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Article Cite This: ACS Omega 2018, 3, 8663−8676

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Au-CGKRK Nanoconjugates for Combating Cancer through T‑CellDriven Therapeutic RNA Interference Suresh Kumar Gulla,†,§,∥ Rajesh Kotcherlakota,†,§,∥ Sahithi Nimushakavi,†,§ Narendra Varma Nimmu,‡ Sara Khalid,‡ Chitta Ranjan Patra,*,†,§ and Arabinda Chaudhuri*,†,§,⊥ †

Division of Applied Biology and ‡Analytical & Mass Chemistry Division, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad 500007, Telangana, India § Academy of Scientific and Innovative Research, CSIR Campus, CSIR Road, Taramani, Chennai 600113, Tamil Nadu, India

ACS Omega 2018.3:8663-8676. Downloaded from pubs.acs.org by 46.148.115.216 on 08/04/18. For personal use only.

S Supporting Information *

ABSTRACT: Numerous prior studies on fighting cancer have been based on using inhibitors of JAK-STAT pathway (signal transducer and activator of transcription 3 (STAT3) inhibitor in particular), a signaling pathway responsible for progression of many types of cancer cells. However, recent studies have shown that STAT3 activation leads to upregulation of program death receptor-ligand 1 (PD-L1, an immune checkpoint protein that plays a major role behind evasion of immune systems by growing tumors) expression levels in tumor cells, leading to enhanced immune suppression. This is why global efforts are being witnessed in combating cancer through use of immune checkpoint inhibitors. Herein, we report on the design, synthesis, physicochemical characterizations, and bioactivity evaluation of novel tumor- and tumor-vasculaturetargeting noncytotoxic Au-CGKRK nanoconjugates (17−80 nm) for combating tumor. Using a syngeneic mouse tumor model, we show that intraperitoneal (i.p.) administration of the AuCGKRK nanoparticles (NPs) complexed with both PD-L1siRNA (the immune checkpoint inhibitor) and STAT3siRNA (the JAK-STAT pathway inhibitor) results in significant (>70%) enhancement in overall survivability (OS) in melanoma-bearing mice (n = 5) when compared to the OS in the untreated mice group. The expression levels of CD8 and CD4 proteins in the tumor lysates of differently treated mice groups (by Western blotting) are consistent with the observed OS enhancement being a T-cell-driven process. Biodistribution study using near-infrared dye-loaded Au-CGKRK nanoconjugates revealed selective accumulation of the dye in mouse tumor. Notably, the overall survival benefits were significantly less (∼35%) when melanomabearing mice were treated (i.p.) with Au-CGKRK NPs complexed with only PD-L1siRNA or with STAT3siRNA alone. The presently described Au-CGKRK nanoconjugates are expected to find future use in therapeutic RNA-interference-based cancer immunotherapy.

1. INTRODUCTION Many of the contemporary cancer treatment modalities including chemotherapy, radiation therapy, surgery, etc. suffer from severe toxic side effects. They not only kill cancer cells but also, due to their nonselective nature, kill noncancerous healthy body cells. Global efforts are being witnessed toward developing tumor-cell-selective treatment strategies for combating cancer. High-affinity ligands for receptors overexpressed on tumor cells are being covalently tethered to the exosurfaces of various biocompatible drug carriers (with loaded drugs) such as biodegradable and injectable polymer-based sustained release microparticles,1−3 cyclodextrin-based systems,4−6 dendrimers,7,8 gold-mesoporous silica hybrid theranostics,9 silkfibroin nanoparticles,10 liposomes,11−17 and metal-based nanoparticles.18,19 The signal transducer and activator of transcription 3 (STAT3) is a transcription factor that plays a pivotal role in tumor cell proliferation20 being constitutively activated © 2018 American Chemical Society

(phosphorylated) in numerous cancer cells. In consequence, STAT3 is emerging as an important target in cancer therapy either through use of RNA interference (RNAi, using STAT3siRNA) or using small-molecule inhibitors of STAT3 phosphorylation such as SU54, WP1066, AG490, curcumin, and analogues of curcumin.20−24 Our immune cells fail to eliminate tumor cells because growing tumors develop strategies to evade our immune system. Such immune evasion happens through immune checkpoint interactions between programmed death receptor 1-ligand 1 (PD-L1) expressed on tumor cells and programmed death receptor 1 (PD-1) on the surface of T-cells (an interaction that inhibits proliferation of cytotoxic T-cells).25,26 This is why unprecedented global efforts are being witnessed in Received: May 18, 2018 Accepted: July 23, 2018 Published: August 3, 2018 8663

DOI: 10.1021/acsomega.8b01051 ACS Omega 2018, 3, 8663−8676

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Figure 1. Synthetic scheme of the CGKRK pentapeptide.

combating cancer through use of immune checkpoint inhibitors.27−31 Recent findings are throwing important new mechanistic insights into why STAT3 inhibitors often fail in fighting cancer. STAT3 activation leads to upregulation of PDL1 expression levels in tumor cells, leading to enhanced immune suppression by tumor cells.32−36 In anti-angiogenic cancer therapy, the nutrient and oxygen supply to tumor cells are shut down by inhibiting angiogenesis, sprouting of new blood vessels (tumor endothelial cells) from pre-existing vessels.14,37−41 In consequence, the tumor cells die out of starvation. A promising anti-angiogenetic cancer therapeutic modality of combating cancer targets potent cytotoxic drugs/ siRNAs selectively to both tumor and tumor endothelial cells by covalent grafting of phage display-study-derived CGKRK ligand onto the nanoparticle surfaces42,43 Taking the above-mentioned upregulation of PD-L1 by STAT3 activation into account, we hypothesized that simultaneous delivery of STAT3siRNA and PD-L1siRNA to tumor tissues using gold-nanoparticle-based delivery systems might be a potent therapeutic RNA interference modality for fighting cancer. Herein, we report on the design, synthesis, physicochemical characterization, and bioactivity evaluation of novel tumor- and tumor-vasculature-targeting noncytotoxic Au-CGKRK nanoconjugates (17−80 nm) for combating tumor. We show that intraperitoneally (i.p.) administration of the Au-CGKRK nanoparticles complexed with both PDL1siRNA (the immune checkpoint inhibitor) and STAT3siRNA (the JAK-STAT pathway inhibitor) results in significant enhancement of the overall survivability (OS) in melanomabearing mice when compared to the OS in the untreated mice group. The expression levels of CD8 and CD4 proteins in the tumor lysates of differently treated mice groups (by Western Blotting) are consistent with OS enhancement for the mice

group treated with both PD-L1siRNA and STAT3siRNA owing to its origin from a T-cell-driven process. The presently described Au-CGKRK nanoconjugates are expected to find future use in combating cancer through therapeutic RNA interference.

