Hypericin - apomyoglobin an enhanced photosensitizer complex for

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Hypericin - apomyoglobin an enhanced photosensitizer complex for the treatment of tumor cells Paolo Bianchini, Marco Cozzolino, Michele Oneto, Luca Pesce, Francesca Pennacchietti, Massimiliano Tognolini, Carmine Giorgio, Santi Nonell, Luigi Cavanna, Pietro Delcanale, Stefania Abbruzzetti, Alberto Diaspro, and Cristiano Viappiani Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00222 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

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Biomacromolecules

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Hypericin - apomyoglobin an enhanced

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photosensitizer complex for the treatment of tumor

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cells

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Paolo Bianchini*1,2, Marco Cozzolino1,3, Michele Oneto1,2, Luca Pesce1,3, Francesca

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Pennacchietti1, Massimiliano Tognolini4, Carmine Giorgio4, Santi Nonell5, Luigi Cavanna6,

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Pietro Delcanale7,§, Stefania Abbruzzetti7, Alberto Diaspro1,2,3, Cristiano Viappiani*7

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1Nanoscopy,

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Nikon Imaging Center, Istituto Italiano di Tecnologia, via Morego 30, Genoa 16163, Italy

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Department of Physics, University of Genoa, via Dodecaneso 33, Genoa 16146, Italy.

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Dipartimento di Scienze degli Alimenti e del Farmaco, Università di Parma, Parco area delle

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Scienze 27/A, 43124 Parma, Italy

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5Institut

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Piacenza, Italy

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7

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delle Scienze 7/A, 43124 Parma, Italy

Istituto Italiano di Tecnologia, via Morego 30, Genoa 16163, Italy

Quimic de Sarrià, Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain

Dipartimento di oncologia-ematologia. Azienda USL di Piacenza. Via Taverna, 49, 29121

Dipartimento di Scienze Matematiche, Fisiche e Informatiche, Università di Parma, Parco area

ACS Paragon Plus Environment

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Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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KEYWORDS

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photodynamic therapy, drug delivery, drug uptake kinetics, live cell fluorescence microscopy,

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STED-nanoscopy.

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ABSTRACT

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Bioavailability of photosensitizers for cancer photodynamic therapy is often hampered by their

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low solubility in water. Here we overcome this issue by using the water-soluble protein

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apomyoglobin (apoMb) as a carrier for the photosensitizer hypericin (Hyp). The Hyp-apoMb

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complex is quickly uptaken by HeLa and PC3 cells, at submicromolar concentrations.

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Fluorescence emission of Hyp-apoMb is exploited to localize the cellular distribution of the

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photosensitizer. The plasma membrane is rapidly and efficiently loaded and fluorescence is

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observed in the cytoplasm only at later times, and to a lesser extent. Comparison with cells

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loaded with Hyp alone demonstrates that the uptake of the photosensitizer without the protein

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carrier is a slower, less efficient process, that involves the whole cell structure without

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preferential accumulation at the plasma membrane. Cell viability assays demonstrate that the

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Hyp-apoMb exhibits superior performance over Hyp. Similar results were obtained using tumor

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spheroids as three-dimensional cell culture models.

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1. Introduction

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Photodynamic therapy (PDT) of tumors is a clinically approved therapeutic procedure that

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relies on the administration of a photosensitizing agent (PS), which is then irradiated with light

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of a suitable wavelength, corresponding to an absorption band of the PS. In the presence of

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oxygen, cytotoxic species are produced causing direct cell death, damage to the vasculature and

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inflammatory reactions 1. The naturally occurring PS Hypericin (Hyp) 2, 3 has been proposed in the treatment of cancer4-

7 8

6,

as an antiviral 7, 8, antibacterial 9-13, and antifungal agent 14. Hyp is brightly fluorescent in polar

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organic solvents

3, 15, 16,

and sensitizes singlet oxygen (1O2) with high yield

17-20.

As many other

10

photosensitizers, Hyp is insoluble in water and forms aggregates in aqueous solutions, which

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dramatically influence the photophysics of the compound, resulting in quenching of the excited

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states that lead to triplet state formation

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properties.

14

21,

consequently impairing the photosensitizing

Binding of Hyp to several proteins increase its solubility in physiological media

22-26,

and

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thanks to their high biocompatibility, proteins appear to be a promising delivery vehicle ensuring

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good bioavailability of the drug and preservation of its photodynamic action against target cells

17

12, 13, 27, 28.

