An effective strategy for development of docetaxel encapsulated gold nanoformulations for treatment of prostate cancer

Nanoformulation based drug delivery is one of the most important research areas in the field of nanomedicine, which provides promising alternatives to the limitations of conventional chemotherapy. Nano drug delivery enables improved pharmacokinetic profile, bioavailability and therapeutic efficiency compared to the regular chemotherapeutic drugs. Herein, we have established a simple method for the synthesis of docetaxel (Dtx) encapsulated poly (ethylene glycol) (PEG) functionalized gold nanoparticles (AuNPs) for targeted drug delivery to prostate cancer. AuNPs were synthesized by the citrate ion reduction method followed by functionalization with thiol-PEG-amine (SH-PEG-NH2). SH-PEG-NH2 functionalized AuNPs were conjugated with the targeting vehicle, folic acid (FA). The anticancer drug, Dtx was encapsulated within AuNPs by the non-covalent linkage method. The physicochemical characteristics of the synthesized nanoformulations were extensively characterized by various spectral and microscopic studies. HR-TEM indicates the average size of the AuNPs is 16 nm and the nanoformulations is 18 nm. The encapsulation efficiency of the Dtx is ~ 96% which is confirmed by the elemental mapping analysis. The in vitro drug release profile of Dtx and AuNPs nanoformulations were studied by the dialysis membrane method. The anticancer activity of docetaxel encapsulated AuNPs were evaluated with prostate cancer cell lines (PC3). The drug encapsulated nanoformulations reduced the cell viability to about 40% (40 µM concentration at 24, 48 and 72 h of treatment). The optical microscopy observation reveals that the damage of prostate cancer cells after exposure to Dtx encapsulated AuNPs. The good cytotoxic activity of the present nanoformulation against prostate cancer cell lines enables its application for targeted drug delivery to prostate cancer.


Methods
Synthesis of gold nanoparticles. The modified procedure has been used for the synthesis of gold nanoparticles from gold (III) chloride trihydrate by chemical reduction method using trisodium citrate as a reducing agent 25,[56][57][58] . Briefly, 5 mL of 1 wt% of trisodium citrate in aqueous medium was added by dropwise to 200 mL aliquot of 1 mM HAuCl 4 ·3H 2 O boiling with stirring the solution under reflux condition. After adding the reducing agent, the reaction mixture color was changed from golden yellow to colorless. This reaction was continued until the solution turned to wine red. This color change appeared within 3 min due to the reduction of Au (III) to Au 0 and the reaction was completed within 5 min. The obtained colloidal suspension was allowed to cool at room temperature and the colloidal suspension was sonicated for 15 min at 37 kHz. Followed by, the loosely bounded citrate ions were removed by centrifugation for 30 min at 0 °C (10,000 rpm). The resulted AuNPs solution was stored at 4 °C and used further characterization. Figure 1 illustrates the mechanism for the synthesis of gold nanoformulations.
Functionalization of gold nanoparticles. Synthesized citrate capped AuNPs were functionalized with SH-PEG-NH 2 . 2 mg of SH-PEG-NH 2 was dissolved in 5 mL Millipore water, then added drop-wise into 100 mL of 1 mM concentration of AuNPs solution and stirred for 12 h at room temperature. After functionalization, the solution was maintained the pH range of 6.5 to prolong the stability of the nanoparticles. Further, the solution was sonicated for 30 min and centrifuged against Millipore water at 10,000 rpm for 30 min at 0 °C. The stable SH-PEG-NH 2 functionalized AuNPs solution was stored at 4 °C and used further characterization.
Fabrication of FA conjugated AuNPs. In order to fabricate FA conjugated AuNPs, 1 mM of FA was dissolved in 10 mL of DMSO and sonicated for 15 min. The sonicated solution was centrifuged at 10,000 rpm for 20 min at 0 °C. The prepared FA solution was mixed with N-(3-dimethyl aminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy succinamide (NHS) and the molar ratio of the FA: EDC: NHS was of 10:1:1 and the reaction solution was maintained the pH at 6.5. The reaction mixture was continuously stirring for 12 h followed by sonication and centrifuged at 10,000 rpm for 30 min. The filtered solution was mixed with SH-PEG-NH 2 functionalized AuNPs and stirred continuously for 6 h. The resulted solution was sonicated and centrifuged at 10,000 rpm for 10 min at 0 °C for three times. The fabricated AuNPs/PEG/FA solution was stored at 4 °C and used further characterization.
