Disintegrable NIR Light Triggered Gold Nanorods Supported

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Disintegrable NIR Light Triggered Gold Nanorods Supported Liposomal Nanohybrids for Cancer Theranostics Deepak S. Chauhan, Rajendra Prasad, Janhavi Devrukhkar, Kaliaperumal Selvaraj, and Rohit Srivastava Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00801 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 28, 2017

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Bioconjugate Chemistry

Disintegrable NIR Light Triggered Gold Nanorods Supported Liposomal Nanohybrids for Cancer Theranostics Deepak S. Chauhana±, Rajendra Prasadbc±, Janhavi Devrukhkara, Kaliaperumal * Selvarajb*and Rohit Srivastavaa

Authors would like to dedicate this work to Sir Fraser Stoddart and Prof. Robert S. Langer. a

Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai, 400076, India Nano and Computational Materials Lab, Catalysis Division, CSIR-National Chemical Laboratory, Pune-411008, India c Academy of Scientific and Innovative Research (AcSIR), National Chemical Laboratory, Pune-411008, India b

± These Authors contributed equally to the work * *

Corresponding Author: [email protected] Co-Corresponding Author: [email protected]

Abstract In this work, facile synthesis and application of targeted, dual therapeutic gold nanorodsliposome (GNR-Lipos) nanohybrid for imaging guided photothermal therapy and chemotherapy is investigated. The dual therapeutic GNR-Lipos nanohybrid consists of GNR supported, and doxorubicin (DOX) loaded liposome. GNRs not only serve as a photothermal agent and increase the drug release in intracellular environment of cancer cells but also provide mechanical strength to liposomes by being decorated both inside and outside of bilayer surfaces. The designed nanohybrid shows a remarkable response for synergistic chemo-photothermal therapy compared to only chemotherapy or photothermal therapy. The NIR response, efficient uptake by the cells, the disintegration of GNR-Lipos nanohybrid and synergistic therapeutic effect of photothermal and chemotherapy over breast cancer cells-

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

MDA-MB-231 are studied for the better development of a biocompatible nanomaterial based multifunctional cancer theranostic agent.

Introduction High dose and heavy radiation of traditional therapies (chemo and radiotherapy) are frequently used in hospitals for the cancer treatment despite of several side effects1–3. Therefore, replacement of radiation therapy and chimeric chemotherapy with inert therapies like magnetic hyperthermia, photothermal therapy or photodynamic therapy, which have significantly less side effects is the current need for cancer theranostics4–10. The nanostructures based on imaging-guided cancer therapy are an alternative for conventional cancer therapies due to their specific binding to cancer cells and thenceforth quick and localized response to affected tissue1,11–15. Nanotechnology has presented numerous inorganic and organic nanostructures for their usage in diagnosis and therapeutics of several diseases16–20. Mesoporous silica, gold nanoparticles, graphene quantum nanodots, metal nanodots, upconversion nanoparticles, core-shell nanoparticles and various lipid assemblies have been researched extensively for their biomedical applications1,2,4–8,19,21–26. In addition, there is also a list of light responsive nanomaterials for imaging and triggered drug delivery4–8,11–15,24,25,27,28. However, engineering of biocompatible, targeted and multi-therapeutic agent for imaging-guided treatment is the current trend towards development of cancer nanomedicines4,29–31. Triggered release of the drug under NIR light exposure and generated hyperthermia work in a synergistic way for precisely controlling the drug diffusion in the cancer environment to regress the tumor11–14,27. Nanomaterials based imaging guided targeted and NIR-responsive synergistic chemo-photothermal therapy is a highly demanding field for cancer research. Inorganic nanocomposites/nanohybrids such as gold 2 ACS Paragon Plus Environment

