Buffer solution for electric field ablation of the urinary bladder wall in urothelial carcinoma

Abstract

The present invention relates to a solution for use in electric field ablation of the urinary bladder wall for electroporation of urothelial carcinoma, as well as to a method for electric field ablation of the urinary bladder wall for electroporation of a urothelial carcinoma, in which the solution according to the invention is employed.

Description

Buffer solution for electrical field ablation of the bladder wall in urothelial carcinoma TECHNICAL AREA

[0001] The present invention relates to a solution for use in the electrical field ablation of the bladder wall for electroporation of urothelial carcinoma and to a method for the electrical field ablation of the bladder wall for electroporation of a urothelial carcinoma, in which the solution according to the invention is used. BACKGROUND

[0002] Bladder cancer, or bladder carcinoma, generally refers to malignant tumors originating in the urinary bladder. Chronic inflammation, including parasitic infections, tobacco use, and other factors are considered to be causes of bladder cancer. Ingestion of certain chemical substances, for example aromatic amines such as 2-naphthylamine, radiation exposure and immunosuppressant drugs.

[0003] Depending on the extent of the cancer, standard treatments for bladder cancer include transurethral resection of the bladder (TLIR-B), complete removal of the bladder, local chemotherapy, or radiation therapy in combination with systemic chemotherapy.

[0004] The European Organisation for Research and Treatment of Cancer (EORTC) estimates that up to 70% of patients with non-muscle-invasive bladder cancer (NMIBC) experience a recurrence after initial surgery. Following standard treatment of non-muscle-invasive bladder cancer with TLIR-B, residual tumor is detected at the resection site in at least 40% of cases. Therefore, further research and development of more effective therapeutic approaches are urgently needed.

[0005] Electroporation-based technologies are increasingly used for tumor ablation. Intense pulsed electric fields (PEF) permeabilize cell membranes, disrupt cellular homeostasis, and thus have a cytotoxic effect on tumor cells. The fundamental mechanism of electroporation is based on a phenomenon called electroporation. Special electrodes are inserted into the bladder wall or placed close to the tissue to be treated. Electrical pulses are applied to the target tissue, generating short but very high voltages. These voltages cause the cell membranes to open, forming electropores. After permeabilization of the cell membrane, the cells are strongly influenced by the extracellular space.Due to the increased permeability of the cell membrane, small intra- and extracellular molecules diffuse freely through the plasma membrane, transported by electrical and concentration gradients. This leads to the loss of intracellular K. + -ions and ATP and for the uptake of Na + - and Ca ++ -ions. Intracellular molecules larger than the membrane pores are trapped into the intracellular space. An excessive influx of Ca ++ A high concentration gradient leads to cell death.

[0006] However, it has been shown that the efficiency of electrical field ablation of the bladder wall for electroporation of urothelial carcinoma is low. Strong electric fields often have to be applied to permeabilize the cell membranes. This is frequently associated with significant disadvantages, including undesirable thermal and electrochemical reactions. These side effects can compromise the safety of the treatment and lead to undesirable effects such as nerve and muscle stimulation or changes in the geometry of the targeted tissue lesions; see, e.g., Gudvangen et al. (2022), Electroporation and Cell Killing by Milli- to Nanosecond Pulses and Avoiding Neuromuscular Stimulation in Cancer Ablation, Scientific Reports, 12(1), Article 1763.

[0007] Against this background, the object underlying the invention is to increase the efficiency of electrical field ablation of the bladder wall for electroporation of urothelial carcinoma, while avoiding or significantly reducing the disadvantages described in the prior art. In particular, a tool is to be provided with which this object can be achieved. BRIEF SUMMARY OF THE INVENTION

[0008] The invention therefore relates to a solution for use in the electric field ablation of the bladder wall for electroporation of urothelial carcinoma, comprising the following components: - approx. 50 - approx. 200 mM sodium ions (Na + ); - approx. 1 - approx. 10 mM potassium ions (K + ); - approx. 0.5 - approx. 7 mM magnesium ions (Mg ++ ); - approx. 0.5 - approx. 20 mM calcium ions (approx ++ ); - approx. 5 - approx. 50 mM buffer with a pH of approx. 7 - approx. 8; - Solvents.

