COMPOSITION COMPRISING AT LEAST ONE NANOBOMB SUITABLE FOR ALTERING A BIOLOGICAL BARRIER

Abstract

A composition comprising at least one nanobomb comprising at least one first particle and at least one second particle in close proximity to the first particle. The at least one first particle is able to absorb electromagnetic radiation so as to generate a vapor bubble. The generation of the vapor bubble causes the at least one second particle to be propelled over a distance D. The composition is suitable to alter a biological barrier, in particular, for deforming, permeabilizing or perforating a biological barrier. A method to alter biological barriers is also disclosed.

Claims

1. A composition comprising at least one nanobomb, said at least one nanobomb comprising n first particles and m second particles, with each of n and m being at least one, at least one of said m second particles being in close proximity to at least one of said n first particles, so as to either be in contact with or be positioned at a distance d smaller than 1 μm, said at least one n first particle being able to absorb electromagnetic radiation such so as to generate a vapor bubble, whereby said generation of said vapor bubble causes said at least one m second particle to be propelled over a distance D away from said at least one n first particle, with said distance D being at least 0.01 μm.

2. The composition according to claim 1, wherein said at least one m second particle of said at least one nanobomb is adapted to alter a biological barrier once propelled upon said generation of said vapor bubble.

3. The composition of claim 1, wherein m is larger than n.

4. The composition of claim 1, wherein said m second particle(s) of said at least one nanobomb has a size ranging between 10 nm and 10 μm and/or a density of at least 1 kg/dm.sup.3.

5. The composition of claim 1, wherein a majority of said n first particles comprises at least p second particles in close proximity, with p being at least 2.

6. The composition of claim 1, wherein said distance D ranges between 0.1 and 100 μm.

7. The composition of claim 1, wherein said at least one n first particle comprises a metal, a metal oxide, carbon, a carbon-based material, a light-absorbing compound or particles loaded or functionalized with one or more light-absorbing compounds or a combination thereof.

8. The composition of claim 1, wherein said at least one n first particle is functionalized with one or more polymer, lipid and/or molecular linker.

9. The composition of claim 1, wherein said m second particle(s) of said at least one nanobomb is selected from the group consisting of polymer particles, metal oxide particle, silicon or silicon oxide particles, liposomes, drug loaded polymer particles, drug loaded silicon or silicon oxide particles an drug loaded liposomes.

10. The composition of claim 1, wherein said at least one m second particle is functionalized with one or more charged polymer or lipid, one or more targeting moiety selected from the group consisting of antibodies, dyes, proteins, nucleic acids, drugs and/or labels and/or one or more functional group to induce a linking strategy with the at least one n first particle in close proximity.

11. A method of altering a biological barrier, the method comprising: using the composition of claim 1 to alter at least one biological barrier.

12. The composition of claim 1 for use in drug delivery, in intracellular delivery of compounds, in drug delivery, in cell therapy, in immunotherapy, in gene therapy and in transfection of cells.

13. An ex vivo or in vitro method for altering a biological barrier, said method comprising: providing the composition of claim 1; introducing the composition in proximity of a biological barrier; and irradiating the composition using electromagnetic radiation so as to generate vapor bubbles, thereby generating a mechanical force to propel at least part of said m second particles of said at least one nanobomb of said composition upon the generation of said vapor bubbles.

14. The method of claim 13, further comprising attracting said at least one nanobomb of said composition to said biological barrier by means of a magnetic field.

15. A method of producing the composition of claim 1, the method comprising: providing first particles able to absorb electromagnetic radiation so as to generate a vapor bubble, providing second particles; and mixing said first particles and said second particles allowing to form at least one nanobomb, said at least one nanobomb comprising n first particles and m second particles, with each of n and m being at least one and with at least one of said m second particles being in close proximity to at least one of said n first particles, with “being in close proximity to” being defined as being either in contact with or being positioned at a distance d smaller than 1 μm.