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nanoparticles, silica coated gold nanoparticles, copper, and manganese-based nanoparticles have been extensively researched out4–8,11–15,18,24,25,27. However, the longterm toxicity, slow degradation, and non-specific uptake of these nanoparticles have always been a major concern for in vivo studies and further clinical applications32–36. Recently, gold nanorods liposome composite has been proposed as a new material for tissue visualization and photo triggered local anesthesia due to high cargo capacity of liposome and photothermal efficiency of GNRs37–39. Herein, we have synthesized biocompatible multifunctional GNRs supported liposome nanohybrid via one pot sonication method for imaging guided synergistic chemophotothermal therapy. The GNRs are found to be assembled on both the surfaces of the liposome (interior as well exterior lipid bilayer) providing strength to liposome and to prevent the premature drug release. To specifically target the overexpressed folate receptors on cancer cells, polyethylene glycol (PEG) functionalized folic acid (FA) was also attached to the surface of GNR-Lipos nanohybrid along with efficient encapsulation of anticancer drug- doxorubicin hydrochloride (DOX.HCL). To the best of our knowledge, synergistic chemo-photothermal therapy, efficient uptake and NIR assisted disassembly directed disintegration of GNR-Lipos nanohybrid are being reported for the first time.

Results and discussion The novel design, multifunctional application and disintegration of NIR-responsive targeted GNR-Lipos nanohybrid for cancer theranostics is illustrated in Fig. 1. Prior to the synthesis of GNR-Lipos nanohybrid, GNRs were prepared according to previously reported seed-mediated growth procedure using cetyltrimethylammonium bromide (CTAB) as a structure-directing agent40 (ESI- Fig. S1). The purified monodispersed 3 ACS Paragon Plus Environment


GNRs were collected by centrifugation and toxic CTAB bilayer was replaced by biocompatible lipid bilayer. The surface modified GNRs were found to be ~ 27 nm in length and ~ 9 nm in width (Fig. 2a, ESI- Fig. S2).

Figure 1. Schematic representation of GNR-Lipos nanohybrid synthesis and its disintegration (a) One pot synthesis of GNR-Lipos nanohybrid. (b) Targeted chemophotothermal therapy and NIR assisted disintegration of GNR-Lipos nanohybrid. The nanohybrid was fabricated via film hydration and sonication process. Selfassembly of lipid (1:9 mixture of dipalmitoylphosphatidylcholine (DPPC) and 1, 2distearoyl-sn- glycero-3-phosphocholine (DSPC) bilayers in the presence of surface modified GNRs assisted the formation of GNR-Lipos nanohybrid (see Experimental details). The framework of designed nanohybrid was confirmed by various characterization techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS), atomic force microscopy (AFM), and UV−VIS-NIR spectroscopy (see Characterization techniques). GNR-Lipos nanohybrid’s spherical morphology with a mean diameter of 170 nm ± 10 nm was determined by microscopic techniques and the hydrodynamic size was found to be ~ 160-180 nm (Fig. 2, ESI- Fig. S3a,b). The presence of GNRs around the lipid 4 ACS Paragon Plus Environment

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bilayer of liposome was established by AFM height profile analysis (Fig. 2e, ESI- Fig. S3c,d) indicates about self-assembly of lipid molecules with GNRs which is also similar to the previous reports37,39. The architecture of GNR-Lipos nanohybrid was further confirmed by TEM analysis where GNRs were found to be decorated on liposome’s interior and peripheral surface (Fig. 2b,c,d&e). The increased height of about 10 nm with homogeneous size distribution of nanohybrid can be correlated with the integration of GNRs on liposome surface which is almost similar to the previous report37 (Fig. 2e inset & ESI- Fig. S3d). The particle size of designed spherical nanohybrid is confirmed through DLS measurement at ambient condition. DLS measurement shows the single peak diameter observation in the range of 160-180 nm indicating good dispersion of spherical nanoparticles (Fig. 2f). The NIR absorbance of nanohybrid was confirmed by UV-VIS-NIR spectroscopy. For GNRs, two absorption bands viz., 525 nm (transverse) and 690 nm (longitudinal) were observed whereas the red shift in the longitudinal band to 758 nm was seen for GNR-Lipos nanohybrid (Fig. 3a). Further, the PEGylated folic acid (PEG-FA) was attached to the surface of GNR-Lipos nanohybrid to target overexpressing folate receptors of cancer cells. The surface functionalization of GNR-Lipos nanohybrid with PEG-FA was confirmed by FTIR spectrum (ESI-Fig. S4). The O-H stretching vibrations (3395 cm-1) and -NH2 bending vibrations (1540 cm-1) confirmed the attachment of the functionalized PEG linker. Vibrations between 1600-1700 cm-1 are assigned to C=O stretching and N-H bending of the CONH group. The bands between 1400-1500 cm-1 (stretching vibrations of the pterin and p-amino benzoic acid rings of folic acid, FA) confirmed the FA functionalization on nanohybrid. To determine the conjugation of PEG with FA, 1H NMR was performed. It was observed that new amide bond was formed between the FA and PEG (ESI-Fig. S5). 5 ACS Paragon Plus Environment


