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Abstract
Laser-induced fluorescence is recently used as an efficient technique in cancer diagnosis and non-invasive treatment. Here, the synergic therapeutical efficacies of the Capecitabine (CAP) chemodrug, photosensitive Phycocyanin (PC) and graphene oxide (GO) under laser irradiation were investigated. The therapeutical efficacies of diverse concentrations of CAP (0.001-10 mg/ml) and PC (0.5-10 mg/ml) alone and with laser irradiation on human breast adenocarcinoma (MCF-7) cells were examined. The interactional effects of 100 mW SHG Nd:YAG laser at 532nm and GaAs laser at 808 nm ranging power of 150 mW- 2.2W were considered. The contribution of graphene oxide (GO) in biocompatible concentrations of 2.5-20 ng/ml and thermal characteristics of laser exposure at 808 nm on GO + fluorophores have been studied. The effects of the bare and laser-excited CAP + PC on cell mortality have been obtained. Despite the laser irradiation could not hold up the cell proliferation in the absence of drug interaction considerably; however, the viability of the treated cells (by a combination of fluorophores) under laser exposure at 808 nm was significantly reduced. The laser at 532 nm excited the fluorescent PC in (CAP + PC) to trigger the photodynamic processes via oxygen generation. Through the in-vitro experiments of laser-induced fluorescence (LIF) spectroscopy of PC + CAP, the PC/CAP concentrations of the maximum fluorescence signal and spectral shifts have been characterized. The synergic effects of the laser exposures and (CAP + PC) treatment at different concentrations were confirmed. It has been shown here that the laser activation of (CAP + PC) can induce the mortality of the malignant cells by reducing the chemotherapeutic dose of CAP to avoid its non-desirable side effects and by approaching the minimally invasive treatment. Elevation of the laser intensity/exposure time could contribute to the therapeutic efficacy. Survival of the treated cells with a combination of GO and fluorophores could be reduced under laser exposure at 808 nm compared to the same combination therapy in the absence of GO. This survey could benefit the forthcoming clinical protocols based on laser spectroscopy for in-situ imaging/diagnosis/treatment of adenocarcinoma utilizing PC + CAP + GO.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Metastatic cancer is the most dangerous and remains the most lethal disease that requires an efficient curative treatment, while most current regimens resemble to be palliative [1,2]. Laser-based methods for non-invasive selective diagnosis/ imaging/treatment of malignant tumors exhibit superior advantages against other traditional techniques [3–5]. Nowadays, the laser is an optically coherent probe in photon-cell interactions through photothermal, photodynamic and photoablation processes [6,7]. Laser spectroscopy methods such as Raman, laser-induced fluorescence (LIF) and laser-induced breakdown spectroscopy (LIBS) provide additional facilities to identify the malignant tumors as the non-invasive early detection tools against the invasive biopsy [8–11]. Fluorescence spectroscopy is an emerging tool for diagnosing neoplasia in the early stages [12]. LIF as an in-vivo/ex-vivo diagnostic technique is based on imaging the distribution of endogenous/exogenous fluorophores and fluorescent chemodrugs [13,14]. Recently, the fluorescent properties of different chemodrugs have been extensively investigated [15,16]. The photothermal/photodynamic properties of optically activated anticancer drugs emphasize their potential for future simultaneous imaging and treatment [17].
Capecitabine is a well-known oral chemodrug for adjuvant treatment of many cancers such as colon and breast, which is also fluorescent active upon excitation of UV photons [18–20]. The success rate for the treatment of metastatic colon cells by intrarectal injection of Capecitabine is ∼ 60% [21], while it resembles to scale up using the simultaneous UV laser exposure. Recently the spectroscopic characteristics of Capecitabine using absorbance, photoluminescence (PL), and NIR techniques have been reported [22]. Besides, LIF spectroscopy of dissolved Capecitabine in deionized water activated by UV beams, i.e., ArF and XeCl excimer lasers, has been extensively studied over a wide range of concentrations (0.001-10 mg/ml) to characterize the fluorescence properties of the chemodrug [22,23].