2. RESULTS AND DISCUSSION 2.1. Synthesis of CGKRK Ligand. Phage display library derived tumor and tumor endothelial cells targeting CGKRK penta peptide ligand was synthesized using Fmoc strategybased solid-phase peptide chemistry (as shown in Figure 1). The structure of the tumor-/tumor-vasculature-targeted CGKRK pentapeptide was confirmed by high-resolution mass spectrometry (HRMS), matrix-assisted laser desorption/ionization (MALDI), and 1H NMR, and its purity was confirmed by reversed-phase analytical high-performance liquid chromatography (HPLC) using two different mobile phases (Figures S1−S4, Supporting Information). 2.2. Fabrication and Characterization of Au-CGKRK Nanoconjugates. The first step in constructing Au-CGKRK nanoconjugates is synthesis of AuNPs. The characteristic surface plasmon resonance peak at 512 nm confirmed the formation of spherical AuNPs. CGKRK peptide conjugation on the surface of the AuNPs was confirmed by change in the absorbance of the nanoconjugates after incubation with increasing concentrations of the CGKRK peptide. The absorbances of the Au-CGKRK nanoconjugates were observed to be increasing till the concentration of added CGKRK reached 10 μg/mL. Notably, the absorbance of the AuCGKRK nanoconjugates was found to decrease when the concentration of the added CGKRK was more than 10 μg/mL (Figure 2). Thus, 10 μg/mL was taken as the saturation point for the synthesis of Au-CGKRK nanoconjugates (saturation 8664


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protein-conjugated AuNPs.44 The percent of added CGKRK peptide (at the saturation point, 10 μg/mL) conjugated to the surface of the AuNPs was found to be 42% by quantitative HPLC analysis of the supernatant after separating the AuCGKRK nanoconjugates by centrifugation (using the standard HPLC calibration graph for CGKRK, data not shown). 2.3. Size and Surface Potential of the Nanoconjugates. The size and charge of the nanoconjugates were measured by the dynamic light scattering (DLS) method. The hydrodynamic diameter of free gold nanoparticles was found to be 17 nm, which increased to 75 nm upon addition of the CGKRK ligand (Figure 3a). Interestingly, the hydrodynamic diameter of the Au-CGKRK nanoconjugates was found to be significantly decreased (42 nm) upon complexation with siRNA presumably due to electrostatic compaction of the positively charged Au-CGKRK nanoconjugates induced by the added negatively charged siRNA. The surface charge of the free gold nanoparticles was found to be −21 mV, which increased to +24 mV upon conjugation with cationic CGKRK (Figure 3b). These findings support the notion that electrostatic interaction between the negatively charged bare AuNPs and the positively charged CGKRK ligand might play an important role behind the formation of Au-CGKRK nanoconjugates. The surface charge (ζ potential) of the AuCGKRK nanoconjugates was further reduced to +15 mV upon complexation with siRNA (Figure 3b) possibly due to additional electrostatic interaction between the negatively charged siRNA and the positively charged Au-CGKRK nanoconjugates. 2.4. X-ray Diffraction (XRD) and TEM Analyses of Nanoconjugates. The surface crystallinity of nanoconjugates

Figure 2. Physicochemical charaterization of the Au-CGKRK nanoconjugates by UV−vis spectroscopy. The saturation point was determined by adding increasing concentrations of the CGKRK peptides (2−20 μg/mL) to a fixed concentration of AuNP (0.4 μg/ μL). The saturation was attained at 10 μg/mL CGKRK. The inset shows the picture of bare AuNP (left) and Au-CGKRK nanoconjugate (right).

point is defined as the maximum concentration of the peptide required to saturate the surface of nanoparticles). Such saturation phenomena are routinely studied by observing the increases in the absorbances as well as the increases in the wavelength maxima (popularly known as “red shift”) of the nanoconjugates (compared to those of bare nanoparticles). The increased absorbance of the Au-CGKRK nanoconjugate and the observed red shift at the saturation point (Figure 2) were fully consistent with a recently reported red shift of

Figure 3. Size, charge, and morphological characterizations of Au-nanoconjugates by DLS and transmission electron microscopy (TEM). The sizes (a) and surface charges (b) of bare AuNPs (blue), Au-CGKRK nanoconjugates (black), and Au-CGKRK-scrambled siRNA nanoconjugates (red) were measured by DLS. The morphology of the negatively charged bare AuNPs (c), positively charged Au-CGKRK nanoconjugates (d), and positively charged Au-CGKRK-siRNA nanoconjugates (e) studied by TEM. 8665


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was confirmed by XRD analysis. The similar XRD patterns observed for both Au-CGKRK nanoconjugates and bare AuNPs (Figure S5) clearly showed that the Au-CGKRK nanoconjugates maintained their integrity as to their nanoparticular nature. The data also corroborates with the earlier published literature. The morphology of nanoconjugates was studied by TEM analysis. The electron micrographs depicted in Figure 3c−e suggest spherical morphology for all of the Aunanoconjugates. Importantly, these TEM pictures (Figure 3c− e) revealed the nanoconjugates to be well dispersed in nature as was the case for bare AuNPs (∼5 nm size nanoparticles free from aggregation). It is worth mentioning at this point of discussion that the size of the AuNPs and Au-nanoconjugates measured by the DLS method differed significantly from that measured by TEM analysis. Notably, these two techniques measure different aspects of nanoparticles. DLS measures the hydrodynamic diameter of the nanoparticles in solution, whereas TEM gives the exact size of the dried nanoparticles without the presence of water of hydration. Because the positively charged CGKRK ligand is expected to bring lots of water molecules on the surface of the Au-CGKRK nanoconjugates, the hydrodynamic diameters of these nanoconjugates measured by the DLS method are found to be significantly higher than the nanoconjugate sizes measured by TEM. Stated differently, under systemic settings, water molecules (and possibly other biomolecules) adhere to the surface of Au-CGKRK nanoconjugates forming hydrated NPs, the average sizes of which are obtained by DLS measurements. Thus, the hydrodynamic diameters of metal-based NPs obtained by DLS measurements provide information on the size of inorganic core along with adhered water and other systemic molecules as the particles move under the influence of Brownian motion. However, such hydration layers are not taken into account while measuring sizes of NPs by TEM. In TEM, we obtain information about only the inorganic core size. This is presumably why the presently described AuCGKRK nanoconjugates (with sizes ∼5 nm as measured by TEM and ∼42 nm as measured by DLS) are not cleared by kidney when administered systemically. Such size difference of AuNPs measured by DLS and TEM has also been reported in our earlier study.44 2.5. siRNA Binding, Serum Compatibility, RNase-1 Sensitivity, and Cell Viability Studies. Conjugation of the cationic CGKRK peptide on the surface of AuNPs imparted a positive charge on the surface of the Au-CGKRK nanoconjugates. The siRNA-binding properties of the positively charged Au-CGKRK nanoconjugates were assessed by the native gel-binding assay (GBA) using increasing amounts of added nonsilencing siRNAs and a constant amount (17.8 μg) of Au-CGKRK nanoconjugates. The observed gel pattern revealed 1.5 μg of siRNA as the optimal amount that binds completely with the Au-CGKRK nanoconjugates (Figure 4a). Use of 2.5 μg of siRNA clearly showed the presence of a noncomplexed free siRNA band in the gel (Figure 4a). Notably, whereas 1.5 μg of siRNA was found to be completely bound to 185.8 μg of Au-CGKRK nanoconjugates (containing 17.8 μg of Au), siRNA did not bind significantly even with a high amount (22.3 μg) of bare AuNPs (Figure S6a,b). From this gel-binding assay, we found that 1.5 μg of siRNA was completely bound to 185.8 μg of the Au-CGKRK conjugate. Thus, the loading efficiency of siRNA was 0.8% (w/w).

Figure 4. siRNA binding, serum compatibility, RNase sensitivity, and cellular cytotoxicity profiles of Au-nanoconjugates. The siRNA binding properties of the nanoconjugates (a) were performed using native polyacrylamide gel electrophoresis (PAGE) and using the same concentration of Au-CGKRK (17.8 μg in 40 μL) and varying concentrations of siRNA (0.5−2.5 μg). Complete binding was observed at 1.5 μg of added scrambled siRNA (fourth lane from the right). The increase in the sizes of the Au-nanoconjugates (measured by DLS) with time (0−100 h) were higher in 10% fetal bovine serum (FBS) compared to those in phosphate-buffered saline (PBS) (b). siRNA (1.5 μg) complexed with Au-CGKRK (17.8 μg in 40 μL) was stable in the presence of RNase (0.5 μg/mL) as determined by native PAGE (c). The cellular cytotoxicity profiles by the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in B16F10 cells (d) revealed noncytotoxic nature of the Au-nanoconjugates up to 10 μL of added nanoconjugtes (nanoconjugate stock 446 μg/mL of gold).

