AU2012201290A1 - Methods of increasing cell porosity - Google Patents

Abstract

C:\NRPonbl\DCCSXD\4 18767 I DOC-2/03/2012 Methods of increasing porosity of cells by exposing the cells to microwave radiation are provided. It is desirable to increase the porosity of cells for a variety of purposes, such as to introduce an agent or agents into a cell or cells, to harvest a product or products produced within a cell or cells, to assist in the preservation of cells and to achieve other physiological effects such as to alter the tone of a vessel, to treat or prevent undesirable muscle contraction, to modulate neural cell stimulation or perception by slowing or preventing neural action potentials and thereby reduce nervous stimulation to an organ or tissue or to prevent or reduce pain, and the like.

Description

Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT (ORIGINAL) Name of Applicant: Swinburne University of Technology Actual Inventors: CROFT, Rodney CRAWFORD, Russell IVANOVA, Elena TAUBE, Alex Address for Service: DAVIES COLLISON CAVE, Patent Attorneys, 1 Nicholson Street, Melbourne, Victoria 3000. Invention Title: Methods of increasing cell porosity The following statement is a full description of this invention, including the best method of performing it known to us: C:\NRPortbl\DCC\SXD\4187512_1 DOC - 2/3/12 C:\NRPortb\DCC\SXD\4157567_1 DOC.2A3/2012 Methods of increasing cell porosity Field of the invention The present invention relates to methods of increasing porosity of cells by exposing cells 5 to microwave radiation, and in particular, but not exclusively, to the use of such methods in introducing agents into cells, in harvesting products from cells, in cell preservation, in medical therapies and in molecular biological or genetic interventions conducted upon cells. 10 Background of the invention As it well understood, microwave (MW) radiation is extensively used in modern society in the heating and cooking of food materials due to the convenience and speed of heating that is able to be generated in this manner through the absorption of MW energy by dielectric molecules, especially water, which is transferred into heat due to the internal resistance of 15 rotation. However, although the effects of MW radiation on biological materials, and microorganisms in particular, have been studied and debated for more than half a century there is still relatively little known about postulated and so-called "specific MW effects" 20 that are non-thermal in nature. Much literature has been published supporting the notion that a range of specific MW effects exist, and can be identified in terms of their manifestation on cell physiology (9). For example, Dreyfuss and Chipley examined the effects of MW radiation (2.45 GHz) at sub-lethal temperatures on the metabolic activity of a range of enzymes expressed by the bacterium Staphylococcus aureus (7). These results 25 suggested that MW radiation affected S. aureus cells in a way that could not have been explained solely by thermal effect theories. It has also been found that Burkholderia cepacia bacteria could be wholly inactivated using MW radiation at sub-lethal temperatures at a frequency of 20 GHz (7). Samarketu et al. (17) examined the effects of MW radiation at a frequency of 9.575 GHz on the physiological behaviour of 30 Cyanobacterium dolium (Anabaena dolium). The authors suggested that MW radiation non-thermally induced different biological effects by changing the protein structures by C :NRPonb\DCC\SXD4 187567_ DOC-2103/2O12 -2 differentially partitioning the ions and altering the rate and/or direction of biochemical reactions. It was also suggested that turbidity of the cell suspension, protein, carbohydrate, chlorophyll A, carotenoids and phycocyanin of microwave-exposed samples were inversely correlated with higher modulation frequencies. Previous studies completed by 5 the present research group have also demonstrated that sterilisation of raw meat (19) as well as transplant biomaterial (18) could be achieved using MW radiation under defined MW settings and solute concentrations. To date, however, no available research has demonstrated a solid understanding of how MW radiation (at a defined range of frequencies) causes certain effects on biological cells such as those of microorganisms, and 10 why they occur. In investigating the effects of MW radiation on a range of different cell types the present inventors have demonstrated that under the appropriate conditions exposure of cells to MW radiation, at a level that substantially avoids a lethal heating effect, causes a 15 temporary increase in cellular porosity, and that this effect can be exploited in a range of useful ways, as will be further described in detail herein. Summary of the invention According to one aspect of the present invention there is provided a method of temporarily 20 increasing porosity in a cell or cells comprising exposing the cell/s to microwave radiation for a time and under conditions selected to substantially avoid lethal heating of the cell/s; which results in a temporary increase in porosity of the cell/s relative to the porosity of conventionally heated equivalent cell/s and does not adversely affect cell viability relative to that of the conventionally heated cell/s. 25 In another aspect of the invention there is provided a method of introducing an agent or agents into a cell or cells comprising exposing the cell/s to microwave radiation for a time and under conditions selected to substantially avoid lethal heating of the cell/s, which results in a temporary increase in porosity of the cell/s relative to the porosity of 30 conventionally heated equivalent cell/s and does not adversely affect cell viability relative to that of the conventionally heated cell/s; and exposing the cell/s to the agent/s to be C:.NPonb\DCC\SXD\4187567 I DOC-2A)3/2012 -3 introduced into the cell/s so that the agent/s is/are available for passage into the cell/s during the temporary increase in porosity of the cell/s. In a further aspect of the invention there is provided a method of harvesting a product or 5 products of interest from a cell or cells comprising exposing cell/s that produce product/s of interest to microwave radiation for a time and under conditions selected to substantially avoid lethal heating of the cell/s, which results in a temporary increase in porosity of the cell/s relative to the porosity of conventionally heated equivalent cell/s and does not adversely affect cell viability relative to that of the conventionally heated cell/s; and 10 washing the cell/s with an appropriate media during or after the temporary increase in porosity of the cell/s and then removing product/s containing media from the cell/s. In another aspect of the invention there is provided a method of preserving a cell or cells comprising exposing the cell/s within a liquid growth media to microwave radiation for a 15 time and under conditions selected to substantially avoid lethal heating of the cell/s; which results in a temporary increase in porosity of the cell/s relative to the porosity of conventionally heated equivalent cell/s and does not adversely affect cell viability relative to that of the conventionally heated cell/s; removing said growth media from the cells during the temporary increase in porosity of the cell/s and freezing the cell/s. 20 An aspect of the invention is also directed to a method of treating or preventing undesirable muscle contraction or spasm in a mammalian subject comprising exposing cell/s subject to or that control muscle contraction or spasm to microwave radiation for a time and under conditions selected to substantially avoid lethal heating of the cell/s, which 25 results in a temporary increase in porosity of the cell/s relative to the porosity of conventionally heated equivalent cell/s and does not adversely affect cell viability relative to that of the conventionally heated cell/s; wherein the increase in porosity of the cell/s prevents muscle contraction.