Fabrication of docetaxel encapsulated AuNPs. 1 mM of Dtx was dissolved in 5 mL ethanol, water and tween 80 (1:1:0.25 ratio) solution and sonicated for 15 min. The sonicated solution was centrifuged at 10,000 rpm for 20 min at 0 °C. This solution was added into the SH-PEG-NH 2 functionalized AuNPs in 50 mL beaker using the non-covalent binding method with continuously stirred for 4 h. The prepared AuNPs/PEG/Dtx/FA solution was sonicated in an ice bath for 30 min, followed by centrifuging at 10,000 rpm for 20 min at 0 °C. The obtained solution was stored at 4 °C and used for further characterization.
Characterization of Dtx encapsulated gold nanoformulations. The physicochemical properties and step-by-step conjugation of the synthesized AuNPs nanoformulations were extensively characterized by various analytical techniques. UV-Vis absorption spectra were carried out by using Agilent diodaris spectrophotometer and Cary-8453, USA. The surface functional groups of the nanoformulations were determined by FT-IR spectrophotometer (Bruker, Vertex, 80 V, USA). The crystallinity and phase purity of the nanoformulations were studied by XRD analysis (Bruker D8 advance PXRD and Rigaku X-ray diffractometer, Smart lab, UK). The chemical composition and oxidation state were evaluated by X-ray photoelectron spectroscopy (XPS, Omicron Nanotechnology, ESCA-14 (Germany). The surface morphology and elemental composition of the nanoformulations were examined by a high-resolution transmission electron microscope (HR-TEM) ((JEOL-2100-JEM) and (Bruker)) and field emission scanning electron microscope (FE-SEM) (Hitachi, SU-6600-Japan) instrument with energy dispersive X-ray spectroscopy (EDS). The chemical bond and surface modification of the nanoformulations were determined by Raman spectrophotometer (Xplora Plus, Raman spectrometer, Horiba, Japan with laser excitation of 785 nm). The anticancer activity of the AuNPs nanoformulations was analyzed by ELIZA Reader (Enzyme-Linked Immunosorbent Assay) microplate reader (Robonik, Mumbai, India) with the excitation wavelength of 460 nm. In vitro drug release. In vitro drug release kinetics of optimized Dtx encapsulated AuNPs nanoformulations and the free drug was investigated by the dialysis membrane method. Briefly, 75 mL of phosphate-buffered solution (pH 7.4) and 5 mL of 0.1% tween 80 was taken as release media in 150 mL borosil glass beaker. After that, 5 mL of nanoformulations were poured into the dialysis membrane (molecular weight cut-off 12-14 kDa) and immersed in an 80 mL PBS solution with the help of a glass rod. The bag was immersed centrally into the release media using a burette stand. The beaker was placed on the magnetic stirrer with maintained a constant rotation of 300 rpm with a magnetic pellet. The release kinetics was predetermined at different time intervals like 1-72 h. At a notable time interval, 3 mL of drug release medium was aspirated and replaced 3 mL fresh drug release media and also maintained the constant bath volume. The absorbance of the samples was recorded in a UV-Vis spectrophotometer at λmax of Dtx at 230 nm, FA at 280 and 344 nm, and AuNPs at 528 nm. The calibration curve was calculated with different concentrations of Dtx in the PBS solution.
The in vitro drug release kinetics were analyzed by different release kinetic models like zero-order, first-order, Higuchi kinetics, Korsmeyer-Peppas model, and Hixon-Crowell model which enables the identification of the R 2 value (correlation coefficient) 60 .

Cytotoxicity analysis of gold nanoformulations.