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(apoMb) and forms a self-assembled, non-covalent complex (Hyp-apoMb, Figure 1a), endowed

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with good fluorescence quantum yield (F = 0.140.02, fluorescence excitation and emission

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spectra are reported in Figure 1b) and efficient singlet oxygen photosensitization (Δ =

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0.140.03)19. For comparison, the fluorescence and singlet oxygen quantum yields of Hyp in

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DMSO are F = 0.350.02 and Δ = 0.280.05, respectively19. The size of the nanostructure is

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the same as that of the monomeric carrier protein, characterized by a diffusion coefficient D =

In particular, Hyp binds with moderate affinity (Kd = 4.2±0.8 M) to apomyoglobin


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120±20 m2s-1, as determined by Fluorescence Correlation Spectroscopy19. Myoglobin is a water

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soluble protein with an isoelectric point of 7.23, which ensures low electrostatic interactions with

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cellular components at physiological pH.

4 5

These properties make Hyp-apoMb a promising theranostic agent and Hyp-apoMb was already exploited to deliver Hyp to bacterial cells for antimicrobial PDT applications 12, 19.

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Because 1O2 is a short-lived (s) species in aqueous solutions, photo-oxidative damage is

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confined within a few hundred nanometers from the site of its generation. Hence, accurate

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localization of the PS molecules as a function of time is important to assess where and when the

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photoinduced cellular damages are expected.

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In this work we show that complexation of Hyp with apoMb improves its efficacy as a

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photosensitizer in vitro on HeLa and PC3 tumor cells. Confocal spinning disk fluorescence

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imaging demonstrates that cells are loaded with the PS within a few minutes as judged from the

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intense fluorescence emission, which is initially mostly localized on the plasma membrane.

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Comparison with cells loaded with Hyp delivered from a concentrated DMSO solution

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demonstrates a much faster uptake when complexed to apoMb. Although Hyp-apoMb allows

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imaging with STED-nanoscopy

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microscope without inducing extensive cellular damage. Application of the Hyp-apoMb to tumor

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spheroids followed by illumination was able to substantially reduce the growth rate of the

19

structure.

19,

it proved impossible to monitor PS uptake using the STED

20 21


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2. Materials and Methods

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2.1 Hyp-apoMb preparation.

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Horse heart myoglobin was purchased from Sigma-Aldrich. Hypericin was obtained from HWI

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Analytik GmbH. ApoMb was prepared from myoglobin using standard biochemical procedures.

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The heme was removed from myoglobin by cold (-30°C) acid acetone extraction 29. The sample

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was washed with cold acetone and centrifuged several times, dried with pure nitrogen, and

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suspended in PBS buffer at pH 7.4. The suspension was then centrifuged, and the supernatant

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was spectroscopically checked to assess sample purity. The concentration of the apoMb stock

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was calculated from the absorption at 280 nm ( = 15,800 cm-1M-1) and residual heme

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concentration was determined from the absorption at 408 nm ( = 179,000 cm-1M-1)

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typical heme contamination in all batches was about 0.5% of the total protein content.

30.

The

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The complex between Hyp and apoMb was prepared by mixing a concentrated DMSO solution

13

of Hyp (prepared from stock solutions ~ 0.5 mM) with a PBS buffered solution containing

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apoMb at the proper concentration (prepared from stock solutions ~ 200 M), so that the final

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ratio between Hyp and protein is 1:3, which warrants complete binding of Hyp to the protein.

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Self-assembly of the complex occurs immediately 12. Concentrations of Hyp-apoMb are given as

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Hyp concentrations, the corresponding protein concentrations being threefold higher. In the

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Results and Discussion sections concentrations refer to the final concentration in the cell medium

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or the imaging buffer. Solutions used in cell viability assays and in imaging experiments

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contained 0.5 % DMSO. The  potential of apoMb (-8±4) mV and Hyp-apoMb (-9±5) mV in

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water were determined with a Malvern Zetasizer Nano Z.

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2.2 Cell cultures.


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All culture media and supplements were purchased from Euroclone. PC3 cells and HeLa cells

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were obtained from ECACC. Dimethyl thiazolyl diphenyl tetrazolium (MTT) was purchased

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from Applichem.

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PC3 human prostate adenocarcinoma cells were grown in Ham F12, supplemented with 5%

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FBS and 1% penicillin-streptomycin solution. HeLa human cervix adenocarcinoma cells were

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grown in EMEM supplemented with 1% antibiotic solution, 1% glutamine, 1% non-essential

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amino acid solution and 10% FBS. Both the cell lines were maintained in a humidified

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atmosphere of 95% air, 5% CO2 at 37°C.

9 10

2.3 Viability assay.

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MTT assay was used to evaluate PC3 and HeLa viability. Cells were seeded in 96-well cell

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culture plates at the density of 3×105 cells/ml and the day after, they were starved and treated

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with different concentrations of Hyp (from a concentrated DMSO solution) or Hyp-apoMb (from

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a concentrated PBS solution of Hyp-apoMb), or control (0.5 % DMSO for Hyp), avoiding

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exposure to ambient light. Thirty minutes after the treatment, cells were exposed to light for

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different times and then maintained for 24 hours in a humidified atmosphere with 5% CO2 at

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37°C, without light exposure. MTT was finally added at the concentration of 1 mg/ml and

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incubated for 2 hours. The resulting formazan crystals were solubilized with DMSO and the

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absorbance was measured at 550 nm using an ELISA plate reader (Sunrise, TECAN,

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Switzerland). Control solution (0.5 % DMSO for Hyp) did not lead to any phototoxicity.