Before establishing the chemo-photothermal synergistic behavior of GNR-Lipos nanohybrid its phototransduction efficiency, biocompatibility and drug release kinetics were evaluated. The photothermal transduction experiment of GNR-liposome

Fig. 2 Characterization of GNRs and GNR-Lipos nanohybrid using TEM and AFM (a) TEM image of surface modified GNRs with size distribution (inset). (b, c) TEM images of nonfunctionalized GNR-Lipos (b), PEG-FA functionalized GNR-Lipos(c); inset- zoomed image. (d) Schematic representation of GNR-Lipos; yellow arrows indicate encapsulated GNRs while red and white arrows represent the supporting GNRs on external and internal surface of lipid bilayers of liposome respectively (e) AFM images of GNR-Lipos nanohybrid with its height profile. (f) Hydrodynamic diameter of liposome and GNR-Lipos in PBS, saline and culture media.

nanohybrid was carried out at various concentrations ranging from 0.1 to 0.5 mg/mL using 750 nm NIR laser. The photothermal response of nanohybrid was recorded at NIR exposure for 5 min after stabilizing the surrounding temperature to 37 °C. At NIR exposure (650 mW), temperature continually increased with the increase of irradiation time. The hyperthermia temperature (43 °C) was recorded in 5 min. of NIR exposure time for 0.1 mg/mL of concentration and within 3 min. for 0.2 mg/mL concentration. Further, the temperature increased up to 53 °C in 5 min, when the concentration was


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raised to 0.5 mg/mL and laser power was set to 1 W (Fig. 3b). Also, the NIR-triggered disintegration of GNR-Lipos nanohybrid was also determined using UV-VIS-NIR spectroscopy and TEM imaging. The absorbance spectra showing both transverse and longitudinal bands of GNRs was almost lost in the designed nanohybrid after NIR exposure due to disturbance in the refractive index of the surrounding environment (Fig. 3a). The destabilization of GNR-Lipos nanohybrid was observed due to the heat generated by NIR light (Fig. 3a,c&d & ESI-Fig. S6). NIR light responsive disintegration concept of nanohybrid is also illustrated for better understanding (Fig. 3g).

Fig. 3 (a) Absorbance spectra of GNRs, GNR-Lipos nanohybrid and NIR exposed GNRLipos nanohybrid. (b) Concentration dependent photothermal transduction of GNR-Lipos nanohybrid. (c, d, g) TEM images of GNR-Lipos nanohybrid before (c) and after (d) NIR exposure along with cartoon representation of NIR assisted disintegration (g); yellow marking signifies the embedded liposomes which are revealed after NIR triggered disassembly of GNR-Lipos nanohybrid. (e) Percentage toxicity of CTAB surfactant stabilized GNRs, lipid functionalized GNRs, GNR-Lipos nanohybrid and PEG-FA_GNR-Lipos nanohybrid over mouse normal fibroblast- L929. (f) Time dependent drug release kinetics at different cellular pH and in presence of NIR.

Biocompatibility of the fabricated nanohybrid was determined with the help of MTT assay over L929 normal fibroblast cell line. For CTAB stabilized GNRs, only 30 % 7 ACS Paragon Plus Environment


