Cobalt Phosphide Nanorods with Controlled Aspect Ratios as

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Cobalt Phosphide Nanorods with Controlled Aspect Ratios as Synergistic Photothermo-Chemotherapeutic Agents Sutanu Kapri, and Sayan Bhattacharyya ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01221 • Publication Date (Web): 31 Aug 2018 Downloaded from http://pubs.acs.org on August 31, 2018

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ACS Applied Nano Materials

Cobalt Phosphide Nanorods with Controlled Aspect Ratios as Synergistic Photothermo-Chemotherapeutic Agents Sutanu Kapri and Sayan Bhattacharyya* Department of Chemical Sciences and Centre for Advanced Functional Materials, Indian Institute of Science Education and Research (IISER) Kolkata, Mohanpur - 741246 India * Email for correspondence: [email protected] ABSTRACT Polydopamine (PDA) capped cobalt phosphide nanorods (Co2P@PDA NRs) with six aspect ratios (ARs) from 1.4 to 10 are synthesized by one step thermal decomposition and micro-emulsion method. The NRs with AR ~6.4 show good dispersibility in physiological solutions exhibited an outstanding near-infrared (NIR) photothermal performance due to high molar extinction coefficient of 13.08 Lg-1cm-1 at 980 nm, excellent photostability, photothermal conversion efficiency (PCE) of ~64% and no obvious cytotoxicity to various types of cells. Coupled with NIR excitation, folic acid conjugation to the doxorubicin loaded NRs (AR ~6.4) helped them in securing targeted and controlled drug release to the cancer cells. The synergistic photochemothermal property of Co2P@PDA NRs offers itself as a worthy candidate in tumor theranostic applications. Keywords: Cobalt Phosphide; Polydopamine; Nanorod; Aspect ratio; Photochemothermal therapy; Photothermal conversion efficiency INTRODUCTION Photothermal therapy (PTT) agents convert laser induced optical energy into heat energy via phonon-phonon or electron phonon coupling, leading to irreversible damage of the biological cells.1 NIR radiation of wavelength 700-1100 nm is particularly fascinating because of better tissue penetration and low absorption of NIR light by the primary absorbents of hemoglobin and water in the biological tissues. PTT treatment increases the rate of biochemical reaction generating reactive oxygen species which subsequently damages 1 ACS Paragon Plus Environment

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the cell organelles.2,3 To increase photothermal conversion efficiency and accumulation of PTT agents within the targeted cancer cells, various NIR absorbing nanostuctures with different ARs of noble metals / alloys / branches,1,4-8 semiconductors,9,10 black phosphorous,11 and carbon,12,13 have been explored. A majority of them however lack desirable PCEs and photostability in aqueous media. For example, with 808 nm laser the efficiency of gold nanorod and nanovesicles is only 21 and 37%, respectively.1,14 However, under laser irradiation, the heat induced by the photothermal agents such as gold nanorods can melt them resulting in lower photothermal effect.15-17 However, PTT agents when used alone have the disadvantage of inhomogeneous heat distribution leading to non-specific cancer cell killing. Therefore to achieve a thermoresponsive on-demand drug delivery system for non-invasive treatment, the combination of PTT with chemotherapy is becoming popular that can also maximize the therapeutic efficacy.18-20 The so far reported nanoplatforms of Au, Fe3O4, graphene oxide and silica suffer from time-consuming complicated synthesis procedures and limited performance.20-23 Development of materials that can simultaneously offer as PTT agent and enable light triggered target-specific drug release with spatiotemporal selectivity is of prior need. The material of interest here, Co-phosphide remains relatively unexplored for this dual functionality. The elements of interest, Co and P have promising biomedical applications.24,25 Cobalt nanoparticles (NPs) however have been explored in drug delivery, as PTT and as magnetic resonance imaging (MRI) contrast agents.26,27 Indeed high concentrations of Co+2 induce DNA damage and activates the reactive oxygen species (ROS) and are therefore toxic to cells. Herein the bio-distribution and bio-safety of each of the elements in Co2P@PDA NRs are well understood. Very recent reports show the use of PDA coated cobalt phosphide nanocomposite as an excellent MRI agent where the nanocomposite shows outstanding bio distribution profile (30 day) without any toxicity and body weight loss in vivo.28,29

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Co2P@PDA NRs with different ARs from 1.4 to 10 are shown to be a cost-effective and superior PTT agent compared to noble metal nanostructures. Intriguingly, the Co2P@PDA NRs with AR ~6.4 show a high PCE ~ 64% among other ARs (viz. 1.4, 4.5, 5, 5.5 and 10) of Co2P@PDA NRs. Since photothermal therapy largely depends on PCE, herein we have employed Co2P@PDA NRs with AR ~6.4 for all biological experiments. In the presence of a semi-graphitic PDA shell with modest surface area for π-π stacking of drug molecules, the as synthesized Co2P@PDA NRs demonstrate a broad NIR absorption, good photostability, decent stability in physiological solutions and minimal cytotoxicity to both tumor and healthy cells. Under NIR light exposure (980 nm, 1 W/cm2) into the aqueous dispersion of 50 µg/mL Co2P@PDA NRs the temperature can be increased by ~31oC within 10 min. While this manuscript was under preparation a recent report by Chen et. al. and Li et. al. described the photothermal property of CoP nanocages,30 Co-P nanocomposite,28 and CoP nanoparticles,29 respectively. Indeed the one-dimensional morphology of the PTT agent plays a vital role in its performance, which is why the PCE of Co2P@PDA NRs is ~64%, far superior than the reported nanocages and nanoparticles, 28.7% and 25.7% under nearidentical conditions. Since, the intracellular microenvironment of cancerous cells is more acidic than normal cells and blood,23 and the cancer cell surface has over expressed folate receptors,31 it is common practice to use folic acid (FA) as the targeting ligand for sitespecific drug release. Through functionalization of DOX, a clinically used model anticancer drug, and FA, the NRs can selectively and efficiently kill the cancer cells assisted by NIR photoexcitation. EXPERIMENTAL SECTION Materials Cobalt chloride hexahydrate (CoCl2.6H2O, 90%, Merck), hexadecylamine (HDA, 90%, Aldrich), tri-n-octylphosphine (TOP, 90%, Aldrich), 1-octadecene (ODE, 90%,



