Copper-Cysteamine Nanoparticles As New Radio-Photosensitizer With Low Sunlight Toxicity
Photodynamic therapy is a new cancer treatment modality that uses singlet oxygen and other reactive oxygen species to kill cancerous cells. However, most conventional photosensitizers which have FDA approval are toxic to not only cancerous cells but also to normal cells. Most of these PSs can be stimulated by ultraviolet-visible wavelength light and thus results in unwanted toxicity under sunlight and bright room light exposure. Therefore, PDT treated patients are required to avoid bright light for up to six weeks. Copper cysteamine(Cu-Cy) nanoparticle, which was invented by our group in 2014, is a new type of photosensitizer and a novel radiosensitizer that can be activated by X-ray, UV-light, microwave, and ultrasound to produce singlet oxygen and other forms of reactive oxygen species for cancer treatment1–4. The Cu-Cy nanoparticle has strong absorption in the UV region but not in the visible region. Therefore, Cu-Cy treated a patient may not have to avoid room light and sunlight. In the present article, we report comparative toxicity studies of Cu-Cy and PPIX (an FDA approved PSs) and show that while Cu-Cy is toxic to cancer cells, it is much safer to normal cells. On the other hand, PPIX is toxic to both cancer and normal cells.
Photodynamic therapy (PDT) is a cancer treatment modality in which photosensitizers (PSs) are practiced to generate highly reactive oxygen species (ROS) by means of photoexcitation, such as hydroxyl radicals (. OH), singlet oxygen (1O2 ), as well as peroxides (R–O–O.) which irreversibly damage cancer cells5–7 . Most conventional photosensitizer such as PPIX can be activated by UV-Visible light6,8 which makes this photosensitizer toxic under sunlight or bright light exposure9. Furthermore, Cu-Cy has an absorption band in the UV region with a peak absorption at about 365 nm. It absorbs little or no light in the visible region which may be advantageous over other conventional photosensitizers such as PPIX which has strong soret band at around 400 nm and other Q-bands between 500 and 630 nm . Protoporphyrin IX(PPIX) is an FDA approved photosensitizer. PPIX has a strong Soret absorption band at 405 nm and four relatively strong Q bands between 500 and 630 nm10.On the other hand, Cu-Cy has only one absorption peak at 365 nm 11. Therefore, Cu-Cy does not produce a significant level of singlet oxygen or ROS under room light or sunlight excitation. Herein, we demonstrate that Cu-Cy is much safer as compared to PPIX at the same concentration under room light or sunlight excitation. Historically, conventional photosensitizer treated patients had to avoid sunlight.
Cu-Cy nanoparticle was invented by our group in 201411. It is a new type of photosensitizer and radiosensitizer that can be activated by X-ray1,11, UV-light11, microwave2, and ultrasound3 to produce singlet oxygen and other forms of ROS species for cancer treatment. The ability of Cu-Cy to be excited by microwave, ultrasound and X-ray make it a promising candidate for new nanomedicine for cancer treatment
If PPIX is taken up by the skin, an acute, painful and mostly non-blistering type of photosensitivity is the most common symptoms12. To prevent symptoms, patients are forced to avoid light which interferes with professional and other daily activities and impairs quality of life. PPIX either in blood in the dermal vessels or after uptake into skin cells absorb light energy and transfers this energy to oxygen, generating reactive oxygen species (ROS) which can lead to cytotoxicity through protein, DNA, and lipid damage13. The requirement that patients needed to avoid sunlight also makes patients vitamin D deficient 9,14.