Taken together, these findings support electrostatic nature of the binding interactions between the negatively charged siRNA and positively charged Au-CGKRK nanoconjugates. The sizes of the Au-CGKRK + siRNA nanoconjugates were found to be fairly stable in PBS buffer for up to 100 h (40−50 nm, Figure 4b). In the presence of 10% added serum, the sizes of the Au-CGKRK + siRNA nanoconjugates were found to be somewhat increased within the same time period (40−70 nm, Figure 4b). Such less than 100 nm sizes in the presence of added serum indicated in vivo compatibility of the presently described Au-CGKRK-siRNA nanoconjugates. 8666


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Figure 5. FAMsiRNA (green) delivered to melanoma cells (B16F10) with the Au-CGKRK nanoconjugate avoids fusion with lysosome (red), as revealed in the cellular uptake study by confocal microscopy. Cells were incubated with Au-CGKRK-FAMsiRNA nanoconjugates for 30 min, 1 h, 3 h, and 6 h. Lysosomes of the incubated cells were labeled with LysoTracker (red). Merged panels (fourth column) clearly showed that the nanoconjugates avoid lysosomes even at 6 h postincubation. Green fluorescence from FAM-labeled siRNA was monitored using excitation and emission wavelengths of 490 and 520 nm, respectively. Red fluorescence from LysoTracker Red was monitored using excitation and emission wavelengths of 650 and 670 nm, respectively. The cell nucleus was labeled with Hoechst-33258 (exλ 352 nm/emλ 461 nm). Magnification: 40×. Scale bar 50 μm.

The findings in the gel-binding assay (GBA) using native PAGE showed the absence of any free siRNA band (Figure 4c). Notably, uncomplexed naked siRNA was completely destroyed in the presence of RNase-1 (Figure 4c). Collectively, these findings demonstrated strong siRNA-binding characteristics of the presently described positively charged Au-CGKRK nanoconjugates. The cellular cytotoxicities of the bare AuNPs and Au-CGKRK nanoconjugates, if any, were examined by the conventional MTT assay in B16F10 cells. Importantly, both AuNPs and Au-CGKRK nanoconjugates were found to be noncytotoxic even when 10 μL of the nanoconjugates (containing 446 μg/mL of gold) was incubated with cells (Figure 4d). These findings are consistent with priorly disclosed noncytotoxic natures of other gold nanoconjugates.45−49 In the present study, we used the CGKRK peptide that specifically targets the tumor cells. The lesser uptake of nanoparticles in NIH3T3 compared to that in B16F10 cells may be due to the presence of the CGKRK peptide. Recent studies also strengthen our observation that CGKRKconjugated nanoparticles specifically internalize into cancer cells. For example, Sharma et al. developed siRNA delivery using fatty acyl-CGKRK peptide conjugates. The authors

demonstrated that these conjugates specifically internalize into cancer cells due to the presence of CGKRK.50 2.6. Cellular Uptake and Subcellular Localization Studies Using Confocal Microscopy. With a view to examine the degree of cellular uptake as well as subcellular localization for the Au-CGKRK-siRNA nanoconjugates in tumor (B16F10) cells, we performed a time-dependent cellular uptake experiment using confocal microscopy. In this experiment, we used a fluorescently (green) labeled FAMsiRNA and we prelabeled the lysosomes of the cells with a commercially available acidotropic LysoTracker (red) dye. Cell nuclei were labeled with Hoechst-33258 (blue). The absence of any significant yellow color even at 6 h post nanoconjugate incubation with cells (Figure 5) revealed the lysosome escaping efficacy of the presently described Au-CGKRKsiRNA nanoconjugates. Presumably, siRNA was released rapidly from the endosomally uptaken Au-CGKRK + siRNA nanoconjugates. Such a rapid release of endosomally trapped siRNA evading lysosomal fusion has also been observed by other groups.51−53 However, the mechanistic origin of such a fast endosomal release of siRNA into the cell cytoplasm remains elusive at this point of investigation. Because our primary objective was combating tumor using both STAT38667


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Figure 6. Au-CGKRK nanoconjugates deliver siRNA selectively to cancer cells. In vitro cellular uptake experiments were performed by flow cytometry using fluorescently labeled FAMsiRNA (green) in both cancer (B16F10, melanoma) and noncancerous healthy NIH3T3 (mouse fibroblast) cells. Significantly higher cellular uptake was observed only for cancer cells (B16F10) (a) treated with Au-CGKRK-FAMsiRNA (pink) when compared with healthy cells (NIH3T3), (b) treated with the same nanoconjugate. Notably, degrees of cellular uptake were significantly less in both the cells when treated either with CGKRK-FAMsiRNA (without AuNP, blue) or with naked FAMsiRNA (violet).

Figure 7. Nanoconjugates of Au-CGKRK and near-infrared (NIR) dye (Dil, red) and Au-CGKRK and fluorescently labeled siRNA (Cy-siRNA, red), upon i.p. administration in tumor (melanoma)-bearing mice, get selectively delivered to tumor tissues. The tumor-selective biodistribution profiles of the Au-CGKRK and NIR dye nanoconjugates were initially confirmed by noninvasive in vivo imaging of mice (n = 2) at 2 and 24 h i.p. post-administration (a). With a view to further confirm the tumor-selective biodistribution profile, mice were sacrificed after 24 h and different organs were isolated and ex vivo imaged. The representative ex vivo images (b) further confirmed the tumor-selective biodistribution of the i.p. administered Au-CGKRK-NIR nanoconjugates. The tumor tissue accumulation of the i.p. administered Au-CGKRK-cy5siRNA nanoconjugates was confirmed by the confocal image of the fixed 10 μm tumor cryosection (c) using excitation and emission wavelengths of 650 and 670 nm, respectively. The nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI) using excitation and emission wavelengths of 351 and 461 nm, respectively. Magnification 60×. Scale bar 50 μm.

positively charged Au-CGKRK nanoconjugates. Importantly, confocal images of B16F10 cells taken after 4 h of incubation of such nanoconjugates (containing dual siRNAs) revealed colocalization of both the siRNAs (Figure S7). This finding demonstrated dual siRNA delivery efficacy of the Au-CGKRK

siRNA and PD-L1siRNA in complexation with the presently described Au-CGKRK nanoconjugates, we first examined their dual siRNA delivery efficacy in tumor cells. To this end, as a model experiment, we used FAMsiRNA (green) and Cy5siRNA (red) both simultaneously complexed with the 8668


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Figure 8. Intraperitoneally administered Cy5siRNA (red)-Au-CGKRK nanoconjugates get targeted to mouse tumor vasculature. Tumor cryosections prepared 24 h post i.p. administration of the nanoconjugates were stained first with the FITC-labeled CD31 antibody (green, widely used marker for tumor endothelial cells). The fixed cryosections were then observed by a confocal microscope. The nuclei were stained with DAPI. The tumor endothelial cells were imaged using excitation and emission wavelengths of 490 and 520 nm, respectively; Cy5siRNA-Au-CGKRK nanoconjugates were imaged using excitation and emission wavelengths of 650 and 670 nm, respectively. Magnification 60×. Scale bar 50 μm.