21 22

2.4 Irradiation of cultured cells.


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Biomacromolecules

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Irradiation of cell cultures was conducted using a RGB LED light source (LED par 64 short,

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Show Tec (Highlite International B.V., Kerkrade, The Netherlands), equipped with 19, 3W RGB

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LEDs), whose output was selected to emit at 460 nm (30 nm FWHM, Figure 1b). The blue

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output (shown as the blue solid line in Figure 1b) has a substantial overlap with the fluorescence

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excitation spectrum of Hyp-apoMb.

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The irradiance at the surface of a 96 well plate was homogeneous and corresponds to 30.7

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mW/cm2. Exposure of cultured cells was performed for 0, 2, 5, and 15 minutes which correspond

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to light fluences of 0, 3.6, 9.2, and 27.6 J/cm2, respectively.

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2.5 STED nanoscopy.

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Cells were washed several times with PBS buffer. Finally, a total volume of 200 l of PBS

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buffer was put in each well. For both cell types, concentrated Hyp in DMSO or Hyp-apoMb in

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PBS buffer (in a ratio Hyp-apoMb 1:3) were added to the culture before imaging at a given

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delay. Final concentrations in the bathing solutions within the wells are stated in the figure

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captions. Stimulated emission depletion (STED) nanoscopy was performed using a custom made

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setup equipped with a supercontinuum pulsed laser source (ALP-710-745-SC, Fianium LTD,

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Southampton, UK) 31. The excitation wavelength was selected by means of an AOTF, while the

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STED wavelength was predefined by the laser outputs. The laser had a repetition frequency of

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20MHz and a pulse width of about 100 ps. In all the experiments we used 566 nm for excitation

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and 715 nm for STED. The doughnut shape of the STED beam was realized by a vortex phase

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plate (RPC Photonics inc., Rochester, NY, USA). The beams were scanned on the sample by

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galvanometer mirrors (Till-photonics, FEI Munich GmbH, Germany), focused by a HCX PL

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APO CS 100x 1.4NA oil (Leica Microsystems, Mannheim, Germany) objective and fluorescence


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was collected by an avalanche photodiode (SPCM-AQRH-13-FC, Excelitas Technologies,

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Vaudreuil-Dorion, Quebec, Canada) in the spectral window 670-640nm. The STED efficiency

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had been previously assessed by measuring the fluorescence depletion curve 19.

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2.6 Spinning disk microscopy.

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Preparation of cultured cells and injection of Hyp or Hyp-apoMb were described in 2.5.

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Spinning disk confocal microscopy utilizes multiple pinholes to project a series of parallel

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excitation light beams onto the specimen in a multiplexed pattern that is subsequently detected

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after fluorescence emission passed through the same pinholes. The main features of this

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technique is the high speed imaging of living cells, lower photobleaching and phototoxicity, due

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multiple excitation that needs only a low level of laser power at the specimen to fully excite

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fluorescence. The microscope is composed by a confocal unit Nikon TiE inverted Microscope

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equipped with an Okolab incubation system, and four excitation lasers (405nm, 488nm, 561nm,

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640nm). The system is equipped with a Yokogawa CSU-X1 spinning disk containing about

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20,000 pinholes and a second spinning disk containing the same number of micro-lenses to focus

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the excitation laser light. The fluorescence light is collected by an Andor EMCCD camera Ixon

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897. Therefore, we imaged the specimens sequentially exciting at 561 nm and 640 nm, and

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detecting in the spectral windows 575 - 625 nm and 670 - 740 nm, respectively.

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2.7 Uptake analysis.

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To study Hyp accumulation we have marked cells with “CellMask™ Deep Red Plasma

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Membrane Stain”, characterized by excitation and emission wavelengths (λex = 649 nm, λem =

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666 nm) at longer wavelengths than Hyp (λex = 550 nm, λem = 600 nm), which makes it possible


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Biomacromolecules

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to separate the two signals. This allowed us to identify the cell plasma membrane and compare

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the mutual distributions of the dyes. With the use of the software Fiji

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extrapolate fluorescence profiles of 10 pixel thickness for each cell on each frame for both

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fluorescence channels (in the red for Hyp and in the deep red for CellMask). These profiles were

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analyzed with a Matlab custom written code. First, it was used the CellMask fluorescence profile

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to identify the external membrane position and use it as a reference point. Using this information,

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it was possible to obtain fluorescence emission intensity for Hyp on the plasma membrane and

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the cytoplasm for each frame. Two different profiles were taken for each cell. The analysis was

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repeated on several cells and from average values the accumulation curves were obtained. All

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measurements were taken both on HeLa and on PC3 cells at the temperature of 37 °C R.H. 95%

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and flow 0.6 l/min.