Material and methods
Copper (II) chloride dihydrate (99.99%), 2-mercaptoethylamine hydrochloride (cysteamine hydrochloride or Cys, 98%), and sodium hydroxide (98%) were purchased from Sigma (USA). All chemicals were used as received. Deionized (DI) water was used as the reaction solvent without further purification. KYSE-30, DM6, HET1A, and HDF cell lines obtained from
Synthesis of Cu-Cy
Cu-Cy nanoparticles were synthesized using a facile green synthesis method. Briefly, CuCl2.2H2O (91mg) was dissolved in DI water, followed by addition of cysteamine hydrochloride (127mg) and stirred for 2 hrs under an N2 atmosphere. After adjusting the pH value to 8 by adding a 2.5M NaOH solution, the solution was stirred for about 2hr at room temperature until the color turned to deep-violet due to oxidation. The solution was then heated to its boiling temperature for 30 mins; the deep-violet color changed to whitish murky color which is red luminescent under UV light excitation. The boiling solution was allowed to cool naturally at room temperature. The Cu–Cy particles were obtained by centrifuging and washing the crude product with a solution of DI water and ethanol (v/v = 5:4) five times followed by sufficient sonication. Cu-Cy particles were then dried by placing them under vacuum at room temperature.
Absorption and photoluminescence spectra measurement
The absorption and photoluminescence spectra were measured using a UV-Vis spectrophotometer (Shimadzu UV-2450) and a photoluminescence spectrophotometer (Shimadzu RF-5301PC, Tokyo, Japan) respectively. Cu-Cy particles were dispersed in DI water by ultra-sonication for several minutes for UV-Vis and PL measurement. PPIX solution was prepared by first dissolving in DMSO and then diluting with DI water.
Live-dead cell assay
KYSE30 and DM6 were chosen as cancer cell lines, whereas HET1A and HDF cell lines were chosen as normal cell lines. Cells were seeded in 35mm Petri-dish at a density of 200,000 cells/plate and were incubated at 37 0C in a humidified atmosphere of 5% v/v CO2 for 24hrs. Then, different concentrations of 1ml Cu-Cy nanoparticles and PPIX were added to the Petri-dishes. After incubating for 24 hrs., these Petri-dishes were exposed to either room light (RL), sunlight (SL) or neither and incubated for 3 hrs at 370C in a humidified atmosphere. Cal AM/EthD-1 staining was performed by incubating SL/RL treated cell samples with 1 μmol/L calcein AM and 8 μmol/L ethidium homodimer-I (Live/Dead® Viability/ Cytotoxicity kit; Invitrogen, France) for 45 mins at 37°C inside the incubator . Nonfluorescent cell-permeant calcein-AM enters the cell and is cleaved by ubiquitous esterase in living cells, producing calcein which is well retained within live cells and gives an intense uniform green fluorescence (excitation/emission ~495/515 nm). Ethidium homodimer-I enters cells with damaged membranes and then binds to nucleic acids, resulting in a 40-fold enhancement of fluorescence and producing a bright red fluorescence in dead cells (excitation/emission ~495/635 nm). The cells were imaged using an Olympus IX-71 fluorescence microscope using a suitable filter.
The cytotoxicity of Cu-Cy and PPIX under sunlight and room light exposure were evaluated by means of MTT assay. 10,000 cells/well were seeded in 96 well plates and incubated for 24 hours for the completion of cell attachment. Then, five different concentrations of each Cu-Cy and PPIX were prepared in cell media and were added to the 96 well plates (8 wells per concentration). One column of plates was left untreated and was considered as a control. Then, the plates were incubated for another 24 hours to allow cells to uptake particles. Cu-Cy and PPIX treated cells were then exposed to sunlight or room light for 10 mins and were then incubated for 3 hours. MTT assay was prepared by diluting 5 mg/ml stock solution with media by a factor of 10. 100 uL of MTT assay was added to each well, replacing old media. The 96 well plates were then incubated for 3 hours at 370C under humidified atmosphere. After incubation for 3 hours, MTT solution was removed and 100 uL DMSO was added to solubilize formazan crystal. The formazan crystal becomes purple-colored with DMSO dissolution. The viability of cells was directly dependent on the absorption of formazan solution.
