a UV–visible spectroscopic method.

Cell Culture

Human breast cancer (MDA-MB-231) and MCF-7 cells were purchased from NCCS (Pune, India) and cultured in Dulbecco’s Modified Eagle’s Medium-high Glucose (DMEM-HG) and MEM, respectively, with 10% fetal bovine serum (FBS) in a fully humidified atmosphere of 5% CO2 at 37 °C. After reaching 80% to 90% confluency, cells were trypsinized for experiments.[38]

In Vitro Cytotoxicity Assay

To evaluate the anticancer activity of prepared nanoparticles, an in vitro cytotoxicity assay was performed. About 7.0 × 103 cells/well were seeded in 96-well plates and incubated for 24 h in a CO2 incubator. After that, cells were treated with pure chrysin, PCNP-1, and PCNP-2 at different concentrations (5, 10, 20, 40, 60, 80, and 100 μg/mL). After the completion of the incubation period (24 and 48 h), media from the wells was discarded, and fresh media containing 0.5 mg/mL of MTT reagent was added to each well. After 4 h of incubation, MTT solution was removed, and crystal formazan was dissolved by adding 100 μL of DMSO to each well. After 10 min of incubation, absorbance was measured at 570 nm using a multimode plate reader (Synergy H1, Biotek, USA). Untreated cells were considered as a control, and % cell viability was determined with respect to the viability of the control cells.

Apoptotic Assay

To examine the cellular changes after treatment with PCNP, an apoptotic assay was performed by acridine orange/ethidium bromide (AO/EtBr) staining, as previously reported.[38] About 4.0 × 104 cells/well were seeded in 12-well plates. After 24 h, the cells were treated with pure chrysin, PCNP-1 and PCNP-2, equivalent to 10 μg/mL of chrysin. After the completion of the incubation period (24 and 48 h), the cells were trypsinized and centrifuged at 1800 rpm for 5 min. The resultant cell pellets were dispersed in 1× PBS, and 10 μL of suspension was taken for further examination. The cell suspension was mixed with 5 μL of AO dye (1 mg/mL) and EtBr dye (1 mg/mL) and mounted on a clean glass slide. Then, cells were observed using a fluorescence microscope (Zeiss, Germany). The cells were counted using ImageJ software and plotted as % cells in a stacked column graph by using Graphpad Prism 6.

Colony Formation Assay

To assess the colony formation potential of MDA-MB-231 cells after the treatment with PCNP, a colony formation assay was performed.[39] For that, about 300 cells/well were seeded in 6-well plates. After 24 h, cells were treated with a concentration of 10 μg/mL of pure chrysin, PCNP-1, and PCNP-2. The plates were kept in a CO2 incubator for 7 days to allow the cells to form colonies. Further, the media was discarded and washed twice with 1× PBS. The cells were fixed with cell-fixing solution (95:5, ethanol and glacial acetic acid) for 15 min. The cell-fixing solution was removed, and the cells were washed with PBS. Finally, the colonies were stained by crystal violet staining solution (5 mg/mL crystal violet in 2% ethanol) for 15 min at room temperature and washed with PBS. The number of colonies were counted under microscope (Carl Zeiss, Germany), and a graph was plotted by using GraphPad Prism software (GraphPad 6.0). Representative images were taken by a digital camera (Nikon 1000D camera, Taiwan).

Cellular Uptake Study

To evaluate the internalization of PCNP into MDA-MB-231 cells, a cellular uptake study was performed, as previously reported.[40] For that, coumarin-6 (C6) was used as a fluorescence probe and encapsulated in PCNP-1 (C6-CNP-1) and PCNP-2 (C6-CNP-1). About 2.0 × 104 cells/well were seeded into 35 mM cell culture dishes and allowed to incubate for 24 h. Further, cells were treated with 50 μg/mL concentration of C6-CNP-1 and C6-CNP-2 for 0, 0.5, 1, 2, 4, and 8 h. At the different time intervals, plates were washed twice with PBS and observed under a fluorescence microscope (Zeiss Axio Scope A1, Germany).

