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Bombesin Peptide Conjugated Water-Soluble Chitosan Gallate—A New Nanopharmaceutical Architecture for the Rapid One-Pot Synthesis of Prostate Tumor Targeted Gold Nanoparticles

Purpose We report herein bombesin peptide conjugated water-soluble chitosan gallate as a template for rapid one-pot synthesis of gold nanoparticles (AuNPs) with capabilities to target receptors on prostate cancer cells. Methods Water-soluble chitosan (WCS), anchored with gallic acid (GA) and LyslLys3 (1,4,7,10-tetraazacyclo dodecane-1,4,7,10-tetraacetic acid) bombesin 1–14 (DBBN) peptide, provides a tumor targeting nanomedicine agent. WCS nanoplatforms provide attractive strategies with built-in capabilities to reduce gold (III) to gold nanoparticles with stabilizing and tumor-targeting capabilities. WCS-GA-DBBN encapsulation around gold nanoparticles affords optimum in vitro stability. Results The DBBN content in the WCS-GA-DBBN sample was ~27%w/w. The antioxidant activities of WCS-GA and WCS-GA-DBBN nanocolloids were enhanced by 12 times as compared to the nascent WCS. AuNPs with a desirable hydrodynamic diameter range of 40–60 nm have been efficiently synthesized using WCS-GA and WCS-GA-DBBN platforms. The AuNPs were stable over 4 days after preparation and ~3 days after subjecting to all relevant biological fluids. The AuNPs capped with WCS-GA-DBBN peptide exhibited superior cellular internalization into prostate tumor (PC-3) cells with evidence of receptor mediated endocytosis. Conclusion The AuNPs capped with WCS-GA-DBBN exhibited selective affinity toward prostate cancer cells. AuNPs conjugated with WCS-GA-DBBN serve as a new generation of theranostic agents for treating various neoplastic diseases, thus opening-up new applications in oncology.


Introduction
The application of nanotechnology in tumor diagnostics and therapy continues to attract considerable attention.  In this context, we have been exploring the utility of green nanotechnology in the design and architecture of tumor-avid gold nanoparticles. 6,9,11,12,14,20,23,26,34 Tumor avidity and selective accumulation of optimum doses of therapeutic gold nanoparticles is of paramount importance in drug delivery. 35,36 Specifically, tumor selectivity and cytotoxicity of NPs depend on many factors, such as particle size, surface chemistry and biological target. 37 Firstly, the particle size should be small enough to escape capture by fixed macrophages that are lodged in the reticuloendothelial system (liver and spleen) but should be large enough to prevent their rapid leakage into blood capillaries. 38 The optimal NP size is considered to be in the range of 10 to 100 nm. 39 Secondly, the effect of surface charge on toxicity and cellular internalization are important considerations in the design and development of nanomedicine agents. As reported herein, functionalization of gold nanoparticles with positive effective surface charges are ideal for disrupting the negatively charged cellular membrane through electrostatic interactions. 40,41 Thirdly, the targeting agents should have the ability to be selective toward tumor cells to reduce off target systemic toxicity. 35 Gold nanoparticles (AuNPs) possess outstanding properties because of high surface area-to-volume ratio and their unique ability to anchor or conjugate specific ligands with myriad functions on their surface-all aimed at improving the effective targeting capabilities that are required in diagnosis and therapy. 42 The surface properties of AuNPs can be changed by conjugating with active targeting molecules, such as tumor-avid small molecules, proteins, peptides, and monoclonal antibodies. 43 For cancer treatment, AuNPs can easily permeate, accumulate, and remain in tumors due to their permeability and retention (EPR) effects, thus resulting in enhanced uptake of diagnostic/therapeutic cargo. 44 Although AuNPs enjoy several advantages for their use in nanomedicine, the synthesis of AuNPs generally involves chemical reduction reactions in the presence of highly toxic reducing agents, such as hexadecyltrimethyl ammonium bromide (CTAB), 45,46 sodium borohydride (NaBH 4 ) and formaldehyde, and sodium hydrazine (N 2 H 4 ). 47 Toxicities of representative examples of reducing agents, capping agents and solvents have been reviewed in the literature. 47 Takahashi et al 41 have shown that AuNPs extracted from CTAB exhibited higher biocompatibility when compared to CTAP-capped AuNPs. AuNPs stabilized with CTAB showed strong cytotoxicity at 0.05 mM (Au atom) due to unreacted free CTAB. 48 It has been reported that polyethylene glycol (PEG) capped-AuNPs would help in enhancing the biodistribution and also to reduce systemic toxicity of CTAB. 48 Therefore, the development of efficient, ecofriendly, and straightforward one-pot synthetic protocols to prepare AuNPs in a water-based system is of paramount importance. Many natural compounds, 13,15 such as those derived from phytochemicals, 49,50 ascorbic acid, 51 sugar, [52][53][54] starch, 55,56 alginate, 57 gum arabic, 15,23 fibroin 16 and chitosan 17,[58][59][60] have been reported for green synthesis of non-toxic AuNPs. The pioneering efforts of Katti et al have demonstrated the extensive utility of electron-rich phytochemicals from tea, cinnamon, mango and various other plant species for the development of tumor-specific diagnostic and therapeutic gold nanoparticles. 26,[31][32][33] Chitosan (CS) is a biopolymer composing of D-glucosamine and N-acetyl-D-glucosamine units linked by β (1, 4)-glycosidic linkages. It has excellent biocompatible and biodegradable characteristics with low toxicity and immune stimulating properties, including excellent antioxidant activity. 60 The development of chitosan and its derivatives is of interest in many areas, especially biomedicine. When considering biological molecules such as lipids, proteins, enzymes, DNA, and RNA, it is important to recognize how free radical reactions of these substances are involved in human health issues, including aging, atherosclerosis, and carcinogenesis. 61,62 Chitosan has been exploited as an alternative natural antioxidant. 63,64 Due to the abundance of amino (-NH 2 ) and hydroxyl (-OH) groups, acting as electron transfer agents, CS has attracted considerable attention for its utility as a natural antioxidant [65][66][67] and as a reducing agent for phytochemical-mediated synthesis of AuNPs. 17 Although the -OH and -NH 2 groups of CS present several advantages, CS still has some limitations of non-reactivity and insolubility in water. Aqueous solutions of CS exhibit pH >6.5 and while in organic solvents the insolubility is dictated by strong inter-and intra-molecular hydrogen bonds. 68 This has limited the use of CS in aqueous solutions at neutral pH profiles relevant in biological media. CS gallate derivative was developed and proposed as a novel and potential antioxidant due to synergistic effects of CS and gallic acid function. 67,69 This novel antioxidant was developed in our laboratory and in this paper we are exploring its utility as an effective reducing agent in the form of a new nano-template for the rapid and efficient one-pot synthesis of functionalized AuNPs.
The CS gallate derivatives promote free radical scavenging by trapping hydroxyl radicals, carbon center radicals and also the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. In particular, CS gallate can suppress the unwanted pro-oxidant metals by chelating agents. Since the first establishment of CS-GA derivative, 67,69 it has been used as an antioxidant in various applications, for example in food packaging films, 70,71 wound healing, 72 tissue engineering, 73 inhibition of hydrogen peroxide-induced oxidative damage, 76 prevention of oxidative stress in Chang liver cells, 77 drug delivery system 18,22,78 and synthesis of silver NPs. 5 In continuation of efforts to capitalize on the antioxidant and reducing capacity of CS, water-soluble chitosan (WCS) has been developed through the green radiolysis processes. 17 Based on the chemical structure of WCS, it is susceptible to depolymerization by radiolysis to form smaller units of D-glucosamine, a derivative of glucose. 28 Therefore, the WCS nanocolloids, without functionalization, serve as efficient CS-containing antioxidants with built-in capabilities, including reducing power and stabilizing efficiency-all serving vital roles in the green synthesis of AuNPs. 8 We hypothesized that WCS gallate would be a good candidate for not only its role as a reducing and stabilizing agent in the green synthesis of AuNPs, but also for providing antioxidant functions for use in cancer treatment. It is known that gallic acid (GA) is an antioxidant extractable from plants 19 which is widely used in foods, drugs, and cosmetics. This antioxidant effect could prove beneficial to address numerous disease states, including cardiovascular disease. On the medical applications front, GA exhibits no cytotoxicity against normal fibroblast and endothelial cells. Therefore, the development of synthetic gallic acid derivatives has attracted considerable attention as a potent chemotherapeutic agent. Polyphenol compounds are well known as an important class of chemopreventive agents because of their acidity and the presence of delocalized π-electrons of benzene rings, leading to effective quenching of free radicals in cancer cells. 21 Our continued interest in exploring the targeting potential of bombesin (BBN) peptide stems from the fact that it is structurally similar to human gastrin-releasing peptide (GRP). 25,79,80 GRP receptors are over-expressed in many neoplastic diseases, including breast, lung and prostate cancers. 27 Several analogues of BBN have been synthesized to target BBN receptors in humans for imaging, diagnosis and treatment of cancer. 1,23,30 Peptide conjugated gold nanoparticles are able to localize on tumor cells with high selectivity with subsequent penetration through interstitial barriers to reach the desired tumor tissues and cells with minimal loss of their activity in the blood circulation. 35 We are, therefore, focusing our efforts on the applications of BBN conjugated gold nanoceuticals for targeting the primary and peripheral tumor targets. 29,81 In this paper, we report the preparation of water-soluble chitosan gallate (WCS-GA) derivative and its conjugation with LyslLys3 (1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid)-bombesin 1-14 (DOTA-BBN or DBBN) peptide. The successful conjugation of WCS-GA with DBBN (CS-GA-DBBN) has been fully elucidated using FT-IR, 1 H-NMR, XRD and SEM-EDX. Nanoparticulate formation and identity has been inferred through a combination of techniques including transmission electron microscopy (TEM) and dynamic light scattering (DLS). The antioxidant activity of WCS-GA-DBBN has been measured using DPPH radical assay. This report also provides full details on the ability of WCS-GA-DBBN to be used as a reducing and stabilizing agent for the one-pot template synthesis of gold nanoparticles (AuNPs). The nanostructure architecture of AuNPs, in the presence of WCS-GA-DBBN, has been further elucidated through UV-Vis, Zetasizer, TEM, HR-TEM, XRD and SEM-EDX. Our investigations have further included in vitro stability of AuNPs-WCS-GA-DBBN toward myriad of biological media through detailed monitoring of hydrodynamic sizes. Prostate tumor receptor avidity of AuNP-WCS-GA-DBBN has been established using PC-3 and LNCaP cell lines. Confocal microscopy and TEM studies have inferred the selective cellular uptake and cytotoxicity of the targeted AuNPs-WCS-GA-DBBN in comparison with the non-targeted AuNPs standards.