In vitro cytotoxicity analysis used to investigate the cell viability or metabolic activity of prostate cancer cell line (PC3) against gold nanoformulations. This colorimetric assay is highly sensitive and reliable to the transformation of the metabolic activity of the cells. Briefly, 100 µL (2500 cells per well) of PC3 cells were harvested, counted, and seeded in flat-bottom 96 well plate (Corning USA) and then incubated for 24 h at 37 °C under 5% CO 2 atmospheric conditions which used to adhere the cells. After 24 h, the cells were washed with PBS (Phosphate Buffer Saline solution) solution twice. Subsequently, the cells were treated with different concentration of synthesized AuNPs nanoformulations (10 to 60 µM) includes free AuNPs (10 to 125 µM), PEG functionalized AuNPs (10 to 125 µM), FA (10 to 60 µM), and Dtx (10 to 60 µM) and incubated for different time intervals like 24 h, 48 h, and 72 h. The AuNPs nanoformulations free cells are considered as control and the experiments were carried out in triplicate. The selected concentration of the nanoformulations which enables the drug particles were reached in the cancerous cells at the plasma level. After the incubation time interval, the HAMS F12 medium was aspirated and washed with PBS two times. Then, 3 µL of Premix WST assay solution was added in each well and the cells incubated for 4 h in dark condition. In this period, the metabolically active cells to transform WST into the formation of insoluble Formazan crystals. Followed by, the optical absorbance of cells was measure immediately at 460 nm using ELIZA (Enzyme-Linked Immunosorbent assay) microplate reader (Robonik, Mumbai, India). All the experiments were carried out in triplicate. The cell viability was calculated by the following equations.
Statistical analysis. The statistical analysis was expressed as a mean ± standard deviation by SPSS 16.0 software (Name: IBM SPSS Software, Version Number: SPSS16.0, and URL Link: https ://www.ibm.com/in-en/ produ cts/spss-stati stics .Chica go, IL,USA).The statistical comparison was carried out all the AuNPs nanoformulations by one-way analysis of variance (ANOVA) by post hoc hypothesis testing (Tukey test). The p-value is less than 0.05 (p < 0.05) was considered to be a significance level.

Results and discussion
Synthesis of Dtx encapsulated AuNPs nanoformulations. The stability of the colloidal AuNPs is a very important feature for surface functionalization. The colloidal suspension of AuNPs was synthesized by chemical reduction method (citrate ion acts as a reducing agent and stabilizing agent). UV-Vis absorption spectra of the citrate capped AuNPs exhibited the Surface Plasmon Resonance (SPR) band at 527 nm as shown in Fig. 2a. The magnitude of the SPR peak depends on the concentration and the morphology of the synthesized AuNPs. This absorption spectrum indicates that the increased concentration of the AuNPs and the SPR peak height also increased to some extent the absorption peak gets broaden. Followed by, AuNPs were functionalized with thiolated PEG amine (SH-PEG-NH 2 ). During the thiolation of AuNPs, AuNPs appeared rapid agglomeration which exhibited the SPR signal of PEG functionalized AuNPs observed at 528 nm (Fig. 2b). To overcome this agglomeration of AuNPs, during the thiolation process the AuNPs solution pH was adjusted at 6.5 61 . This pH condition, PEG functionalized AuNPs are highly stable to prevent the agglomeration 62 . The stable solution of PEG functionalized AuNPs were conjugated with Au-S bond and amine (-NH 2 ) groups. Thereafter, PEG functionalized AuNPs were conjugated with FA by using EDC/NHS coupling method (the free amine (-NH 2 ) groups were bonded with the carboxylic group (-COOH) and formed a strong bond between PEG-AuNPs and FA). UV-Vis absorption spectra of FA were observed in the two bands at 277 and 367 nm (Fig. 2d). The FA conjugated PEG functionalized AuNPs, it was observed that the one molecule of FA was attached per six molecules of PEG functionalized AuNPs. This result indicates that the FA was partially attached over the surface of PEG functionalized AuNPs, which exhibited the steric hindrance of the bulky structure of FA. The SPR signal of the FA conjugated PEG functionalized AuNPs indicated that the 528 nm, 344 nm, and 277 nm (Fig. 2e). These results suggest that the FA were strongly attached over the surfaces of PEG functionalized AuNPs. Subsequently,  (Fig. 3e). It is evident that the peak at 3454 cm −1 , 1659 cm −1 and 1031 cm −1 corresponds to the formation of primary amine and sulfoxide groups are conjugated over folic acid using EDC/NHS