cell viability was noticed due to the toxic effect of CTAB surfactant whereas, after CTAB replacement by lipid molecules, viability increased up to 85 %. Both bare GNRLipos nanohybrid and PEG-FA_GNR-Lipos nanohybrid also showed more than 80 % biocompatibility even at concentrations as high as100 µg/mL (Fig. 3e). In this study, doxorubicin hydrochloride (DOX) was used as a model drug. 10 µg of it was loaded during film hydration process and it was confirmed via UV-Vis absorption spectra. After loading drug, the obtained nanohybrid was purified through dialysis process and excess drug was removed via same procedure. About 85 % entrapment and 39 % of loading efficiency of drug into liposomal nanohybrid were calculated (ESI Eq. S1). The drug release kinetic was tested both at extracellular and intracellular conditions (i.e. pH 7.4, 5, 4, 2 and 2 with NIR exposure respectively). At neutral pH (7.4), negligible (~ 4 %) drug release was observed whereas more than 50 % drug release was observed in the cancer cell interior environment (pH 4, 5) and in the late endosomal conditions (pH 2) in 24 h study due to the destabilization of nanohybrid in the cancer mimicked environment. In addition, the noticed enhance drug release ability in cancer cell interior indicates the disintegration of nanohybrid due to the destabilization of lipid self assembly and detachment of nanorods from liposomal nanoparticles at the high strength of protonation in lower pH (pH 2 to 5). The enhance drug release is further corroborated with fluorescence microscopic images of DOX loaded nanohybrid treated cancer cells. Further, about 100 % drug release is observed within 12 h kinetic time in case of NIR exposed drug loaded nanohybrid at pH 2 due to the complete disintegration of nanohybrid. In addition, the complete disintegration of designed nanohybrid in above mentioned condition can be correlate with the effect of generated heat during NIR exposure and the high potential of protonation at lower pH. It clearly indicates the controlled and efficient destabilization of lipid self-assembly in cancer mimicked environment (Fig. 3f).

To affirm the targeting ability and


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intracellular localization of GNR-Lipos nanohybrid, 100 µL of DOX-loaded PEGFA_GNR-liposome nanohybrid (200 µg/mL) was incubated with MDA-MB 231.

Fig. 4 Fluorescence microscopy and assessment of time dependent internalization of DOX loaded PEG-FA_ GNR-Lipos nanohybrid over breast cancer cells- MDA-MB 231. In 4 h incubation, the DOX loaded PEG-FA_ GNR-Lipos nanohybrid showed fluorescence in cell cytoplasm, and after 12 h incubation, red fluorescence was also observed in nucleus. DAPI stained nucleus (blue), DOX (red) and merged fluorescence of DAPI and DOX (purple); red marking indicates the zoomed image. Scale bar: 100 µm

Red fluorescence was observed within 4 h of incubation indicating the DOX release in the cell interior (cytoplasm) and in 12 h of incubation, fluorescence spread in the nucleus as well. The significant strong red fluorescence in cell interiors (i.e. both cytoplasm and nucleus) clearly indicates GNR-Lipos nanohybrid ability to be efficiently taken up by cells overexpressing the folate receptor. Also, its ability to release the drug inside the cells in order to minimize the side-effects on the surrounding healthy tissues. (Fig. 4). To determine the uptake and fate of GNR-Lipos nanohybrid, Bio-TEM was also performed. It was seen that GNR-Lipos efficiently taken up by the cells within 12h, and its morphology seems to be distorted (Fig. S7).



To establish the synergistic effect of combined chemo-photothermal therapy various formulations were used (see Experimental details). The DOX-loaded and PEG-FA functionalized nanohybrid (100 µL of 200 µg/mL concentration) at NIR exposure of 5 min. (i.e. targeted chemo-photothermal therapy) rendered more than 90 % cell death (determined by both quantitative and qualitative analysis) as shown in Fig. 5a and b. In the case of standalone targeted photothermal therapy (i.e. NIR + PEG-FA_GNRLipos nanohybrid) and only targeted chemotherapy (i.e. DOX-loaded PEG-FA_GNRLipos nanohybrid) about 58 % and 35 % cell death was observed respectively. Percentage cell death was further poor in case of non-targeted alone photothermal (i.e. NIR + GNR-Lipos nanohybrid), chemotherapy (i.e. DOX-loaded GNR-Lipos nanohybrid). Even therapeutic efficacy obtained in non-targeted chemo-photothermal therapy wasn’t satisfactory. The low cell death establishes the paltry therapeutic effect of standalone chemo and photothermal therapy and sets up the relevance of efficient uptake of nanoparticles for the therapies mentioned above. Percentage therapeutic efficacy was also determined and it was found that therapeutic efficacy of combined targeted chemo-photothermal therapy to be significantly higher than the additive therapeutic efficacy of chemotherapy and photothermal therapy (ESI- Fig. S8). Additive therapeutic efficacy was estimated by the formula Tadditive = 100 - (ftargeted photothermal

x ftargeted

chemotherapy)

x 100, where f is the fraction of viable cells in each

treatment group.