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it was possible to

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In these experiments, we injected the solutions containing either Hyp-apoMb or Hyp alone and

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started collecting images immediately after. There was no pre-incubation in order to follow

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uptake from the very beginning.

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2.8 HeLa cells transfection and tumor spheroids preparation.

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A stable cell-line expressing miRFP703

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fused to histone H2B obtained from HeLa cell

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culture was selected with 0.7 mg/ml of antibiotic G418. The positive cells were sorted by BD

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FACSAria III (BD Biosciences San Jose, CA, USA) using 633 nm laser line and 780/60 BP

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emission filter. Such a stable cell-line was used to obtain multicellular tumor spheroids using

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suitable microplates which use non-adhesive surfaces to maintain the cells in an unattached state


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(Corning) (Figure 5). The number of cells seeded was around 103 – 2x103 and they were left in

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the incubator at 37 °C and 5% CO2 for at least 48 h before imaging.

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3. Results and Discussion

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3.1 The construct Hyp-apoMb can act faster and has higher efficacy than Hyp.

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Cell viability of PC3 and HeLa cells in the presence of Hyp or Hyp-apoMb was determined as

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a function of PS concentration and light fluence (i.e. illumination time). Clear concentration- and

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light fluence- dependent cytotoxic effects can be observed both for HeLa and PC3 cells when

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treated with either Hyp or Hyp-apoMb (Figure 1, panels c and d). At high Hyp or Hyp-apoMb

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concentrations, some dark toxicity is observed (Figure 1, panels c and d).

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By comparing the light-fluence and the concentration dependent data for Hyp and Hyp-apoMb

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it is possible to appreciate the higher efficiency of the latter compound in reducing cell viability.

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The concept is made quantitative by estimating the EC50, the concentration which causes 50%

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decrease in cell viability, see representative plots in Figure 1 e. EC50 of Hyp decreased from

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1330 nM to 130 nM (10-fold) when we increased treatment from 2 to 15 minutes on PC3 cells.

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We observed a similar effect in HeLa cells where EC50 decreased from 1520 nM to 210 nM (7-

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fold). Complexation of Hyp with apoMb significantly increased the phototoxic effect of Hyp,

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reducing the EC50 for 2-minute exposure from 1330 to 190 nM (7-fold) and from 1520 to 230

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nM (7-fold) for PC3 and HeLa cells, respectively. At longer exposure times, the difference

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between Hyp and Hyp-apoMb becomes progressively smaller. Using Hyp-apoMb, EC50 for 15-

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minute exposure is reduced from 130 to 50 nM (2.5-fold) and from 210 to 63 nM (3-fold) for

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PC3 and HeLa cells, respectively Thus, under the employed conditions, Hyp-apoMb outperforms

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Hyp for in vitro PDT at low PS concentrations and short irradiation times.

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Figure 1. Cell viability and fluence dependency for PC3 and HeLa cells incubated with Hyp or

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Hyp-apoMb and exposed to blue light.

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(a) Model of the three dimensional structure of Hyp-apoMb (Autodock)

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excitation (green) and emission (orange) overlaid on the emission spectrum from the blue LED

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lamp (blue line) used in the photoinactivation studies.

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Fluence- and time-response graphs of PC3 (c) and HeLa (d) cells viability treated with increasing

7

concentrations of Hyp or Hyp-apoMb and exposed to increasing irradiation times. Data are the

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means of at least three independent experiments. The blue light output (460 nm, 30 nm FWHM,

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30.7 mW/cm2) from a LED lamp was used.

12.

(b) Fluorescence

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(e) Fluence-dependent PDT effects of Hyp (red) and Hyp-apoMb (black) on viability of PC3

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(left) and HeLa (right) cells (range 0.03-3 M at exposure times of 2 minutes (filled symbols)

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and 15 minutes (open symbols), corresponding to light fluences of 3.6 and 27.6 J/cm2,

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respectively). Data are the means of at least three independent experiments ± st. err.

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The higher efficiency of Hyp-apoMb over Hyp in inhibiting cell viability demonstrated in

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Figure 1 may be associated with an improved (in rate and extent) binding of the PS with the

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investigated tumor cells, thanks to the presence of the protein scaffold or to a different sub-

18

cellular localization, as derived from a different mechanism of association and/or internalization.

