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. 2022 Mar 1;13(4):641–647. doi: 10.1021/acsmedchemlett.1c00691

Improvement of Photodynamic Activity by a Stable System Consisting of a C60 Derivative and Photoantenna in Liposomes

Risako Shimada †, Shodai Hino ‡, Keita Yamana †, Riku Kawasaki †, Toshifumi Konishi §, Atsushi Ikeda †,*
PMCID: PMC9014432  PMID: 35450358

Abstract

graphic file with name ml1c00691_0008.jpg

A dyad system comprising a lipid membrane-incorporated fullerene derivative with an N,N-dimethylpyrrodinium group (C60-1) and a photoantenna molecule (DiD) did not exhibit the high photodynamic activity expected based on its singlet oxygen generation ability. Comparison with a fullerene derivative with an amide substituent (C60-2) suggested the cause to be that some of the fullerene derivative had been released from the liposomes, partly disrupting the dyad system. The dyad system of C60-2 and DiD exhibited about twice the photodynamic activity toward HeLa cells as that of C60-1 and DiD, due to the suppression of the release of the fullerene derivative from the liposomes. The hydrophobicity/hydrophilicity balance of the substituent in fullerene derivatives was shown to be very important to obtain a dyad system in liposomes characterized by high photodynamic activity.

Keywords: Fullerenes, liposomes, energy transfer, photodynamic therapy, and photosensitizers

Photodynamic therapy (PDT) has been attracting a great deal of attention for cancer treatment, given its minimal invasiveness.1−4 Porphyrin derivatives and their analogues have been used as photosensitizers (PSs) in PDT; however, [60]fullerene (C60) derivatives have recently come to be used for the same purpose,5−7 because photoactivated C60 is characterized by a high quantum yield in the generation of singlet oxygen(1O2).8 The hydrophobic C60 needs to be dissolved in water via solubilizing agents like polyvinylpyrrolidone,9 γ-cyclodextrin (γ-CDx),10−12 and liposomes.13−17 Although the lipid-membrane-incorporating C60 (LMIC60) and the γ-CDx·C60 complex exhibit a strong tendency to generate 1O2 following irradiation with near-ultraviolet light (350–400 nm wavelength), this tendency was observed to substantially decrease under photoirradiation at λ > 400 nm.14−17 The reason for this behavior has to do with the low absorbance of C60 at wavelengths above 400 nm, which results from a transition that is symmetry-forbidden. In order to address this shortcoming, the LMIC70 and LMIC60 derivatives were used as PSs because C70 and C60 derivatives have higher absorbance at wavelengths above 400 nm due to a lowering symmetry. Indeed, these PSs exhibited a higher photodynamic activity than LMIC60 when subjected to photoirradiation at λ > 400 nm.16−18 Recently, we prepared C60, a C60 derivative (Figure 1, C60-1), and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD), a dialkylated carbocyanine lipid membrane probe (Figure 1) acting as a light-harvesting antenna molecule. Importantly, the mentioned fullerene derivatives were included in liposomes alongside DiD to produce the dyad systems labeled LMIC60–DiD and LMIC60-1–DiD, comprising C60 or C60-1, respectively.19−21 These dyad systems in the liposomes were able to generate large amounts of 1O2 under photoirradiation at wavelengths longer than 620 nm with high biological tissue permeability. DiD employed as a photoantenna absorbed light energy at around 650 nm, which was subsequently transferred to C60 or C60-1. The efficiency of the energy transfer between DiD and C60-1 was much higher than that between DiD and C60, as inferred based on data on the fluorescence quenching of DiD in these dyad systems.20,22 Therefore, the 1O2 generation ability and the photodynamic activity of LMIC60-1–DiD was observed to be much higher than that of LMIC60–DiD. Remarkably, despite the large difference in 1O2 generation ability between LMIC60-1–DiD and LMIC60-1, the photodynamic activity of LMIC60-1–DiD was only 1.4 times higher than that of LMIC60-1. In this context, we expected that the release of part of C60-1 from the liposomes as a result of the high hydrophilicity of N,N-dimethylpyrrodinium group in C60-1, before the photoreaction would lead to a decrease in the photodynamic activity of LMIC60-1–DiD. In the present study, in order to prove the mentioned hypothesis and to improve on the photodynamic activity of the dyad system, we prepared a novel dyad system comprising DiD and C60-2, which does not include a cationic group (Figure 1).