C :RPorb\DCCSXDWl87567_ DOC-2/03/12 -4 Brief description of the figures The invention will be further described with reference to the following non-limiting figures, wherein: 5 Fig. I shows a graph of temperature (degrees Celsius) against time for the cells subject to MW processing (squares) and the Peltier Plate heating/cooling control treatment (diamonds). Fig. 2 shows typical SEM images of E. coli. MW treated cells immediately following 10 radiation exposure (a, b); untreated control cells (c, d); MW treated cells, 10 minutes following radiation (e, f; thermally heated control cells immediately following Peltier Plate treatment (g, h), wherein the arrow indicates a leak of cytosolic fluids out of the E. coli cell. 15 Fig. 3 shows typical 2D confocal microscopy images of E. coli, untreated control cells (a); MW treated cells (b); and Peltier Plate treated control cells (c). Fig. 4 shows typical scanning electron micrographs of the super high frequency microwave radiation effect on the four Gram positive coccoid bacteria; P. maritimus KMM 3738, 20 S. aureus ATCC 25923, Staphylococcus aureus CIP 65.8 T and S. epidermidis ATCC 14990"', after 1 minute and 10 minutes following MW treatment. No significant change of cell morphology was observed. Scale bars are 10 tm, inset scale bars are 200 nm. Also provided are two-dimensional CLSM images showing intake of 23.5 nm nanospheres (above) and 46.3 nm nanospheres (below) after 1 minute following MW 25 radiation. Notably, after 10 minutes following MW radiation, both types of the nanospheres were not detected inside the bacterial cells. Scale bars in all fluorescence images are 5 pm. Fig. 5 shows a bar graph of percentage recovery rate for each of the four Gram positive 30 coccoid bacteria after either exposure to heat treatment (clear) or MW treatment (shaded), C:\NRPertbl\DCOSXD\4187567_1.DOC-20312012 -5 to demonstrate the effect of super high frequency microwave radiation and heat on cell viability. Fig. 6 shows typical scanning electron micrographs of the super high frequency microwave 5 radiation effect on the rigidity of the four Gram positive coccoid bacteria: P. maritimus KMM 3738, S. aureus ATCC 25923, S. aureus CIP 65 .8T and S. epidermidis ATCC 14990T, on the insect Clanger cicada Psaltoda claripennis wing membrane (control) and the same bacteria after MW radiation (MW treated samples). Irradiated cells appeared ruptured by the wing nanopillars due to decreased inner pressure and cell rigidity. Scale 10 bars on all electron micrographs are 200 nm. Fig. 7 shows a schematic outline of the experimental design showing the super high frequency microwave radiation effect on four selected Gram positive coccoid bacteria. (a) Under normal conditions, nanospheres cannot penetrate inside bacterial cells. 15 (b) Following microwave radiation, bacterial cell membranes undergo reversible poration resulting in the internalizing nanospheres inside the cells. Only MW treated bacterial cells are ruptured by the insect wing nanopillars as the consequence of the decreased inner pressure and rigidity of the cells. (c) Non-treated bacterial cell are not ruptured by the nanopillars. 20 Fig. 8 shows typical scanning electron micrographs of untreated and heat treated controls for the four Gram positive coccoid bacteria: Planococcus maritimus KMM 3738, Staphylococcus aureus ATCC 25923, Staphylococcus aureus CIP 65.8T and Staphylococcus epidermidis ATCC 14990'. A dehydrated appearance of heat treated 25 bacteria cells was observed. Scale bars are 10 pm, inset scale bars are 200 nm. Also shown are 2D CLSM images confirming there were no poration on the membrane of the untreated and heat-treated controls, through the absence of 20 nm nanospheres in green (middle row) and 50 nm nanospheres in red (bottom row) of the four selected Gram positive coccoid bacteria. Scale bars in all fluorescence images are 5 pm. 30 C:NRPortb\DCC\SXDu I87567-DOC-2I3/2012 -6 Fig. 9 provides a calibration curve of relative fluorescence units (RFU) against nanosphere concentration (mg ml- 1 ) for (a) 23.5nm green FITC-conjugated silica and (b) 46.3nm red Rhodamine B-conjugated silica nanospheres. 5 Detailed description of the invention Bibliographic details of documents referred to in this specification are listed at the end of the specification. The disclosures of all documents referred to in this specification are included herein in their entirety by way of reference. 10 The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. 15 Throughout this specification and the accompanying claims, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or 20 integers. As used herein the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell, as well as two or more cells; reference to "an agent" includes one agent, as well 25 as two or more agents; and so forth. The present invention relates generally to methods of increasing porosity of cells by exposing the cells to microwave radiation. It is desirable to increase the porosity of cells for a variety of purposes, such as to introduce an agent or agents into a cell or cells, to 30 harvest a product or products produced within a cell or cells, to assist in the preservation of cells and to achieve other physiological effects such as to alter the tone of a vessel, to treat C:NRPonbNlDCC\SXD\4 87567 .DOC-2/103/2012 -7 or prevent undesirable muscle contraction, to modulate neural cell stimulation or perception by slowing or preventing neural action potentials and thereby reduce nervous stimulation to an organ or tissue or to prevent or reduce pain, and the like. 5 The methods according to the present invention can be conducted in conjunction with single selected cells or populations of cells that are located in vitro, ex vivo (with the intention of returning cells so treated to an organism) or in vivo. It is possible to apply the methods according to the present invention to both prokaryotic and eukaryotic cells, including cells of bacterial, fungal, protozoa, helminth, algae, plant and animal origins. In 10 conducting the methods of the invention it is, however, desirable to expose the cells in which increased porosity is desired to a relatively even microwave radiation energy in order that heating of the cells due to microwave energy absorption can be controlled within desired parameters. This may require the adoption of a suitable platform, stage or pedestal upon which in vitro or ex vivo cells can be located. In the case of cells located within an 15 organism such as a plant or animal it will be desirable to utilise a microwave radiation generating device that allows for focused and even exposure to the desired cells. For example, in the case of exposure of in vivo mammalian cells to microwave radiation it may be appropriate to adopt an endoscopic microwave generating device to ensure local and even microwave energy exposure to the desired cells. In the case of harvesting of a 20 product or products from cells this can conveniently take place in a commercial scale fermentation facility, wherein it is possible to pass broth including the cells through a conduit or pipe in which they are exposed to microwave radiation and wherein the flow speed of the cells within the broth and residence time within the zone within which the cells are exposed to the radiation can be controlled within desired parameters. 25 Poration of cell membranes is known to be caused by a rearrangement of the molecular structure of the membrane, which can be physically induced through exposure to stimuli such as mechanical stress, ultrasound (sonoporation), electrical field (electroporation) and laser light (photoporation). Membrane poration is believed to lead to the relaxation of 30 surface tension of cell membranes as well as a reduction in the osmotic pressure of a cell due to the release of cytosolic components through the formed pores. The initial rupture of C:NRPortbDCC\SXD4 197567 I.DOC-2/03/2012 -8 the cell membrane leads to the formation of cylindrical pores which keep increasing in size until reaching a zero surface tension (24). It is possible to identify and to quantify an increase in cellular porosity in a variety of 5 conventional ways, such as for example by scanning electron microscopy (SEM) and the use of confocal laser scanning microscopy (CLSM) in conjunction with the use of a fluorescent dye probe that has either previously been introduced into the cells prior to MW radiation exposure or is introduced into the media prior to MW radiation exposure, the passage of which either into or out of the cells can be detected and/or quantified. In a 10 similar way it is possible to identify and/or measure an increase in porosity of cells through the use of coloured, reactive, radioactive and/or magnetic agents, for example, that are of an appropriate size to allow passage into or out of cells when the cells are in a porous state. For example, such agents may have a diameter of from about 5 nm to about 60 nm, such as from about 10 nm to about 50 nm, from about 15 nm to about 45 nm or from about 20 nm 15 to about 30 nm. Microwave (MW) radiation is a component of the electromagnetic spectrum. There is some conjecture regarding the delineation of different components of the electromagnetic spectrum such that depending upon the field of interest the understanding of what 20 constitutes MW radiation can differ. MW radiation is sometimes defined to include that radiation having a wavelength ranging from approximately 3000 m to 1 mm, with a corresponding frequency of approximately 100 KHz to 300 GHz, respectively. However, in the context of the present invention it is generally more convenient understand MW radiation as meaning that radiation in the Super High Frequency range of from about 1.0 25 GHz to around 40 GHz and the Extremely High Frequency range of above about 40 GHz. In specific aspects of the invention the MW radiation utilised can have a frequency of from about 1.0 GHz to about 300 GHz, such as from about 5 GHz to about 100 GHz, from about 15 GHz to about 25 GHz, from about 17 GHz to about 22 GHz or about 18 GHz, 19 GHz or 20 GHz. 30 The present inventors have determined that MW radiation has a direct effect upon cells to C:WRPonb\DCC\SXD\4187567_ .DOC-213/2012 -9 increase their porosity, that is to increase the extent and rate at which agents are able to enter and/or exit cells subject to the treatment, which is often determined by identifying leakage of cytosolic components from the cells. The inventors understand this effect to be a specific effect of the MW radiation that is not related to the heating effect that 5 microwave radiation also has upon cells. Clearly, however, excess heating of cells is generally deleterious as a result of irreparable damage to lipid membranes and denaturation of proteins. While the specific parameters of predisposition to the effects of heating vary between cell types, it is generally desirable to limit the heating of cells when conducting the invention to a maximum temperature of about 40*C. To avoid undue heating of the 10 cells that may adversely affect viability it may also be appropriate in specific aspects of the invention to control the rate of heating of the cells, such as for example within the parameters of about 5 0 C/min to about 80'C/min, alternatively between about 10'C/min to about 40*C/min, such as for example a heating rate of about 20*C/min. Clearly the time of exposure of the cells to MW radiation will depend upon the energy of the microwave 15 radiation adopted and other conditions such as the nature of the cells, the media being utilised, the ambient temperature and the heat transfer characteristics of the vessel in which the MW radiation exposure is conducted. For example, however, the exposure to microwave radiation can be conducted for a period of from about 15 seconds to about 4 minutes, such as from about 30 seconds to about 2 minutes, from about 45 seconds to 20 about 90 seconds or about 1 minute. In another aspect of the invention, which is independent of the purpose for which the increase in cellular porosity is desired, the exposure to MW radiation can be conducted repeatedly, such as for example from 2 to 5 times. Between cycles of MW radiation 25 exposure the cells can be left to cool or may be exposed to some form of cooling treatment, such as by passing the cells within a fermenting apparatus into a cooling chamber, or conduit or another form of heat exchanger through which temperature can be lowered, by including a flask or vessel in which the cells are contained within an ice bath, a refrigerator or freezer or by exposing the vessel to a current of a cooling fluid. In the case of cells 30 being treated in the in vivo context it is possible to apply a cooling agent directly to the relevant location within the organism.

