Article type : Special Issue Research Article

Accepted Article
Photodynamic Therapy in HeLa Cells Incubated with Riboflavin and

Pectin-coated Silver Nanoparticles†

María Belén Rivas Aiello1, Daniel Castrogiovanni2, Julieta Parisi2, Julio C. Azcárate3,

Fernando S. García Einschlag1, Thomas Gensch4, Gabriela N. Bosio1,4, Daniel O.

Mártire*1

1
Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Facultad de Ciencias

Exactas, Universidad Nacional de La Plata, C. C. 16, Suc. 4, (1900) La Plata, Argentina

2
Instituto Multidisciplinario de Biologia Celular (IMBICE), CCT-La Plata-CONICET, Camino General

Belgrano y 526, B1906APO, La Plata, Argentina

3
(CAB), CONICET, Avenida Bustillo 9500, San Carlos de Bariloche, Río Negro

R8402AGP, Argentina

4
Institute of Complex Systems (ICS-4 (Cellular Biophysics)), Forschungszentrum Jülich, Leo-Brandt-

Str., 52428 Jülich, Germany.

†This article is part of a Special Issue dedicated to Dr. Norman Andi García.

*Corresponding author e-mail: [email protected] (Daniel O. Mártire)

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/php.12974

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 ABSTRACT

Riboflavin (Rf) is an endogenous photosensitizer, which can participate in Type I and Type II
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processes. We have recently shown that the yield of the triplet excited states of Rf is enhanced in the

presence of pectin-coated silver nanoparticles (Pec@AgNP) due to formation of a complex between

Rf and Pec@AgNP (Rf-Pec@AgNP). Consequently, under aerobic conditions the amounts of singlet

molecular oxygen and superoxide radical anion generated are also larger in the presence of the

nanoparticles. This result made us suspect that the nanoparticles could have a beneficial effect in Rf-

based PDT. To prove this hypothesis, we here compared the photodamage in HeLa cells incubated

with Rf in the presence and in the absence of Pec@AgNP applying several optical assays. We used

fluorescence imaging of irradiated HeLa cells incubated with Annexin V and Propidium Iodide to

evaluate the occurrence of apoptosis/ necrosis, the reduction of the tetrazolium dye MTT to

formazan and Neutral Red uptake to prove cell viability, as well as synchrotron infrared microscopy

of single cells to evaluate possible structural changes of DNA and nuclear proteins. The enhanced

photodamage observed in the presence of Pec@AgNP seems to indicate that Rf enters into the cells

complexed with the nanoparticles.

INTRODUCTION

Photodynamic therapy (PDT) consists of tumor irradiation with visible or NIR light after application of

a photosensitizer (1). Riboflavin (vitamin B2, Rf) is present in biological systems as a constituent of

proteins binding flavin mononucleotide and flavin adenine dinucleotide as cofactors (2). Electron

transfer from numerous donor molecules present in cells to the triplet state of Rf, 3Rf*, yields the Rf.-

radical anion, which initiates free radical reactions (Type I photosensitization) (3). In addition, upon

irradiation of Rf solutions under aerobic conditions, singlet molecular oxygen, O2(1Δg), is also formed

by energy transfer from 3Rf* to ground state oxygen (Type II photosensitization). Consequently, the

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 Type I and Type II photooxidation of several substrates sensitized by Rf was the topic of several

publications (4,5).
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The photoinduced generation of reactive oxygen species (ROS) motivated the testing of the

photosensitizing ability of Rf for a number of applications. For instance, light irradiation of Rf in

aqueous medium was demonstrated to be effective as antibacterial agent (6,7), as crosslinker for

corneal stiffening (5,8), for blood product sterilization (9), and for the treatment of skin lesions (10).

The improved generation of O2(1Δg) by Rf in the presence of pectin-coated Ag nanoparticles

(Pec@AgNP) was previously demonstrated by an indirect method (11). More recently, we have

shown by time-resolved absorption spectroscopy that the decay of the excited state of the complex

formed by Rf with pectin-coated Ag nanoparticles (Pec@AgNP) in aqueous solution feeds 3Rf*

excited state. As a result, the amounts of 3Rf*, O2(1Δg), and superoxide radical anion, O2.-, generated

by Rf are enhanced in the presence of the nanoparticles (12).