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Aldrich), oleylamine (Sigma Aldrich, > 98%), oleic acid (Sigma Aldrich, technical grade 90%), Cobalt (II) carbonate (99 %, Aldrich), ethanol, chloroform, acetone and toluene (Merck, India) were used without further purification. All reactions were carried out in the Schlenk line under nitrogen gas flow. Dopamine hydrochloride (Sigma Aldrich), 98-102% doxorubicin hydrochloride (DOX, HPLC, Sigma Aldrich), ≥97% folic acid (FA, Sigma Aldrich),

98%

N-hydroxysuccinamide

(NHS,

Sigma

Aldrich),

>98%

N-(3-

dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC, Sigma Aldrich), dialysis membrane (MWCO 3500, Fischer Scientific), Fluorescein diacetate (FDA, Sigma Aldrich), ≥94% propidium iodide (PI, HPLC, Sigma Aldrich), potassium bromide (KBr, IR grade), Trypsin/EDTA solution (Gibco), dulbecco’s modified eagle media (DMEM, Gibco), fetal bovine serum (FBS, Hyclone, Thermo Scientific), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT, USB Corporation), 99.9% dimethyl sulphoxide (DMSO, molecular biology

grade,

Sigma

Aldrich),

poly-D-lysine

(Sigma

Aldrich),

99%

paraformaldehyde (PFA, Sigma Aldrich), 4ˊ,6-diamidino-2-phenylindole (DAPI, USB Cooperation), antifade reagent (Invitrogen), were used without further purification. Instrumentation The zeta potential of the NPs was measured with dynamic light scattering (DLS) instrument of model Malvern Zetasizer Nano ZS. JASCO V-670 spectrophotometer was used to record the ultraviolet-visible (UV-vis) absorption spectra. Horiba Scientific Fluoromax-4 spectrofluorometer using a Xe lamp as the excitation source with a wavelength of 309 nm was used for the fluorescence spectra. Rigaku (mini flex II, Japan) powder X-ray diffractometer having Cu Kα = 1.54059 Å radiation was used for recording the X-ray diffraction (XRD) patterns. Carl Zeiss SUPRA 55VP FESEM was used to obtain the field emission scanning electron microscope (FESEM) images while energy dispersive analysis of X-ray (EDAX) analyses were carried out with the Oxford Instruments X-Max with INCA


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software coupled to FESEM. Transmission electron microscopy (TEM) images were recorded with JEOL, JEM-2100F, the DST-FIST facility of IISER Kolkata. Scanning TEM high angle annular dark field (STEM-HAADF) analyses were taken in the same instrument. The Fourier transform infrared (FTIR) spectroscopy studies were carried out with a Perkin Elmer spectrum RX1 using KBr pellets. Raman spectra were recorded with 633 nm line of a He-Ne ion laser as the excitation source (LABRAM HR800) to characterize the nanorods and PDA coating. N2 adsorption−desorption isotherms were obtained on a Micromeritics Gemini VII surface area analyser and reported by Barrett-Joyner-Halenda surface/volume mesopore analysis. Samples were degassed at 100°C under N2 atmosphere for 3 h. The specific surface area was determined as per the Brunauer-Emmett–Teller (BET) method. Flow cytometry experiments were performed in Fluorescence-activated cell sorting (FACS) instrument (BD FACSVerseTM). Carl Zeiss structured illumination microscope (SIM) technique using Apotome module with Axio Observer was used to obtain the fluorescence images. Live dead cells experiments were performed Olympus IX83 microscope with Hamamatsu ORCA-Flash 4.0 camera. Synthesis of Cobalt Phosphide Nanorods (Co2P NRs) with different Aspect Ratios (ARs) and Spherical Cobalt Phosphide NPs (Co2P NPs) Co2P NRs and spherical Co2P NPs were prepared as per a previous report albeit with modifications.32,33 Briefly, 0.5 mmol CoCl2, 6 H2O, 2 mmol HDA and 6 mL ODE were taken in a three-neck round-bottom flask and degassed at room temperature for 20 min. Thereafter, 2 mmol TOP was injected to above mixture and the total mixture was heated at 120oC for 45 min under nitrogen to remove residual water and volatile impurities. The temperature was then increased to 3000C at a heating rate of 50C min-1 and was kept at this temperature under N2 atmosphere for 10 min, 30 min, 1 h, 2.5 h, 4 h, 5 h and 10 h. The NRs with shorter length (smaller AR) are synthesized at shorter reaction time and the AR can be precisely increased