16. A composition comprising a nanobomb, wherein the nanobomb has n first particles and m second particles, wherein each of n and m is at least one, and wherein at least one of the m second particles is in close proximity to at least one of the n first particles, so as to either be in contact with one another or to be positioned at a distance d of less than 1 μm from one another, wherein the at least one n first particle absorbs electromagnetic radiation and thereby generates a vapor bubble, wherein generation of a vapor bubble causes said at least one m second particle to be propelled over a distance D away from said at least one n first particle, wherein distance D is at least 0.01 μm.

17. The composition of claim 16, wherein m is greater than n.

18. The composition of claim 17, wherein the at least one m second particle has a size of between 10 nm and 10 μm and/or a density of at least 1 kg/dm3.

19. The composition of claim 18, wherein distance D is between 0.1 and 100 μm.

20. The composition of claim 19, wherein the at least one n first particle comprises a metal, a metal oxide, carbon, a carbon-based material, a light-absorbing compound, a particle loaded or functionalized with one or more light-absorbing compounds, or a combination of any thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0132] The present disclosure will be discussed in more detail below, with reference to the accompanying drawings, in which:

[0133] FIG. 1 illustrates the synthesis of a nanobomb and the irradiation of such nanobomb followed by the generation of a vapor bubble to propel the second particles of a nanobomb, thereby inducing pore formation in a biological barrier;

[0134] FIG. 2 illustrates a first type of a nanobomb according to the present disclosure comprising a first particle surrounded by one layer of second particles;

[0135] FIG. 3 illustrates a second type of a nanobomb according to the present disclosure comprising a container with first and second particles;

[0136] FIGS. 4A and 4B show dark field microscopy images of nanobombs before and immediately after irradiation with pulsed laser light;

[0137] FIG. 5 shows a confocal image of Hela cells and nanobombs according to the present disclosure after laser irradiation;

[0138] FIGS. 6A and 6B compare confocal images of Hela cells using nanobombs comprising first and second particles and using first and second particles (uncoupled);

[0139] FIG. 7 illustrates the efficiency of transfection with mRNA using traditional photoporation with gold nanoparticles and using nanobombs according to the present disclosure;

[0140] FIG. 8 shows the size (bars) and zeta potential (black dots) of different first particles, second particles and nanobombs according to the present disclosure determined by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM) (for the case of nanobomb s);

[0141] FIG. 9 shows the effect of laser fluence on the number of generated vapor nanobubbles;

[0142] FIG. 10 shows the transfection efficiency of FITC dextran FD500 (500 kDa) in Hela cells using different types of nanobombs having second particles of increasing mass density;

[0143] FIG. 11 shows the efficiency of transfection (in transfected cells) with mRNA in Hela cells as well as the cell viability (in %) of a method according to the present disclosure using nanobombs having 200 nm PLGA nanoparticles as second particles, compared to non-transfected cells and compared to traditional photoporation;

[0144] FIG. 12 shows the efficiency of transfection (in transfected cells) with mRNA in Jurkat cells as well as the cell viability (in %) using a method according to the present disclosure using nanobombs having 200 nm PLGA nanoparticles as second particles, compared to non-transfected cells and compared with photoporation.

DETAILED DESCRIPTION

[0145] 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 drawings 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.

[0146] When referring to the endpoints of a range, the endpoint values of the range are included.

[0147] When describing the disclosure, the terms used are construed in accordance with the following definitions, unless indicated otherwise.

[0148] 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.

[0149] 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.

[0150] The term “generation of a vapor bubble” includes either expansion of the vapor bubble, either 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.

[0151] The term “microparticle” refers to particles having a diameter or equivalent diameter ranging between 1 μm and 100 μm. The term “nanoparticle” refers to particles having a diameter or equivalent diameter ranging between 1 nm and 1000 nm.

[0152] 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 μm. Vapor bubbles comprise water vapor bubbles, although embodiments are not limited thereto.

[0153] FIG. 1 illustrates the synthesis of a nanobomb (step a) as well as the irradiation of such nanobombs (step b), followed by the generation of the vapor bubble (step c) and the perforation of a cell by the propelled second particles of nanobombs (step d).