C:\NRPonbDCC\SXD\41577_.DOC-2/3/2012 - 10 The inventors have identified that in conducting the methods according to the invention porosity of the cells exposed to the MW radiation treatment is temporarily increased before substantially returning to original levels. Depending upon the specific cells being treated, 5 the nature of the media in which they are growing, the energy of MW radiation exposure, the time of exposure and extent and rate of heating of the cells during this exposure there is likely to be variation over the duration of increased porosity. For example, however, increased porosity is likely to persist for a period of from about 2 minutes to about 20 minutes, such as from about 5 minutes to about 15 minutes, from about 8 minutes to 10 about 12 minutes or, for example, about 10 minutes. It is, however, within the capabilities of a skilled person to optimise the time and conditions of exposure to the MW radiation in view of the parameters referred to above, in order to achieve a desired extent and duration of increased porosity. 15 In referring to the increased porosity of cells subject to the treatment of the present invention it is to be understood that the identification of an increase and its quantification is relative to the porosity that is achieved in respect of an equivalent cell or population of cells that is heated at the same rate and extent using conventional thermal heating means. As would be well understood by a skilled person, in conducting this comparison it is 20 important for all parameters other than the means of heating, such as the type and concentration of cells, the media in which they are growing, the vessel in which they are contained, the ambient temperature, etc., to be equivalent for both the cells exposed to MW radiation and the equivalent cells exposed to conventional heating. 25 A similar analysis is conducted in comparing the viability of the cells subject to MW radiation exposure with equivalent cells subject to conventional heating. In this comparison the viability of the cells, that is whether the cells remain alive and retain the ability to grow and divide, can be measured directly by microscopic observation and conventional cell counting techniques and can also be inferred by the conduct of 30 conventional cell viability assays. These for example include determining the ratio of potassium to sodium, assaying for lactate dehydrogenase which is an indicator of cell lysis, C:\NRPornbl\DCC\SXD\4187567- IDOC-21/112012 - 11 assaying for mitochondrial activity, motility, deformability, osmotic fragility, haemoglobin content or the like, as appropriate depending on the nature of the cells concerned. By the phrase "does not adversely affect cell viability relative to that of the conventionally 5 heated cell/s" it is to be recognised that while on a direct comparison of the cells exposed to the MW radiation against those exposed only to equivalent heating it may be that there is a small reduction in cell viability within the MW radiation treated cell population on an absolute basis. However, in preferred aspects of the invention the reduction of viability in the MW treated cell population will be less than 20% more than any reduction in viability 10 in the heat only treated cell population, preferably less than 15%, less than 10% or less than 5%. However, it is desirable that there is no statistically significant variation in viability between the MW radiation exposed cell population and the equivalent cells exposed to conventional heating. As is readily apparent, in order to make a determination of statistical significance it is appropriate to conduct duplicate tests, for example at least 5, 15 preferably at least 10 duplicates. Conventional statistical analyses can conveniently be conducted. Statistical data processing can for example be performed using SPSS 15.0 (SPSS Inc., Chicago, Illinois, USA), where three independent T-tests can be conducted to compare consistency across conditions, experiments and bacterial test strains. 20 In methods of introducing an agent or agents into cells according to the invention there are a variety of agents that are comprehended. Such agents include, but are not limited to, pharmaceutically and veterinarally active agents, dyes, radioactive, fluorescent or magnetic labelling agents, nutrients, proteins, peptides, polysaccharides, contrast agents, imaging 25 agents, reagents for cellular processes, nucleic acids, infectious agents, antibodies, immunoglobulins or fragments thereof, plasmids, enzymes, neurotransmitters, cell signalling agents, modulators of gene expression or regulation; nanoparticles and devices, and the like. In introducing such agents into cells it is appropriate for the agents to be made available for passage into the cells during the period in which the cells are 30 experiencing a temporary increase in porosity, such as by administering or otherwise placing the agent into the environment in which the cell exists. For example, this can be C:\NRPorbl\DCC\SXDW1X7567_I DOC.2/)3/2012 - 12 by including the agent within a media or broth in which the cells are located, by placing the cells into the blood stream of an animal or into the extracellular fluid adjacent the cells of interest, for example including the agent within xylem or phloem of a plant. In the case where the cells are located in vivo within an animal such as a mammal or human subject, to 5 which it is desired to assist uptake of the agent into a specified cell population, it is possible to administer the agent via conventional drug administration routes such as by oral, rectal, nasal, topical (including buccal and sublingual), vaginal, intravesical or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. In the case of administration of agents, such as pharmaceutical agents, to 10 humans they may conveniently be formulated into compositions in unit dosage form and may be prepared by methods well known in the art of pharmacy. Such methods include the step of bringing into association an active ingredient with a carrier, which constitutes one or more accessory ingredients. In general, compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers, diluents, 15 adjuvants and/or excipients or finely divided solid carriers or both, and then if necessary shaping the product. Further details of conventional pharmaceutical compositions are explained in Remington's Pharmaceutical Sciences, 18 th Edition, Mack Publishing Co., Easton, PA. USA, the disclosure of which is included herein in its entirety by way of reference. 20 For example, methods according to the invention can conveniently be conducted to assist transfer of pharmaceutically active agents transdermally, for example by formulating the agent in the form of a cream, ointment, jelly, solution or suspension for topical administration, by formulating the agent for topical application to the eye in the form of a 25 solution or suspension in a suitable sterile aqueous or non-aqueous vehicle, by formulating the agent for rectal administration by presentation in the form of a suppository with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at a rectal temperature and that will melt in the rectum to release the active ingredient. Such excipients include cocoa butter or a salicylate. Similarly, agents can be formulated for 30 nasal compositions for administration across the nasal mucosa in the form of nose drops or sprays and may similarly be formulated for administration across the alveolar cells in the C:\NRPonbtlDCC\SXD\4187567_l DOC-2A3/2012 -13 lungs by formulation as an inhalable spray or appropriately dispersed finely divided powder. It is also possible to administer agents utilising methods according to the invention across 5 the blood brain barrier, with appropriate exposure of the cells of the blood brain barrier to the MW radiation treatment as outlined herein. In one aspect of the invention the method of increasing cellular porosity can be conducted on a mammalian, or in particular a human, patient as a means of treating or preventing 10 undesirable muscle contraction or spasm. Such therapy may be appropriate in cases of muscle cramping or spasm resulting from fatigue, injury or other diseases or disorders, wherein relief can be obtained by exposing the cells in question, or neural cells controlling them, to targeted MW radiation exposure, within the parameters previously discussed herein. In this way the increased porosity of the muscle cells or of controlling neural cells 15 will prevent or reduce the extent of muscle contraction to thereby offer relief to the patient's symptoms. In this context the muscle cells can be muscular skeletal cells such as those in the back, legs, arms, neck or thorax of a human patient. However, the cells can equally be smooth muscle cells, for example located within the vasculature, gastrointestinal system or respiratory system. In one aspect of the invention the therapy 20 can be conducted in relation to alveolar smooth muscle cells to thereby reduce the constriction associated with respiratory complaints such as chronic obstructive pulmonary disease (COPD) and asthma. The present invention also provides particular advantage in conjunction with harvesting 25 products produced within cells. In the in vivo context within animals it may be desired to harvest naturally produced materials such as milk, immunoglobulins, hormones, enzymes and the like. Similarly, in the case of genetically modified organisms the techniques of the present invention can be used to assist in the harvest of specialty chemicals, particular antibodies or immunoglobulins or other products that may be produced for example within 30 milk. The techniques of the invention can also be utilised in harvesting products produced by cells in vitro or indeed in large scale commercial fermentation facilities, such as C:\NRPonblDCCSXDW 175671 .DOC-2/3/2012 -14 specialty chemicals, antibodies or immunoglobulins, enzymes, vitamins and biofuels or agents from which they can be produced. In this context the exposure to MW radiation and resultant increase in porosity of the cells will assist in the removal of the desired product from the cells, which will result from removal of the media during or after the 5 temporary increase in porosity. Efficient harvesting of the product can be assisted by washing the cells with media to ensure the existence of an appropriate concentration gradient. In the case of production of products in vivo in an animal it may be appropriate, for example, to expose the mammary tissue of an animal to MW radiation under the parameters of the invention, prior to milking to assist with harvesting of product, 10 particularly in the case of larger product molecules that may otherwise be difficult to harvest. Methods of the invention can also be used in conjunction with approaches for improving preservation of cells. One problem associated with conventional cryogenic preservation 15 methods is that water within cytosolic fluids can crystallise and cause cell damage during freezing. By utilising the methods of the invention it is possible to dehydrate the cells at or just prior to the time of freezing to reduce this problem of crystallisation induced damage. In this context it is convenient to expose the cells to be preserved to the MW radiation prior to removing the growth media from the cells, which may conveniently be achieved 20 by evaporative techniques. In one aspect a freeze drying or lyophilisation process is conducted during the period in which the cells are undergoing the temporary increase in porosity. In particular, it is possible to preserve sperm, ova, early stage embryos, stem cells and blood cells, including red blood cells, utilising this technique. 25 The present invention will now be described further, with reference to the following non limiting examples. Examples Example 1 - Study of specific electromagnetic effects of microwave radiation on 30 Escherichia coli Materials and methods C:\NRPonb\DCCtSXDWI7567_L DOC-2A3/2012 - 15 Bacterial strain, cultivation procedure and sample preparation. E. coli ATCC 15034 was used as test strain in all experiments. The bacterium was obtained from the American Type Culture Collection (USA). Pure cultures were stored at -80"C in nutrient broth (NB) (Oxoid) supplemented with 20% (v/v) of glycerol. The bacteria were routinely cultivated 5 for 24 hours on nutrient agar (NA), (Oxoid). Working bacterial suspensions were freshly prepared for each independent experiment as described elsewhere (18, 19). The cell density was adjusted to 108 colony forming units (CFU per mL) (OD600 = 1.0 in phosphate buffered saline (PBS), 10 mM, pH 7.4, using a spectrophotometer (Amersham Biosciences - Gene Quant Pro) from a bacterial culture grown overnight in 100 mL in NB. 10 Bacterial cells were collected during the logarithmic phase of growth as confirmed by growth curves (data not shown). The bacterial cell suspensions were further subjected to direct counting using a haemocytometer to confirm the number of bacterial cells as described elsewhere (9). Bacterial samples for MW analysis comprised of 2 mL of working suspensions that were transferred into a micro Petri dish (35 mm diameter, 15 Griener). Microwave apparatus. The MW apparatus that was used in the present study had the option of a variable frequency ranging from 5 to 18 GHz (Lambda Technologies Vari Wave Model LT 1500). The LT1500 is a computer controlled variable frequency 20 processing cavity for delivering excellent levels of control and uniformity of energy distribution into a multi-mode microwave cavity. A schematic diagram of the MW apparatus setting has been reported elsewhere (19). Both the amplitude and frequency of the microwave power could be varied, allowing a significant expansion of the parameter space within which the system could be optimized. A data logging option allowed 25 processed data capture from the embedded computer system over a standard RS-232-C serial interface. A cavity characterization option was also available which allowed an evaluation of the performance of a material in the cavity to assist in determining the optimum processing conditions. 30 Microwave settings. Each bacterial sample was transferred into the MW chamber. The latter had its core temperature monitored through the attachment of a fibre optic probe. In C:NRPOnb\DCC\SXDi8767_ .DOC-M/2012 -16 order to minimise thermal MW effects, the bulk temperature rise of the bacterial suspension during exposure was maintained below 40'C, since this is the temperature at which the bacteria were determined to be unaffected by heat. Given that MW frequency is inversely correlated to wavelength, the highest available frequency (18 GHz) was used in 5 all experiments as it produced the shortest wavelength which is comparable to the bacterial cell diameter and would therefore have the maximum effect on cell kinetics. For uniformity of exposure, each sample was placed onto a ceramic pedestal (Pacific Ceramics Inc. PD160, s' = 160, loss tangent < 10-) within the same position in the 10 chamber which had been determined by electric field modelling using CST Microwave Studio 3D Electromagnetic Simulation Software. The experimental apparatus for the MW treatment involved placing the sample in a predetermined location within the MW chamber and subjecting it to a specified radiation 15 treatment at a heating rate of 20'C/min for 1 minute, thereby maintaining the temperature rise from 20'C to 40'C. In order to maximise the specific electromagnetic MW effect, a previously optimised "repeated exposure" technique was employed (18, 19). Each sample was exposed to MW radiation for three consecutive exposures allowing the sample to cool to 20*C on ice (at a rate of I 0C per min), in between exposures. The temperature profile 20 (by time) is illustrated in Fig. 1. Thermally heated control samples. In order to ensure that any effects caused by MW radiation were not purely a result of thermal heating, a control sample was used. A Peltier plate heating/cooling system (TA Instruments) was used to replicate the temperature 25 gradients being experienced by the bacteria during MW processing. A total volume of 2 mL of working bacterial suspension was placed onto the Peltier plate and subjected to heating from 20 0 C to 40'C at a rate of 20*C per min for three consecutive exposures, with identical cooling times as for MW treatment (at a rate of 10*C per min) in between trials (Fig. 1). All experiments using MW treated samples (in triplicate) were performed in 30 parallel with the Peltier plate heated samples.