by increasing the growth time in the reaction container. With 10 min reaction, Co2P NPs were formed and after 10 h, Co2P NRs with the desired AR were obtained. When the reaction temperature was increased, the colour of the solution changes from blue to dark blue and finally blackish at 3000C which indicates the nucleation of cobalt phosphide NPs. Post reaction the solution was cooled down to room temperature, after which the black coloured products Co2P NRs and spherical Co2P NPs, respectively were precipitated by adding ethanol. The precipitate was separated by centrifugation followed by drying in high vacuum. Finally the product was re-dispersed in chloroform and stored at 40C for further use. Synthesis of PDA Coated Co2P NRs (Co2P@PDA NRs) and Spherical Co2P NPs (Co2P@PDA NPs) To prepare Co2P@PDA NRs with different ARs and spherical NPs, 2 mg each of the as-prepared Co2P NRs and NPs were separately dispersed in 10 mL of CHCl3 followed by 1 mL of IGEPAL (CO-630), added drop by drop into the above solution under vigorous stirring. After 30 min, ~50 µL of NH4OH solution was added drop wise into the above solution until the pH of the solution reached ~8.5. Thereafter, 100 µL of dopamine hydrochloride solution was injected to the above solution and the reaction was allowed to proceed for 6 h under stirring at room temperature. The Co2P@PDA nanostructures were precipitated by adding ethanol and separated by centrifugation at 13000 rpm for 5 min. The rinse-centrifugation cycles were repeated with ethanol several times. Finally the product was dried in vacuum overnight and re-dispersed in PBS for further use. Synthesis of PDA Coated CoP NRs (CoP@PDA NRs) CoP NRs were prepared as per previous reports.34 Briefly, 1.5 mmol cobalt (II) carbonate and 4.5 mmol oleic acid in 5 mL trioctylphosphine were taken in a 50 mL threeneck round-bottom flask and heated at 80oC under stirring for 20 min, followed by degassing for 15 min in vacuum. Then the temperature of the solution was increased to 320oC for


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another 30 min in the presence of N2 atmosphere. When the temperature reached 320oC, oleylamine (3.0 mmol) was injected immediately into the reaction mixture. Thereafter the reaction was further carried out for 1 h at this temperature. The solution was cooled down to room temperature, after which the black coloured products were precipitated by adding ethanol and separated by centrifugation for three times and dried in high vacuum. Finally the product was re-dispersed in chloroform for further use. The CoP NRs were coated with PDA in the same way as that of Co2P NRs (mentioned above). Synthesis of PDA Nanosphere PDA nanospheres were prepared as per earlier report albeit with slight modifications.35 Briefly, 1mL of aqueous ammonia solution (28-30% NH4OH) was mixed with 20 mL of ethanol and 40 mL of distilled water under stirring in a closed condition. 0.25 g of dopamine hydrochloride was dissolved in distilled water (5 mL) and then slowly injected into the above mixture solution with vigorous stirring. Upon addition of dopamine hydrochloride the colourless solution turned pale yellow and changed further to dark brown colour. After 16 h, the as-prepared PDA nanospheres with ~125 nm diameter were collected by centrifugation and washed three times with ethanol and distilled water. Finally the product was dried in vacuum for further use. Photothermal Performance of Co2P@PDA NRs Firstly, different concentrations (10, 25 and 50 µg/mL) of Co2P@PDA NRs were dispersed in PBS solution. To check the photothermal performance of Co2P@PDA NRs, 1 mL of the dispersions inside a quartz cuvette were irradiated with 980 nm laser at a power density of 1 W/cm2. PBS solution was used as a control. The solution temperature which increased over time, was recorded by a thermocouple probe digital thermometer every 20 s. To measure the photothermal stability of Co2P@PDA NRs, the PBS dispersion of Co2P@PDA NR solution (50 µg/mL) was irradiated with a laser (980 nm, 5 min, 1 W/cm2).



After the laser irradiation was switched off and cooled to room temperature for another 5 min, the laser irradiation was repeated for another five on/off cycles. The solution temperature was recorded every 20 s. To test the possibility of cobalt leaching from the PBS dispersion of Co2P@PDA NRs, at first UV-vis absorption spectrum was recorded with a reference solution prepared by adding 50 µl of NaOH and HCl (1:1) to the PBS solution containing 1 ppm of CoCl2. From this spectrum, the peaks arising from the characteristic d-d transition in tetrahedral CoCl42and octahedral Co(H2O)62+ complexes were identified. These signatures were cross-checked from the absorption spectra recorded on the PBS dispersion of 0.2 mg/ml NRs which was previously irradiated with 980 nm laser for 30 min (1 W/cm2), dialyzed overnight and the dialyzed solution treated with 50 µl NaOH and HCl (1:1). Preparation of FA Functionalized Co2P@PDA NRs FA was dissolved in 10 mL PBS buffed solution of pH 7.4 at a concentration of 0.5 mg/mL and EDC.HCl was dissolved in 5 mL PBS buffered solution of pH 7.4 at a concentration of 0.2 mg/mL. EDC.HCl solution was added drop wise in the FA solution under stirring, followed by sonication for 20 min. Thereafter, 5 mL of Co2P@PDA NRs (concentration of 1 mg/mL) was added to the above solution and stirred for 24 h at room temperature. Excess FA was removed by dialysis membrane and the samples were washed thrice with buffer solution and dried in high vacuum. The resultant sample was named Co2P@PDA-FA. Loading of DOX onto Co2P@PDA-FA 3 mg of Co2P@PDA-FA was dissolved in 6 mL PBS buffered solution. Separately 6 mg of DOX was dissolved in 6 mL of PBS buffer solution under dark conditions. Both the solutions were vortexed for 5 min. Different volumes (0.2 mL, 0.6 mL, 1 mL and 2 mL) of the DOX solution were added into 1 mL of the NR solution, respectively and mixtures were