coupling method. FTIR spectra of AuNPs nanoformulations exhibits the major signals at 3308 cm −1 corresponds to the OH stretching vibration of carboxylic groups (Fig. 3f). The band at 2907 cm −1 corresponds to the CH stretching vibration of alkanes. The band at 2118 cm −1 corresponds to the atmospheric CO 2 /CΞC week stretching vibration of alkyne. The strongest peak at 1630 cm −1 attributed to the C=C stretching vibration of alkane. The intense band at 1439 cm −1 and 1316 cm −1 ascribed to the C-H and O-H plane bending vibration of alkanes. The weak band observed at 1009 cm −1 is attributed to the C-N/S=O stretching vibration of amine and sulfonate are present in the nanoformulations 65 . A small band at 905 corresponds to the C=C bending vibration of alkane is present in the nanoformulations. Evidently, the FTIR spectra exhibited the FA conjugation with PEG functionalized   www.nature.com/scientificreports/ The size of the nanoparticles was measured indirectly using a broad bottom width of (111) reflection which indicates the smaller size of the nanoparticles. Debye-Scherrer's equation was used to determine the size of the nanoparticles by (111) width of the Bragg reflection signal. The size of the AuNPs was found to be ~ 18 nm. The (111) reflection peak exhibited the strong shoulder peak rest of the peaks are weak peaks which indicate the predominant orientation. There is no small impurities peaks were observed which shows the synthesized AuNPs are highly pure. Interestingly, the XRD pattern of the SH-PEG-NH 2 functionalized AuNPs exhibited diffraction band is similar to AuNPs (Fig. 4c), whereas, the XRD pattern of the characteristics diffraction peak of FA at 10.9°, 13.0°, 16.2°, 16.90°, and 27.2° corresponds to the (110), (011), (101), (220) and (002), respectively (Fig. 4d) 68 (Fig. 4f). On comparison with FA, Dtx and AuNPs, the diffraction peaks of the FA and Dtx signals are slightly suppressed due to the strong attachment between FA-AuNPs-PEG-Dtx. Thus, the XRD analysis confirmed the obtained AuNPs were FCC crystal structure and the step-by-step functionalization of SH-PEG-NH 2 and FA over AuNPs and Dtx encapsulated over the surface of AuNPs-PEG-FA. The synthesized AuNPs nanoformulations were highly stable in the aqueous medium over the period of one year at room temperature.
Morphological and elemental mapping analysis of AuNPs nanoformulations. The surface morphology and microstructure of the synthesized gold nanoformulations were determined by HR-TEM analysis. Typical TEM micrographs of the citrate capped AuNPs indicates that the particles are spherical in shape and uniformly distributed over the copper substrate with an average dimension of 16 nm (Fig. 5a-d). AuNPs were uniformly attached over the SH-PEG-NH 2 (Fig. 5e-h). The average diameter is 24 nm, the slight increase in the dimension of AuNPs is due to the strong interaction between (Au-S) AuNPs-PEG. Figure S1a-d exhibits the FA particles are spherical in shape and the particles are observed the average dimension of 90 nm. After, FA conjugate over the surface of AuNPs-PEG exhibited particles are spherical in shape with an average dimension of 16 nm with slight changes in their shape (Fig. 5i-l). This result confirms that the FA conjugated over the AuNPs-PEG and the interaction between FA and AuNPs through hydrogen bonding. Figure S1e-h depicts the Dtx particles observed the core-shell morphology and uniformly distributed over the substrate with an average dimension of 290 nm. Figure 5m-p exhibits the Dtx encapsulated PEG functionalized AuNPs observed the particles are spherical in shape with slight changes in the size of the particles. Finally, the gold nanoformulations (AuNPs/PEG/Dtx/FA) exhibits that the particles are spherical in shape with uniformly distributed over the substrate with an average dimension of 18 nm, without any aggregation (Fig. 5q-t). Figure 5d shows the SAED pattern of the AuNPs and the particles are highly crystalline in nature. The crystal lattice spacing distances of AuNPs is about 4.0786 Å. These results are in accordance with the (111), (200), (220) and (311) diffraction planes of gold (JCPDS ID # 00-002-1095). Thus, the results indicate that the gold particles are arranged in a face centered cubic crystals system with λ = 1.5406 Å. These results were compared with the XRD pattern. The SAED pattern of the gold nanoformulations was observed the particles are highly crystalline. Hence, this result confirms the step-by-step conjugation of Dtx encapsulated gold nanoformulations are highly crystalline nature without changing the crystalline morphology.