96 well plates then either exposed to room light or sunlight for 10 mins or neither room light or sunlight exposure. After exposing to RL or SL, 96 well plates were incubated for 3 hrs. 0.5 mg/ml MTT solution added to each well and incubated for 3hrs. Yellow colored MTT converted into purple colored formazan crystal. Formazan is insoluble in aqueous solution. Formazan was solubilized by adding 100 Ul DMSO to each well. The absorbance of formazan is directly proportional live cell counts and can be employed to present a relative cell viability as compared to control. The absorbance of Formazan solution was measured using a multiskan FC microplate photometer (Fisher Scientific) at 540 nm.
Cell viability was calculated as follows:
Cell viability=The absorbance of the treatment groupThe absorbance of the control group *100%
Results and discussion
Cu-Cy nanoparticles were coincidentally invented by our group in 201411. It can produce singlet oxygen and reactive oxygen species when excited by UV-light, X-rays, ultrasound, and microwave 1–3,11 and can be used to kill cancer cells effectively. Detailed synthesis method and optical properties of Cu-Cy were reported in our other papers 11. Modified facile synthesis method along with its optical properties was reported in our recent paper (Pandey et. al).
Absorption and photoluminescence spectra measurement
The absorption spectrum of Cu-Cy and PPIX were measured by using UV-Vis absorption spectrophotometer. Figure 1 shows the UV-vis spectrum of Cu-Cy and PPIX. Cu-Cy has absorption in the UV region with peak absorption at 365nm which suggests that Cu-Cy may not be activated by visible light. PPIX has soret band peak at 405 nm and four relatively stronger Q-bands invisible wavelength range 500-700 nm.
Cellular uptake of Cu-Cy on HET1A and MCF7 cells
We chose two cancer cell lines (DM6 and KYSE30) and two normal cell lines (HDF and HET1A). MTT assay was conducted to investigate the cytotoxicity of Cu-Cy and PPIX under dark, room light, and sunlight excitation. Cells were allocated to different groups: Control group (no Cu-Cy and PPIX applied), Cu-Cy (0.23-120 mg/L)) and PPIX (0.23-30mg/L) in 96 well plates. All these groups were either exposed to no light (dark toxicity), room light or sunlight for 10 minutes. Following the treatment, cells were incubated around 4 hours and 0.5 mg/ml MTT solution applied to each well of the 96 well plates and incubated for 3.5 hours. Formazan crystals were then dissolved in 150ul DMSO.
Figure 3 (a-c) represents the toxicity of Cu-Cy to different cell lines under sunlight, room light, and no light excitation, respectively. Figure 3a depicts the sunlight toxicity of Cu-Cy to various cell lines. It was found that Cu-Cy has very less toxicity to the normal cell lines (HET1A and HDF) as compared to the cancer cell lines (KYSE30 and DM6). At 30 mg/L, Cu-Cy treated HET1A cell lines has…… cell viability, which is significantly higher than that of KYSE30 cell lines. Similarly, at 30 mg/L, Cu-Cy treated HDF cell lines has cell viability ……. which is significantly higher than that of DM6 cell lines …. Even at 15 mg/L, Cu-Cy treated normal cells has significantly higher cell viability than corresponding cancer cell lines. Figure 3b represents cell viability of Cu-Cy treated cell lines under room light excitation. Normal cell lines have significant higher cell viability than cancer cell lines. At 30 mg/L, HET1A and HDF cell lines have……… cell viability, which is significantly higher than KYSE30 and DM6 (……). Cell viability of room light excited cell lines is not significantly different than that of sunlight excited cell lines. Figure 3c represents the dark toxicity of Cu-Cy treated cell lines. Similar to sunlight and room light excited cell lines, the viability of normal cell lines was significantly higher than that of cancer cell lines. At 30 mg/L, Cu-Cy treated HET1A and HDF cell has viability …………….. which are significantly higher than that of KYSE30 and DM6 respectively. At 15 mg/L, HET1A and HDF cell lines have viability……….. which are significantly higher than that of KYSE30 and DM6 respectively. The cell viability of all cell lines are not significantly different from that of sunlight or room light exposed cell lines. Figure 3d represents the EC50 value of each cell line under sunlight, room light and no light conditions. It should be noted that the EC-50 values of cancer cells were significantly lower than that for normal cells. The EC-50 values for given cell lines were not significantly different.
