Hybrid Au@alendronate nanoparticles as dual chemo-photothermal agent for combined cancer treatment

A gold therapeutic nanoplatform with the same molecule used as reductant, coating and therapeutic agent has been developed in a one-pot, one-phase process using alendronate, a drug from the bisphosphonate family known for its antitumor effects. In addition, the core made of gold nanoparticles (NPs) brings thermal functionalities under irradiation within the first biological window (650–900 nm). The Au@alendronate nanoplatform thus provided a combined antitumor activity through drug delivery and photothermal therapy. Au@alendronate NPs inhibited in vitro the proliferation of prostate cancer cells (PC3) in a dose-dependent manner, with an IC50 value of 100 µM. Under NIR irradiation a temperature increase was observed leading to a reduction of the IC50 value to 1 µM, with total tumor cell death at 100 µM.


Alendronate synthesis
Alendronate was synthesized according to the general procedure for linear aliphatic BPs and characterized by 1 H and 31 P NMR [1,2]. BPs was prepared from the corresponding carboxylic acid precursor according to the following reaction carboxylic acid (150 mmol) and H 3 PO 3 (150 mmol) were introduced in a three-necked round-bottom flask under inert atmosphere followed by 30 mL of methanesulfonic acid. After heating at 65 °C for 1 h, PCl 3 (40 mmol) was added slowly and the reaction allowed to proceed overnight at 65 °C. The resulting yellow viscous reaction mixture was cooled to room temperature, quenched with 500 mL of ice-cold water. The pH was adjusted to 4.3 with a NaOH aqueous solution (0.5 M) and the obtained white precipitate was collected by filtration. This solid was washed five times with a mixture of methanol/water (95:5), dialyzed for 3 days and freeze-dried to finally obtain linear aliphatic BPs (82%). Carboxylic acid precursor: 4-aminobutyric acid. RMN 31 P (80.9 MHz,

Au@alendronate NPs synthesis
The procedure was adapted from previous study [3] instead of the HMBP reducing/ligands agent is replaced by alendronate. Two precursor solutions were prepared. Solution A consisted in 1 mL of 20 mM chloroauric acid (HAuCl 4 ·3H 2 O, +99.9%, Sigma-Aldrich, St Louis, MO). Solution B consisted in 2 mL of alendronate dissolved in Milli-Q water at pH adjusted at 10 with NaOH. Solution A is added to 17 mL of Milli-Q water in a round bottom flask equipped with a condenser. The solution was brought to the boil while being stirred.
Solution B was added leading to a red color after 10 minutes, denoting for the apparition of nanoparticles. pH is adjusted at 7. Nanoparticles were washed with Amicon® Ultra centrifugal filters (100 K).

Au@alendronate NPs characterisations -ICP analysis
The average number of alendronate molecules at the surface of the particles was determined using an ICP-AES spectrometer (iCAP 6200 duo, Thermofisher). Samples were digested in a HNO 3 and HCl solution (10 mL) evaporated then solubilized in HNO 3 2% for the analysis of phosphorus concentrations.
-TEM analysis S3 grids covered by a carbon film. Observation was carried out with a Philips Tecnai 12 transmission electron microscope. Images were processed with Fiji is just Image J software on 250 particles in order to determine the nanoparticles diameter.
-Absorbance analysis UV-vis spectra were recorded on a Varian Cary 50 Scan UV-vis spectrophotometer.
-Infrared spectroscopy The grafting of the molecules at the surface of the particles was confirmed with Fourier transformed infrared spectroscopy (FTIR) analysis. Spectra were recorded through the form of thin KBr pellets on a Thermo Scientific Nicolet 680 FTIR.
-Hydrodynamic diameter and zeta potential SAR was calculated using the following equation: . d with C is the specific heat capacity of the sample (C water = 4.185 J/g/K), m Au is the total mass of gold in the sample (g), m s is the total mass of the sample (g) and dT/dt is the temperature increase at the initial linear slope (30 s).  Figure S1: Stability of Au@alendronate: Hydrodynamic diameter of Au@alendronate determined by DLS measurement (a) as a function of pH (b) according to synthesis age (comparison of the diameter on the day of the synthesis and after 120 days).