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Fig. 5. Therapeutic assessment of in vitro chemotherapy, photothermal therapy and chemophotothermal therapy over breast cancer cells MDA-MB 231 (a) Quantitative analysis obtained by MTT assay, ****p < 0.0001; ***p < 0.001; *p < 0.05. (b) Qualitative analysis concluded using propidium iodide (dead cell’s stain); (1) only cells, (2) NIR + Cell, (3) GNRLipos nanohybrid treated Cells, (4) NIR + GNR-Lipos nanohybrid treated cell (i.e. alone photothermal therapy), (5) DOX loaded GNR-Lipos nanohybrid treated cells (i.e. alone chemotherapy), (6) NIR + DOX loaded GNR-Lipos nanohybrid treated cells (i.e. nontargeted chemo-photothermal therapy) (7) NIR + PEG-FA_GNR-Lipos nanohybrid treated cells (i.e. targeted photothermal therapy), (8) DOX loaded PEG-FA_GNR-Lipos nanohybrid treated cells (i.e. targeted chemotherapy), and (9) NIR + DOX loaded PEG-FA_GNR-Lipos nanohybrid treated cells (i.e. targeted chemo-photothermal therapy).

Diagnostic potential of GNR-Lipos nanohybrid was determined with the help of computed tomography (CT).

The radio density of GNR-Lipos nanohybrid was



enhanced as the conc. increased from 0.05 to 0.5 mg/ml due to increased X-ray attenuation and gold’s high atomic number 41 (Fig. 6).

Fig. 6 Computed Tomography of GNR-Lipos nanohybrid before and after NIR exposure; and at different concentrations. Yellow marking signifies the zone of contrast obtained.

Although, after NIR exposure, contrast zone was significantly reduced in volume which further supports the points that GNRs-Lipos nanohybrid dissembled after NIR exposure due to aggregation of GNRs, the same was observed in TEM images (Fig. 3c & d).

Conclusions The facile synthesis of multifunctional NIR light responsive DOX-loaded GNR-Lipos nanohybrid for imaging-guided cancer theranostic is reported. The plasmonic heating of GNRs upon NIR exposure is the key factor for synergistic chemo-photothermal 12 ACS Paragon Plus Environment

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therapy and disassembly directed disintegration of GNR-Lipos nanohybrid. The disintegration of GNR-Lipos nanohybrid potentiate its body clearance. The fabrication of nanohybrid ensures (1) dual therapeutic GNRs supported liposome nanohybrid, (2) stealth nature, (3) prevention of premature drug release and (4) targeted internalization of nanohybrid followed by drug release in the cytoplasm as well as the nucleus. Moreover, the designed nanohybrid shows good contrast in CT imaging as well as drug release in mimicked intracellular cancer environment which paves the path of the biocompatible nanocontrast agent. The designed nanohybrid provides imaging guided chemo-photothermal therapy through a single system due to its contrast and cargo carrying ability that proves its potential for application in cancer nanomedicine.

Experimental Section Materials and characterization techniques N-cetyltrimethylammonium bromide (CTAB), silver nitrate (AgNO3, 99.9 %), sodium borohydride (NaBH4, 99 %), ascorbic acid (AA, 99.5 %) and chloroform were purchased from Merck Limited, Mumbai, India. Tetrachloroauric acid (HAuCl4.3H2O, 99.9 %), 1-ethyl3-(3-dimethylaminopropyl) carbodiimide (EDC), n-hydroxysuccinimide (NHS), folic acid (FA), doxorubicin hydrochloride (DOX·HCl, 98%), poly(ethylene glycol) 2-aminoethyl ether acetic acid (average Mn 2,100), propidium iodide, 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT), and 4, 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma–Aldrich Pvt. Ltd., USA. Dipalmitoylphosphatidylcholine (DPPC) and 1, 2-distearoyl-sn- glycero-3-phosphocholine (DSPC) were purchased from Avanti Polar lipids. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), phosphatebuffered saline (PBS), and antibiotic-antimycotic solution were procured from HiMedia Laboratories Pvt. Ltd., India. Synthesized nanomaterials were characterized by various physico-chemical techniques. The size and morphology of nanostructures were examined by 13 ACS Paragon Plus Environment


