Method to selectively permeabilize and/or fragmentize cells

Abstract

A method to selectively permeabilize and/or fragmentize cells. A structure comprising a material and particles able to absorb electromagnetic radiation and cells are brought at close distance from each other. Part of the particles embedded in the structure are exposed to the free surface of the structure. The structure and, in particular, the particles in the structure are irradiated with electromagnetic radiation to selectively permeabilize and/or fragmentize the cells. Furthermore, the disclosure relates to a structure for use in a photothermal process to selectively permeabilize and/or fragmentize cells.

Claims

1. An in vitro or ex vivo method to selectively permeabilize and/or fragmentize cells, the method comprising: providing a structure comprising a material and comprising particles able to absorb electromagnetic radiation, the structure defining a volume V and a free surface S, the free surface S having a free surface area A.sub.S, at least a portion of the particles being partially exposed to the free surface S, each of the particles partially exposed to the free surface S defining a particle-free surface P, the particle-free surface P has a particle-free surface area A.sub.P, the particles being embedded in the structure in such a way that the sum of the particle-free surface area A.sub.P of all particles ranges between 0.0001% and 50% of the free surface area A.sub.S; providing at least one cell; bringing the structure and the at least one cell at a distance d less than 100 ?m from each other, the distance d being the shortest distance between the at least one cell and the free surface S of the structure; and irradiating the structure with electromagnetic radiation.

2. The method according to claim 1, wherein at least part of the particles being partially exposed to the free surface S are protruding from the material.

3. The method according to claim 1, wherein the particles have sizes ranging from 1 nm to 40,000 nm.

4. The method according to claim 1, wherein the particles are arranged in clusters with a cluster comprising between 2 and 1,000 particles.

5. The method according to claim 1, wherein the at least one cell comprises a suspension of cells or a tissue of cells.

6. The method according to claim 1, wherein a density of particles being partially exposed to the free surface S of the structure ranges between 1 and 10 particles/100 ?m.sup.2.

7. The method according to claim 1, wherein the particles comprise particles selected from the group consisting of metal particles, metal oxide particles, carbon or carbon-based particles, particles comprising at least one light-absorbing compound, and particles loaded or functionalized with at least one light-absorbing compound.

8. The method according to claim 1, wherein the material comprises an inorganic material or an inorganic-based material, a ceramic material or a ceramic-based material, an organic material or an organic-based material, a hydrogel or a composite material comprising at least one of these materials.

9. The method according to claim 1, wherein irradiating the structure with electromagnetic radiation comprises irradiating using a pulsed radiation source having pulses with a pulse duration in a range of 1 fs to 1 ms and/or with a fluence per pulse ranging between 0.001 and 10 J/cm.sup.2.

10. The method according to claim 1, wherein the structure comprises a film defining a first surface S1 having a first free area surface A.sub.S1 and a second surface S2, opposite to the first surface S1, having a second free area surface A.sub.S2, wherein the particles embedded in the structure in such a way that the sum of the particle-free surface area A.sub.P of all particles partially exposed to the first surface S1 ranges between 0.0001% and 50% of the first free area surface A.sub.S1.

11. The method according to claim 1, wherein the structure comprises a porous structure comprising fibers, particulates, a combination of the fibers and the particulates or a foam, with the particles being embedded in the fibers, the particulates or the foam.

12. A photothermal process to selectively permeabilize and/or fragmentize cells, which photothermal process utilizes a structure, the structure comprising a material and comprising particles able to absorb electromagnetic radiation, the structure comprising a material and comprising particles able to absorb electromagnetic radiation, the structure defining a volume V and a free surface S, the free surface S having a free surface area A.sub.S, at least a portion of the particles being partially exposed to the free surface S, each of the particles partially exposed to the free surface S defining a particle-free surface P, the particle-free surface P has a particle-free surface area A.sub.P, the particles being embedded in the structure in such a way that the sum of the particle-free surface area A.sub.P of all particles ranges between 0.0001 and 50% of the free surface area A.sub.S, the photothermal process comprising: bringing the structure and the at least one cell at a distance d less than 100 ?m from each other, the distance d being the shortest distance between the at least one cell and the free surface area A.sub.s of the structure; and irradiating the structure with electromagnetic radiation to selectively permeabilize and/or fragmentize the at least one cell.

13. The photothermal process according to claim 12, wherein the photothermal process comprises a method of therapy in a subject to selectively permeabilize and/or fragmentize cells of the subject.

14. The photothermal process according to claim 13, wherein the method of therapy comprises nanosurgery.

15. The photothermal process according to claim 12, wherein the at least one cell comprises a suspension of cells or a tissue of cells and/or wherein the electromagnetic radiation comprises pulsed radiation generated by a laser.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The disclosure will be discussed in more detail below, with reference to the attached drawings, in which:

(2) FIG. 1A shows a schematic illustration of a structure to selectively permeabilize and/or fragmentize cells according to the disclosure;

(3) FIG. 1B shows the cross-section of the structure shown in FIG. 1A with a cell positioned at a distance d from the structure;

(4) FIGS. 2 and 3 schematically illustrate the method to selectively permeabilize and/or fragmentize cells according to the disclosure, whereby cells are grown on a film (cells on top of film) as shown in FIG. 2 and a film is applied on a cell layer (film on top of cells) as shown in FIG. 3;

(5) FIG. 4 illustrates a method whereby a structure comprising particles is applied on the cornea of a bovine eye before being irradiated with a pulsed laser;

(6) FIG. 5 shows the average size of a cluster of particles in a PLA film in function of the concentration of IONPs;

(7) FIG. 6 shows the cluster density (number of clusters of particles/100 ?m.sup.2) in a PLA film in function of the concentration of IONPs;

