WO2018085542A1

WO2018085542A1 - Cellular poration using continuous laser radiation

Info

Publication number: WO2018085542A1
Application number: PCT/US2017/059720
Authority: WO
Inventors: Eric Mazur; Nabiha SAKLAYEN; Valeria NUZZO
Original assignee: President And Fellows Of Harvard College
Priority date: 2016-11-03
Filing date: 2017-11-02
Publication date: 2018-05-11

Abstract

In one aspect, the present invention provides methods of cell processing, which include placing at least one cell on or near a metalized surface having a plurality of projections. The cell(s) can be disposed on the substrate in a variety of different ways. In some embodiments, the cell(s) can be grown on the metalized surface or chemically attached to the metalized surface. In some embodiments, the cell(s) can be entrained in a medium (e.g., a liquid medium) and the medium can be dispersed over the metalized surface. In some cases, the medium can further include one or more cargos to be internalized by the cells. The metalized surface, and in particular the metalized projections, can be irradiated with a continuous laser radiation, e.g., for a time duration of at least about 1 ms, so as to modulate at least one attribute of the cell.

Description

CELLULAR PORATION USING CONTINUOUS LASER RADIATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] The present application claims the benefit of U.S. Provisional Application No.

62/416,857, filed November 3, 2016; the disclosure of which is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

[2] The present invention was made with United States government support under Grant Nos. PHY-1219334 and PHY-1205465 from the National Science Foundation. The United States government has certain rights in this invention.

FIELD

[3] This disclosure relates generally to substrates and methods for changing cellular attributes, e.g., for causing a transient change in the permeability of a cell's membrane. In some aspects, this invention relates to cellular poration and intracellular delivery methods using continuous laser radiation and thermoplasmonic substrates.

BACKGROUND

[4] The direct intracellular delivery of biological and non-biological materials is a powerful way to manipulate cell behavior for a wide range of applications. For example, functional proteins are delivered to ablate genes in hematopoietic stem cells with high precision for research and therapeutic applications. Enzymes are delivered for their ability to bind targets with high affinity and specificity and siRNA delivery has created opportunities in gene silencing for biomedical applications. In the case of blood disorders such as human immunodeficiency virus (HIV) or leukemia, delivering functional molecules into a patient's stem cells for transplantation therapy shows promise for curing such disorders, circumventing the side effects of chemotherapy and the search for a matching donor. [5] The ability to effectively deliver large and diverse cargos such as, without limitation, amino acids, peptides, proteins, protein cages, antibodies, polysaccharides, nucleic acids, viruses, or DNAs/RNAs directly into cells would be a great boost for biomedical research. However, no current intracellular delivery method, either biological, chemical or physical, can offer all desirable "high-performance" features for intracellular delivery at once: (1) high efficiency, viability, and throughput, (2) diverse cargo delivery capability, (3) spatial selectivity (delivering to specific cells on a surface), scalability and reproducibility, (4) no post-delivery immunotoxicity, and (5) cost-effectiveness.

[6] Viral transduction is the most popular biological method due to decades of extensive research, but has limitations. For example, viral methods offer limited cargo-carrying capacity, only deliver genetic cargo, and include immunotoxicity risks. Gold nanoparticles irradiated by pulsed lasers have proven to be an interesting alternative to viral techniques, but have limitations too. For instance, they can potentially pose cytotoxicity challenges as entire nanoparticles or their fragments may enter cells and cause harmful DNA mutations.

[7] Accordingly, there is a need for enhanced substrates and methods for intracellular delivery of diverse cargos into cells.

SUMMARY

[8] In one aspect, the present invention provides methods of cell processing, which include placing at least one cell (e.g., a mammalian cell, an epithelial cell, a neuron, a fibroblast, a stem cell, an immune cell, or a blood cell) on or near a metalized surface having a plurality of projections. The cell(s) can be disposed on the substrate in a variety of different ways. In some embodiments, the cell(s) can be grown on the metalized surface or chemically attached to the metalized surface. In some embodiments, the cell(s) can be entrained in a medium (e.g., a liquid medium) and the medium can be dispersed over the metalized surface. In some cases, the medium can further include one or more cargos to be internalized by the cells. The metalized surface, and in particular the metalized projections, can be irradiated with a continuous laser radiation, e.g., for a time duration of at least about 1 millisecond (ms), so as to modulate at least one attribute of the cell(s). [9] The modulated attribute of the cell can be any physical and/or chemical property of the cell. By way of example, the modulated attribute of the cell can correspond to a change in permeability of the cell's membrane. In some embodiments, the modulated attribute can initiate cell death. In some embodiments, the method of cell processing includes exposing the cell to at least one external cargo such that the modulated attribute facilitates the uptake of the cargo by the cell. Without being bound to any theory, the mechanisms for modulating the attribute of the cell can include electrical and/or heating effects.