Phycocyanin is a low-toxic bioagent that benefits antitumor properties in favor of several cancerous cells alongside the anti-oxidative and immune enhancement functions [24,25]. Even high doses of this medication can be prescribed as an FDA-approved safe oral medication [26]. In the case of MCF-7 malignant breast cells, the inhibition rate of PC at 0.5mg/ml is estimated to be up to 23.6% using the MTT assay [27]. In addition, the PC represents evident photodynamic characteristics which makes this phototherapeutic alga a preferred biological drug for neoplastic diseases [28]. This makes the PC an adaptable photosensitizer for selective cancer treatment with minimum damage to the surrounding normal tissues compared to other chemotherapeutic agents. The chemotherapeutic potential of PC + chemodrug conjugate is more effective than the chemodrug alone due to the chemoresistance properties of this marine agent [29]. In-vivo and in-vitro photodynamic therapies by PC under He-Ne laser exposure in a murine tumor model and cultured MCF-7 cells were studied. Accordingly, the anti-cancer effects of PC treatment were further enhanced when combined with He–Ne laser irradiation [30]. The spectral characteristics of fluorescence emission such as peak wavelength, quantum yield, spectral shift, extinction, and self-quenching coefficients were extracted for both anti-cancer agents of PC and CAP. As the key parameters, the concentrations with maximum fluorescence emissions (Cp)s were driven for both medications [22,23,31].
Dynamic drug delivery and imaging were achieved by nanoparticle labeling of chemodrug under laser excitation, leading to selective drug release. The latter prevented unwanted damage to healthy tissue by reducing the side effects [32]. These controllable nanoparticles contribute to the treatment of malignant adenocarcinoma cells by carrying and releasing an effective dose of the drug into the target cells [33]. Graphene oxide (GO) provides high cytotoxicity against various types of cancers due to its thermal characteristics [34]. The GO as a low-cost nanoparticle (NP) act as a photothermal agent and could be used for anti-cancer drug delivery [35].
Regarding the high absorbance band around the therapeutic window over 700-1000 nm, GO enhances the photothermal effects under NIR exposure [36]. The hybrid effect of doxorubicin (DOX) loaded GO alongside activation by Diode laser at 808nm has been vastly investigated [37]. Accordingly, the viability of MCF7 cells was assessed under the chemo and hyperthermia treatments of photoactivated (DOX + GO). This exhibited the high selectivity for demolishing the malignant against healthy cells. The quenching and spectral shift properties of GO were also reported according to LIF spectra of the chemodrugs of interest, such as Capecitabine, Bleomycin and DOX [23,38,39].
In this study, the fluorescence properties of (CAP + PC) conjugate at various concentrations of CAP (0.5-10 mg/ml) and PC (1-10 mg/ml) were measured. Fluorescence peak intensity and the spectral shift of (CAP + PC) suspension were recorded versus PC concentration at different CAP densities. Accordingly, PC and CAP concentrations were found at which maximum peak intensity and highest spectral shift take place. Moreover, the therapeutic efficacies of different concentrations of CAP (001-10 mg/ml) and PC (0.5-10 mg/ml) medications on MCF7 cells were studied separately. The viability assessments of cancerous cells were carried out under the photoactivation of different concentrations of (CAP + PC) and (CAP + PC + GO) using the various doses of lasers at 532 and 808 nm (within the therapeutic window).
2. Materials and methods
2.1. Materials
Phycocyanin was synthesized in department of plant science, faculty of physiology, agricultural technology research institute of Iran University. In general, the dilution was systematically performed in deionized water to prepare various PC concentrations. Capecitabine with MW = 359.354 g/mol is supplied (Roche GmbH) in 500mg tablets. Graphene oxide (C20O154) nanosheets were provided by Sigma-Aldrich Co. with monolayers thickness of 0.7-1.4 nm and lateral size of 5-100 µm dispersed in deionized water by 1 mg/ml.
2.2. Instrumentations
LIF measurements of CAP + PC were carried out using the fluorescence spectroscopy set-up. CAP + PC solution in deionized water was irradiated in the standard cubic quartz cell. The SHG Nd: YAG laser at 532 nm, 100 mW power was employed to activate the compound. The spectrometer of Avaspec-2048 with 0.5 spectral resolution was exploited to collect the fluorescence emission over 200-1100 nm. Figure 1(a) depicts the photothermal interaction of laser 808 nm with GO and photodynamic activation of PC using laser 532nm in conjugation with chemotherapy by CAP in order to optimal cancerous cell treatment.
