Enhanced Efficiency of Pd(0)-Based Single Chain Polymeric Nanoparticles for in Vitro Prodrug Activation by Modulating the Polymer’s Microstructure

Bioorthogonal catalysis employing transition metal catalysts is a promising strategy for the in situ synthesis of imaging and therapeutic agents in biological environments. The transition metal Pd has been widely used as a bioorthogonal catalyst, but bare Pd poses challenges in water solubility and catalyst stability in cellular environments. In this work, Pd(0) loaded amphiphilic polymeric nanoparticles are applied to shield Pd in the presence of living cells for the in situ generation of a fluorescent dye and anticancer drugs. Pd(0) loaded polymeric nanoparticles prepared by the reduction of the corresponding Pd(II)-polymeric nanoparticles are highly active in the deprotection of pro-rhodamine dye and anticancer prodrugs, giving significant fluorescence enhancement and toxigenic effects, respectively, in HepG2 cells. In addition, we show that the microstructure of the polymeric nanoparticles for scaffolding Pd plays a critical role in tuning the catalytic efficiency, with the use of the ligand triphenylphosphine as a key factor for improving the catalyst stability in biological environments.


Materials and Methods
All chemicals were purchased either from Merck or TCI chemicals.Deuterated solvents were purchased from Cambridge isotope laboratories.The dialysis membrane was regenerated cellulose tubing purchased from Spectra/Por® with a molecular weight cut-off of 6-8 kDa.All solvents were purchased from Biosolve.Gibco DMEM (glucose concentration 1 g/L, with sodium pyruvate, without L-glutamine, without phenol red) and MEM medium were purchased from Fischer scientific.Dialysis of triphenylphosphine (TPP) functionalised polymer was done in degassed solvents in a wide screw-capped 1 L container, keeping it tightly closed.Dialysis solvents were refreshed regularly with new degassed solvents to prevent oxidation of phosphines.Chloroform used for triphenylphosphine polymers' complexation was dried using Mbraun solvent purification system (MB-SPS 800) and was degassed thoroughly by six freezepump thaw cycles.All the flasks and needles used for TPP polymers were pre-dried in the oven at 135 °C overnight, and experiments were performed under an argon atmosphere.Automated column chromatography was performed on Grace Reveleris X2 Flash Chromatography System using Flashpure BUCHI prepacked silica columns.The polymers were synthesized according to previously reported protocol in our group.p-PFPA180 and p-PFPA200 were synthesized according to previously reported protocol. 1Polymer PBTA and PJ were same polymers as reported before. 2 PCOOH and Pcontrol were synthesized following the same protocol. 2Nile red amine was synthesized according to previously reported protocol. 3[6] Fluorescence measurements were performed on an Agilent Cary Eclipse fluorescence spectrophotometer using 1 cm × 1 cm pathlength quartz cuvettes.Liquid chromatography -UV was performed using Shimadzu UFLC-XR with PDA detector with water + 0.1% formic acid and ACN + 0.1% formic acid as eluents on Kinetex column C18 5 mm EVO 100 Å. HPLC Method for Heck coupling, Suzuki-Miyayura coupling and depropargylation reactions: eluent A: water (0.1% formic acid); eluent B: acetonitrile (0.1% formic acid); and A/B = 90:10 isocratic 2 min, 90:10 to 0:100 in 2 min, isocratic 2 min, 0:100 to 90:0 in 2 min, and isocratic 2.0 min (flow = 0.2 mL/min).High-Performance Liquid Chromatography -HPLC-UV/MS was performed on a SHIMADZU Nexera-I LC-2040C 3D coupled with LC-MS 2020 for detection.Method 2 for pro-5FU: A/B = 95:5 isocratic 15 min on Hypercarb column.DMF-SEC measurements of functionalised polymers were performed using PL-GPC-50 plus (Varian Inc.Company) equipped with a refractive index detector.DMF with 10 mM LiBr was used as eluent at a flow rate of 1 mL min -1 on the Shodex GPC-KD-804 column at 50 °C.Exclusion limit = 100.000Da, 0.8 cm i.d.× 300 mm calibrated using poly (ethylene oxide) from polymer laboratories.Dynamic light scattering experiments were performed using Malvern Zetasizer with 830 nm laser and an angle of scattering 90°.For cell experiments, analysis of the microplate was performed using a Tecan MC-SPARK.Confocal microscopy images of HeLa cells were obtained with a Leica SP5 confocal microscope with a HyD2 detector.Identical conditions were followed throughout all measurements.ImageJ was used for processing images.
High angle annular dark field scanning transmission electron imaging (HAADF-STEM) with energy-dispersive X-ray spectroscopy (EDS) analysis was performed at the LMA-ELCEMI ICTs with a field emission gun microscope (Analytical XFEG FEI Titan, 300 kV) equipped with Cs-probe allowing 0.09 nm mean size electron probe formation (CEOS).

