Polyethylene Glycol-Encapsulated Histone Deacetylase Inhibitor Drug-Composite Nanoparticles for Combination Therapy with Artesunate.

Combination drug therapy has become an effective clinical practice for cancer treatment because of low cytotoxicity by the synergistic effect of each medicine. Luminescent Au nanoclusters (Au NCs) were formulated into spherical polyethylene glycol (PEG)-Au NC-encapsulated drug-sodium butyrate (NaB) composite nanoparticles (PEG-Au NC-NaB-NPs) in the presence of PEG and NaB. Their effect on cancer cells was investigated using bio imaging, unravelling the mechanism of the endocytosis pathway and combination therapeutic interventions with a plant-based antimalarial drug artesunate (ART). PEG-Au NC-NaB-NPs showed bright red luminescence in the lysosomal compartment of the cells upon uptake predominantly through a caveolae-mediated pathway. Combination of PEG-Au NC-NaB-NPs with ART displayed enhanced therapeutic activity at a reduced dose compared to its individual doses and revealed heightened synergistic activity as identified from the combination index. The mechanism of synergism revealed elevated generation of reactive oxygen species with both NaB and ART, which disrupts mitochondrial membrane potential as evident from JC-1 staining. Remarkably, the histone deacetylase (HDAC) assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay enlightened the role of NaB and ART in HDAC inhibition and DNA fragmentation, respectively. Thus, induction of apoptosis with the synergistic effect of both NaB and ART with its meticulous mechanism makes it a promising tool for combinational cancer therapy. In vivo activity of the NPs was evaluated on Daltons lymphoma ascites bearing mice, which exhibited significant reduction of tumor volume and viable tumor cells with a prolonged life span.

Introduction

With various treatment options such as surgical intervention, chemotherapy, and radiotherapy for cancer, the most widely used single chemotherapeutic activity (monotherapy) has become less attractive for clinical applications because of its association with many drawbacks, such as high toxicity, nonspecificity, and drug resistance.[1−9] This is mainly because of the physiological heterogeneity of cancers with a complex tumor microenvironment, which harbor abnormalities in multiple signal pathways and are often associated with drug resistance.[8−11] Along with this, the conventional chemotherapeutic drugs like doxorubicin, paclitaxel, and cisplatin are associated with adverse side effects, which induced a paradigm shift toward alternative drugs or combination drug therapy. Combination therapy is the co-administration of two different drugs with different pharmacological mechanisms, which achieves therapeutic synergy efficacy, reduced toxicity, and long-term prognosis.[12−16] Thus, the suitable combinations of drugs are essential to overcome the drug resistance and enhanced therapeutic activity for preclinical and clinical cancer treatment.[6,7,9,13,16] At the same time, use of alternative drugs for combinational therapy as the new effective therapeutics for cancer treatment remains an important endeavour to reduce overall cytotoxicity associated with standard chemotherapeutic drugs.[1,17−19] The shortcomings associated with general administration of different therapeutics (drugs) for combination therapy lies in its distinct pharmacokinetic profiles, leading to inconsistent biodistribution and thus an inefficient therapy. In addition, therapeutic drugs are not directly delivered into targeted tumor sites, which induce adverse effects on normal cells and result in killing of normal cells as well.[4,20,21] Therefore, novel approaches that can overcome the biological barriers and work upon the malignant cells selectively without affecting the normal cells by delivering the optimal dosage of the therapeutics in the complex tumor environment competently are desired.

In the last few years, with advanced understanding of development of cancer, remarkable breakthroughs were unveiled for its diagnosis and treatment in the field of nanomedicine.[4,14,22−25] For the nanoparticle (NP)-based therapeutics, nanocarriers and nanodrugs were poised to significantly improve the aforementioned shortcomings as revealed by some prominent reports, where some were already approved for clinical practices.[4−6,26] The NP-based therapeutics and nanocarriers of various metallic NPs like gold, silver, copper, and silica have many advantages like better solubility, advanced delivery, protection from a tumor microenvironment, and controlled release associated with it.[6,23,27,28] In addition, surface modification of NPs with poly ethylene glycol (PEG) can augment these activities and have improved drug delivery properties and prolonged circulation life time with enhanced protection from clearance by mononuclear phagocytic systems.[29−31] In fact, PEG and many other polymers are formulated into drugs containing NPs[29,32] and have longer in vivo circulation time as well. In this regard, the use of biocompatible luminescent metal nanoclusters (NCs), which has been growing as a promising tool for theranostic applications was mostly anticipated where their luminescence property could also be explored for probing and understanding of the mechanism of uptake and activity.[25,27,28] Metal NCs of Au, Ag, and Cu have been extensively used for drug delivery purposes where Au NCs because of its stability, potentially less toxicity or noncytoxicity, significant luminescence properties, and low photobleaching arose as a new option, overcoming the limits associated with organic dyes and quantum dots.[25,27,33] Thus, we have synthesized polymer PEG-coated nanodrugs by slightly modifying chitosan-based Au NCs as reported earlier[33] with the help of a negatively charged drug sodium butyrate (NaB). As mentioned earlier, because of the drug resistance and side effects associated with standard chemotherapeutic drugs, novel drugs for cancer therapeutics are of intense interest.

As many epigenetic pathways are associated with cancer, one of the prime regulatory mechanisms is the acetylation status of genes controlled by histone acetyltransferases and histone deacetylases (HDACs) for monitoring the gene expression and chromatin structure.[34] At the same time, development of HDAC inhibitors (HDACi) has been received as a promising new class of antineoplastic therapy for anticancer targets, which works by reactivation of tumor suppressor genes.[35,36] One of the common HDACi drugs is sodium butyrate (NaB), which is a short fatty acid chain. NaB is present in the human colon as a product of carbohydrate metabolism and is produced by bacterial fermentation naturally.[37] Moreover, the promising results of NaB as an anti-tumour drug, possibly through inhibition of HDAC, have led to phase I and phase II clinical trials.[38] NaB acting as an HDACi, is known to exhibit anticancer effects via differentiation of carcinoma cells and instigate apoptosis in cancer cells both in vivo and in several pre-clinical models.[35,38] However, HDACi, although being a potent anti-cancer drug, has the limitation of short half-life that is easily metabolized, and therefore, it has better activity in combination with other therapeutic drugs in clinical studies.[36,39] Moreover, there are reports of NaB delivery by solid lipid NPs to overcome the constraints associated with fast metabolism of NaB.[38] Hence, NaB was used for nanodrug formulations for its enhanced delivery and is intended for combination therapy for which a plant-based drug was chosen.