Instruments and Characterization
The 60 Co from a Gamma cell 220 irradiator was used as a γray source with an absorbed dose rate of 3.3 kGy/h at room temperature. Chemical structure was characterized using Fourier transform infrared spectrophotometer (FT-IR) in a Bruker Tensor 27 (USA) with 32 scans at 2 cm −1 resolution in a frequency range of 4000-400 cm −1 . Proton nuclear magnetic resonance 1 H NMR were determined on a Bruker Ultrashield 500 Plus (500 MHz) using D 2 O as solvent. Elemental composition of the conjugated sample was measured using a scanning electron microscope with an energy dispersive X-ray spectroscopy (SEM/EDX) and the elemental mappings were collected on a FET QUANTA 450 (Netherlands). The signals are quantified and plotted against the concentrations of the standard sulfur (S) added into the sample for quantifying the amount of DBBN conjugate. The standard sulfur solution with different concentrations of 0, 0.02, 0.04, 0.06, 0.08 and 0.10 μg/mL were added to the sample solution (7.5 μg/mL, 25 μL) for preparing addition standardization. The set of sample solutions with different sulfur standard concentrations was dropped onto a copper grid and determined using SEM/EDX. Powder X-ray diffraction (XRD) patterns were taken on a Bruker AXS (Germany) D8 Advance X-ray diffractometer with CuKα radiation, operating at 50 kV and 100 mA. The powder of the samples was placed on a polymethyl methacrylate (PMMA) specimenholder ring. The pattern of the samples was recorded from 10° to 80°, 0.0389° = 2θ. The optical extinction spectra of AuNPs formation was operated using a 1-cm path length quartz cuvette with a Varian Cary 50 UV-Vis spectrophotometer, Shimadzu (USA). The intensity-average hydrodynamic diameter and zeta potentials were measured using Zetasizer Nano S90, Malvern Instruments Ltd. (USA) with a 659 nm diode laser source. The morphologies of the dried samples were taken by transmission electron microscope (TEM) on a JEOL JEM 1400 TEM and operated at 120 kV. The AuNPs sample was prepared by centrifuging at 15,000 rpm, 15 °C for 15 min. The supernatant was discarded and the solution was brought to initial volume with deionized water. The sample was sonicated for 15 min and dropped onto a carbon coated copper grid. The sample was allowed to dry at ambient temperature before TEM measurement. Cellular internalization was investigated using a Confocal Laser Scanning Microscope from Leica Microsystems (USA). In vitro cytotoxicity evaluation was investigated using a bright field microscope and SpectraMax M2 microplate reader from Molecular Devices LLC (USA).