C:\NRPorb6DCC\SXDW 17567 _.DOC-2/0312012 - 17 Scanning Electron Microscopy (SEM) analysis. A FeSEM - ZEISS SUPRA 40VP was used to obtain high-resolution images of the bacterial cells according to previously developed protocol (9). Primary beam energies of 3 to 15 kV were used, which allowed features on the sample surface or within a few microns of the surface to be observed, 5 respectively. Immediately following radiation and Peltier plate treatment, 100 pL aliquots of the bacterial suspensions were transferred onto a glass cover slip. Half of the samples were immediately washed with distilled water, air dried (this process took less than 60 seconds 10 to complete) and then sputter coated with gold prior to imaging according to a previously developed laboratory protocol (14). The remainder of the samples were left to stand for 10 minutes and then transferred to a cover slip, washed, dried and sputter coated. This was performed to identify any changes in cell morphology following MW radiation, and to determine whether these changes were reversible with time. The control for these 15 experiments consisted of 100 pL of untreated bacterial suspension that was transferred onto a glass cover slip, washed with distilled water, air dried and sputter coated with gold. Approximately 20 SEM images taken at x5000 magnification were analysed. Recorded densities have estimated errors of approximately 10% due to local variability in the 20 coverage. The number of cells observed from the SEM images prior to and following treatment was transformed into a number of bacteria per unit area and the percentage of affected cells was calculated accordingly. Statistical data processing was performed using the SPSS 16.0 software (SPSS Inc., Chicago, Illinois, USA). 25 Confocal Laser Scanning Microscopy (CLSM) analysis. CLSM analysis was performed in order to determine whether the bacterial cell membrane was damaged as a result of MW radiation exposure, thus allowing cell permeability. A fluorescent dye probe (FITC Dextran; 150 kDa; Sigma-Aldrich) was added to the bacterial suspension at a concentration of 25 pg/mL. Bacterial suspensions were then subjected to MW radiation 30 and Peltier Plate treatment at the optimised settings. Following radiation and Peltier Plate treatment, 1 mL of each suspension was washed twice and resuspended (centrifuged at C :NRPonbl\DCC\SXD4i87%57_LDOC-2/03120)I2 - 18 5000 rpm for 3 minutes and having supernatant removed). A volume of 20 PL of each suspension was then transferred to a glass slide and viewed using an Olympus FluoViewTM FV1000 Spectroscopic Confocal System, which includes an inverted Microscope System OLYMPUS IX81 (20x, 40x (oil), 100x (oil) UIS objectives) and 5 operates using multi Ar and HeNe lasers (458, 488, 515, 543, and 633 nm). The control for these experiments consisted of I mL of untreated bacterial suspension mixed with the fluorescent probe and processed simultaneously with treated bacterial suspensions. Approximately 15 CLSM images were analysed (5 images per treatment group). 10 In order to tentatively determine the size of formed pores, an experimental equation (Eq. 1.) was used, which takes into account the radius of dextran molecules in relation to its molecular weight (10). Radius of dextran molecule (A) = 0.33(MM) 0.46 (1) where: Radius of dextran molecule (A) - Angstrom (1 x 10-10 metres) 15 MM - Molar Mass (Da) Cell Viability. In order to investigate the effects of MW processing on bacterial cell growth/viability, working bacterial suspensions were subjected to MW radiation or Peltier Plate treatment. Sampl es were then diluted to a concentration of approximately 300 20 cfu/mL as described elsewhere (6), then 100 pL of each suspension was spread onto NA plates and incubated for 36 hours at 37 0 C. The control for these experiments consisted of an untreated bacterial suspension that was processed simultaneously with the treated samples. Evaluation of the colony formation (in cfu) and corresponding statistical analysis were performed as described elsewhere (6). 25 Theoretical aspects This study was designed using accurately controlled experimental conditions and well defined MW radiation parameters. The modelling of the distribution of the electric field using CST Microwave Studio 3D Electromagnetic Simulation Software allowed the C:\NRPonbl\DCC\SXD\4 17567_ DOC-2013/2012 - 19 electric field absorbed by the sample to be determined. This value was approximated to be 1500 kW/m 3 . Theoretical calculations were also employed to validate the modelling analysis. 5 Equation 2 depicts a theoretical calculation using the general thermo-dynamic formula for absorbed power. P~b _ cpAT V t (2) where: AT - the increase of the mean temperature of the heated body P - microwave power used for heating (W) 10 V, c, p - volume, heat capacity, density (m 3 , J/kg x K, kg/m 3 ) t - time of heating (s) Using Equation 2 and the values c = 4200 J/kg/*C, p = 1000 kg/m 3 , AT = 20 *C and t = 60 s the power absorbed by the sample was calculated to be approximately 1600 kW/m 3 . A 15 further calculation was completed to verify the absorbed power in a MW processing system. The value of the MW power absorbed in the unit volume of the heated load (Pv) is described theoretically in Equation 3. Pv =2 x 7E x f x EO x s''E1 2 (3) where: Pv - power absorbed in the unit volume of the load (W/m 3 ) 20 f - microwave frequency (Hz) so - permittivity of free space (F/m) C'' - dielectric loss factor of the load - relative value (1) E - strength of the electric field inside of the load (V/m) 25 By substitution of values: f = 18 GHz, so = 8.85 x 10-12 F/m, s'" = 36.34, E = 300 V/m and using Equation 3, the absorbed power was calculated to be at approximately 1400 kW/m 3 . A comparison of these analyses (absorbed power calculated using Equations (2) and (3) and the Software simulation) highlighted consistency between the experimental and modelling results. 30 C NRPonbl\DCC\SXDWI 17567_ .DOC-2A32012 - 20 Furthermore, it was determined that the total biomass of the bacteria in the suspension was significantly lower than the total mass of water in the suspension, and therefore could be taken as negligible for the purposes of modelling calculations. (Multiplying the approximate number of cells in the suspension (0.8 x 108 cells/mL) by the generally 5 accepted average mass of a single E coli cell (9.5 x 10-1 grams (15)) indicated that the overall biomass in the bacterial suspension was approximately 1.52 x 10-7 grams (in 2 mL suspension) which could be regarded as an insignificant mass). Based on previously reported data regarding the dielectric properties of bacterial cells and 10 organelles (8), it was assumed that the dielectric loss factor of the bacterial sample was lower than for the surrounding liquid. The heating within a heterogeneous system can be expressed by the ratios of the temperature rise rate: TAT] ~AT] C bacteria - + - = ..""'"___ x bactera __e___ At Jwafer _ At - bacteria bacic. Civaler Pvaer (4) where: AT/At - rate of temperature rise 15 Cwater - specific heat capacity (kJ/kg K) for water and bacteria pw and Pb - density (kg/m 3 ) of water and bacteria Thus, rate of change in temperature of the bacteria would be lower than that for the surrounding liquid. The change of dielectric properties during the microwave processing 20 was considered insignificant due to the fact that in the temperature interval from 20'C to 40*C, dielectric loss will not change by more than 10% (20). The average dielectric constant of the bacterial suspension was therefore assumed to be that of water at 18 GHz, Er = '+ j" = 44 + j36 (36). Similarly, the specific heat of the 25 total sample was taken as that of water. In order to determine whether the temperature inside the bacterial cells was the same as the surrounding medium, the depth of penetration of MW radiation was also determined using C:W\RPorbl\DCC\SXD\4187567_1.DOC-2/03/2012 -21 Equations 5-7. At the frequency of 18 GHz, the wavelength of the microwave in a vacuum was calculated using Equation 3 to be 16.7 mm. 3 x 10 (m/s) s18 x 101 (1/s) where: k18 - wavelength of microwave in a vacuum at 18GHz 5 m - length in metres s - time in seconds The wavelength in water was determined to be 2.34 mm using equation (6) w 1+ tan2' +1 (6) 10 where: water - wavelength of microwave in water ?I - wavelength of microwave in a vacuum (16.7 mm) Er' - static relative permittivity of water, C'=44 at 25*C and 18 GHz tan 6 - loss tangent, tan 8 = 0.821 at 25'C and 18 GHz 15 From this calculation the depth of penetration of microwave radiation into the sample was determined to be 1.04 mm (Equation 7). 2 22 2;r s'V1 +tan T3 - I ) where: Z - depth of penetration (power / energy) k - wavelength of microwave in a vacuum 20 Er - static relative permittivity of water, E'=44 at 25'C and 18 GHz tan 8 - loss tangent, tan 6 = 0.821 at 25'C and 18 GHz Given that the total depth of the sample in the micro Petri dish was calculated to be 1.00 mm, it was assumed that complete penetration was achieved. In light of the assumptions C :\RPonbKlDCC\SXDW 187567_ DOC-2/03/2012 - 22 and calculations, the temperature inside the bacterial cells was suggested to be the same as the temperature in the surrounding medium. Results and discussion 5 The SEM analysis in the present study revealed that E. coli cells that were dried for 60 seconds following MW radiation exhibited a different cell morphology compared to that of all other treatment groups (Fig. 2). As can be seen from the images in Fig. 2 (a and b), up to 98% ± 1% the cells had a dehydrated appearance in contrast to all control groups where no change in morphology was observed (Fig. 2 c - f). A Statistical analysis revealed a 10 significant difference between this data t(18) = 8.77, p < 0.05. Cell viability experiments revealed up to 87.7% ± 4% of MW treated E. coli cells and 89.9% ± 2% of E. coli cells after Peltier Plate treatment remained viable and were recovered on nutrient agar plates. A statistical analysis of the data revealed that no 15 significant differences existed between the two treatment groups (p > 0.05). It can be inferred from these results that the specific MW effect was not bactericidal under the current experimental conditions. The small reduction in bacterial cell numbers following both MW and Peltier plate processing was therefore most likely due to thermal shock. It has been reported previously that even slight increases in temperature can have weak 20 bactericidal effects (1). Our previous studies (18, 19) which accomplished near complete bacterial inactivation following MW radiation were performed at 45*C, suggesting that by varying certain experimental conditions such as temperature and/or osmolarity, a diversity of specific MW effects may be manifested. 25 Our current observations are consistent with the explanation that the application of MW radiation causes a disruption to the cellular membrane, such that the cytosolic fluids within the E. coli cells are able to pass through the cellular membrane. This effect however, appeared to be temporary, as the morphological shape of cells appears to have recovered within 10 minutes after the application of MW radiation (Fig. 2 c and d). This is most 30 likely due to the re-absorption of the fluids back into cells. The morphology of all Peltier Plate treated cells (Fig. 2 g and h) examined immediately following treatment appeared C:4RPorb\DCC\SXD\4187567_ DOC-23/2012 -23 unaffected and identical to those of non-treated control cells. To our knowledge, no data is currently available regarding the observation of specific effects of MW radiation on the morphology of prokaryotic cells. 5 Interestingly, our findings appear to be consistent with the results reported for eukaryotic cells by Chang and Reese (5) who observed the shrinkage of human red blood cells following electroporation caused by the application of a pulsed radiofrequency electric field in a hypo-osmotic medium (notably, a continuous wave electric field was generated as a result of the MW treatment in the present study). Using SEM, the authors also 10 observed that within 30 seconds, all cells had swelled back to their original morphology. It was concluded that the shrinkage was a result of a rapid escape of cellular material via pores formed in the membrane (5). The study did not report data pertaining to the resulting cell physiological conditions. 15 In order to evaluate whether MW radiation affected the cell membrane integrity and caused pore formation within cellular membranes, high molecular weight (150 kDa) FITC Dextran fluorescent probes were used in the present study. Dextran molecules are relatively uncharged and therefore predominantly unaffected directly by the presence of an electric field. This simple assay was used to determine whether pores were formed during 20 MW exposure, thus allowing the free floating dextran molecules to penetrate the cell membrane during the re-hydration process and remain trapped inside the cell. As can be seen in Fig. 3b, all MW treated cells appeared to have ingested the dextran molecules, whereas only 17% ± 3% of the untreated controls (Fig. 3a) and 13% ± 4% of the Peltier treated cells (Fig. 3c) appeared fluorescent. A subsequent statistical analysis indicated that 25 there was a significant difference in the number of fluorescent MW treated cells compared to the thermally heated and untreated controls, t(13) = 6.34, p < 0.05. It is proposed that the minor amount of fluorescence observed for the control and Peltier Plate treated cells was most likely a result of cell membrane damage caused by the centrifugation of the cell suspensions. 30 C:\NRPonblDCCSXD\. 1875671 DOC-2/03/2012 - 24 The size of the dextran probes was calculated to be 15.9 nm. Given that the dextran molecules were able to pass through the cell membrane, it can therefore be concluded that the size of pores formed within the E. coli cell membrane were equal to or larger than the diameter of each dextran probe, i.e. 15.9 nm. The actual size of pores formed within the 5 cell membrane was not determined in the present study. This finding is consistent with previous pulse-induced reversible electroporation studies, where pore sizes of approximately I1 nm were detected using 70 kDa FITC-dextran probes. Images obtained using rapid freezing electron microscopy, together with estimations using theoretical models (13) have suggested that pore sizes as large as 120 nm in diameter can be induced 10 using electric fields where reversible electroporation occurs. The overall conclusion of this section of the study supported the SEM results indicating that MW radiation exposure under the specified settings caused reversible MW-induced poration of the E. coli cell membrane and that this behaviour appeared to be similar to that observed for traditional reversible electroporation processes. 15 The vast majority of the data reported in previous literature has been inferred from experiments completed at temperatures in the range of 45*C - 60*C that are near or greater than the thermal degradation points of the target organisms, thus making it very difficult, if not impossible to make a clear distinction between the thermal and non-thermal, specific, 20 effects of MW radiation (12, 21). At these temperatures protein denaturation, breakdown of cell membrane structures as well as the disruption of replication machinery occur (2, 19). While the mentioned studies reported different findings when using MW's compared to conventional heating methods, the results of many of these publications cannot be used for interpreting the heat independent effects of MW radiation because of the experimental 25 temperatures employed. Furthermore, replication of studies that have suggested the existence of specific MW effects at sub-lethal temperatures has generally been unsuccessful due to the great variability in the experimental conditions, such as MW frequency, power and exposure times. It has been shown that the manifestation of specific MW effects on microorganisms has only been observed when particular combinations of 30 these factors are present (2, 18).