There are many literature reports showing the beneficial role of heavy metals on the photodynamic

activity of organic photosensitizers. In particular, several nanohybrids of organic sensitizers with

noble metal nanoparticles were prepared and their performances in PDT in carcinoma cells were

tested. For instance, studies performed in HeLa cells and Protoporphyrin IX with gold nanoparticles

(13, 14) showed a higher phototoxicity of the sensitizer when complexed with the nanomaterial. The

enhanced performance of nanohybrids in PDT compared to the free sensitizers was also shown for

other systems, including Au nanoparticles functionalized with zinc phthalocyanine and a lactose

derivative employing SK-BR-3 breast cancer cells (15), meso-tetrahydroxy-phenylchlorin conjugated

to Au nanoparticles in SH-SY5Y human neuroblastoma cells (16), pluronic coated gold nanoparticles

incorporating the hydrophobic dye IR780 in murine colon carcinoma C-26 cells (17), and gold

nanocomposites with hematoporphyrin in MT-4 leukemia cells (18). In addition, in vitro photo-

activity assays performed with a hybrid nanomaterial of Au nanoparticles decorated with

Pheophorbide-A (PheoA), and the cancer-targeting agent hyaluronic acid in the lung cancer cell line

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 (A549) showed that over 95% of the cells were dead upon laser irradiation (19). Porphyrin-decorated

Au nanoparticles containing a targeting agent that recognizes the erbB2 receptor overexpressed on
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the surface of SK-BR-3 were shown to elicit targeted PDT of these cells (20). Au nanoclusters and

Chlorine 6 loaded into pH-sensitive liposomal nanocomposites were proven to be efficient

nanohybrids for PDT in vivo (21). Examples of nanohybrids employing two-photon excitation can be

found in the review article by Shen et al (22).

Motivated by the enhanced generation of ROS by Rf in the presence of Pec@AgNP and by the

beneficial role of metallic nanoparticles on PDT treatments, the main goal of the present work is to

prove the presumable enhancing effect of Pec@AgNP on PDT when Rf is employed as a sensitizer. To

this end, we here compare the photodamage induced in HeLa cells incubated with Rf in the presence

and in the absence of Pec@AgNP. In order to evaluate the effect of PDT on the cells, we performed a

series of biological assays to monitor apoptosis, necrosis, mitochondrial activity, the integrity of

lysosome membrane, and structural changes in DNA and nuclear proteins.

MATERIALS AND METHODS

Materials. Riboflavin, pectin from apple, furfuryl alcohol (FFA), 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT), Neutral Red, Annexin V, and PI were purchased from Sigma-Aldrich.

Silver nitrate was obtained from Biopack and sodium hydroxide from J. T. Baker. All experiments were

performed with deionized water.

Synthesis of Pec@AgNP. The reported method was employed (11).

TEM imaging. The size of Pec@AgNP was characterized by Transmission Electron Microscopy (TEM)

using a Phillips CM200-UT (LaB6) operated at 200kV. The size of the particles was fitted with lognormal

distributions.

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 Oxygen uptake experiments. The consumption of oxygen upon irradiation was measured with an

oxygen-sensitive electrode (Consort SZ10T) in initially air-saturated solutions of Rf with and without
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Pec@AgNP in the presence of FFA, a well-known quencher of O2(1Δg) (23).

u u : Hu H L sw g w M d f d E g ’s du (MEM) (GI O)

containing 10% FBS (Internegocios S.A.) and 100 µg.mL−1 of penicillin. Cell cultures were performed in an

incubator with 5% CO2 and 95% air at 37°C. 1.5x104 cells were seeded in 96-well plates and grown for 24

h until confluence.

Cellular uptake of riboflavin. OPTIMEM media (GIBCO) were prepared with and without addition of

Rf (final concentration: 50 µM). An aliquot of 1 mL volume of these media were kept in the dark for 24 h.

T s d w b d “b f ”. T g d - containing 50 µM or 0 µM- were employed

for the incubation of HeLa cells for 24h. Then, cells were counted and the fluorescence (exc = 488 nm,

em = 530 nm) of every sample was measured (before and after cell growth) and corrected by the

number of cells.

Cytotoxicity assay in dark. HeLa cells (1.5x104) were plated in 100 µL MEM (GIBCO) containing 10%

FBS (Internegocios S.A.) and 100 µg.mL−1 of penicillin, and incubated overnight at 37°C with 5% CO2 until

confluence. The medium was then removed and the cells were incubated for 24 h with different

concentrations of Rf, Pec@AgNP or a mixture in MEM. Every experiment was compared to a control

culture. Cell survival was assessed by MTT and NRU assay as described previously (24,25,26).