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again vortexed for 5 min. Thereafter, these mixtures were again stirred for 6 h in dark at room temperature. After 6 h, the mixtures were collected and centrifuged for 5 min at 13000 rpm. Finally the products were collected by repeated washing with PBS buffer and dried under inert atmosphere. The DOX adsorbed complex was re-suspended in 5 mL PBS buffer solution of pH 7.4 and stored at 4oC. For the quantification of DOX, the PBS dispersion of DOX loaded samples were taken in a quartz cuvette and the absorbance at 490 nm was monitored by UV-vis spectrophotometry after subtracting the absorbance of Co2P@PDA-FA at that wavelength. The drug loading capacity was calculated by the following formula:31 Drug loading (w/w %) = (mass of drug loaded / mass of nanostructures) × 100 Drug Release Study The study of drug release from DOX adsorbed Co2P@PDA NRs was carried out by the dialysis method at room temperature. Co2P@PDA/DOX and Co2P@PDA-FA/DOX were dispersed in PBS buffer with different pH of 5.5 and 7.4, and then introduced into a dialysis bag. The dialysis bag was introduced into 200 mL PBS buffer solution and stirred at 100 rpm at 37oC. 3 mL aliquots of the released media were taken at different time intervals and the DOX concentration was checked by measuring the absorbance at 490 nm after subtracting the Co2P@PDA absorbance with molar extinction co-efficient (ɛ) values of 0.01992 and 0.01687 (µg/mL)-1 cm-1 at pH 5.5 and 7.4, respectively. Beer-Lambert Law was used to determine the ɛ of DOX at different pH values. After each sampling, 3 mL of fresh buffer was added to keep the total volume of released media constant. The drug release percentage was calculated according to the equation: drug release (%) = (mDOX-release/mDOX-loaded) × 100, where DOXrelease implies DOX released from Co2P@PDA/DOX.31 Photothermally Triggered Drug Release In this experiment, Co2P@PDA/DOX (5 mg) was dispersed in PBS solution with pH 5.5 and 7.4 at room temperature. The solutions were stored in the dark at room 9 ACS Paragon Plus Environment


temperature. The solutions were irradiated with a 980 nm laser (1W/cm2, 5 min) at predetermined time intervals (laser power was 80 mW, the measured spot diameter was 3.2 mm and calculated spot area was 0.0803 cm2). At different time intervals, the suspensions were centrifuged at 13000 rpm for 10 min. The amount of DOX release was quantified by UV-vis spectra. In Vitro Cytotoxicity and Photothermal Cancer Therapy To check the cytotoxicity of Co2P@PDA towards HeLa and HEK 293 cells (with or without laser irradiation), MTT assay was conducted to determine cell viability. Cells were seeded in 24-well plate at 2 × 104 cells per 1 mL of 10% FBS containing DMEM media (MEM for HEK 293 cell) and allowed to grow at 37oC for 24 h in 5% CO2. After 24 h incubation, different concentrations of Co2P@PDA NRs were dispersed in DMEM media and added into different wells. After 48 h incubation with Co2P@PDA NRs, cells were washed by PBS solution and 100 µL of freshly prepared 5 mg/mL MTT solution was added to each well for another 4 h incubation time at 37oC. Minimum three parallel samples were performed in each group and these sets were tested with triplicates. The culture media was discarded and 500 µL of DMSO was added to each well and samples were shaken for 10 min to dissolve the formazan crystals. After diluting the resultant solution 10 fold, absorbance was measured at 570 nm. The absorbance was correlated with cell viability by assuming 100% viability for untreated control cells. The cell viability (%) was calculated according to the equation: Cell viability (%) = [{OD@570 nm (sample)}/{OD@570 nm (Control)}] × 100, where OD is the optical density. For in vitro cancer therapy, HeLa and HEK 293 cells were seeded and incubated with different concentrations of Co2P@PDA NRs and Co2P@PDA-FA/DOX, respectively, into 96 well plate. After 4 h incubation, to remove excess Co2P@PDA NRs and Co2P@PDAFA/DOX, the cells were washed with PBS solution twice and replaced with fresh cultured 10 ACS Paragon Plus Environment

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media. Thereafter the cells were irradiated with 980 nm laser for 10 min (power density 1, 2 W/cm2), cultured again for next 24 h. Finally, the aforementioned MTT assay was conducted to measure the cell viability. Cellular Imaging Experiments For cellular imaging, HeLa cells (and both HeLa and HEK 293 cells for Co2P@PDAFA/DOX) were seeded (1 × 105 cells per well) on glass cover slip coated in a 24-well plate with poly-D-lysine and cultured at 37oC with 5% CO2. The cells were washed with PBS twice and incubated with Co2P@PDA, free DOX, Co2P@PDA/DOX and Co2P@PDAFA/DOX after 24 h. Before addition to each well, all samples were mixed with 1 mL culture media. Minimum three parallel samples were performed in each concentration and all the sets were tested in triplicates. After incubation at 3 and 6 h time intervals, the samples were washed thrice with PBS buffer to remove the dead cells and impurities. These cells were fixed for 30 min with 4% PFA and stained for 5 min in the dark with DAPI for monitoring their fluorescence. FDA and PI Co-stain Assay Fluorescein diacetate and PI double-staining were used to evaluate the cell death under irradiation with NIR light.36 HeLa cells were seeded in 35 mm petri dishes (2 mL, 30,000 cells per well) and incubated overnight at 37oC to allow the cells to adhere. After treatment with 50 µg/mL of Co2P@PDA for 4 h, cells were irradiated with NIR laser for 10 min and continued to culture for 12 h. Thereafter, the cells were washed with PBS for two times. After staining with a mixture of FDA/PI in FBS free media for 15 min and after washing with PBS twice, images of the four samples were captured by using epifluorescence microscope. Flow Cytometry Assay