nanoconjugates. The findings summarized in Figure S7 demonstrated colocalization of both the siRNAs. Toward confirming the cytoplasmic localization of the siRNA, we performed an additional confocal microscopic experiment for taking the transmitted light differential interference contrast images (TD image) 4 h postincubation of B16F10 cells with the Au-CGKRK + FAMsiRNA nanoconjuate. The TD image depicted in Figure S9 (Supporting Information) clearly demonstrated the cytoplasmic localization of the siRNA. Toward confirming that the nanoparticle-associated siRNA did not get localized within the lysosomes, we have performed an additional confocal microscopy experiment for taking the three-dimensional (3D)-z-stack images of the lysosome-labeled (with commercially available LysoTracker, red) treated cells. The 3D-z-stack images of lysosome-labeled B16F10 cells treated with the Au-CGKRK + FAMsiRNA nanoconjuate (Figure S10) clearly demonstrated that the delivered siRNA did not fuse with lysosomes. 2.7. Cellular Uptake Study by Flow Cytometry. The degree of cellular uptake of Au-CGKRK-FAMsiRNA nanoconjugates was assessed quantitatively by flow cytometry. Both cancer cells (B16F10) and noncancerous healthy cells (NIH3T3) were separately treated with (1) Au-CGKRK + FAMsiRNA nanoconjugates, (2) CGKRK + FAMsiRNA complex (without gold), and (3) naked FAMsiRNA. Results summarized in Figure 6a revealed Au-CGKRK nanoconjugates to be the most efficient in delivering siRNA to cancer cells. Notably, the degree of cellular uptake was significantly less in NIH3T3 cells (Figure 6b), thereby demonstrating tumor-cellselective siRNA delivery efficacy of the Au-CGKRK nanoconjugates. Prior in vivo phage display studies demonstrated that cell-penetrating peptide CGKRK is highly selective for entering tumor and tumor endothelial cells (and not healthy noncancerous cells) presumably via heparan sulfate receptors.50,54 The findings summarized in Figure 6 are consistent with these prior reports. However, in-depth studies need to be carried out in future to gain further mechanistic insights into whether or not poorly expressed heparan sulfate receptor profiles could be playing an important role behind the observed significantly less cellular internalization of the AuCGKRK nanoconjugates in the healthy noncancerous NIH3T3 cells. 2.8. Biodistribution Studies. Tumor-cell-selective siRNA delivery efficacy of the Au-CGKRK nanoconjugates (Figure 6a,b) in the in vitro study described above prompted us to

carry out an in vivo biodistribution study in melanoma-bearing C57BL/6J mice (n = 2) involving i.p. administration of both near-infrared (NIR) dye (Dil)-loaded Au-CGKRK and AuCGKRK + Cy5siRNA nanoconjugates. The representative noninvasive images (PerkinElmer IVIS spectrum animal imager) showed tumor-tissue-selective accumulation of the Au-CGKARK nanoconjugate-associated NIR dye 24 h post i.p. administration (Figure 7a). Next, to further confirm tumorselective in vivo delivery of the Au-CGKARK nanoconjugateloaded NIR dye, we sacrificed the mice 24 h post i.p. administration and recorded the ex vivo images of different organs. The ex vivo images of different organs (Figure 7b) also confirmed the tumor-tissue-selective biodistribution profile of the i.p. administered Au-CGKARK nanoconjugate-loaded NIR dye. Next, with a view to confirm the efficacy of Au-CGKRK nanoconjugate in delivering siRNA to mouse tumor, melanoma-bearing C57BL/6J mice (n = 2) were i.p. administered with Au-CGKRK + Cy5siRNA nanoconjugates. Fixed tumor tissues were cryosectioned (10 μm) 24 h post i.p. treatment. Confocal images of the representative tumor cryosection (Figure 7c) revealed significant accumulation of Cy5siRNA (red) in tumor tissue. 2.9. Tumor-Vasculature-Targeting Property of AuCGKRK Nanoconjugates. Despite significant progresses in the field of therapeutic RNA interference (RNAi) for combating cancer, clinical success of RNAi remains critically dependent on (a) the use of efficient delivery of systemically administered RNAi therapeutics selectively to tumor cells (by protecting them from assault by systemic RNases, by preventing their rapid renal filtration, and by evading their phagocytotic uptake); (b) successful crossing of vascular barrier followed by efficient internalization of RNAi therapeutics in cancer cells; and (c) efficient endosomal escape of therapeutic siRNA payload into the cell cytoplasm form RNA-induced silencing complex.55,56 Large-sized drug-/ siRNA-delivery vehicles are more susceptible to phagocytosis. Presumably, the small size (∼42 nm) of the presently described siRNA and Au-CGKRK nanoconjugates prevented their phagocytosis. Furthermore, this size range, being much larger than the pore size of the glomerular filtration barrier (∼8 nm), is likely to play a key role in preventing their fast renal clearance. The tumor-tissue-selective biodistribution of the siRNA and Au-CGKRK nanoconjugates was accomplished by the use of priorly reported tumor- and tumor-vasculaturetargeting CGKRK ligands in the molecular architecture of the 8669


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Figure 9. Intraperitoneally administered Au-CGKRK + PD-L1siRNA + STAT3siRNA nanoconjugates elicit enhanced overall survivability of melanoma tumor-bearing mice. (a) Relative tumor growth inhibition profiles observed in tumor-bearing mice (n = 5) i.p. treated with 5% aqueous glucose (UT, pink, group I); CGKRK + PD-L1 + STAT3 (without any AuNPs, green, group II); Au-CGKRK (red, without using any therapeutic siRNA; group III); and Au-CGKRK + PD-L1siRNA + STAT3siRNA nanoconjugates (blue, group IV). Arrows underneath the x axis indicate the days of total 5 i.p. injections post-tumor inoculations. The inset shows the average weights of the tumors excised from these differently treated mice groups. ** P < 0.005 compared to untreated control group I. (b) After the last i.p. injection on day 20 post-tumor inoculation, mice were observed for overall survivability (OS) from day 21. Group IV mice treated with Au-CGKRK + PD-L1siRNA + STAT3siRNA nanoconjugates showed significantly enhanced OS compared to that of other mice groups.

nanoconjuate. Because prior studies demonstrated tumor endothelial-cell-targeting properties of the CGKRK ligand,57,58 we carried out an additional immunohistochemical staining study toward examining such tumor-vasculature-targeting property of the presently described Au-CGKRK nanoconjugates. Melanoma-bearing C57BL/6J mice (n = 3) were i.p. administered with Au-CGKRK + Cy5siRNA nanoconjugates. Fixed tumor cryosections (prepared 24 h post i.p. treatment) were immunostained with mAb against CD31 (a widely used marker for tumor endothelial cells) using a fluorescein isothiocyanate (FITC)-labeled secondary antibody. Significant colocalization of Cy5siRNA and tumor endothelial cells (Figure 8) clearly demonstrated the tumor-vasculaturetargeting nature of the Au-CGKRK nanoconjugates. 2.10. Tumor Growth Inhibition Studies. Tumorselective biodistribution profile of the i.p. administered AuCGKRK-NIR nanoconjugate (Figure 7a,b) as well as the dual siRNA delivery efficacy of the Au-CGKRK nanoconjugate under in vivo conditions (Figure S7) finally prompted us to examine its therapeutic promise in inhibiting established tumor through RNAi. To this end, we envisaged that i.p. administration of both STAT3siRNA and PD-L1siRNA in complexation with tumor- and tumor-endothelial-cell-targeting Au-CGKRK nanoconjugate might harness synergistic therapeutic effect in inhibiting established mouse melanoma tumor growth. The rationale behind this hypothesis is based on two important earlier findings: (i) inhibition of STAT3 activation pathways through use of STAT3 inhibitors has been well studied in the past for the treatment of many kinds of cancers; and (ii) STAT3 activation leads to upregulation of PD-L1 expression levels in tumor cells, which in turn leads to enhanced immune suppression.32−36 Thus, simultaneous delivery of both STAT3siRNA and PD-L1siRNA to tumor cells will not only inhibit tumor cell growth but also shut down the immune evasion mechanism due to downregulation of immune checkpoint protein PD-L1. Importantly, significant tumor growth inhibition was observed in established melanoma-bearing C57BL/6j mice (n = 5) upon i.p. administration of Au-CGKRK + PD-L1siRNA + STAT3siRNA nanoconjugates (Figure 9a). Notably, tumor growth inhibition