The elemental compositions of the synthesized gold nanoformulations were analyzed by EDS analysis (Fig. 5t). The results indicate the carbon of 34.09 wt%, nitrogen of 10.06 wt%, oxygen of 43.22 wt%, Sulfur of 0.22 wt% and gold of 12.40 wt% elements are present in the sample. These results suggest that the synthesized AuNPs nanoformulations are highly pure with good crystallinity. Hence, TEM and EDS analyses confirmed the microstructure, size, morphology and elemental composition of the AuNPs nanoformulations during the stepwise conjugation and drug formulation. The synthesized nanoformulations were highly stable in aqueous medium over the period of one year at room temperature.
Field emission scanning electron microscope. Elemental mapping analysis of the synthesized nanoformulations was evaluated by FESEM analysis. Figure 6a shows the AuNPs nanoformulations exhibited low and high magnification. The high magnification image of nanoformulations exhibited the particles are uniformly arranged over the substrate which is used for elemental mapping analysis. These images could be used as a selected area for the mapping analysis. Elemental mapping analysis of the nanoformulations was clearly indicating the Dtx and FA particles embedded with PEG functionalized AuNPs (Fig. 6b). The mapping images of nanoformulations showed the embedded elements of CK = 41%, NK = 9%, OK = 30%, metallic gold (AuM = 12%), and Sk = 8%. Figure 6c depicts the mapping image of gold particles in the metallic form which indicates the gold particles are present in the sample. The mapping images of carbon, sulfur, nitrogen, and oxygen were exhibited in Fig. 6d-g, respectively. This image represents the synthesized gold nanoformulations are highly pure without any impurities.
The elemental composition of the gold nanoformulations was evaluated by EDS spectroscopy as shown in www.nature.com/scientificreports/ Dtx, AuNPs-PEG-Dtx, and AuNPs-PEG-FA-Dtx, which observed the strongest band of gold, sulfur, carbon, nitrogen, and oxygen (Au4f, S2p, C1s, N1s, and O1s), respectively. Figure 7b depicts the survey spectrum of the AuNPs. The valence band and binding energy of 4f core level spectrum of AuNPs have observed the peaks at 85.3 eV and 88.9 eV corresponds to the Au 4f 7/2 and Au 4f 5/2 spin orbitals, respectively (Fig. 7c) 25 . These binding energies were compared to the respective core levels of bulk Au crystals. Evidently, this result exhibited the metallic types of gold (Au 0 ) were present in the sample. Moreover, the resulted value of Au 4f 7/2 and Au 4f 5/2 exhibits the narrow width revealed that only a single element of gold was present in the system which indicates the synthesized AuNPs are highly stable. The Au4f core level spectrum of the SH-PEG-NH 2 functionalized AuNPs exhibited the two components at 85.22 eV and 89.01 eV corresponds to the Au 4f 7/2 and Au 4f 5/2 of spin www.nature.com/scientificreports/ orbitals of PEG-AuNPs bonds with alkyl thiol self-assembled AuNPs (Au-S), respectively ( Fig. 7f) whereas, the FA-PEG-AuNPs exhibited the two components at 83.55 eV and 86.88 eV corresponds to the core level of Au 4f 7/2 and Au 4f 5/2 spin-orbital, respectively and this value confirms the