HeLa cells and and HEK 293 cells were seeded into six well plates at a density of 1 × 106 cells per well and incubated at 37°C in DMEM medium. Then the cells were allowed to adhere on the well. After 24 h, DMEM medium was removed and replaced with media containing free DOX, Co2P@PDA/DOX and Co2P@PDA-FA/DOX. Cells without sample were taken as the control. After 3 h and 10 h incubation, the cells were washed with PBS thrice. After that cells were harvested with trypsin–EDTA and incubated at 37°C until the cells were detached from the well. Then, 2 mL media was added into the trypsinized cell suspensions and centrifuged at 1000 rpm for 5 min followed by decantation of the supernatant. The cell pellet was resuspended in 500 µL ice cold PBS. Finally, the resultant cell suspensions were taken in FalconTM tubes and analyzed by flow cytometry experiments with FL2-H channel (excitation 488 nm and emission 575 nm). The mean fluorescent signals were taken for 10000 cells. RESULTS AND DISCUSSION NR Characterization The Co2P NRs were prepared from CoCl2.6H2O in the presence of 1-octadecene and hexadecylamine with tri-n-octylphosphine as the phosphide source (see Figure 1a and experimental section). The average length and width of the monodispersed NRs are 90 ± 3.5 nm and 14 ± 2 nm (average of 50 Co2P NRs), respectively, with AR ~6.4 (Figure 1b). High resolution TEM image and the corresponding diffraction spots in Figure 1c indicate high crystallinity of the NRs where (130) and (002) reflections of Co2P are visible. PDA shell was incorporated onto Co2P NRs via self-polymerization of dopamine in the presence of hexadecylamine and an amphiphilic compound IGPAL-630 in alkaline microemulsion.37


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Figure 1: (a) Schematic illustration of the synthesis of Co2P@PDA NRs, their folic acid (FA) functionalization and doxorubicin (DOX) loading; HDA: hexadecylamine, TOP: tri-noctylphosphine. (b, d) Low and (c, e) higher magnification TEM images of Co2P NRs and Co2P@PDA NRs, respectively, inset showing the thickness of PDA shell. Insets of (c, e) show the SAED patterns. (f) HADDF-STEM image of Co2P@PDA NRs and the corresponding maps show the elemental distribution of Co, P and C. (g) XRD patterns. (h) FTIR spectra of (i) Co2P, (ii) Co2P@PDA NRs, (iii) Co2P@PDA-FA and (iv) Co2P@PDAFA/DOX, respectively. Owing to the small molecule adsorption capability, NIR absorption and self polymerization properties, the PDA shell improves the stability and functionality of nanostructures both in vitro and in vivo.38 Also the surface functional groups of PDA, amine and catechol allow further conjugation and adsorption of biomolecules.23,39 In this case, the uniform ~4 nm thick PDA shell retains monodispersity and high crystallinity of the NRs showing (130) and (111) reflections (Figure 1d,e). The NR dimensions remain intact without agglomeration even when stacked in the undispersed powder form (Figure S1 and Figure S2). Figure 1f shows the elemental mapping obtained by HAADF-STEM where homogeneous distribution of Co, P 13 ACS Paragon Plus Environment


and C are observed.34 The AR of Co2P@PDA NRs could be tuned by changing the reaction time as shown in Figure S3. For comparison, highly crystalline CoP@PDA NRs with AR ~6.4 were also synthesized (Figure S4). Co2P crystallizes in the orthorhombic crystal structure according to JCPDS card No. 32-0306 (Figure 1g). Since the PDA shell is amorphous, a weak feature is observed at 2θ ≈ 25o. In the Raman spectrum of Co2P NRs (Figure S5) the A1g Raman active mode at 670 cm-1 affirms the good sample quality. Post PDA coating, this band vanishes and the Raman bands corresponding to sp3 and sp2 hybridized carbon at 1354 and 1594 cm-1, respectively become prominent. Co2P has a negligible surface area of 6 m2/g which after PDA coating slightly improves (27 m2/g) with an average pore diameter of 3.7 nm (Figure S6). The zeta potential measurements in aqueous solution show that Co2P NRs are positively charged (+6 mV) whereas Co2P@PDA NRs are negatively charged (-17.4 mV) due to the presence of -OH and catechol groups on the surface of Co2P@PDA NRs. These negative charges can enhance the adsorption of positively charged drug molecules for better loading via electrostatic interactions.23 FTIR spectroscopy was used to study the changes in chemical bonding at the NR surface after each modification (Figure 1h and Figure S7). In Co2P NRs the bands at 2977 and 2856 cm-1 are due to C-H stretching vibration of methylene group whereas those at 1467 and 1022 cm-1 are due to -CH3 and Co-P vibrations, respectively.40 After PDA coating, the new broad band at 1800-1000 cm-1 is due to C-O stretching of phenolic hydroxyl group and aromatic ring in the PDA shell. The bands at 1634 and 1384 cm1

arise from C-N and C-C stretching vibrations, respectively. After binding the –COOH group

of FA with -NH2 group at the Co2P@PDA NR surface, new bands appear at 1500 to 500 cm1

, 3385 and 1630 cm-1 corresponding to the characteristic vibrations of FA and -NH bond

stretching, respectively.39 Post DOX adsorption onto Co2P@PDA/FA, the characteristic bands of DOX aromatic ring skeleton are observed at 1560 and 1059 cm-1. 14 ACS Paragon Plus Environment