Statistical Analysis

All the experimental measurements were performed in triplicate, and the values are expressed as the mean ± standard deviation (SD). A p-value less than 0.05 was considered statistically significant.

Results and Discussion

Synthesis and Characterizations of pH-Sensitive PEGylated Chrysin Conjugate

The synthesis procedure of PEGylated chrysin conjugate is schematically shown in Figure S1. These amphiphilic conjugates of chrysin were prepared by a two-step reaction. In the first step, the PEG4000 was reacted to succinoyl and cis-aconityl linkers via ring-opening mechanism. In the second step, chrysin was conjugated to succinoyl–PEG4000 or cis-aconityl–PEG4000 conjugates through esterification. The formation of the PEGylated chrysin conjugates was confirmed by 1H NMR and FTIR techniques. Figure S2 shows the 1H NMR spectra of PEG4000, PEG4000–SA, and PEG4000–CAA where PEG4000 shows the characteristic chemical shift values at 3.65–3.68 and 4.26 ppm due to CH2–CH2 and −OH groups, respectively.[41] The chemical shift value observed at 2.64 ppm confirmed the formation of succinoyl–PEG4000, while the peaks observed at 2.10 and 5.31 ppm confirmed the formation of cis-aconityl–PEG4000. The pure chrysin shows the characteristic peaks at 6.20–6.22 ppm (6H), 6.41 ppm (8H), 6.57–6.59 ppm (3H), 7.48–7.49 ppm (4′H), 7.56 ppm (3′ and 5′H), 7.84 ppm (2′ and 6′H), 10.26 ppm (7OH), and 12.65 ppm (5OH)[21,42] (Figure S3). The 1H NMR of a succinoyl linkage containing PEGylated chrysin shows the peaks of both PEG and chrysin. The peak observed at 3.65 ppm was due to −CH2 of the PEG chain while the peaks corresponding to the protons of chrysin appeared at 6.35–6.52 ppm (6H and 8H), 6.56–6.63 ppm (3H), 7.51–7.89 ppm (2′, 3′, 5′ and 6′H), and 8.22–12.70 ppm (5H and 7OH) (Figure ).

Figure 1

1H NMR spectra of chrysin, PEG4000–chrysin via succinoyl linker (PEG4000–SA–CHRY), and PEG4000–chrysin via cis-aconityl linker (PEG4000–CAA–CHRY).

Similarly, the synthesis of cis-aconityl linkage containing PEGylated chrysin conjugate was confirmed by peaks observed at 3.65 ppm due to −CH2 of the PEG chain and 6.38–6.54 ppm (6H and 8H), 6.65 ppm (3H), 7.53–7.90 ppm (2′, 3′, 5′ and 6′H), and 12.70 ppm (7OH) of chrysin. In FTIR analysis, pure PEG4000 shows characteristic peaks at 3456 cm–1 (−OH stretching), 2829 cm–1 (C–H stretching), 1286–1112 cm–1 (C–O stretching), and 951 and 844 cm–1 (C–O bending).[43] The FTIR peaks observed at 1736 cm–1 confirmed the formation of PEG4000–SA while the peaks at 1964, 1576, and 1467 cm–1 confirmed the formation of PEG4000–CAA. The pure chrysin molecule shows the characteristic peaks at 2727–2633 cm–1 (C–H stretching), 1650–1606 cm–1 (>C=O stretching), 1573–1446 cm–1 (C=C stretching), and 1164–1032 cm–1 (C–O–C stretching).[44] The succinoyl linkage containing PEGylated chrysin was confirmed by observed FTIR peaks at 2881 cm–1 (C–H stretching of PEG), 1734–1646 cm–1 (>C=O stretching of ester bond and chrysin), 1569–1464 cm–1 (C=C stretching of chrysin), and 1144–1062 cm–1 (C–O–C stretching of chrysin and PEG). Similarly, the cis-aconityl linkage PEGylated chrysin conjugate shows the peaks at 2881 cm–1 (C–H stretching of PEG), 1733–1651 cm–1 (>C=O stretching of ester bond and chrysin), 1562–1469 cm–1 (C=C stretching of chrysin), and 1149–1060 cm–1 (C–O–C stretching of chrysin and PEG) (Figure S4). The UV–vis spectra of both PEGylated chrysin conjugates exhibited a shift in λmax of chrysin (Figure S5). The observed λmax for chrysin, PEG4000–SA–CHRY, and PEG4000–CAA–CHRY was 266, 279, and 285 nm, respectively.