(8) FIG. 7 shows images obtained by dark field microscopy before and after irradiation of a PLA-IOC film (0.1% IONs, 2% PA) with a single laser pulse of respectively 0.3 J/cm.sup.2 and 1.6 J/cm.sup.2;

(9) FIG. 8 shows the number of clusters per Hela cell grown on PLA-IOC films having different concentrations of IONP;

(10) FIG. 9 shows the uptake of FD500, the cell viability and rMFI (relative mean fluorescence intensity) of Hela cells grown on PLA films with 0.1% IONPs;

(11) FIG. 10 shows images obtained by confocal microscopy showing the uptake of FD500 grown on PLA films with 0.1% IONPs irradiated with a single laser pulse of 0.3 J/cm.sup.2;

(12) FIG. 11 shows the cell viability of Hela cells grown on PLA-IOC films (without or with 0.1% IONPs) as a function of the laser fluence after a single irradiation;

(13) FIG. 12 shows the cell viability of Hela cells grown on PLA-IOC films (0.1% IONPs) irradiated with one or two laser pulses of 0.3 J/cm.sup.2;

(14) FIG. 13 shows the selective radiation of predefined area (circle with a diameter of 6.5 mm) of a PLA-IOC film (2% PLA, 0.1% IONPs) irradiated with a single pulse at different laser fluences (scale bar is 1000 ?m);

(15) FIG. 14 shows the percentage of retrieved cells after selective radiation as shown in FIG. 13, in function of different laser fluences;

(16) FIG. 15 shows the ratio of living cells (stained with calcein AM) to dead cells (stained with propidium iodide (PI)) after selective radiation as shown in FIG. 13 in function of different laser fluences;

(17) FIG. 16 shows selective cell killing upon local irradiation of a PLA-IOC film (2% PLA, 0.1% IONPs) (scale bar is 10 ?m);

(18) FIG. 17 shows the success rate of single cell ablation for cells grown on PLA-IOC film (2% PLA, 0.1% IONPs) irradiated with a single laser pulse (0.3-1.3 J/cm.sup.2). For each value of laser fluence, 30 single cell ablation experiments were done (n=30);

(19) FIG. 18 shows spatial selective laser-induced killing of Hela cells covered with a PLA-IOC film (2% PLA, 0.1% IONPs);

(20) FIG. 19 shows the result of illumination of Hela cells covered with a PLA-IOC (2% PLA, 0.1% IONPs) as shown in FIG. 18, illuminated with a pulsed laser following a pre-defined pattern (Ghent University logo), stained with calcein AM, PI and the merged picture. The scale bar is 1000 ?m;

(21) FIGS. 20 and 21 respectively show the ratio of living cells to dead cells and the total amount of cells in the treated area as shown in FIG. 18, following a consecutive number of laser pulses using a laser fluence of 1.6 J/cm.sup.2;

(22) FIG. 22 is a schematic representation of pore formation during cell killing if a PLA-IOC films is positioned on top of the cells (as shown in FIG. 18) (left) and the resulting uptake of FD500 by Hela cell (right) after irradiation (1.6 J/cm.sup.2; four consecutive times);

(23) FIG. 23 shows the ICP-MS analysis (iron content) on the Hela cells following four consecutive irradiations (1.6 J/cm.sup.2) of the PLA-IOC film; cells refer to untreated cells; IONPs and IONPs+laser refer to cells in the presence of free IONPs (1 g/L), respectively, without or with laser treatment; PLA-IOC and PLA-IOC+laser concern cells covered with a PLA-IOC film, respectively without and with laser treatment. Data are shown as mean?SD. Statistical significance: Student's t test, *indicates p<0.05, ** indicates p<0.01, ns indicates nonsignificant;

(24) FIG. 24 shows laser-induced killing of cells in bovine cornea covered with a PLA-IOC film (2% PLA, 0.1% IONPs). The corneas were excised and stained with propidium iodide. The whole cornea of the enucleated bovine eye was illuminated four times with a laser pulse of 1.6 J/cm.sup.2;

(25) FIG. 25 shows the number of stained cells (dead cells) resulting from the laser-induced killing of cells shown in FIG. 24, using a PLA-IOC film compared to control experiments (PLA film without IOCs);

(26) FIG. 26 shows laser-induced killing of cells in bovine cornea covered with a PLA-IOC film (2% PLA, 0.1% IONPs). Corneas were excised and immersed in FD500 solution. IOC film (2% PLA, 0.1% IONPs). The whole cornea of the enucleated bovine eye was illuminated four times with a laser pulse of 1.6 J/cm.sup.2;

(27) FIG. 27 shows the uptake of FD500 resulting from the laser-induced killing of cells shown in FIG. 26 using a PLA-IOC film compared to control experiments (PLA film without IOCs); and

(28) FIG. 28 shows corneal epithelium of bovine eyes stained with calcein, isolated and placed on top of a PLA-IOC film, followed by illumination of a single cell, four times at a fluence of 1.6 J/cm.sup.2 and 4.5 J/cm.sup.2.

DETAILED DESCRIPTION

(29) The disclosure will be described with respect to particular embodiments and with reference to certain drawings, but the disclosure is not limited thereto but only by the claims. The drawings are only schematic and are non-limiting. The size of some of the elements in the drawing may be exaggerated and not drawn on scale for illustrative purposes. The dimensions and the relative dimensions do not correspond to actual reductions to practice of the disclosure.

(30) When referring to the endpoints of a range, the endpoints values of the range are included.

(31) When describing the disclosure, the terms used are construed in accordance with the following definitions, unless indicated otherwise.