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Optical and Photothermal Properties The Co2P@PDA NRs show broad absorption in the NIR range covering both biological window I (λ~800-1000 nm) and II (λ~1000-1300 nm) (Figure 2a). At a given length (L) of the quartz cuvette, absorbance (A) at 980 nm increases with increasing NR concentration (c). The molar extinction coefficient (ϵ) is 13.08 Lg-1cm-1 obtained from the slope of the linear plot of A/L as a function of c (Figure 2a inset). Owing to a high ϵ, the photothermal properties were evaluated by 980 nm NIR laser (1W/cm2) irradiation on different NR concentrations in PBS solution for 5 min. The solution temperature was measured by a thermocouple probe digital thermometer as a function of irradiation time and NR concentration (Figure 2b). Noteworthy, at 50 µg/mL temperature rises from 28oC to 57.3oC in 5 min and to 62.1oC after 10 min in comparison to the PBS control where the rise is only up to 32.7oC in 10 min. The efficient ability of Co2P@PDA NRs to convert laser radiation into heat energy is further elucidated from the increase in solution temperature with increasing laser power density (Figure S8). When compared with spherical Co2P@PDA NPs of diameter 15 nm (Figure S9), the NR absorbance is six times higher than the NPs due to a larger absorption cross-section in the NRs.41 The significance of employing a NR shape is evident from the limited temperature increase of the spherical NPs and only PDA nanosphere (125 nm) to 37.3 and 36.2oC, respectively, after 5 min of laser irradiation. The Co2P@PDA NRs show high colloidal stability (Figure S10) and photostability with no obvious deterioration in aqueous medium under six on/off cycles of 5 min irradiation followed by cooling (Figure 2c). This observation is particularly interesting since in earlier experiments of electron beam exposure for 1 min, the luminescence intensity of carbon coated inorganic semiconductor nanocrystals decreased by a factor of 10 due to disruption of graphitic shell bonds.42 The PCE (η) of the NRs was calculated using data fitting from the



cooling period (see discussion S1),43 when the laser was switched off after 10 min of irradiation (Figure 2d). The η for Co2P@PDA NRs is ~64% which is superior than other PTT agents (see comparison in Table S1). The structural and optical properties of Co2P@PDA NRs do not change after 60 min of irradiation (Figure S11), which further manifest the indubitable photostability of Co2P@PDA NRs. Furthermore, the PDA coating introduces excellent biocompatibility to the NRs since even after 30 min of continuous laser irradiation into the PBS dispersion of Co2P@PDA NRs, leaching of cobalt is not observed (Figure S12).

Figure 2: (a) Absorbance spectra of Co2P@PDA NRs in phosphate buffered (PBS) solution. The straight line shows data fitting to the plot of A/L at 980 nm as a function of NR concentration. (b) Photothermal heating curves, (c) photostability and (d) photothermal response of the NRs in PBS dispersion (50 µg/mL) under NIR irradiation from a laser of power density 1.0 W/cm2. In (d), the laser was switched off after 10 min of continuous radiation and the system was cooled to room temperature from a steady state. The inset represents the linear time versus negative logarithm of the temperature obtained from the cooling period. 16 ACS Paragon Plus Environment

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When the AR of Co2P@PDA NRs is increased from 1.4 to 6.4 the absorbance at 980 nm increases too, along with a gradual increase in PCE from 26 to 64% (Figure S13a). However this increase is not monotonic since with AR ~10, the PCE drops albeit with a characteristic higher absorbance since the slight decrease in surface area to volume ratio of higher AR NRs, make heat transfer to the surrounding biological medium relatively difficult. Keeping AR the same at 6.4, the PCE of CoP@PDA NRs is 54%, 10% lower than Co2P@PDA NRs (Figure S13b), due to reduced electron-phonon and phonon-phonon interactions in CoP containing lower Co content that restricts heat dissipation from the crystal lattice. The bare PDA nanospheres display a low PCE of 12% suggesting minimal photothermal effects of the PDA shell alone in Co2P@PDA NRs (Figure S13b, inset). Among the wide spectrum of Co2P@PDA NRs with length ranging from 20 to 130 nm synthesized for this work (Figure S3), it is crucial to choose the most suitable NRs for in vitro studies. While the NRs with larger dimensions absorb more incident radiation with monotonous increase of their recorded optical absorbance (Figure S13a), maximum PCE is only observed for the NRs with length 80 to 90 nm (AR 5.5-6.4). Therefore since the 90 nm long NRs (AR ~6.4) have better absorbance and demonstrate the highest PCE of 64% (Figure 2d), this set of Co2P@PDA NRs have been considered for the extensive in vitro studies. Moreover the chosen NRs have a length below 100 nm, ideal for biomedical applications.

In Vitro Cytotoxicity and Photothermal Effect The Co2P@PDA NRs demonstrate excellent cell viability towards HeLa cells (human cervical carcinoma cells) and HEK 293 cells (human embryo kidney cells) for 48 h incubation in the absence of laser irradiation (Figure S14). When HeLa cells co-stained with fluorescein diacetate for live cells and propidium iodide (PI) for dead cells, are cultured and incubated with 50 µg/mL Co2P@PDA NRs alone in vitro, green fluorescence of live HeLa cells, without any red fluorescence, is observed suggesting that cell vialibility is not



compromised (Figure 3a). In fact, more than 95% cells are retained with 50 µg/mL NRs, the typical concentration range for photothermal and photo-chemotherpy experiments. It reduces to 80% only with a high concentration ~320 µg/mL. The same holds true for NIR radiation only, without the NRs (Figure S15). In this study a laser radiation of 980 nm wavelength is used which is well justified from the absorption spectra in Figure 2a where although the absorbance shows a hump at 750 nm the tail extends beyond 980 nm up to the measured 1300 nm. While 750 nm light can completely defer the absorption by hemoglobin and H2O etc., 980 nm is equally viable for PTT which is also shown earlier.44,45 The use of 980 nm wavelength laser is further validated from Figure S15 since in the absence of NRs, negligible cell damage is observed with only laser irradiation. However after 6 h incubation, when the cells are exposed to the laser at 1 W/cm2 for 5 min in the presence of NRs, intense red emission of PI is observed suggesting cell death (Figure 3b). With increase in laser power the intensity of red emission increases due to dissipation of more heat beyond focus area of the laser (Figure S15). The in vitro studies show that under 980 nm NIR laser irradiation the cell viability reduces dramatically with increasing NR concentrations causing photothermal damage of HeLa cells (Figure 3c). For example, the cell viability reduces to 25% within 24 h incubation at a NR concentration of 40 µg/mL with 1 W/cm2 laser, which decreases further upon increasing the laser power density (Figure S16) showing potent photothermal cell killing efficacy of the Co2P@PDA NRs. The high PTT efficiency of Co2P@PDA NRs is likely due to their NR shape which facilitates cellular internalization inside cancerous cells.46 Drug Loading and Release Studies In Vitro DOX was loaded non-covalently onto Co2P@PDA NRs at neutral pH 7.4 since at lower pH the solubility of DOX increases due to protonation of amino groups.19 As one of the criteria for an efficient drug loading vehicle, the PBS solution of Co2P@PDA/DOX shows no 18 ACS Paragon Plus Environment