Because of the adverse side effects associated with standard chemotherapeutic drugs, plant-based and naturally available potent anti-cancer agents offer better alternatives and their use for cancer treatment is in demand.[40] In the last few decades, artemisinin, which is a plant derivative from Chinese medical plant Artemisia annua L. (sweet wormwood), has been used as an well-established antimalarial drug.[41] Its semisynthetic derivatives are dihydroartemisinin, artesunic acid, and artemether where artemisinin and sodium artesunate (ART) are water-soluble salts of artesunic acid. The World Health Organization (WHO) have recently recommended use of artemisinin and sodium ART for chemotherapy of humans (artemisinin-based combination therapy, ACT) in combination with conventional antimalarial drugs.[41] Many reports suggest DNA fragmentation leading to DNA damage as a prime reason for anticancer activity of ART.[42] As ART is a prescribed remedy for malarial treatment without any adverse effects and has potent anticancer activity, combination therapy will aid to reduce the overall dose of ART and show enhanced activity along with NaB. There is a report of using ART and NaB together, but detailed understanding of their fate and anticancer mechanism was not well studied.[39] Thus, for combination therapy with ART, a smart theranostic material was desired, having cytotoxic activity with reduced side effects and which can be probed to elucidate the mechanism.

Herein, polymer PEG-coated luminescent NPs were synthesized with Au NCs and the negatively charged drug NaB. NaB and PEG reacted with Au NCs and formulated into spherical polymer drug-encapsulated composite NPs, which is referred to as (PEG–Au NCs–NaB-NPs). The luminescent NPs were used as a probe for uptake studies. Here, to reduce the side effects associated with regular chemotherapeutic drugs, a plant-based drug (ART) with an anticancer property and a naturally available short chain fatty acid (NaB) were chosen for combinational cancer therapy. This resulted in enhanced synergistic therapeutic activity, and the mechanism of synergy was elucidated in detail. To the best of our knowledge, for the first time, polymer-coated HDACi drug-encapsulated composite NPs synthesized herein were studied in detail for combination therapy with ART. The synergy of action of both the drugs, that is, NaB and ART was studied, and the mechanism has been studied meticulously. In vivo studies were also conducted for its efficacy in Swiss albino mice with Dalton’s lymphoma Ascites (DLA) to endorse it for real life applications.[28] The schematic representation of formulation of luminescent PEG–Au NC-coated drug-encapsulated composite NPs and their synergistic combinational therapeutic activity in vitro as well as in vivo highlighting the probable mechanism is elucidated below (Scheme ).

Scheme 1

Schematic Representation of Formulation of Luminescent PEG–Au NCs Drug-Encapsulated Composite NPs (PEG–Au NCs–NaB-NPs) and Their Synergistic Combinational Therapeutic Activity in Vitro and in Vivo Systems

Results and Discussions

The polymer-coated drug-encapsulated NPs were fabricated with red-emitting chitosan-stabilized Au NCs by slightly modifying the previous protocol.[33] The transmission electron microscopy (TEM) image revealed formation of an average of 1.4 ± 0.4 nm of Au NCs (Figure S1a,b, Supporting Information), which were transformed into polymer-coated drug-encapsulated NPs with the help of negatively charged drug sodium butyrate (NaB) and nominally charged PEG molecules. Moreover, the absence of any surface plasmon resonance peak in the visible region rules out the formation of gold NPs (Figure S2, Supporting Information). The formation of spherical polymer-coated drug-encapsulated composite NP (PEG–Au NC–NaB-NPs) has been confirmed with the TEM image (Figure a) with Au NCs embedded on its surface (Figure b), while no characteristic selected area electron diffraction (SAED) pattern for Au metal (Figure c) was observed. The average particle size of the composite NPs was calculated to be 99.62 ± 18.25 nm (Figure d) using ImageJ software. TEM images showing spherical homogenous NPs were also observed (Figure S3, Supporting Information). The particle size obtained herein is greater than the established chitosan TPP nanocarrier with Au NCs (72.1 ± 21.8), mainly because of PEG coating as PEGylation increased the size along with its colloidal stability and reduced the surface charge density.[29−31] The NP formation would take place because of the electrostatic interaction between positively charged chitosan Au NCs and negatively charged NaB, and PEG might have helped in modifying its surface. To corroborate this, potential studies were conducted, which revealed a surface charge of 34.65 ± 8.35 and −5.10 ± 3.24 mV for Au NCs and NaB, respectively, the value of which was 8.97 ± 5.8 mV after polymeric NP formation (Figure S4, Supporting Information). Synthesis of polymeric NPs with chemotherapeutic drugs is well established where PEG has been extensively used for NP formation with some of the known standard drugs (e.g., doxorubicin).[29,32] Polymeric nanocomposites are known to have better drug encapsulation capacity and delivery and help in sustained drug release with prolonged circulation half-life.[43] The as-synthesized PEG–Au NC–NaB-NPs displayed strong red emission at 610 nm (λex = 320 nm) as shown in Figure e and in its inset image. Further, the atomic composition of Au NCs was calculated using the Jellium model using the following equation.[44]

Figure 1

(a) TEM image of PEG–Au NC–NaB-NPs (scale bar 100 nm) with the portion to be magnified is marked in red. (b) Magnified image of the same sample clearly showing embedded Au NCs on its surface. (c) SAED pattern obtained from image in (b) showing no characteristic peaks of Au metal. (d) Histogram showing the particle size distribution of PEG–Au NC–NaB-NPs (as calculated from (b) and from the others images taken from different sets of experiments). (e) Photoluminescence spectra of the as-synthesized Au NCs and PEG–Au NC–NaB-NPs (λex = 320 nm) and the digital photograph in the inset showing bright red luminescence of PEG–Au NC–NaB-NPs under UV transilluminator.

As emission maximum of Au NCs was observed at 610 nm, Eemission was calculated to be 2.032 eV. Further, EFermi of gold is reported to 5.53 eV. Thus, on the basis of eq , N was calculated to be 20.1–20 atoms. The quantum yield of the composite NP was calculated to be 6.8%, using quinine sulphate as the standard and thus can be used for theranostic. The hydrodynamic size of the PEG–Au NC–NaB-NPs was found to be 147.62 ± 10.21, measured with the help of dynamic light scattering (DLS)-based studies (Figure S5, Supporting Information). This increment in size with respect to the size measured from TEM may be due to the presence of chitosan and PEG polymer surrounding the composite NPs.

To determine the percentage of drugs encapsulated on PEG–Au NC–NaB-NPs, binding assay was carried out by probing the luminescence of the as-synthesized composite NPs. Probing the luminescence of the composite NPs is one of the common methods used for enumerating drug binding (%).[27] Here, a negatively charged drug NaB served as an ion-gelating agent for NP synthesis and PEG helped in its encapsulation, which was carried out based on the solvent exchange method and precipitation.[45,46] For this, various concentrations (10–60 mM) of drugs were used for NP synthesis as mentioned in the Experimental Section. After synthesis, the aqueous phase was collected and centrifuged at 10 000 rpm for 10 min. The pellet was carefully mixed in 2 mL Milli-Q water, and pH was maintained at ∼7, and the luminescence was measured (λex = 320 nm and λem = 610 nm). It was found that NP synthesis with 50 mM concentration of NaB retained 65% of drugs in the nanocomposite after which it became saturated (Figure S6, Supporting Information). Thus, dispersion of PEG–Au NC–NaB-NPs (50 mM) has 32.5 mM of NaB in it after encapsulation.