(32) The terms first, second, and the like, used in the description as well as in the claims, are used to distinguish between similar elements and not necessarily describe a sequence, either temporally, spatially, in ranking or in any other manner. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the disclosure described herein are capable of operation in other sequences than described or illustrated herein.

(33) The term and/or when listing two or more items, means that any one of the listed items can by employed by itself or that any combination of two or more of the listed items can be employed.

(34) The term area refers to the measurement of the size of a flat surface in a plane, i.e., the size of a two-dimensional object.

(35) The term surface area refers to the measurement of the size of the surface of a three-dimensional shaped object.

(36) The term cell refers to all types of biological cells, including eukaryotic cells an prokaryotic cells. The cell may refer to a human cell, an animal cell and a plant cell.

(37) The term fragmentize refers to any way to break, cut or otherwise separate something into fragments. Fragmentize cells refers to any way to break, cut or otherwise separate cells into fragments and includes killing of cells and ablation of cells.

(38) The terms increase the permeability of, permeabilize, permeabilizing and permeabilization refer to any way to alter the permeability of a cell, in particular, the permeability of a membrane or barrier of a cell, for example, the plasma membrane of a cell, at least partially or locally. After permeabilization, the membrane or barrier, for example, the plasma membrane of a cell is altered in such a way that it is more permeable for one or more types of compounds as, for example, molecules, macromolecules, particles or nanoparticles.

(39) The terms perforate, perforating or perforation refer to any way to provide a membrane or barrier, for example, the plasma membrane of a cell, with one or more openings, holes or pores. By perforating a membrane or barrier, for example, the plasma membrane of a cell, openings are created into the membrane or barrier, for example, the plasma membrane of a cell, allowing the transport of compounds, such as molecules, macromolecules, particles or nanoparticles across the membrane or barrier, for example, across the plasma membrane of a cell.

(40) For the purpose of this disclosure, the terms increase the permeability of, permeabilize, permeabilizing and permeabilization and the terms perforate, perforating and perforation are interchangeably used. Similarly, the terms opening, hole and pore may be used interchangeably.

(41) The term generation of a vapor bubble includes expansion of the vapor bubble, collapse of the vapor bubble or a combination of expansion and collapse of the vapor bubble and secondary effects that can be the result of the bubble expansion and collapse, such as pressure waves and flow of the surrounding medium.

(42) The term vapor bubble or bubble refers to vapor nanobubbles and vapor microbubbles. Preferably, the term vapor bubble or bubble refers to vapor bubbles having a diameter in the range of 10 nm to 100 ?m. Vapor bubbles comprise water vapor bubbles, although embodiments are not limited thereto.

EXPERIMENTAL RESULTS

Example 1

(43) An embodiment of a structure 10, according to the disclosure, comprising a film comprising polymer material 11 and particles 12 able to absorb electromagnetic radiation is shown in FIG. 1A. A portion of the particles is partially exposed to one surface, for example, the top surface 13 of the structure. The particles are embedded in such a way that the sum of the particle-free surface area A.sub.P of all particles ranges between 0.0001% and 50% of the free surface area A.sub.S, more preferably between 5% and 25% of the free surface area As or between 15% and 25% of the free surface area A.sub.S. More particularly, the particles are embedded in such a way that the sum of the particle-free surface area A.sub.P of all particles ranges between 0.0001% and 50% of the top free surface area A.sub.S1, more preferably between 5% and 25% of the free surface area As1 or between 15% and 25% of the free surface area A.sub.S1.

(44) FIG. 1B shows the structure of FIG. 1A with a cell 15 or a plurality of cells 15 at short distance d from the structure 10.

(45) The below described examples comprise polylactic acid (PLA) as material of the structure and comprise iron oxide particles as particles able to absorb electromagnetic radiation.

(46) It is clear that other materials and other particles can be considered as well. To evaluate whether vapor bubbles generated at the surface of such structure can permeabilize and/or fragmentize cells in contact or at short distance d from a cell or from a plurality of cells (FIG. 1B), two types of experiments were performed: cells were grown on top of the structure and the structure (more particularly, the particles in the structure) are irradiated with (pulsed) electromagnetic radiation (cells on top of the structure) (FIG. 2); a structure was applied on a cell layer and the structure (more particularly, the particles in the structure) is radiated with (pulsed) electromagnetic radiation (structure on top of the cells) (FIG. 3).

(47) Furthermore, a structure comprising particles was applied on the cornea of a bovine eye and irradiated with a pulsed laser (FIG. 4).

1. Materials and Methods

(48) 1.1 Materials

(49) Iron oxide (Fe.sub.3O.sub.4) nanoparticles (IONPs) with a size of 50-100 nm, polylactic acid (PLA) with a molecular weight (Mw) of 80 kDa, FITC-dextrans of 500 kDa and chloroform were all purchased from Sigma-Aldrich (Belgium). Calcein AM and propidium iodide (PI) were purchased from Fisher Scientific (USA). CELLTITER-GLO? was purchased from Promega (USA).

(50) 1.2 Preparation of PLA-IOC (Iron Oxide Cluster) Films

(51) PLA films, with or without IONPs, were prepared by a one-step spin coating method. The films were prepared on a square cover glass (22 mm?22 mm) that was then placed in the center of the spin coating device. Briefly, IONPs were dispersed in a PLA solution (2%, 4% and 8% (w/v) in chloroform); the concentration of IONPs in the PLA solution varied between 0.01%, 0.1%, and 0.2% (w/v). The IONPs-PLA dispersions were first sonicated for 1 minute with a tip sonicator (Branson digital sonifier, Danbury, USA) at an amplitude of 10%. Next, 0.5 mL of a dispersion was applied onto a cover glass that was then placed in the center of the spin coating device. The speed and time for spin coating was set at respectively 2000 rpm and 20 seconds. After spin coating, the films were placed overnight in an oven at 50? C. to allow evaporation of any residual organic solvent.