Preparation of Water-Soluble Chitosan Nanoparticles (WCS)
WCS was originally prepared following a procedure reported by Pasanphan et al. 17 Briefly, CS solution (1% v/v, 1000 mL) was prepared by dissolving CS powder into aqueous acetic acid solution (1% v/v, 1000 mL) and stirring overnight at room temperature. The CS solution was γ-rays irradiated with an absorbed dose of 80 kGy under ambient temperature in air. The samples were neutralized in NaOH solution (1% w/v, 600 mL) by precipitation method. The sample was dialyzed in a dialysis bag (MWCO = 1000 Da) in deionized water for 24 h. Finally, the CS solution was frozen and lyophilized to obtain water-soluble chitosan nanoparticle (WCS) powder. Functionalization of WCS with GA WCS (0.169 g, 1 mol) was dissolved in deionized water (20 mL) and stirred for 15 min. GA (0.510 g, 3 mole, 3 mole equiv. to WCS) was dissolved in ethanol (30 mL) and EDC (1.725 g, 3 mole equivalent to GA) was reacted with GA. NSH (1.036 g, 3 mole equivalent to GA) was further added. The reaction was stirred in an ice bath for 1 h in ethanol to obtain reactive GA/EDC/NSH system. The WCS solution was added into GA/EDC/NHS solution and the reaction was carried out in an ice bath for 15 min and further performed at room temperature for 24 h with magnetic stirring. The sample was dialyzed using a dialysis membrane (MWCO 3500 Da) at room temperature for 24 h and lyophilized to obtain WCS-GA powder.

The Antioxidant Activity of WCS-GA-DBBN
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging capacity was measured according to a previous protocol. 25 Briefly, the aqueous solutions of WCS-GA-DBBN (0.04, 0.12, 0.2, 0.4, 0.6, 0.8, 10, 16, 2.0, 3.0 and 4.0 mg/mL, 500 µL) were added to methanolic DPPH solution (300 µM, 500 µL). The mixture was stirred for 30 min in a dark at room temperature, followed by absorbance measurement at 515 nm by spectrophotometer. The percentage of DPPH free radical scavenging ability was calculated using the following equation: where A 0 and A c are absorbances of the bank solution and the sample solution at different concentrations, respectively. Similarly, study of the antioxidant activity of WCS, GA, WCS-GA samples was also carried out using the same procedure.

One-Pot Synthesis of AuNPs in WCS-GA-DBBN
One-pot synthesis of AuNPs was carried out via reduction reaction of HAuCl 4 1.0 mM using different concentrations of polymer and reaction times. HAuCl 4 (1.0 mM, 1 mL) was prepared in aqueous solution and the solution was added into WCS-GA-DBBN aqueous solution (0, 1, 2, 3, 4 mg/mL, 1 mL). The mixture was then stirred for 0, 1, 2, 3, 4, 5, 6, 24, 48, 72 and 96 h at ambient temperature and atmosphere. Similarly, the WCS-GA aqueous solution was mixed with HAuCl 4 aqueous solution using the abovedescribed protocol. The AuNPs solution was quantified using UV-vis spectrophotometer in a range of the visible light (400-800 nm). The selected sample of AuNPs-WCS-GA-DBBN was purified by ultracentrifugation at 15,000 rpm at 15 °C for 15 min and re-dispersed in distilled water before further characterization.

In vitro Stability Assessment of AuNPs
In

Cellular Internalization
Cell internalization of AuNPs into prostate cancer cells was determined by a confocal laser scanning microscope and transmission electron microscope. Human prostate cancer cell line (PC-3) and lymph node carcinoma of the prostate cell line (LNCaP) were sub-cultured in media RPMI and RPMI customized, respectively. To evaluate cellular internalization of AuNPs, PC-3 cells were placed at 4 × 10 4 cells/ mL in a 6-well plate and let to adhere overnight. The cells were then treated with AuNPs (50 µg/mL) and incubated for 6 h. The treated cells were washed twice with PBS and fixed in 2% glutaraldehyde (2% v/v in 0.1 M PBS). The PC-3 cells were stained with wheat germ agglutinin (WGA) stain solution (1 µg/mL) and washed with Hank's balanced salt solution (HBSS) and Triton solution. The cells were placed onto a glass slide and covered with a cover slip. The images of PC-3 cells were detected with a confocal laser scanning microscope. For TEM measurement, the PC-3 cells were placed at 4 × 10 4 cells/mL in a 6-well plate, and let to adhere overnight. The cells were then treated with AuNPs (50 µg/ mL) and incubated for 6 h. The PC-3 cells were washed twice with PBS and fixed in glutaraldehyde (2% v/v in 0.1 M PBS). The samples were then rinsed twice in buffer solution, post-fixed in 1% osmium tetroxide in 0.1 M PBS for 1 h and dehydrated through a graded ethanol series. The cells were cut on a Leica UCT ultramicrotometer and transferred to copper grids. The TEM images of the cells were acquired with TEM. Similarly, LNCaP cells were prepared and measured using the same procedure.

Cytotoxicity of AuNPs to Prostate Cancer Cells
Human prostate cancer cell lines, ie, PC-3 and LNCaP, were sub-cultured in media RPMI and RPMI customized, respectively supplemented with 10% FBS and gentamicin and stored in a CO 2   chain scission, through high energetic radiation, the original WCS contains -NHCOCH 3 of the chitin unit and -NH 2 and -OH of CS units. Additional functionalities include C-O-C bonds of the glycosidic linkage and the carbonyl (C=O) group of its degraded chain end. After preparation, a detailed characterization of the WCS nanocolloids was performed to gain full insights into its structure prior to functionalization. As a first observation, the chemical structures were characterized by FT-IR and 1 H NMR. The FTIR spectrum of WCS ( Figure 1A(a)) shows the characteristic peaks at 898, 1020, 1029, 1423, 1562, 1630, 2900 and 3350 cm −1 , which are attributed to pyranose ring, C-O-C bridge and C-O stretching of C 3 and C 6 , C-N stretching, N-H stretching (amide ll), C=O stretching of acetyl group (amide l), C-H stretching and O-H stretching, respectively. The chemical structure of WCS was also established using 1 H-NMR. As shown in Figure 1B, the characteristic peaks of WCS exhibited a signal at 2.1 ppm (H-Ac, three protons of the N-acetyl group), 3.3 ppm (H at carbon-2 position), multiple peaks around 3.7-3.9 ppm (H at carbon-3 and carbon-6 position) and a peak at 5.4 ppm (H at carbon-1 position). The FT-IR and 1 H-NMR results confirmed the chemical structure of WCS, which has aided our efforts for further chemical functionalization and biomolecular conjugations, as discussed in the following sections.