C VaRPonbl\DCC\SXDu2x7567_ I DOC-2A3/2012 - 25 The challenge in investigating the specific effects of MW exposure on microorganisms at sub-lethal temperatures surrounds the inherent difficulty in measuring the temperature of a sample during microwave processing and the inaccuracies in measurement that can result. The first source of inaccuracy is caused by the presence of an uneven electric field 5 distribution inside the microwave cavity. The microwaves within the enclosed cavity experience numerous reflections and produce a constructive-destructive interference pattern within the cavity which creates localised "hot spots". A hot spot is a thermal irregularity that arises because of the non-linear dependence of the electromagnetic and thermal properties of the material on temperature. This effect leads to a non-uniform 10 power absorption by the sample, which in turn leads to non-uniform heating and temperature distribution within the sample. In the present study, this effect was diminished by placing the sample on a ceramic pedestal that made the field distribution relatively even. This arose from the dielectric properties of the ceramic block, which allowed for the concentration of the electric field around the sample causing a uniform power distribution 15 throughout the sample. Furthermore, as the calculated wavelength of microwaves in the cavity (2.34 mm) was much greater than the dimensions of each bacterial cell, the possibility of non-even heating due to non-uniform field distribution was negligible. Since the cell morphology following Peltier Plate heating appeared identical to that of the 20 control, we conclude that the specific effect of MW radiation was due to a direct electromagnetic interaction with the bacterial cells. In the experiments reported in this study, such direct electromagnetic interactions affected the cellular membrane, causing the reversible development of cell membrane pores, accompanied by the leakage of cytosolic fluids through these pores. This observation has led to a suggestion that a possible specific 25 effect of MW radiation at sub-lethal temperatures on bacteria is similar to that of electroporation of the cell membrane. Electroporation is the production of pores in a cell membrane by the application of transverse electric fields. It has been determined from observations of substances being transported into and out of cells (16), freeze-fracture studies (5), and measurements determining the change in membrane impedance after direct 30 current treatment of cells (11), that electroporation allows normally non-permeant matter to diffuse freely through the membranes. While the exact mechanisms by which the C;WRfonb \DCC\XD4187 567 1.DOC-2)/2012 - 26 electrical pulses cause the cell membrane to be permeabilised are not yet fully understood, it is thought that the electric field causes localised structural rearrangements within the cell membrane, resulting in the formation of pores which allow for the free transport of ionic and molecular material through the cell wall. Depending on the field strength and 5 exposure time, the subsequent removal of the electric field may then allow the cell membrane to regain its structural integrity. Analysis of the SEM images obtained for E. coli in the present study, the visual reversibility of the MW effect and the uptake of fluorescent dextran probes in MW treated 10 cells suggests that under current experimental conditions MW radiation caused reversible MW-induced poration, the morphological manifestation of which was similar to that observed in traditional electroporation using pulsed fields. Electromagnetic fields in the MW frequencies are known to significantly increase conductivity and permeability of materials (3), particularly due to enhanced diffusion (1) and enhanced mobility of ions (4). 15 In comparison to traditional electroporation caused by direct currents, high frequency electromagnetic fields amplify all of the kinetic processes compared to low frequency fields. This is because ions cross the cell membrane under two influences: diffusion and the application of an electric field (6). High frequency electromagnetic fields which change the direction of ion movement at an approximate rate of 1010 times per second 20 enhance both of these effects. This study is, to the best of our knowledge, the first of its kind to provide evidence of the reversible leakage of cellular cytosolic fluids and a visual representation of the morphological changes occurring in E. coli cells following continuous wave MW radiation 25 at sub-lethal temperatures. Given that electro-osmosis occurred as a result of the pores formed by the exposure to MW radiation, the MW-bacterial cell interaction was described as electrokinetic in nature. An electrokinetic phenomenon, in particular electro-osmosis, refers to a motion of liquid in a porous body under the influence of an electric field. We propose that molecular transport caused by the MW radiation is controlled by 30 electrokinetic mechanisms that increase transport rates, i.e. a dynamic interaction of microwave field and cell involving both membrane charging and discharging control C:NRPonbl\DCC\SXD\4187367_1 DOC.2A)/2012 -27 electrical behaviour and molecular transport. The important role of electrokinetics is supported by the analyses based on electro-hydrodynamic theory, our numerical simulations and experimental results. 5 In summary, the results of this study advocate that the interaction of continuous wave MW radiation at sub-lethal temperatures with bacteria appears to be electro-kinetic in nature. Temporary changes in cell morphology and the uptake of fluorescent probes indicate that during MW treatment, pores are formed within the bacterial cell membrane. The observed reversibility of the dehydration effect, together with the lack of bactericidal effects, 10 demonstrates that this treatment method will be applicable to a range of bio-industrial and biomedical applications. Example 2 - Poration of Gram positive coccoid bacterial cells induced by super high frequency microwave radiation 15 Materials and methods Bacterial strains, cultivation procedures and sample preparation. Four Gram positive coccoid bacterial strains, P. maritimus KMM 3738, S. aureus ATCC 25923 and S. aureus CIP 65.8T and S. epidermidis ATCC 14990T were used in these experiments. E. coli ATCC 15034 was used as test strain. Bacterial strains were obtained from the American 20 Type Culture Collection (USA), the Culture Collection of the Pasteur Institute (France) and the Collection of Marine Microorganisms (KMM, Russian Federation). Pure cultures were stored at -80 *C in 80 % nutrient broth (NB, Oxoid) or marine broth (MB, BD) for P. maritimus KMM 3738 supplemented with 20 % (v/v) of glycerol. All strains were routinely cultivated on nutrient agar (NA, Oxoid) and marine agar (MA, BD) for 25 P. maritimus KMM 3738. Prior to each experiment, the strains were grown overnight at 37 *C for pathogenic bacteria and 25 0 C for marine bacterium up to the stationary phase as confirmed by growth curves. Working bacterial suspensions were freshly prepared for each independent experiment. The cell density was adjusted to OD600 = 0.1 in phosphate buffered saline (PBS), 10 mM, pH 7.4, using a spectrophotometer (Dynamica - Halo RB 30 10 UV-Vis). Each initial suspension was subjected to four 1:10 serial dilution steps and a further 1:2 dilution step in order to obtain the recovery of approximately 500 bacterial C:\NRPortbl\DCC\SXD\4 7567 I DOC-2/3/2012 - 28 colony forming units (cfu) from 100 gL of bacterial suspension on the plate. Bacterial suspensions were further subject to MW exposure and Peltier plate heat treatment at the optimised settings. 5 MW treatment. Bacterial samples for MW exposure comprised 2 mL of working suspension that were transferred into a micro Petri dish (35 mm diameter, Griener). The MW apparatus was Lambda Technologies Vari-Wave Model LT 1500 with the fixed frequency of 18 GHz and the settings as described in Example I above. In order to minimise thermal effects of MW, the bulk temperature rise of the bacterial suspension 10 during exposure was maintained below 40 *C, so that the bacteria were unaffected by heat. Each sample was exposed to MW radiation for three consecutive exposures (from 20 *C to 40 'C at a heating rate of 20 'C per min) for 1 minute, allowing the sample to cool to 20 'C on ice (at a rate of 10 *C per min) between exposures to MW radiation. 15 Heat treated controls. A Peltier plate heating/cooling system (TA Instruments) was used to replicate the temperature gradients attained during MW processing as described in Example I above (see also ref 30 by the present research group). A total volume of 2 mL of each working bacterial suspension was placed onto the Peltier plate and subject to heating from 20 "C to 40 "C (at a rate of 20 *C per min) in 1 minute for three consecutive 20 exposures, with identical cooling times as for MW treatment (at a rate of 10 *C per min) between trials. All experiments using MW treated samples (in triplicate) were performed in parallel with the heated samples. The working bacterial suspensions that underwent neither MW radiation nor heat treatment were used as the negative controls in all experiments. 25 Confocal Laser Scanning Microscopy (CLSM). Two types of fluorescent silica nanospheres (23.5 ± 0.2 nm Green (FITC) fluorescent silica nanospheres; 46.3 ± 0.2 nm Red (Rhodamine B) fluorescent silica nanospheres; Corpuscular Inc.) were added to the bacterial suspension at a concentration of 50 ptg/mL after 1 minute and 10 minutes 30 following MW radiation and heat treatment. I mL of each suspension was washed twice (centrifuged at 4500 rpm for 5 minutes and had the supernatant removed) and resuspended.