[10] In some embodiments, the continuous laser radiation can have a wavelength in a range of about 200 nm to about 5 microns, and an intensity in a range of about 10² W/cm² to about

5 2 3 2 4 2

10^J W/cm^z, or in a range of about 10 W/cm to about 10 W/cm . By way of example, in some embodiments, a laser intensity of at least about 10⁴ W/cm² can be used for delivery of a cargo into one or more cells. It has been unexpectedly discovered that even at such high intensities and fluences, many of the processed cells survive and divide.

[11] In some embodiments, the continuous laser radiation can be applied to the substrate for a time duration of at least about 1 ms, e.g., for a time duration in a range of about 1 ms to about 500 ms.

[12] In some embodiments, the metalized substrate surface comprises a metal layer having a thickness in a range of about 10 nm to about 1 micron, e.g., in a range of about 50 nm to about 1 micron. By way of example, the metal layer can comprise gold, silver, copper, platinum, chromium, titanium, or metallic titanium nitride.

[13] In some embodiments, the metalized surface is formed on a surface of an underlying substrate (e.g., a dielectric substrate, a semiconductor substrate, a glass substrate, or a polymer substrate) having a plurality of projections. In some embodiments, the metalized surface can also be reused.

[14] In some embodiments, a plurality of cells are disposed on the metalized surface and are processed in a manner discussed above. In some such cases, the change in the cells' attribute can be caused at an efficiency of at least about 40%, or at least about 50%, or at least about 60%), or at least about 70%, or at least about 80%, or greater than 90% and with a cell viability of at least about 60%, at least about 70%, or at least about 80%, or greater that about 90%.

[15] The methods of the present teachings can be employed to deliver a variety of cargos into cells. Some examples of such cargos include, without limitation, a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a plasmid, a protein, a dye, a polymer, a quantum dot, a nanoparticle, a protein, or a protein complex, e.g., a Cas9-gRNA complex. In some embodiments, the cargo can have a size in a range of about 0.5 kDa to about 2000 kDa, or in a range of about 0.6 kDa to about 200 kDa.

[16] In some embodiments, the metalized surface has a plurality of projections having at least one dimension of at least about 20 nm, or at least about 40 nm, or at least about 60 nm. In some embodiments, the metalized surface has a plurality of projections in the form of pyramids. In some such embodiments, the pyramids can have a height in a range of about 100 nm to about 100 microns and a base length in a range of about 100 nm to about 10 microns.

[17] The step of irradiating the substrate surface can be performed in a variety of different ways. For example, a substrate can be mounted on a movable x-y stage, which can be moved relative to a laser beam so as to expose different portions of the substrate surface to laser radiation. In some embodiments, a sample containing cells can be positioned in a petri dish and immersed in a medium containing a cargo (e.g., dissolved cargo or suspended cargo) to be delivered to the cell. The cells can then be exposed to continuous laser radiation to facilitate the uptake of the cargo by the cells.

[18] In some embodiments, small and large dye molecules (e.g., calcein green, calcein AM, dextran) can be used to probe the efficiency of the delivery of a cargo to a cell as well as the cell's viability after such delivery.

[19] In another aspect, the present invention provides methods of cell processing, which include disposing at least one cell on or near a structured metal surface, exposing the cell to at least an external cargo, and irradiating the metal surface with a continuous laser radiation at an intensity in a range of about 10² W/cm² to about 10⁵ W/cm² so as to facilitate uptake of the cargo by the cell. By way of example, the metal surface can be irradiated for at least about 1 ms. In some embodiments, the structured metal surface includes a plurality of metalized projections, e.g., metalized pyramids. In some such embodiments, upon irradiation of the metalized pyramids, at least some of the cells in proximity of the pyramids (e.g., cells positioned within hundreds of nanometers (e.g., 1000 nm)) can undergo a physical and/or chemical change, which can facilitate uptake of a cargo by those cells.

[20] In some embodiments, the structured metal surface comprises a plurality of projections, e.g., in the form of pyramids, having a height in a range of about 100 nm to about 100 microns.