19

A similar effect was observed for Hyp associated with Human Serum Albumin (HSA), and it

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was concluded that besides acting as a passive Hyp carrier, HSA may also actively contribute to

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the selective localization of the compound 26. It was found that when Hyp is solubilized by HSA,

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an excellent differentiation in distribution of Hyp in normal urothelial spheroids and malignant


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spheroids is observed, a fact that suggests the capability of HSA of leading to specific

2

localization of Hyp in non-muscle-invasive bladder tumors. Importantly, PDT results showed

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that PDT effect on tumor spheroids and the selective character of the treatment were significantly

4

increased by the presence of the protein. However, to the best of our knowledge, no specific

5

interactions of the apoMb scaffold with cellular components is reported in the literature.

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3.2 Cellular distribution of Hyp-apoMb by STED imaging and consequent photodamage.

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Intracellular distribution of the PS may play a key role in the different degrees of phototoxicity

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of Hyp and Hyp-apoMb. Fluorescence microscopy is one of the methods of choice to investigate

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the localization and diffusion of molecules or proteins in a living system 34, and it is effective in

11

the characterization of PS uptake by cultured cells 35, 36.

12

The development of super-resolution microscopy methods

37-40

in principle opens new

13

possibilities for identifying the PS cellular distribution of fluorescent species with high spatial

14

resolution. We recently demonstrated that the inherent bright fluorescence emission by Hyp can

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be exploited to collect STED images of bacterial cells loaded with Hyp-apoMb 19, 27, 31. We thus

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applied STED nanoscopy with the aim of following the uptake of the PS and distinguishing

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possible different mechanisms for Hyp and Hyp-apoMb.

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STED images of HeLa and PC3 cells incubated with Hyp-apoMb (0.5 µM Hyp, 1.5 µM

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apoMb) for 30 minutes are reported in Figure 2. Panels a and b (at time 0 min) evidence

20

accumulation of the PS on the plasma membrane as well as within the cytoplasm, whereas the

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cell nucleus is devoid of fluorescence emission. This behaviour is very similar in both types of

22

cells. The intense fluorescence emission indicates the presence of monomeric, well solubilized

23

PS molecules, which represents a major advantage over other nanostructured PS systems for


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Biomacromolecules

41.

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which a clear tendency to aggregate was sometimes evidenced

Since only fluorescent

2

molecules are photoactive, the photo-oxidative damages are expected to reflect the fluorescence

3

distribution.

4 5

Figure 2. Photoinduced damage to HeLa and PC3 cells incubated with Hyp-apoMb.

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Effect of illumination by STED imaging on HeLa (Panels a) and PC3 cells (Panels b) incubated

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for 30 minutes with Hyp-apoMb (0.5 µM Hyp, 1.5 µM apoMb) after 0, 5 and 10 minutes of


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irradiation. STED images collected under excitation at 566 nm and detection at 605\70 nm. The

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STED beam was at 715 nm, power 8 mW and dwell time 0.05 ms. Panels c show close-up

3

images on swelling and vesicle formation as a consequence of the photoinduced damage on the

4

plasma membrane on HeLa cells. Panels c, left and center. The plasma membrane swells towards

5

the extracellular medium at those points marked by a white arrow. Spherical vesicles are

6

detaching from the plasma membrane in the area surrounded by the dashed line. Panels c, right.

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Intense vesicle formation as a consequence of the photoinduced damage on the plasma

8

membrane of HeLa cells. Scale bars, 10 µm.

9 10

Exposure of the cells to the intense excitation laser beam in the STED microscope rapidly

11

leads to extensive photoinduced cell damage which first appears at the level of the plasma

12

membrane just a few minutes after illumination, as shown in central panels of Figure 2 (a and b)

13

when compared to non-irradiated HeLa and PC3 cells (Figure 2 a and b, left panels). At first, the

14

plasma membrane shows localized swelling areas that increase in size and result in vesicles

15

formation, which eventually detach from the cell. Since membrane permeability is increased, the

16

cell swells and after just a few minutes, an intense fluorescence emission also appears on all

17

internal membranes (Figure 2 a and b, center and right panels). In particular, Figure 2, panels c,

18

offers close-up views on areas of extensive swelling where the photo-oxidative damage

19

concentrates. The left and center images of panel c show regions where vesicles are forming (at

20

points indicated by the arrows) and detach (within the dashed line box). The right image of

21

panels c reports an area of intense formation of vesicles, whose diameter shows large variations.

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In the same panel, it is evident the loss of integrity of several regions of the plasma membrane.

23

Time lapse movies, available as Supporting Information (Supporting Information movie 1 and


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movie 2), show damages induced on PC3 and HeLa cells while collecting STED images. Much

2

faster and extensive damage is observed when higher concentrations of Hyp-apoMb are used

3

(data not shown).

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3.3 Spinning disk imaging reveals that photosensitizer uptake is remarkably different for Hyp and Hyp-apoMb.