in mice was not accomplished through i.p. administration of (i) only 5% aqueous glucose (vehicle control group) and (ii) STAT3siRNA and PD-L1siRNA in complexation with the bare CGKRK peptide (Figure 9a). Tumor growth inhibition was found to be significantly less in mice i.p. administered with only Au-CGKRK + STAT3siRNA and only Au-CGKRK + PDL1siRNA nanoconjugates (Figure S8a). As expected, no tumor growth inhibition was observed in mice i.p. administered with only Au-CGKRK nanoconjugates (Figure 9a) and Au-CGKRK + nonsilencing scrambled siRNA nanoconjugates (Figure S8a). Most importantly, the overall survivability (OS) benefit was ∼75% higher in mice treated with Au-CGKRK + PD-L1siRNA + STAT3siRNA nanoconjugates compared to that in all of the control mice groups mentioned above (Figures 9b and S8b). An issue is worth discussing at this point. The in vivo research findings summarized in Figure 9a revealed that free siRNA combinations (group II) were much less efficient in inhibiting tumor growth compared to that by Au-CGKRK (group III), whereas in the in vitro MTT assay (Figure 4d), Au-CGKRK demonstrated almost no cytotoxicity. The dose-dependent in vivo toxicity of AuNPs has been reported.59 A similar slight in vivo toxicity of the presently described positively charged AuCGKRK nanoconjugates (showing nontoxic nature under the in vitro MTT assay) may not be ruled out at this point of investigation. Such slight in vivo toxicity of the Au-CGKRK nanoconjugates may play some role behind the observed difference between the tumor growth inhibition properties of i.p. administered Au-CGKRK nanoconjugates and that of i.p. administered free siRNA and CGKRK combination. 2.11. Expression Profiles of PD-L1, STAT3, CD4, and CD8 Proteins in Tumor Tissues. The observed significant established melanoma growth inhibition and overall survivability enhancement described above are likely to originate from (i) simultaneous downregulation of STAT3 and PD-L1 expression levels in the tumor tissue of mice treated with AuCGKRK + PD-L1siRNA + STAT3siRNA nanoconjugates and (ii) enhanced accumulation of T-cells in the tumor microenvironment due to blockade of immune checkpoint protein PD-L1. With a view to gain some mechanistic insights to this end, we performed a Western blotting experiment with tumor 8670


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shown to be blocked by the use of STAT3 inhibitors.62,63 Thus, less accumulation of Treg cells in the tumor microenvironment might also play a crucial mechanistic role behind the presently observed tumor growth inhibition. Clearly, further in-depth mechanistic investigations need to be undertaken in future toward understanding the exact nature of such T-cell-driven tumor growth inhibition.

lysates prepared from differently treated mice groups using antibodies against STAT3 and PD-L1 as well as antibodies against CD4 and CD8 (two distinguishing markers for T-cells). Findings in the Western blotting experiment (Figure 10)

3. CONCLUSIONS In summary, we have reported herein on the design, physicochemical characterization, and bioactivities of an effective tumor- and tumor endothelial-cell-targeting positively charged Au-CGKRK nanoconjugate system for combating cancer via therapeutic RNA interference. The nanometric size range (17−80 nm) of the presently described system was confirmed by dynamic light scattering and transmission electron microscopy. Findings in the degree of cellular uptake by both confocal microscopy and flow cytometry experiments showed tumor-cell-selective targeting properties of AuCGKRK nanoconjugates. Biodistribution studies involving i.p. administration of the Au-CGKRK nanoconjugate-associated NIR dye revealed significant accumulation of the dye in tumor tissues in established melanoma-bearing C57BL/6J mice. Importantly, co-delivery (i.p.) of both STAT3siRNA (as an inhibitor of tumor and tumor endothelial cell progression) and PD-L1siRNA (as an immune checkpoint inhibitor) in complexation with the presently described Au-CGKRK nanoconjugate significantly inhibited the growth of established mouse melanoma. The treatment strategy also led to significant (>70%) enhancement in the overall survivability (OS) of the tumor-bearing mice compared to the OS of untreated tumorbearing mice. Notably, i.p. administration of single siRNA (i.e., either only STAT3siRNA or only PD-L1siRNA in complexation with Au-CGKRK nanoconjugates) was found to be less effective in inhibiting the growth of established mouse melanoma. Findings in the Western blotting experiments with tumor lysates support the notion that the observed tumor growth inhibition is a T-cell-driven process. The Au-CGKRK nanoconjugate system described herein opens a new door for simultaneously targeting therapeutic siRNA against tumor growth and siRNA against immune checkpoint to tumor and tumor endothelial cells. The present approach is expected to find future use in the emerging field of cancer immunotherapy.

Figure 10. PD-L1siRNA (the immune checkpoint inhibitor) and Stat3siRNA (the JAK-STAT pathway inhibitor) harness T-cell-driven synergistic therapeutic effects upon i.p. delivery with Au-CGKRK NPs. Expression levels of the indicated proteins in the tumor lysates prepared from differently treated mice groups were measured by Western blotting. The house-keeping protein β-actin was used as a control in the experiment. Lane 1, mice treated with 5% aqueous glucose; lane 2, mice treated with CGKRK; lane 3, mice treated with AuNP-CGKRK; lane 4, mice treated with AuNP-CGKRK + scrambled siRNA; lane 5, mice treated with AuNP-CGKRK + PDL1siRNA; lane 6, mice treated with AuNP-CGKRK + STAT3siRNA; and lane 7, mice treated with AuNP-CGKRK + PD-L1 + STAT3siRNA.

clearly revealed pronounced downregulation of STAT3 and PD-L1 in the tumor lysate prepared from mice treated with Au-CGKRK + PD-L1siRNA + STAT3siRNA nanoconjugates compared to that in the other control mice groups. Consistent with the observed downregulation of PD-L1, significant upregulations of CD8 and CD4 expression levels were also observed (Figure 10), indicating that the established melanoma growth inhibition in mice treated with AuCGKRK + PD-L1siRNA + STAT3siRNA nanoconjugates is likely to be T-cell-driven. The possible role of interleukin 6 (IL6) behind trafficking of T-cells in the tumor microenvironment cannot be ruled out. Prior studies demonstrated IL6 to play the role of a double sword. On one hand, IL6 promotes proliferation of tumor cells (tumorigenesis) by upregulating the STAT3 activation pathway, whereas it also promotes T-cell infiltration in the tumor microenvironment.60,61 Use of STAT3siRNA in the present study is likely to inhibit IL6mediated tumorigenesis. The findings in the Western blotting experiments (Figure 10) are consistent with enhanced accumulation of CD8+ and CD4+ T-cells in the tumor microenvironment. IL6 may play some role behind such increased T-cell infiltration in the tumor tissue. Clearly, further in-depth mechanistic studies need to be carried out in future for obtaining more insights into the role of IL6 in the observed tumor growth inhibition mediated by Au-CGKRK + PDL1siRNA + STAT3siRNA nanoconjugates. Accumulation of T-regulatory (Treg) cells in the tumor microenvironment has been shown to deactivate tumor-killing efficiencies of CD8+ cells, and migration of Treg cells in tumor tissues has been