For effective application of this composite NP in theranostics, the size and its luminescence plays a pivotal role. First, the bright luminescence of the PEG–Au NC–NaB-NPs was probed. There are many reports of use of metal NCs for theranostics because of their intense luminescence, enhanced quantum yields, tunable fluorescence emission, large Stokes shift, and good photostability as opposed to conventional dyes, which undergo photobleaching.[27,28] To elucidate its role in internalization and tracking of cancer cells, PEG–Au NC–NaB-NPs were incubated with a cervical cancer cell line (HeLa cells) for 4 h, and confocal laser scanning microscopic (CLSM) studies were carried out. Bright red luminescence was evidently visible inside the cells as shown in Figure b, substantiating their uptake by HeLa cells. This was further confirmed by depth projection (Z-stack) studies which validated their successful internalization inside the cells (Figure c). Moreover, the emission spectrum was also measured inside the live cells (Figure S7, Supporting Information) which corroborate the red emission obtained by the composite NPs. The control cells, that is, cells without treatment with PEG–Au NC–NaB-NPs, did not reveal any luminescence (Figure S8, Supporting Information). This shows the potential of use of PEG–Au NC–NaB-NPs in cancer theranostics.

Figure 2

CLSM images of HeLa cells after incubation with PEG–Au NC–NaB-NPs for 4 h. (a) Bright-field image of HeLa cells. (b) HeLa cells showing distinct red emission because of uptake of luminescent PEG–Au NC–NaB-NPs and (c) Z-stack projection of the cell imaged in (b) confirming the uptake of the NPs.

We have also explored the intracellular localization of the synthesized composite NPs on HeLa cells to find out whether they penetrated the nuclear membrane and entered the nucleus or were localized in the lysosomes. For this, the treated cells were incubated with 2 μg/mL of DAPI (for 10 min) and cytopainter green (for 2 h). The lack of colocalization of the composite NPs (PEG–Au NC–NaB-NPs) with the nucleus marker DAPI was observed after 30 min of incubation as can be seen in the merged image (Figure S9d, Supporting Information), whereas Figure S9a,b in the Supporting Information shows red and blue luminescence of the probe and DAPI, respectively. This was carried out using CLSM with the simultaneous mode where λex and λem of the composite NPs and nuclear marker were fixed in different channels (λex = 405 nm and λem = 620 and 460 nm). Comparatively, noticeable colocalization was observed with cytopainter green that specifically stains the lysosomes. It is to be mentioned here that the cells (i.e., those treated with the composite NPs and without cytopainter green) showed only red luminescence (Figure b), and nothing was observed in the green channel (Figure c). However, the cells treated with the PEG–Au NC–NaB-NPs and cytopainter green clearly revealed colocalization of the probe with cytopainter green in the merged image (Figure h), confirming its presence in the lysosomes. In Figure f, red luminescence represents internalization of the composite NPs, and the green luminescence in Figure g is due to staining of only lysosomes with cytopainter green. Thus, the internalized PEG–Au NC–NaB-NPs were randomly distributed in the perinuclear region and the lysosomal compartments. The samples were analyzed using CLSM with (λex = 405 nm) and (λex = 488 nm) for the composite NPs and cytopainter green, respectively. Chitosan used as a template for synthesis of Au NCs in our system has N-acetylglucosamine units, which have an affinity toward N-linked glycans present on the lysosomal membrane, and this might be the prime reason for its localization in the lysosomes.[47]

Figure 3

Confocal laser scanning microscopy images of HeLa cells showing intracellular distribution of PEG–Au NC–NaB-NPs after 4 h of incubation. (a) Bright-field image of the cell, (b) luminescence observed under the red channel (λex = 405 nm and λem = 610 nm), (c) luminescence observed under the green channel (λex = 488 nm and λem = 520 nm), and (d) merged image of (b,c). CLSM images of HeLa cells incubated along with counter stain cytopainter green. (e) Bright-field image, (f) luminescence observed under the red channel (λex = 405 nm and λem = 610 nm), (g) luminescence observed under the green channel (λex = 488 nm and λem = 520 nm), and (h) merged image of (f,g) showing yellow color, confirming its localization in the lysosome.

Localization of PEG–Au NC–NaB-NPs on lysosomes mainly suggests their internalization through the endosomal pathway as transfer to the lysosomes validates the characteristic transport pathway of NPs internalized by encytotosis.[48] As mentioned earlier, the size of the NP also plays an crucial role in its uptake. NPs with size <200 nm are usually taken by the endocytic pathway, which is mainly divided into clathrin-dependent, caveolae, macropinocytosis, and phagocytosis pathways.[49] To shed light in the cellular uptake mechanism of PEG–Au NC–NaB-NPs, two inhibitors were used, namely, chlorpromazine (50 μM) and methyl-β-cyclodextrin (10 mM), which are known clathrin- and claveolae-mediated endocytosis inhibitors. In this context, HeLa cells were incubated with the respective inhibitors for 1 h prior to their treatment with PEG–Au NC–NaB-NPs for 4 h. Quantitative analysis revealed significant decrease in the luminescence intensity to 15.85 ± 2.9 and 61.22 ± 3.4% with chlorpromazine and methyl-β-cyclodextrin, respectively, from that of control (Figure a). This was further corraborrated with CLSM studies where less uptake of PEG–Au NC–NaB-NPs was observed in chlorpromazine (Figure b(iii))-treated cells as compared with that of PEG–Au NC–NaB-NP-treated cells without inhibitors (Figure b(i)). Negligible luminescence was observed in methyl-β-cyclodextrin-treated cells (Figure b(iv)), which emphasizes that the uptake of PEG–Au NC–NaB-NPs was primarily through claveolae-mediated endocytosis.

Figure 4

(a) Quantitative analysis of cellular uptake of PEG–Au NC–NaB-NPs by HeLa cells after 1 h incubation with chlorpromazine (50 μM) and methyl-β-cyclodextrin (10 mM) inhibitors. All the measurements were carried out in triplicates, and luminescence was measured in Tecan (λex = 405 and λem = 610 nm). Here, control stands for HeLa cells treated with PEG–Au NC–NaB-NPs (without inhibitors) for 4 h. (b) Confocal laser microscopy images of HeLa cells treated with PEG–Au NC–NaB-NPs. (i) Luminescence of the cells in the red channel without any inhibitors (λex = 405 and λem = 610 nm), (ii) bright-field image of (i), (iii) luminescence of the cells after pretreatment with chlorpromazine (50 μM for 1 h), (iv) bright-field image of (iii), (v) luminescence of the cells after pretreatment with methyl-β-cyclodextrin (10 mM for 1 h), and (vi) bright field image of (v).

