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Nanosalina: A tale of saline-loving algae from the lake’s agony to cancer therapy Fatemeh Ostadhossein, Santosh K. Misra, Aaron Schwartz-Duval, Brajendra K. Sharma, and Dipanjan Pan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01483 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 16, 2017

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ACS Applied Materials & Interfaces

Nanosalina: A tale of saline-loving algae from the lake’s agony to cancer therapy Fatemeh Ostadhossein, Santosh K. Misra, Aaron S. Schwartz-Duval, Brajendra K. Sharma† and Dipanjan Pan*

Departments of Bioengineering, Materials Science and Engineering and Beckman Institute, University of Illinois at Urbana-Champaign, Mills Breast Cancer Institute, and Carle Foundation Hospital, Urbana, IL 61801, USA.

†

Illinois Sustainability Technology Center, Prairie Research Institute, University of Illinois at

Urbana-Champaign, Urbana, IL 61801, USA

E-mail: [email protected]

Keywords: Inherently therapeutic nanoparticles, Reactive oxygen species, Apoptosis, Dunaliella Salina microalgae, Carbon dot

1 ACS Paragon Plus Environment


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Abstract The nanoparticles (NPs) that contain the therapeutic agent within themselves without further modifications can be coined as ‘self-therapeutic’ NPs. The development of these agents especially when derived from natural resources can lead to a paradigm shift in the field of cancer nanotechnology as they can immensely facilitate the complex chemistry procedures and the follow up biological complications. Herein, we demonstrate that inherently therapeutic NPs ‘integrating’ β-carotene can be synthesized from Dunaliella Salina microalgae in a single step without complicated chemistry. The facile synthesis involved microwave irradiation of aqueous suspension of algae which resulted in water dispersible NPs with hydrodynamic diameter of ~80 nm. Subsequently, extensive physiochemical characterizations were performed to confirm the integrity of the particles. The pro-oxidant activities of the integrated β-carotene were triggered by photoexcitation under UV lamp (362 nm). It was demonstrated that after UV exposure, the C32 human melanoma cells incubated with NPs experienced extensive cell death as opposed to nonilluminated samples. Further cellular analysis revealed that the significant reactive oxygen species (ROS) and in particular singlet oxygen was responsible for the cells’ damage while the mode of cell death was dominated by apoptosis. Moreover, detailed endocytic inhibition studies specified that UV exposure affected NPs’ cellular uptake mechanism. These inherently therapeutic NPs can open new avenues for melanoma cancer treatment via ROS generation in vitro.


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1. Introduction The hypersaline lake Urmia in the northwest of Iran has been reported to morph from green to bloody red, based on the images released by NASA that captured the changes from April-June 2016.1 The riddle of the ‘bloody pool’ doomed to death knell was attributed to the genus of microalgae called ‘Dunaliella Salina’ (D. Salina).2-3 This microalgae is known to accumulate massive carotenoids content especially β-carotene under the stress conditions such as high salinity, excessive irradiance, and nutrient deprivation.4 Under these environmental stresses, the amount of reactive oxygen species (ROS) generated in the cell increase. In order to protect and quench high levels of ROS in the algae, the enzymatic and non-enzymatic pathways are activated and among them is the upregulation of carotenogenic gene.4 Carotenoids are terpenoid-based antioxidants with unique optical absorptive properties and benefit from preferential uptake by the cellular membrane due to their ability to be deeply fused with the lipid hydrophobic center.5-7 β-carotene, in particular has been implicated to have effective protective antioxidant role in the free radical-mediated diseases such as cancer, cardiovascular disease, and macular degeneration.8-11 Despite the highly documented antioxidant properties of β-carotene, the pro-oxidant property of this molecule is less emphasized.12-13 The intracellular free radical modulation has been observed in high concentration of β-carotene and in the presence of an external oxidative stress.12, 14-15 Several mechanisms have been proposed as to the ROS generation of β-carotene such as the induction of P450 isoforms, Fenton reaction modulation and auto- oxidation.5, 15-17 The utilization of the naturally occurring compounds as anticancer agents has garnered researchers’ interest specially when combined with nanotechnology approach.18-19 The 3 ACS Paragon Plus Environment