Experimental Procedures
Table S1: Overview of the copolymer composition (a-f), degree of polymerisation (n), molecular weight (Mn,SEC) and molar mass dispersity (Đ) of PBTA-Pcontrol before complexation to Pd(II).Dynamic light scattering results of the polymers complexed to Pd(II) and after CO reduction to Pd(0) in H2O.
. In all cases, RH was determined after the filtration of particles using a 100 nm PVDF filter.The RH was determined from the volume plot of the DLS results, the values in the bracket correspond to a small fraction of larger aggregates present as follows a 3%, b 4%, c 2%, d 3%.
Synthesis of PCOOH: p-PFPA180 (100 mg, 1 eq, 0,0023 mmol) was dissolved in dry and degassed DMF in a Schlenk flask kept in a preheated oil bath at 50 °C.To this solution Nile red amine 15 (3 mg, 3 eq, 0.0069) was added and stirred overnight, the reaction was monitored using 19 F NMR by comparing the peaks of free pentafluorophenol with those in the polymer backbone.The incorporation of Nile red amine was found to be 1% after overnight stirring.To this solution, TPP ligand N-(6-aminohexyl)-4-(diphenylphosphaneyl) benzamide (33 mg, 36 eq, 0,082 mmol) was added and stirred overnight.Followingly, 100 µL triethylamine (not dried) was added to the reaction mixture to trigger hydrolysis of the poly-(pentafluorophenol acrylate) backbone.After monitoring the amount of displaced pentafluorophenol using 19 F NMR, Jeffamine® M-1000 (414 mg, 180 eq, 0.41 mmol) was added.The reaction mixture was then left overnight under argon and the completion of the reaction was again monitored using 19 F NMR.Then, the reaction mixture was purified by dialysis (1 x 1 L methanol, 2 x 1 L THF) for 3 days in a tightly closed screw-capped container.Degassed solvents were used for dialysis which was refreshed as frequently as possible (> 6 h time gap) to prevent oxidation of triphenylphosphine.After dialysis, the THF volume was reduced to ~ 3 mL using a rotary evaporator and the polymer was precipitated into ice-cold pentane (800 mL).The precipitated polymer was washed again with ice-cold pentane, dried under argon flow and was then transferred to a small glass vial.The polymer was then dried under vacuum overnight at 50 °C to yield a bright pink solid and was stored at -19 °C wrapped with aluminium foil.Yield: 42 mg.Mn, SEC-DMF = 36.5 kD.Đ = 1.26.Synthesis of Pcontrol: Synthesis was performed similar to above protocol with varying ligand ratio as follows: p-PFPA200 (100 mg, 1 eq, 0,0020 mmol), dodecyl amine (22 mg, 60 eq, 0.12 eq, Jeffamine® M-1000 (360 mg, 180 eq, 0.36 mmol).Dialysis was performed same as PCOOH but degassing was not performed.The polymer was dried under vacuum overnight at 50 °C to yield a pale solid and was stored at -19 °C.Mtheoretical = 181 kD, Mn, SEC-DMF = 24.4kD, Đ = 1.16.
Preparation of P@Pd(0): All polymers were formulated to nanoparticles by dissolving 10 mg of polymer PBTA-Pcontrol in 9979 µL degassed MilliQ water, followed by the addition of 21 µL of Pd(COD)Cl2 stock solution (100 mM in DMSO) to reach a final concentration of 210 µM Pd(II) in 1 mg/mL polymer solution.Reduction of P@Pd(II) to P@Pd(0) was done using a gas-phase reduction process in a stainless steel Teflon lined autoclave.The resulting homogeneous solution was introduced into the autoclave and gently stirred with a magnetic stirrer.The autoclave was flushed under CO and pressurized to 6 bar.The autoclave was kept at 30 °C for 60 min.After the CO treatment, N2 gas was introduced in the reaction vessel and the solution was further used.The samples after reduction were immediately transferred to a glove box under a nitrogen atmosphere.Samples kept under atmospheric conditions were found to show decreased reactivity after 1 week.