Physicochemical Characterization of PEGylated Chrysin Nanoparticles

Critical micelle concentrations (CMC) of prepared PEG4000–chrysin conjugates are shown in Figure a. The ratio of fluorescence intensities at 383 and 373 nm (I383/I373) was plotted against the concentration of conjugates to determine their CMC values. The fluorescence intensity increased suddenly and sharply at a concentration of 300 μg/mL of the conjugates, indicating the formation of micelles by the PEG4000–chrysin conjugates. The same CMC values of both conjugates suggested that there was no significant role of linkers in the formation of PEGylated nanoparticles. Therefore, the observed results also depict the role of PEG4000 in the formation of stable and compact nanoparticles of chrysin. Figure b shows the predictive model representing a possible arrangement of chrysin, linkers, and PEG4000 molecules in self-assembled PCNP formed by PEG4000–chrysin conjugates. The observed chrysin content was found to be 4.05% and 4.11% in PCNP-1 and PCNP-2, respectively (Figure S6). The TEM images and particle size distribution of PCNP-1 (Figure c,d) and PCNP-2 (Figure e,f) show the formation of spherical nanoparticles with a particle size of about 60 nm. The hydrodynamic diameter and surface charge of nanoparticles were measured by DLS technique (Figure S7). The particle size, polydispersity index, and zeta potential values were 77.3 nm, 0.173 ± 0.003, and −4.25 mV for PCNP-1 and 75.5 nm, 0.185 ± 0.040, and −3.47 mV for PCNP-2, respectively. These results suggested the formation of nanosized and monodisperse particles.

Figure 2

Characterization of PEGylated chrysin nanoparticles (PCNPs): (a) critical micelle concentration of different PEGylated chrysin conjugates; (b) a predictive model of PCNP formation; (c) TEM image and (d) particle size distribution histogram of succinoyl linkage containing PCNP (PCNP-1); (e) TEM image and (f) particle size distribution histogram of cis-aconityl linkage containing PCNP (PCNP-2); (g) DSC spectra of pure chrysin, PEG4000, PCNP-1 and PCNP-2; and (h) XRD patterns of pure chrysin, PEG4000, PCNP-1, and PCNP-2.

The thermal behavior and physical state of chrysin after formation of the PCNPs were confirmed by DSC and XRD analysis. Figure g shows the DSC scans of chrysin, PEG4000, PCNP-1, and PCNP-2. The pure chrysin shows a sharp endothermic peak at 289 °C[45] while pure PEG4000 shows an endothermic peak at 64 °C, corresponding to their melting points. The PCNP-1 and PCNP-2 show endothermic peaks at 55 and 59 °C, respectively, because of the presence of PEG4000. However, the typical endothermic peak of pure chrysin was not observed in the scans of both PCNP-1 and PCNP-2, which specifies the phase conversion of chrysin. This change in the physical state of chrysin was further confirmed by PXRD analysis (Figure h). The XRD pattern of pure chrysin shows sharp peaks at 2θ values 7.2°, 12.2°, 13.2°, 14.7°, 17.5°, 20.3°, 22.3°, 24.6°, and 27.4° because of the crystalline nature of chrysin.[45] The pure PEG4000 shows three peaks at 2θ values 13.8°, 19.2°, and 23.3°. The PCNP-1 and PCNP-2 show the 2θ values at 18.8° and 23.05°, and 18.4° and 22.7°, respectively. So, because no sharp peaks of pure chrysin were observed in the XRD spectra of PCNP-1 and PCNP-2, the formation of amorphous nanoparticles was confirmed.[35]