XPS traces of alkyl thiol self-assembled bonded over AuNPs (Fig. S3h). Figure S3a depicts the XPS survey spectrum of the free docetaxel which observed the traces of C1s, N1s, and O1s, whereas, Fig. 7k exhibits the XPS survey spectrum of gold nanoformulations. The Au4f core level XPS spectrum of the AuNPs-PEG-FA-Dtx exhibited the two components at 83.21 eV and 87.56 eV corresponds to the Au 4f 7/2 and Au 4f 5/2 spin-orbital, respectively (Fig. 7l). Hence, these XPS results confirm the functionalization of AuNPs nanoformulations. The C1s core level spectra of the AuNPs exhibited the two XPS traces at 286.93 eV and 290.92 eV attributed to the C-O-H, C=C/C-O-C groups of the carbon atom, respectively (Fig. 7d), whereas, PEG functionalized AuNPs exhibited three XPS signals at 286.76 eV, 287.74 eV and 290.68 eV correspond to the C−O or C−S, C=O (amide) and O-C=O group of the carbon atom, respectively (Fig. 7h) [70][71][72] . The appearance of the C1s XPS spectra of FA-AuNPs-PEG exhibited the signals at 284.02 eV, 285.69 eV ascribed to the C=C/C=N and C−C/C−H groups confirmed the attachment of FA, respectively (Fig. S3j). The Shifting of carbonyl carbon bond towards the lower binding energy level compared to PEG-AuNPs indicated the conversion of acid carbonyl group into amide carbonyl group and hence, the FA conjugation over the AuNPs-PEG via the formation of the amide bond. The C1s spectrum of FA exhibited the three XPS traces at 286.63 eV, 288.03 eV and 290.52 eV correspond to the C-N, C=O, and O-C=O, respectively (Fig. S3e), whereas, the Dtx spectrum exhibits the two signals at 297.96 eV and 301.02 eV corresponds to the O-C=O and C-O-C groups of carbon atoms (Fig. S3b). The Dtx encapsulated with FA conjugated PEG functionalized AuNPs exhibited the two components corresponding to the 284.05 eV and 286.69 eV corresponds to the C=C/C=N and C-C/C-H groups of the carbon atom, respectively (Fig. 7p). www.nature.com/scientificreports/ The O1s XPS spectra of the AuNPs exhibited a band at 534.37 eV attributed to C-O/C=O groups of the oxygen atom (Fig. 7e) 25,58,73 . PEG functionalized AuNPs exhibited the two XPS traces at 534.19 eV and 535.53 eV corresponds to the C=O and C-O-H groups of the oxygen atom, respectively (Fig. 7j). The appearance of O1s XPS spectra of the AuNPs-PEG-FA exhibited the signals at 532.65 eV corresponds to the carbonyl group of FA and the minor component at 533.78 eV attributed to the C-O-C group of the oxygen atom (Fig. S3l). The O1s traces of FA exhibited the components at 533.83 eV and 534.18 eV corresponds to the C-O-C and C-O-H groups of the oxygen atom, respectively (Fig. S3g). The XPS spectra of Dtx exhibited the signals at 544.9 eV and 545.66 eV corresponds to the C-O and C=O groups of the oxygen atom present in the samples, respectively (Fig. S3d). The gold nanoformulations exhibit the O1s traces at 533.09 eV and 534.26 eV correspond to the free carboxylic acid and C-O-H group of the oxygen atom (Fig. 7o). This result showed that the O1s trace of the amide carbonyl groups of Dtx was encapsulated over AuNPs-PEG-FA via amide carbonyl groups at 533.09 eV, which confirms the step-by-step conjugation of Dtx encapsulated AuNPs-PEG-FA nanoformulations.