7

Although the high-resolution images obtained with STED nanoscopy give a dramatic view on

8

the details of the photo-oxidative damage inflicted to the plasma membrane and other internal

9

membranes under intense illumination, they appear unsuitable to follow the cell uptake of the PS

10

under conditions close to the ones normally met in PDT applications. On one hand, the rate of

11

photo-oxidation induced by the intense laser beam (fluence rate ∼10MW/cm2 in the focal plane)

12

by far exceeds the rate normally encountered in the usual PDT experiments on cultured cells

13

where the light fluence rate is on the order of 10-30 mW/cm2. On the other hand, the

14

photoxidative damage is convoluted with the progressive and concomitant change in the

15

transport properties of the system and results in a greatly accelerated PS uptake.

16

We therefore sacrificed spatial resolution in favour of a less invasive imaging technique.

17

Spinning disk confocal microscopy was chosen because, while keeping sectioning capability and

18

a good resolution, this method allowed substantial reduction in the frame acquisition time (one

19

frame in 0.2 s every minute for the first 11 minutes and one frame in 0.2 s each 5 minutes for 40

20

minutes ), the number of averages (only 2 to optimize the signal-to-noise ratio) and laser power

21

(132 W). Additionally, we used low PS concentrations (50nM), but still high enough to observe

22

a detectable action against cells. Overall, this decreased the photo-oxidative damage during

23

image acquisition and allowed us to follow the uptake of the PS by cells over a ~50-minute time


17


Page 18 of 37

1

window, without any interfering photo-oxidative damage. We show examples of Hyp and Hyp-

2

apoMb uptake time sequences by HeLa cells in Figure 3 panels a and b, respectively. Simple

3

visual inspection of the images allows to appreciate a remarkable difference in the PS loading

4

mechanism. In the case of Hyp administered from a concentrated DMSO solution, the

5

fluorescence grows homogeneously in the whole cell structure. On the other hand, when Hyp-

6

apoMb is delivered to the cell culture, fluorescence appears to be initially concentrated mostly at

7

the plasma membrane, where the PS concentration appears higher than in the cytoplasm at all

8

times. Similar results were obtained on PC3 cells.

9 10

Figure 3. Uptake dynamics of Hyp and Hyp-apoMb.


18

Page 19 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

Comparison between the accumulation of Hyp (50 nM) in HeLa cells at selected delays when the

2

PS is delivered bound to apoMb (Panels a) and when delivered without a carrier (Panels b).

3

Experiments on PC3 cells gave similar results. Images were collected with a spinning disk

4

microscope with excitation at 561 nm (power 132 W). We acquired one image (0.2 s frame

5

acquisition time) every minute for the first 10 minutes and one image (0.2 s frame acquisition

6

time) every 5 minutes for the last 40 minutes. Scale bar 10μm (Panels a and b).

7

Panels c-f. Fluorescence intensity for the plasma membrane and for the cytoplasm calculated

8

from images collected at increasing delay after Hyp or Hyp-apoMb was added to the solution.

9

Plots refer to HeLa cells (c, d) and PC3 cells (e, f) treated with Hyp (c, e) and with Hyp-apoMb

10

(d, f). Intensity values are calculated for several cells and the error bars represent cell-to-cell

11

variability. The intensity curves are normalized to the maximum fluorescence value observed for

12

the internal part of the cell.

13 14

In order to quantitatively study the variations in time of the fluorescence intensity on the

15

plasma membrane after injection of Hyp or Hyp-apoMb to the buffer where cells are held, we

16

labelled it by “CellMask™ Deep Red Plasma Membrane Stains”. Such a stain allows

17

maintaining a reference for the position of the membranes while preventing excessive excitation

18

of Hyp (Supporting Information Figure 1). Using this reference signal, it was possible to

19

determine the fluorescence emission intensity at selected positions (Supporting Information

20

Figure 2). We then plotted the fluorescence intensity at the plasma membrane and in the interior

21

of the cell (cytoplasm and inner membranes) as a function of time (Figure 3 panels c-f).

22

The trends of the Hyp uptake when delivered in complex with apoMb or without a carrier

23

show a significant difference. In particular, it is evident that the fluorescence rise at the plasma


19


Page 20 of 37

1

membrane (red symbols in Figure 3 panels d and f) is much faster when cells are treated with

2

Hyp-apoMb. The time scale of the concentration increase at the plasma membrane is about 5-10

3

minutes from administration. Moreover, the presence of the protein carrier leads to higher

4

concentrations of the PS on the plasma membrane than on the inner membranes. This represents

5

an advantage when it is important to induce the photodynamic action in short times after the drug

6

has been administered.