[0154] Nanobombs 1 are synthesized by mixing first particles 2 (acting as vapor nanobubble source) and second particles 3 (acting as nanoprojectiles) (step a). The nanobombs 1 are irradiated, preferably using short pulsed laser light 8 of sufficient intensity (step b). Upon irradiation, the nanobombs 1 and, in particular, the first particles 2 of the nanobombs 1 heat up. The temperature exceeds the surrounding medium's boiling temperature, thereby evaporating the surrounding medium and forming vapor bubbles 9. Alternatively or additionally, the temperature exceeds the evaporation temperature of the first particle 2 or of part of the first particle 2 and the first particle 2 or part of the first particle 2 evaporates forming vapor bubbles 9. The vapor bubbles 9 are quickly expanding around the first particles 2. The expanding, and possibly the collapsing, of the vapor bubble 9 causes that the second particles 3 are propelled away from the first particle (step c). The propelled second particles are indicated by reference number 10. The propelled second particles 10 may cause pore formation in the membrane of a nearby cell 11 (step d).

[0155] FIG. 2 illustrates a first type of a nanobomb 1 according to the present disclosure. The nanobomb 1 comprises an iron oxide nanoparticle (IONP) as first particle 2 and fluorescent polystyrene nanospheres as second particles 3. The IONP has, for example, an average diameter of 500 nm and may generate a vapor bubble upon irradiation with a single laser pulse, for example, a 7 ns laser pulse with a fluence of 1 J/cm.sup.2 and a wavelength of 561 nm. The IONP can be functionalized with streptavidin molecules 5. A detail of a second particle 3 is given in box a of FIG. 2. The second particles 3 comprise fluorescent nanospheres having an average diameter of 100 nm and functionalized with biotin 4. The second particles 3 are, for example, attached to the first particle 1 by biotin-streptavidin linker moieties 4 as shown in box b of FIG. 2.

[0156] It is clear for a person skilled in the art that other linking strategies such as bioconjugation, complexation, electrostatic connection, physisorption, chemical connection, for example, by one or more covalent bonds (click chemistry) can also be considered.

[0157] Furthermore, it is clear for a person skilled in the art that a nanobomb according to the disclosure may comprise more than one first particle, for example, in contact with or connected to each other such as by bioconjugation, complexation, electrostatic connection, physisorption, or chemical connection.

[0158] It is also clear for a person skilled in the art that a nanobomb according to the disclosure may comprise a first particle or a plurality of first particles surrounded by more than one layer of second particles, for example, surrounded by two or three layers of second particles.

[0159] Table 1 mentions further examples of nanobombs according to the present disclosure by specifying the type of the first particle (functioning as vapor nanobubble (VNB) source), the surface functionalization of the first particle, the type of the second particle (functioning as projectile), the surface functionalization of the second particle and the linking strategy between the first particle(s) and the second particle(s).