Figure 1.

Figure 1

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Structures of (a) [60]fullerene derivatives C60-1 and C60-2, (b) lipids DMPC and 3 used to obtain liposomes, (c) photoantenna molecules DiD and DiI, and (d) dyad system LMIIC60-2–DiD and mechanism of singlet oxygen (1O2) generation.

Aqueous solutions of LMIC60-1–DiD and LMIC60-2–DiD were prepared by the fullerene exchange method starting from γ-cyclodextrin (γ-CDx), C60-1 or C60-2, and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)–DiD liposomes (Scheme S1).14,15,19,20 The transfer of C60-2 from γ-CDx to liposomes was confirmed by the broadening of the UV–vis absorption spectrum in the mixture between the γ-CDx·C60-2 complex12,23−25 and DMPC-based liposomes or DMPC–DiD-based liposome (LMIDiD) after heating at 80 °C for 2 h (Figure 2). Furthermore, 1H NMR spectrometry evidence confirmed that all C60-2 had been released from the cavities formed by two γ-CDx groups (Figures 3 and S1). Indeed, all peaks assignable to γ-CDx in the γ-CDx·C60-2 complex disappeared after heating at 80 °C for 2 h in the presence of DMPC-based liposomes or LMIDiD (Figures 3b and c), suggesting the formation of LMIC60-2–DiD. The contemporary presence of C60-2 and DiD in the liposomes led to quenching of DiD fluorescence, because C60 and C60 derivatives act as fluorescence quenchers (Figure S2). The value for the fluorescence quenching by C60-2 was estimated to be 61%, which is intermediate between those of C60 and C60-1 (55 and 87%, respectively).20 This evidence indicates that the light energy absorbed by DiD is transferred to fullerene derivatives in the following order of decreasing efficiency: C60-1 > C60-2 > C60. The size distributions of the liposomes were determined performing dynamic light scattering (DLS) analyses. Notably, since DiD adsorbs light in a wavelength range that includes the laser wavelength utilized in DLS measurements, the average liposome diameters were measured using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI) instead of DiD as the photoantenna. The values of the average diameters of all the liposomes before and after the exchange reaction of C60-2 are listed in Table S1. The average diameter of LMIC60-2–DiI-containing liposomes (67 nm) was smaller than that of LMIC60-2-containing liposomes (96 nm). This difference in size is due to the presence of DiI, as indicated by the average diameters of DMPC-based liposomes and LMIDiI being determined to be 82 and 62 nm, respectively.

Figure 2.

Figure 2

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UV–vis absorption spectra of the γ-CDx·C60-2 complex and DMPC–liposome mixture before (black) and after (red) heating at 80 °C for 2 h and of the γ-CDx·C60-2 complex and DMPC–DiD-liposome mixture before (blue) and after (green) heating at 80 °C for 2 h. All spectra were recorded at 25 °C (1 mm cell).

Figure 3.

Figure 3

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Sections of the 1H NMR spectra of the γ-CDx·C60-2 complex (a) before the addition of the DMPC-based liposomes, (b) after such addition, and (c) after the addition of the DiD-liposome in D2O. Black circles indicate peaks due to free γ-CDx; red circles indicate peaks due to γ-CDx in the γ-CDx·C60-2 complex; the blue circle indicates the peak due to C60-2 in the γ-CDx·C60-2 complex; green circles indicate the peak due to DMPC. (a) [C60-2] = 0.05 mM; [γ-CDx] = 0.7 mM; (b, c) [DMPC] = 1.0 mM; [C60-2]/[DMPC] = 5.0 mol %; [DiD]/[DMPC] = 2.5 mol %.