To evaluate the potential therapeutic impact of PEG–Au NC–NaB-NPs and combination therapy with drugs (ART), the cell viability assay with 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) was carried out. The cytotoxic activity was tested on cervical cancer cells (HeLa) with 48 h of treatment. The drug involved herein for combination therapy was NaB, which was encapsulated on the composite NPs (32.5 mM) and in conjunction with ART (100 μM) added separately. First, the cytotoxic effect of NaB and ART were checked individually. After 48 h of treatment with NaB and ART, MTT assay exhibited cell viability of 43.7 and 40.23% at 11.1 mM and 51.3 μM concentration of NaB and ART, respectively (Figure a,b). To calculate the IC50 values, a nonlinear regression curve fit using Graphpad Prism software was used with which the IC50 values were calculated to be 10.52 mM for NaB and 51.40 μM for ART. The cytotoxic activity of control Au NCs showed viability of 87.38% (Figure S10, Supporting Information), whereas after synthesis of PEG–Au NC–NaB-NPs, the viability was found to be 22.24%, which is possibly because of encapsulation of the drug NaB (11.1 mM). The IC50 value for PEG–Au NC–NaB-NPs was found to be 8.69 mM with respect to NaB, which is lower than IC50 dose of control NaB (10.52 mM) as observed in Figure c. For combinational therapy, different ratios of PEG–Au NC–NaB-NPs (32.5 mM) and ART (100 μM) were used, and it was found to have enhanced activity. It is to be mentioned here that after several trials, PEG–Au NC–NaB-NPs of a fixed concentration (1.8 mM) were given to all the cells. With the fixed concentration of PEG–Au NC–NaB-NPs, varied concentrations of ART (4.4, 8.8, 13.3, 17.7, and 22.2 μM) were added. The results after 48 h of treatment exhibited significant reduction in the cell viability percentage (Figure d). The IC50 value for the combination therapy of PEG–Au NC–NaB-NPs with ART was calculated to be 1.8 mM and 17.32 μM for NaB and ART, respectively, which were prominently less than individual IC50 doses of NaB (10.52 mM) and ART (51.40 μM). At the same time, the cytotoxicity was also checked in the normal lung cell line L132 where at the highest concentration of combinational dose, 69% of cells were found to be viable (Figure S11, Supporting Information). Thus, the combination therapy helped to use drugs at a lower dose, which reduces the cytotoxic activity of its individual doses; however, it enhances the efficacy, suggesting the augmentation of synergistic activity. To establish the synergistic effect between PEG–Au NC–NaB-NPs and ART, the isobologram and combination index (CI) were calculated by using the CalcuSyn software (Biosoft, Version 2.1). The isobologram plot can be explained based on the median-effect equation (Chou) and the CI theorem (Chou-Talalay). The CI values (CI values) were used to characterize as synergy (CI < 1), additivity (CI = 1), and antagonism (CI > 1).[50] The isobologram plot of combination therapy of PEG–Au NC–NaB-NPs and ART (Figure d) revealed that the obtained values of all the data points were <1 (CI < 1), which signified drug–drug interactions (Figure e). The CI values for all the combinations are given in Table S1, Supporting Information. Hence, combination therapy with NaB and ART augmented the efficacy with heightened synergistic activity, outweighing the benefits of monotherapy and a prevalent combination therapy associated with many side effects, and it paves a new way for plant-based combinational therapy with naturally available drugs.

Figure 5

MTT assay was carried out to find out the number of viable HeLa cells after 48 h of treatment. All sets of experiments were carried out in triplicates and are represented as the mean ± SD. The ANOVA test was also carried out for each set where “*” (p < 0.05), “**” (p < 0.005), “***” (p < 0.001) and “****” (p < 0.0001) represent statistical significance. (a) Cell viability assay of NaB (50 mM), (b) cell viability assay of artesunte (200 μM), (c) cell viability assay of PEG–Au NC–NaB-NPs (32.5 mM with respect to sodium butyrate), (d) cell viability assay of combination therapy of PEG–Au NC–NaB-NPs–ART, and (e) isobologram analysis and CI values of combination therapy doses. All the values were obtained below the line of additivity to indicate the synergistic effect. Isobologram analysis was calculated using Calcusyn software.

To investigate the mode of impact exerted by combination therapy of PEG–Au NC–NaB-NPs–ART and the possible mechanism of synergistic therapeutic activity, fluorescence-activated cell sorting (FACS)-based experiments were carried out. First generation of reactive oxygen species (ROS) was evaluated by measuring the DCF fluorescence in HeLa cells following treatments with Au NCs, NaB, ART, PEG–Au NC–NaB-NPs, and PEG–Au NC–NaB-NPs–ART. It was found that a trivial amount of ROS was generated with Au NCs only, but significant enhancement of the fluorescence of DCF was observed with NAB, ART, PEG–Au NC–NPs, and PEG–Au NC–NaB-NPs–ART-treated cells from that of the control (Figure S12, Supporting Information). Here, we would like to mention that the maximum shift of the peak for PEG–Au NC–NaB-NPs–ART-treated cells was mainly because of synergistic action of NaB and ART where both the drugs are known to generate ROS.[37,41,42] In ART-treated cells, oxidative cleavage occurs, leading to ROS production.[41] Elevated ROS production with NaB and ART might be associated with dysfunction of the mitochondria.[37,51] To enlighten the fact, mitochondrial membrane potential damage was measured with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide JC-1 dye and observed under CLSM. It was found that the merged image of HeLa cells treated with PEG–Au NC–NaB-NPs–ART primarily showed green luminescence (Figure t) as opposed to that of control that showed red luminescence (Figure d). The primary reason was due to high mitochondrial membrane potential of the control cells (without treatment) that allowed to form J-aggregates and exhibited red emission, whereas in the cells treated with PEG–Au NC–NaB-NPs–ART, they failed to undergo aggregation (because of low mitochondrial membrane potential) and remained in the monomeric form to show green emission. As clearly observed from the merged images, enhanced green luminescence was observed with ART, and PEG–Au NC–NaB-NPs–ART-treated cells (Figure p,t) were mainly due to mitochondrial dysfunction. In cells treated with NAB and PEG–Au NC–NaB-NPs, less dysfunction of mitochondria had occurred (Figure h,i), and hence, the role of ART in mitochondrial damage could be stated here. At the same time, HDAC activity was also checked as NaB is known to induce HDAC inhibition (HDACi), which is an upcoming treatment domain of cancer inhibition.[37,38] For this HDAC assay kit, fluorometric was used for detection of HDAC activity. HDAC activity analysis of HeLa cell lysates treated with NaB (10.52 mM), PEG–Au NC–NaB-NPs (8.69 mM with respect to NaB), and PEG–Au NC–NaB-NPs–ART (1.8 mM of NaB and 17.32 μM of ART) revealed significant decrease in the luminescence intensity as compared with that of untreated HeLa cell lysate luminescence (Figure S13, Supporting Information). Hence, the role of NaB in HDACi might have contributed in enhanced synergistic activity.