The S2p XPS spectra of the PEG functionalized AuNPs exhibited the two signals at 161.18 eV and 162.38 eV along with a 1.2 eV gap corresponds to the S2p 3/2 and S2p 1/2 , (Fig. 7g) 74 of the SH-PEG-NH2 functionalized AuNPs, respectively. Spampinato et al. reported that the unbounded sulfur observed at 164 eV corresponds to S2p 3/2 , while, the sulfur traces bound over the surface of AuNPs appeared at 162 eV. The appearance of the FA conjugated PEG functionalized AuNPs exhibited the S2p 3/2 and S2p 1/2 XPS signals at 162.02 eV, 163.56 eV and 164 eV correspond to the Au-S, R-SH and S groups of sulfur atoms, respectively (Fig. S3i). The gold nanoformulations exhibit the S2p traces at 162.18 eV and 163.80 eV corresponds to the Au-S and S groups of sulfur atoms present in the sample, respectively (Fig. 7m) 75 . Similarly, the entire spectrum exhibited the S2p 3/2 XPS traces at 162 eV confirm the conjugation of PEG-FA-Dtx over AuNPs. The AuNPs did not show any N1s of XPS traces, whereas, N1s spectrum of the PEG-Functionalized AuNPs exhibited the XPS components at 401.27 eV and  (Fig. 8f). From the spectra, the shoulder peak at 260 cm −1 corresponds to the Au 0 strongly observed in the surface. The peaks at 451 cm −1 , 1078 cm −1 and 1253 cm −1 are attributed to the C-NH, C-N, C-H of carbonyl and amine groups are present in the sample, respectively. These peaks indicates the conjugation of AuNPs with FA and Dtx. The major peaks at 1385 cm −1 , 1455 cm −1 and 1597 cm −1 are ascribed to the C-H/NH, C-H and C=C of bending frequency of amine and carbonyl groups due to the formation of FA and Dtx. The combined Raman spectra of gold nanoformulations are provided due to comparison between the nanoparticles (Fig. 8a). Thus, the results suggest that the synthesized AuNPs are highly pure.
In vitro analysis of gold nanoformulations. The encapsulation efficiency of gold nanoformulations was observed at approximately ~ 95%. Here, 1 mM con of Dtx solution was prepared and encapsulated with PEG functionalized FA conjugated AuNPs by the non-covalent method. The resulted nanoformulations were sonicated and centrifuged at 10,000 rpm at 0 °C for 15 min. The obtained solution was used for further characterization and drug release studies. www.nature.com/scientificreports/ In vitro drug release studies. The controlled drug discharge profile exhibited the drug was smoothly circulated in blood over the period and which minimizes side effects, reduces drug usages, and maximizes therapeutic efficacy. In vitro, drug release pattern of free docetaxel and Dtx encapsulated gold nanoformulations were evaluated using the dialysis membrane diffusion method. The release pattern was expressed by the percentage of cumulative drug release against time in hours as shown in Fig. 9. Initially, a very fast and continuous drug (Dtx) release behavior was observed (i) burst discharge up to 45 to 50% in 1.5 h followed by (ii) the sustained discharge at the maximum level of 96% in 18 h (Fig. 9a). The cumulative drug discharge behavior of Dtx encapsulated gold nanoformulations were observed during the prolonged discharge. The Dtx encapsulated AuNPs nanoformulations exhibited the tri-phasic release pattern was observed. At first, burst discharge was observed about 28% of Dtx released from the nanoformulations in 16 h than, sustained discharge up to 35 h and finally slow with constant discharge up to 72 h at the maximum of the release of 96%. These results clearly indicate that the maximum Dtx release in gold nanoformulations was observed up to 96% and the discharge behavior (rate and style) varies which exhibited in Fig. 9b. This drug release system contains a tri-phasic discharge system, initial burst release due to the release of drugs from the surface of nanoparticles followed by the second phase which shows a sustained release due to the release of drugs from the matrix which represents the Fickian diffusion and finally, slow with a constant discharge of drugs from the nanoformulations. This release pattern has followed the characteristics of Higuchi's square root kinetics with a correlation coefficient of r = 0.9996. This drug discharge as follows the diffusion with erosion mechanism.