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integration of multiple functionalities on a single platform has been enabled using the nanoparticles where multiple therapeutic agents are combined together under the umbrella of ‘nanomedicine’ utilizing these small entities.20 However, the introduction of additional functionalities requires complex chemistry, added cost, more convoluted characterizations in vitro and in vivo. This trade-off between additional functionality and complexity has led to extensive efforts to develop intrinsically therapeutic NPs (without further modification). Intrinsically therapeutic NPs that preclude the use of additional cytotoxic agent obtained by green chemistry route have been coveted for long. The elevated amount of ROS is established in cancer cells, which is attributed to the altered biochemistry of the cells as a result of abnormal aerobic glycolysis aka. Warburg’s effect.21-22 Moreover, cancer cells lack the full functionality of ROS-scavenging enzymes.23 While reasonable amount of ROS is required to maintain the cellular activity, the excessive production of ROS can lead to cellular damage.24 This vulnerability of cancer cells has been curbed to researchers’ benefit to develop strategies for the cancer treatment by ROS induction via exogenous chemicals while sparing the normal cells.25 For instance, in photodynamic therapy, the photosensitizer agents would induce oxidative stress when activated by the light source of appropriate wavelength.26 The inflammatory and immune response resulting from the generated ROS by the reaction would lead to tumor control.27-29 These effects can be extended to 2D culture leading to cell growth regression. Herein, the synthesis of nanosalina particles has been accomplished via facile microwave irradiation using microalgae as the sole precursor. The use of algae as a natural source of NPs is attractive due to their fast growing rate and easy harvesting. 30-31 The resultant NPs were ~80 nm in hydrodynamic diameter with high aqueous dispersibility. Furthermore, these NPs were found


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to contain elements indispensable to maintain the nutrients’ balance for the algae such as Na, P, Si, and Fe. With additional high luminescent efficiency, these NPs were utilized for site-specific and UV-mediated therapy. Interestingly, the derived NPs exhibited unique anticancer properties against human skin melanoma’s cells (C32) upon photoexcitation at 362 nm without any exogenous drug use. This extraordinary property is hypothesized to originate from the ‘integrated’ β-carotene in the NPs leading to the burst of ROS and phototoxicity. Further biological assays pointed to the contributing role of ROS as the cell death-inducing mechanism. Specifically, the ample generation of singlet oxygen was detected over extended time on the cellular level after photoexcitation for ~30 min. The mode of cell death was found to be dominated by apoptosis as the preferred mode of growth regression and cancer cell therapy. Overall, these NPs are offered as novel cancer therapeutics for skin cancer nurtured by Mother Nature (Figure 1A). 2. Results and Discussion The nanosalina particles were synthesized utilizing microwave-based hydrothermal method.32 The characterizations of the raw dried algae are shown in Figure 2 where the microstructure of the algae is shown in Figure 2A, B. A dispersion of dried D. Salina algae in water was irradiated with microwave for 45 min at 150 ºC (Figure 1B, C). The algae itself had limited dispersibility in water (Figure 1B) due to the presence of non-soluble carbohydrates, fat, etc. while the resultant NPs yielded orange-tinted homogenous suspension (Figure S1). The as-obtained NPs were passed through 0.45 and 0.22 µm filters to remove small percentage of large particles/aggregates and were used for further characterizations. The number-averaged hydrodynamic size of the NPs was measured to be 76±10 nm as shown in Figure 3A. TEM images (Figure 3B) revealed the spherical morphology of the NPs without any distinct crystallographic features. The distribution