PDT phototoxicity assay. The procedure was the same as in the dark assay. After de incubation with

Riboflavin, Pec@AgNP or the mixture, the medium was changed for DMEM/F12 (GIBCO) without phenol

red and the samples were irradiated with two RPR-3500 A lamps with emission centered at 350 nm for 2

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 h into the cell incubator. The emission spectrum of the lamps is shown in Figure S1 (see Supporting

Information). The irradiance measured using potassium ferrioxalate as actinometer (27) with the same
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irradiation geometry was 9.6 mW.cm−2. Photocytotoxicity was evaluated by MTT and NRU assay (28,29).

Annexin V/ Propidium Iodide (PI) staining assay evaluated by fluorescence microscopy.

HeLa cells exposed to Pec@AgNP in the presence as well in the absence of Rf, or to Rf alone and without

any additives were cultivated as indicated above into a glass bottom 35 mm Petri dish (ibidi, Martinsried,

Germany), previously treated with Poly-L-Lysine (PLL) (0.1 mg.mL−1) (1 mL for 5 min). After 24h of

incubation, the cell culture was washed twice with PBS buffer of pH 7.4. Samples were then placed

directly inside a stage top incubator (Okolab S.R.L., Pozzuoli, Italy; environmental parameters set to 37

ºC, 85% humidity, and 5% CO2) on the stage of an inverted microscope (Ti-E, Nikon) and 1 mL of Binding

Buffer BD Pharmingen was added. The inverted microscope was used as a spinning disk confocal

microscope (30) (ACALBFI, Groeben, Germany) with a spinning disk unit (CSU-W1; Yokogawa Electric

Corporation, Tokyo, Japan) as the central part, and an EMCCD camera (Ixon Ultra 897; Andor

Technologies Ltd., Belfast, UK) as a detector and an image splitting device (Optosplit II; Cairn Research,

Fversham, UK) for simultaneous observation of two spectral regions of the emitted light (with the option

of using one or two simultaneous excitation light sources). Bright field images, as well as spinning disk

confocal fluorescence images with excitation at 488 nm and 561 nm were taken with a 100X

magnification immersion objective (plan apo chromat, NA=1.40, Nikon). Appropriate dichroic beam

splitter and bandpath filters in the Optosplit unit allowed the simultaneous detection of fluorescence in

two spectral regions, 500 - 550 nm (Annexin V-FITC excited by 488 nm laser) and 590-650 nm (PI excited

by 561 nm laser), respectively. The software Andor IQ2 was used for image acquisition. Exposure time for

single image was set 200 ms and the frame rate was set the minimal. Irradiation for photodynamic

action: HeLa cells were then illuminated with a 405nm laser (417 mW cm−2), for 5 min to induce

photodamage. Afterwards, 1mL Annexin V/PI solution (50 uL Annexin V-FITC - BD Pharmingen 10X + 20

uL PI 50 ug.mL−1 BD Pharmingen + 930 uL Binding Buffer) was carefully added without moving the

sample. After 15 min of incubation in the dark, both PI fluorescence (excitation at 561 nm (116 mWcm−2,

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 200ms), emission maximum at 620 nm) and Annexin V-FITC fluorescence (excitation at 488nm (60.9

mWcm−2, 200ms), emission maximum at 530 nm) images were recorded. Control experiments were
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performed in HeLa cells without nanoparticles but with Rf and without neither of the two.

FTIR microspectroscopy. The experiments were performed at the infrared SMIS beamline (SOLEIL

Sy , L’O d sM s s, G f su Yv ,F ). T sy based Fourier transform

infrared (SR-FTIR) microspectroscopy was employed to investigate structural changes of DNA and nuclear

proteins of HeLa cells grown on CaF2 slides. Spectra were taken specifically on the cells nuclei in

transmission mode. The instrumentation, methods, and conditions used have been previously described

(31). Cell spectra recorded one by one, with 50 to 100 individual cells per sample were analyzed.

Principal Component Analysis (PCA) was used for outliers detection within samples and also for

comparison between samples. The preprocessing methods of the spectra have been described

elsewhere (31). Briefly, the Savitzky-Golay algorithm was applied either to perform a baseline correction

of the raw data (zero order spectra) or to evaluate the second derivative of the data (second order

spectra) (31). Before executing PCA routines, the spectra obtained by the latter procedures were scaled

to unit norm within the desired wave number range.