Figure 6

CLSM images of control HeLa cells along with JC-1 dye. First row: (a) bright-field image, (b) red channel, (c) green channel, and (d) merged image of (a–c). Second row: Hela cells treated with NaB (10.52 mM). (e) Bright-field image, (f) red channel, (g) green channel, and (h) merged image of (e–g). Third row: Hela cells treated with PEG–Au NC–NaB-NPs (8.69 mM). (i) Bright-field image, (j) red channel, (k) green channel, and (l) merged image of (i–k). Fourth row: Hela cells treated with ART (51.40 μM). (m) Bright-field image, (n) red channel, (o) green channel, and (p) merged image of (m–o). Fifth row: HeLa cells treated with PEG–Au NC–NaB-NPs–ART. (q) Bright-field image, (r) red channel, (s) green channel, and (t) merged image of (q–s).

Further, the effect of combination therapy on cell cycle progression of HeLa cells was also examined by flow cytometric analysis with PI staining, where arrest in the G0/G1 phase of the cell cycle was observed in the case of cells treated with PEG–Au NC–NaB-NPs–ART. The cell population (%) in the G0/G1 phase after treatment with PEG–Au NC–NaB-NPs–ART was considerably higher (76.7%) than that of the cells treated with individual drugs NaB (53.64%) and ART (68.47%) (Figure S14, Supporting Information). This can be attributed to DNA damage caused by ART, which was more in the case of combinational therapy. Additionally, terminal deoxynucleotidyl transferase (TdT) dUTP nick end labelling assay (TUNEL assay) was also performed, which is a method employed for detection of apoptotic DNA fragmentation and is a hallmark of late apoptosis. In this, the addition of bromolated deoxyuridine triphosphates (Br-dUTP) takes place on the broken or fragmented 3′-hydroxyl (OH) termini of DNA and is catalyzed by TdT enzyme. These were analyzed by staining the cells with a fluorescein isothiocyanate (FITC)-labeled anti-BrdU mAb and were observed under flow cytometry in the green channel. From the histogram obtained (Figure S15, Supporting Information), it was evident that there was a shift in green fluorescence intensity in the PEG–Au NC–NaB-NP-treated cells, which is proportional to fragmented DNA. After 48 h of treatment with PEG–Au NC–NaB-NPs, 25.46% of TUNEL-positive cells were found, whereas a negligible amount of TUNEL-positive cells of 5.78% was found in the case of PEG–Au NC–NaB-NPs that indicates the role of ART on DNA damage. To ensure apoptosis as the mechanism of cell death, FACS-based Annexin V-7AAD assay was performed where a considerable higher percentage (52.73%) of apoptotic cells were obtained after treatment with an IC50 dose of PEG–Au NC–NaB-NPs–ART followed by observation of 34.64, 27.18, and 14.24% of apoptotic cells following treatment with ART, PEG–Au NC–NaB-NPs, and NaB, respectively (Figure ).

Figure 7

FACS-based assay with annexin-V-7-AAD stained HeLa cells. The dot plots showing progression of cell death are divided into four quadrants, where quadrant 2 and 3 represent early and late apoptotic cell population, respectively. (a) Control cells. Annexin-V-7-AAD-stained HeLa cells after treatment with (b) Au NCs, (c) NaB, (d) PEG–Au NC–NaB-NPs, (e) ART, and (f) PEG–Au NC–NaB-NPs–ART.

Finally, the efficacy of combination therapy observed in vitro was tested in vivo mice bearing DLA tumor. For induction of tumor, adult male Swiss albino mice were used where subcutaneous inoculations of DLA cells were performed in mice belonging to group G-II to group G-VII. Following inoculation, the size of tumour growth (belly swelling) was monitored for 8 days that were used for various experiments. First, the uptake of PEG–Au NC–NaB-NPs on DLA cells was measured for the use of these materials on the DLA model. The CLSM studies revealed the uptake of PEG–Au NC–NaB-NPs (red luminescence) upon incubation for 4 h (Figure S16, Supporting Information) and thus, the DLA model was chosen for determining the potent therapeutic activity with combination therapy. The toxicity effects of PEG–Au NC–NaB-NPs and ART were pursued, which showed the absence of deleterious effects on animal health at 20 mg/kg (NaB and ART) till 14 days without any mortality. As no observed adverse effect level of PEG–Au NC–NaB-NPs and ART was observed, 1/10th of its doses (2 mg/kg for NaB and 2 mg/kg for ART) were used for cytotoxic assessment and tumor suppression activities for in vivo experiments. The Swiss albino mice used for the experiments were first distributed into seven distinct groups (n = 10) for respective treatments which includes a control group as well. As already mentioned for DLA tumorogenesis (tumour growth with swollen belly) that is, after 8 days of inoculation, treatment with the nonlethal dose was given in the peritoneal cavity of the mice at fixed intervals (every 24 h). The nonlethal doses of Au NC (2 mg/mL), NaB (2 mg/kg), PEG–Au NC–NaB-NPs (2 mg/kg), ART (2 mg/kg), and PEG–Au NC–NaB-NPs–ART (2 mg/kg) were given, and after completion of 8 days of drug treatment, the trypan blue cell viability test was conducted for the cells collected from the mice (on the 16th day). The results, as shown in Table S2 in the Supporting Information, revealed that combination therapy with PEG–Au NC–NaB-NPs and ART treatment resulted in significant reduction in the percentage of viable DLA cells. Interestingly, PEG–Au NC–NaB-NPs have shown distinct enhanced therapeutic activity in contrast with other treated groups (NaB and ART only). The viable numbers of DLA cells were similar in the case of Au NC-treated cells to those of control cells showing almost no toxic effects of Au NCs. At the same time, prominent reduction in tumor volume was observed in both PEG–Au NC–NaB-NP- and PEG–Au NC–NaB-NPs–ART-treated mice, which was confirmed by the body weights measured on respective days (0, 8th, and 15th day) as in Figure a. It can be inferred from Figure S17, Supporting Information, that treatment with PEG–Au NC–NaB-NPs and combination therapy of PEG–Au NC–NaB-NPs with ART showed clear potential in tumor volume reduction (Figure S14d,f). Among all the treatments with PEG–Au NC–NaB-NPs and in combination therapy of PEG–Au NC–NaB-NPs with ART showed less increment in body weight 2.44 ± 0.36 and 1.85 ± 0.31 g, respectively, as calculated from the initial body weight of the mice. There was an upsurge in body weight of DLA-induced mice (11.28 ± 1.62 g) and Au NC-treated group (10.52 ± 1.52). The results are illustrated in Table S2 in the Supporting Information.