Fig. 1. (a) The photothermal interaction of GO and photo activation of PC using two laser beam exposures at different wavelengths of 808 and 532 nm respectively (b) The experimental schematic of LIF spectroscopy arrangement to collect the fluorescence emission of PC + CAP from the MCF7 cells in microplates.
Figure 1(b) represents the experimental configuration of LIF spectroscopy by the means of fiber-coupled laser and spectrometer.
2.3. Methods
2.3.1. Cell culture
MCF-7 cells were cultured in population of 50,000 cells per T25 flask in Dulbecco modified low-glucose Eagle (DMEM) medium containing 10% FBS and 1% penicillin-streptomycin at 37°C in 5% CO2. The culture medium was changed every 2 to 3 days and the cells were passaged when 65-80% confluent.
2.3.2. Cell exposure
After passage 3, the MCF7 cells were precultured in 48 and 96-well microplates (15000 cells per microwell). These cells were incubated with different anti-cancer agents of CAP, PC and GO under laser exposures at two distinct wavelengths i.e., 808 and 532 nm. The laser at 532 nm of 100 mW CW-power was used to treat the cells under 3 min exposure. The laser beam was set at the center of the well and 5mm afar to prevent the beam scattering from the borders. In addition, the OELabs diode laser at 808 nm, 0-2.2 W enjoys a pig tail fiber with radius of 1000 micron.
2.3.3. Cell death detection
The MTT assay has been widely used to assess cell viability. It is a colorimetric reaction that can be easily measured from cell monolayers that have been plated in multiwell plates. Here, the cytotoxicity of considered agents on MCF7 breast cells was evaluated using the MTT assay after 24h and 48h. Briefly, cells were incubated for 4 h in culture medium containing 0.5 mg ml−1 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT). After 4 h, the incubation buffer was removed and the blue MTT–formazan product is extracted with acidified isopropyl alcohol (0.04 N HCl). After 30 min extraction at room temperature, the absorbance of the formazan solution was read at 570 nm by spectrophotometr. Tests were repeated 5 times for every protocol of treatment and the average standard error of mean (±SEM) was reported. Statistical analysis was conducted using SPSS software version 26 and P-Value < 0.05 was achieved represented the viability results statistically significant. All the figures were plotted using the Origin V.06 software.
3. Results and discussions
3.1 Fluorescence characteristics of PC + CAP
The fluorescence properties of PC using LIF technique has been investigated previously. PC shows a broad absorbance band over visible region ∼ 614 nm. The fluorescence peak appears ∼ 656 nm with stokes shift of 42 nm [31]. The peak signal intensity at a certain concentration Cpc ≈20 mg/ml exhibits a distinct fluorescence property of the chromophore of interest. The red shift of LIF signal is measured to be ∼ 12 nm corresponding to the concentrations ranging 1-10 mg/ml [31]. The spectroscopic properties of CAP have been recently studied [22,23]. UV-VIS spectroscopy of CAP attests high absorbance over UV region having three notable peaks at 216, 237 and 302 nm. The LIF spectra were taken at various CAP concentrations up to10 mg/ml. This reveals that the maximum intensity takes place at 408 nm for CCAP = 2.5 mg/ml [22]. Here, the (PC + CAP) compound at different concentrations were prepared. Figure 2(a) represents the LIF spectra of (CAP + PC) solution at CCAP = 2 mg/ml alongside different concentrations of PC i.e., 1, 2, 5 and 10 mg/ml.
Fig. 2. (a) LIF spectra of different concentrations of (CAP + PC) at CCAP= 2 mg/ml and several PC concentrations of 1, 2, 5 and 10 mg/ml (b) LIF peak intensity versus CAP concentration ranging 0.5-10 mg/ml. This indicates that maximum fluorescence signal takes place at CPC = 10 mg/ml and CCAP = 3 mg/ml. Note that only PC is excited by laser at 532 nm.