We investigated the 1O2 generation ability of LMIC60-2 in the absence and presence of DiD under irradiation with visible-light at wavelengths longer than 620 nm. We have reported that C60, C70, and C60 derivatives in CDxs or liposomes generate 1O2 by reaction with dissolved oxygen molecules (3O2) via the energy-transfer pathway (Type II), rather than by reaction with the superoxide anion (O2•–) via the electron-transfer pathway (Type I).8,19,25,26 The various systems’ 1O2 generation ability was determined by measuring the decrease in absorbance at 400 nm resulting from the conversion of 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) to the corresponding endoperoxide, as a consequence of ABDA photoreacting with 1O2 (Scheme S2).27,28 Specifically, after O2 bubbling, changes in the UV–vis absorption spectra of the aqueous solutions of LMIC60-2 and LMIC60-2–DiD were measured as a result of the addition of a dimethyl sulfoxide solution of ABDA (Figure S3); indeed, the intensity of the absorption at 400 nm, corresponding to an absorption maximum of ABDA, was plotted as a function of the photoirradiation time (Figure 4). The 1O2 generation ability of LMIC60-2–DiD was much higher than that of LMIC60-2, similarly to what was observed in the case of LMIC60-1–DiD versus LMIC60-1 (Figure S4a). Therefore, evidence suggests that DiD acted as a light-harvesting antenna and LMIC60-2–DiD functioned as a dyad system (Figures S5 and S6).29−31 Additionally, generating 1O2 as the final product by the energy-transfer pathway (Type II) proves the energy transfer between DiD and C60-2. Moreover, the energy transfer between the C60 derivative moiety and a light-harvesting antenna moiety connected by a covalent bond was observed.32,33 To further confirm the energy transfer from DiD to C60-2, the level of 1O2 generated by LMIC60-2–DiI was measured under light irradiation at a wavelength over 620 nm, because DiI, which has a similar structure to DiD, exhibits an absorption maximum at 551 nm and scarcely absorbs light at wavelengths longer than 600 nm (Figure 1). As can be seen from Figure 4, LMIC60-2–DiI scarcely generated any 1O2 under the photoirradiation conditions applied to LMIC60-2–DiD (λ > 620 nm). Consequently, in LMIC60-2–DiI, DiI scarcely acted as a photoantenna, similarly to what has previously been reported for LMIC60-1–DiI.20

Figure 4.

Figure 4

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Time-dependent bleaching of 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) caused by reaction with the singlet oxygen generated by LMIC60-2 (blue line), LMIC60-2–DiD (red line), and LMIC60-2–DiI (black line) prepared in DMPC liposomes upon photoirradiation (λ > 620 nm, 15 mW cm–2). A dimethyl sulfoxide solution of ABDA was injected into an aqueous solution of the liposomes. Changes in the ABDA absorption at 400 nm were monitored as a function of time (Abs0: initial absorbance). [DMPC] = 0.3 mM; [C60-2]/[DMPC] = 5.0 mol %; [DiD or DiI]/[DMPC] = 0 or 2.5 mol %; [ABDA] = 25 μM. Experiments were conducted under an oxygen atmosphere at 25 °C. Error bars represent the values for the standard deviation for n = 3.

The levels of 1O2 generated in the presence of LMIC60-2 were lower than those generated in the presence of LMIC60-1 (Figure S4a). This observation can be understood based on the fact that 1O2 generation ability of the γ-CDx·C60-2 complex was lower than that of the γ-CDx·C60-1 complex under direct photoexcitation of fullerenes as previously described.25 Furthermore, the 1O2 generation ability of LMIC60-2–DiD was lower than that of LMIC60-1–DiD (Figure S4a), but it was much higher than that of LMIC60-2 (Figure 4). In addition to the higher 1O2 generation ability of C60-1, this result is consistent with the inferior efficiency of the energy transfer from the photoactivated antenna molecule DiD to C60-2 as compared with that to C60-1 described above. By contrast, the levels of 1O2 generated by LMIC60–DiD were much lower than those generated by LMIC60-1–DiD and LMIC60-2–DiD, because pristine C60 tends to localize in the area near the center of the lipid bilayer.20,22 Therefore, one of the reasons for the high 1O2 generation ability of LMIC60-2–DiD compared with LMIC60-2 has to do with C60-2’s tendency to be located at or near the surface of the lipid membrane as a result of the presence of the polar amide group; indeed, similar considerations apply to C60-1 (Scheme 1b). To confirm the values for the fluorescence quenching without the influence of the lipid membranes, we measured the fluorescence quenching value of DiD via the γ-CDx·C60-2 complex in the absence of liposomes (Figure S7). The value of C60-2 (66%) was intermediate between those of C60 (78%) and C60-1 (61%),20 indicating that the primary quenching effect of C60-2 is higher than that of C60-1. Although a reverse result for the quenching effects was excepted from the energy levels (Figure S5), it can be explained via electrostatic repulsion between C60-1 and DiD and it is expected that same repulsion occurs in lipid membranes. However, the result that the quenching effect of C60-1 is higher than that of C60-2 in the lipid membranes indicates that C60-1 is located at the surface of the lipid membrane and the neighborhood of DiD compared with C60-2.