Furthermore, under sunlight excitation, Cu-Cy treated cells has higher cell viability as compared to PPIX treated cells.
Figure 4 (a-c) represents the cell viability of PPIX treated cell lines under sunlight, room light, and no light conditions. Figure 4a represents the cell viability of PPIX treated cell lines. Both cancer and normal cell lines have viability under 10 % indicating that PPIX can easily be activated by sunlight exposure. Furthermore, the cell viability of PPIX treated cells are significantly low as compared to the Cu-Cy treated cell under sunlight excitation. However, the cell viability of PPIX treated cell lines were not significantly different among different cell lines. Figure 4c represents cell viability of PPIX treated cell lines under dark conditions. Except for DM6 cell lines, all other cell lines have cell viability above 60%, even at 30 mg/L. It should be noted that the cell viability of different cell lines is not significantly different from each other. EC20, EC50 and EC80 values of PPIX to different cell lines under different excitation condition are shown in figure 4d. Calculation and fitting parameters of EC20, EC50, and EC80 are shown in supporting information (figure s……). The result suggests that there is no significant difference between these values among cell lines. The cell viability under dark condition was significantly higher than under sunlight and room light excitation for all cell lines and all concentration used. Thus we can conclude that PPIX can easily be activated by sunlight and even by room light excitation. The cell viability of PPIX treated cell lines were significantly low as compared to Cu-Cy treated cell lines under sunlight excitation.
Live dead cell assay
Figure 3a and b represent live-dead cell assay on Cu-Cy and PPIX treated HDF and DM6 cells exposed to room light for 10 mins. As expected, Cu-Cy did not show significant toxicity to the normal HDF cells, but they posed significant toxicity to DM6 cell lines. The result is quite similar to that of sunlight. More interestingly, PPIX shows minimal toxicity to both HDF and DM6 cell lines under room light excitation. This result is consistent with the MTT assay. However, very few PPIX treated cells were found dead under room light excitation. This does not match the result from MTT assay where under room light or dark condition, PPIX posed significant toxicity. This may be because of limitation of both MTT and live-dead cell assay. Live-dead assay stains dead cell only if it is completely dead. It may not stain cells that are in early apoptosis. MTT assay is based upon esterase activity of cells. Any low activity due to dying cells will be reflected in MTT assay but it cannot be stained dead by live-dead cell assay. However, Cu-Cy toxicity matches well for both MTT assay and live-dead assay.
It can be activated by X-ray, MW, US or UV light 2,11. It is also safer as it does not absorb in the visible wavelength region. PPIX treated patients may not have to avoid light at all. PPIX is an effective photosensitizer that can be activated by UV light, visible light, X-rays, and microwaves. Moreover, the singlet oxygen production efficiency excited by UV at 365nm in PPIX nanoparticles is almost the same as in protoporphyrin IX (PPIX) which is a well-known, FDA approved commercially available photosensitizer17. We used RNO-IMD as a probe to measure singlet oxygen. This probe is not specific to singlet oxygen but can also measure other reactive oxygen species (ROS) such as hydroxyl radical. To confirm whether PPIX produces singlet oxygen or other ROS, we applied sodium azide (NaN3), an effective singlet oxygen quencher to PPIX containing RNO-IMD probe. After applying sodium azide, ROS production measured by RNO-ID decreased significantly, which indicates that most ROS produced by PPIX was singlet oxygen.
PPIX also has very long decay life times: 7.399 microseconds and 0.363 milliseconds which are in the same range of luminescence decay lifetimes from triplet states of photosensitizers.2 these lifetimes are in the same range as for other conventional photosensitizer18. These indicate that PPIX has a triplet state which is a very important requirement to be a photosensitizer.
