Post-functionalization of drug-loaded nanoparticles prepared by polymerization-induced self-assembly (PISA) with mitochondria targeting ligands

Herein, the postfunctionalization of different non-fouling PISA particles, prepared from either poly(oligo ethylene glycol methyl ether methacrylate) (pPEGMA) and the anticancer drug PENAO (4-(N-(S-penicillaminylacetyl)amino)phenylarsenonous acid) or zwitterionic 2-methacryloyloxyethyl phosphorylcholine (MPC) and PENAO were reported. Both PISA particles were reacted with triphenylphosphonium (TPP) as mitochondria targeting units in order to evaluate the changes in cellular uptake or the toxicity of the conjugated arsenic drug. Attachment of TPP onto the PISA particles however was found not to enhance the mitochondrial accumulation, but it did influence overall the biological activity of pMPC-based particles in 2D and 3D cultured sarcoma SW982 cells. When TPP was conjugated to the pMPC PISA particles more cellular uptake as well as better spheroid penetration were observed, while TPP on PEG-based PISA had only little effect. It was hypothesized that TPP on the micelle surface may not be accessible enough to allow mitochondria targeting, but more structural investigations are required to elucidate this.


Dynamic light scattering (DLS).
As described in ref. [1], DLS was performed on aqueous solutions (approximately 1 mg mL -1 polymer in milli-Q water) which were analysed by a Malvern Zetasizer Nano ZS instrument equipped with a 4 mV He-Ne laser operating at λ = 632 nm and non-invasive backscatter detection at 173°. Measurements were carried out in a disposable cuvette at 25 °C, provided 15 scans equilibration period prior to each set of measurements. For a given sample, a total of three measurements were conducted with the number of runs, attenuator, and path length being automatically adjusted by the instrument, depending on the sample quality.

Critical micelle concentration (cmc)
The cmc was obtained by dynamic light scattering measurements, quantifying the scattering intensity for different micelle concentrations. The aqueous micelle solutions (approx. 1 mg mL -1 ) were serially diluted with milli-Q water to 0-100 µg mL -1 working solutions. For each sample, a total of two measurements with a 10 scan equilibration period prior to each set of measurements were conducted. The number of runs was set to 11 (run duration 10 sec.), attenuator and path length was fixed to 7 and 4.65.

Laser scanning confocal microscopy (LSCM)
As described in ref. [1], LSCM (Zeiss LSM 780) was used to observe the cellular localization and spheroid penetration of the PENAO micelles. imaging software (Zeiss) was used for image acquisition and processing.

Statistical analysis
As described in ref. [1], all data for in vitro studies were reported was mean ± standard deviation (SD). A one-way analysis of variance was performed for the statistical analysis followed by a Turkey's post hoc test for pairwise comparison. A P value <0.05 was considered statistically significant. All statistical analysis was done with GraphPad Prism 7.

IC50 Determination via WST-1 assay
As described in ref. [1], cytotoxicity test of polymeric micelles PPM-NP4, MPM-NP2 and PENAO were determined by a WST-1 based assay. Synovial sarcoma (SW982) cell lines were seeded in a 96-well plate (8000 cells/well) and incubated at 37 °C in a 5% CO2 atmosphere for 24 h. For the cytotoxicity assay, the medium in the cell culture plate was discarded and 100 µL of fresh 2x concentrated RPMI 1640 medium was added to each well of the 96-well culture plate. The specimens were sterilized by UV irradiation for 20 min before serially diluting (sequential halved dilution) with sterile water. They were then added into the plate at 100 µL per well, respectively. As a control, sterile water (100 µL) was added to the nontreated cells. The cells were incubated with the particles and PENAO for 72 h. The old medium was discarded, and cells were washed with PBS. 100 µL of fresh warm medium along with 5 µL WST-1 was added to each well, and the plates were incubated for further 3 h at 37 °C before reading the absorbance on a Bio-Rad BenchMark microplate reader at 440 nm with a reference wavelength of 650 nm. Dose-response curves were plotted accordingly where the values were S1 expressed as percentage of control (non-treated cells were used as controls). The optical density was used to calculate cell viability. The experiments were carried out in triplicate.

3D Sarcoma MCTS formation
As described in ref. [1], the sarcoma MCTSs were prepared by a liquid overlay method [2].
The 143B and SW982 cells were suspended in RPMI-1640 media at a density of 12.5 x 10 3 cells/mL and 15 x 10 3 cells/well respectively. The cells were seeded (200 µL) into each well of an ultralow attachment 96-well plate (Coming) and the plate was centrifuged for 5 min at 1900 rpm and incubated at 37 °C for 5 (143B) and 6 days (SW982). The spheroid formation was recorded by an inverted microscope with CCD camera (Leica).

Micellar treatment of sarcoma MCTS
As described in ref. Penetration studies into sarcoma MCTS As described in ref. [1], the formed SW982 and 143B MCTSs were washed with 150 µL PBS as described above before the particles (100 µL mL -1 in 200 µL media) were loaded into each well and incubated for 3 hours at 37 °C and 5% CO2. The spheroids were washed with Hanks' Balanced Salt Solution twice and imaged using a Zeiss LSM780 laser scanning confocal microscope. The excitation wavelengths were set as 488 nm and the images were captured and processed using ZEN software.

Particle localization via confocal microscopy
As described in ref. [1], SW982 cells were seeded into a fluoro dish at a density of 5 x 10 4 cells in 2 mL RPMI medium per dish and incubated at 37 °C at a 5% CO2 atmosphere for 24 h. The medium was discarded, and the cells were washed with PBS (1 mL hours. The cloudy mixture was filtered and the solvent was removed under reduced pressure. The crude product was then purified by silica gel column chromatography using a 1:4 solvent mixture of ethylacetate/hexane. The product was obtained as pink sticky liquid (yield = 57%).

S1
As described in ref. [1], the PENAO containing copolymer MP2 was prepared by The previous described copolymers PP3 and MP2 were used as macro RAFT agents for the particle formation by the PISA process with methyl methacrylate (MMA). As described in ref. This content is not subject to CC BY 4.0