Scheme 1. Illustration of the (a) Intracellular Uptake of LMIC60-1-DiD or LMIC60-2-DiD by Endocytosis, (b) 1O2 Generation by the Dyad System, and (c) Transfer of C60-1 and C60-2 from the Liposome Membrane to the Cell Membrane.

Scheme 1

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The photodynamic activities of LMIC60-2–3 and LMIC60-2–DiD–3 prepared in DMPC-3 liposomes (i.e., liposomes obtained applying a [DMPC]:[3] = 9:1 mol/mol ratio for the two lipids DMPC and 3 whose structures are reported in Figure 1) were assessed by measuring the viability of human cervical cancer HeLa cells. Aqueous solutions of LMIC60-2–3 and LMIC60-2–DiD–3 were added into HeLa cell cultures, and the obtained mixtures were exposed to light in the 610–740 nm wavelength range. Cell viability was determined as a percent ratio compared with cells untreated with PSs by the WST-8 method.34 No cell toxicity was detected in the dark for both LMIC60-2–3 and LMIC60-2–DiD–3 (Figure 5). By contrast, HeLa cell viability was reduced in a drug dose-dependent manner in the case of LMIC60-2–3 and LMIC60-2–DiD–3 under photoirradiation. The medium inhibitory concentrations (IC50 values) of LMIC60-2–3 and LMIC60-2–DiD–3 were estimated to be ca. 0.90 and 0.41 μM for the fullerenes, respectively (Figure 5). Based on the IC50 values, the photodynamic activity of LMIC60-2–DiD–3 was ∼2.2 times as large as that of LMIC60-2–3. Indeed, the higher photodynamic activity of LMIC60-2–DiD–3 was consistent with the high 1O2 generation ability because the cationic liposomes used as drug carriers were observed to exhibit intracellular uptake by HeLa cells similar to LMIC60-2–3. Notably, the mentioned difference (2.2 times) is larger than that measured for LMIC60-1–3 versus LMIC60-1–DiD–3 (1.4 times; Figure S4b).15 Furthermore, the photodynamic activity of LMIC60-2–DiD–3 was ∼2.1 times higher than that of LMIC60-1–DiD–3 (IC50 value for LMIC60-1–DiD–3: 0.87 μM). These results were in contradiction with those according to which LMIC60-1–DiD exhibits higher 1O2 generation ability than LMIC60-2–DiD, which are discussed in detail in the following section.

Figure 5.

Figure 5

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Viability of HeLa cells in the dark and after photoirradiation. Cells were treated with various concentrations of LMIC60-2–3 or LMIC60-2–DiD–3 and either kept in the dark or subjected to light irradiation. Cell viability was evaluated 24 h after treatment as described in the Experimental Section. The black and orange lines reflect cell viability in the presence of LMIC60-2–3 and LMIC60-2–DiD–3, respectively, in the dark. The blue and red lines reflect HeLa cell viability in the presence of LMIC60-2–3 and LMIC60-2–DiD–3, respectively, after photoirradiation (610–740 nm) for 30 min. Cell viability data were confirmed implementing the WST-8 method. Error bars represent the values for the standard deviation for n = 3.

The photodynamic activity of the various systems is dictated by both the extent of intracellular uptake of LMIC60-1–DiD–3 or LMIC60-2–DiD–3 and the amount of 1O2 generated in cells. The intracellular uptake of LMIC60-1–DiD–3 and LMIC60-2–DiD–3 could not be determined directly based on the fluorescence signals of HeLa cells, because of the very weak emission of LMIC60-1–DiD–3 and LMIC60-2–DiD–3. Therefore, after the intracellular uptake of LMIC60-1–DiD–3 and LMIC60-2–DiD–3, we measured the fluorescence spectra of DiD in the ethyl acetate extracts of HeLa cells. As can be seen from Figures 6a and S8, similar fluorescence intensities were observed for LMIC60-1–DiD–3 and LMIC60-2–DiD–3. Consequently, evidence indicates that the extent of intracellular uptake of LMIC60-1–DiD–3 and LMIC60-2–DiD–3 was about the same, and it was determined by the properties of the cationic liposomes which interact with the anionic surfaces of HeLa cell membranes.15,35,36

Figure 6.