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aggregation even after several weeks. Both Co2P@PDA/DOX and free DOX show absorption around 493 nm due to π-π* transition of the naphthacenedione component,47 suggesting successful DOX loading onto Co2P@PDA NRs (Figure S17). The fluorescence intensity of DOX in Co2P@PDA/DOX is significantly quenched mainly due to photoinduced electron transfer between DOX and PDA shell (Figure S17).31 The highest DOX loading capacity of Co2P@PDA NRs is quantified up to ~35% by UV-vis spectroscopy (Figure 3d inset) implying hydrogen bonding interaction and π-π stacking between DOX and the PDA shell. The pH dependent DOX release profile from this proficient delivery vehicle was evaluated in presence or absence of laser irradiation. Without laser, ~39% and ~7% DOX is released at pH 5.5 and 7.4, respectively (Figure 3d).

Figure 3: (a) Epifluorescence microscopy images of HeLa cells co-stained by fluorescein diacetate (green emission for live cells) and PI (red emission for dead cells) with (a) only Co2P@PDA NRs and (b) Co2P@PDA + NIR laser irradiation (1 W/cm2). (c) The viability of HeLa cells in presence of different concentrations of Co2P@PDA NRs and 1 W/cm2 laser radiation. Data is represented as mean values ± standard deviations (SD) (n = 3). (d) Drug release study of Co2P@PDA/DOX in PBS buffer at pH 7.4 and 5.5 in the presence or absence of 980 nm laser irradiation. Inset shows the drug loading capacity of Co2P@PDA NRs. 19 ACS Paragon Plus Environment


After NIR laser irradiation (980 nm, 5 min, 1 W/cm-2), drug release escalates by more than five times at pH 7.4 and almost doubled at pH 5.5 within 12 h. NIR light induces a local temperature which increases the kinetic energy of DOX resulting in a burst release of the drug molecules.48 Therefore pH and NIR responsive drug release processes are evident in Co2P@PDA/DOX, useful for synergetic photothermal and chemotherapy.

In Vitro Cytotoxicity of Co2P@PDA/DOX and Co2P@PDA-FA/DOX A vital aspect of any nanocarrier is the targeted delivery towards cancer cells preventing side effects. Without FA tagging, this is not followed by Co2P@PDA/DOX (Figure S14). With Co2P@PDA-FA/DOX the viability of normal HEK 293 cells is prominently retained at a high 8 µg/mL DOX concentration (Figure 4a), since the intracellular pH is non acidic and the cell surface contain very few folate receptors. The maximum DOX used in common practice is less than 4 µg/mL where the cell viability is ~85%. However, free DOX can easily enter and diffuse inside the normal cells and cause significant cell damage by binding with DNA base pairs. Inside Hela cells however Co2P@PDA/DOX release more DOX molecules as compared to free DOX (Figure 4b). The cell viability of free DOX in HEK 293 and Hela cells is different may be their individual cellular microenvironment and the half maximal inhibitory concentration (IC50) value (24 h) of doxorubicin (DOX) in HeLa cells is 37.2 µM and HEK 293 cells is 4.15 µM. Efficient cell killing is observed after 24 h incubation since the folate receptors of HeLa cells help to internalize the Co2P@PDA-FA/DOX and DOX is released in the cytosol region by the influence of intracellular pH and NIR irradiation absorbed by the NRs. As expected the highest cell death is observed with Co2P@PDA-FA/DOX combined to NIR radiation in HeLa cells whereas no obvious cell death was observed for HEK 293 cells under NIR irradiation (Figure S18). These results suggest a feasible mechanism of drug delivery inside the HeLa


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cells (Figure 4b inset) demonstrating the efficacy of Co2P@PDA-FA/DOX as an ideal candidate for targeted safe delivery.

Figure 4: (a) Viability of (a) HEK 293 cells against free DOX and Co2P@PDA-FA/DOX, and (b) HeLa cells against free DOX, Co2P@PDA/DOX, Co2P@PDA/DOX-NIR, Co2P@PDA-FA/DOX and Co2P@PDA-FA/DOX-NIR incubated with different DOX concentrations for 24 h. Data is represented as mean values ± SD (n = 3). (Inset) The schematic illustration shows FA receptor mediated cellular internalization of Co2P@PDAFA/DOX NRs followed by NIR irradiation and pH triggered release of DOX in intracellular lysosome. The overlay fluorescence microscopy images of HeLa cells incubated with (c) free DOX, (d) Co2P@PDA/DOX (e) Co2P@PDA/DOX+NIR for 6h at 37oC. The overlay fluorescence microscopy images of Co2P@PDA-FA/DOX in (f) HEK 293 cells and (g) HeLa cells. Blue and red emissions indicate DAPI and DOX, respectively. (h) The quantitative analyses of DOX uptake by flow cytometry in HeLa cells. x-axis shows fluorescent intensity and y-axis shows the cell counts. Cells were treated with PBS, free DOX, Co2P@PDA/DOX and Co2P@PDA-FA/DOX, for 10 h each.



