Cancer-mitochondria-targeted photodynamic therapy with supramolecular assembly of HA and a water soluble NIR cyanine dye

Herein, we introduce an indocyanine derivative (IR-Pyr) that is highly water soluble, exhibiting higher mitochondrial targetability and better photostability than IR-780.


General Information:
The reagents and materials for the synthesis were used as obtained from Sigma -Aldrich and Alfa Aesar chemical suppliers. Sodium hyaluronate was purchased from Acros Organic (Belgium). All solvents were used after drying by standard methods prior to use. The NMR solvents were used as received and the spectra were recorded with Agilent 400 MHz spectrometer. Spectra were referenced internally by using the residual solvent ( 1 H δ =3.34 and 13 C δ = 49.86 for CD 3 OD-d4) resonances relative to SiMe4. The ESI-MS spectra were recorded on Bruker, 1200 Series & HCT Basic System. The electronic absorption spectra and steady state fluorescence spectra were recorded on Jasco V-670 spectrophotometer and Hitachi F-7000 fluorescence spectrophotometer respectively. DLS was measured in a Malvern-Zetasizer nano system.

Cell culture and cell viability:
Human cervical cancer HeLa cells and non-cancerous fibroblast HeK293T cells were cultured (using DMEM medium) supplemented with 10% fetal bovine serum (FBS), 100 µgmL -1 streptomycin and 100 U mL -1 penicillin in sterile 96-well Nunc (Thermo Fisher Scientific Inc.) microtitre plate at a seeding density of 5 x 10 3 cells/well and they were allowed to settle for 24 h under incubation at 37 °C and 5% CO 2 . In-order to check cell viability, the cells was then treated with different concentrations of Ir-780, IR-Pyr and HA-IR-Pyr (2.5, 5.0, 10.0 and 20 µM) for 12 h incubation, washed, replaced with fresh media and checked the cell viability after another 12 h incubation using the alamar blue dye assay by setting the excitation wavelength at 565 nm and monitoring emission at 590 nm excitation under dark and in presence of laser irradiation after 3 min at 200 mWcm -2 .

Cellular uptake analysis:
HeLa and HeK293T cells were seeded in one well glass cover glass (Lab Tek II, Thermo Scientific) at a seeding density of 2 x 10 5 cells/well. After 24 h, cells were treated with 2.5 µM for a period of 4 h and replaced with fresh media. The cellular uptake was monitored periodically using Carl Zeiss LSM 780 NLO multiphoton microscope connected to CO 2 incubator setting the excitation at 720 nm and emission between 725-758 nm along with the colocalization analysis with mitotracker green FM setting excitation at 488 nm and emission between 500-550 nm.

Endocytic pathway analysis.
To check the endocytosis mediated uptake, HeLa cells were seeded in chambered cover glass and pretreated with different endocytosis inhibitors including sucrose (clathrin-mediated uptake, 400 nM), methyl-β cytodextrin (caveolae mediated uptake) and amilorin (macropinocytosis) in serum-free DMEM for 1 h and replaced with fresh media. Afterwards, the uptake pathway for HA-IR-Pyr at 2.5 µM for a period of 4h and analyzed using the Carl Zeiss LSM 780 NLO Multiphoton microscope connected to CO 2 incubator setting the excitation at 720 nm and emission between 725 nm to 758 nm.

MitoSox ROS generation analysis.
HeLa and HeK293T cells were seeded on a Lab Tek II chamber cover glass at 90% confluence in DMEM media supplemented with 10% FBS, 100 µg mL -1 streptomycin, 100 UmL -1 penicillin and incubated at 37 °C under 5% CO2. After incubation with 2.5 µM of HA-IR-Pyr for different time intervals, by following the manufacturer's protocol (MitoSox, M36008); the cell culture medium was then replaced with media containing 5 μM MitoSox reagent working solution to cover the adherent cells. The cells were then incubated for 10 minutes at 37 °C, protected from light and irradiated using 200 mWcm -2 . The cells were then analyzed under a FV1000 laser confocal scanning microscope.

TMRM depolarization analysis:
Confocal imaging of TMRM depolarization in HeK293T and HeLa cells were pre-incubated 200 nM of TMRM for 30 min and incubated with 2.5 μM of HA-IR-Pyr and analyzed using an FV1000 laser confocal scanning microscope at different time intervals.

Composition and CMC of HA-IR-Py
To prepare HA-IR-Pyr, HA (10 mg) was dissolved in 15 ml of DI water. To this solution, IR-Pyr (5 mg, dissolved in 1 ml DI water) was added slowly with continuous stirring. Further, diluted by adding another 5 ml DI water and allowed to stir for overnight at room temperature.
The solution was centrifuged to remove any precipitated particles and dialyzed using a 3.5KD cut off membrane tube in DI water for 24h. The composition of micelles was 1:2 weight ratio of IR-Pyr and HA.

NMR spectral analysis:
Fig. S1 1 H-NMR spectrum of 1 in CD 3 OD.       To measure the CMC, we have tried several methods to evaluate the CMC of HA-IR-Pyr. We tried the well-known dye encapsulation methods (Nile red or Pyrene encapsulation), but failed to obtain meaningful data and CMC value. We have performed concentration dependent size distribution analysis. Interestingly, we found that there was no difference in the size distribution in accordance with the concentration of the micelle ( Figure S14a). Further, we observe that the correlation functions have not disappeared even below 0.25 μM of IR-Pyr concentration in the micelle ( Figure S14b). These observations suggest that once the micelle formed by the electrostatic interaction (between the positively charged IR-Pyr and negatively charged HA) and hydrophobic-hydrophilic interactions, the micelle shows strong stability and the CMC might be very low.

In-vitro experiments
Hek HA-Ir-Pyr BF Merged