C :NRPonbl\DCC\SXDuI87567 I DOC-2A)3/2012 -29 Each suspension was then observed and analysed using a Fluoview FV10i-W inverted microscope (Olympus, Japan). The controls for these experiments consisted of I mL of untreated bacterial suspension mixed with both of the fluorescent silica nanospheres and processed in parallel with the treated bacterial suspensions. Approximately 15 CLSM 5 images were analysed (5 images per treatment group). A fluorescent probe FITC-Dextran (150 kDa; Sigma-Aldrich) was also used as described in Example 1 to confirm the regularity of SHF MW effect in respect to E. coli and other coccoid bacteria (data not shown). 10 Quantification of internalizing nanospheres. A POLARstar Omega microplate reader (BMG Labtech) was used to measure the fluorescence intensity of nanospheres in each bacterial suspension as prepared above for use in CLSM analysis. A standard curve was also constructed to determine the correlation of fluorescence intensity as relative 15 fluorescence units (RFU) with the silica nanosphere concentration. A total of eight nanosphere concentrations were prepared, which included: 0.0005 mg ml-', 0.005 mg ml- , 0.05 mg ml', 0.25 mg ml- 1 , 0.6 mg ml~ 1 , 0.875 mg ml~1, I mg ml-' and 1.25 mg ml-'. The loading capacity with nanospheres of four studied coccoid bacterial cells after MW 20 treatment was based on the numbers of fluorescent silica nanosphere internalized within the target bacteria. The mass of a silica nanosphere can thus be determined using Equation 1. m=pX V (I) where: m - mass of silica nanosphere (g) 25 p - mass density of silica (g cm- 3 ) V - volume of a silica nanospheres (cm~3) Furthermore, the volume of a silica nanosphere can be calculated using Equation 2. 4 V = 3 E r3 (2) 30 where: V - volume of a silica nanosphere (cm 3

)

C:\NRPonbl\DCCSXD\4 187567 _.DOC-2A)312012 -30 r - radius of a silica nanosphere (cm) The radii of 23.5 nm and 46.3 nm nanospheres are 11.75 x 10-7 cm and 23.15 x 10- 7 cm respectively (Corpuscular Inc.). According to Equation 2, the volumes of 23.5 nm and 5 46.3 nm silica nanosphere were calculated to be 6.8 x 10-18 cm 3 and 5.2 x 10-7 cm 3 respectively. By substituting the volumes of nanosphere into Equation 2, the masses of each 23.5 nm and 46.3 nm silica nanosphere were determined to be 1.8 x 10-17 g and 1.38 x 10~16 g respectively. The mass of a single nanosphere was used to calculate the numbers of nanosphere internalized within the target bacterial cells. 10 A calibration curve of relative fluorescence units (RFU) with the 23.5 nm nanosphere concentration (Fig. 9a) reveals that the amount of nanospheres internalized into the target bacteria cells was approximately 0.31 mg mlr for P. maritimus, 0.29 mg ml-' for S. aureus ATCC 25923, 0.47 mg mlE for S. aureus CIP 65.87 and 0.38 mg ml" for S. epidermidis. 15 By dividing these obtained concentrations by the total concentration of cells in the bacteria suspension, the mass of 23.5 nm green nanospheres which were internalized into a single bacterial cell was obtained, which was approximately 4.2 pg/cell of P. maritimus, 3.8 pg/cell of S. aureus ATCC 25923, 6.3 pg/cell of S. aureus CIP 65.8' and 5.1 pg/cell of S. epidermidis. By dividing the mass obtained above by the calculated mass of a single 20 green nanosphere, the number of green nanospheres which were internalized into a single bacterial cell was obtained, which was approximately 2.3 x 108 spheres/cell of P. maritimus, 2.1 x 108 spheres/cell of S. aureus ATCC 25923, 3.5 x 108 spheres/cell of S. aureus CIP 65.8 and 2.8 x 108 spheres/cell of S. epidermidis. Similarly, the number of 46.3 nm nanospheres that were internalized MW treated cells was also quantified. - This 25 data is shown in Table I below. Cell Viability. A total volume of 100 1 .L of each suspension was spread onto nutrient agar (NA, Oxoid) for pathogenic bacteria and marine agar (MA, Difco) for marine bacterium and then incubated; the pathogenic strains for 24 hours at 37 "C and the marine strains for 30 48 hours at 25'C. Ten plates were used for each sample for conducting the statistical analysis as described in Example 1.