Cellular Internalization and in vitro Synergistic Therapy The synergistic chemo- and photothermal therapy of Co2P@PDA/DOX was studied by the DOX release behaviour of Co2P@PDA/DOX with or without NIR light. HeLa cells were incubated with free DOX, Co2P@PDA and Co2P@PDA/DOX at 37oC and the DOX fluorescence was analyzed. The fluorescence of DOX was quenched when loaded onto Co2P@PDA NRs (Figure S17) and a retrieval of their fluorescence indicates drug release from Co2P@PDA/DOX. After 6 h incubation, the HeLa cells treated with only Co2P@PDA NRs do not show any DOX fluorescence (Figure S19) and no significant change is observed even when the cells are treated with free DOX with or without NIR light (Figure 4c). After 3 h incubation with Co2P@PDA/DOX, a low intense red DOX fluorescence is observed in both cytosol and cell nucleus whereby the red fluorescence invades the nucleus after prolonged incubation for 6 h. From an overlay of blue and red channels corresponding to the DAPI stained cell nucleus and that of DOX in TRITC channel, respectively, it is evident that with NIR light irradiation for 5 min, the red fluorescence increases significantly (Figure 4d, e). When calculated from 15 cells per sample, indeed the fluorescence intensity is much higher in the presence of NIR irradiation (Figure S20). The drug loaded NRs can efficiently enter the cancerous cells and release the drug molecules to organelles such as endosome/lysosome by the influence of acidic microenvironment along with NIR light irradiation.49 As a cross-check for the internalization capacity of the NRs, a cell impermeable dye propidium iodide, PI loaded onto Co2P@PDA NRs is successfully released inside the HeLa cells (Figure S21). In accordance with the MTT assay shown in Figure 4a and b, the cancer cell specific delivery of the Co2P@PDA-FA/DOX was examined by fluorescence microscopy. After 3h incubation, HEK 293 cells with low folate receptors show a minimal DOX fluorescence in comparison to the intense red fluorescence signal of DOX inside the HeLa cells under similar experimental conditions (Figure 4f, g). The specific folate receptor targeting of Co2P@PDA-


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FA/DOX is further confirmed by a competitive inhibition assay, where the folate receptors on HeLa cell surface were saturated by pre-treatment with FA (5 µM), before incubating with Co2P@PDA-FA/DOX (Figure S22). Due to scarcity of free folate receptors on the HeLa cell surface, the DOX fluorescence is reduced significantly. The cellular uptake was studied by flow cytometry analysis towards HeLa cells whereby the fluorescence intensity for free DOX and Co2P@PDA/DOX is almost similar after 3h incubation. The higher cellular uptake of Co2P@PDA-FA/DOX by folate receptors is manifested from its slightly higher intensity (Figure S23). After 10 h incubation, the fluorescence intensity increases ~5 fold as compared to Co2P@PDA/DOX and free DOX (Figure 4h), implying time controlled drug release from Co2P@PDA-FA/DOX nanocarrier. HEK 293 cells show the reverse effect (Figure S24) whereby free DOX can accumulate inside the HEK 293 cells by inward diffusion but Co2P@PDA/DOX and Co2P@PDA-FA/DOX release minimal quantity of DOX inside HEK 293 cells with non-acidic intracellular pH and absence of enough folate receptors. These results clearly substantiate the Co2P@PDA NRs as a practical nanocarrier system for chemophotothermal therapy. Conclusions In summary, a highly efficient theranostic nanoplatform based on Co2P@PDA NRs has been developed for chemo-photothermal therapy. Along with a high molar extinction coefficient and photothermal conversion efficiency, the NRs exhibit outstanding photostability and minimal cytotoxicity. The DOX loaded NRs allow photocontrolled drug release under 980 nm NIR laser irradiation in vitro. Also, FA functionalization helps in targeted release inside the cancer cells whereas the low folate receptor expressing healthy cells remain majorly unaffected. Alongside the photothermal agents such as Au and metal chalcogenide nanostructures, black P, Fe3O4 and carbon dots prevalent in the literature, Co2P NR is a



worthy candidate demonstrating effective photochemothermal therapeutic applications with reduced side effects. AUTHOR INFORMATION Corresponding Author * Email: [email protected]

Supporting Information Additional FESEM, TEM images, Raman spectra, N2 sorption isotherms, photothermal temperature measurments, PCE measurments, UV-vis-NIR spectrum, cell viability, microscopy images and FACS results. This material is available free of charge via the Internet at http://pubs.acs.org. ACKNOWLEDGEMENTS The Department of Science and Technology (DST) - Science and Engineering Board, Government of India is duly acknowledged for the financial support under grant no. EMR/2016/001703. SK thanks Council of Scientific and Industrial Research (CSIR), New Delhi for his fellowship. The authors acknowledge Dr. Sankar Maiti for extending the laboratory facilities for cell culture and Dr. M. Venkataramanan for providing access to the laser set up. SK acknowledges the help of Rahul Majee for STEM-HAADF imaging and Swagata Das for microscopy imaging. REFERENCES (1) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. Cancer Cell Imaging and Photothermal Therapy in the Near-Infrared Region by Using Gold Nanorods. J. Am. Chem. Soc. 2006, 128, 2115−2120. (2) Ferrari, M. Cancer Nanotechnology: Opportunities and Challenges. Nat. Rev. Cancer 2005, 5, 161–171. (3) Pérez-Hernández, M.; del Pino, P.; Mitchell, S. G.; Moros, M.; Stepien, G.; Pelaz, B.; Parak, W. J.; Gálvez, E. M.; Pardo, J.; de la Fuente, J. M. Dissecting the Molecular Mechanism of Apoptosis during Photothermal Therapy Using Gold Nanoprisms. ACS Nano 2015, 9, 52– 61. 24 ACS Paragon Plus Environment

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Cobalt Phosphide Nanorods with Controlled Aspect Ratios as

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