RESULTS

TEM Images of Pec@AgNP

We have prepared pectin-coated silver nanoparticles according to the reported procedure (12). A

typical TEM image and its corresponding histogram is shown in Figure 1. The measured nanoparticles

size can be described by a bi-modal distribution with an average of 2.3 ± 0.7 nm (major fraction; >

95%) and 9 ± 6 nm.

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 Effect of Pec@AgNP in Oxygen Uptake
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From now on, the concentration of Ag will be specified instead of that of Pec@AgNP. Note that from

the average diameter of the spherical nanoparticles (2.3 nm) and neglecting the contribution of the

bigger particles, an average volume of the nanoparticles of 6.37×10-21 cm3 is obtained. Taking the

reported metal density of 10.5 g.cm−3 (32), the molar concentration of the nanoparticles results 3.73

×102 times lower than that of Ag.

Oxygen uptake experiments with samples containing FFA were performed. The results are

shown in Figure S2 of the Supporting Material. The rate of oxygen uptake is slightly enhanced by the

presence of Pec@AgNP ([Ag] = 1 µM), which is within the concentration range used in the biological

assays. This result is in line with the increase of O2(1Δg) yield produced by Rf in the presence of

Pec@AgNP, which was previously demonstrated in time-resolved phosphorescence experiments.

According to the phosphorescence assays, a larger increment in rate of oxygen uptake is expected

for concentrations of Pec@AgNP higher than those employed in the present work (12).

Cellular Uptake of Riboflavin

The uptake of Rf in HeLa cells was measured by fluorescence. Comparison of the fluorescence

emission spectra (exc = 488 nm) of the Rf solutions before and after incorporation of the dye in the

cells are shown in Figure 2. We tested different Rf concentrations and the uptake was always

significant and in the range of 50%, as shown here for OPTIMEM medium containing 50 µM Rf.

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 MTT and NRU tests
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Two different tests for cyto/phototoxicity were employed: 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) reactivity and neutral red uptake (NRU). The former assay

determines mitochondrial dehydrogenase activities in living cells. In metabolic active cells MTT is

reduced to formazan crystals by nicotinamide adenine dinucleotide hydride (NADH). The purple

crystals of formazan extracted from lysed cells are then dissolved in an organic solvent and the

absorbance is measured at 570 nm (33). The NRU test is based on the ability of viable cells to

incorporate and enrich the dye in lysomes via the endocytotic route. After extraction of the dye with

an acidified ethanol solution, its concentration is determined spectrophotometrically (24).

MTT and NRU assays performed with non-irradiated HeLa cells incubated with Rf in the

concentration range from 0 to 100 µM or Pec@AgNP containing silver in the concentration range

from 0 to 1 µM delivered no evidence that, under our experimental conditions, Rf or the Pec@AgNP

have an inherent adverse effect on HeLa cells (see Figure S3). Similar results were obtained with cells

incubated with Rf (50 µM) and different amounts of the nanoparticles (Figure 3D).

Assays performed with irradiated HeLa cells showed that increasing the concentration of Rf

in the medium from 0 25 μM s ff ’s b y du MTT f z .

higher Rf concentrations (25- 100 μM) d s f ´s v b y p d f

control is observed (Figure 3A). NRU assays, which test the dye uptake by active cells and its

incorporation into lysosomes, showed a decrease of the number of viable cells for Rf concentrations

qu g 25 μM (F gu 3 ).

The presence of Pec@AgNP ([Ag] in the range from 0.25 to 1 µM) in the pre-incubation

medium of irradiated HeLa cells has no effect on the ability of the cells to reduce MTT to formazan

nor on the incorporation of neutral red to lysosomes (Figure 3B).

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 In order to check the possible enhanced photodamage caused by the nanoparticles in the

presence of Rf, MTT and NRU assays were performed with cells incubated with Rf (50 µM) and
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different amounts of Pec@AgNP ([Ag] ranging from 0.25 to 1 µM). The addition of the nanoparticles

produced a further decrease of cell´s viability as sensed by MTT, but not by the NRU assay (Figure

3C).