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of anhydrous diameter of NPs is shown in Figure 3A inset. X-ray diffraction (XRD) patterns (Figure 3D) further indicated that these NPs possessed shared diffraction patterns with carbon dots (CDs) with the broad diffraction peak happening at 2θ = 24° and a small shoulder peak at 44° that would correspond to (002) and (100) planes of graphitic carbon while the NPs are consisting of highly disordered carbon atoms in nature.33 This is in contrast to the crystalline features found in the raw algae diffraction pattern (Figure S5). In addition, the anhydrous state morphology of the NPs were observed by atomic force microscopy and was determined to be spherical (Figure 3C). Zeta potential measurements of the NPs revealed that they were negatively charged with electrophoretic potential of -21±3 mV thus rendering them with colloidal stability (Figure S2). In addition, the optical properties of the prepared NPs are demonstrated in Figure 3E. The broad absorption band around 280 nm could signify the n–π* transition of the C=O bond and the π–π* transition of the conjugated C=C bond.34 It is important to notice that the characteristic broad absorption peak of β-carotene at ca. 450 nm is absent in the UV-Vis patterns of the NPs implying that the β-carotene molecules are presumably ‘integrated’ within the NPs. However, the innate presence of the carotenoid was further corroborated through 1H NMR analysis (vide infra). In spectral plot, the photo luminescent (PL) emission spectrum of NPs upon excitation at 360 nm is shown (Figure 3E). The emission band appeared in 400-500 nm hence emitting in the green region. The digital camera images of the dispersed NPs under ambient white light and UV lamp (λexcitation=362 nm) indicated the strong green fluorescence of the NPs (Figure 3E inset). The 2D excitation-emission fluorescence contour (Figure 3F) further demonstrated that the NPs were highly fluorescent in the wide range of excitation wavelengths (260-400 nm) while


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showing excitation- dependent PL pattern as could be inferred from the shape of the fluorescent region. Moreover, to gain insight to the surface chemical composition of the algae and NPs, x-ray photoelectron spectra (XPS) were obtained as indicated in Figure 2C-E and Figure 4A-B as well as Figure S3 and S4. The survey spectrum (Figure 4A) demonstrated that the NPs were rich in C (61.0±0.3%) and O (37±0.3%) while they contained elements such as Na (0.4±0.1%), Si (0.6±0.2%) as detected by XPS. The presence of the elements other than C and O would make the composition of these NPs distinct from CD and/or graphene dot (GD) while partially sharing similar characters with the mentioned NPs.35 The detected elements could be attributed to the natural mineral required for maintaining nutritional balance of algae since the raw algae itself had the similar elements available in them as observed in its XPS spectrum (Figure 2C).36-37 It could be inferred from the decomposed C1S peaks (Figure 4B) that the aliphatic (C-C) bonds, carbonyl (C=O), ethereal (C-O), hydroxyl (C-OH) and ester (O=C(O)) would be present which conferred the NPs with water dispersibility. Further evidence of the availability of the elements in addition to C, H and O came from energy dispersive spectroscopy (EDS) as shown in Figure 4C. The peaks could be assigned to P, S, Cl, K and Cu as observed along with the formerly identified elements on the XPS spectrum. X-ray fluorescence (XRF) (Figure 4D) as a powerful elemental analysis tool also corroborated the trace amounts of minerals such as Fe, Na, Cu, and Si which can open up avenues for the applications of these NPs for other purposes. 1

H NMR spectra of β-carotene and NPs are shown in Figure 4E. Besides, the comparison

in the spectra between raw algae and β-carotene is made in Figure S6 while the comparison between raw algae and nanosalina particles are made in Figure S7. The peaks were assigned in β-carotene spectrum as could be seen in the Figure 4E a. It should be noted that the symmetry in