By using the excitation of the coherent source at 532 nm, the fluorescence emission of CAP + PC appears in VIS region ranging 620-780 nm. The maximum LIF signal is revealed at 671 nm for CPC = 10 mg/ml.As expected, the fluorescence spectra in different PC concentrations undergo a spectral shift. Figure 2(b) depicts LIF peak intensity of PC + CAP versus CAP concentration between 0.5-10 mg/ml at certain PC concentrations of 1, 2, 5 and 10 mg/ml.
The LIF signals of PC + CAP compound elevates versus PC concentration and the maximum LIF signal gives out Cmax around CCAP = 3 mg/ml and CPC = 10 mg/ml. Figure 3(a) shows the LIF peak wavelength of CAP + PC compound versus PC concentration ranging 1-10 mg/ml at CCAP= 1, 2 and 5 mg/ml. A lucid red shift appears in favor of CPC. Moreover, the wavelength shift also depends on the CAP concentration and the maximum red shift of 19.5 nm could be seen at CCAP= 2 mg/ml. Figure 3(b) depicts the LIF peak wavelength of CAP + PC solution at different CAP concentrations between 1-10 mg/ml and CPC = 10mg/ml.
Fig. 3. (a) Fluorescence peak wavelength in terms of various PC concentrations in CAP + PC solution at CCAP= 1, 2 and 5 mg/ml (b) LIF peak wavelength of CAP + PC solution in terms of CCAP: 1-10 mg/ml and for typical CPC of 10 mg/ml. Note that PC undergoes red shift at dense solution. Similarly, a lucid blue shift takes place in terms of CAP concentration.
Otherwise, it is seen that the fluorescence peak wavelength will be reduced from 678 to 655 nm by increasing the CAP concentration ranging 1:10 mg/ml. Conjugated formation of (CAP + PC) obliterates fluorophore population leading to a notable reduction of fluorescence emission signal and corresponding blue shift (∼23nm) in agreement to Ref. [38]. This arises from the most likely of binding CAP to PC. Thus, LIF spectra can be used to carefully monitor the dynamic function of the fluorophores/chemodrugs according to the red/blue shifts during cancer therapy.
3.2 MCF7 cell viability under laser treatment
3.2.1. Laser exposure at 808 nm
A fiber coupled CW diode laser at 808nm with maximum power of 2W was employed to investigate the effect of thermal treatment on viability of MCF7 cells. For this purpose, 24 wells of the 48-well microplates were filled with MCF7 cells in order to preculture them and 24 empty wells were allocated to avoid the exposure overlap. Besides, the fiber was fixed 5mm above the well to irradiate the whole well in a steady state condition according to the fiber numerical aperture (NA). The laser irradiated 3 separate wells of the same surface. The exposure time was set for 3 min and cell viability was measured after 48 h.
Figure 4 illustrates the viability of the cells under different laser fluences ranging 4.8-70 W/cm2 (Laser powers over 150 mW-2.2 W). In fact, 0.15-2.2 W is equivalent to 4.8-70 W/cm2 at the output fiber tip. However, we fixed the power density at the target to be 0.01-2 W/cm2 by setting output power and the position of the fiber tip above the well plate. Furthermore, the received power at the target (reported by others) varies between 0.5-10 W/cm2 [40,41]. On the other hand, the damage threshold depends on the laser wavelength and thermal effects. Here, the irradiation at 808 nm is within therapeutic window where NIR emission could not induce notable thermal effects which lies much below the damage threshold [42].
Fig. 4. MCF7 cell viability versus different laser fluences ranging 4.8-70 W/cm2 at 808 nm for 3 minutes exposure time and 48 h incubation.
The cell viability increases nonlinearly in terms of the laser fluence (w/cm2). This confirms the MCF7 malignant cell proliferation under laser exposure at 808nm particularly at larger laser fluences. Several articles report the effect of laser irradiation on cancerous cell growth, emphasizing that laser exposure at some wavelengths can enhance the proliferation of the malignant cells. For instance, laser exposure at 780 nm (within therapeutic window) shows an incremental dose exposure relationship [43]. The laser at 830 and 904 nm (within therapeutic window) enhances MCF7 cell proliferation [44,45]. Furthermore, MCF7 cell viability increases under 1 J/cm2 laser fluence using He-Ne laser at 633 nm [46]. In confirmation of these studies, it was also observed that the proliferation of breast cancer stem cells and breast cancer cells lucidly take place under laser treatment at 636, 825 and 1060 nm with 5-40 J/cm2 [47].