[0009] The inventors recognized that when performing ablation in the bladder, the effectiveness and safety of the ablation can be increased not only by the parameters of the electric field but also by the properties of the intravesical fluid. According to the inventors, the solution according to the invention, also referred to here as "ISEA" (Intravesical Solution for Pulsed Electric Field Ablation), has an optimized ion content for performing intravesical ablation with pulsed electric fields. For this purpose, the bladder is emptied before the ablation, and the empty bladder is then filled with the solution according to the invention. The buffer solution according to the invention is suitable for performing electroporation of ablation cells using a bipolar or monopolar pulse, preferably with a field strength of up to 2 to 15 kV*cnv. 1 and a duration of 100 ns to 200 ps.

[0010] In vitro and ex v / vo studies show that effective cell ablation can be achieved with the solution according to the invention using a lower electric field strength. The total electric field strength required to achieve the same ablation is reduced with the solution according to the invention by the Ca ++ -Content many times lower than in ablation with a reference solution without or with lower concentrations of Ca ++To generate the same lesion, the number of pulses in the solution according to the invention is significantly reduced. Undesirable thermal and electrochemical reactions are thus avoided or significantly reduced. By reducing side effects, the safety of the treatment is improved and undesirable changes in the geometry of the targeted tissue lesion are avoided. The generation of a higher cell death rate in the weak electric field of the solution according to the invention also significantly reduces the risk of unwanted neuromuscular stimulation.

[0011] Following permeabilization of the carcinoma cells, an influx of Ca occurs. ++-ions into the cells. This influence disrupts cellular homeostasis and leads to various adverse effects. According to the inventors' findings, the use of the solution according to the invention leads not only to programmed cell death processes in irreversibly electroporated cells, but also in cells that are only reversibly electroporated due to a low electric field, typically due to more distant electrodes, and thus also to cell death there due to ATP depletion and other processes. Metabolic disorders. These effects are further enhanced by the solution according to the invention, which makes the cytotoxicity of the therapy more effective.

[0012] The high buffering capacity of the solution according to the invention reduces pH changes near the electrodes during pulse delivery, thereby avoiding uncontrolled damage to the urothelium and providing better control over the lesion geometry.

[0013] Furthermore, the solution according to the invention is composed in such a way that it largely corresponds to extracellular conditions. This prevents dilution of the protein and electrolyte concentrations of the body fluids after damage to the urothelium by electroporation or overdistension of the bladder and increases the safety of the procedure.

[0014] Electroporation was traditionally developed as a method for gene transfection. However, the buffers used there, e.g., those described in WO 02 / 086134 and WO 2008 / 134200, are unsuitable for use in electrical field ablation of the bladder wall for electroporation of urothelial carcinoma. These buffers are formulated to maintain high cell survival rates and maximize transfection efficiency in vitro by preventing cell death after plasma membrane permeabilization. They are not intended for use with high-voltage short pulses in humans, and their chemical composition, conductivity, and formulation are not designed for this purpose. They typically contain inorganic phosphate, sugars, and high concentrations of magnesium. ++and other substances that are intended to maintain cell viability and would therefore counteract the purposes of the invention. However, the solution according to the invention contains no cytoprotective ingredients or only extremely small amounts thereof. Preferably, the solution according to the invention therefore contains no phosphate or inorganic phosphate, and / or no sugar and / or other such cytoprotective substances.