C RPonbrDCC\SXDW187567_ .DOC-2A1/2012 -31 Scanning Electron Microscopy (SEM) analysis. A FeSEM - ZEISS SUPRA 40VP with primary beam energy of 3 kV was used to obtain high-resolution images of the bacterial cells. Approximately ten SEM images per sample were taken at x5000 and x70000 5 magnifications for statistical analysis. Immediately following MW radiation and heat treatment, 100 pL aliquots of the bacterial suspension were transferred onto glass cover slips (used as control surfaces) and insect wing membrane. Cicada (Psaltoda claripennis) specimens were collected from the greater 10 Brisbane parkland areas (typically on flora such as eucalypts). All cell regions of the dorsal and ventral sides of the wings possess a homogeneous nano-structuring. Wing sections of approximately 0.5 cm x 0.5 cm were excised using a scalpel or scissors attached by adhesive tape onto circular discs. They were then briefly rinsed with MilliQ H 2 0 (resistivity of 18.2 MQ cm' 1 , Millipore, U.S.A.) and finally dried using 99.99% purity 15 nitrogen gas. After 1 minute, half of the samples were washed with nanopure H 2 0 (with resistivity of 18.2 MW cm~'), dried with 99.99 % purity nitrogen gas. All experimental steps were completed within less than 60 seconds. The SEM samples were then sputter coated with 6 20 nm thick gold film using JEOL's NeoCoater model MP-1902ONCTR. The remainder of the samples were left to stand for 10 minutes and transferred onto insect wing membrane, following the same procedure. This step was performed to identify any changes in cell morphology following MW radiation, and to determine whether these changes were reversible with time. 25 Results and discussion Our investigation of the effect of SHF MW at 18 GHz on four Gram positive coccoid bacteria demonstrated that pores were formed in all studied strains, with 97% ± 8% of the P. maritimus cells, 99% ± 4% of the S. aureus ATCC 25923 cells, 99% ± 3% of the 30 S. aureus CIP 6 5 .8T cells and 99% ± 5% of the S. epidermidis affected, as confirmed by C:\NRPonb\DCC\SXDwi75(2_ .DOC-2/03/l2 - 32 fluorescent nanosphere intake (Fig. 4) and in a similar manner as in Gram negative E. coli cells as shown in Example 1. Notably, that while the pores of approximately 23.5 nm in diameter were formed on the 5 cell membranes of all four bacteria, the pores of up to 46.3 nm in diameter were formed in P. marilimus (80% ± 9% affected cells) and two strains of S. aureus (40% ± 7% of the S. aureus ATCC 25923 cells and 44% ± 7% of the S. aureus CIP 65 .8T cells); and not observed in S. epidermidis cells. This observation suggested that the maximum size of the pores induced by the SHF MW is approximately 46 nm. 10 The lifetime of the pores formed within bacterial cell membranes as a result of SHF MW appeared to be approximately 10 minutes. CLSM analysis (Fig. 4) indicated that none of the nanospheres were detected after 10 minutes following MW treatment, which is equivalent to the results obtained for E. coli cells in Example 1. 15 The loading capacity of the cells with the nanospheres was also evaluated for all four studied coccoid bacterial strains (Table 1) and it was found that up to approximately 3 x 108 of 23-nm nanospheres and 2 x 10 7 of 46-nanospheres can be internalized by the coccoid cells. 20 Table 1. Quantification of internalized nanospheres. Silica nanospheres, x 106 nanospheres* Bacterial strains 23.5 nm# 46.3 nm" P. maritimus KMM 3738 230 9 S. aureus ATCC 25923 210 19 S. aureus CIP 65.8T 350 12 S. epidermidis ATCC 14 9 9 0 T 280 n/a * per a single bacterium cell # nanosphere diameter C:W4RPonbrDCC\SXD\ 1875%7_.DOC-2/33/2012 -33 Cell viability experiments were conducted using SHF MW treated and thermally heated (up to 40 'C) cells. The results (presented in Fig 5) showed that up to 85% SHF MW treated cells remained viable with 85% ± 8% of the P. maritimus cells, 85% ± 5% of the 5 S. aureus ATCC 25923 cells, 89% ± 5% of the S. aureus CIP 6 5 .8T cells and 84% ± 9% of the S. epidermidis cells recovered on the plates. Thermally heated cells, maintained up to 99% + 6% of the P. maritimus cells, 98% ± 7% of the S. aureus ATCC 25923 cells, 99% ± 9% of the S. aureus CIP 65 .8T cells and 99% ± 8% of the S. epidermidis cells as viable. The statistical analysis showed that there was no statistically significant difference between 10 the thermally heated cells and the MW treated cells; P. maritimus (p > 0.05), of S. aureus ATCC 25923 (p > 0.05), of S. aureus CIP 65.8T (p > 0.05) and S. epidermidis (p > 0.05). Scanning Electron Microscopy (SEM) analysis showed that following SHF MW exposure, coccoid bacteria, surprisingly, did not exhibit any changes in cell morphology; although 15 some traces of leaking cytosol surrounding the MW treated cells could be seen (Fig.4 and Fig. 8). This was not as expected because Example I showed that under the identical SHF MW treatment, E. coli cells exhibited a dehydrated appearance in contrast to the untreated cells. In order to reconfirm that electro-osmosis occurred due to the pores formed and as the result of SHF MW exposure, another experiment was performed. The insect Clanger 20 cicada (Psaltoda claripennis) wing membrane was employed to test whether or not the coccoid bacteria is ruptured by the nanopillars of the wing membrane after SHF MW radiation exposure. R ecently it was reported that the cocci, e.g. P. maritimus, were relatively unaffected by the wing nanopillars due to coccoid cell rigidity, which arises from a composite of morphology, cell wall structure and turgor pressure (32). 25 The results of this experiment are summarised in Fig. 6, which clearly showed that the all four coccoid bacteria were ruptured by the nanopillars. This study provides evidence SHF MW radiation induces pore formation in both Gram 30 positive and Gram negative bacterial cells, despite the considerable difference in their cell walls. In Example I we demonstrated that under specific experimental conditions and sub- CRPonbrDCC\SXDu L87567_1 DOC-203/20l2 -34 lethal temperatures, electromagnetic fields at MW frequencies directly interact with bacterial cells, inducing reversible pores within cell membrane, and that this is a pore forming phenomenon similar to that observed in traditional electroporation using pulsed electrical fields and in photoporation using lasers with wavelengths in the ultraviolet (UV), 5 visible (VIS), and infrared (IR) regions (30). We propose that the mechanisms by which the electromagnetic fields interact with the cell membrane are similar to those of electroporation and photoporation. Without wishing to be bound by theory, the electrical field components of electromagnetic fields may induce dipole interactions with polar molecules such as water leading to the alignment of the polar ends of these molecules as 10 well as oscillation, resulting in an increase in cell membrane surface tension. This increase in surface tension may lead to local-disruption of the membrane which in turn generates the formation of transient hydrophilic pores within the membrane. In this study, an electro-osmosis phenomenon was observed to occur in coccoid cells as a 15 result of the pores formed by the exposure to SHF MW radiation. Silica nanospheres with 23.5 nm in diameter were internalized in all studied Gram positive bacterial cells with the degree of efficiency being more than 95%, indicating that the diameter of pores formed by MW radiation is larger than 23.5 nm. While sonoporation requires a liquid environment and micro-bubbles as contrast agents, this is not the case for MW-induced poration. 20 MW can travel through vacuum, liquid and gas environments, so that MW radiation can therefore be used in specific cases where ultrasound cannot work. The high degree of internalizing efficiency (more than 95%) caused by SHF MW radiation is also very attractive in comparison to that of the photoporation, with its reported efficiency of less than 30% (27, 28, 29). 25 In summary, the finding that the pores are observed to have a long life time of 10 minutes, demonstrates that MW radiation induced cell poration will have important applications as an alternative method to the traditional poration techniques in a variety of contexts, such as biomedical engineering, cell drug delivery and gene therapy (23, 25, 26, 31). 30 C:\NRPonblDCCSXD 1875671 DOC-2/)3/2012 - 35 References 1. Antonio, C., and R. T. Deam. 2007. Can "microwave effects" be explained by enhanced diffusion? Physical Chemistry Chemical Physics 9:2976-2982. 2. Banik, S., S. Bandyopadhyay, and S. Ganguly. 2003. Bioeffects of microwave - a brief review. Bioresource Technology 87:155-159. 3. Bober, K., R. H. Giles, and J. Waldman. 1997. Tailoring the microwave permittivity and permeability of composite materials. International Journal of Infrared and Millimeter Waves 18:101-123. 4. Challis, L. J. 2005. Mechanisms for interaction between RF fields and biological tissue. Bioelectromagnetics 26:S98-S106. 5. Chang, D. C., and T. S. Reese. 1990. Changes in membrane structure induced by electroporation as revealed by rapid freezing electron microscopy. Biophysical Journal 58:1-12. 6. Despa, S. 1995. The influence of membrane permeability for ions on cell behaviour in an electric alternating field. Physics in Medicine and Biology 40:1399-1409. 7. Dreyfuss, M. S., and J. R. Chipley. 1980. Comparison of effects of sublethal microwave radiation and conventional heating on the metabolic activity of Staphylococcus aureus. Applied and Environmental Biology 39:13-16. 8. Foster, K. R., and H. P. Schwan. 1995. "Dielectric Properties of Tissue" in Handbook of Biological Effects of Electromagnetic Fields vol. 2. CRC Press. 9. George, D. F., M. M. Bilek, and D. R. McKenzie. 2008. Non-Thermal Effects in the Microwave Induced Unfolding of Proteins Observed by Chaperone Binding. Bioelectromagnetics 29:324-330. 6. Granath, K., and B. Kwist. 1967. Molecular weight distribution analysis by gel chromatography on sephadex. Journal of Chromatography 28:69-81. 11. Huang, Y., and B. Rubinsky. 2000. Micro-electroporation: improving the efficiency and understanding of electrical permeabilization of cells. Biomedical Microdevices 2:145-150. 12. Kim, S. Y., E. K. Jo, H. J. Kim, K. Bai, and J. K. Park. 2008. The effects of high power microwaves on the ultrastructure of Bacillus subtilis. Letters in Applied Microbiology 47:35-40.