7

Since apoMb is a water soluble protein with no known specific affinity for plasma membrane

8

components, it remains to be understood whether the higher PS uptake involves unreported

9

interaction with the cell structure. In order to further comprehend the action of the complex Hyp-

10

apoMb, we covalently labelled apoMb with Fluorescein-5-Isothiocyanate (FITC). Such a

11

labelling allowed us to study independently the fate of the carrier apoMb and the payload Hyp

12

during the uptake process in HeLa and PC3 cells.

13

As can be appreciated in Figure 4 for PC3 cells, green fluorescence from FITC-labelled

14

ApoMb is appreciable only from the buffer medium and is not detectable either on the cell

15

membrane or inside the cell. In contrast, the red fluorescence emission from Hyp is observed

16

from the plasma membrane as well as from the internal membranes, and reflects the distribution

17

(after incubation and uptake) reported in Figures 2 and 3. Similar results were obtained for HeLa

18

cells. These findings suggest that apoMb mostly acts as a passive carrier that facilitates delivery

19

of Hyp to the membranes by preventing aggregation in the aqueous phase. The higher affinity of

20

the PS for the lipid phase leads to fast partitioning of Hyp, leaving the unloaded protein in

21

solution.

22

Thus, we envision the following mechanism. The water soluble protein carrier is very effective

23

in preventing Hyp aggregation, and when the Hyp-apoMb solution is injected into the buffer


20

Page 21 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

medium covering cells in the wells, the monomeric, apoMb-bound Hyp is rapidly and efficiently

2

transferred to the plasma membrane. This speeds up cell loading and allows to increase

3

bioavailability of the PS compound. This mechanism is not present when Hyp is added to the

4

solution without the protein carrier. In these conditions, right after injection to the cell-bathing

5

buffer, the PS is extensively aggregated and rescuing the aggregated PS molecules is a slower

6

and less efficient process. Thus, Hyp-apoMb remarkably increases prompt PS bioavailability.

7

8 9

Figure 4. Hyp-apoMb-FITC imaging. Spinning Disk Fluorescence Imaging of PC3 cells treated

10

with Hyp-apoMb (a) in the FITC channel (500-550 nm) shows the distribution of apoMb, in

11

comparison with (b) the red channel (575-605 nm) that shows Hyp distribution. (c) Overlay of

12

the two images [ApoMb] = 10 M, [Hyp] = 3 M. Incubation time was 1 hour. Scale bars are

13

10μm.

14

FITC channel is excited at 488nm, Exposure time 400ms, power 324W; Hypericin is excited

15

with laser at 561nm, exposure time 100ms, power 567W, for both channels the pixel size is

16

0.14m.

17


21


Page 22 of 37

1

3.4 Tumor spheroid treated with PS.

2

While we have demonstrated that uptake of the PS is faster and more efficient for Hyp-apoMb

3

than for Hyp in bi-dimensional cell cultures, it is not obvious that this advantage is preserved in

4

tumor tissues as well. Thus, we used tumor spheroids as a three-dimensional model system to

5

study the uptake of the PS. Spheroids are considered a good model for small solid tumors prior to

6

neovascularization

7

miRFP703

8

spheroid, as well. Since such a fluorescent protein has excitation and emission spectra in the far

9

red, it is particularly suitable for imaging thick samples and scattering objects as tumor

10

spheroids. In Figure 5, we show the effects of Hyp and Hyp-apoMb on HeLa spheroids. As

11

expected, in the control spheroids which are not exposed to the microscope light (Figure 5 a), we

12

could not see any evidence of morphological damage induced by phototoxicity, and the spheroid

13

grows normally. Although the size of Hyp-apoMb is significantly larger than that of Hyp, steric

14

effects do not impair penetration of the drug. Thus, uptake through the spheroid occurs both for

15

Hyp (Figure 5 b) and for Hyp-apoMb (Figure 5 d). However, as noted for cultured single-layer

16

cells, the concentration of the fluorescent species is higher when Hyp-apoMb is used (Figure 5

17

d), the increase in the case of spheroids being at least by one order of magnitude. After

18

irradiation, the cells in the outer layer die and in the short term (1 week), the growth of the

19

spheroids is completely blocked (right images in panels c, and e). A more quantitative estimate

20

of the efficiency of the phototoxicity on the spheroid is hampered by a few documented

21

difficulties. These include low oxygen content in the inner parts of the spheroid

22

distribution of the PS, which is found in higher concentration at the spheroid rim

23

accumulation at the spheroid rim was reported also for treatments of spheroids with hypocrellin-

33

42-45.

We transfected HeLa cells with the far red-emitting fluorescent protein

fused to histone H2B to visualize the cell nucleus and to monitor the size of the

46

and uneven 47-49.