[21] In some embodiments, the structured metal surface is formed on an underlying substrate. The underlying substrate can be formed of a variety of different materials. Some examples of suitable materials include, without limitation, glass, a semiconductor or a dielectric. In some embodiments, the underlying substrate is a polymeric substrate. A variety of polymers can be employed, such as, epoxy glue, UV glue, and

polydimethylsiloxane. In some embodiments, the underlying substrate is silicon, sapphire, diamond, and/or glass. In some embodiments, the metal layer can have a thickness in a range of about 50 nm to about 1 micron.

[22] In another aspect, the present invention provides methods for delivery of a cargo to a cell, which include placing at least one cell on or near a metalized surface having a plurality of projections, and irradiating the metalized surface (and in particular the projections) with a continuous laser radiation at an intensity of at least about 10² W/cm² (e.g., an intensity in a range of about 10² W/cm² to about 10⁵ W/cm²) while exposing the cell to an external cargo so as to cause the cell to internalize said cargo. In some embodiments, the continuous laser radiation can be applied for a time duration of at least about 1 ms, e.g., for a time duration in a range of about 1 ms to about 500 ms.

[23] In some embodiments, the projections have a pyramidal shape, and can be uniformly or non-uniformly distributed across the metalized surface. By way of example, the metalized projections can be in the form of a plurality of metalized pyramids distributed according to a regular array (e.g., a regular two-dimensional array).

[24] In some embodiments, the metalized surface comprises a metal layer, e.g., a gold layer, a silver layer, a copper layer, or a metallic titanium nitride layer, that is at least partially coating a surface of an underlying substrate.

[25] Further understanding of the invention can be obtained by reference to the following detailed description in conjunction with the associated drawings, which are described briefly below.

BRIEF DESCRIPTION OF THE DRAWINGS

[26] The present disclosure is described with reference to the following figures, which are presented for the purpose of illustration only and are not intended to be limiting.

[27] In the Drawings:

[28] FIG. 1 is a flow chart depicting various steps in an exemplary embodiment for modulating at least one attribute of cells;

[29] FIG. 2 is a flow chart depicting various steps in an exemplary embodiment for delivering cargos to cells;

[30] FIG. 3 is a schematic representation of a perspective view of a cell-processing structure in accordance with an embodiment of the present teachings;

[31] FIG. 4 is a schematic cross sectional view of a cell -processing structure in accordance with an embodiment;

[32] FIG. 5 is a schematic representation of a pyramid of a cell -processing structure in accordance with an embodiment of the present teachings; [33] FIGs. 6A - 6J schematically depict various steps for fabricating a cell-processing substrate in accordance with an embodiment of the present teachings;

[34] FIG. 7 is a schematic diagram of an exemplary apparatus for performing cell processing according to an embodiment of the present teachings;

[35] FIG. 8 is a scanning electron microscopy image of a side view of a thermoplasmonic substrate in accordance with an embodiment of the present teachings;

[36] FIG. 9A is a fluorescence image of calcein green delivered to cells using continuous wave laser excitation in accordance with an embodiment of the present teachings;

[37] FIG. 9B is a fluorescence image of calcein AM illustrating the viability of cells exposed to continuous wave laser excitation in accordance with an embodiment of the present teachings;

[38] FIG. 10A presents fluorescence images depicting the delivery efficiency of dextran (green) into cells and the viability of cells using calcein AM (magenta) at day 0, day 1 and day 2 following continuous wave laser excitation of increasing intensity (arrow direction) in accordance with an embodiment of the present teachings; and

[39] FIG. 10B is a fluorescence image depicting cargo (dextran) retention during cell division at day 2 in accordance with aspects of the present disclosure.

DETAILED DESCRIPTION

[40] The present invention relates generally to substrates and methods for processing cells, and more specifically, it relates to substrates and methods for changing one or more cellular attributes, e.g., in a transient manner. By way of example, in some embodiments, the substrates and methods according to the present teachings can be used to facilitate the delivery of cargos to cells. It has been unexpectedly discovered that continuous laser radiation at intensities in a range of about 10² W/cm² to about 10⁵ W/cm² can be employed to process cells disposed on a metalized surface having a plurality of projections, e.g., to facilitate delivery of a variety of cargos to the cells and/or change a cellular attribute.

[41] Various terms are used herein consistent with their common meanings in the art. The following terms are defined for additional clarity:

[42] The term "continuous-wave (CW) laser radiation," refers to laser radiation generated by a laser operating in the continuous-wave mode, rather than pulsed mode.