Results and Discussion
To improve the antioxidant activity and thereby enhance the reducing capacity of WCS, we anchored the polyphenolic group onto WCS. This approach affords the development of an effective reducing and stabilizing agent for rapid one-pot synthesis of gold nanoparticles (AuNPs). In this way, gallic acid derived polyphenol (GA) polyphenol was functionalized onto WCS. GA was functionalized onto WCS via amide and/or ester linkages using EDC/NHS coupling reaction. The EDC/NHS initiated the reactive ester species on GA and the nucleophilic reaction possibly occurs at the NH 2 -group at C-2 and the -OH group at C-6 and C-3 on WCS. In the presence of NHS, the long-lived GA-NHS ester is formed to improve the efficiency of EDC coupling reactions with WCS to form WCS-GA. 69 Functionalization of GA onto WCS was further confirmed by FT-IR. The FTIR spectra ( Figure 1A(c)). FT-IR spectrum of WCS-GA shows peaks of amide bond at 1643 cm −1 implying the formation of amide bonds between -NH 2 of WCS and -COOH of GA. In addition, new peaks at 1761 cm −1 were found due to the ester linkage formation between the -OH group of WCS and the -COOH group of GA. In addition, the peaks around 700-900 cm −1 were assigned to the aromatic ring of GA. The FT-IR results agree with the conjugation of GA onto CS as indicated in previous literature. 82,83 Further characterization of WCS-GA was performed by 1 H NMR. 69 The 1 H NMR spectrum of WCS-GA ( Figure 1B(c)) exhibited all peaks of WCS and an additional new signal at 7.1 ppm (assigned as H-a') corresponds to non-H-atom substituents on the benzene ring. The 1 H NMR spectrum of GA revealed a chemical shift at 7.6 ppm (assigned as H-a) belonging to two protons at the ortho positions on benzene ring of GA at a position ( Figure 1B 84%. The DS of GA on WCS was comparable to that reported in the previous work when GA was functionalized on CS (DS ~15%). 67,69 The DS of GA on WCS in the present work is slightly higher than that of GA on CS in the previous report owing presumably to higher reactivity of the short WCS chains than the longer CS chains.

Conjugation and Characterization of DBBN Onto WCS-GA
In order to conjugate tumor targeting biomolecules on to WCS-GA, DBBN was conjugated onto WCS-GA via the EDC/NSH coupling system (Scheme 1). The chemical structure of WCS allows the reactions of its -OH as well as -NH 2 groups with the COOH on DOTA in DBBN. EDC reacts with the -COOH group of DOTA, forming a reactive ester intermediate. However, this intermediate is prone to hydrolysis and might convert back into the parent carboxylic group. Hence, NHS was added which replaces the previous unstable ester into a much more stable NHS-ester species. With the conjugation on WCS-GA, the NHS-ester species then reacted with the primary -NH 2 or -OH groups to form an amide or ester bond, respectively. Confirmation of the conjugation of DBBN on WCS-GA has come from detailed analysis of the FTIR ( Figure 1A) and 1 H NMR ( Figure 1B Figure 1B(d)). It was confirmed that the DS of GA was ~19.80%, agreeing with the above calculations. Our BBN conjugation results from 1 H NMR also agree with previous literature. For example, the BBN conjugated N-acetyl histidine-glycol chitosan nanoparticles (BC-NAHis-GC NPs) also showed chemicals shift around 7.5-9.0 ppm attributed to the protons of the aromatic regions of BBN, confirming the conjugation of BBN onto NPs. 84 The chemical analysis of WCS-GA-DBBN was further performed by the energy dispersive X-ray (SEM-EDX) (Figure 2A). The WCS-GA-DBBN showed the characteristic X-ray of S at 2.3 keV) as attributed to a Kα X-ray of S in the BBN peptide structure, while WCS-GA did not show an S peak. The S peak was measured from several focal areas under a longer period of measurement with the SEM-EDX. In order to quantify the DBBN content in the WCS-GA-DBBN sample, we used a standard addition technique through SEM-EDX measurements. A series of S standardizations was prepared for determining the S amount in DBBN in the WCS-DBBN sample. The amount of DBBN peptide in the conjugated sample was calculated to be 27.04% (w/w). The S peak and its weight percentage found in the SEM-EDX result demonstrated that DBBN was successfully conjugated with WCS. The S peak and its weight percentage, determined from the SEM-EDX data, also confirmed the conjugation of DBBN peptide on WCS-GA. Our results corroborate with findings from recent reports showing the presence of S, C, N and O in the 177 Lu-dendrimer conjugated to BBN with AuNPs. 85

Morphology and Particles Size of WCS-GA-DBBN
The morphological information of WCS-GA-DBBN was elucidated through TEM. Both WCS-GA ( Figure 2B(a)) and WCS-GA-DBBN ( Figure 2B(b)) revealed spherical shapes and uniform size distribution ( Figure 2B

Antioxidant Activity of WCS-GA-DBBN
Based on our previous work, it has been found that functionalization of a radical scavenging group (ie, GA) onto the high molecular weight CS is beneficial to enhance not only solubility but also antioxidant efficiency. 67,69 Even with 15% of GA moiety on CS, the galloyl group provided effective transferring of H-atom and subsequently the forming of stable semiquinone radicals. It is important to note that an equivalent GA moiety of CS-GA could quench hydroxyl radical and exhibited synergistic function of CS with GA to retard the pro-oxidation of free molecular GA in the Fenton reaction. Besides CS gallate derivative, short chain WCS nanocolloids also showed superior antioxidant activity than the conventional CS in aqueous acetic acid solution. 17 WCS also has powerful reducing capacity to assist the template synthesis of AuNPs. 8 The antioxidant activities of WCS-GA and WCS-GA-BBN were determined in order to compare them with free WCS and GA molecules. Using DPPH standard free radical assay, it is possible to measure their antioxidant activities due to both hydrogen and electron transfer mechanisms. 86,87 The interaction of samples with DPPH radicals resulted in fast fading of the DPPH solution color from purple, corresponding to the UV−vis spectrum at 515 nm, to yellow ( Figure 2C in set). 88 Figure 2C shows a plot of the DPPH free radical scavenging activities of WCS, WCS-GA, WCS-GA-DBBN and GA against a wide range of concentrations. The WCS reached 100% radical scavenging activity at its concentration of 0.8 mg/ mL. The EC 50 values are expressed as an effective antioxidant concentration to reduce the radicals by 50%. The EC 50 of WCS was ~0.025 mg/mL. The EC 50 of WCS agrees with our previous report 17 and this also supports the high reproducibility of radiation-induced depolymerization of CS to create water-soluble nanocolloids of WCS.