PCNP Releases Chrysin in a pH-Dependent Manner

To evaluate the effect of succinoyl and cis-aconityl linkers on the release of chrysin from PCNPs, PCNP-1 and PCNP-2 were put into dialysis bags and dialyzed against SAB (pH 5.0), PB (pH 6.5), and PBS (pH 7.4). As shown in Figure a, the amount of chrysin released from PCNP-1 and PCNP-2 was 23.18 and 20.18%, respectively, after 24 h at pH 7.4. The chrysin release was 42.13% and 46.98% from PCNP-1 and PCNP-2, respectively, at pH 6.5. Hence, at the slightly acidic pH of the tumor microenvironment, the amount of chrysin released was higher than at the physiological pH 7.4. However, the chrysin release was about 60% in the first 3 h at pH 5.0 from both PCNP-1 and PCNP-2. Further, more than 90% of chrysin was released from PCNP-2 at pH 5.0 in 24 h, while 72.9% of chrysin was released from PCNP-1. As expected, a higher release of chrysin was observed at the lower pH 5.0 while a slow chrysin release was observed at pH 7.4. However, at pH 5.0 the accelerated chrysin release was observed because of the rapid hydrolysis of cis-aconityl linker. Hence, the observed results from this study suggest the effect of the pH-responsive (cis-aconityl) linker on the release of chrysin from the prepared PCNP-2 nanoparticles. Importantly, no burst release of drug was observed at pH 6.5 and pH 7.4 from either PCNP-1 or PCNP-2. Therefore, this pH-sensitive response of PCNP-2 may help in the site-specific release and delivery of chrysin.

Figure 3

(a) Chrysin release patterns of succinoyl linkage containing PCNP (PCNP-1) and cis-aconityl linkage containing PCNP (PCNP-2) in sodium acetate buffer pH 5.0, phosphate buffer pH 6.5, and phosphate buffer saline pH 7.4. (b) Anticancer activity of pure chrysin, PCNP-1, and PCNP-2 against MDA-MB-231 cells after 24 and 48 h of treatment (*p < 0.05, **p < 0.01, ***p < 0.001). (c) Anticancer activity of pure chrysin, PCNP-1, and PCNP-2 against MCF-7 cells after 24 and 48 h of treatment (*p < 0.05, **p < 0.01).

Stability of PCNP

In order to check the stability of PCNP, particle size and drug content were measured at freeze temperature. Figure S8 shows the stability results of PCNP-1 and PCNP-2. Initially the particle sizes were observed as 77 and 75 nm for PCNP-1 and PCNP-2, respectively. After a period of 96 h, the particle sizes were observed as 91 and 78 nm for PCNP-1 and PCNP-2, respectively. Hence, no significant particle size changes were observed for either PCNP, which confers physical stability of the nanoparticles. Similarly, % chrysin content was observed as 91 and 98% over the period of 96 h. Hence, the observed physical as well as chemical stability results clearly confers the stability of the prepared PEGylated nanoparticles.

