The primary targets of Hematoporphyrin monomethyl ether‑PDT are reactive oxygen species (ROS) including singlet oxygen and hydroxyl radical, which are generated upon light irradiation.
Intracellular calcium signaling pathways: elevation of cytosolic free calcium concentration ([Ca²⁺]ᵢ) mediated by degradation of sarco/endoplasmic reticulum Ca²⁺‑ATPase (SERCA2) on the endoplasmic reticulum membrane.
Mitochondrial apoptotic pathway: cytochrome c release from mitochondria into cytosol and subsequent caspase‑3 activation. [1]

For photodynamic treatment (PDT), hematoporphyrin monomethyl ether (HMME) is a unique and promising porphyrin-related photosensitizer. In HeLa cells, HMME-PDT can cause both necrosis and apoptosis to cause cell death. HeLa cell-produced ROS, such as hydroxyl and singlet oxygen, are essential for HMME-PDT-induced cell death[2].

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Hematoporphyrin monomethyl ether‑PDT (10 µg/ml HMME, 2 h incubation, then irradiated with 510.6 nm light at 18 kJ/m² for 3 min) induced HeLa cell death through both necrosis and apoptosis. At 24 h post‑irradiation, cell survival rate measured by MTT assay was 19.73 ± 3.98% (vs. untreated control). [1]
Sodium azide (10 mM, singlet oxygen quencher) or D‑mannitol (40 mM, hydroxyl radical scavenger) significantly increased cell survival to 38.14 ± 2.85% and 31.46 ± 3.67%, respectively (P < 0.01 vs. PDT). BAPTA/AM (100 µM, intracellular calcium chelator) increased survival to 42.67 ± 5.67% (P < 0.01 vs. PDT). [1]
Apoptosis was assessed by sub‑G1 population (PI staining) and annexin V/PI flow cytometry. At 12 h post‑PDT, the apoptotic rate was 48.5 ± 2.85%. Sodium azide reduced it to 40.45 ± 3.56%, D‑mannitol to 39.43 ± 4.37%, and BAPTA/AM to 30.56 ± 3.84% (P < 0.05 vs. PDT). [1]
Hematoporphyrin monomethyl ether‑PDT immediately elevated [Ca²⁺]ᵢ: basal level 108.4 ± 7.5 nM; at 0 min post‑PDT 164.5 ± 11.6 nM; at 30 min 315.2 ± 12.6 nM; at 60 min 597.5 ± 41.7 nM (P < 0.01 vs. control). These elevations were significantly blocked by sodium azide or D‑mannitol. [1]
Western blot analysis showed that HMME‑PDT induced cytochrome c release from mitochondria into cytosol at 6 h and 12 h after PDT. Sodium azide, D‑mannitol or BAPTA/AM partly prevented this release. Densitometric analysis at 6 h: decrease in cytochrome c release by 18.78 ± 2.31% (sodium azide), 38.37 ± 5.47% (D‑mannitol), and 43.88 ± 6.73% (BAPTA/AM) (P < 0.05 vs. PDT). At 12 h: decreases of 28.28 ± 3.51%, 21.99 ± 2.47%, and 37.10 ± 4.73%, respectively. [1]
Caspase‑3 activity was measured using DEVD‑AFC substrate. At 6 h post‑PDT, activity increased 7.19 ± 0.43‑fold over control; at 12 h, 5.58 ± 0.48‑fold. Sodium azide reduced the fold increase to 4.50 ± 0.35 (6 h) and 3.79 ± 0.41 (12 h); D‑mannitol to 5.02 ± 0.51 (6 h) and 4.07 ± 0.33 (12 h); BAPTA/AM to 3.03 ± 0.38 (6 h) and 3.61 ± 0.46 (12 h) (P < 0.01 vs. PDT). [1]
Hematoporphyrin monomethyl ether‑PDT induced rapid degradation of SERCA2 (the ubiquitously expressed SERCA2b isoform in HeLa cells) as shown by Western blot. Degradation was partly inhibited by sodium azide or D‑mannitol. [1]

Caspase‑3 activity assay: Cells were collected at various post‑PDT times by centrifugation, resuspended in 50 µl cell lysis buffer, and incubated for 10 min at 0 °C. Lysates were centrifuged to remove debris. Then 50 µl of 2× reaction buffer/DTT mix and 5 µl of caspase‑3 substrate (DEVD‑AFC) were added to the supernatants. After 1 h incubation at 37 °C, fluorescence was measured with a fluorescence spectrophotometer using 400 nm excitation and 505 nm emission filters. Caspase‑3 activity was expressed as fold of treated cells’ emitted fluorescence over normal cells. Protein concentration was measured using a protein assay. [1]
SERCA2 Western blot: HeLa cells were collected, washed with cold PBS, and lysed with lysis buffer for 30 min on ice, then centrifuged at 10,000×g for 10 min. Detergent‑soluble proteins (100 µg) were separated by 5% SDS‑PAGE under reducing conditions and transferred to membranes. Membranes were incubated with SERCA2 antibody (1:1000) for 2 h at room temperature, followed by anti‑mouse HRP‑conjugated secondary antibody (1:1000). Chemiluminescent signals were imaged on X‑ray films and quantified by densitometric scanning. [1]
Cytochrome c release assay: Cells were harvested by centrifugation at 1000×g for 5 min at 4 °C, washed with cold PBS, and resuspended in five volumes of buffer A (250 mM sucrose, 20 mM HEPES‑KOH, 10 mM KCl, 1.5 mM MgCl₂, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 10 µg/ml aprotinin, pH 7.5). Homogenization was performed by 15‑20 passages through a 22‑gauge needle. Homogenates were centrifuged twice at 750×g for 10 min at 4 °C to remove debris, then supernatants were centrifuged at 10,000×g for 15 min at 4 °C to obtain mitochondrial pellets. Cytosolic extracts were obtained by centrifuging the above supernatants at 100,000×g for 1 h at 4 °C. Protein concentration was determined, and 100 µg of mitochondria or S‑100 fraction was loaded onto 15% SDS‑PAGE. Western blot used mouse monoclonal IgG1 anti‑cytochrome c antibody (1:500) and secondary anti‑mouse HRP‑conjugated antibody (1:1000). [1]