3.2.2. Treatment under different CAP concentrations
The viability of MCF7 cancerous cells was examined at various CAP concentrations. Figure 5 illustrates the viability of MCF7 cells in terms of CAP concentrations ranging 0.001-10 mg/ml after 24 (and 48) hours treatments respectively. In fact, the chemodrug molecules were located at the vicinity of the MCF7 cells for a certain period of interaction time. Note that no laser exposure was carried out in this stage. In the case of CAP concentrations below 0.2 mg/ml, the viability above 95% is obtained after 24/48 h treatment. However, for the concentrations above 0.2 mg/ml, the viability notably reduces at larger concentrations. The half maximal inhibitory concentration (IC50) is a measure of the effectiveness of a compound in inhibiting biological function. Here, the calculated IC50 is 10 mg/ml after 48h treatment indicating to be 10 times larger than the obtained IC50 of 1 mg/ml after 72h treatment of CAP on MCF7 cells [48].
Fig. 5. MCF7 cell viability after (a) 24 h and 48 h chemotherapy by CAP in various concentrations ranging 0.001-10 mg/ml. According to the statistical analysis, the measured typical P-Value is ≤ 0.044.
3.2.3. Hybrid treatment using PC and laser exposure at 532 nm
PC is considered to inhibit the progress of the cell cycle according to the cell apoptosis which promotes the complement-mediated cytolysis [49]. The synergic effects of laser exposure at 532 nm on the viability of PC treated cells were examined here. Regarding the spectral absorbance of PC, the fluorescent properties of PC could be provoked by a visible coherent line of 532 nm contributes to the selective treatment of unhealthy cells. As a result, the stimulation of the cytotoxicity trigged by laser exposure at 532nm was investigated. Figure 6(a) displays MCF7 viability under the PC treatment and PC+532 nm laser exposure in terms of PC concentrations ranging 0.5-10 mg/ml.
Fig. 6. MCF7 cell viability after (a) 48 h treatment in terms of PC concentration ranging 0.5-10 mg/ml without (with) laser exposure at 532 due to photoactivation of anticancer drug (b) 24 (48) h treatment by 5 and 10 mg/ml PC concentrations (c) after 48 h laser exposure at 808 nm, 150 mW at 532 nm, 30-100 mW power with (without) PC ≈ 5 mg/ml treatment (d) Emitted LIF peak intensity versus time by the treated cells using 5 mg/ml of PC. Inset up: LIF peak wavelength versus time representing a lucid blue shift. Inset down: Collected LIF emission from treated cells using 5 mg/ml of PC after T = 0 and 24 h incubation. According to the statistical analysis, the measured typical P-Value is ≤ 0.003.
The cytotoxicity of PC and PC+532 nm laser exposure against MCF7 cells undergoes a linear descending function in terms of PC concentrations, however the slope varies from -1.8 due to -3.4 under laser exposure. Thus, it enlarges two times in favor of PC + laser exposure at 532 nm against PC treatment without the laser provoking. Laser excites the anti-cancer drug to enhance the reactivity in order to demolish the unhealthy cells efficiently. The excited PC expedites the cell mortality at larger PC concentrations regarding its dominant PDT effect. The same result appeared on the MDA-MB-231 breast cancer cells using laser exposure at 625 nm [50]. Figure 6(b) plots the viability of the precultured MCF7 cells under 24(48) h treatment at PC concentrations of 5 and 10 mg/ml. The cell viability of 75% is achieved after 24 h treatments for 5 mg/ml PC. Otherwise, the survival rate is leveled down to 60% after 48 h treatment using 10 mg/ml of PC. Figure 6(c) displays the cell viability with (without) PC treatment (CPC = 5 mg/ml) under laser exposures at 532 (and 808 nm too). Furthermore, the neutral density filters (NDFs) were used to attenuate the laser irradiation at 532 nm giving out different powers of 30, 90 and 100 mW. It is shown that cell viability of 90% (82%) is obtained under the bare laser exposure at 532 (808 nm) respectively.