PCNP Causes Concentration- and Time-Dependent Cytotoxicities for Human Breast Cancer Cells

The cytotoxicity of prepared PCNP was evaluated against MCF-7 and MDA-MB-231 human breast cancer cells. The results of cell viability after treatment with chrysin, PCNP-1, and PCNP-2 at concentrations of 5, 10, 20, 40, 60, 80, and 100 μg/mL are presented in Figure b. It was observed that the PCNP-1 and PCNP-2 showed a significant decrease in the viability of breast cancer cells compared to chrysin in both dose- and time-dependent manner. In addition, PCNP-2 showed a higher inhibitory effect (p value < 0.05) as compared with PCNP-1 or chrysin after 24 and 48 h. The half-maximal inhibitory concentration (IC50) of the pure chrysin, PCNP-1, and PCNP-2 after 24 and 48 h incubation time are presented in Table . The IC50 values (at 24 h) were found to be 52.2, 49.6, and 34.8 μg/mL for pure chrysin, PCNP-1, and PCNP-2, respectively. After 48 h, the IC50 values were decreased to 19.4, 15.2, and 6.2 μg/mL for pure chrysin, PCNP-1 and PCNP-2, respectively. In the case of MCF-7 cells, the IC50 values were 89.2, 48.3, and 35.7 μg/mL after 24 h of incubation with chrysin, PCNP-1, and PCNP-2, respectively (Figure c). The IC50 values were decreased after 48 h of incubation and observed to be 68.4, 44, and 26.1 μg/mL for chrysin, PCNP-1, and PCNP-2, respectively. The results of the cytotoxicity studies depict the potent and significantly enhanced anticancer activity of chrysin-conjugated nanoparticles in comparison to pure chrysin. The values of IC50 also suggest that PCNP-2 had comparatively higher cytotoxicity than PCNP-1.

Table 1

IC50 (μg/mL) Values for Prepared PCNPsa

time	chrysin	PCNP-1	PCNP-2
MDA-MB-231
24 h	52.2 ± 1.4	49.6 ± 4.5	34.8 ± 3.8*,#
48 h	19.4 ± 1.3	15.2 ± 0.7*	6.2 ± 2.3***,##
MCF-7
24 h	89.2 ± 10.8	48.3 ± 1.6**	35.7 ± 0.5***,#
48 h	68.4 ± 3.7	44.0 ± 1.3**	26.1 ± 3.5***,##

Comparisons with chrysin are denoted with an asterisk (*) symbol while comparisons between PCNP-1 and PCNP-2 are denoted with a hash (#) symbol. Data are the mean ± SD of three replicates. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

Induction of Apoptosis by PCNP

Cytotoxicity of prepared PCNP was further confirmed by an apoptosis induction assay using AO/EtBr staining. This assay helps to check the changes at the cellular level and to differentiate between apoptotic, necrotic, and live cells after treatment with pure chrysin, PCNP-1, and PCNP-2. The AO dye gives green fluorescence after staining the nuclei of the live cells that have an intact cellular membrane, whereas EtBr emits red fluorescence after staining the nuclei of dead cells that have a compromised/damaged cellular membrane. Figure represents the fluorescent images of MDA-MB-231 cells treated with 10 μg/mL concentration of pure chrysin, PCNP-1, and PCNP-2. After 24 h of incubation, MDA-MB-231 cells treated with pure chrysin did not show any significant signs of apoptosis and necrosis.

Figure 4

Apoptotic study by AO/EtBr staining: effect of pure chrysin, PCNP-1, and PCNP-2 on apoptosis induction in MDA-MB-231 cells. Cells were treated with 10 μg/mL of pure chrysin, PCNP-1, and PCNP-2 for 24 and 48 h. Images were captured by a fluorescence microscope. Scale bar = 200 μm.