C:NRPortbl\DCC\SXM 175%7 I DOC-2/03f202 -36 13. Krassowska, W., and P. D. Filev. 2007. Modelling electroporation in a single cell. Biophysical Journal 92:404-417. 14. Mitik-Dineva, N., J. Wang, V. K. Truong, P. R. Stoddart, F. Malherbe, R. J. Crawford, and E. P. Ivanova. 2009. Differences in colonisation of five marine bacteria on two types of glass surfaces. Biofouling 25:621-631. 15. Neidhardt, F. C., J. L. Ingraham, and M. Schaechter. 1990. Physiology of the bacterial cell: a molecular approach. Sinauer Associates Inc. 16. Neumann, E., and K. Rosenheck. 1972. Permeability changes induced by electric impulses in vesicular membranes. Journal of Membrane Biology 10:279-290. 17. Samarketu, S. P., S. P. Singh, and R. K. Jha. 1996. Effect of direct modulated microwave modulation frequencies exposure on physiology of cyanobacterium Anabena dolilum. Asia Pacific Microwave Conference 2:155-158. 18. Shamis, Y., S. Patel, A. Taube, Y. Morsi, I. Sbarski, Y. Shramkov, R. Croft, R. J. Crawford, and E. P. Ivanova. 2009. A new sterilization technique of bovine pericardial biomaterial using microwave radiation. Tissue Engineering - Part C: Methods 15:445-454. 19. Shamis, Y., A. Taube, Y. Shramkov, N. Mitik-Dineva, B. Vu, and E. P. Ivanova. 2008. Development of a microwave effect for bacterial decontamination of raw meat. Journal of Food Engineering 4:1-15. 20. van der Veen, M. E., A. J. van der Goot, C. A. Vriezinga, J. W. G. De Meester, and R. M. Boom. 2004. On the potential of uneven heating in heterogeneous food media with dielectric heating Journal of Food Engineering 63:403-412. 21. Woo, I. S., I. K. Rhee, and H. D. Park. 2000. Differential damage in bacterial cells by microwave radiation on the basis of cell wall structure. Applied and Environmental Microbiology 66:2243-2247. 22. Zaghloul, H., and H. A. Buckmaster. 1985. The complex permittivity of water at 9.356 GHz from 10 to 40 degrees C. Journal of Physics D: Applied Physics 18:2109-2118 23. Chen, C., S. W. Smye, M. P. Robinson, and J. A. Evans. 2006. Membrane electroporation theories: A review. Medical and Biological Engineering and Computing 44:5-14.

C:NRPorbl\DCC\SXDWXI7567.1 DOC-2/03/2012 - 37 24. Farago, 0., and C. D. Santangelo. 2005. Pore formation in fluctuating membranes. Journal of Chemical Physics 122:1-9. 25. Granot, Y., and B. Rubinsky. 2008. Mass transfer model for drug delivery in tissue cells with reversible electroporation. International Journal of Heat and Mass Transfer 51:5610-5616. 26. Hofmann, G. A., S. B. Dev, S. Dimmer, and G. S. Nanda. 1999. Electroporation therapy: A new approach for the treatment of head and neck cancer. IEEE Transactions on Biomedical Engineering 46:752-759. 27. Palumbo, G., M. Caruso, E. Crescenzi, M. F. Tecce, G. Roberti, and A. Colasanti. 1996. Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation. Journal of Photochemistry and Photobiology B: Biology 36:41-46. 28. Paterson, L., B. Agate, M. Comrie, R. Ferguson, T. K. Lake, J. E. Morris, A. E. Carruthers, C. T. A. Brown, W. Sibbett, P. E. Bryant, F. Gunn-Moore, A. C. Riches, and K. Dholakia. 2005. Photoporation and cell transfection using a violet diode laser. Optics Express 13:595-600. 29. Schneckenburger, H., A. Hendinger, R. Sailer, W. S. L. Strauss, and M. Schmitt. 2002. Laser-assisted optoporation of single cells. Journal of Biomedical Optics 7:410-416. 30. Shamis, Y., A. Taube, N. Mitik-Dineva, R. Croft, R. J. Crawford, and E. P. Ivanova. 2011. Specific electromagnetic effects of microwave radiation on Escherichia coli. Apple. Environ. Microbiol. 77:3017-3022. 31. Song,' Y., T. Hahn, I. P. Thompson, T. J. Mason, G. M. Preston, G. Li, L. Paniwnyk, and W. E. Huang. 2007. Ultrasound-mediated DNA transfer for bacteria. Nucleic Acids Research 35. 32. Arnoldi M, et al. (2000) Bacterial turgor pressure can be measured by atomic force micrscopy. Phys Rev E 62:1034-1044

Claims (22)

1. A method of temporarily increasing porosity in a cell or cells comprising exposing the cell/s to microwave radiation for a time and under conditions selected to substantially avoid lethal heating of the cell/s; which results in a temporary increase in porosity of the cell/s relative to the porosity of conventionally heated equivalent cell/s and does not adversely affect cell viability relative to that of the conventionally heated cell/s.

2. A method of introducing an agent or agents into a cell or cells comprising exposing the cell/s to microwave radiation for a time and under conditions selected to substantially avoid lethal heating of the cell/s, which results in a temporary increase in porosity of the cell/s relative to the porosity of conventionally heated equivalent cell/s and does not adversely affect cell viability relative to that of the conventionally heated cell/s; and exposing the cell/s to the agents to be introduced into the cell/s so that the agent/s is/are available for passage into the cell/s during the temporary increase in porosity of the cell/s.

3. A method of harvesting a product or products of interest from a cell or cells comprising exposing cell/s that produce products of interest to microwave radiation for a time and under conditions selected to substantially avoid lethal heating of the cell/s, which results in a temporary increase in porosity of the cell/s relative to the porosity of conventionally heated equivalent cell/s and does not adversely affect cell viability relative to that of the conventionally heated cell/s; and washing the cell/s with an appropriate media during or after the temporary increase in porosity of the cell/s and then removing products containing media from the cell/s.

4. A method of preserving a cell or cells comprising exposing the cell/s within a liquid growth media to microwave radiation for a time and under conditions selected to substantially avoid lethal heating of the cell/s; which results in a temporary increase in porosity of the cell/s relative to the porosity of conventionally heated equivalent cell/s and does not adversely affect cell viability relative to that of the conventionally heated cell/s; removing said growth media from the cells during the temporary increase in porosity of the C :RPorb\DCCSXD4187567_IDOC.2//3f/012 - 39 cell/s and freezing the cell/s.

5. A method of treating or preventing undesirable muscle contraction or spasm in a mammalian subject comprising exposing cell/s subject to or that control muscle contraction or spasm to microwave radiation for a time and under conditions selected to substantially avoid lethal heating of the cell/s, which results in a temporary increase in porosity of the cell/s relative to the porosity of conventionally heated equivalent cell/s and does not adversely affect cell viability relative to that of the conventionally heated cell/s; wherein the increase in porosity of the cell/s prevents muscle contraction.

6. The method of any one of claims I to 3 wherein the cell/s are in vivo in a plant or animal.

7. The method of any one of claims I to 3 wherein the cell/s are in vitro.

8. The method of any one of claims 1 to 7 wherein the cell/s exposed to microwave radiation are heated to a maximum temperature of about 40"C.

9. The method of any one of claims 1 to 8 wherein microwave radiation exposure conditions are selected to result in a rate of heating of the cell/s of from about 5*C/min to about 80*C/min.

10. The method of any one of claims 1 to 8 wherein microwave radiation exposure conditions are selected to result in a rate of heating of the cell/s of about 20*C/min.

11. The method of either claim 9 or claim 10 wherein the exposure to microwave radiation is for a period of from about 15 seconds to about 4 minutes.

12. The method of either claim 9 or claim 10 wherein the exposure to microwave radiation is for a period of about 1 minute. C:\ PorbhDCC\SXD\4 87567 I DOC.2A3/2012 - 40

13. The method of any one of claims 1 to 12 wherein the microwave radiation is at a frequency of from about 5 GHz to about 100 GHz.

14. The method of any one of claims 1 to 12 wherein the microwave radiation is at a frequency of from about 15 GHz to about 25 GHz.

15. The method of any one of claims I to 12 wherein the microwave radiation is at a frequency of about 18 GHz.

16. The method of any one of claims 1 to 15 wherein exposure of the cell/s to microwave radiation is conducted from 2 to 5 times, with the cell/s being exposed to cooling between exposures to microwave radiation.

17. The method of claim 2 wherein the agent/s is/are selected from one or more of pharmaceutically and veterinarally active agents, dyes, radioactive, fluorescent or magnetic labelling agents, nutrients, proteins, peptides, polysaccharides, contrast agents, imaging agents, reagents for cellular processes, nucleic acids, infectious agents, antibodies, immunoglobulins or fragments thereof, plasmids, enzymes, neurotransmitters, cell signalling agents, modulators of gene expression or regulation, nanoparticles and devices.

18. The method of claim 3 wherein the product/s is/are selected from one or more of milk, immunoglobulins, hormones, enzymes, specialty chemicals, antibodies, immunoglobulins or fragments thereof, vitamins and biofuels or agents from which they can be produced.

19. The method of claim 4 wherein removing said growth media from the cells during the temporary increase in porosity of the cell/s and freezing the cell/s are achieved by freeze drying.

20. The method of claim 5 wherein the undesirable muscle contraction or spasm in a mammalian subject is skeletal muscle contraction or spasm. C:NRPortbl\DCC\SXD\4187567. lDOC-2/03/202 -41

21. The method of claim 5 wherein the undesirable muscle contraction or spasm in a mammalian subject is smooth muscle contraction.

22. The method of claim 21 wherein the smooth muscle contraction is associated with asthma.