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the molecule has been considered and therefore, only the first set of numbers are shown on the spectrum. As could be seen in the spectrum of NPs (Figure 4E b), the characteristic features of βcarotene have been retained even after undergoing microwave irradiation. In addition, other characteristic features emerged in the spectrum. Namely, the signals in 3-4 ppm could be due to protons in the vicinity of hydrophilic functional groups such as hydroxyl, carbonyl and ether.38 The intact nature of β-carotene in the NPs and literature reports on therapeutic nature of βcarotene prompted us to seek its effect in an in vitro investigation. Light-sensitive drugs that absorbs ultraviolet (long wave - UVA) light are frequently used in the clinic together with ultraviolet light to treat skin conditions such as psoriasis and lymphomas. Methods such as PUVA and/ or PUVB therapy has emerged in the clinic for the treatment of severe skin diseases such as Psoriasis, dermatitis with little or no concern about skin cancer after short term exposure.39 The viability of the C32 (human skin melanoma) cells was assessed based on the loss in the cell growth density and mitochondrial respiration via the MTT assay with/without UV irradiation. C32 cells were chosen since the depth of penetration of UV is limited in biological tissues and therefore skin is a good target for UV therapy. Initially, the nanosalina particles were tested in a range of concentrations where the decrease in the cell viability was evident in the UV treated vs. non-UV treated samples as shown in Figure S8. The concentration of the treatment was optimized at 1.25 mg.ml-1 and was maintained in the followup experiments. The MTT results attested to the safety of the NPs for cells in absence of UV where nearly 90% of the cells survived and is consistent with the prior observations to the cytocompatibility of the CDs.35, 40 By contrast, upon UV irradiation, the cell viability markedly decreased and led to the mortality of ~80% of the cells suggesting the intrinsic therapeutic effects of the NPs activated by UV irradiation after 30 min of exposure (Figure 5E). The bright field


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images also showed the loss of cell growth density and induced cell shrinkage in the NP-treated samples under UV exposure (Figure 5D). In contrast, the NP treated samples in absence of UV exposure (Figure 5C) did not show noticeable change in the morphology and preserved their spindle like shape, similar to the control cells (Figure 5A). After establishing the cancer cell growth regression benefits of the NPs as UV therapy agents, we investigated the potential mechanisms governing their efficacy. We hypothesized that ROS generation could be one of the plausible routes for the cell death. This hypothesis was further corroborated with the ROS induction assay where a significant time-dependent enhancement in the ROS level (up to 44 h after irradiation) was noticed in the UV treatment group compared with the non-UV irradiated cells (Figure 6A). Ample generation of singlet oxygen is one of the effective strategies to overcome the downsides of the conventional photodynamic agents.41 The nanosalina particles may have potential as photosensitizer which requires high singlet oxygen quantum yield and plays the most prevalent role in the molecular processes initiated by photodynamic therapy.41-44 Therefore, we investigated whether singlet oxygen generation could be ascribed as a mechanism involved in the cells’ death (Figure 6B and C). For the Singlet Oxygen Sensor Green (SOSG) detector, the fluorescent signal from the chromophore is quenched prior to the reaction with 1O2 due to intramolecular electron transfer. Upon the reaction of the 1O2 with the reporter, the electron transfer would be inhibited and endoperoxides would form by the reaction of 1O2 with the anthracene component of SOSG thus strong green fluorescence would emerge.45 The confocal images of the SOSG and the propidium iodide (PI) staining which identifies the dead cells are shown for the NP treatment (Figure 6C) and control cells (with UV exposure) (Figure 6B). It could be inferred from the images that the control cells indicated no significant green signal nor have they experienced extensive cell death. However, for the UV irradiated cells, it