(52) To prepare fluorescent PLA-IOC films, PLA (2%, w/v) and Rhodamine B (0.01% w/v) (Sigma-Aldrich, Belgium) were dissolved in chloroform overnight. Then, iron oxide nanoparticles (0.1% w/v) were added to the PLA-Rhodamine B solution. Next PLA-Rhodamine B films were prepared as described in the paragraph above.

(53) 1.3 Surface Morphology of PLA-IOC Films

(54) The surface morphology of the PLA-IOC films was characterized by scanning electron microscopy (SEM, FEI Quanta 200F (Thermo Scientific)). The SEM images were acquired with an acceleration voltage of 20 kV. It should be noted that depending on the concentration of the IONPs, iron oxide clusters (IOCs) of different sizes could be observed.

(55) As the iron oxide particles are arranged in clusters of iron oxide particles (IOCs) the films are referred to as PLA-IOC films (instead of PLA-IONP films). The size and the distribution of the IOCs were calculated based on SEM images.

(56) 1.4 Laser-Induced Formation of Vapor Bubbles (VNBs) and VNB Threshold

(57) To evaluate whether PLA-IOC films have the ability to generate VNBs, they were irradiated with a pulsed laser (HE 355 LD laser, OPOTEK Inc.; 7 ns, 561 nm). VNBs can be easily visualized by dark-field microscopy as they scatter light during their lifetime. Dark-field images of the films (covered with a thin layer of water) were recorded before and during irradiation of the films with laser pulses with a varying fluence. The VNB threshold, which is the fluence of a single laser pulse at which 90% of the IOCs form a VNB, was determined by plotting the number of bubbles as a function of the laser fluence.

(58) 1.5 Cell Culture

(59) Hela cells (ATCC? CCL-2?) were cultured (at 37? C. and 5% CO.sub.2) in DMEM/F-12 (Gibco-Invitrogen) supplemented with 10% fetal bovine serum (FBS, Biowest), 2 mM glutamine and 100 U/mL penicillin/streptomycin (Gibco-Invitrogen). For splitting, cells were first washed with Dulbecco's Phosphate Buffered Saline (DPBS) and then detached using a trypsin-EDTA solution (Gibco-Invitrogen).

(60) For the cells on top experiments (FIG. 2) Hela cells were seeded on top of PLA-IOC films present at the bottom of wells in a 6-well plate; the Hela cell concentration was 1?10.sup.6 cells/mL; in each well 2 mL of cells was applied. After one day of incubation, the PLA-IOC films on which cells adhered were removed from their initial wells and placed into new wells (of a 6-well plate); this to remove the cells that were not in contact with the films but just adhered to plastic bottom of the wells.

(61) For the experiments where the PLA-IOC films were placed on top of the cells (structure on top of the cells) (FIG. 3), Hela cells were seeded in the wells of a 6-well plate at a concentration of 0.5?10.sup.6 cells/mL (2 mL). The following day, the Hela cells in the wells were covered with a PLA-IOC film. To maximize the contact between the cells and the film, 90% of the cell medium was removed before applying the film so that only a small layer of medium remained on top of the cells.

(62) 1.6 Cell Killing by PLA-IOC Films Exposed to Pulsed Laser Light

(63) Cells were seeded onto a PLA-IOC film or cells were covered with a PLA-IOC film as described above.

(64) The PLA-IOC films were then irradiated with a nanosecond pulsed laser (pulse duration <7 ns, 532 nm wavelength). A galvano scanner was used to allow a fast scanning of the laser beam so that each location essentially received a single laser pulse (or two in the overlapping region between neighboring spots); a single scanning of the whole surface of a PLA-IOC film in a well of a 6-well plate took around 2 minutes. In some cases, the films were scanned multiple times as indicated in the text.

(65) The extent of cell killing by PLA-IOC films exposed to light pulses was evaluated by measuring the metabolic activity of the cells using the CELLTITER-GLO? assay. The assay was performed as recommended by the manufacturer, though with some slight modifications. In brief, following the treatment of the cells on the PLA-IOC films with light pulses, they were placed in a cell incubator for 2 hours to allow sufficient time to die. After 2 hours, the cell medium was removed and replaced by 1 mL of new cell medium (DMEM/F-12) supplemented with 1 mL of CELLTITER-GLO? solution. The well plate was then put on a shaker at 120 rpm for 10 minutes at room temperature. Finally, 150 ?L of the supernatant was transferred into the wells of a 96-well plate and the absorbance was measured with a microplate reader (Victor 3, PerkinElmer).

(66) 1.7 Measuring the Permeabilization of Cell Membranes

(67) To evaluate to what extent applying laser pulses on the PLA-IOC films permeabilized the membranes of cells, cells were seeded onto a PLA-IOC film or cells were covered with a PLA-IOC film as described above. In case cells were grown on a film, 1 mL DMEM/F-12 cell culture medium containing FITC-dextran (molecular weight 500 kDa; FD500) at a concentration of 2 mg/mL was added. In case a film was applied on top of the cells, 300 ?l of DMEM/F-12 cell culture medium containing FD500 (2 mg/ml) was added on the cells before applying the film. The (whole surface of the) films were scanned with the laser beam (as described above; at a fluence of 0.3 J/cm.sup.2 or 1.6 J/cm.sup.2) and the cells were extensively washed with DPBS for several times to remove the excess of FD500. The cells were then trypsinized (using a 0.25% trypsin solution) and neutralized with cell medium. The suspension of cells was collected and washed by centrifugation for several times (500 g; 5 minutes). Finally, the pellet was resuspended in flow buffer (DPBS, 1% Bovine Serum Albumin (BSA) and 0.1% NaN3). The cytosolic delivery of FD500, which only happens after membrane permeabilization, was measured by flow cytometry (Cytoflex, Beckman Coulter, Krefeld, Germany); the fluorescent dye was excited at 488 nm while the fluorescence intensity was detected at 530 nm (bandpass filter 530/30).