[43] The cell viability is defined herein as the percentage of cells that survive after thermoplasmonic intracellular delivery of a cargo in accordance with the present teachings.

[44] The efficiency at which at least one attribute, e.g., the membrane's permeability, is changed is defined herein as the percentage of cells exposed to continuous-wave radiation in accordance with the present teachings that exhibit said attribute change. The efficiency of delivery of cargo to a cell is defined herein as the percentage of cells that contain the cargo after undergoing thermoplasmonic intracellular delivery according to the present teachings.

[45] The term "thermoplasmonic substrate" as used herein refers to a substrate containing at least one micron-sized and/or nano-sized projection having a thin metallic film interfacing with a dielectric material. The projection absorbs light energy and converts it into thermal energy.

[46] The term "polymer" is used herein consistent with its common meaning in the art to refer to a macromolecule formed by the chemical union of five or more repeating chemical units, e.g., by repeating monomers.

[47] The term "cargo" and "agent" as used herein refer to any compound, molecule, molecular complex, and/or biological organisms, such as plasmid or viruses. [48] The term "about" as used herein denotes a variation of at most 10% around a numerical value.

[49] The term "substantially" denotes a deviation of at most 5% relative to a complete state and/or condition

[50] In some embodiments, a plurality of cells can be disposed on a thermoplasmonic substrate according to the present teachings having metal-coated micropyramids and the micropyramids can be irradiated with continuous laser radiation with an intensity of at least about 10² W/cm² so as to cause a change in at least one cellular attribute, e.g., a transient change in the permeability of the cell's membrane, of at least some of the cells, e.g., those cells that are in proximity of the micropyramids (e.g., at a distance of hundreds of nanometers (e.g., within 1000 nm of the micropyramids)). Without being bound to any theory, the thermoplasmonic substrates according to the present teachings having a plurality of micro- sized pyramids produce a strong electrical and/or thermal effect under laser illumination by focusing energy in a small volume in the vicinity of the tip of each pyramid. In some embodiments, such localized focusing of the energy can lead to the poration (e.g., a transient poration) of the membranes of one or more cells in the vicinity of the tip, thus allowing a variety of cargos, e.g., dyes, proteins, or RNA/DNA strands, to which the cells are exposed to diffuse into the cells' cytoplasm. In many embodiments, the porated cells show recovery and cell viability for at least a few days after such processing. In some cases, such processing of cells can induce cell death, as discussed in more detail below. This intracellular delivery technique is scalable and allows the delivery of a variety of different cargos to living cells with high efficiency and high viability.

[51] With reference to the flow chart of FIG. 1, in an embodiment of a method for cell processing according to the present teachings, one or more cells are disposed on or near a metalized surface having a plurality of projections, and the cells are irradiated with a continuous laser radiation at an intensity in a range of about 10² W/cm² to about 10⁵ W/cm², e.g., in a range of about 10³ W/cm² to about 10⁴ W/cm², so as to modulate at least one cellular attribute. By way of example, in some embodiments, the cells are irradiated with the continuous laser radiation for a time duration of at least about 1 ms, e.g., for a time duration in a range of about 1 ms to about 500 ms. In some embodiments, the modulation of the cellular attribute can be achieved at an efficiency of at least about 60%, e.g., in a range of about 60% to about 98%, and a cell viability of at least about 60%, e.g., in a range of about 60% to 98%). In some embodiments, the fluence of the applied laser radiation can be in a range of about 10² J/cm² to about 10⁵ J/cm².

[52] With reference to flow chart of FIG. 2, in some embodiments, a medium containing a plurality of cells and at least one cargo to be internalized by the cells is disposed on or a near a substrate surface having a plurality of metalized projections and the substrate surface is irradiated with continuous laser radiation at an intensity in a range of about 10² W/cm² to about 10 5 W/cm 2 , e.g., in a range of about 103 W/cm 2 to about 104 W/cm 2 , so as to facilitate the uptake of the cargo by at least some of the cells. In some embodiments, the irradiation of the substrate can cause a change in the permeability of the cells' membranes, e.g., via formation of bubbles and/or pressure waves around the cell membrane, which can in turn facilitate the cellular uptake of the cargo(s) by the cells. In some such embodiments, the fluence of the laser at the substrate surface can be in a range of about 10² J/cm² to about 10⁵ J/cm².