transmission electron microscope (TEM, FEI Tecnai T-20) and scanning electron microscope (SEM, FEI Quanta-200). Samples for TEM were prepared by evaporating a droplet of the sample onto 200 mesh carbon coated copper grid. Optical properties of GNR-Lipos nanohybrid were understood by UV-VIS-NIR spectrophotometer (Jasco V570) using standard quartz cuvette having a path length of 1 cm. Fourier transform infrared (FT-IR) spectrum was recorded by Perkin-Elmer FT-IR Spectrum GX instrument. KBr crystals were used as the matrix for preparing the samples. Height profile and resolution at z-axis were determined by atomic force microscope (AFM, PSIA XE- 100) on tapping mode. The samples for AFM measurements were prepared by sample drop casting on clean silicon wafers. Elemental analysis was done by energy-dispersive X-ray module (EDAX) integrated to TEM. Computed tomography images were performed on a clinical CT scanner (Toshiba 64 slice, 0.5 mm slice per rotation) with 120 kV, 50 mA. The Hounsfield unit (HU) was measured by using RadiAnt DICOM Viewer software (V3.10ER009). Photothermal transduction experiments were completed using 750 nm CW NIR laser source with 1 W power. Bio-imaging and intracellular localization of drug and carrier were performed with the help of fluorescence microscope (Axio Observer Z1, Carl Zeiss).

Synthesis of gold nanorods Gold nanorods (GNRs) were prepared according to a previously reported42 seed-mediated growth procedure with few modifications. - Seed solution The preparation of gold seed solution was carried out by adding 5 mL of 0.2 M CTAB solution in 3 mL of 1.0 mM HAuCl4 solution. To the stirring solution, 1.2 mL ice cold 0.01 M NaBH4 was added, which resulted in the formation of brownish-yellow solution. Vigorous stirring of the solution was continued for 2 min and thereafter kept at 25 °C for maximum of 2 h before further use. 14 ACS Paragon Plus Environment

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- Growth solution The growth solution was prepared by adding 100 mL of 0.2 M CTAB solution to 5.6 mL of 0.004 M AgNO3. 10 mL of 15 mM HAuCl4 and 2.5 mL of 0.08 M ascorbic acid was added into the above solution and left to mix gently for 10 min. - Surface modification After complete mixing of growth solution, 2 mL of seed solution was added to the growth solution at 33 °C. The color of solution gradually changed into dark blue within 10 min of mixing. The temperature of the growth medium was kept constant at 33 °C throughout the procedure. The GNRs were collected by three cycles of centrifugation. For replacement of CTAB surfactant, 1 mL of CTAB stabilized GNRs were mixed with the aqueous solution of 10 mM of DPPC lipid (1 mL) for 2 h under gentle stirring at room temperature. The purified surface modified GNRs were collected via centrifugation and stored at 20 °C for further usage.

Synthesis of GNR-Lipos nanohybrid, conjugation of PEG-FA and loading of doxorubicin In a round bottom flask, mixture of DPPC: DSPC (1: 9) were dissolved in chloroform and the organic solvent was evaporated using rotary evaporator. The resulting lipid film was hydrated in the presence of surface modified GNRs dispersed in PBS solution (10 mL). Further, lipidGNRs hydrated solution was sonicated for 15 min. to obtain the GNRs supported liposomes. To load the anticancer drug in the nanohybrid, another set of experiment was carried out where 5 µg of DOX was added during film hydration process and rest of procedure was similar as mentioned earlier. To functionalize the exterior surface of nanohybrid, EDC:NHS (1 : 1 molar ratio) activated folic acid (100 mg in 20 mL PBS) was attached with Poly(ethylene glycol) 2-aminoethyl ether acetic acid (1 mg/mL) by 6 h coupling reaction and thereafter mixed with DOX-loaded GNR-Lipos/ GNR-Lipos nanohybrid (1 mg/mL). The PEG-FA functionalized GNR-Lipos nanohybrid was collected via centrifugation and washed repeatedly to remove unbound PEG-FA moiety. 15 ACS Paragon Plus Environment


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Phototransduction and disintegration study Phototransduction ability of GNR-Lipos nanohybrid was assessed by placing the GNR-Lipos nanohybrid along with controls in a 96-well plate. Laser irradiation was performed over samples of varying concentration for different time intervals. Temperature was recorded using probe thermometer after stabilizing the surrounding temperature to 37 oC.