(68) 1.8 Spatial Selective Cell Killing by PLA-IOC Films Exposed to Pulsed Laser Light

(69) In a first set of experiments, Hela cells were seeded in a 6-well plate on top of PLA-IOC films (0.1% IONPs). Laser pulses, with increasing fluences, were applied on the films, in pre-defined zones. After laser irradiation, the cells on the PLA-IOC films were placed in an incubator (37? C., 5% CO.sub.2) for 2 hours. Then 1 ?l of calcein AM (0.5 mmol) and 10 ?l propidium iodide (1 mg/ml) were added into each well. Calcein AM was used to stain living cells and propidium iodide to stain dead cells. After 10 minutes of incubation, the cells were imaged by confocal microscopy (C1-si, Nikon, Japan). The extent of cell killing in the treated zones was evaluated.

(70) In a second set of experiments, PLA-IOC films (without or with 0.1% of IONPs) were applied on top of Hela cells seeded as described above. Four consecutive laser irradiations at 1.6 J/cm.sup.2 were performed according to a pre-defined pattern (similar to logo of Ghent University). After laser scanning, 2 mL DMEM/F-12 was added to the wells and the films were carefully removed from the cells. After 30 minutes, 2 ?l calcein AM (0.5 mmol) and 20 ?l propidium iodide (1 mg/ml) were added to stain both the living and dead cells.

(71) 1.9 Single Cell Killing by PLA-IOC Films Exposed to Pulsed Laser Light

(72) To evaluate to which extent the irradiation of PLA-IOC films with pulsed laser light allows specific killing of a chosen target cell (single cell killing), cells were seeded the cells on top of PLA-IOC films as described above. First, the (living) cells were stained by adding 2 ?l of calcein AM (0.5 mmol) into each well. A single cell was then randomly selected as the target and imaged by fluorescence microscopy. Subsequently one laser pulse (varying fluence) was applied on the target cell. After 30 minutes, 10 ?L propidium iodide (1 mg/ml) was added into each well and cells were imaged again by fluorescence microscopy. The red and green fluorescence of the cells was analyzed using ImageJ software. Cells in the wells were also imaged by transmission microscopy; movies were recorded and processed by ImageJ software.

(73) 1.10 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Measurements

(74) Hela cells were seeded into a 6-well plate as described above. In the absence of laser treatment, either cell culture medium (1.5 ml), a suspension of free IONPs (1.5 ml; 1 g/l) or a PLA-IOC film (keeping of very thin layer of medium between the film and the cells) was added/applied to/on the cells in a well. After 20-minute incubation (37? C., 5% CO.sub.2), the cell medium/free IONPs (or) the film in the well was/were removed. Then 1.5 mL nitric acid (65%) was added for 10 minutes to each well to digest the cells. After 10 minutes, 1 mL was collected from each well for ICP-MS analysis.

(75) To measure the effect of laser treatment, 20 minutes after applying cell medium/free IONPs/PLA-IOC film (see paragraph above), each well was scanned four times (1.6 J/cm.sup.2). After laser treatment, the film was removed and the well was gently washed with fresh cell culture medium twice. Subsequently, 1.5 mL nitric acid (65%) was added to each well for 10 minutes. After digestion of the cells, 1 mL solution was collected from each well for ICP-MS analysis.

(76) (Ultra-)trace element determination of iron (Fe) was carried out using an Agilent 8800 ICP-MS/MS instrument (ICP-QQQ, Agilent Technologies, Japan). The sample introduction system comprises a concentric nebulizer (400 ?L min?1) mounted onto a Peltier-cooled (2? C.) Scott-type spray chamber. This instrument is equipped with a tandem mass spectrometry configuration consisting of two quadrupole units (Q1 and Q2) and a collision/reaction cell (CRC) located in-between both quadrupole mass filters (Q1-CRC-Q2). As a result of a much better control over the in-cell chemistry (chemical resolution), the MS/MS mode provides additional means to deal with spectral overlap in a more straightforward way compared to traditional single-quadrupole ICP-MS instrumentation. In this work, the CRC was pressurized with a mixture of NH3/He (10% NH3 in He) to overcome the spectral interferences seriously hampering (ultra-)trace element determination of Fe, such as the overlap between the signals of 40ArO+ and 40CaO+ polyatomic interferences and those of the most abundant Fe isotope (56Fe+, 91.7%) at mass-to-chargem/z56. To overcome spectral overlap, 56Fe.sup.+ ions were mass-shifted to 56Fe(NH.sub.3).sup.2+ reaction product ions upon introduction of 3.0 mL min?1 of NH.sub.3/He. After mass-shifting, 56Fe.sup.+ can be detected free from spectral interferences at the m/z ratio of the newly created reaction product ion (56Fe(NH.sub.3).sup.2+, m/z=90).

