Supplementary materials

Results

Photophysical Measurements

The three bacteriochlorins have a strong ( ~ 100,000 M1cm1) absorption feature (the Qy band) between 720 and 750 nm in the near-infrared spectral region. Due to the limited supply of 1, the photophysical properties appropriate for this bacteriochlorin were measured for two analogs described previously (16), 5-methoxy-8,8,18,18-tetramethyl-2,12-di-p-tolylbacteriochlorin and 8,8,18,18-tetramethyl-2,12-di-p-tolylbacteriochlorin. The results are shown in Table S1.

Table S1. Photophysical Properties and Molecular-Orbital Energies of Bacteriochlorins.a

Cmpd	Qy(0,0) b absorption	Qy(0,0) b fluorescence	fc	S d	isce	Tf	Orbital Energy
λ (nm)	fwhm (nm)	λ (nm)	fwhm (nm)	(ns)	(s)	HOMO (eV)	LUMO (eV)
1	739	23	739	24	0.16g	4.1g	0.51g	55g	4.39	2.20
2	721	15	727	21	0.093	4.0	0.58	180	4.36	2.12
3	737	24	745	20	0.12	3.7	0.41	200h	4.46	2.28

aAll measurements performed on compounds at room temperature. bPeak wavelength (λ) and full width at half maximum (fwhm) of spectral feature for compound in aerated solution of DMSO, except the fluorescence data for bacteriochlorin 1 is for the compound in methanol. cFluorescence quantum yield for compound in Ar-purged methanol. dLifetime of the lowest singlet excited state for compound in Ar-purged methanol determined using fluorescence detection. eMeasured yield of the lowest triplet excited state for the compound in Ar-purged methanol. fLifetime of the lowest triplet excited state for the compound in Ar-purged MeTHF unless indicated otherwise. gThe average values from two analogs of bacteriochlorin 1 have the following characteristics (all measurements in Ar-purged toluene except for T which utilized the compound in Ar-purged MeTHF): f = 0.14, s = 3.4 ns, isc = 0.53, T = 45 s for 8,8,18,18-tetramethyl-2,12-di-p-tolylbacteriochlorin (determined here; f = 0.18, s = 4.8 ns, isc = 0.50, T = 65 s for 5-methoxy-8,8,18,18-tetramethyl-2,12-di-p-tolylbacteriochlorin determined previously (reference S1). hThe triplet lifetime of bacteriochlorin 3 in deoxygenated (by freeze-pump-thaw procedure) aqueous Cremophore micellar solution is 190 s.

Cellular destruction upon in vitro PDT

Supplementary Figure S1 provides TEM micrographs that show morphological changes observed after PDT with bacteriochlorin 2. The figure also compares PDT killing effects of Photofrin and bacteriochlorin 3.

Supplementary Figure S1.

Legend to Figure S1. TEM micrographs of morphological changes observed after PDT with bacteriochlorin 2. A) Control, non-treated cells; B) B16F10 melanoma cells 8 h after PDT with bacteriochlorin 2. Scale bar is 5 µm. Comparison of Photofrin (C) and bacteriochlorin 3 (D) PDT killing effects measured by MTT metabolic assay and also by crystal violet (CV) cellular integrity assays.

Melanin levels

The melanin absorption of 1x106 cells of all tested melanoma cell lines were measured, including variants of the same cell line mouse B16 melanoma, in 1 mL of 0.1 M NaOH/1%SDS after being lysed by overnight incubation at 37 °C. The absorbance was measured with spectrophotometer (Evolution 300, UV-VIS, Thermo Fisher Scientific, Franklin, MA). B16-G4F lacks the -MSH receptor and has low melanin levels (OD at 400 nm of 0.055) (37). B16F1 has moderate levels of melanin (OD at 400nm of 0.5214); however, after growth for 7 days in low glucose-containing medium the melanin level was greatly increased (B16F1 LG, OD at 400 nm of 1.1841). Supplementary Figure S2 shows absorption spectra between 300 and 700-nm for the described cell lines.

Supplementary Figure S2.

Legend to Figure S2. Visible absorption spectra of cell lysates containing equal numbers of melanoma cells of B16G4F (low melanin), B16F1 growing in RPMI medium (moderate melanin) and B16F1 growing in low glucose DMEM medium (high melanin).

Colocalization of Lysotracker and TRP-1

Since lysosomes and melanosomes share common origin we had to determine the difference in staining pattern of Lysotracker and TRP-1. Supplementary Figure 3 shows that Lysotracker RED stained both Lysosomes (red color on micrograph) as well as melanosomes since Lysotracker RED overlayed with the TRP-1 staining resulted in yellow. The TRP-1 stained only melanosomes. This result together with the results presented in Figure 3 support our finding that the bacteriochlorins examined herein localize in both lysosomes and melanosomes.

Supplementary Figure S3.

Legend to Figure S3. Fluorescence micrographs of B16F10 cells. (A) Green fluorescence from FITC-anti-TRP1 antibody that stains melanosomes; (B) Red fluorescence from Lysotracker that stains lysosomes; (C) Overlay showing that Lysotracker stains Lysosomes (red) as well as melanosomes (yellow from red and green overlay).

Reference

S1.	Kee, H. L., Diers, J. R., Ptaszek, M., Muthiah, C., Fan, D., Lindsey, J. S., Bocian, D. F., and Holten, D. (2009) Chlorin–bacteriochlorin energy-transfer dyads as prototypes for near-infrared molecular imaging probes: Controlling charge-transfer and fluorescence properties in polar media, Photochem. Photobiol. 85, 909–920