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International Journal of Nanomedicine 2021:16 exhibited 5 times higher than the non-functionalized WCS.
It is important to note that the non-functionalized WCS also exhibited ~5 times more powerful antioxidant and reducing activities over the conventional CS in aqueous acetic acid. 17 These results corroborate well with our previous work when GA was functionalized on insoluble CS under dissolution in an acetic acid condition. 17 It has also been reported that the DPPH-scavenging activities were positively correlated with polyphenol contents in the chitosan-based conjugates. In addition, formation of ester or amide bonds of the synthetic GA derivatives could adapt to increase the antioxidant efficiency of the galloyl group based on a push-pull effect. 89 Our present work proves that GA moieties potentiate antioxidant activity of WCS. The EC 50 of CS-GA in our previous work was 0.14 mg/ mL whereas the EC 50 of WCS-GA in the present work is 0.012 mg/mL. The result suggests an enhancement in the antioxidant potential of WCS to almost 12 times greater antioxidant capacity than our original CS-GA reported in 2008 and 2010. 67,69 We then focused on the effects of BBN conjugation on antioxidant capacity. We found that DBBN peptide conjugation onto WCS slightly reduced the antioxidant capacity of WCS-GA. This may be attributed to the presence of polyphenol groups in GA, and the amino acid groups in the DBBN peptide. Our findings of antioxidant capacities, as outlined above, are rationalized in terms of the antioxidant capacities of polyphenol-protein complexes, which are always lower than the sum of the antioxidant capacities of the individual components. 90

One-Pot Synthesis of AuNPs
Green and one-pot synthesis of AuNPs were carried out using the WCS-GA and WCS-GA-DBBN as a template for reducing and stabilizing nanoparticles. The WCS plays dual roles of reduction and transformation of Au(III) to the corresponding Au nanoparticles, while at the same time the corona of the chitosan polymer stabilizes the surface atoms of gold nanoparticles from a plethora of its -NH 2 and -OH groups through electron transfer mechanism. Besides the main functional groups of CS, the WCS prepared from radiation-induced chain scission process also contains C=O ended groups. Considering the GA function on WCS (see in Scheme 2A) on the overall reduction of Au(III) to the corresponding nanoparticles, three main mechanisms of the polyphenolic antioxidant processes are important: the first one involves H atom transfer (route b1); the second one involves single electron transfer (route b2), and the third one involves metals chelation (route b3). We infer that all three mechanisms act in

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Tangthong et al synergy in the overall reduction of Au(III) to the WCS stabilized gold nanoparticles. 91,92 Various functional groups of the WCS structure, ie, -NH 2 (a1), -OH (a2), -C-O-C-(a3) and -C=O (a4) (see Scheme 2A) support the stabilization of surface atoms on AuNPs through strong electrostatic (see in Scheme 2A(a)) and steric mechanisms. 28 We also suggest that WCS chains possibly form complexation with Au(III) ions thus controlling the particle size of AuNPs in the overall polymer template effect. 8,93 To demonstrate the reduction capacity of WCS-GA due to GA moiety, AuNPs were synthesized in WCS-GA and the results compared with WCS as a synthetic template ( Figure 3A). The representative UV-vis spectra of AuNPs prepared in WCS-GA and WCS at 6 h are shown in Figure 3A. At 6 h, the AuNPs prepared in WCS-GA exhibited the characteristic surface plasmon resonance (SPR) at 530 nm, indicating successful formation of AuNPs. It is clear that WCS-GA exhibited superior reducing potential over WCS. In order to probe whether the WCS-GA could create AuNPs faster than non-functionalized WCS, the kinetic plot between the absorbance at maximum wavelength (λ max ) and reaction times was plotted ( Figure 3B). We observed that AuNPs were rapidly formed and almost reached the steady state at a short reaction time of 15 min. The AuNPs formation through the WCS-GA solution was evident within 15 min and reached a steady state at 1 h. The AuNPs formation also reached a steady state when they were prepared in nonfunctionalized WCS aqueous solution; however, it took a longer duration, of 24 h. 94 Considering the first steady state point, the ability of WCS-GA to promote rapid creation of AuNPs was 24-fold faster than WCS. As expected, the result confirms the significant ability of GA moiety on WCS to assist rapid reduction of Au(III) to AuNPs in this green one-pot nanotechnology synthesis route.
Formation of AuNPs using WCS-GA and WCS-GA-DBBN as templates was also assessed using UV-vis absorption ( Figure 3C and D). At a concentration of 0.5 mg/mL (filled color) of WCS (in gray) and WCS-GA-DBBN (in yellow), the narrow UV-vis peaks corresponding to the surface plasmon resonance (SPR) were found at 530 nm. An SPR absorption band at ~530 nm implied spherical particles of AuNPs. 95 When the concentrations increased to 1.0-2.0 mg/mL, the UV signal intensity reduced and the UV-vis spectra of both WCS-GA ( Figure 3C) and WCS-GA-DBBN ( Figure 3D) became broader and red shifted to a longer wavelength of ~560-620 nm. Figure 3E Figure 3F. The λ max increased from 530 nm to longer wavelengths of 600-620 nm for AuNPs prepared in WCS-GA at 1.0 and 1.5-2.0 mg/mL, respectively. Similarly, the λ max of AuNPs prepared in WCS-GA-DBBN also gradually increased from 530 nm to longer wavelengths of 560-620 nm when the concentrations increased from 0.5 to 1.0-2.0 mg/mL. The physical appearance of AuNPs solutions are depicted in Figure 3D (in set). The solution color changed from red wine appearance to almost purple, indicating the larger particle size of AuNPs. 17 The red color of gold colloid is attributed to SPR arising from the collective oscillation of free conduction electrons induced by an interacting electromagnetic field. 96 The UV absorption characteristic of metallic nanoparticles provides accurate insights on changes in particle sizes of AuNPs. 97 It is known that the position and intensity of the band depends on the size, surface morphology and aspect ratio of nanostructures. 98 The particle size is large due to agglomeration of AuNPs when prepared using high concentrations of reducing and stabilizing functionalized and conjugated polymers. It is well known that certain biomolecules not only act as reducing agents, but also provide capping corona for the stabilization of AuNP surface atoms. When the concentrations of Au(III) ions are high, the strong reducing ability of WCS-GA and WCS-GA-DBBN results in a rapid formation of numerous small metallic particles. The lack of polymeric biomolecules, needed for capping surface atoms, results in a continuous assembly of small nanoparticulates causing their aggregation. 99 Particle Morphology and Sizes of AuNPs Morphologies and particle sizes of AuNPs were visualized using TEM, HR-TEM and SEM-EDX. The TEM images and histograms of AuNPs size distribution are illustrated in Figures 4A and B. Most of the AuNPs prepared in WCS-GA ( Figure 4A(a)) and AuNPs-WCS-GA-DBBN ( Figure 4A(b)) exhibited spherical and uniform shapes with the equivalent average sizes of 21±5 and 19±4 nm, respectively. AuNPs with the other shapes, ie, round, triangle, pentagon, hexagonal, diamond and rod, were also observed in minor quantities, especially with WCS-GA-DBBN peptide conjugate template. The different particle shape of AuNPs is due to molecular template effects presented by the WCS-GA-DBBN structures. The templatebased rapid synthesis, as described above, has produced well-defined spherical-shaped AuNPs. Spherical-shaped AuNPs are taken up by cells more efficiently than othershaped nanostructures. 100 AuNPs were also analyzed using HRTEM coupled with XRD to confirm Au nanostructures constructed within the WCS-GA and WCS-GA-DBBN templates. The representative HRTEM images clearly showed the Au nanostructure ( Figure 4C). The XRD patterns of both AuNPs, prepared through WCS-GA and WCS-GA-  Figure 4D). These are attributed to the planes (111), (200), (220) and (311) of the face-centered cubic Au, respectively, in agreement with previously reported data. 24 The high intensity diffraction peak at 38.3° is attributed to the Au crystal lattice plane (111) with an inter-planar distance of ~2.3 °A.
In order to establish the conjugation of sulfur from the BBN peptide on the surface of AuNPs through Au-S bonding, we used the SEM-EDX technique. Figure 4E(a) provides the overall morphology of AuNPs-WCS-GA-DBBN. The mapping profile of the Au and S elements including their overlay are presented in Figure 4E(b-d).
A characteristic spectrum of Au Lα X-rays at 9.711 keV and S K α X-rays at 2.309 keV were observed and used to formulate their mapping profiles. The red color profile reveals the Au element of the AuNPs, whereas the green profile indicates the S element of the BBN structure. As shown in the overlay elemental mapping ( Figure 4E(d)), the AuNPs containing the S element on the surface implies that the BBN targeting peptide was anchored with the AuNPs through the WCS-DOTA-DBBN conjugate polymer template. The overall SEM-EDX results demonstrated successful formation of AuNPs conjugated to the tumor-specific BBN peptide for applications as targeted diagnostic, therapeutic or theranostic agents in treating tumors.