As a result, the bare laser exposure by itself do not contribute to damage the MCF7 cells, while the synergic effects of PC treatment alongside laser irradiation notably reduce the cell viability. Note that PC + laser exposure at 808 nm, 150 mW demonstrates the viability reduction mainly due to the contribution of thermal processes. However, the treated cells undergo more cytotoxicity under PC+ laser at 532 nm, 100 mW against laser exposure at 808 nm, 150 mW mainly due to the pronounced photon activation of the PC. This arises from the fact that PDT is the major mechanism here in conjugation with laser power. The mortality notably elevates under (PC + laser) and the optimal case demonstrates a decrease to 62% of cell viability for the PC + laser exposure at 532nm, 100 mW amongst the trials. Simultaneously, monitoring of LIF signal is carried out at the onset of laser exposure and once at the end of the incubation T= 0 (24) h. Figure 6(d) depicts the fluorescence signal intensity captured at T = 0 and 24 h incubations from the precultured cells by 5 mg/ml PC. Figure 6(d) inset up illustrates the corresponding emission wavelength in terms of incubation time. Note that the fluorescence spectra attest signal reduction and corresponding blue shift at the end of incubation against the process onset which emphasizes the conjunction of PC + MCF7 cells leading to obliteration of PC population. On the other hand, the Fig. 6(d) inset down shows the LIF spectrum of the precultured cells using 5 mg/ml PC after T = 0 and 24 h incubation. The peak wavelength takes place at 653 nm at T = 0 h. That is in agreement with LIF spectra of PC during previous tests [31]. While, the fluorescent PC spectrum demonstrates some noise due to the photon-scattering at the brink of the wells which exhibits a blue shift of ≈ 7 nm due to the solvent.
3.2.4. Hybrid effect of CAP + PC under laser exposure
Phycocyanin (PC) as the marine pigment exhibits same significant inhibitory characteristics in favor of angiogenic activities of the chemoresistant cells [51]. Consequently, it is desirable to study the combination of the oral bioagent with different chemodrugs to upgrade their therapeutical aspects. Accordingly, the effects of various concentrations of CAP + PC and simultaneous laser irradiation at 532 (and 808nm) on MCF7 cells are to be inspected.
Figure 7(a) displays the viability of cells under the treatments of PC + CAP + laser at 532nm in terms of different concentrations of CAP ranging 0.25-5 mg/ml for three typical PC concentrations of 1, 2.5 and 5 mg/ml. As expected, the viability levels down versus CAP concentration. Besides, the photodynamic effect at PC + CAP is pronounced at lower concentrations of CAP≈ 1 mg/ml as to the viability notably drops at larger PC concentrations elucidating the excited PC gives rise to the cell death.
Fig. 7. Viability of MCF7 cells under CAP + PC treatment in terms of various CAP concentrations (0.25-5 mg/ml) (a) for different concentrations of PC at ▪ 1, ●2.5 and▴5 mg/ml under laser exposures at 532 nm, 100 mW (b) for certain PC concentration of 5 mg/ml without/under the laser exposures at 532 and 808 nm. According to the statistical analysis, the measured typical P-Value is ≤ 0.03.