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was clear that the green signal could be co-localized with the red signal implying that the cells’ death was the result of 1O2 species. Nevertheless, the PI staining results revealed the loss of cell membrane integrity in the oxidatively stressed cells. Based on these findings, we hypothesize that the prevalent mechanism in the cell death here is the ROS and singlet oxygen generation. The lifetime of singlet oxygen is very short (approximately 10-320 ns) and therefore the diffusion is only expected to occur to the immediate biological entities, i.e. at only 10-55 nm in the cells, Therefore, the damage can be restricted intracellularly to a localized region.46 On the other hand, we hypothesize that the other elements found in the system might not have contributed to the cytotoxic effects. The NPs seemed to be safe without UV activation while their cytotoxicity emerged after UV exposure. To quantify the apoptotic and necrotic cells, Annexin-V FITC/PI staining was utilized (Figure 7). At the early stages of apoptosis, phosphotidylserine (PS) would emerge in the outer leaflet of plasma membrane which could be detected by Annexin-V/FITC labeled protein. On the other hand, PI is known to intercalate DNA upon cell membrane damage and permeation. Here, DMSO (10% v/v) was used as a positive control, known as cause for inducing considerable apoptosis in human cancer cells.47 The analysis revealed that the cells treated with NP in absence of UV exposure (Figure 7B) indicated negligible cell damage (4.9±1.9% cell apoptosis) and the results were in agreement with the control group (Figure 7A) with minimal apoptosis (6.6±1.6% cell apoptosis). In contrast, the cells treated with NPs under UV (Figure 7E) presented late stage apoptotic features (35.6±2.0% cell apoptosis) as the dominant death mode similar to DMSO treatment (Figure 7F). Finally, the effect of UV treatment on the NPs entry pathway was investigated by subjecting the cells to various endocytic pharmacological inhibitors (dynasore: inhibitor of


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dynamin-dependent endocytosis (80 µM), 2-deoxyglucose (DOG)/NaN3: energy mediated pathway inhibitor (10/50 mM), chlorpromazine (CPM): clathrin-mediated endocytosis inhibitor (28 nM), nystatin: caveolin/lipid raft mediated endocytosis endocytosis inhibitor (180 nM)48-50) as shown in Figure 8. The cell viability was chosen as the criterion for comparison. Upon the inhibition of a particular pathway, it was predicted that the cell viability would increase due to the lowered level of cellular internalization. In general, the cellular internalization of the NPs is occurring via various pathways such as micropinocytosis, phagocytosis, passive diffusion, and pinocytosis and is highly dependent on the chemistry of the NPs.51-52 C32 cells were pretreated with the inhibitors for an hour and then were treated with the NPs and the remaining procedures were followed as described previously for the UV treated and dark samples. The cells with no treatment, cells pretreated with only inhibitors, and those treated with only NPs were adopted as the negative control. For the NP treated cells in absence of UV (Figure 8A), none of the investigated pathways was active and the difference between the negative control (NP treatment only) and the pre-incubated samples with inhibitors was marginal. However, upon photoexcitation (Figure 8B) all of the investigated pathways were found to be contributing effectively to the NPs uptake by the cells. Of particular interest, was the cells treated with NaN3/DOG. NaN3 is a well-known ROS quencher. It is evident that upon ROS generation, some of the cytotoxic effects of the ROS would be diminished in the presence of NaN3 hence enhancing the cell viability. Our results are in good agreement with this explanation. Overall, this is the first report to study anti-cancer potential of a novel bioactive carbonaceous NP derived from Dunaliella Salina microalgae in vitro. Our results demonstrated that NP can be derived using biological waste, which in turn retains some of the key functionalities that are present in the precursor materials that can potentially be used for the ROS



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generation for cancer therapy. Although out of the scope of this present study, the eventual clinical translation against melanoma cancer should be addressed by future experiments in vivo models. 3. Conclusions In the current study, we synthesized NPs based on D. Salina which is a rich source of β-carotene using the facile microwave irradiation. The structural properties of the NPs were characterized utilizing DLS, TEM, AFM, and XRD. In addition, the optical properties of NPs were investigated. It was demonstrated that the NPs would absorb strongly around 280 nm attributed to the n–π* transition of the C=O bond and the π–π* transition of the conjugated C=C bonds. Moreover, the NPs had high fluorescence in a wide range of wavelengths. The 1H NMR spectroscopy confirmed that β-carotene remained intact even after MW synthesis. These NPs were found to induce extensive cells’ death in human skin melanoma cells when photoexcited in the UV range while this effect was absent otherwise. The hypothesized ROS-mediated death as a result of β-carotene was corroborated by the in vitro ROS assay and singlet oxygen sensor green combined with PI staining assay. Annexin-V FITC/PI assay revealed significant cell apoptosis in NP-treated samples triggered with UV. The altered mode of NP internalization was concluded from the inhibitor studies. Overall, these NPs would offer a self-contained inherent green solution to skin cancer and would open up new pathways in cancer therapy using self-therapeutic NPs from natural resources while avoiding complicated synthesis. 4. Experimental Section Physiochemical Characterizations: The spray dried food grade Dunaliella salina microalgae was obtained from Illinois Sustainable Technology Center and was procured from buyalgae.com. A dispersion of 100 mg.ml-1 of algae (2.5wt% of beta carotene content) was made in water and was