Annexin V/PI tests

It is accepted that apoptosis is the main mechanism of cell death when cells are treated with PDT in

vitro (34). In particular, it was shown that irradiation of HL-60 and murine NS0/2 tumor cells in a

culture medium enriched with Rf induces cell death by apoptosis (35,36). On the basis of these

results, we performed here Annexin V / Propidium Iodide (PI) tests with fluorescence detection with

HeLa cells previously incubated with Rf with and without Pec@AgNP. Annexin V reveals the presence

of phosphatidylserine on the cell surface, which is indicative of an early stage of apoptosis. On the

other hand, PI is used as a marker of membrane disintegrity and necrosis (37,38).

Irradiated HeLa cells pre-incubated with 50 µM Rf yielded a positive result to Annexin V, but

negative to PI (see Figure 4) indicating early apoptosis. In contrast, when Pec@AgNP were

additionally included in the culture medium, not only Annexin V but also PI yielded positive results,

thus indicating late apoptosis or necrosis. These results show a higher photodamage achieved in the

presence of Pec@AgNP and Rf.

SR-FTIR microspectroscopy

Synchrotron based Fourier transform infrared (SR-FTIR) microspectroscopy was used to detect any

intracellular biochemical modification following the photoirradiation of HeLa cells containing Rf

and/or the nanoparticles. Comparative assays with cells incubated with the sensitizer 5,10,15,20-

Tetrakis (1-methyl-4-pyridinio) porphyrin tetra (p-toluenesulfonate), TMPyP, were performed. The

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 visible light irradiation conditions were similar to those previously employed to show changes in the

protein secondary structures in cells incubated with TMPyP (31).

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Recorded FTIR data were preprocessed to yield zero order and second order spectra. PCA

scores, the corresponding scree plots and sample mean zero order spectra in the region of the

Amide I and Amide II bands (1480–1720 cm−1) are shown in Figure 5 for: non-irradiated and

irradiated HeLa cells without the addition of Rf or Pec@AgNP in the incubation medium (controls),

irradiated HeLa cells pre-incubated with 50 µM Rf, irradiated HeLa cells pre-incubated with 1 µM Ag,

irradiated HeLa cells pre-incubated with 50 µM Rf and 1 µM Ag, and irradiated HeLa cells pre-

incubated with 10 µM TMyP for comparison.

PC1 (explaining 87% of the variation) seemed to distinguish between PDT-treated HeLa cells

employing TMPyP as the sensitizer from all the other samples (31). Irradiation of HeLa cells

incubated with TMPyP, which tends to accumulate in the cell nucleus, was reported to induce

pronounced changes in the s u u d Ib d (d s f p f α-helix) (31).

These results indicate that it was not possible to detect by SR-FTIR microspectroscopy any

significant change in the secondary structure of nuclear proteins of irradiated HeLa cells pre-

incubated with Rf and/or Pec@AgNP. Similar results were obtained after submission of the spectra

to different pre-treatments (see the analysis of the derivative of the spectra in Figure S4). In order to

inspect any change in the region of the Amide III peak and also include the absorption of nucleic

acids, the PCA analysis was also performed in an extended wavenumber region (1748–952 cm−1)

(39). In this region, independent of the data pre-treatment employed it was also observed that only

the sample incubated with TMPyP showed a different behavior, confirming that under the conditions

of our experiments only the use of the porphyrin as photosensitizer affected the IR absorption of the

protein amides of the proteins as well as nucleic acids (see Figures S5 and S6).

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 Although SR-FTIR spectra of HeLa cells pre-incubated with Rf show no changes after PDT

treatment, MTT and NRU assays evidenced lower viability of irradiated cells. The MTT test also
Accepted Article
indicated a decreased viability when the nanoparticles were added along with Rf to the incubation

medium. However, we should consider that the MTT assays reflects the mitochondrial activity, NRU

the lysosomal activity, whereas SR-FTIR microspectroscopy analyzes global structural changes of the

biological molecules in the cells nuclei. Under the conditions of our experiments, these latter

alterations were in fact detected when TMPyP was used as the sensitizer, but not with Rf. This could

be related to the different localization of TMPyP and Rf. The porphyrin accumulates in the cell

nucleus (31), whereas endocytic compartments are involved in the uptake and intracellular

trafficking of Rf (41).