Stability of AuNPs
In order to explore further utility of BBN bound AuNPs in imaging and therapy, we studied the kinetic behavior of AuNPs conjugated to WCS-GA and WCS-GA-DBBN. The stability was measured as a function of surface plasmon resonance by monitoring λ max , D H as well as zeta potential in various biologically relevant media over a 0-96 h time period (Figure 5A and B). The AuNPs prepared using 0.5 mM of HAuCl 4 and 0.5 mg/mL of WCS-GA (or WCS-GA-DBBN) and the reactions conducted for various time periods and several batches of AuNPs were subjected to stability measurements. Figure 5A shows that the SPR absorbance λ max dramatically increased when the duration of reactions increased from an initial time interval of 15 min to 1 h. At 15 min, the formation of AuNPs using the WCS-GA template was ~3-fold faster than the WCS-GA-DBBN template. At the steady state, the amount of AuNPs-WCS-GA is ~1.5-fold higher than that of AuNPs-WCS-GA-DBBN. Although the amount of AuNPs when using the WCS-GA-DBBN was lower than that produced through the WCS-GA template, the rate of formation of AuNPs through the WCS-GA-DBBN template was significantly higher than that of the WCS-GA template. The results suggested that the lower amount of AuNPs in WCS-GA-DBBN was due to the lower reducing power of WCS-GA-DBBN than the WCS-GA template, as described above.
The yields of AuNPs from both WCS-GA and WCS-GA-DBBN templates reached a maximum amount at around 1 h. After 1 h, the intensities of the SPR bands of AuNPs-WCS-GA remained unchanged when aliquots of reaction mixtures at 2, 4 and 24 h were monitored, thus suggesting complete transformation of Au (III) ions in to the corresponding AuNPs within 1 h. Our results corroborate with previous investigations where it has been reported that the SPR wavelength and maximum absorbance of AuNPs synthesized in aqueous extract oils from palm leaves reached a plateau after 1 h, indicating complete reduction of Au(III) precursor to AuNPs. 94 We observed that the AuNPs prepared using the WCS-GA template were stable within the time intervals of 1-6 h and the stability gradually reduced by ~30% at 96 h. In the case of AuNPs prepared using the WCS-GA-DBBN template, although the absorbances at λ max gradually declined after 1 h, their stability reduced by only ~8% at 96 h. Overall, our extensive investigations of stability of nanoparticles revealed that the AuNPs-WCS-GA-DBBN nanoparticles exhibited prolonged stability for over 3 days. The results suggested that WCS-GA-DBBN corona on AuNPs exert significantly higher stability than that of the WCS-GA corona. Figure 5B shows the D H results of AuNPs-WCS-GA and AuNPs-WCS-GA-DBBN within the same time intervals of 15 min to 96 h. The increase in D H ( Figure 5B) corresponded with the maximum absorbance λ max data ( Figure 5A). Generally, the D H increased because of the particle agglomeration leading to a decrease in the particulate amounts which was reflected in the reduction of absorbance. The The size of the NPs is of pivotal significance as they dictate blood circulation time and tumor accumulation. The NPs smaller than 20 nm are better candidates for deep tumor penetration, but they are easily removed from blood circulation. 101,102 The size of NPs used in any drug delivery system should be small enough to escape capture by fixed macrophages that are lodged in the reticuloendothelial system, such as the liver and spleen, but should be large enough to prevent their rapid leakage into blood capillaries. The size of the sinusoid in the spleen and fenestra of the Kupffer cells in the liver varies from 150 to 200 nm. 23 Thus, the size of AuNPs should be appropriate not exceeding 150 nm. In addition, NPs of about 100 nm have longer blood circulation and better tumor accumulation due to the enhanced permeability and retention effects. These data, taken together, suggest that the AuNPs-WCS-GA-DBBN exhibiting D H in the range of 40-60 nm are highly suited to a myriad of applications as diagnostic and therapeutic nanomedicine agents. Furthermore, zeta potential is an important indicator for assessing the stability of nanoparticles. 103 Therefore, zeta potentials of AuNPs were measured to investigate their surface charges and stabilities ( Figure 5C). At 15-30 min, the zeta potential of AuNPs-WCS-GA was +12-13 mV while for the AuNPs-WCS-GA-DBBN it was +15-17 mV. For AuNPs-WCS-GA-DBBN, the zeta potential was +25 mV within the time intervals of 1-5 h and approximately +20 mV within the time intervals of 6-96 h. The results suggest that AuNPs which were developed rapidly at 15 min exhibited lower stability than those formed at 1 h. It is therefore important to recognize that the AuNPs-WCS-GA-DBBN exhibited optimum in vitro stability lasting up to 3 days. Generally, those AuNPs that exhibit zeta potentials greater than +20 mV or less than -20 mV possess sufficient electrostatic repulsions to remain stable in solutions for extended periods. 104 The positive zeta potential values from the highly charged biomolecular corona on the surface of AuNPs enhance electrostatic repulsive forces between AuNPs and also exert steric effects, thus causing an overall increase in the in vitro stability of AuNPs. 105 This is clearly reflected for AuNPs-WCS-GA-DBBN which exhibit enhanced in vitro stability attributable to the WCS-GA-DBBN biomolecular corona that avoids nanoparticles from aggregation.