4. MATERIALS AND METHODS 4.1. Reagents. H-Lys(Boc)-2-ClTrt resin, Fmoc-Arg(Pbf)OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Boc-Cys(Trt)-OH, Cl ion exchange Amberlyst resin, O-(benzotriazol-1-yl)N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIPEA), piperidine, dimethylformamide (DMF), trifluoroacetyl (TFA), thioanisole, ethanedithiol, anisole, sodium borohydride (NaBH4), chloroauric acid (HAuCl4), Dulbecco’s modified Eagle’s medium (DMEM), trypsin from bovine pancreas, radioimmunoprecipitation assay (RIPA) buffer, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protease inhibitor cocktail (PIC), Hoechst-33258, and fluoroshield with DAPI mounting medium were purchased from Sigma (St. Louis, MO). Mouse PD-L1siRNA, mouse STAT3siRNA, nonsilencing siRNA (scrambled siRNA), carboxy fluorescein (FAM), and Cy5-labeled siRNAs were purchased from Eurogenetic, Belgium. CD8 (Cat# SC-1177) were purchased 8671


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separated. The precipitate upon chloride ion exchange chromatography over Amberlyst IRA-400 and repeated precipitation from MeOH/acetone afforded the pure CGKRK pentapeptide (VII) as a white fluffy solid (15 mg, 60% yield based on intermediate VI; Rf = 0.2 in 15% MeOH/ CHCl3, v/v). The purified CGKRK pentapeptide was found to be essentially insoluble in chloroform and was dissolved in 3:1 (v/v) methanol/chloroform. The structural characterization was performed using high-resolution mass spectrometry (HRMS), matrix-assisted laser desorption/ionization (MALDI), and 1H NMR (Figures S1−S3, respectively). The purity of the peptide was confirmed by reverse-phase HPLC analysis (Figure S4) using two mobile phases (methanol and 95:5 methanol/water, v/v). 1H NMR characterization of compound VII (400 MHz, CDCl3 + CD3OD): δ/ppm = 1.5 (m, 4H, a + a′), 1.6−1.9 (m, 12H, b + b′ + b″ and c + c′ + c″), 2.9 (m, 4H, d + d′), 3.2 (m, 2H, e), 3.4 (m, 2H, f), 3.8 (m, 2H, g), 4.2−4.5 (m, 4H, h + h′ + h″ + h‴). HRMS and MALDI of compound VII: m/z = 591 [M + 1]+ for C23H46N10O6S. 4.6. Synthesis of Gold Nanoparticles (AuNPs) and Conjugation of the CGKRK Peptide. The gold nanoparticles were synthesized by a previously described sodium borohydride reduction method.64 Briefly, 1 mL of HAuCl4 stock solution (10−2 M) was diluted to 100 mL with sterile Milli-Q water (18.2 mΩ), 50 mL of sodium borohydride solution (0.05 mg/mL) was added, and the reaction mixture was stirred overnight. The tumor-targeting CGKRK peptide was conjugated to the resulting AuNPs as follows: briefly, series of 1 mL AuNP solutions (freshly prepared) were separately incubated with increasing amounts of CGKRK peptides (2−20 μg) for 10 min at room temperature. The changes in both the absorbances and the wavelength maxima (λmax) of the Au-CGKRK nanoconjugates and bare AuNPs were monitored using a spectrophotometer (JASCO dualbeam spectrophotometer, model V-650). The saturation curve constructed using the absorbance versus wavelength provided the maximum CGKRK concentration (10 μg/mL, Figure 2) for preparing the tumor-targeting Au-CGKRK nanoconjugates described below. 4.7. Preparation of Nanoconjugates. The CGKRK peptide (10 μg/mL) was added to 30 mL of AuNPs and was first centrifuged using an Eppendorff centrifuge (12 000 rpm at 20 °C) for 1 h. The resulting intense red pellet upon further ultracentrifugation (20 000 rpm, Beckman Coulter) at 4 °C for 1 h afforded 30 μL of Au-CGKRK nanoconjugates. The amount of CGKRK peptide conjugated to AuNPs was measured by HPLC analysis, and the concentration of gold in the Au-CGKRK nanoconjugates (loose pellet) was determined by the inductively coupled plasma optical emission spectrometry method. The loose pellet (Au-CGKRK) was used for all characterization and biological studies. 4.8. XRD Analysis. The surface crystallinity of the nanoconjugates was characterized by the XRD method. Briefly, both bare AuNPs and Au-CGKRK nanoconjugates were separately coated on the same place of a glass slide at regular time intervals to allow formation of thin films. The dried coated glass slides were used for XRD studies with a Bruker D8 Advance Powder X-ray diffractometer. 4.9. TEM Analysis. The size and morphological features of the gold nanoconjugates were analyzed by TEM. Briefly, 5 μL of diluted (1:200) nanoconjugates was placed on a carboncoated copper grid (glow-discharged for 45 s using a Tolaron

from Santa Cruz Biotechnology Inc. CD4 (Cat# 14-9766-82), CD31 (Cat# MA5-13188) monoclonal antibodies, goat antimouse immunoglobulin G (IgG) (H + L) FITC (Cat# 31569) secondary antibodies, and super signal west pico chemiluminescent substrate (Cat# 34077) were purchased from Thermo Fisher Scientific. STAT3 (Cat# 9139), PD-L1 (Cat# 29122), β-actin (Cat# 4967), and horseradish peroxidase (HRP)-linked anti-mouse IgG (Cat# 7076) and anti-rabbit IgG (Cat# 7074) antibodies were purchased from Cell Signalling Technology. 4.2. Cell Lines. B16F10 and NIH3T3 cell lines were procured from the National Centre for Cell Science (Pune, India). These cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. 4.3. Stock Solution Preparation. Gold stock: 10−2 (M) chloroauric acid solution was prepared by dissolving 1 g of chloroauric acid in 294 mL of Milli-Q water and stored at 4 °C. Peptide stock: 1 mg of CGKRK was dissolved in 1 mL of Milli-Q water and stored at 4 °C. 4.4. Animals. Female C57BL/6J mice (6−8 week old, each weighing 20−22 g) were procured from the animal house of our Institute. All animals were maintained in filtered-top autoclavable cages provided with sterilized water, food, and bedding. All of the experiments were conducted in accordance with the protocols approved by our Institutional Animal Ethics committee vide approval (IICT/52/2016) of the Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, India. 4.5. Synthesis of the CGKRK Pentapeptide. The Fmoc solid-phase peptide synthesis strategy (as shown schematically in Figure 1) was used to synthesize the CGKRK pentapeptide. Briefly, 500 mg of H-Lys(Boc)-2-ClTrt resin (I) (Nε-BocLysine preloaded 2-chlorotrityl resin, 0.72−0.77 mmol/g loading) in the peptide synthesizer (CS Bio) vessel was first allowed to swell in 5 mL of DMF for 30 min. The α-COOH group of the Fmoc-Arg(Pbf)-OH arginine (3 equiv) residue was then coupled with the α-NH2 group of the resin-attached lysine, HBTU (3 equiv), and DIPEA (6 equiv) in dry DMF (10 mL) by agitating the peptide synthesizer vessel under nitrogen for 40 min. The same coupling method was repeated twice to ensure complete conjugation. The resin was then washed with DMF (2 × 5 mL) for 5 min, and the Fmoc group of the resulting intermediate II was removed by stirring with a solution of piperidine/DMF (1:4, v/v, 10 mL) under nitrogen for 10 min (2×). The same peptide coupling strategy was repeated by sequentially coupling with Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, and Boc-Cys(Trt)-OH (3 equiv each) using HBTU (3 equiv) and DIPEA (6 equiv) in dry DMF (10 mL) under nitrogen for 40 min. As before, the coupling at each stage was repeated two times to ensure completion of reaction, which eventually provided the resin-bound intermediate V via intermediates III and IV (Figure 1). The resin-bound intermediate was then washed thoroughly with dry dichloromethane (DCM) (2 × 10 mL) for 5 min and dried well. The resulting dried resin-bound intermediate (V) upon treatment with 0.5% TFA in dry DCM (100 mL) for 3 h at 0 °C afforded the resin-free CGKRK pentapeptide intermediate VI in the reaction mixture. The solvent was evaporated in the rotary evaporator at 30 °C, and the residue was dried under high vacuum for 30 min. The dried intermediate VI (60 mg) was treated with TFA−thioanisole−ethanedithiol−anisole (90:5:3:2, v/v, 1 mL) for 3 h at 0 °C. Dry ether (10 mL) was added to the reaction mixture until a white precipitate 8672