(77) For ICP-MS/MS analysis, only high-purity reagents were used. Ultra-pure water (resistivity 18.2 M? cm) was obtained from a Milli-Q Element water purification system (Millipore, France). Pro-analysis purity level 14 M HNO.sub.3 (Chem-Lab, Belgium) further purified by sub-boiling distillation and ultra-pure 9.8 M H2O2 (Sigma Aldrich, Belgium) were used for sample digestion. Appropriate dilutions of 1 g L?1 single element standard solutions of Fe and Ga (Inorganic Ventures, USA) were used for method development, optimization and calibration purposes. For quantitative element determination of Fe, external calibration was relied on as calibration approach (0, 0.5, 1.0, 2.5, 5.0, 10 and 20 ?g L.sup.?1 Fe). 5.0 ?g L.sup.?1 of Ga were used as internal standard to correct for instrument instability, signal drift and matrix effects.

(78) Prior to sample preparation for ICP-MS/MS analysis, all samples were transferred from Eppendorf tubes to Teflon Savillex beakers, which had been pre-cleaned with HNO.sub.3 and HCl and subsequently rinsed with MILLI-Q? water. After evaporation until dryness (80? C.), the samples were digested via acid digestion with a mixture of 750 ?L of 14 M HNO.sub.3 and 250 ?L of 9.8 M H.sub.2O.sub.2 at 110? C. on a hot plate for approximately 18 hours. Prior to ICP-MS/MS analysis, the digested samples were appropriately diluted in MILLI-Q? water or HNO.sub.3 (final acid concentration ranging between 0.35 and 0.70 M HNO.sub.3). To avoid contamination, only metal-free tubes were used for standard and sample preparation (15 or 50 mL polypropylene centrifuge tubes, VWR, Belgium). The complete sample preparation procedure, including digestion and adequate dilutions, was carried out in a class-10 clean room.

(79) 1.11 Killing of Superficial Corneal Cells by PLA-IOC Films Exposed to Pulsed Laser Light

(80) Fresh excised bovine eyes were collected from a local slaughterhouse (Flanders Meat Group, Zele). After enucleation, the eyes were maintained in cold CO.sub.2 independent medium (Gibco-Invitrogen) during transit and were processed within one hour. The excess of tissue was removed and the eyes were washed in cold DMEM. PLA films (2% PLA) without or with 0.1% IONPs (w/v) were used. The PLA-IOC films were placed into a 55 mm glass bottom dish with a 30 mm micro-well (Cellvis, USA) and filled with 3 mL cell culture medium. Subsequently, the eye was transferred into the dish and fixed with parafilm to immobilize it, making sure that the cornea was in close contact with the PLA-IOC film. To know whether applying laser pulses permeabilized the membranes of the corneal cells, the cell culture medium was supplemented with FD500 (4 mg/mL).

(81) The whole cornea was repeatedly (4 times) scanned with the pulsed laser at a fluence of 1.6 J/cm.sup.2. After the irradiation, the eyes were washed three times with DPBS. To assess the killing efficiency, the eyes were placed in a new dish, supplemented with fresh DMEM/F-12 medium containing propidium iodide (0.01 mg/ml) and incubated during 15 minutes at room temperature.

(82) Finally, a 12 mm button was punched into the cornea using a trephine blade (Beaver Visitec International, Abingdon, UK). The thus isolated corneal tissue was placed on a glass bottom dish with the epithelial side down and imaged by confocal microscopy (Nikon A1R). Z-stacks with steps of 1 ?m were recorded and 3D images were obtained using available software.

(83) For spatial selective cell killing experiments, the excess of tissue from fresh bovine eyes was removed and eyes were washed in DPBS. A solution of calcein AM (5 ?g/ml) was applied at the level of the cornea to stain living epithelial cells and eyes were incubated at room temperature for 10 minutes. The eyes were then washed three times in DPBS and the cornea was isolated using a trephine blade. The corneas were then placed on top of the film PLA-IOC film. Epithelial cells could be observed by fluorescence microscopy and a single cell was targeted before being irradiated with the laser (561 nm; <7 ns).

2. Results

(84) 2.1 Physicochemical Characterization of PLA-IOC Films

(85) To be able to target each single cell in the cell culture (or tissue), ideally each single cell should be in contact with/in close proximity of at least one IOC. A well-controlled distribution of IOCs in the PLA films is therefore requested. SEM-imaging of the PLA-films revealed that IONPs were embedded as clusters (IOCs) within the films; some IOCs were partially protruding out of the films. The average size and the distribution of IOCs in the films depended on the IONP concentration. Indeed, a higher concentration of IONPs (for a fixed concentration of PLA) resulted in larger clusters (FIG. 5). The cluster density equaled 5.6?2.4 IOCs/100 ?m.sup.2 for a concentration of 0.1% IONPs, while it was lower at a higher IONP concentration (FIG. 6), which can be explained by the formation of larger clusters.

(86) 2.2 Laser-Induced Vapor Bubble Formation by PLA-IOC Films

(87) To evaluate whether applying pulsed-laser light to PLA-IOC films resulted in the formation of vapor bubbles (VNBs) at the surface of the films, the PLA-IOC films were irradiated with a single laser pulse of respectively 0.3 J/cm.sup.2 and 1.6 J/cm.sup.2. Irradiation of a PLA-IOC film (0.1% IONs, 2% PA) with such laser pulsed gave rise to VNBs, as localized flashes of (scattered) light were detected by dark field microscopy (FIG. 7). No VNBs could be observed from PLA films without IOCs. At the lower fluence, the number of VNBs was clearly lower than at the higher fluence.

(88) Subsequently, the number of VNBs as a function of the fluence of the laser pulse was measured and the VNB threshold of the PLA-IOC films (0.1% IONPs; 2% PLA), commonly defined as the laser fluence at which 90% (T90) of the IOCs form VNB, was determined to be 0.56 J/cm.sup.2. T10, corresponding to the laser fluence at which 10% of the IOCs result in the formation of VNB, was determined to be 0.1 J/cm.sup.2. For fluences lower than T10, heat generation is predominant (heating mode); between T10 and T90 both confined heat and bubbles are generated; for fluences higher than T90, VNB formation is the predominant photothermal effect (bubble mode).