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|80 Um||40 uM|
Figure: Dark toxicity of PPIX and PPIX to KYSE cell lines.
). cell lines however it produced substantial toxicity; at 40 um, Cu-Cy killed more than 50% cells whereas, at 80 um, Cu-Cy killed more than 70% cells. This implies that Cu-Cy might be selective to cancer cells over normal cells. It must be further noted that PPIX kills more normal cells than cancer cells at the same concentration.
Figure 3a represents sunlight toxicity of Cu-Cy and PPIX to HET1A and KYSE30 cell lines. Under sunlight excitation, Cell viability of both kyse30 and HET1A are less than 10%. The sunlight toxicity of PPIX to HET1A and kyse30 are not significantly different. Cu-Cy has significantly less sunlight toxicity to HET1A and KYSE30 cell lines as compared to PPIX toxicity at all concentrations used. Furthermore, cell viability of Cu-Cy+sunlight treated HET1A and KYSE30 are significantly different at …… Cu-Cy+sunlight has ….. to kyse30 and HET1A respectively. Figure 3b and 3c represents room light and dark toxicity of PPIX and Cu-Cy to kyse30 and HET1A cell lines. Cu-Cy has very good cell viability to HET1A cell lines under both room light and dark condition. On the other hand, Cu-Cy treated KYSE30 cell lines has much lower cell viability as compared to HET1A cell lines under both room light and dark condition. The difference in toxicity is significant at concentrations…………… However the toxicity of Cu-Cy to both HETA and kyse30 under sunlight are not significantly different from that of room light and dark conditions. This indicates that Cu-Cy may not be excited by sun light. PPIX treated cell has much higher cell viability to both KYSE30 and HET1A cell lines under both room light and dark conditions. PPIX treated cells has significantly lower cell viability under sunlight than that of room light and dark conditions. At 30 mg/L, PPIX has 4.48+-0.20%, 61.06+-1.95% and 5.47+-0.27% cell viability to HET1A cell lines under sunlight, roomlight and dark conditions. In similar fashion, PPIX has 5.69+- 0.19, 78.70+-0.84 and 25.27+- 1.26 % cell viability to KYSE30 cell lines under sunlight, room light toxicity. There was no significant difference between PPIX toxicity to KYSE30 and HET1A cell lines. However, PPIX toxicity to both HETA and KYSE30 are significantly different under room light and sunlight excitation. P<0.05 was calculated between roomlight and sunlight whereas between sunlight and dark light p<0.01.
To determine if the selective toxicity of Cu-Cy is sunlight excitable, we performed MTT assay of Cu-Cy to Kyse30 and HET1A under room light or dark condition. Cu-Cy+ SL, Cu-Cy+RL, and Cu-Cy+NL has cell viability to kyse30 ………… and HET1A ………….. at 30 mg/L. The difference in toxicity was not significant to both kyse30 and HET1A at all concentration used. This result implies that Cu-Cy can not be excited by sunlight to produce significant toxicity. Also, the Cu-Cy kills significantly more cancer cells than normal cell at the given concentration. The mechanism is not clearly understood yet.
Under room light excitation, kyse30 and HET1A cell lines shows 80% and 60% cell viability at 30 mg/L respectively. For Cu-Cy treated group, it showed very good cell viability to normal HET1A cell lines. At 30 mg/L, Cu-Cy has 26.04+- 1.03 %, 82.43+- 2.77% and 85.21+- 0.19% cell viability to HET1A cell lines under sunlight, room light and dark conditions.
This shows that Cu-Cy has minimal toxicity to HET1A cell lines whereas under sunlight excitation, PPIX has significant toxicity to HET1A cell lines. We performed the one-way anova to Compare of PPIX to KYSE30 and HET1A, we do not find any significant differences between the sunlight toxicity of PPIX to KYSE30 and HET1A cell lines.