[53] A variety of different cargos can be delivered to cells using the methods of the present teachings. Some examples of such cargos include, without limitation, a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a plasmid, a protein, a dye, a polymer, a quantum dot, a nanoparticle, a protein, and a protein complex, among others. In some embodiments, the protein complex can be a Cas9-gRNA complex. Further, the methods of the present teachings can be used to process a variety of different cell types. Some examples of cell types include an epithelial cell, a neuron, a fibroblast, a stem cell, an immune cell (e.g., a T cell), and a blood cell. In some embodiments, the present methods can be applied to mammalian cells to cause a change in their physical and/or chemical attributes.

[54] In some embodiments, the present methods can be employed to cause a change in one or more physical and/or chemical attributes of cells. For example, the modulated attribute of the cell can correspond to a change in permeability of the cell's membrane or may initiate cell death. By way of example, cell death can occur before, during or after laser radiation, or before, during, or after cargo delivery. Without being bound to any theory, the cell death can be due to one or more factors, such as the laser intensity, the laser duration, the cargo type, the cargo size, and the cargo dispersing medium.

[55] FIGs. 3 and 4 schematically depict a cell -processing substrate (herein also referred to as a thermoplasmonic substrate) 300 that can be used in methods according to the present teachings for processing cells, for example, as discussed above. More specifically, the cell- processing substrate 300 includes a support substrate 302 across a top surface 304 of which a plurality projections 306 in the form of pyramids are distributed. In this embodiment, the pyramids 306 are distributed over the surface of the substrate 300 as a regular two- dimensional array. In other embodiments, the pyramids 306 may be randomly distributed across the substrate surface. In some embodiments, the pyramids can have a height (H) in a range of about 100 nm to about 100 microns, e.g., in a range of about 1 μπι to about 10 μπι. Further, in some embodiments, pyramidal surfaces can form an angle (a) of about 35.3° with a putative vertical line extending from the tip of the pyramids to its base, as shown schematically in FIG. 5. In this embodiment, the pyramids 306 have a square base characterized by a base length, e.g., in a range of about 100 nm to about 10 microns. In other embodiments, the pyramids 306 can have triangular bases. In some embodiments, the spacing between neighboring pyramids can be, for example, in a range of about 10 nm to about 100 /mi. In some embodiments, the surface density of the pyramids (i.e., the number of pyramids per unit area of the substrate surface on which the pyramids are distributed) can be in a range of about 1/cm² to about 500 millions/cm², e.g., in a range of about 10 millions/cm² to about 500 millions/cm², or in a range of about 100 millions/cm² to about 500 millions/cm².

[56] With continued reference to FIGs. 3 and 4, in this embodiment, a thin electrically conductive layer 400, such as a thin metal layer, coats the top surface of the support substrate including the exposed pyramidal surfaces, thereby forming a structured metalized surface on which a plurality of cells can be disposed. In some embodiments, the thickness of the metal layer can be, for example, in a range of about 1 nm to about 100 nm. A variety of metals can be used to form the metal layer 400. Some suitable examples include gold, silver, and copper. In some embodiments, the coating 400 can be formed of metallic TiN. The underlying substrate 302 can also be formed of a variety of different materials. In this embodiment, the substrate 302 is formed of a polymeric material. Some examples of suitable polymeric materials include, without limitation, epoxy glue, UV glue, and

polydimethylsiloxane. In some other embodiments, the underlying substrate 302 can be formed of a semiconductor (e.g., silicon, diamond, sapphire), or it can be formed of glass. In some embodiments, the substrate 302 can have a thickness in a range of about 10 nm to about 500 μιη.