Figure 8

(a) Effects on body weight of tumor-bearing mice as monitored till 16 days following treatment with Au NC, NaB, PEG–Au NC–NaB-NPs, ART, and PEG–Au NC–NaB-NPs–ART. (b) Survival rate of DLA-induced mice monitored up to 50 days after treatment with Au NC, NaB, PEG–Au NC–NaB-NPs, ART, and PEG–Au NC–NaB-NPs–ART, as represented by the Kaplan–Meir curve.

The enhanced therapeutic activity with combinational therapy can be attributed to synergistic activity of NaB and ART, where PEG-encapsulated drug composite NPs provide a steric barrier, which prevents NP opsonization, thereby increasing the blood circulation time and avoiding nonspecific cellular uptake. Thus, it helps in EPR and sustained drug release. The increased life span of DLA mice was found in PEG–Au NC–NaB-NPs–ART-treated groups that emphasized superior therapeutic activity in combinational treatment (Figure b). All the mice died within 17th and 18th days of post-tumour development in the case of control and Au NC-treated group (Figure b). It is to be noted here that there was upsurge in the mean survival time (MST) in the case of NaB- and ART-treated groups, where mice died on 39th, 40th, and 47th day (3 out of 5) and on 43rd and 46th day (2 out of 5), respectively. In an allied vein, interestingly, having potent therapeutic activity PEG–Au NC–NaB-NPs and PEG–Au NC–NaB-NPs–ART resulted in increasing the survivability of mice in the course of study (50 days), where systemic toxicity of NaB and ART was significantly reduced as observed in Figure b.

For haematological parameters, increase in WBC counts was observed in DLA-induced mice with reduced RBC and Hb counts (Table S3, Supporting Information). At the same time, similar effects were observed on Au NC-treated groups. Treatment with NaB, PEG–Au NC–NaB-NPs, and ART also showed elevated WBC counts, but less than only DLA-induced mice (i.e., without treatment). The RBC and Hb counts though had improved in these groups, but they still were in the lower range. Importantly, treatment with PEG–Au NC–NaB-NPs–ART indicated better outcomes with counts almost close with that of normal levels of WBC, RBC, and Hb as shown in (Table S3, Supporting Information). To evaluate the hepatotoxicity in DLA mice after various treatment conditions, the liver function tests were carried out. It was found that there was a rapid elevation of liver function marker enzymes (SGOT, SGPT, and ALP) in the case of DLA-induced mice as compared to that of normal mice (Table S4, Supporting Information). The treatment with Au NCs also revealed enhanced liver function marker enzymes. Though treatment with NaB and ART assisted in reduction of these enzymes to some extent, it was still in a higher range. However, treatments with PEG–Au NC–NaB-NPs and PEG–Au NC–NaB-NPs–ART showed substantial reduction and were almost near to the levels of control animals (without DLA). The details of the obtained liver function marker enzymes after various treatments are included in Table S4, Supporting Information.

Further histopathological studies were also carried out in the kidney of DLA-induced mice that resulted in tubular congestion, glomerular atrophy, and deformed epithelial cells along with inflammatory cell infiltrates (Figure a). Administration of Au NCs failed to ameliorate the pathological condition to that of DLA animals (Figure b). Intraperitoneal administration of NaB and ART exhibited pronounced improvement in renal pathology where reduced occurrences of glomerular and blood vessel congestion were recorded (Figure c,e), whereas PEG–Au NC–NaB-NPs and PEG–Au NC–NaB-NPs–ART treatment ensured the significant protection from DLA-induced renal deformities (Figure d,f), where pronounced reduction of epithelial desquamation, tubular congestion, and hyperaemia of the medullary part of kidney resembling the normal renal physiology of mice was observed. Additionally, liver histology analysis portrayed the pathological changes of liver in DLA mice. Abnormal hepatocytes along with hepatic fibrillation, hemorrhage, polymorphic neutrophil infiltration, and perinuclear clumping of the cytoplasm were observed in DLA mice, whereas similar changes were observed on Au NC treatment (Figure g,h). In contrast, NaB, ART, PEG–Au NC–NaB-NPs, and PEG–Au NC–NaB-NPs–ART (Figure i–l) treatment demonstrated significant protection with normal hepatocellular ultra-structures. Among all the treatments, the PEG–Au NC–NaB-NPs–ART (Figure l) was found to be the best in combating hepatic damage during the DLA condition.

Figure 9

Histopathology of the kidney and liver collected on the 17th day from different treatment groups (a,g), DLA mice without any drug treatment; (b,h), DLA mice with Au NC treatment; (c,i), DLA mice with NaB treatment; (d,j), DLA mice with PEG–Au NC–NaB-NP treatment; (e,k), DLA mice with ART treatment; and (f,l) and DLA mice with PEG–Au NCs–NaB-NPs–ART treatment.

Conclusions

In brief, spherical PEG-coated luminescent drug-encapsulated composite NPs (PEG–Au NC–NaB-NPs) were synthesized in the presence of negatively charged sodium butyrate as an ion-gelating agent. The internalization of the luminescent PEG–Au NC–NaB-NPs in Hela cells occurred through receptor-mediated endocytosis as was evident from the experiments with specific inhibitors. The work emphasized the use of combination therapy with a plant-based antimalarial drug ART and small fatty acid chain (NaB) to enhance therapeutic activity. The detailed studies on the mechanism of synergy of ART and NaB revealed enhanced generation of ROS, which resulted in depolarization of mitochondria. At the same time, the work highlighted the role of ART and NaB in DNA damage and HDACi, resulting in cell death. Our findings were well supported with in vivo studies to decipher synergistic therapeutic activity and provided a promising combination platform for an effective cancer therapy.

Experimental Section

TEM Analysis

TEM analysis was carried out to find out the size and morphology of the as-synthesized gold NCs (Au NCs) and PEG–Au NC-encapsulated drug NPs (PEG–Au NC–NaB-NPs) by using JEOL, JEM 2100 TEM (Peabody, MA, USA), which operates at the maximum accelerating voltage of 200 kV. For analysis, the as-synthesized Au NCs were diluted in 1 mL of water and TEM was conducted. For PEG–Au NC–NaB-NPs, first, 1 mL of it was taken and centrifuged at 10 000 rpm for 5 min. The supernatant was discarded, and the pellet was dispersed in autoclaved Milli-Q water (1 mL). Thereafter, it was diluted in a 1:10 ratio with autoclaved Milli-Q water, from which 8 μL of the PEG-AuNC-NaB-NPs was drop-cast onto a copper-coated TEM grid. The sample was left overnight for drying before analysis.