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Korea) confocal dishes with 1 mL of growth medium 12 h prior to treatment. To monitor the extent of lysosomal release of gold nanoconjugates, lysosomes of cells were first labeled with LysoTracker Red DND-99 (Thermo Fisher Scientific) for 1 h. Cells were then incubated with Au-CGKRK-FAMsiRNA (2 μg) nanoconjugates for increasing time periods (30 min, 1 h, 3 h, and 6 h) and washed thoroughly with PBS (3 × 1 mL). Fresh culture media were added to each well and cells were observed with a confocal microscope (Nikon Ti Eclipse) after staining cell nuclei with Hoechst-33258. With a view to examine the efficiency of the gold nanoconjugates for dual siRNA delivery, 1 × 105 B16F10 cells were treated with AuCGKRK-FAMsiRNA (2 μg) and Au-CGKRK-Cy5siRNA (2 μg) nanoconjugates in 35 mm Petri dishes for 2.5 h at 37 0 °C. Cell nuclei were then stained with Hoechst-33258, incubated for additional 30 min, and washed with PBS (3 × 1 mL). Fresh culture medium (1 mL) was added, and images were taken with a confocal microscope. Green fluorescence from FAMlabeled siRNA (exλ 490 nm/emλ 520 nm), red fluorescence from Cy5-labeled siRNA (exλ 650 nm/emλ 670 nm), and blue fluorescence from Hoechst-33258 (exλ 352 nm/emλ 461 nm) were obtained using a 40× objective. The results were analyzed using Nikon A1 software. Similarly, confocal images of the untreated cells and cells treated with either naked siRNA or free AuNPs were also recorded. 4.14. Quantitative Cellular Uptake Study with Flow Cytometry. The degree of cellular uptake was also studied quantitatively using flow cytometry. To examine the cancer cell selective internalization, both cancer (B16F10) and normal (NIH3T3) cells were cultured in six-well plates at a density of 1 × 106 cells overnight. The cells were then incubated with AuCGKRK-FAMsiRNA (2 μg), CGKRK + FAMsiRNA (2 μg), and free FAMsiRNA (2 μg) for 4 h. Cells were harvested by trypsinization, washed with PBS (3 × 1 mL) to remove unbound conjugates, and analyzed using a flow cytometer (BD FACSCanto II) under an FITC channel. The shift of the fluorescently labeled cells was compared with that of untreated cells using FCS software. 4.15. Establishment of the Syngenic Mouse Melanoma Tumor Model. Female C57BL/6J mice (6−8 week old, each weighing 20−22 g, n = 5) were subcutaneously (s.c.) injected with 1 × 105 B16F10 cells in the left flank. On day 12, when tumor became palpable, the treatment was initiated for either biodistribution or tumor regression studies. 4.16. Biodistribution Study. The biodistribution pattern of the nanoconjugates was monitored by the noninvasive imaging method. Dil (25 μg; a near-infrared dye) was added to 320 μL of the Au-CGKRK nanoconjugate (containing 143 μg of gold). The solution was centrifuged at 20 000 rpm and 4 °C for 1 h to obtain the NIR-dye-labeled nanoconjugates. The resulting 320 μL of NIR-dye-labeled Au-CGKRK nanoconjugates was divided into two equal parts. Two B16F10 tumor-bearing 6−8 week old female C57BL/6J (each weighing 20−22 g) mice were i.p. administered with NIR-labeled nanoconjugates (each mouse received 160 μL of NIR-labeled nanoconjugates). Animals were anesthetized with ketamine− xylazine 2 h post i.p. treatment of NIR-labeled nanconjugates, and the biodistribution profile of the NIR dye in mice was monitored using a PerkinElmer IVIS spectrum animal imager. Such biodistribution profile was also measured 24 h post i.p. treatment. With a view to further confirm the biodistribution patterns, mice were sacrificed after the completion of noninvasive imaging 24 h post i.p. treatment, vital organs

Hivac Evaporator) for 10 min. The excess sample was blotted with Whatman filter paper. The dried coated grids were vacuum-dried, and the electron micrographs of the gold nanoconjugates were recorded with a FEI Tecnai 12 TEM instrument. 4.10. DLS Measurements (Size and Surface Charge). The hydrodynamic diameter and surface charges (ζ potentials) of various nanoconjugates including AuNPs, Au-CGKRK, and Au-CGKRK-siRNA were measured by photon correlation spectroscopy and electrophoretic mobility using an Anton-Paar Litesizer 500 instrument and using diluted (1:200) samples. Each experiment was carried out in triplicate. For studying the stability of the nanoconjugates in the presence of added serum, nanoconjugates were dissolved in 10% FBS and size measurements were carried out every 25−100 h. 4.11. siRNA Binding Studies and RNase Sensitivity Assay. The siRNA binding properties of the nanoconjugates were studied by the gel-binding assay using native PAGE. Briefly, increasing amounts (2.2−22.3 μg) of freshly prepared nanoconjugates were incubated with 0.5 μg of nonsilencing siRNA for 30 min at room temperature and the resulting electrostatic complexes were electrophoresed (native PAGE) for 1 h at 80 V in TAE (1×) buffer. siRNA binding properties of bare AuNPs were studied as control. Similar native PAGE was also carried out by incubating the same amount (17.8 μg) of Au-CGKRK nanoconjugates with increasing amounts (0.5− 2.5 μg) of nonsilencing siRNA. After electrophoresis, the gels were stained with EtBr and observed under a gel docking system (Vilber Lourmart). Naked nonsilencing siRNA (0.5 μg) was used as a control. With a view to evaluate the RNase sensitivity of the presently described Au-CGKRK-siRNA nanoconjugates, the Au-CGKRK-siRNA nanoconjugates containing 1.5 μg of siRNA were incubated with RNase-1 (0.5 μg/ mL) for 1 h at room temperature. Nanoconjugates were then extracted with phenol/chloroform/isoamyl alcohol (25:24:1, v/v, 1 mL), and siRNA from the supernatant was precipitated with ice-cold isopropyl alcohol (1 mL). The siRNA pellet was dissolved in RNase-free water and loaded on the native gel. Samples of naked siRNA and siRNA incubated with RNase-1 were used as controls. 4.12. In Vitro Cytotoxicity Assay. The cellular cytotoxicities of the nanoconjugates were measured using the conventional MTT assay. Briefly, B16F10 cells were seeded in 96-well plates at a density of 4 × 103 cells/well and incubated overnight. Cells were then treated with increasing volumes (1, 2.5, 5, 7.5, and 10 μL) of the nanoconjugate stock solution (446 μg/mL of gold) of AuNPs and Au-CGKRK nanoconjugates for 48 h. The stock 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) reagent (10 μL, 3 mg/mL) was added to each well and incubated for 4 h under dark. The purple formazan crystals were dissolved in 50 μL of dimethyl sulfoxide/methanol (1:1, v/v), and absorbances of the resulting clear solutions at 575 nm were recorded using a microplate reader (Biotek Synergy). The results were expressed as percent cell viability using the following formula: % cell viability = [A575 (treated cells) − background/A575 (untreated cells) − background] × 100. Each experiment was performed in triplicate. 4.13. Cellular Uptake Study Using Confocal Microscopy. The degrees of cellular uptake for the Au-CGKRK nanoconjugates in cancer cells were studied qualitatively by confocal microscopy. Briefly, 1 × 105 B16F10 cells were seeded in 35 mm coverglass-bottom (SPL Life Sciences Co., Ltd., 8673