Cell culture and PDT: HeLa cells were maintained in DMEM with 10% heat‑activated bovine serum, 100 IU/ml penicillin, and 100 mg/ml streptomycin at 37 °C in 5% CO₂. For PDT, medium was replaced with fresh medium containing 2% FBS with or without 10 µg/ml Hematoporphyrin monomethyl ether for 2 h in the dark. Cells were then irradiated with 510.6 nm light from a copper vapor laser at a fluence of 18 kJ/m² (light exposure time 3 min, frequency 6 kHz) at room temperature. After irradiation, cells were kept in 5% CO₂ at 37 °C until assay. Sodium azide (10 mM), D‑mannitol (40 mM), or BAPTA/AM (100 µM) were added to cultures 2 h prior to light irradiation as indicated. [1]
Cell survival assay (MTT): Cells were seeded into 96‑well plates at 1×10⁴ cells/well in 200 µl culture medium. After overnight growth, cells received PDT treatment as described. At 24 h post‑irradiation, 20 µl MTT (final concentration 0.5 mg/ml) was added to each well and incubated for 4 h at 4 °C. Medium was replaced with 200 µl DMSO to dissolve formazan crystals. Plates were shaken for 30 min at room temperature, and optical density was read at 570 nm. Survival rate was expressed as percent of treated cells’ absorbance relative to normal cells. [1]
Measurement of [Ca²⁺]ᵢ: Cells were washed with D‑PBS, harvested by trypsinization, and resuspended in RPMI‑1640 with 10% FBS to 2×10⁷/ml at various post‑treatment times. Cells were loaded with Fura‑2/AM (final concentration 5 µM) for 45 min at 37 °C. After loading, cells were centrifuged, washed three times with Ca²⁺‑free HEPES‑buffered HBSS, and resuspended in 2 ml Ca²⁺‑free HBSS to 10⁶/ml. Fluorescence was measured at 37 °C with a fluorescence spectrometer set to alternate between excitation wavelengths 340 and 380 nm every 4 s; emission was 500 nm. [Ca²⁺]ᵢ was calculated using the formula: [Ca²⁺]ᵢ (nM) = (F – F_min) / (F_max – F) × 224, where F is the 340/380 ratio. F_max was obtained by adding 0.1% Triton X‑100 and 1.25 mM CaCl₂, and F_min by adding 10 mM EGTA. [1]
Apoptosis detection (annexin V/PI): About 0.5×10⁶ treated or untreated cells were scraped, washed twice with cold PBS, and resuspended in 480 µl annexin V binding buffer. Cells were incubated with 5 µl annexin V and 10 µl propidium iodide for 20 min at room temperature in the dark, then analyzed by flow cytometry (1×10⁵ cells per sample). Lower right quadrant (annexin V⁺/PI⁻) = early apoptosis; upper right quadrant (annexin V⁺/PI⁺) = late apoptosis. Total apoptotic rate = sum of lower right and upper right quadrants. [1]
Apoptosis detection (sub‑G1 population): Cells were harvested by trypsinization, resuspended in 200 µl PBS, and fixed in 2 ml 75% ice‑cold ethanol overnight at 4 °C. Cells were spun down, resuspended in 950 µl PBS containing 0.1 µg/ml RNase A and 40 µg/ml PI, stained at 37 °C for 30 min, and examined by flow cytometry (10,000 cells/sample). Apoptotic cells were quantified as percentage of cells in sub‑G1 phase. [1]

[1]. Ding X, et al. Hematoporphyrin monomethyl ether photodynamic damage on HeLa cells by means of reactive oxygen species production and cytosolic free calcium concentration elevation. Cancer Lett. 2004;216(1):43-54.

Hematoporphyrin monomethyl ether is a second‑generation porphyrin‑related photosensitizer developed to overcome drawbacks of first‑generation photosensitizers such as long‑term skin photosensitization (up to 10 weeks), suboptimal activation wavelength for tissue penetration, and poorly defined chemical composition. HMME consists of two monomer porphyrins that are positional isomers. The photodynamic therapy mechanism involves generation of reactive oxygen species (singlet oxygen and hydroxyl radical) upon light activation, which then induce ER stress via SERCA2 degradation, leading to cytosolic calcium elevation, cytochrome c release, caspase‑3 activation, and subsequent apoptosis. [1]

C70H80N8O12

1225.43

612.294

148471-91-4

Typically exists as solid at room temperature

1.3±0.1 g/cm3

1123.6±65.0 °C at 760 mmHg

633.3±34.3 °C

0.0±0.3 mmHg at 25°C

1.622

6.87

CC(C1=C(C)C2=NC1=CC1=C(C)C(CCC(=O)O)=C(C=C3C(CCC(=O)O)=C(C)C(C=C4C(C)=C(C(OC)C)C(=C2)N4)=N3)N1)O

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 62.5 mg/mL (51.00 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (1.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)