Initial


22

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Biomacromolecules

1

loaded liposomes, followed by a more even distribution of the PS at longer times 48. This delayed

2

kinetics most likely derives from the high affinity of Hyp for liposomes that may transiently hold

3

the photosensitizers and prevent binding to the plasma membrane of tumor cells. Hyp-apoMb

4

potentially offers advantages in terms of delivery rate, since fluorescence is detected also in the

5

center of the spheroid (although at lower concentration than at the periphery) after just one-hour

6

incubation time with Hyp-apoMb (Figure 5d). The moderate affinity of Hyp for apoMb and the

7

high affinity for lipid membranes mean that, when Hyp-apoMb is in the presence of tumor cells,

8

Hyp will be quickly released by the protein in favour of the lipid phase. a

b

c

d

e

9 10

Figure 5. Effects of Hyp and Hyp-apoMb on HeLa tumor spheroids upon light irradiation.

11

Spinning disk imaging of HeLa cells permanently transfected with miRFP703-H2B (shown in


23


Page 24 of 37

1

cyan-hot) (a - e) and treated with Hyp (1 μM) (b and c), and with Hyp-apoMb (1 μM) (d and e).

2

The samples in a, b, and c were incubated 1h with Hyp. The samples in d and e were incubated

3

1h with Hyp-apoMb. Panels a, c and e are time sequences at day 0, 1, and 6. Samples were

4

illuminated at day 0 for 5 minutes at 561 nm with power 3.37 mW. Each image is composed by

5

an inset with a 3x magnification of the squared area defined with a dashed line, and by an

6

orthogonal projection of middle plane, yz. Scale bar is 50 μm, voxel size is 1.6 × 1.6 × 1.4 μm.

7 8

4. Conclusions

9

PS shows a quick accumulation at the plasma membrane even at low concentrations (~10-7 M)

10

inducing extensive oxidative damages and leading to cell death. At longer incubation times, the

11

cellular distribution of the PS becomes similar to the one observed when Hyp is delivered from

12

concentrated solutions in organic solvents.

13

Recent reports demonstrated that activation to induce necrosis by compromising the plasma 50-52.

14

membrane has the benefits of fast cell death and shorter irradiation times

15

dealing with Hyp based PDT showed that when delivered at sub-micromolar concentrations,

16

similar to the ones reported in our time lapse experiments, after 16 h incubation the PS

17

dominantly associates with the endoplasmic reticulum membranes

18

by low (100 nM) Hyp concentrations, apoptosis was shown to be the main death pathway,

19

whereas at higher concentrations ( 1M) necrosis prevails due to the excessive photodamage 54.

20

The improvement observed in EC50 after just a few minutes of incubation of cells with Hyp—

21

apoMb may open new perspectives in PDT applications. Fast and efficient distribution of the PS

22

is observed also for tumor spheroids, an indication that the transport system maintains a good

23

performance also for arrangements of cells that mimick a three dimensional tissue. Taken

53.

Previous studies

For cells photosensitized


24

Page 25 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

together, the present data suggest that photodynamic treatment with the photosensitizing

2

structure Hyp-apoMb could offer remarkable advantages when irradiation after a short

3

incubation time is needed.

4 5


25


1

Page 26 of 37

ASSOCIATED CONTENT

2

The following files are available free of charge.

3

Confocal imaging of Hyp and Hyp-apoMb distribution in HeLa and PC3 cells labelled with

4

CellMask (PDF).

5

Time-lapse STED imaging of PC3 cells treated with Hyp-apoMb (SI_movie1_PC3.avi)

6

Time-lapse STED imaging of HeLa cells treated with Hyp-apoMb (SI_movie2_HeLa.avi).

7 8

AUTHOR INFORMATION

9

Corresponding Authors

10

Paolo Bianchini, Nanoscopy, Istituto Italiano di Tecnologia, via Morego 30, Genoa 16163, Italy,

11

email: [email protected]

12

Cristiano Viappiani Dipartimento di Scienze Matematiche, Fisiche e Informatiche, Università di

13

Parma, Parco area delle Scienze 7/A, 43124 Parma, Italy, email: [email protected]

14

Present Addresses

15

§Present

16

Science and Technology (BIST), Barcelona, Spain

17

Author Contributions

18

The manuscript was written through contributions of all authors. All authors have given approval

19

to the final version of the manuscript.

address: Institute for Bioengineering of Catalonia (IBEC), the Barcelona Institute of


26

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Biomacromolecules

1

Funding Sources

2

LC, CV and SA acknowledge support from Azienda USL di Piacenza, Italy, and Fondazione di

3

Piacenza e Vigevano.

4

ACKNOWLEDGMENT

5

We thank Vladislav V. Verkhusha, Department of Anatomy and Structural Biology, Albert

6

Einstein College of Medicine, Bronx, New York 10461, USA, for providing the plasmid of

7

miRFP703 fused to the histone H2B.

8


27


1

Page 28 of 37

Table of Contents Graphics

2 3 4


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1 2

Biomacromolecules

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Hypericin - apomyoglobin an enhanced photosensitizer complex for

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