(89) For the experiments that follow, PLA films containing 0.01% IONPs were selected. Attractive as well is that such PLA-IOC films are as transparent as PLA films without IOCs.

(90) 2.3 Cell Killing by PLA-IOC Films Exposed to Pulsed Laser Light

(91) Subsequently, the capacity of PLA-IOC films to kill Hela cells that were grown on the surface (cells on top) of the films (FIG. 2) was evaluated. IOCs in the films could be easily observed as they scatter light strongly (refractive index of iron oxide is 2.9). Based on images of cells grown on the PLA-IOC films, it was estimated that each cell was in contact with 4.6+/?0.5 IOCs (FIG. 8) when the concentration of IONPs in the films equaled 0.1%; at a lower (0.01%) concentration of IONPs, each cell was in contact with 1.2+/?0.4 IOCs.

(92) FIG. 9 shows that (most) cells grown on PLA-IOC films with 0.01% IONPs survive (as measured by flow cytometry; calcein AM staining) when the films are scanned with a laser at low fluence (single scan; 0.3 J/cm.sup.2). With a higher amount of IONPs in the PLA films (0.1%) and at the same fluence, a significant number of cells became killed (cell viability 52.8?12.6%). Also note that all cells grown on a PLA film without IONPs survived the laser treatment. To visualize changes in cell membrane permeability following laser irradiation (which might cause cell killing), fluorescent dextrans (FD500) were added to the cells. Due to their large size (around 30 nm), FD500 do not diffuse over cell membranes and have therefore been reported to be well suited to assess significant permeability changes of plasma membranes. As FIGS. 9 and 10 show, FD500 entered into Hela cells following the irradiation of PLA-IOC films with a laser pulse; note that for PLA-films without IONPs, FD500 did not enter the cells.

(93) Subsequently, it was evaluated how the killing of cells could be maximized by using respectively higher laser fluences and repeated laser scanning of the films. As FIG. 11 shows, increasing the laser fluence improved the cell killing capacity of PLA-IOC films: the cell viability dropped to 30% at a laser fluence of 0.8 J/cm.sup.2. This was not the case for PLA films without IOCs, which did not induce any cell killing, even not at a very high laser fluence of 1.6 J/cm.sup.2. As it was observed that the IOCs survived a single laser pulse (i.e., did not fragment), it was hypothesized that applying a second pulse could further enhance the cell killing capacity of the films. To ensure that an effect of a second pulse could be observed, a (low) fluence of 0.3 J/cm.sup.2 was selected as after a single pulse of 0.3 J/cm.sup.2 approximately 50% of the cells remained viable. Films were thus scanned for two consecutive times at a fluence of 0.3 J/cm.sup.2. As can be seen from FIG. 12, the cell viability dropped from 46%?6% (single scan) to 25%?3% (two consecutive scans).

(94) 2.4 Spatial Selective Cell Ablation and Cell Killing by PLA-IOC Films Exposed to Pulsed Laser Light

(95) Given (i) the fact that one can control the density and size of the IOCs in the films and (ii) the inherent ability to scan specific areas in the films, it was evaluated to what extent bubble-films allow to kill target cells with high spatial precision. Therefore, pre-defined circular areas in the PLA-IOC films were scanned with laser pulses with increasing fluences (FIG. 13).

(96) Cells present in the pre-defined areas (circles having a diameter of 6.5 mm) became selectively killed, as could observed by the red fluorescence of propidium iodide (PI) entering dead cells only. It was observed however that at a fluence of 1.3 J/cm.sup.2, the total number of cells in the treated areas (living plus dead cells) significantly decreased (FIG. 14), indicating that cells were lost (ablated) upon laser treatment, highly likely by the mechanical forces exercised by the vapor bubbles. As shown in FIG. 15, staining the cells with calcein AM and propidium iodide allowed to determine the ratio of living to dead cells remaining in the treated areas; clearly, this ratio gradually dropped upon increasing the laser fluence.

(97) FIG. 16 shows single cell killing (the scale bar is 10 ?m) upon local irradiation of a PLA-IOC film with a pulsed laser. Single cell killing was obtained, as could observed by the red fluorescence of propidium iodide (PI) entering dead cells only. Transmission images of selective cell treatment of cells on top of films showed single cell ablation at fluences of 1.3 J/cm.sup.2. Using a fluence between 0.3 and 0.8 J/cm.sup.2 VNBs could be generated though cell ablation did not occur.

(98) The success rate of single cell ablation increased with the fluence (FIG. 17). Besides, it was observed that while a fluence of 0.3 J/cm.sup.2 does not allow single cell ablation, at a fluence of 1.3 J/cm.sup.2 single cell ablation was always successful.

(99) In all experiments above, cells were grown on the PLA-IOC films. To further explore the clinical potential of structures comprising particles according to the disclosure, the cell killing capacity of the films when placed on top of a cell layer (FIG. 18) was evaluated. This mimics the intended use of the films to cover the surface of a target tissue.

(100) As outlined in the experimental section, after seeding the cells in a 6-well plate the cell medium was removed, though leaving a thin layer of medium on top of cells; subsequently a PLA-IOC film was placed on top of the cells. The distance between the cells and the film was estimated to be 40 ?m. A pre-defined area in the film (Ghent University logo) was then exposed to the laser (FIG. 19). As a larger distance between the film and the cells as compared to experiments in FIG. 13 where cells were cultured directly on top of the film was estimated, a higher fluence of 1.6 J/cm.sup.2 was used. As FIG. 20 shows, cells became killed, though four pulses were required to realize a ratio of living to dead cells of around 25%. Also note that the total number of cells did not change (FIG. 21), likely because of the longer distance between the cells and the IOCs, lowering the mechanical forces by VNBs acting on the cells.