[0015] N / a + is primarily an extracellular ion that plays a crucial role in maintaining the conductivity and osmolarity of the solution according to the invention. The significance lies in the fact that, according to the inventors, minor fluctuations in sodium concentration (+ / - 20 mM) do not significantly affect cytotoxicity. However, an excessively low sodium concentration could reduce the conductivity and osmolarity of the solution, which in turn could compromise the safety of the ablation and control over the lesion geometry. Therefore, maintaining the concentration range for the sodium according to the invention is crucial. + -Ion crucial. The main task of Na + The advantage of the solution according to the invention lies in adapting the conductivity of the solution to that of the surrounding tissue. This ensures that when the current is applied, a homogeneous electric field is generated which ablates a specific area of ​​tissue. By adjusting the conductivity, the space between the electrodes becomes electrically homogeneous, resulting in a uniform distribution of the electric field.

[0016] K +is considered an intracellular ion. In the solution according to the invention, the concentration of K + maintained at an mM level corresponding to the physiological ^-concentration in the extracellular fluid. The risk of depolarization, which occurs at very high K + -concentrations could occur, and an extensive vascular K + This prevents influx of blood, which is associated with life-threatening arrhythmias.

[0017] The inventors have discovered that Mg ++ must be present within the specified concentration range. Thus, the absence of Mg has been shown to be a significant factor. ++ In electroporative media for transfection, the prior art has a detrimental effect on the permeabilization efficiency for small molecules. Higher concentrations of Mg ++ However, they could increase cell survival after electroporation. The concentrations used by the inventors have therefore proven advantageous.

[0018] To stabilize the pH during the process, a physiological, non-toxic buffer is used, preferably one suitable for and, if necessary, approved for parenteral injection. The influence of pH changes on cell survival during electroporation has been demonstrated; specifically, a slightly acidic extracellular pH enables more efficient repair of damage caused to the cell membrane during electroporation. To prevent pH shifts and thus changes in the efficiency of electroporation, the solution according to the invention contains a buffer at a concentration that ensures pH stability.

[0019] Any solvent suitable for use in mammals, in which the specified ions and salts can be dissolved, is acceptable. Preferred solvents include water, glucose solution, glycerol, saline solution, Ringer's lactate solution, propylene glycol, fat solutions, etc.

[0020] In a preferred embodiment of the invention, the solution contains approximately 50 - approximately 200 mM sodium chloride (NaCl), preferably approximately 100 - approximately 150 mM NaCl.

[0021] This measure involves the use of a salt approved for medical applications, which has proven itself as a sodium source and with which the desired Na can be achieved. + - The concentration can be adjusted easily and precisely. The specified concentrations have proven to be particularly advantageous.

[0022] In a further preferred embodiment of the solution according to the invention, it is provided that approximately 1 - approximately 10 mM potassium chloride (KCl), preferably approximately 4 - 6 mM KCl, is used.

[0023] This utilizes a potassium source that is particularly suitable for physiological applications and is readily available. The specified concentrations have proven to be especially advantageous.

[0024] In another embodiment, the solution according to the invention contains approximately 0.5 - approximately 7 mM magnesium chloride (MgCh), preferably approximately 2 - 4 mM MgCh.

[0025] In this configuration, a magnesium source is used to adjust the required Mg level. ++ The ion concentration is well-suited and physiologically compatible, and is characterized by good bioavailability. According to the inventors, the specified concentrations yield particularly good results.

[0026] A preferred solution according to the invention comprises approximately 0.5 - approximately 20 mM calcium chloride (CaCh), more preferably approximately 2 - 10 mM CaCh, and more preferably approximately 5 mM CaCh.

[0027] Calcium chloride is a particularly suitable calcium chloride according to the invention. ++ -Source for the preparation of the solution. In vitro and ex v / o studies by the inventors have shown that the specified Ca ++ - or rather, CaCh concentrations allow for effective cell ablation with a lower electric field strength. The total electric field strength required to achieve the same ablation is, for example, higher with a Ca ++ - Concentration of 5 mM up to 2 times lower than with ablation using a Ca-free solution ++ To create the same lesion, the number of pulses in the solution with 2 mM Ca is ++ or CaCh twice and with 5 mM Ca ++ or CaCh three times lower. After permeabilization, an influx of Ca occurs.++ -ions into the carcinoma cells. This influence disrupts cellular homeostasis and leads to cell death.