Before Reduction
After Reduction

General procedure for reactions in water :
Depropargylation reaction: Substrate stock solution (1) was prepared in DMSO at 30 mM concentration.PBTA@Pd(0) was at 210 µM Pd(0) concentration.The stock solution was diluted in 3 mL water in 10 mm fluorescence cuvette to a reach a final concentration of Pd(0) = 30 µM; [1] = 30 µM.Cuvettes were then transferred to fluorescence spectrophotometer at 37 °C with stirring and the reaction progress was monitored in real-time.Aliquots from the sample were taken at specified intervals and diluted with 50% ACN by volume which was then analysed using HPLC-UV.

General procedure for reactions in complex media:
Pro-rho 1 activation in different media: Substrate stock solution (1) was prepared in DMSO at 30 mM concentration.PBTA@Pd(0) stock solution was prepared at 210 µM concentration.Depending on each experiment, all stock solutions were diluted in 3 mL water, PBS, DMEM or PBS supplemented with 10% FBS serum, in 10 mm fluorescence cuvette to a reach a final concentration of Pd(0) = 30 µM; [1] = 30 µM.Cuvettes were then transferred to fluorescence spectrophotometer at 37 °C with stirring and the reaction progress was monitored in real-time.Aliquots from the sample were taken at specified intervals and diluted with 50% ACN by volume which was then analysed using HPLC-UV.

Cell experiments
Assessment of cell viability: HepG2 cells were cultured and seeded in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and phenol red.Cytotoxicity of P@Pd(0) was studied using the cell counting kit-8 (CCK-8) assay.A 96-well plate was used seed HepG2 cells.Wells were filled with 100 µL of cell suspension containing 8000 cells.The plate was then placed in an oven at 37 °C with 5% CO2 flow for 24 h.Then, the P@Pd(0) to be tested was added to the cells by varying Pd(0) from 20 -100 µM.The amount of stock solution volume to be added was first removed from the well to keep concentrations constant.Followingly, the plate was placed in the oven.After 24 h, the medium was removed and 100 µL DMEM with 10% CCK8 were added to each well.The plate was then placed back in the oven at 37 °C for 2 to 4 h.The absorbance of each well containing cells was measured at 450 nm at the microplate reader.Cell viability was determined as a fold change of the absorbance with respect to untreated cells.Error bars represent the standard deviation of 3 different wells incubated with same sample.Procedure for pro-rhodamine activation in HepG2 cells: Cultured HepG2 cells were seeded in a µ-Slide 18 well from Ibidi.Wells were filled with 100 µL of cell suspension containing 8000 cells.Cells were incubated with P@Pd(0) and 1 for the incubation time as mentioned from the corresponding stock solutions.Later, the µ-Slide was placed back in the oven.The cells were then monitored in a confocal microscope at an excitation wavelength of 485 nm.For the control experiment, only pro-rho 1 was incubated.It is good to note here that the hydrophobic pro-rho 1 by itself tends to precipitate in aqueous solution at concentrations > 100 µM as large aggregates, which reduces its ability to enter cells.Procedure for pro-drug activation in HepG2 cells: The procedure was followed as explained above for pro-5FU 3, pro-DiFU 5, pro-dox 6 as substrates.After the indicated reaction times the compound containing medium was removed and 100 µL DMEM with 10% CCK-8 was added to each well.The plate was then placed back in the oven at 37 °C for 2 to 4 h.The absorbance of each well containing the cells was measured at 450 nm at the microplate reader.Cell viability was determined as explained before.

Figure S2 :
Figure S2: 31 P NMR of polymer PCOOH in CDCl3.The peak at -5 ppm belongs to triphenylphosphine (TPP) ligands attached to the polymer backbone.Slight oxidation of TPP ligands was also observed as from the peak at 29 ppm indicating the presence of triphenylphosphine oxide.