For

disassembly directed disintegration study, GNR-Lipos nanohybrid (200 µg/mL) was exposed to NIR light for 5 min at 1 W power. The sample was collected and drop casted on TEM grid for structural analysis. Further, the absorbance spectra of nanohybrid were also recorded using standard quartz cuvette.

Drug release study Dialysis was done to remove the excess drug from the GNR-Lipos nanohybrid before performing release kinetics. The amount of drug loaded into GNR-Lipos nanohybrid was calculated by subtracting the amount of drug in the supernatant from the total amount of drug used. For drug release study, 1.0 mL of DOX loaded GNR-Lipos nanohybrid in dialysis bag (molecular weight cut-off 5 KD) was immersed in 50 mL of different pH buffer solutions (e.g. 7.4, 5.0, 4.0, 2.0 and 2.0 under NIR exposure) and left to stir at 37 °C for 24 h. At different time intervals, 2 mL solution was collected and replaced with the same volume of fresh PBS solution to keep the volume constant. The amount of DOX released was measured by UV-Vis spectroscopy at a wavelength of 480 nm.

Cell culture The mouse normal fibroblast (L929) and human breast cancer cells (MDA-MB-231) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum and 1 % penicillin/ streptomycin, under 5 % CO2 atmosphere at 37 °C. 16 ACS Paragon Plus Environment

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-In vitro cytotoxicity- MTT assay 24 h in vitro cytotoxicity studies were carried out with L929 cells using MTT assay. Cells were seeded at a density of ~ 1 × 105 cells per well in a 96 well plate. After overnight incubation at 5 % CO2 and 37 °C, 100 µl of different concentration (10-100 µg/mL) of (CTAB) GNR, surface modified GNRs, GNR-Lipos nanohybrid and PEG-FA_GNR-Lipos nanohybrid dispersed in media were added into the wells. Following 24 h incubation, wells were washed off with PBS and 20 µl of MTT dye was added. Formazan crystals formed after 4 h were dissolved in 200 µL of DMSO. Optical absorbance was recorded at 570 nm and 690 nm using microplate reader (Tecan Infinite 200 PRO). Percentage cell viability was calculated with reference to untreated cells (negative control) according to the following equation (1). Optical density (OD) of the treated cells × 100

% Cell viability = OD of untreated cells

Eq.1 Calculation for % Cell viability

-Targeting ability of GNR-Lipos nanohybrid and DOX distribution MDA-MB-231 breast cancer cells were seeded in 96 well plate at density of ~ 1 × 105 cells/well and incubated overnight in 5 % CO2 at 37 °C. Next day, wells were washed off with PBS and 100 µl of 200 µg/mL of DOX loaded PEG-FA_GNR-Lipos nanohybrid were added to the wells. After 4 h and 12 h, wells were washed off with PBS three times to remove unbound particles. Thereafter, 4 % paraformaldehyde solution was added to the cells, left for 10 min of incubation, and nuclei were stained with DAPI. The coverslip was then mounted over a drop of 70 % glycerol on glass slide to fix the phase of the cell. Images were captured using fluorescence microscope (Axio Observer Z1, Carl Zeiss). For the Bio-TEM, same experiment was repeated except adding stains after primary fixation, osmimum tetraoxide was added and subsequently graded (30, 50, 70, 90, 100%) dehydration with ethanol was done. 17 ACS Paragon Plus Environment


Finally, it was embeded in epoxy resin and cured for 72 h at 65 oC to cut the sections with microtome.

-Synergistic chemo-photothermal therapy Synergistic effect of chemo-photothermal therapy was determined over MDA-MB-231 breast cancer cell line. Following groups were categorized (1) only cells, (2) NIR + Cell, (3) GNRLipos nanohybrid treated Cells, (4) NIR + GNR-Lipos nanohybrid treated cell (i.e. only photothermal therapy), (5) DOX loaded GNR-Lipos nanohybrid treated cells (i.e. only chemotherapy), (6) NIR + DOX loaded GNR-Lipos nanohybrid, (7) NIR + PEG-FA_GNRLipos nanohybrid treated cells (i.e. targeted photothermal therapy), (8) DOX loaded PEGFA_GNR-Lipos nanohybrid treated cells (i.e. targeted chemotherapy) and (9) NIR + DOX loaded PEG-FA_GNR-Lipos nanohybrid treated cells (i.e. combined targeted chemophotothermal therapy). The cells were seeded into 96 well plates at a density of 2×104 cells/well and incubated for 24 h in 5 % CO2 atmosphere at 37 ºC. After rinsing the wells, cells were incubated with 200 µg/mL of various formulations of GNR-Lipos nanohybrid for 4 h. After incubation, the cells were again washed with PBS three times to remove all the unbound nanohybrid before laser irradiation. For quantitative analysis- percentage cell viability was determined by MTT assay as described earlier. For qualitative analysis- other procedures were same except after careful washing, each group of cells were stained with propidium iodide to mark the dead cells.