UV–Visible and Fluorescence Spectroscopic Measurements

UV–vis spectra of the composite NPs were recorded using a PerkinElmer Lamda 25 UV–vis spectrophotometer. For fluorescence measurements, Fluorolog-3, Horiba Jobin Yvon, Edison, NY, USA was used.

DLS-Based Measurements

The Malvern Zetasizer Nano ZS-90 instrument was used to measure the hydrodynamic diameter of the PEG–Au NC–NaB-NPs and their net surface charges before and after drug encapsulation. All experiments were performed at a temperature of 25 °C and with a fixed run time of 11 s where the scattering angle was fixed at 90°. For sample preparations, a ratio of 1:10 (Au NC–NPs/H2O) was maintained, and experiments were conducted in triplicates in three different sets. Data analysis was carried out using the Malvern DTS 5.10 software.

Synthesis of PEG-Coated Composite NPs of Chitosan–Au NCs–NaB

For the synthesis of PEG-coated composite NPs, first, Au NCs were synthesized by slightly modifying the previous protocol. Thus, 1.5 mL of 0.5% (w/v) chitosan was added in 8 mL of H2O in a reaction vessel under constant stirring. Into this, 180 μL of 10 mM HAuCl4 and 60 μL of 0.11 M MPA were added and stirred for 10 min. Chitosan used as a template was first prepared by dissolving 0.5% (w/v) of chitosan in 0.1% (v/v) acetic acid and kept for overnight stirring, which was then filtered by using Whatman filter paper to remove the residuals. The pH of the chitosan solution was adjusted at 6.2 prior to synthesis of Au NCs. For the synthesis of the composite NPs, 50 mM NaB (negatively charged drug), which served as an ion-gelating agent was added into 2 mL of Au NC solution. For the coating of PEG, 200 μL of PEG-400 was taken in 1 mL of chloroform and added drop-wise into the as-synthesized Au NC solution under probe sonication (50 amplitude) in an ice cold condition for 10 min. Thus, PEG-coated drug-encapsulated composite NPs (PEG–Au NC–NaB NPs) were formed in the aqueous phase, which was collected carefully and centrifuged at 10 000 rpm for 10 min. Finally, the precipitate was re-dispersed in 2 mL phosphate buffered saline (PBS) solution (pH 7.4) and stored at 4 °C for further use.

Drug Encapsulation Efficiency

The encapsulation efficiency of the drug was calculated based on the luminescence of Au NCs.[27] For this, in a fixed concentration of Au NCs (v/v), varied concentrations of the NaB drug (10, 20, 30, 40, 50, and 60 mM) were added and incubated for 3 h. This was followed by PEG coating and drug-encapsulated NP synthesis with the help of a probe sonicator as already mentioned. The aqueous phase was collected followed by centrifugation, and the precipitate was re-dispersed in water. To find out the drug encapsulation efficiency, the luminescence of Au NCs was probed using fluorescence spectrophotometer LS55 PerkinElmer, at excitation and emission wavelengths of 320 and 610 nm, respectively. Therefore, the luminescence of control Au NCs (without drugs) was measured and Au NCs (with different concentration of drugs) was calculated as followswhere Au NCsi refers to luminescence intensity of control Au NCs (without drug) and Au NCsf refers to luminescence intensity of Au NCs after drug binding (10–60 mM).

In Vitro Experiments (Mammalian Cell Culture)

All experiments were carried out in human cervical cancer cell lines (HeLa), which were procured from National Centre for Cell Sciences, Pune. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, high glucose) along with 10% FBS, 10 000 units of penicillin and 10 mg/mL streptomycin. The cultured cells were maintained in a CO2 incubator with 5% carbon dioxide in humidified atmosphere.

Cell Viability Assay

The cytotoxic activity of composite NPs along with various controls was carried out with the help of MTT assay. For this, the treatments with the respective composites and individual drugs were given for 48 h followed by MTT assay. Briefly, 96-well plates were seeded with 10 × 103 cells/well and were allowed to attach for 24 h. Treatments were given for 48 h, in triplicates with varied concentrations of Au NCs, NaB (50 mM), PEG–Au NC–NaB-NPs (32.5 mM) with respect to NaB, ART (200 μM), and PEG–Au NC–NaB-NPs–ART (where NaB concentration is fixed, i.e., 1.8 mM and varied ART concentration, i.e., 100 μM). MTT was added after the treatment period of 48 h in each well and incubated at 37 °C for 3 h which resulted in formazon formation. The formed formazon was dissolved in DMSO and gives purple color having absorbance at 550 nm. The measurements were taken at 550 nm with the background reference measured at 655 nm in a multiplate reader (Tecan) and were calculated based on the following formula

Confocal Laser Scanning Microscopy

Confocal laser microscopy studies were carried out for uptake studies along with inhibition and mitochondrial damage assay using the Zeiss LSM 880 microscope. For the uptake studies, 10 × 103 cells were seeded on a coverslip in 35 mm culture plates and were incubated in a CO2 incubator for 24 h. After attaining the required morphology and confluency, cells were treated with the IC50 dose of PEG–Au NC–NaB-NPs (8.69 mM with respect to NaB) for 4 h. Following incubation, the cells were thoroughly washed with PBS and thereafter fixed with 4% formaldehyde (10 min at 37 °C). The coverslips were slowly taken out from the plate with the help of tweezers and were placed on a glass slide (upside down). The sides of the coverslips were sealed. The prepared samples were observed under Zeiss microscope LSM 880 with λex = 405 nm and λem = 610 nm. For DAPI staining after incubation with PEG–Au NC–NaB-NPs (8.69 mM) for 4 h, cells were thoroughly washed and incubated with DAPI (2 μg/mL) for 10 min followed by fixing. To carry out imaging, the simultaneous mode was used [(λex = 405 nm and λem = 460 for DAPI) and (λex = 405 nm and λem = 610 nm for PEG–Au NC–NaB-NPs)].

Colocalization Study

To confirm the prevalence of PEG–Au NC–NaB-NPs on lysosomes, the cytopainter green lysosome staining kit (Abcam) was used. Similarly, the cells were grown in a coverslip, and following treatment with composite NPs for 4 h, 1 mL of cytopainter green was added and incubated for 1 h. Thereafter, the cells were washed with PBS twice followed by fixing and transferring into glass slides, where the edges were sealed before analysis. The samples were then analyzed; (λex = 405 nm) and (λex = 488 nm) for the composite NPs and cytopainter green, respectively.