However, this effect is mitigated at larger CAP concentration and three plots would be intersected at CAP = 5 mg/ml regarding the dominated chmodrug efficacy. The viability plot is linear vs CAP concentration for the CAP + PC + laser treatment at PC≈ 1 mg/ml. However, nonlinearity of the viability plots takes place at higher concentrations of PC≈ 2.5 and 5 mg/ml. Figure 7(b) illustrates the treatments of CAP + PC indicating the viability of MCF7 cells at different CAP concentrations (≤ 5 mg/ml) under the laser exposures. The laser excites the medications at 532 nm, 100 mW for 6 min exposure time. The second laser at 808 nm is also employed with 500 mW and 6 min exposure time. The cytotoxicity of CAP + PC (without laser exposure) is less than pure PC (or CAP) according to the statistical significance. However, the CAP + PC + laser treatment could be considerably more effective in demolishing the cancerous cells than CAP + PC. The lasers at 532 and 808 nm both could contribute to elevate the cytotoxicity during CAP + PC treatment. The laser irradiation at 808 nm demonstrates to be more effective than those at 532 nm. Besides, both laser exposures experience dominant destructive effect in favor of treated MCF7 cells under PC + CAP medications with low CAP concentrations, whereas this effect gets faint at higher chemodrug concentrations. The laser exposure at CAP concentration of 1 mg/ml resembles to be an optimal condition
3.2.5 GO treatments under laser exposures in vicinity of CAP and PC
The outstanding applications of GO nanostructure in drug delivery and cancer treatment is inevitable. Recently, the cell viability is investigated under DOX and GO treatment at various GO concentrations [6]. Here, the effect of the low GO concentrations on the viability of MCF7 cells has been studied. The GO at lower concentrations below 20 ng/ml is safe for body regarding the biocompatibility of the nanoparticle. Figure 8(a) depicts the viability of the cells after 48 (and 72) h treatment versus 10 and 20 ng/ml of GO. According to the scheme, the cell viability still remains high 90%. Otherwise, the viability reduces by 80% after 72 h at the neighborhood of GO≈20 ng/ml.
Fig. 8. MCF7 cell viability (a) after 48 and 72 h treatment in terms of GO concentrations of 10 and 20 ng/ml (b) versus GO concentration between 2.5-20 ng/ml with 48 h treatment by ▴GO without laser exposure, ▪GO + laser 532 nm/100 mW exposure, ●GO+ laser 808 nm/1.5 W exposure. According to the statistical analysis, the measured typical P-Value is ≤ 0.01.
Figure 8(b) depicts the MCF7 cell viability in terms of GO concentration ranging 2.5-20 ng/ml after 48 h treatment of GO under laser exposures at 532nm (808 nm), 100mW (1.5 W) for 6 min respectively. The cell viability reduces with GO concentration in all cases. The cytotoxicity of low GO concentrations over MCF7 cells is negligible. However, the laser exposure notably decreases the cell viability by scaling up the cytotoxicity of nano medication to a great extent. As it is anticipated, CW diode laser at 808 nm with higher power exhibits better efficacy than 532 nm laser to reduce cell viability imposing synergic thermal effect on GO. The photothermal effect would enhance at higher GO concentrations too [6]. The optimum case is achieved for 20 ng/ml of GO under laser exposure of 808 nm demonstrating a 17% drop in cell viability after 48 h incubation. Here, the low biocompatible dosage of GO accompanying the laser irradiation is realized as a local treatment method of malignant tumors. Eventually, the experiments are performed with low CAP chemodrug concentration facing the kinetic processes in the body.
Figure 9 illustrates the viability of the MCF7 cells after 48 h incubation preculturing by CAP concentrations ≈ 2.5 µg/ml and GO with different concentrations of GOI (II) ≈ 1 (2.5 ng/ml) respectively under simultaneous laser irradiation at 808nm with powers of 1.5 (and 2 W) and various exposure times of 3 (and 6 min). The compound of CAP + GO + PC is examined under the laser irradiation of 532 nm too. Some parameters such as GO concentration and laser power significantly reduce the cell viability. However, extending the exposure time is more effective than elevation of laser power. In this case, the CAP + GO and laser/808 nm/1.5W with 6 min exposure time is more effective than CAP + GO and laser/808 nm/2 W with 3 min exposure time to reduce the cell viability.
Fig. 9. MCF7 cell viability after 48 h incubation in vicinity of (from left to right): 2.5 µg/ml of CAP treatment GOI with 1 ng/ml concentrations+ laser at 808 nm, 2W with 3 min exposure time and laser 808 nm, 1.5W with 3 and 6 min exposure time; 2.5 µg/ml of CAP + GOI(II) with (2.5 ng/ml) concentrations; 2.5 µg/ml of CAP +1.5 mg/ml of PC + GOI(II) with 1 (2.5 ng/ml) concentration; CAP with 1 ng/ml; GOI + PC with 0.3 mg/ml under 532 nm laser exposure with 3 min exposure time and 100 mW power. Inset: Microscopic image of MCF7 cells with 20 times magnitude -Up: before treatment, Down: After 48 h incubation under the treatment by CAP + PC + GOI. According to the statistical analysis, the measured typical P-Value is ≤ 0.013.