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in sodium dodecyl sulfate (SDS)-PAGE sample buffer, and the components were separated using 12% SDS-PAGE. Proteins were transferred to the poly(vinylidene difluoride) membrane by wet blotting, and membranes were blocked for 2 h at room temperature with 5% nonfat milk in PBS containing 0.05% Tween-20 (PBS-T). The blots were incubated with monoclonal antibodies against β-actin, PD-L1, STAT3, CD8, and CD4 (using dilution factors mentioned in the manufacturer’s instructions) overnight at 4 °C, washed with PBS-T (3 × 10 mL), and incubated with the HRP-conjugated secondary antibody for 1 h at room temperature. The membranes were developed using the enhanced chemiluminescent method in Vilber Lourmat. 4.20. Statistical Analysis. Error bars represent mean values ± SD. For comparison between two treatment groups, two-tailed Student’s t-test was used. For the in vivo experiments, * P < 0.05 was considered significant.

(including brain, heart, lungs, liver, kidney, spleen, and tumor tissue) were collected, and ex vivo imaged with the same imager. In addition, with a view to confirm accumulation of i.p. administered fluorescently labeled Au-CGKRK + Cy5siRNA nanoconjugates in melanoma-bearing mice, tumor tissues were cryosectioned (10 μm) at 24 h post i.p. treatment and observed under a confocal microscope at 60× magnification using DAPI for nucleus staining. 4.17. Tumor-Vasculature-Targeting Property of AuCGKRK Nanoconjugates. Tumor-vasculature-targeting properties of the Au-CGKRK nanoconjugate were evaluated by CD31 (one of the most widely used markers for tumor endothelial cells) staining of tumor endothelial cells. AuCGKRK + Cy5siRNA nanoconjugates were i.p. administered to melanoma tumor-bearing mice (n = 3). After 24 h, mice were sacrificed, and tumors were excised. Tumor cryosections (10 μm thick) were prepared on glass slides using a cryostat instrument (Leica). The slides were fixed in isopropyl alcohol (15 mL) for 15 min and washed with PBS (2 × 5 mL). The fixed slides were incubated in citrate buffer at 65 °C for 10 min for antigen retrieval and blocked with 5% bovine serum albumin in TBST for 1 h. Slides were washed with TBST (2 x 5 mL) and incubated with CD31 monoclonal primary antibody (1:100 dilution) overnight at 4 °C. All of the slides were washed thoroughly with TBST buffer and incubated with the FITC-conjugated goat anti-mouse IgG secondary antibody (1:200 dilution) at room temperature for 30 min to mark the tumor endothelial microvessels. Slides were mounted with DAPI mounting media and analyzed by a confocal microscope under a 60× objective. 4.18. Tumor Growth Inhibition Study. Female C57BL/ 6J mice (6−8 week old; each weighing 20−22 g, n = 5) were s.c. inoculated in the left flanks with 1 × 105 B16F10 cells in 200 μL of Hank’s buffer salt solution. On day 12, melanoma tumor-bearing mice were randomly divided into four groups and were then i.p. administered with 5% aqueous glucose (group I); CGKRK + PD-L1siRNA + STAT3siRNA (each siRNA 3 μg) (group II); Au-CGKRK nanoconjugate (160 μL) containing 71.3 μg of gold (group III); and Au-CGKRK + PDL1siRNA + STAT3siRNA containing 3 μg of each siRNA (group IV) on days 12, 14, 16, 18, and 20. In addition, with a view to check the therapeutic efficacy of Au-CGKRK nanoconjugates in complexation with only STAT3siRNA or only PD-L1siRNA, melanoma tumor-bearing mice (n = 5) were i.p. administered with 5% aqueous glucose (group I); AuCGKRK + scr.siRNA containing 6 μg of scr.siRNA (group II); Au-CGKRK + PD-L1siRNA containing 6 μg of PD-L1siRNA (group III); and Au-CGKRK + STAT3siRNA containing 6 μg of STAT3siRNA (group IV) on days 12, 14, 16, 18, and 20. Animals were monitored during the entire course of the experiment. The tumor volumes (V = 1/2·ab2, where “a” represents the maximum length of the tumor and “b” represents minimum length of the tumor measured perpendicular to each other) were measured with a slide caliper for up to 24 days. Results represent the means ± standard deviation (SD, n = 5). 4.19. In Vivo Expression Profiles of PD-L1, STAT3, CD4, and CD8 by Western Blotting. The tumor tissues were collected in ice-cold PBS 24 h post i.p. treatment and lysed by homogenization in RIPA buffer containing protease inhibitor cocktail (PIC). The lysate was centrifuged at 12 000 rpm for 30 min, and the protein concentrations were estimated by the Bradford reagent. Total proteins (50 μg) were dissolved

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01051.

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HRMS electrospray ionization spectrum of the CGKRK pentapeptide, MALDI mass spectrum, 1 H NMR spectrum, HPLC chromatograms, the X-ray diffraction (XRD) pattern of the Au-CGKRK conjugate, siRNA binding properties, confocal imaging studies, tumor volumes and survivability of nanoconjugates, AuCGKRK-FAMsiRNA nanoconjugate uptake in B16F10 cells, 3D-z-stack images of Au-CGKRK-FAMsiRNA nanoconjugates (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected], [email protected]. Phone: 040-27191480. Fax: +91-40-27160387 (C.R.P.). *E-mail: [email protected], [email protected]. Tel: 91-4027156755, 91-9440040582 (A.C.). ORCID

Chitta Ranjan Patra: 0000-0001-9453-6524 Arabinda Chaudhuri: 0000-0002-0734-8320 Present Address ⊥

Superannuated from CSIR-Indian Institute of Chemical Technology on January 31, 2018 (A.C.). Author Contributions ∥

S.K.G. and R.K. contributed equally to this article.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Harikrishna from CSIR-Centre for Cellular and Molecular Biology for his kind help in taking TEM images. S.K.G and R.K. thank the Council of Scientific and Industrial Research (CSIR), Government of India, New Delhi, for their doctoral research fellowship. A.C. and C.R.P. thank CSIR for sponsoring this research work (project codes: CSC0302 and BSC0123). IICT manuscript communication number is IICT/ Pubs./2018/233. 8674


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