Cellular Uptake Mechanism Study

To understand the endocytosis mechanisms of composite NPs, clathrin and claveolar inhibitors were used and luminescence based assays were carried out on a multiple plate reader (Tecan) and using CLSM. The inhibitors used for studying the endocytosis mechanism were chlorpromazine (50 μM) and methyl-β-cyclodextrin (10 mM) for clathrin-based endocytosis and caveolae-mediated endocytosis, respectively. For Tecan-based assay, 10 × 103 cells were grown in 96-well plates, whereas for CLSM studies, cells were grown on a coverslip in 35 mm culture plates for 24 h at 37 °C. After attachment, first, the cells were incubated with the inhibitors for 1 h followed by treatment with nanocomposites for 4 h. Then, the cells were washed with PBS and readings were taken in Tecan (λex = 320 nm and λem = 610 nm), whereas for CLSM studies, after washing, the samples were fixed on glass slides and were analyzed (λex = 405 nm and λem = 610 nm).

JC-1 Staining

Apoptosis involves series of events like release of caspase activators such as cytochrome c, alterations in electron transport, and damage or change of mitochondrial transmembrane potential, which eventually resulted in cellular death. For early stages of programmed cell death, many changes happen in mitochondria like variations in its membrane potential. For this, a membrane-permeant cationic lipophilic dye (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide) (JC-1) is widely used for effective distinction between apoptotic and healthy cells based on mitochondrial health. In normal healthy cells (having high mitochondrial transmembrane potential), JC-1 forms complexes known as J-aggregates in a mitochondrial matrix and gives red fluorescence. However, in apoptotic or unhealthy cells, JC-1 remains in the monomeric form as low mitochondrial transmembrane potential prevents its accumulation in the mitochondria. Therefore, the dye is disseminated throughout the cell, which results in shifting from red (J-aggregates) to green fluorescence (JC-1 monomers), and hence, gives green fluorescence. For this, the cells (10 × 103) were grown in life cell-imaging plates (from Thermofisher), and treatments with an IC50 dose of NaB (10.52 mM), PEG–Au NC–NaB-NPs (8.69 mM), ART (51.40 μM), and PEG–Au NC–NaB-NPs–ART (concentration of NaB is 1.8 mM and ART is 17.32 μM) for 48 h were given. The cells were washed, and phenol red free DMEM medium was added along with incubation with JC-1 dye (2.7 μM) for 10 min, and then life cell imaging was conducted. CLSM was carried out in the simultaneous mode for red (λex = 525 nm) and green emission (λex = 480 nm).

HDACi Assay

HDACi was estimated using the fluorometric HDAC activity assay kit (Sigma), which offers an easy method for the detection of HDAC activity based on enzymatic reaction. For this, cells were incubated with NaB (10.52 mM), PEG–Au NC–NaB-NPs (8.6 9 mM with respect to NaB), ART (51.40 μM), and PEG–Au NC–NaB-NPs–ART, where the NaB concentration is fixed, that is, 1.8 mM and ART concentration is 17.32 μM for 4 h. Following treatment, the cells were lysed with buffer containing 50 mM HEPES, 150 mM NaCl, and 0.1% Triton X-100 supplemented with protease inhibitor cocktail. The obtained cell lysates were then sonicated, and further experiments were performed as per the protocol provided with the kit. The Tecan plate reader (Infinite 200 PRO, Tecan, Switzerland) was used to measure the luminescence intensity with λex = 350 nm and λem = 440 nm.

TUNEL Assay

TdT dUTP nick end labeling (TUNEL) is an established method for determination of DNA fragmentation, which is a characteristic hallmark of apoptosis. In TUNEL assay, Br-dUTPs were added to the 3′-hydroxyl (OH) termini of double- and single-stranded DNA, which is catalyzed by the TdT enzyme. Thereafter, the sites are stained with FITC-labeled anti-BrdU mAb and is identified with the help of a flow cytometer. For this, 10 × 103 cells were seeded in 6-well plates and were allowed to grow for 24 h followed by treatment with Au NCs, sodium butyrate, PEG Au NC–NaB-NPs, ART, and PEG–Au NC–NaB-NPs–ART for 48 h. The cells were collected and fixed with 1% paraformaldehyde and incubated in ice for 30–60 min. For further steps, protocols provided by the manufacturer were followed.

In Vivo Experiments

For animal studies, adult male Swiss albino mice weighing 22–25 g were used, which were obtained from Chakraborty enterprise (1443/PO/b/11/CPCSEA), Kolkata, India. The mice were retained in polypropylene cages at room temperature, and relative humidity maintained at 22 ± 2 °C and 60–70%, respectively, with a 12–12 h light–dark cycle. They were sheltered at Central Animal Facility, Institute of Advanced Study in Science and Technology (IASST), Guwahati, Assam, and standard Rodent pellet diet (Provimi Animal Nutrition India Pvt. Ltd., India) with water ad libitum was provided. The mice were closely monitored for a week to acclimatize. The protocols for various experiments were designed and implemented as per guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India and was approved (IASST/IAEC/2016–17/2301) by the Institutional Animal Ethics Committee (IAEC) of IASST.

In Vivo Tumor (Dalton’s Ascites Lymphoma Cells) Development

DLA cells were acquired from IASST Central Animal Facility and maintained in Swiss albino mice through intraperitoneal (I.P.) injection of viable cells (1 × 106 cells/mice). Abnormal belly swelling and increased body weight were observed within 8–10 days after induction, confirming the growth of tumor.

Animal Grouping and in Vivo Experimental Design

For in vivo experiments, first, the acute toxicity of PEG–Au NC–NaB-NPs–ART was carried out and the doses of NaB and ART were selected from already established results. DLA cells (1 × 106 cells/mL in 0.2 mL of PBS/mice) were injected intraperitoneally on day 0 for the growth of tumor, keeping aside the normal group. After growth of tumor, drug treatments were started (9th day) and were given in the interval of 24 h for 8 days. For the present work, mice were divided in 7 groups with 10 mice in each group as follows:

1st group: Mice with no DLA tumor + no drug treatment.

2nd group: DLA-bearing mice + 0.2 mL PBS (I.P) for 8 days.

3rd group: DLA-bearing mice + Au NCs at 2 mg/kg (I.P) for 8 days.

4th group: DLA-bearing mice + NaB at 2 mg/kg (I.P) for 8 days.

5th group: DLA-bearing mice + PEG–Au NCs–NaB-NPs at 2 mg/kg (I.P) for 8 days.

6th group: DLA-bearing mice + ART at 2 mg/kg (I.P) for 8 days.

7th group: DLA-bearing mice + PEG–Au NCs–NaB-NP–ART at 2 mg/kg (I.P) for 8 days.

Effect of Treatment on Tumor Growth

The mice were monitored and measured daily for body weight changes during the experimental period (30 days). For cell viability assay, cells from all the respective groups were collected after the drug treatment period of 16 days. For biochemical and histopathological analysis, on the 17th day, five mice from each group were sacrificed by decapitation to collect the blood, liver, and kidney. To estimate the MST and percentage increase in life span (% ILS), all the remaining 5 mice from each group were monitored up to 50 days.