[57] With reference to FIG 6, in one exemplary method of fabricating the cell-processing substrate 300, a silicon master template 612 can be used for fabricating a cell-processing substrate 610. Specifically, the silicon master template 612 can be fabricated using the following steps. A silicon wafer 600 can be sonicated in acetone (e.g., 5 min) and methanol (e.g., 5 min) before being rinsed in isopropyl alcohol (IP A) (FIG. 6A). 0₂ plasma cleaning of the wafer can then be performed (100 W, 20 mTorr, 1 min) before a chromium (Cr) hard lithographic mask 602 is deposited on the wafer via thermal evaporation (Cr thickness = 150 A). The wafer can be baked (200 °C, 3 min) to evaporate all solvents before processing. An SPR 700-1 photoresist 604 can then be spin-coated onto the wafer (3000 RPM, 45 s, ramp of 1000 RPM/s) and the wafer can be soft baked (115 °C, 60 s) (FIG. 6B). The entire area of the silicon wafer can be exposed in an autostepper to form a grid-based pattern before performing a post-exposure bake (115 °C, 60 s). The wafer can then be developed in CD-26 developer (1 min), then rinsed with deionized (DI) water (20 s). Development can be repeated until no residue is released into the developer. A plasma stripper can then be used to descum the wafer (100 W, 20 mTorr, 15 s) (FIG. 6C) and a chromium etch can be performed (12 s, 15 angstroms/s etched, room temperature) to remove the Cr in the exposed squares. The sample can then be washed with DI water and dried with an N₂ gun. The photoresist can be removed in acetone. An 0₂ plasma clean can be performed (100 W, 20 mTorr, 3 min) to completely remove residual photoresist (FIG. 6D). An HF etch (4.9% HF) can be used to remove oxide formed on the silicon (15 s). A KOH etch (2 parts water and 1 part of 45% KOH) can be performed on a hotplate with a thermometer to form a plurality of inverted pyramids in the substrate (80 °C, 3 min) (FIG. 6E). Chromium etching can be performed to remove the hard mask (20 s, room temperature) and obtain the silicon master template 612 (FIG. 6F). [58] The thermoplasmonic substrates 610 can be formed in the following way. A gold layer 606 can be deposited via an electron beam evaporator on the silicon master template 612 (e.g., gold thickness = 50 nm) (FIG. 6G). A coverslip 608 (no. 1.5) formed of glass can be glued to the gold-coated master template with UV curable glue (Norland Adhesive 61) and cured under the UV lamp overnight (FIG. 6H). The thermoplasmonic substrate can be peeled off ("template-strip") from the template using a razor blade (FIG. 61) resulting in a final substrate with a plurality of gold-coated pyramids, e.g., 10 million pyramids (FIG. 6J). By repeating gold deposition and template-stripping, large quantities of thermoplasmonic substrates can be fabricated with high precision. The master template for the fabrication of the thermoplasmonic substrate can be re-used hundreds of times for template-stripping. This approach allows for low-cost, highly-precise, and highly-reproducible fabrication of thermoplasmonic substrates.

[59] FIG. 7 schematically depicts a system for illuminating a thermoplasmonic substrate according to the present teachings so as to process one or more cells disposed on or in proximity of a metalized surface of the substrate, e.g., to deliver one or more cargos to the cells. The system 700 includes a laser source 702 that generates a continuous laser radiation. The laser radiation can have a central wavelength, for example, in a range of about 900 nm to about 1060 nm. The laser radiation is focused by an objective 706 onto a thermoplasmonic substrate contained in a petri dish 708 and on which a plurality of cells are disposed (e.g., the cells can be in a medium containing one or more agents to be delivered to the cells). The petri dish 708 is in turn mounted on a stage 710 with x-y mobility and coupled with a microscope 712. A camera 714 and a video monitor 716 allow obtaining images of a substrate undergoing laser processing and displaying those images to a user. The laser 702 is controlled through a laser controller 704. An attenuator 718 allows adjusting the laser power reaching a substrate under processing. The laser is focused by lenses 722 and 724 and reflected toward the substrate using mirror 726. A shutter 720 turns the laser exposure on and off. The substrate can be seeded with a plurality of cells, typically disposed in a medium that contains one or more agents to be internalized by the cells. The x-y stage 710 allows the motion of the substrate 708 in two dimensions, e.g., at an average speed of about 500 /mi/second so as to expose different cells disposed on different portions of the substrate to the laser radiation.

[60] In some embodiments, the central wavelength of the laser radiation can range from about 10 nm to about 2000 nm.

[61] In some embodiments, a plurality of cells is disposed on the cell-processing substrate 708 and the substrate is exposed to continuous laser radiation at an intensity of at least about 10² W/cm² so as to initiate chemical and physical processes in the cells that will lead to cell death, e.g., within 24 or 48 hours of such cell processing.

[62] To further elucidate various aspects of the invention, the following working example is provided. The example is provided only for illustration purposes and is not intended to present necessarily optimal practice of the invention and/or optimal results that may be obtained by practicing the invention.

Example 1

[63] Thermoplasmonic substrates containing plasmonic micron-sized pyramid arrays were fabricated using photolithography, anisotropic etching of silicon, metal deposition, and template stripping techniques in a manner discussed above. FIG. 8 shows a scanning electron microscopy image of a side view of a thermoplasmonic substrate containing pyramidal structures made of glass coverslip, polymer and a 50 nm gold layer, and having a base length of about 2 μπι. The silicon template used for the fabrication of the thermoplasmonic substrate can be used repeatedly.