DISCUSSION

In this paper we proved that incubation of HeLa cells with normal cell medium containing

additionally Rf and Pec@AgNP lead to the incorporation of both the dye and the nanomaterial. We

have previously demonstrated that a 1:1 complex (Rf-Pec@AgNP) between Rf and Pec@AgNP is

formed (12). While it is not possible for us to determine directly, i.e., by spectroscopic means,

whether and to which extent the complex exists, when incorporated into the cells, we have here

given evidence for the synergistic photodamage effect by the nanoparticles and Rf (see Figures 3 and

4). This is interpreted as a consequence of the presence of Rf and Pec@AgNP in the same

intracellular microenvironment. According to a previous publication of our lab, the enhancement of

the amount of ROS (O2(1Δg) and O2.-) generated by Rf in the presence of Pec@AgNP is a consequence

of the decay of the excited state of the complex Rf-Pec@AgNP*, which feeds the triplet state of Rf

(3Rf*) (12) (Scheme 1). Thus, we here propose that the uptake of the complex is the responsible for

the increased photodamage effect observed when Pec@AgNP and Rf are present (see MTT in Figure

3 and Annexin V/PI in Figure 4).

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 In summary, our results clearly show the beneficial role of Pec@AgNP on the photodynamic activity

of Rf in mammalian cells. Given the nature of Rf, which is a natural vitamin able to cross biological
Accepted Article
membranes, the phototreatment proposed here looks promising for many other biological

applications.

ACKNOWLEDGEMENTS: This manuscript is part of a collection of articles to honor the

work of our colleague and friend Norman A. García (Andi), who actively contributed in the past 40

years to the development of basic and applied photochemistry. We acknowledge SOLEIL for

provision of synchrotron radiation facilities and we would like to thank Christophe Sandt for

ss s us g “SMIS” b .T sw k sb supp d by g s PI T 2012-1817 and

PICT 2016-0974 from ANPCyT, Argentina, and by Proposal 20160848 on SMIS beamline from SOLEIL.

G.N. .’s w k the lab of T.G. was financed by a grant from the Federal Ministry of Education and

Research (OptoSys, FKZ 031A16) of the Federal Republic of Germany. M.B.R.A. thanks CONICET for a

graduate studentship. G.N.B., J.C.A., and F.S.G.E. are permanent research staff of CONICET,

Argentina. D.O.M. is permanent research staff of CIC, Buenos Aires, Argentina.

SUPPORTING INFORMATION

Additional Supporting Information can be found in the online version of this article:

Figure S1. Emission spectrum of the 8 RPR-3500 A lamps with emission centered at 350 nm [S1].

Figure S2. Oxygen uptake experiments with samples containing 54 µM Rf and 10 mM FFA:

Without Nanoparticles (brown dots), with 0.5 µM of Ag (orange dots) and with 1 µM of Ag (green

dots).

Figure S3. Histograms showing the results of the MTT (green) and NRU (orange) assays on non-

irradiated HeLa cells treated with different concentrations of Ag (A) or Rf (B) in the incubation

medium. Values of formazan and neutral red absorbance were normalized against average values

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 obtained from no irradiated cells to which neither Rf nor Pec@AgNP had not been added (control).

Error bars refer to one standard deviation; in each case, the number of samples examined was at least
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4 and as large as 8. No statistically significant difference between any number and the number

obtained from the control was observed. (one-way ANOVA with Fisher’s posthoc test; p < 0.05).

Figure S4. PCA scores: PC2 vs. PC1 (upper left panel), PC3 vs. PC1 (lower left panel), scree plot

(upper right panel) and sample mean of second order derivative spectra (lower right panel) in the

region of the Amide I and Amide II bands (1480–1720 cm−1) of the following samples: non-irradiated

(blue) and irradiated (red) HeLa cells without the addition of Rf or NPs in the incubation medium

(controls); irradiated HeLa cells pre-incubated with 50 µM Rf (magenta), irradiated HeLa cells pre-

incubated with 1 µM M Ag (black), irradiated HeLa cells pre-incubated with 50 µM Rf and 1 µM Ag

(cyan), and irradiated HeLa cells pre-incubated with 10 µM TMyP for comparison (green). Vertical

lines show the wavenumbers expected for the peaks of α-helix (1654 cm−1), β-sheet (1632 cm−1), and

unordered random coil (1641 cm−1).

