Design of Pluronic-based formulation for enhanced
Supporting Information for
Design of Pluronic-based formulation for enhanced redaporfin-photodynamic therapy against pigmented melanoma Barbara Pucelik, † Luis G. Arnaut,§ Grażyna Stochel† and Janusz M. Dąbrowski†*
†
§
Faculty of Chemistry, Jagiellonian University, 30-060 Kraków, Poland
CQC, Chemistry Department, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal
* Correspondence should be sent to Dr. Janusz M. Dąbrowski, [email protected], +48126632293, +48126340515 (fax)
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Cellular uptake studies Cellular uptake of redaporfin and its Pluronic-based was investigated in B16F10 cells exposed to 5 μM of redaporfin in culture medium for various time intervals, from 1 hour up to 24 hours. The retention of cell-associated redaporfin was detected by fluorescence (λexc=505,
λem=750 nm) with the microplate. The fluorescence spectra of redaporfin and redaporfinP123 at each incubation time are presented in Figure S1.
Figure S1. The fluorescence spectra of redaporfin (a) and redaporfin-P123 (b) registered at each point of incubation time with B16F10 melanoma cells. Dark cytotoxicity studies The cytotoxicity of redaporfin in the dark against B16F10 cells was also tested using the AlamarBlue assay (Figure S2). Negligible toxicities were found in the redaporfin concentration range from 0 – 20 μM. Pluronics F127 and P123 alone exhibit no dark toxicity in the same concentration range (see Figure S3).
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Figure S2. Dark cytotoxicity of redaporfin, redaporfin-F127 and redaporfin-P123 investigated in B16F10 cells after 4 h (a) and 20/24 h (b) of incubation time estimated by Alamar Blue assay.
Figure S3. Dark cytotoxicity of Pluronic F127 and Pluronic P123 empty micelles investigated in B16F10 melanoma cells after 24 h of incubation.
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Photodynamic effect The PDT efficacies of the redaporfin and redaporfin-F127 or redaporfin-P123 were tested against B1610 mouse melanoma cells after incubation for 4 h, 20 h or 24h with photosensitizer concentrations of 5 μM. Figure S4 shows the decrease of cell viability with an increase of illumination time using a 735±20 nm light source determined by the Alamar Blue assay which measures cellular integrity, and found to be consistent results obtained by MTT assay.
Figure S4. Photodynamic effect of redaporfin, redaporfin-F127 and redaporfin-P123 against B16F10 after 4h (a) and 20 h or 24 h (b) incubation with estimated by Alamar Blue assay.
Photostability experiments under in vitro biological conditions Solutions containing samples of redaporfin were dissolved in DMSO/PBS or encapsulated in P123 and F127 micelles and irradiated with a 735±20 nm LED irradiation system in 96-plate microplate for various time intervals. The absorption spectra were recorded before the irradiation and immediately after irradiation between 1 and 60 minutes. The experimental conditions were the same as in photodynamic effect experiments.
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As illustrated in Fig. S5, after 60 min. of irradiation the relative redaporfin content inside the P123 and F127 formulation decreased ca. 10 %, while that of redaporfin in PBS (DMSO
Design of Pluronic-based formulation for enhanced redaporfin-photodynamic therapy against pigmented melanoma Barbara Pucelik, † Luis G. Arnaut,§ Grażyna Stochel† and Janusz M. Dąbrowski†*
†
§
Faculty of Chemistry, Jagiellonian University, 30-060 Kraków, Poland
CQC, Chemistry Department, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal
* Correspondence should be sent to Dr. Janusz M. Dąbrowski, [email protected], +48126632293, +48126340515 (fax)
S-1
Cellular uptake studies Cellular uptake of redaporfin and its Pluronic-based was investigated in B16F10 cells exposed to 5 μM of redaporfin in culture medium for various time intervals, from 1 hour up to 24 hours. The retention of cell-associated redaporfin was detected by fluorescence (λexc=505,
λem=750 nm) with the microplate. The fluorescence spectra of redaporfin and redaporfinP123 at each incubation time are presented in Figure S1.
Figure S1. The fluorescence spectra of redaporfin (a) and redaporfin-P123 (b) registered at each point of incubation time with B16F10 melanoma cells. Dark cytotoxicity studies The cytotoxicity of redaporfin in the dark against B16F10 cells was also tested using the AlamarBlue assay (Figure S2). Negligible toxicities were found in the redaporfin concentration range from 0 – 20 μM. Pluronics F127 and P123 alone exhibit no dark toxicity in the same concentration range (see Figure S3).
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Figure S2. Dark cytotoxicity of redaporfin, redaporfin-F127 and redaporfin-P123 investigated in B16F10 cells after 4 h (a) and 20/24 h (b) of incubation time estimated by Alamar Blue assay.
Figure S3. Dark cytotoxicity of Pluronic F127 and Pluronic P123 empty micelles investigated in B16F10 melanoma cells after 24 h of incubation.
S-3
Photodynamic effect The PDT efficacies of the redaporfin and redaporfin-F127 or redaporfin-P123 were tested against B1610 mouse melanoma cells after incubation for 4 h, 20 h or 24h with photosensitizer concentrations of 5 μM. Figure S4 shows the decrease of cell viability with an increase of illumination time using a 735±20 nm light source determined by the Alamar Blue assay which measures cellular integrity, and found to be consistent results obtained by MTT assay.
Figure S4. Photodynamic effect of redaporfin, redaporfin-F127 and redaporfin-P123 against B16F10 after 4h (a) and 20 h or 24 h (b) incubation with estimated by Alamar Blue assay.
Photostability experiments under in vitro biological conditions Solutions containing samples of redaporfin were dissolved in DMSO/PBS or encapsulated in P123 and F127 micelles and irradiated with a 735±20 nm LED irradiation system in 96-plate microplate for various time intervals. The absorption spectra were recorded before the irradiation and immediately after irradiation between 1 and 60 minutes. The experimental conditions were the same as in photodynamic effect experiments.
S-4
As illustrated in Fig. S5, after 60 min. of irradiation the relative redaporfin content inside the P123 and F127 formulation decreased ca. 10 %, while that of redaporfin in PBS (DMSO