[64] Samples containing HeLa cells and a cargo were seeded on the thermoplasmonic substrates, and the substrates were irradiated with continuous laser radiation using a fiber coupled JDSU diode laser at a wavelength of 980 nm, a power of 500 mW, and an intensity of 10⁴ W/cm² to deliver the cargo to the cells. More specifically, the samples were positioned in a petri dish and immersed in a solution containing dissolved cargos to be delivered to the cells. The laser beam was focused on the substrate and the petri dish was fixed on an x-y movable stage to scan the sample at a determined speed (which is related to the laser exposure time). The laser parameters were optimized for high-efficiency delivery of small dye molecules like calcein at high-cell viability, using fluorescence microscopy. Alongside small dyes, different-sized fluorescently labeled dextrans were delivered. This method allows for the delivery of molecules in different types of cells. The thermoplasmonic substrate can be reused for repeated high efficiency poration as the substrate undergoes no damage after laser irradiation.

Efficiency and Viability of Molecular Delivery

[65] Small dye fluorescent molecules were delivered into cells using continuous wave laser excitation. FIG. 9A shows the intracellular delivery of calcein green (648 Da) and FIG. 9B shows the viability of the cells using calcein AM (magenta), which fluoresces only in living cells where the nonfluorescent calcein AM is converted to a fluorescent calcein (shown here in magenta) after acetoxymethyl ester is hydrolyzed by intracellular esterases.

Modification of Cell Metabolism

[66] Large areas of thermoplasmonic substrates with seeded cells were scanned while changing the laser intensity in order to change the cell metabolism. FIG. 10A shows the change in cell metabolism of HeLa cells using intense continuous wave laser illumination where the efficiency of FITC-dextran 150 kDa delivery (green) and viability (magenta) are presented for 3 days, i.e., day 0, day 1, and day 2. The columns were scanned from top to bottom while the laser intensity was increased in discrete steps. On day 0, the cells exhibited a decrease in enzymatic activity (shown in magenta) as the laser intensity increased. On day 1, some of the cells recovered. On day 2, some of the cells underwent cell death. Many cells retained the FITC-Dextran 150 kDa that was delivered to them over several days. (Scale bar: 1500 μιη)

Cargo Retention and Cell Division

[67] Large dye molecules (FITC-Dextran, 150 kDa) were delivered and retained into HeLa cells for 48 hours after continuous wave laser excitation. FIG. 10B shows the long-term retention of larger dye molecules (FITC-Dextran, 150k Da) as well as cell division. (Scale bar: 20 um)

[68] Those having ordinary skill in the art understand that various changes can be made to the above embodiments without departing from the scope of the invention.

Claims

What is claimed:

1. A method of cell processing, comprising:

placing at least one cell on or near a metalized surface having a plurality of projections, and

irradiating the metalized surface with a continuous laser radiation at an intensity in a range of about 10² W/cm² to about 10⁵ W/cm² so as to modulate at least one attribute of the cell.

2. The method of claim 1, wherein the cell is irradiated with said continuous laser radiation for a time duration of at least about 1 ms.

3. The method of claim 2, wherein said time duration is in a range of about 1 ms to about 500 ms.

4. The method of claim 1, wherein said continuous laser radiation has a wavelength in a range of about 200 nm to about 5 microns.

5. The method of claim 1, wherein the intensity of said laser radiation is in a range of about 10³ W/cm² to about 10⁴ W/cm².

6. The method of claim 1, wherein said metalized surface comprises a metal layer having a thickness in a range of about 10 nm to about 1 micron.

7. The method of claim 6, wherein said metal layer comprises any of gold, silver,

copper, platinum, titanium, and metallic titanium nitride.

8. The method of claim 1, wherein said attribute is any of a physical or chemical

property of the cell.

9. The method of claim 1, wherein said modulated attribute corresponds to a change in permeability of the cell's membrane.

10. The method of claim 1, wherein said at least one cell comprises a plurality of cells and said change of the attribute is caused with a cell viability of at least about 60%.

11. The method of claim 10, wherein said cell viability is at least about 70%.

12. The method of claim 10, wherein said cell viability is at least about 80%.

13. The method of claim 1, wherein said modulated attribute initiates cell death.

14. The method of claim 1, further comprising exposing said irradiated cell to at least one external cargo such that said modulated attribute results in the cargo being

internalized by the cell.