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then subjected to microwave irradiation (Biotage® Initiator+ microwave synthesizer, Charlotte, NC, USA) at 150 ºC and 150 watts for 45 min. The temperature was chosen to be below the degradation temperature of β-carotene.53 The as-synthesized NPs were passed through 0.45 and 0.22 µm syringe filters. Subsequently, extensive physiochemical characterizations were conducted. The morphological features of the samples were observed under the transmission electron microscopy (TEM) on a JEOL 2100 Cryo TEM machine (Tokyo, Japan) equipped with Gatan UltraScan 2k × 2k CCD camera. The holey carbon coated copper grid was utilized for TEM sample preparation. The hydrodynamic diameter and electrophoretic potential of the NPs were measured on a Malvern Zetasizer ZS90 instrument (Malvern Instruments Ltd, United Kingdom) at fixed angle of 90º. Atomic force microscopy (AFM) was conducted for the sample deposited on a mica wafer secured on a stainless steel disc in the tapping mode (Asylum Cypher (Asylum Research, Santa Barbara, CA)). The absorbance of the NPs in the UV and visible spectrum was recorded on a GENESYS 10 UV–vis spectrometer (Thermo Scientific, MA, USA) while the 2D fluorescence-emission spectra of the samples were measured on a Horiba Aqualog Scanning Spectrofluorometer (Horiba scientific, Edison, NJ, USA). After each measurement, the firstorder Rayleigh scattering was corrected and all spectra were normalized to 1 mg.l−1 quinine sulfate. X-ray photoluminescent spectrum (XPS) was obtained on a thick vacuum dried layer of the NPs applied on the glass surface using Physical Electronics PHI 5400 spectrometer with Al Kα (1486.6 eV) radiation. The spectrum was referenced to the adventitious C 1s feature at 284.8 eV. The chemical components of the NPs and raw algae were determined on an X-ray fluorescence (XRF) spectrometer/energy-dispersive X-ray (Shimadzu EDX 7000/8000 energy dispersive XRF spectrometer, Japan). Other elemental analysis included SEM/EDS using Hitachi



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(Schaumburg, Illinois) S-4700 SEM with Oxford Instruments (Abingdon, Oxford shire) ISIS EDS X-ray Microanalysis System and Centaurus BSE detector. The chemical characteristics of the NPs was inferred from 1H NMR analysis using a 500 MHz machine (Varian VXR 500 (Varian, Inc., Palo Alto, CA)) equipped with a 5-mm Nalorac QUAD probe in deuterium oxide (D2O) (Cambridge Isotope Laboratories, Inc., MA, USA). The data was analyzed using MestRenova™ 8.1 software (Mestrelab Research SL; Santiago de Compostela, Spain).54 Raw algae and β-carotene (Cayman chemical, Ann Arbor, Michigan, USA) were initially dissolved in small amount of DMSO and diluted in D2O. X-ray diffraction (XRD) patterns were collected on a Siemens-Bruker D5000 (Madison, WI, USA) using Cu-Kα (1.54 Å) radiation in the θ-2θ configuration operated at 30 kV and 40 A with the scan rate of 1°/min, and the step size of 0.02°. The spectrum was smoothed using a 10-point Savitzky–Golay algorithm. Cell Viability Assessments: C32 human melanoma cell lines obtained from American Type Culture Collection (ATCC, VA, USA) were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 1% pen-strep under 99% humidity and 5% CO2. The cells were passaged via trypsinaztion (0.1%) containing 0.02% ethylenediaminetetraacetic acid (EDTA) after being washed with Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4). 10,000 cells in 200 µl were seeded in each well of a 96-well plate and were allowed to reach 80% confluence. The cells were incubated with NPs at concentration of 1.25 mg.ml-1 for 4 h and were subsequently irradiated with a UV lamp operating at 362 nm (Avantco W62, China, 60 Hz, 500 watts) for 30 min while maintaining the plate to source distance 10 cm. The cells without NPs were considered as control. A parallel experiment was conducted under similar conditions while no UV exposure was applied. After 44 h of incubation, the cells were imaged using bright field microscope. Next, 20 µl of MTT solution (5 mg.ml-1)