After 48 h, the cells started to emit red fluorescence that implies chrysin exhibited cell death after 48 h. However, cells treated with PCNP-1 and PCNP-2 showed apoptosis and necrosis after 24 h, which were further increased after 48 h of incubation. The number of cell deaths was higher for PCNP-2 than PCNP-1 or pure chrysin at 10 μg/mL after the 24 and 48 h periods of time. The viable, apoptotic, and necrotic cells were also counted for each group represented in Figure S9. More than 95% of the control cells produced green fluorescent, implying healthy cells. The number of apoptotic and necrotic cells were observed higher for PCNP-2 (33.02 and 62.99%) as compared with PCNP-1 (29.45 and 48.93%) and chrysin (17.92 and 28.85%) after 48 h. The AO/EtBr staining results revealed that PCNP-1 and PCNP-2 can induce apoptosis in a time-dependent manner in MDA-MB-231 cells, which also corroborates the results of the in vitro cytotoxicity assay. Therefore, these findings suggest that prepared PCNP have higher anticancer activity and can induce more apoptosis in MDA-MB-231 cells as compared with pure chrysin.

Colony Formation Assay

To further validate the cytotoxicity and growth inhibitory effects of chrysin and PCNP, the clonogenic assay was performed. As shown in Figure , with a 10 μg/mL concentration of chrysin, PCNP-1, and PCNP-2, the number of colony formations by MDA-MB-231 cells was decreased as compared with untreated control cells. Quantitatively, the colony formation activity of cells was observed to be 31.4%, 7.3%, and 2.9% after incubation with chrysin, PCNP-1, and PCNP-2, respectively. Further, the cells treated with PCNP-1 and PCNP-2 showed fewer colonies in comparison with the cells treated with pure chrysin. Following the patterns observed in the cytotoxicity assay and apoptosis study, PCNP-2 also showed more inhibition than PCNP-1 of colony formation by MDA-MB-231 cells.

Figure 5

(a) The effect of pure chrysin, PCNP-1, and PCNP-2 on colony formation inhibition assay in MDA-MB-231 human breast cancer cells treated with 10 μg/mL for 24 h. (b) Quantitative assessment of colony formation experiments. (**p < 0.01).

Uptake of PCNP-1 and PCNP-2 by MDA-MB-231 Cells

To determine the uptake of the prepared PCNP-1 and PCNP-2 by MDA-MB-231 cells, PCNP-1 loaded with coumarin-6 (C6-PCNP-1) and PCNP-2 loaded with coumarin-6 (C6-PCNP-2) were prepared. Figure shows the fluorescent images of MDA-MB-231 cells after treatment with native C6, C6-PCNP-1, and C6-PCNP-2 at 0.5, 1, 2, 4, and 8 h time intervals. Cells treated with C6-PCNP-1 and C6-PCNP-2 showed higher fluorescent intensity compared with native C6, suggesting that prepared nanoparticles were taken up more than hydrophobic C6. Further, it was found that the uptake of C6-PCNP-2 was faster and higher than the uptake of C6-PCNP-1. The results suggest that the synthesized PCNP could enhance the delivery of hydrophobic chrysin. Thus, increased delivery of chrysin could be responsible for the enhancement of anticancer activity and apoptosis of PCNP-1 and PCNP-2 against MDA-MB-231 cells.

Figure 6

Cellular uptake of coumarin-6 (C6), C6-loaded PCNP-1 (C6-PCNP-1), and C6-loaded PCNP-2 (C6-PCNP-2) in MDA-MB-231 cells after different time intervals. Scale bar = 200 μm.

Conclusion

In the present work, pH-sensitive PEGylated chrysin nanoparticles (PCNPs) were prepared and evaluated for their anticancer activity against MDA-MB-231 human breast cancer cells. The conjugation of chrysin with PEG via different pH-sensitive groups was confirmed by FTIR and 1H NMR analysis. The prepared conjugates were self-assembled into nanoparticles with a particle size of about 60 nm. Both the PCNPs showed a pH-dependent drug release, that is, a faster and more complete drug release at acidic pH than physiological pH. The PCNPs with cis-aconityl linkage showed higher toxicity toward MDA-MB-231 cells as compared with pure chrysin or succinoyl linked PCNPs. Further, these PCNPs caused induction of apoptosis and a decrease in the colony formation ability of breast cancer cells. Therefore, this study highlights the development and significance of PEGylated chrysin as an anticancer agent.