Figure S5. PCA scores: PC2 vs. PC1 (upper left panel), PC3 vs. PC1 (lower left panel), scree plot

(upper right panel) and sample mean of zero order spectra (lower right panel) in the region of the

Amide I and Amide II bands (1480–1720 cm−1) of the following samples: non-irradiated (blue) and

irradiated (red) HeLa cells without the addition of Rf or NPs in the incubation medium (controls);

irradiated HeLa cells pre-incubated with 50 µM Rf (magenta), irradiated HeLa cells pre-incubated

with 1 µM M Ag (black), irradiated HeLa cells pre-incubated with 50 µM Rf and 1 µM Ag (cyan),

and irradiated HeLa cells pre-incubated with 10 µM TMyP for comparison (green). Vertical lines

show the wavenumbers expected for the peaks of α-helix (1654 cm−1), β-sheet (1632 cm−1), and

unordered random coil (1641 cm−1).

Figure S6. PCA scores: PC2 vs. PC1 (upper left panel), PC3 vs. PC1 (lower left panel), scree plot

(upper right panel) and sample mean of second order derivative spectra (lower right panel) in the

region of the Amide I and Amide II bands (1480–1720 cm−1) of the following samples: non-irradiated

(blue) and irradiated (red) HeLa cells without the addition of Rf or NPs in the incubation medium

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 (controls); irradiated HeLa cells pre-incubated with 50 µM Rf (magenta), irradiated HeLa cells pre-

incubated with 1 µM M Ag (black), irradiated HeLa cells pre-incubated with 50 µM Rf and 1 µM Ag

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(cyan), and irradiated HeLa cells pre-incubated with 10 µM TMyP for comparison (green). Vertical

lines show the wavenumbers expected for the peaks of α-helix (1654 cm−1), β-sheet (1632 cm−1), and

unordered random coil (1641 cm−1).

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FIGURE CAPTIONS

Figure 1. TEM bright field image of Pec@AgNP and the size distribution histogram of 956 particles.

Figure 2. Emission spectra (λexc = 488 nm) of Rf in OPTIMEM medium before and after

incorporation into HeLa cells, as indicated. The emission of the control media without Rf before and

after incubation is also shown.

Figure 3. Histograms showing the results of the MTT (green) and NRU (orange) assays on

irradiated (A, B, C) and non-irradiated (D) HeLa cells. Values of formazan and neutral red absorbance

were normalized against average values obtained from the control (irradiated cells to which neither

Rf nor Pec@AgNP had not been added). The data shown were obtained from experiments in which

Rf (A), Pec@AgNP (B), and Rf + Pec@AgNP (C, D) were present in the incubation medium. Error bars

refer to one standard deviation; in each case, the number of samples examined was at least 4 and as

large as 8. The asterisk indicates that there is a statistically significant difference between this

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 particular number and the number obtained from the control. The double asterisk means a

statistically significant difference from the irradiated sample with Rf (one-w y NOV w Fs ’s
Accepted Article
post hoc test; p < 0.05).

Figure 4. Fluorescence images taken after 405 nm (142 µW) irradiation for 5 min. The excitation

wavelength was 561 nm (40 µW) and the maximum emission wavelength was 620 nm for PI test. For

the Annexin V-FITC (AnnV) fluorescence the excitation was at 488nm (21 µW), and the maximum

emission wavelength was 530 nm. (a) Control (b) 50 µM of Rf (c) 50 µM of Rf and 1 µM of Ag.

Figure 5. Figure 5: PCA scores: PC2 vs. PC1 (a), PC3 vs. PC1 (c), scree plot (b) and sample mean of

zero order spectra (d) in the region of the Amide I and Amide II bands (1480–1720 cm−1) of the

following samples: non-irradiated (blue) and irradiated (red) HeLa cells without the addition of Rf or

Pec@AgNP in the incubation medium (controls); irradiated HeLa cells pre-incubated with 50 µM Rf

(magenta), irradiated HeLa cells pre-incubated with 1 µM Ag (black), irradiated HeLa cells pre-

incubated with 50 µM Rf and 1 µM Ag (cyan), and irradiated HeLa cells pre-incubated with 10 µM

TMyP f p s (g ). V ss w w v u b s xp df p ks f α-helix

(1654 cm−1), β-sheet (1632 cm−1), and unordered random coil (1641 cm−1) (26,40).

Scheme 1. Effect of Pec@Ag on the generation of 3Rf*. 1Rf* is the singlet excited state of Rf.

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Accepted Article

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Accepted Article

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Accepted Article

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