Figure 6

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(a) Mean fluorescence intensity (λex: 630 nm) of ethyl acetate extracts of HeLa cells treated with LMIC60-1–DiD–3 (blue) and LMIC60-2–DiD–3 (red) ([DMPC] = 0.09 mM; [3] = 0.01 mM; [C60-1 or C60-2] = 5.0 μM; [DiD] = 2.5 μM) for 24 h at 37 °C. Error bars represent the values for the standard deviation for n = 3. (b–d) Overlay of phase contrast and fluorescence images of HeLa cells. HeLa cells stained by DiI were treated (b) without fullerene derivatives and with (c) LMIC60-1–3 or (d) LMIC60-2–3 (scale bars: 10 μm). (e) Values for the fluorescence intensity of HeLa cells stained by DiI and treated with LMI–3 without fullerene derivatives (gray), LMIC60-1–3 (blue: The fluorescence intensity was nearly zero.) or LMIC60-2–3 (red) for 6 h at 37 °C. Fluorescence images were analyzed using the ImageJ software. Statistical analysis was carried out via Student’s t testing (n = 50). The samples were observed by fluorescence microscopy. (f) Changes in intensity of the DiD absorption maximum at 650 nm in LMIDiD–3 (black), LMIC60-1–DiD–3 (blue), and LMIC60-2–DiD–3 (red), and of the C60-1 (320 nm) or C60-2 (340 nm) absorption maxima in LMIC60-1–DiD–3 (purple) and LMIC60-2–DiD–3 (orange) in 10% blood serum after 24 h incubation at 37 °C.

Based on the results discussed above and the fact that LMIC60-1–DiD–3 generated larger amounts of 1O2 than LMIC60-2–DiD–3 (Figure S4a), we hypothesized the following: as opposed to C60-2, C60-1 is gradually released from liposomes into HeLa cells, so that the dyad system partially loses its ability to function. To confirm this hypothesis, we measured the fluorescence spectra of the mixed solution of LMIDiD and either LMIC60-1 or LMIC60-2. Compared with LMIDiD, a system comprising no fullerene derivatives, the fluorescence intensity of LMIC60-1 and LMIC60-2 decreased by 59% and 20%, respectively (Table 1 and Figure S9). Since the quenching values of C60-1 and C60-2 toward DiD differed in the liposomes (87 and 61%, respectively), the corrected quenched efficiencies were calculated to be 0.68 and 0.33 for LMIC60-1 and LMIC60-2, respectively (Table 1). These values mean that C60-1 can be transferred more easily between liposomes than C60-2. The reason for this difference in behavior has to do with the ammonium group of C60-1 being more hydrophilic than the amide group of C60-2. To determine whether the fullerene derivatives are transferred from liposomes to HeLa cells in a fashion similar to the way they are transferred between liposomes, the cell membranes of HeLa cells were stained by DiI and the HeLa cells were treated with LMIC60-1 or LMIC60-2 (Figures 6b–e). After 6 h of incubation, the HeLa cells treated with LMIC60-1 exhibited weaker fluorescence than those treated with LMIC60-2, and C60-1 was concluded to be released from liposomes also in HeLa cells, as compared with C60-2. The release of C60-1 from liposomes results in a collapse of the dyad system comprising photoantenna and fullerene, and it causes a lowering of the photodynamic activity of the said dyad system.