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was added to the growth medium, and then, the cells were incubated for an additional 4 h. The old media was replaced with 200 µl of DMSO to solubilize the formed formazan salt and the absorbance at 570 nm measured on multi-well plate reader (BioTek Synergy HT, USA) was correlated to the cell viability. Reactive Oxygen Species (ROS) Assays: The treated and non-treated (with UV and/or NPs) C32 cells were investigated for the ROS generation using the Fluorometric Intracellular ROS Kit (Green Fluorescence, Catalog Number MAK143, Sigma- Aldrich, MO, USA). The cells (10,000 cells/well in 90 µl) grown for 24 h were treated with the master reaction mix based on the prescribed protocol for one hour. Next, the cells were treated with the formulations (13.75 mg in 20 µl) for 4 h and irradiated with UV for 30 min. Finally, at the specified time points, the samples were analyzed under λex = 490/λem = 525 nm on a multi-well plate reader. The generation of the singlet oxygen was assessed utilizing the Singlet Oxygen Sensor Green Reagent (Molecular probes, S36002). The cells grown on the coverslips for 24 h, were treated with 1.5 µl of 5 mM reagent for 30 min. The cells were treated with the NPs (1.25 mg.ml1

) for 1 h to allow internalization. Subsequently, UV treatment was conducted for 30 min. The

cells were then thoroughly washed with DPBS and fresh media was supplemented. Further incubation for 2 h was conducted before the cells were stained and incubated with 2 µl of PI for 30 min in dark. The cells were incubated with paraformaldehyde as previously mentioned followed by washing with DPBS. The imaging of the SOSG component was performed under the 514 nm excitation laser adopting the FITC channel for the imaging purposes. Apoptotic Assay: To quantify and differentiate between the apoptotic and necrotic cells, the Annexin-V labeled FITC and PI double staining (ThermoFisher Scientific, Catalog number: V13242) have been implemented on the C32 cells in the absence or the presence of UV (30 min)



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with various formulations. DMSO (10% vol/vol)-treated cells were considered as positive control. The treatment lasted for 44 h. The staining has been carried out based on the manufacturer’s protocol. Briefly after harvesting the cells with trypsin, 5 µl of FITC labelled Annexin-V and 1 µL of the 100 µgml-1 PI working solution were added to each 100 µl of cell suspension and incubated in dark for 15 min. Immediately afterwards, the cells were analyzed on a Guava® easyCyte Single Sample Flow Cytometer (Guava Technologies, Billerica, Massachusetts, USA). Study of the Nanoparticles’ Entry Pathway: C32 cells grown for 24 h in 96 well plates were treated with various endocytic inhibitors (NaN3/DOG: 10/50 mM, Dyansore: 80 µM, CPM: 28 nM, nystatin: 180 nM) for 1 h. The old media was replaced with the NP suspensions (1.25 mg.ml-1) and the cells were incubated for 4 h before being irradiated with UV for 30 min. MTT assay was conducted as previously described to assess the cell viability after 48 h in the absence or presence of NPs and/or UV. The fold change was calculated with respect to the nonpreinhibitor treated sample. Statistical Analysis: The tests were done in triplicate and the results were expressed as the mean±standard deviation. The data were analyzed on the GraphPad Prism 6.0 software using analysis of variance (ANOVA) Bonferroni correction for post hoc. The p

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