Furthermore, adding the PC to the low concentrations of CAP + GO under the laser exposure at 532 nm makes an optimal condition to treat MCF7 cells by almost 15% reduction in viability against the alternative of CAP + GO with laser exposure at 808 nm. Figure 9 insets show the microscopic image of MCF7 cells before and after the treatment. There are many live cells before the treatment; inset (up), whereas the number of the cancerous cells would be notably reduced after the treatment in favor of low doses of CAP + PC + GO with CAP ≈2.5 µg/ml, GO≈1 ng/ml and PC≈ 0.3 mg/ml; inset (down).
In summary, treatment of MCF7 cells through the conjugated photodynamic/photothermal/chemo therapies are studied here. These interactions lead to the irreversible cell damage. The efficient treatment of target tumor cells is carried out by the synergic effects of the activated PC, CAP and GO compounds under the localized fiber-coupled laser exposure. The fluorescence properties of hybrid PC + CAP at different concentrations are examined. The LIF peak intensity and corresponding spectral shift are determined under various concentrations of both medications. The cytotoxicity of CAP, PC and GO as the hybrid anti-cancer agents is evaluated alongside laser exposures at 532(and 808 nm) over the MCF7 cells using the MTT assay. The maximum fluorescence emission appears at the concentrations of CAP≈ 3 mg/ml and PC≈ 10 mg/ml. Besides, maximum spectral shift for hybrid PC + CAP combination is realized in favor of 2 mg/ml of CAP and 0-10 mg/ml of PC. It is shown that the photon exposures at 808 nm is not cytotoxic by itself for MCF7 cells, while the cell proliferation takes place subsequently. The cell viability is investigated after 24 (and 48 h) after treatment adding different concentrations of CAP as an efficient chemodrug. The half maximal inhibitory concentration (IC50) is estimated to be 10 mg/ml after 48h treatment. Besides, the cell viability at different concentrations of PC as a bio-anticancer agent is determined with and without laser provoking. According to the photodynamic reactions, the laser induced fluorescence could enhance the therapeutic efficacy of the photosensitizer to a great extent. Regarding the PD properties of PC, its mortality enhances up to 30% under the laser irradiation.
4. Conclusion
The significant reduction in cell viability is trivial at dense anti-cancer drugs. Here, low dose chemodrugs are examined to level down the lateral cellular damage applying hybrid agents under laser activation. The synergic efficacy of hybrid (PC + CAP) treatment under laser exposures at 532 (and 808 nm) are assessed. The laser activation at the certain PC/CAP concentrations is crucial since the viability of treated cells reduces by 20% after laser exposure. Optimal PDT is achieved using dilute CAP (≈ 1 mg/ml) and dense PC (≈ 5 mg/ml) concentrations under laser irradiation. However, the hybrid PD/PT effects reduces at larger CAP concentrations due to the light scattering which attest the efficient treatment could take place at dense PC but dilute CAP concentrations. This affords a privilege for selective treatment of tumors avoiding the side effects of high dose chemotherapy to the neighboring normal tissues.
On the other hand, hybrid CAP/PC treatment by laser at 808 nm exhibits dominant impact against laser exposure at 532 nm regarding the thermal consequences. It is deducted that LIF emission induced by visible laser can contribute to the targeted imaging and effective therapy of the cancerous cells. In fact, it was shown here that the hybrid medication including low dose of CAP and high dose of PC concentration under laser activation at 532 and 808 nm is a promising episode to act as an alternative treatment of breast cancer. To the best of our knowledge, there is no report available to demonstrate the efficient curative characteristics of laser activated PC + CAP so far. The conjugated PTT of GO at 808 nm would enhance the destructive efficacy too.
Acknowledgments
We are thankful to PhD students of Department of Energy Engineering and Physics of Amirkabir University of Technology, Tehran, Iran (Ms. Leyla Shirafkan Dizaj and Ms. Fatemeh Shahi), for their voluntary collaborations over SPSS statistical analyses and experimental calculations, respectively.
Disclosures
The authors declare no conflicts of interest.
Data availability
The datasets obtained by experiments and/or analyzed during the current research are not publicly available because we do not have consent from all patients to publish the raw data.
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