Cytotoxicity analysis of gold nanoformulations. The anticancer activity of the synthesized gold nanoformulations includes AuNPs, AuNPs-PEG, FA, Dtx and Dtx encapsulated gold nanoformulations (AuNPs-PEG-FA-Dtx) were investigated against prostate cancer cell of PC3 cells (Fig. 10a-h). Figure 10a and b clearly indicates that the cell viability of the AuNPs and PEG-AuNPs was observed the 75% and 60%, respectively at 72 h post treatment, this difference due to the amine and thiol groups are attached over the AuNPs surface. The above 60% of cell viability was detected at 72 h treatment of FA (Fig. 10d). This result indicates that the AuNPs-PEG-FA showed less cytotoxicity effect on PC3 cells up to 60 μM of AuNPs and 50 μM of FA over 72 h. The anticancer drug of docetaxel was detected the 50% cell viability of 40 μM (32.28 μg/mL) at 72 h treatment. IC 50 was determined to be 40 µM for free docetaxel at 48 h (Fig. 10c). At the same time point the concentrations for AuNPs, AuNPs-PEG and FA were also determined at which we have maximum cell death, thus contributing to an optimum value for the formation of nanoformulations. We have determined that at the maximum concentration of 60 µM for AuNPs and the cell death has approximately 25% of the control, while FA has almost 80% cell viability in the 50 µM concentrations. The results provide an idea of the optimal concentrations of the components of the nanoformulation with better cytotoxic activity. Interestingly, AuNPs-PEG-FA-Dtx exhibited only 60-70% of cell viability at the concentration of 10 µM to 60 µM at 24 h treatment, and the PC3 cells proliferation was decreased about 75%, 68%, 56%, 47 and 39% over 73 h (Fig. 10e). The result indicates that the potential cytotoxic effect of AuNPs-PEG-FA-Dtx against PC3 cells at 10 µM to 60 µM. Moreover, this results compared with Dtx to an increase in the cytotoxicity up to 50% and the significance (p < 0.05) results also investigated. The comparison between Dtx, FA and nanoformulations were observed different cytotoxic activity against prostate cancer cell lines as shown in Fig. 10f, g. The conjugation of FA into AuNPs-PEG-Dtx could increase the targeting efficacy of the obtained nanoformulations due to the relatively higher occurrence of folate receptors present on the surface of the PC3 cancer cells. However, the anticancer activity of the gold nanoformulations possesses to their anti-folate activity of folate-Dtx released from the AuNPs-PEG-FA-Dtx under lysosomal pH condition in PC3 cells. Hence, the anticancer activity of the AuNPs-PEG-FA-Dtx possesses potential nanoformulations for the promising chemotherapeutic agents for the treatment of various cancers. The morphological analysis of AuNPs nanoformulations, based on the cytotoxicity effect of the PC3 cell lines treated against nanoformulations has been visualized by optical microscope. The corresponding untreated cells as well as cells treated images are shown in Fig. 10. Figure 10h depicts the normal

Conclusion
In this work, we have established a simple, stable and highly promising AuNPs nanoformulations for targeted drug delivery to prostate cancer. The SH-PEG-NH 2 was successfully functionalized over citrate capped AuNPs without any agglomeration at pH 6.5 and FA conjugated onto AuNPs-PEG by EDC/NHS coupling and covalent linkage method. The anticancer drug, Dtx was encapsulated within AuNPs-PEG-FA by the non-covalent linkage method. The FTIR, Raman and XPS structural analysis confirmed the step-by-step chemical bonding of FA over AuNPs-PEG and Dtx over AuNPs-PEG-FA nanoformulations. The XRD and SAED pattern confirms the AuNPs nanoformulations exhibited the face centered cubic crystal structure of AuNPs. The HR-TEM and FE-SEM confirm the morphology and microstructure of the gold nanoformulations are spherical in shape with an average size of 16 nm and 18 nm for AuNPs and nanoformulations, respectively. The elemental mapping analysis also evident that the encapsulation of Dtx over AuNPs and the encapsulation efficiency of the Dtx was found to be ~ 96%. The drug release profile of the nanoformulations was found three different kinetics of slow, burst and sustained release which mainly follows the Higuchi kinetics. The cell viability of the gold nanoformulations treated against PC3 cells increases with increasing time, indicating that the nanoformulations decelerate cell proliferation of PC3 cells. Hence, the synthesized AuNPs-PEG-FA-Dtx nanoformulations could be used as an effective and alternative drug delivery system for prostate cancer treatment to achieve improved therapeutic efficacy with decreased drug dosage.