TABLE-US-00001 TABLE 1 Surface Second Surface First particle functionalization particle functionalization Linking (VNB source) of first particle (Projectile) of second particle strategy Iron oxide Coating or surface Polystyrene Coating or surface Covalent bond Gold ligands exposing - Polymeric NP ligands exposing - formation Titanium oxide NH.sub.2 groups Polyplexes COOH groups through Carbon Liposomes Carbodiimide Nanotubes Silica Crosslinker Graphene oxide Titanium oxide Chemistry Polydopamine Poly(N- phenylglycine) Iron oxide Coating or surface Polystyrene Coating or surface Covalent bond Gold ligands exposing - Polymeric NP ligands exposing - formation Titanium oxide COOH groups Polyplexes NH.sub.2 groups through Carbon Liposomes Carbodiimide Nanotubes Silica Crosslinker Graphene oxide Titanium oxide Chemistry Polydopamine Poly(N- phenylglycine) Iron oxide Coating or surface Polystyrene Coating or surface Covalent bond Gold ligands exposing - Polymeric NP ligands exposing formation Titanium oxide N.sub.3 groups Polyplexes propargyl groups catalyzed by Carbon Liposomes Cu.sup.+ Nanotubes Silica Graphene oxide Titanium oxide Polydopamine Poly(N- phenylglycine) NP Iron oxide Coating or surface Polystyrene Coating or surface Covalent bond Gold ligands exposing Polymeric NP ligands exposing - formation Titanium oxide propargyl groups Polyplexes N.sub.3 groups catalyzed by Carbon Liposomes Cu.sup.+ Nanotubes Silica Graphene oxide Titanium oxide Polydopamine Poly(N- phenylglycine) Iron oxide Coating or surface Polystyrene Coating or surface electrostatic Gold ligands exposing Polymeric NP ligands exposing Titanium oxide positively charged Polyplexes negatively charged Carbon groups Liposomes groups Nanotubes Silica Graphene oxide Titanium oxide Polydopamine Poly(N- phenylglycine) Iron oxide Coating or surface Polystyrene Coating or surface electrostatic Gold ligands exposing Polymeric NP ligands exposing Titanium oxide negatively charged Polyplexes positively charged Carbon groups Liposomes groups Nanotubes Silica Graphene oxide Titanium oxide Polydopamine Poly(N- phenylglycine) Iron oxide Coating or surface Polystyrene Coating or surface bioconjugation Gold ligands exposing Polymeric NP ligands exposing Titanium oxide proteins Polyplexes specific ligands Carbon (e.g., streptavidin, Liposomes (e.g., biotin, Nanotubes antibody, Silica antigens, etc.) Graphene oxide nanobody, etc.) Titanium oxide Polydopamine Poly(N- phenylglycine) Iron oxide Coating or surface Polystyrene Coating or surface bioconjugation Gold ligands exposing Polymeric NP ligands exposing Titanium oxide specific ligands Polyplexes proteins Carbon (e.g., biotin, Liposomes (e.g., streptavidin, Nanotubes antigens, etc.) Silica antibody, Graphene oxide Titanium oxide nanobody, etc.) Polydopamine Poly(N- phenylglycine) Iron oxide DNA strand Polystyrene Complementary bioconjugation Gold Polymeric NP DNA strand Titanium oxide Polyplexes Carbon Liposomes Nanotubes Silica Graphene oxide Titanium oxide Polydopamine Poly(N- phenylglycine)

[0160] FIG. 3 illustrates a further embodiment of a nanobomb 1′ according to the disclosure. The nanobomb 1′ comprises first particles 2′ and second particles 3′ held together by a matrix 6′ or a shell 7′. It is clear that the matrix material 6′ or the shell 7′ may comprise one nanobomb or a plurality of nanobombs.

[0161] FIGS. 4A and 4B illustrate the optical triggering of a nanobomb as visualized with dark field microscopy. FIG. 4A shows a dark field microscopy image of a dispersion of nanobombs in water as illustrated in FIG. 1 before irradiation (at time to) and FIG. 4B shows a dark field microscopy image right after irradiation of the nanobomb indicated by the arrow in FIG. 4A with a single 7 ns laser pulse (at time ti). FIG. 4B clearly illustrates the generation of the vapor bubble from the first particle of the nanobomb (indicated by arrow 20) as well as the propelling of the second particles (indicated by arrows 22). The second particles 22 are thereby propelled over tens of micrometers in the surrounding medium.

[0162] Penetration of nanospheres of a nanobomb into cells after irradiation of a nanobomb could be demonstrated by confocal images. To demonstrate penetration of the nanospheres into the cells' nanobombs according to the disclosure, in particular, nanobombs as illustrated in FIG. 1, are added to cultured cells, together with Propidium Iodide (PI), as a marker for membrane permeabilization. The confocal image of FIG. 5 shows that after laser irradiation, the nanospheres had successfully penetrated into the cells with a concomitant influx of PI into the cell's cytoplasm. The second particles 40 are found partially in the cells 41.

[0163] FIGS. 6A and 6B show confocal images of cultured cells together with Propidium Iodide as marker in the presence of nanobombs according to the present disclosure, i.e., nanobombs comprising first and second particles (FIG. 6A) and in the presence of the uncoupled first and second particles (FIG. 6B). As shown in FIGS. 6A and 6B, PI could be delivered into most cells using the nanobombs according to the disclosure, while this was clearly not the case in the control experiment where IONP and nanospheres were added to the cells as a mixture of both components, i.e., without the first and second particles being in contact to one another to form the actual nanobombs.