In vitro Stability of AuNPs
In the present study, we are interested in the development of highly stable AuNPs for use in biomedical applications. Therefore, we have examined the in vitro stability of AuNPs-WCS-GA-DBBN in distilled water in various biologically relevant media, including NaCl, Cysteine (Cys), Histidine (His), BSA, HSA and phosphate buffer at pH 5, 7 and 12. The in vitro stability of AuNPs in these biological fluids was monitored through changes in D H ( Figure 5D). The AuNPs solution samples were incubated in different biological media for 72 h before measurement. The D H of AuNPs-WCS-GA was 50 nm in DI water, Cys, His, BSA, HSA and PBS (pH 12) ( Figure 5D(a)), whereas the D H of AuNPs-WCS-GA dramatically increased in NaCl, PBS pH 5 and pH 7 solutions. The AuNPs-WCS-GA underwent agglomeration as reflected in their enhanced particle sizes of 735, 284 and 655 nm when incubated in NaCl ( Figure 5D (vi)), PBS pH 5 ( Figure 5D(vii)), and pH 7 ( Figure 5D (viii)), respectively. In contrast, the D H of AuNPs-WCS-GA-DBBN ( Figure 5D(b)) was 55-75 nm in all biological media. These results unequivocally suggest that AuNPs-WCS-GA-DBBN are stable in all biological media for 72 h with no observable changes in D H . It is thus conceivable that the WCS-GA-DBBN is not only creating AuNPs efficiently but also exerts stabilization architecture around the AuNPs in various biological fluids. Nanoparticles (NPs) of metal readily aggregate because of their relatively high surface energy and low atomic coordination number. NPs aggregation strongly depends on various parameters, including chemical composition, size, shape, surface coating, and physicochemical characteristics. 106 As shown in our investigations, AuNPs-WCS-GA-DBBN exhibited optimum stabilities in all biologically relevant media, whereas the AuNPs-WCS-GA showed instability in NaCl, PBS pH 5 and pH 7. It may be noted that functionalized gold nanoparticles in PBS buffer, with relatively high ionic strength, exhibit particle aggregation causing dispersion behavior of NPs and thus leading to destabilization and adhesion to surrounding substrates. 107 The solution pH also strongly affects the surface charge of AuNPs, which can alter the inter-relationship between the van der Waals attraction forces and electrostatic repulsive forces. 106 Therefore, nanoparticles solution pH is an important consideration for various pH-responsive cationic nanoparticles. 108 In this context, it is well known that NaCl plays an important role in increasing the particle size of CSbased NPs. 109 Additionally, CS can be deprotonated at pH 12, thus rendering CS-GA strongly capped and thus stabilizing the corresponding AuNPs. In the presence of DBBN peptide, the protonation of CS is hindered, and the steric effects of AuNPs-WCS-CS-GA appear to be significant, resulting in greater stability for these NPs. The outstanding stability of AuNPs-WCS-GA-DBBN is rationalized in terms of synergistic effects of GA and DBBN functions. The pKa of phenolic hydrogens of GA has been reported to be ~ 5, 110,111 thus the phenolic -OH of semiquinone is ionized at pH >5. 111 The phenolic -OH and their ionized forms would help in stabilizing additional H + at pH 5, thus precluding H + from protonation of CS. At higher pH of 7 and 12, the ionized phenolic -OH groups would cause electrostatic repulsions between individual NPs-thus aiding excellent stability in vitro and in vivo. Overall, the optimum nanoparticulate size and outstanding in vitro stability, as exhibited by the AuNPs-WCS-GA-DBBN, makes them ideal nanoplatforms for use in molecular imaging and therapy.

Cellular Uptake of AuNPs
A major advantage of gold nanoparticles is that they can be engineered to sizes to facilitate tumor cell-specific selectivity and thereby achieve tumor targeting. Toward this objective, we used bombesin peptide (BBN), which has selective and high affinity toward gastrin-releasing peptide (GRP) receptors that are overexpressed in a host of tumors, including breast, small cell lung, prostate and pancreatic cancers. The BBN conjugated AuNP-WCS-GA-DBBN nanoconjugate has been tested for its efficacy in targeting two different prostate cancer cells, the PC-3 and LNCaP. We treated these cancer cells with AuNPs (50 μg/mL) for 6 h and the cellular uptake and localization of AuNPs were visualized using confocal microscopic images and TEM images. The uptake of AuNPs-WCS-GA-DBBN in PC-3 and LNCaP cell lines is illustrated in Figure 6. Figure 6A shows the images of PC-3 cells before and after exposing them to AuNPs-WCS-GA ( Figure 6A cells. In comparison, we observed highly efficient internalization of AuNPs-WCS-GA-DBBN at 6 h, as shown in Figure 6A(c). This image shows that AuNPs have surrounded the nucleus and are bound by the plasma membrane. High magnification of cellular internalization, as shown in Figure 6A(c), indicated the presence of a significant amount of AuNPs in the PC-3 cells, as seen in position numbers 1, 2, 3 and 4 and presented in yellow spots. It is conceivable that AuNPs-WCS-DBBN internalization in the prostate cancer cells is occurring through GRP receptor mediated endocytosis (Scheme 2B) because prostate tumor cells have an abundance of GRP surface receptors, and BBN targets such receptors. It is interesting to note that AuNPs-WCS-GA-DBBN showed a lower propensity to internalize the LNCaP prostate tumor cells as compared to the PC3 cells. It is well known that AuNPs in sizes less than 200 nm are internalized into the cells through micropinocytosis (clathrin-mediated, caveolae/lipid raft-mediated, and clathrin/caveolae-independent). 112 In general, AuNPs are internalized from the cell surface through endocytosis and confined to the endosome (Scheme 2). The uptake of AuNPs in PC-3 cells is predominantly due to the surface chemistry of the targeted AuNPs correlating to the number of GRP receptors on the cells. 113 This can occur through either receptor-mediated endocytosis, which was expected for the targeted AuNPs, or through pinocytosis, which is known for the nonfunctionalized AuNPs. On the other hand, the particles can avoid the endosomal pathway by being taken up directly into the cytosol using cell-penetrating peptides. However, the BBN peptide used is not a cell-penetrating peptide. Therefore, we infer the mechanism of cell internalization to be receptor-mediated endocytosis for our AuNPs. The highly efficient cellular uptake of AuNPs demonstrates GRP receptor affinity and validates our hypothesis on the creation of prostate tumor-specific AuNPs via a BBN peptide conjugated WCS biopolymer nanocolloid template. Detailed TEM images have been analyzed in order to elucidate the uptake of AuNPs into the cells, and gain insights into the mechanism of cell internalization (Figure 7). We observed dark spots corresponding to AuNPs (eg, areas (a) and (b)) in the cytoplasm of both PC-3 ( Figure 7A) and LNCaP ( Figure 7B) cells. AuNPs were found surrounding the nucleus and bound by the plasma membrane. With a high magnification of a 100 nm scale, we observed a significant amount of AuNPs as dark spots in the areas (a) and (b) in Figure 7 in both PC-3

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Tangthong et al and LNCaP cells. The particle shapes and sizes of AuNPs corresponded to those observed by TEM, as described in Figure 4, suggesting that the particles retained their size (~20 nm) and shape after internalization in tumor cells.