15. The method of claim 14, wherein said cargo is a macromolecule.

16. The method of claim 14, wherein said cargo comprises any of a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a plasmid, a protein, a dye, a polymer, a quantum dot, a nanoparticle, and a protein complex.

17. The method of claim 16, wherein said protein complex comprises a Cas9-gRNA

complex.

18. The method of claim 1, wherein said cell comprises a mammalian cell.

19. The method of claim 1, wherein said cell comprises any of an epithelial cell, a neuron, a fibroblast, a stem cell, an immune cell, and a blood cell.

20. The method of claim 1, wherein said projections comprise a plurality of pyramids.

21. The method of claim 20, wherein said pyramids have a height in a range of about 100 nm to about 100 microns.

22. The method of claim 20, wherein said pyramids have a base length in a range of about 100 nm to about 10 microns.

23. The method of claim 1, wherein said metalized surface comprises a surface of a

substrate having a plurality of projections and a metal layer at least partially coating said substrate surface.

24. The method of claim 23, wherein said substrate comprises a dielectric.

25. The method of claim 23, wherein said substrate comprises a semiconductor.

26. The method of claim 23, wherein said substrate comprises glass.

27. A method of processing cells, comprising:

disposing at least one cell on or near a structured metal surface,

exposing the cell to at least an external cargo, and

irradiating the structured metal surface with a continuous laser radiation at an intensity in a range of about 10² W/cm² to about 10⁵ W/cm² so as to facilitate uptake of the cargo by the cell.

28. The method of claim 27, wherein said continuous laser radiation is applied for a time duration of at least about 1 ms.

29. The method of claim 27, wherein said structured metal surface comprises a plurality of projections having a height in a range of about 100 nm to about 100 microns.

30. The method of claim 29, wherein said projections have a pyramidal shape.

31. The method of claim 27, wherein said cargo comprises a biological agent.

32. The method of claim 31, wherein said biological agent comprises a macromolecule.

33. The method of claim 27, wherein said structured metal surface comprises a metal layer at least partially coating a structured surface of an underlying substrate.

34. The method of claim 33, wherein said structured surface of the underlying substrate exhibits surface height variations in a range of about 100 nm to about 100 microns.

35. The method of claim 33, wherein said metal layer has a thickness in a range of about 10 nm to about 1 micron.

36. The method of claim 27, wherein said underlying substrate comprises any of glass and a semiconductor.

37. The method of claim 27, wherein said at least one cell comprises a plurality of cells and said continuous laser radiation concurrently irradiates said cells.

38. The method of claim 37, wherein application of said continuous laser radiation results in the uptake of the cargo by the cells with an efficiency of at least about 60%.

39. The method of claim 38, wherein said efficiency is at least about 70%.

40. The method of claim 38, wherein said efficiency is at least about 80%.

41. The method of claim 37, wherein application of said continuous laser radiation results in the uptake of the cargo by the cells with a cell viability of at least about 60%.

42. The method of claim 41, wherein said cell viability is at least about 70%.

43. The method of claim 41, wherein said cell viability is at least about 80%.

44. A method of delivering a cargo to a cell, comprising:

irradiating the metalized surface with a continuous laser radiation at an intensity of at least about 10² W/cm² while exposing the cell to an external cargo so as to cause the cell to internalize said cargo.

45. The method of claim 44, wherein the intensity of the continuous laser radiation is in a range of about 10² W/cm² to about 10⁵ W/cm².

46. The method of claim 44, wherein said continuous laser radiation is applied for a time period in a range of about 1 ms to about 10 ms.

47. The method of claim 44, wherein said projections have a pyramidal shape.

48. The method of claim 44, wherein said projections are distributed uniformly across said metalized surface.

49. The method of claim 44, wherein said projections are distributed non-uniformly across said metalized surface.

50. The method of claim 44, wherein said projections have at least one dimension of at least about 20 nm.

51. The method of claim 44, wherein said metalized surface comprises a metal layer at least partially coating a surface of an underlying substrate.

52. The method of claim 51, wherein said metal layer comprises any of gold, silver, copper, and metallic titanium nitride.

53. The method of claim 44, wherein said continuous laser radiation modulates at least one attribute of the cell.

4. The method of claim 53, wherein said modulated attribute corresponds at least to a change in the cell's membrane.