[0164] Transfection of cells with mRNA encoding for GFP using traditional photoporation was compared with transfection of cells with mRNA using nanobombs as illustrated in FIG. 1.

[0165] The traditional photoporation was performed by 30 minutes incubation with 70 nm gold nanoparticles (AuNPs) (8.5×10.sup.7 AuNPs/mL), positively charged. After this period, the AuNPs are washed and medium containing mRNA was added. The cells were immediately irradiated. For the transfection using nanobombs according to the disclosure, a mixture of the nanobombs (6.4×10.sup.8 nanobombs/mL) and the mRNA in medium was added to the cells and incubated for 5 minutes before laser treatment. For both methods, the cells were washed and new medium was added after the laser treatment and the green fluorescence protein (GFP) was checked after 24 hours. For each experiment, 15,000 cells were seeded in 96-well plates 24 hours prior to the experiment. The results are shown in FIG. 7. The efficiency of transfection with mRNA using nanobombs according to the disclosure is considerably higher than the efficiency with mRNA using traditional photoporation: 70% transfected cells vs. 20% transfected cells without additional cytotoxicity.

[0166] FIG. 8 shows the size (bars) and zeta potential (black dots) of different first particles, second particles and nanobombs according to the disclosure determined by DLS (Dynamic Light Scattering) and SEM (Scanning Electron Microscopy (for the case of nanobombs). The first particles, the second particles and the nanobombs that are considered are: [0167] iron oxide nanoparticles (IONPs) having a diameter of 0.5 μm; [0168] polystyrene beads having a diameter of 200 nm; [0169] polystyrene beads having a diameter of 200 nm functionalized with Biotin; [0170] nanobombs comprising a core of IONP having a diameter of 0.5 μm surrounded with an average of 35 polystyrene beads having a diameter of 200 nm functionalized with Biotin; [0171] iron oxide nanoparticles (IONPs) having a diameter of 1 μm; [0172] nanobombs comprising a core of IONP having a diameter of 1 μm surrounded with polystyrene beads having a diameter of 200 nm functionalized with Biotin.

[0173] FIG. 9 shows the effect of laser fluence on the number of generated vapor nanobubbles using nanobombs comprising a core of 0.5 μm IONP surrounded by 200 nm polystyrene beads. The fluence threshold (90% probability) was determined to be 1.05 J/cm.sup.2.

[0174] FIG. 10 shows the delivery efficiency of FITC dextran FD500 (500 kDa) in Hela cells using different nanobombs having either a core of 0.5 μm or a core of 1 μm. The nanobombs (with a core of 0.5 μm and with a core of 1 μm) have second particles (nanoprojectiles) of 200 nm of one of the following materials: [0175] polystyrene: density of 1.04 g/cm.sup.3 [0176] PLGA (poly(lactic-co-glycolic acid): density 1.37 g/cm.sup.3 [0177] TiO.sub.2: density of 4.30 g/cm.sup.3

[0178] PLGA has the advantage of being a biodegradable, biocompatible and FDA- and EMA-approved material.

[0179] FIG. 11 shows the efficiency of transfection (in %) with mRNA in Hela cells as well as the cell viability (in %) using a method according to the disclosure compared with non-transfected cells and compared with photoporation. The nanobombs were irradiated with a single laser pulse at the previously determined fluence threshold using 1.3×10.sup.8 nanobombs/mL with an incubation time of 5 minutes. For photoporation, a concentration of 4×10.sup.7 gold nanoparticles/mL was used.

[0180] FIG. 12 shows the efficiency of transfection (in %) with mRNA in Jurkat cells as well as the cell viability (in %) using a method according to the disclosure compared to non-transfected cells and compared with photoporation. The nanobombs were irradiated with a single laser pulse at the previously determined fluence threshold using 1.3×10.sup.8 nanobombs/mL with an incubation time of 20 minutes. For photoporation, a concentration of 4×10.sup.7 gold nanoparticles/mL was used.