Computed Tomography Compute Tomography (CT) was performed at 120 kV by taking different conc. ranging from 0.05 to 0.5 mg/l of GNR-Lipos nanohybrid along with PBS (negative control) in Eppendorf. CT imaging was performed both before and after NIR laser irradiation. Data were procured and analyzed using Toshiba 64 followed by DICOM viewer. 18 ACS Paragon Plus Environment

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Supporting Information Following additional figures are available free of charge on the ACS publication website. Figure S1: TEM image along with aspect ratio of gold nanorods synthesized using CTAB as a structure directing agent. Figure S2: Elemental analysis via EDAX of lipid functionalized GNRs Figure S3: Characterization of GNR-Lipos nanohybrid using DLS, SEM and AFM Figure S4: FTIR spectrum of PEG-FA functionalized GNR-Lipos nanohybrid Figure S5: 1H NMR of a) PEG, b) FA and c) FA-PEG. Figure S6: TEM image and aspect ratio of GNR-Lipos after NIR exposure Figure S7: Percentage therapeutic efficacy in different treatment groups. Figure S8: Bio-TEM image of PEG-FA_GNR-Lipos uptaken MDA-MB 231. Figure S9: Lipase driven disintegration study of GNR-Lipos.

Notes The authors declare no conflict of interest and have approved final version of the manuscript.

ORCID Prof. Rohit Srivastava: 0000-0002-3937-5139 Dr. Kaliaperumal Selvaraj: 0000-0002-9996-8112 Dr. Rajendra Prasad: 0000-0001-9851-8630 Deepak S. Chauhan: 0000-0001-8602-6006 Acknowledgements The authors are grateful to acknowledge Infosys Foundation and CFN EMPOWER (OLP002626), CSIR New Delhi for supporting the project. Authors also acknowledge Mr. Manoj Kumar, IIT-Bombay for generous support in several experiments. DSC acknowledges the IITB for Senior Research Fellowship, and RP acknowledges the UGC, New Delhi for Senior Research Fellowship. Abbreviations NIR, near-infrared; DOX, doxorubicin; DOX.HCL, doxorubicin hydrochloride; GNR-Lipos, gold nanorods-liposome; PEG, polyethylene glycol; FA, folic acid; CTAB, cetyltrimethylammonium bromide; DPPC, dipalmitoylphosphatidylcholine; DSPC, 1, 2distearoyl-sn- glycero-3-phosphocholine; TEM, transmission electron microscopy; SEM, scanning electron microscopy; DLS, dynamic light scattering; AFM, atomic force microscopy; PEG-FA, PEGylated folic acid; MTT, (3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide); DAPI, (4, 6-diamidino-2-phenylindole); EDC, 1-ethyl-3-(3dimethylaminopropyl) carbodiimide; NHS, n-hydroxysuccinimide; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum, PBS, phosphate-buffered saline; CW, continuous wave; CT, computed tomography References (1) Ding, X., Liu, J., Li, J., Wang, F., Wang, Y., Song, S., and Zhang, H. (2016) Polydopamine coated manganese oxide nanoparticles with ultrahigh relaxivity as nanotheranostic agents for magnetic resonance imaging guided synergetic chemo/photothermal therapy. Chem. Sci. 7, 6695–6700. (2) Dykman, L., and Khlebtsov, N. (2012) Gold nanoparticles in biomedical applications: recent advances and perspectives. Chem. Soc. Rev. 41, 2256–2282. 19 ACS Paragon Plus Environment


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Disintegrable NIR Light Triggered Gold Nanorods Supported

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