In vitro Cytotoxicity Evaluation of AuNPs on Prostate Cancer Cells
In our efforts to understand the effects of composition of various AuNPs on tumor cells, we investigated dosedependent cytotoxicity of various AuNPs on prostate cancer cell viability. Our objective was to evaluate cell viability of AuNPs conjugated WCS with and without GA functions. We performed MTT assays on PC-3 ( Figure 8A) and LNCaP ( Figure 8B) cells with both AuNPs-WCS and WCS-GA solutions at various concentrations (6.25, 12.5, 25, 50 and 100 µg/mL). The results indicated that AuNPs-WCS-GA exhibited significant reduction of cell viability as compared to AuNPs-WCS, suggesting that the GA functional group exerts significant effects on damaging of PC-3 prostate cancer cells. The inhibition concentration to reduce cell viability by 50% (IC 50 ) of AuNPs-WCS-GA was found to be 6.13 µg/mL. For AuNPs-WCS, no IC 50 value and no significant reduction of PC-3 cells viability was observed within the concentration range of 6.25-100 µg/mL. The IC 50 of GA is approximately 150 μM (10.45 µg/ mL) and 50 μM (3.48 µg/mL) for human breast cancer cells for 24 and 48 h. 114 In a similar study using the PC-3 cells, it was found that the IC 50 calculated for GA was 50 μM (3.48 µg/mL) at 48 h. These results suggest that GA is a potent anticancer agent in the treatment of prostate cancer. 115 Although pure GA exhibited a 1.70-fold higher efficiency in damaging PC-3 cells than the AuNPs-WCS-GA-DBBN (IC 50 = 5.94 µg/mL) nanoconjugate, it is important to note that WCS-GA contained only ~15% GA conjugated on the WCS. 115 In addition, it has been reported that free GA may also promote prooxidation and that the CS gallate derivative provides synergistic effects for antioxidation which may result in reduction of toxicity or side effects in the healthy cells. 67 In order to verify how the BBN targeting peptide might influence prostate cancer cell viability, we performed MTT assays using AuNPs with and without the BBN peptide ( Figure 9). Various concentrations of AuNPs-WCS-GA and AuNPs-WCS-GA-DBBN were treated with both PC-3 and LNCaP cells, as shown in Figures 9A and B, respectively. For PC-3 cells, we observed that the IC 50 values of AuNPs-WCS-GA-DBBN were 5.05, 5.94 and 3.65 µg/mL. These values were lower as compared to the AuNPs-WCS-GA, which showed IC 50 values of 6.13, 12.05 and 8.75 µg/mL at 24, 48 and 72 h, respectively. At 24 h, both AuNPs-WCS-GA and AuNPs-WCS-GA-DBBN showed significant damage to both PC-3 and LNCaP cells. It was found that the PC-3 cell viability dramatically reduced even at a low concentration of 6.25 µg/mL, whereas the LNCaP cell viability showed no significant change even at concentrations higher than 6.25 µg/ mL. The cell viability significantly reduced at 72 h for both types of AuNPs. Compared to AuNPs-WCS-GA (IC 50 : 8.74 µg/mL), AuNPs-WCS-GA-DBBN showed In our investigations with LNCaP cells (Figure 9B), we observed that the cell viability for AuNPs-WCS-GA was lower as compared to the AuNPs-WCS-GA-DBBN nanoconjugate at the initial time of 24 h and showed no significant difference in cell viability at 48 h. However, no IC 50 was observed for AuNPs-WCS-GA at 24 and 48 h. Compared to AuNPs-WCS-GA, considering cell viability at 72 h ( Figure 9B(c)), AuNPs-WCS-GA-DBBN showed more impact in damaging LNCaP cells with IC 50 5.49 µg/mL. It is important to note that AuNPs-WCS-GA exhibited no IC 50 at all incubation times studied.
Overall, our MTT assay results infer that BBN, in the AuNPs-WCS-GA-DBBN nanoconjugate, exerted significant damage to both the PC-3 and LNCaP prostate cancer cells as compared to the AuNPs-WCS-GA which has no BBN peptide. This enhanced tumor cell killing effect may be rationalized in terms of tumor cell receptor specificity of the BBN peptide functionalized AuNPs-WCS-GA-DBBN nanomedicine agent, which enhances internalization of AuNPs into tumor cells more than does the non-BBN conjugate AuNPs-WCS-GA. The potent anticancer activities, against the PC-3 and LNCaP cell lines, for AuNPs-WCS-GA-DBBN is presumably occurring via induction of apoptosis because the cationic NPs are able to bind the negatively charged DNA. Moreover, CS also exerts a strong electrostatic interaction with the negative charge of the tumor cells through the positive charge of amine groups of CS. When DNA binds to highly positive NPs, it wraps around the NPs and bends. This bending generally causes the DNA damage.

Conclusions
Our discovery, as outlined in this paper, encompasses the creation of a multifunctional WCS-GA-DBBN nanomedicine agent containing: (i) antioxidant/reducing functions due to WCS and polyphenolic GA moieties, (ii) gold nanoparticle stabilizable functions due to the steric hindrance of the cationic biopolymer of WCS, and (iii) tumor cell specific targeting function afforded by the BBN peptide vector in AuNPs-WCS-GA-DBBN. The WCS-GA and WCS-GA-DBBN templates exhibited a propensity for the rapid, efficient and reproducible synthesis of functionalized AuNPs within 15 min and achieved a steady state at 1 h. We discovered that incorporation of gallic acid (GA) functionality in WCS-GA and WCS-GA-DBBN enhanced the reduction power to transform Au(III) to the corresponding functionalized biocompatible gold nanoparticles.
Our investigations have unequivocally demonstrated that incorporation of tumor cell specific bombesin (BBN) peptide within the AuNPs-WCS-GA-DBBN nanomedicine agent enhanced intracellular uptake selectively with uniform distribution within the cytoplasm of prostate cancer cells (PC-3 and LNCaP). The higher density of GRP receptors in PC-3 cells resulted in an efficient endocytosis of AuNPs-WCS-GA-DBBN within tumor cells. Overall, our detailed investigations on the discovery of a new nanomedicine architecture consisting of a chitosan DovePress platform conjugated to both theranostic gold nanoparticles and a tumor-specific BBN peptide, AuNPs-WCS-GA-DBBN, will provide new opportunities for the selective and efficient delivery of antitumor nanopharmaceuticals for applications in oncology.