Table 1. Quenching Efficiency of LMIDiD and LMIC60-1 Mixture and LMIDiD and LMIC60-2 Mixture Compared to LMIDiD 2 h after Mixing Two Liposome Solutions at 37°C (λex: 630 nm).

  quenching efficiencyb corrected quenching efficiencyc
LMIDiD and LMIC60-1 mixturea 0.59 0.68
LMIDiD and LMIC60-2 mixturea 0.20 0.33
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a

The final concentrations after mixing were [DMPC] = 0.2 mM, [C60-1 or C60-2] = 5.0 μM, and [DiD] = 2.5 μM.

b

Values were obtained dividing the fluorescence intensity of the mixture by that of LMIDiD.

c

The quenching efficiencies of C60-1 and C60-2 toward DiD in the liposomes were different. Therefore, the values of the quenching effect were corrected by quenching value of LMIC60-1–DiD (87%) and LMIC60-2–DiD (61%).

The stabilities of LMIC60-1–DiD–3 and LMIC60-2–DiD–3 in the blood serum were determined by UV–vis spectroscopy. From the data reported in Figures 6f and S10, it is shown that, compared with the value of the same parameter determined for LMIDiD–3 and LMIC60-2–DiD–3, the absorbance of DiD in LMIC60-1–DiD–3 decreased. These results show that not only C60-1 and C60-2 but also a portion of DiD were released from liposomes. Additionally, C60-1 absorbance decreased faster in LMIC60-1–DiD–3 than C60-2 absorbance did in LMIC60-2–DiD–3. This shows that LMIC60-2–DiD–3 was more stable in blood serum than LMIC60-1–DiD–3.

Under the same conditions applied to LMIC60-2–DiD–3 to compare photofrin (Figure S11a) with LMIC60-2–DiD–3, an IC50 value of 3.02 μM was determined for photofrin (Figure S11b),18,20 which is currently the principal drug utilized as a PS in PDT.37−39 when the number of moles was converted to the number of porphyrin units; notably, photofrin consists of porphyrin oligomers containing two to eight units (Figure S11a).40 Therefore, the photodynamic activity of LMIC60-2–DiD–3 was approximately 7.4 times higher than that of photofrin (Figure S11b).

In summary, an aqueous solution of LMIC60-2–DiD was prepared by the exchange method starting from the γ-CDx·C60-2 complex and LMIDiD. Although the LMIC60-2–DiD dyad system exhibited lower 1O2 generation ability than the LMIC60-1–DiD dyad system, LMIC60-2–DiD displayed higher photodynamic activity toward HeLa cells than LMIC60-1–DiD. This reversal is attributed to the fact that fullerene derivatives with a polar group can be transferred between lipid membranes, so, in the presence of other lipid membranes that do not comprise DiD, they may end up being too far from DiD molecules for the fullerene derivative–DiD energy transfer to take place. As the N,N-dimethylpyrrodinium group of C60-1 is more hydrophilic than the amide group of C60-2, the transfer of C60-1 between lipid membranes occurred more frequently. On the other hand, although an increase in the hydrophobicity of the C60 derivative inhibited fullerene transfer between lipid membranes, the 1O2 generation ability of the hydrophobic C60 derivatives dropped as a result of the low polarity of the molecules25 and preferential location close to the center of the lipid membrane, as is the case for pristine C60 as mentioned in the previous paper.20 Therefore, in order to develop liposomal dyad systems, striking the right balance between hydrophobicity and hydrophilicity of the substituent of C60 derivatives is very important.

Acknowledgments

The authors would like to thank Enago (www.enago.jp) for the English language review.

Glossary

Abbreviations

1O2

singlet oxygen

ABDA

9,10-anthracenediyl-bis(methylene)dimalonic acid

C60

[60]fullerene

CDx

cyclodextrin

DiD

1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine

DiI

1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine

DMPC

1,2-dimyristoyl-sn-glycero-3-phosphocholine

IC50

medium inhibitory concentration

LMI

lipid-membrane-incorporating

PDT

photodynamic therapy

PS

photosensitizer

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00691.

  • Experimental procedure, average liposome hydrodynamic diameter, representation of the exchange method, conversion of ABDA by 1O2, 1H NMR spectra before and after the exchange method, fluorescence spectra, time-dependent bleaching of ABDA by 1O2, HeLa cell viability, schematic illustration of the Jablonski diagram, fluorescence spectra of ethyl acetate extracts of HeLa cells, absorption changes of DiD, and the cytotoxicity of photofrin (PDF)

Author Contributions

All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ml1c00691_si_001.pdf (873.6KB, pdf)

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