(101) To evaluate whether poration of cell membranes is also involved in the killing of the cells if PLA-IOC films are positioned on top of the cells (FIG. 22 (left)), cells were incubated with FD500 and covered with a film. The film was then subsequently irradiated (1.6 J/cm.sup.2; four consecutive times). As shown in FIG. 22 (right), green (FD500) and red (PI) fluorescence could be observed in cells, while fluorescence was much lower in cells covered with a PLA film without IOCs and irradiated with laser pulses.

(102) To determine the amount of intracellular iron after laser irradiation of PLA-IOC films covering cells, ICP-MS experiments were performed. As shown in FIG. 23, exposing the cells to free IONPs (1 g/L, being equivalent to the concentration of iron in the films) leads to a higher iron content of the cells. Laser irradiation of the free IONPs further increases the iron levels in the cells. This is likely attributed to (i) the fragmentation of IONPs into smaller fragments and (ii) VNBs (as generated by the free IONPs), which porate cell membranes, thereby facilitating iron uptake. In cells covered with a PLA-IOC film (without laser treatment) the iron content remains as low as in untreated cells. Upon irradiating the PLA-IOC films an increase in intracellular iron content could be observed. However, the intracellular iron content remains far below the amount in cells treated with free IONPs and laser.

(103) 2.5 Killing of Superficial Corneal Cells by PLA-IOC Films Exposed to Pulsed Laser Light

(104) As efficient cell killing could be achieved when films were positioned on top of a cultured cell layer (FIG. 18), it was in a next step evaluated whether superficial cells of a tissue (cornea) could be killed by a structure according to the disclosure. As illustrated in FIG. 24, bovine eyes were positioned on the films; the whole cornea was subsequently illuminated four consecutive times at 1.6 J/cm.sup.2. Corneas were then excised, stained with PI and imaged by confocal microscopy. The first observation was that, somewhat unexpectedly, PI positive cells were present in the control eyes, i.e., eyes without PLA films or covered with PLA films without IOCs. This is likely because (i) ex vivo bovine corneal epithelial cells already start dying few hours after enucleation and (ii) it is known that epithelial cell death can physiologically occur due to shear forces caused by blinking, for instance. However, confocal microscope images confirmed that a significant increase in PI positive cells was observed in corneas covered with PLA-IOC films irradiated four times with 1.6 J/cm.sup.2 laser pulses compared to control experiments using PLA films without IOCs (FIG. 25).

(105) To confirm membrane poration of the corneal cells, the experiments were repeated in the presence of green fluorescent FD500. Confocal microscope images show a clear difference in green fluorescence in corneas covered with PLA-IOC films and treated with four pulses of 1.6 J/cm.sup.2 compared to control experiments using PLA films without IOCs. This observation indicates that poration of corneal cell membranes indeed occurred. Important as well is that the green fluorescence (FD500) did not co-localize with the red fluorescence of naturally died cells, indicating that the formation of pores is due to laser irradiation of the PLA-IOC film covering the cornea. It was observed that FD500 did not penetrate untreated corneal cells.

(106) To check if spatial selective cell killing could be achieved at the level of the cornea, epithelial cells were stained with calcein AM (5 ?g/ml). The stained corneas were then placed on top of films (FIG. 28) with the epithelial side down. A region of interest was selected and laser irradiation was applied (<7 ns; 561 nm) four times. After four irradiations at a fluence of 1.6 J/cm.sup.2, a local decrease of fluorescence can be observed and irradiated cells lost their staining. At a higher fluence (4.5 J/cm.sup.2, four times), cells in the targeted area were not visible anymore suggesting ablation occurs. For both fluences, the surrounding cells (i.e., located outside of the laser beam) did not lose staining after laser irradiation suggesting they were left untouched.

Example 2

(107) A second embodiment of a structure, according to the disclosure, comprises a porous structure comprising nanofibers and particles able to absorb electromagnetic radiation embedded in the nanofibers, whereby a portion of the particles are partially exposed to the free surface S of the structure, in particular, to the free surface of the nanofibers. The below described examples comprise polycaprolactone as material of the structure and iron oxide nanopowder as particles able to absorb electromagnetic radiation. It is clear that other materials and other particles can be considered as well.

1. Materials and Methods

(108) 1.1 Materials

(109) The following materials are used for the synthesis of the web of nanofibers: Polycaprolactone (PCL, Mw?70,000 g/mol); N,N-Dimethylformamide (DMF); Tetrahydrofuran (THF); iron oxide (Fe.sub.3O.sub.4) nanopowder (IONP) (#MKBW3262, Sigma-Aldrich, Belgium); Poly(allylamine hydrochloride) (PAH, Mw=17,560 g/mol, #MKBZ2824V, Sigma-Aldrich, Belgium); concentrated sulfuric acid solution (96%) (Sigma-Aldrich); Collagen I Rat Protein (Thermo Fisher Scientific, #A1048301, Gibco?, Belgium).
1.2 Preparation of Structure Comprising Nanofibers and Particles

(110) IONP was re-dispersed in a 1:1 DMF/THE solution to which PCL in different concentrations between 0 vol % and 1.15 vol % was added.

(111) The thus obtained mixture was used to manufacture nanofibers by electrospinning. The nanofibers were collected on microscope glass slides (#1000912, Marienfeld, Germany) mounted on a grounded rotating collector.

(112) IONP was embedded in the nanofibers with a portion of the particles being partially exposed to the free surface S of the nanofibers.