[0028] In a further preferred embodiment, the solution according to the invention comprises approximately 10 to approximately 30 mM buffer, preferably Tris or Tris-HCI and / or HEPES buffer, with a pH value of approximately 7 to approximately 8, more preferably of approximately 7.2 to approximately 7.6.

[0029] These measures have the advantage that a non-toxic, physiological buffer, possibly approved for parenteral use, is employed at a concentration sufficient to ensure pH stability. In particular, a drop into the acidic range, and thus undesirable repair of damage in the carcinoma cells, is prevented.

[0030] In a further embodiment, the solution according to the invention has an osmolarity of approximately 200 to 400 mOsm / kg, preferably approximately 250 to 350 mOsm / kg, and more preferably approximately 290 to 300 mOsm / kg. Furthermore, the solution according to the invention preferably has a conductivity of approximately 1 to 2 S / m, preferably approximately 1.2 to 1.6 S / m, preferably at approximately 36.6°C.

[0031] In a further preferred embodiment, the solution according to the invention has a temperature of approximately 4 - approximately 50°C, preferably of approximately 20 - approximately 40°C, more preferably of approximately 36 - approximately 37°C, and most preferably of approximately 36.6°C.

[0032] Another object of the present invention relates to a method for electrical field ablation of the bladder wall for electroporation of a urothelial carcinoma in a mammal, preferably a human, which comprises introducing the solution according to the invention into the bladder, preferably the emptied bladder, and administering electrical impulses to the bladder wall, preferably to the urothelial carcinoma.

[0033] The solution according to the invention is preferably introduced into the empty bladder via a catheter before ablation is performed using inserted electrodes and a pulsed electric field. The buffer solution according to the invention is suitable for performing electroporation of ablation cells using a bipolar or monopolar pulse, preferably with a field strength of up to 2 to 15 kV*cnv. 1 and a duration of 100 ns to 200 ps.

[0034] The features, properties, advantages and embodiments of the solution according to the invention apply accordingly to the method according to the invention.

[0035] Further advantages and features of the invention will become apparent from the following description and the accompanying figures. It is understood that the features mentioned above and those to be explained below can be used not only in the combinations specified, but also in other combinations without departing from the scope of the present invention. BRIEF DESCRIPTION OF THE FIGURES

[0036] The following section provides a detailed explanation of non-limiting embodiments with reference to the figures. The figures show the following: Fig. 1 Experimental workflow (not shown: data analysis workflow). Extracellular Ca ++and efficiency of cell death in nsPEF urothelial cells. In subfigure A, cell death is shown as a function of electric field strength. SV-HUC-1, T24, and UC3 cells were measured with 200 and 300 ns PEFs at 10 Hz in ISEA at different Ca ++ -concentrations. The data points were fitted with sigmoidal curves constructed using the Hill equation (dashed lines), with shaded areas representing the 95% confidence interval for the best fit. Values ​​are given as mean ± SE, with n > 4 independent experiments for each fit. The addition of Ca 2+ The ISEA solution shifted the dose-response curves to lower electric field strengths, indicating a more effective treatment. LD50 of the electric field for 100, 200 and 300 pulses (300 ns duration, 10 Hz frequency) in ISEA at 0, 2 and 5 mM Ca ++, calculated from the dose-response curves in subfigure A. The error bars represent 95% confidence intervals for ED50 (mean ± Kl, n = 4-10 experiments per data point). In ISEA, which uses 2 of 5 mM Ca ++ When enriched, the LD50 after exposure to 100, 200 and 300 PEF was significantly lower in all cell lines than the LD50 in Ca ++ depleted medium (P < 0.05 for all data points). The significance of this observation has not been shown in the graph for clarity, but can be assessed based on non-overlapping 95% confidence intervals. The LD50 (electric field dose that kills 50% of the cells) for SV-HLIC cell death was 2 mM Ca ++ 1.2 times and at 5 mM Ca ++ 1.5 times lower than in the gas-free medium (p<0.001). For the cancer cell line UC3, the LD50 was 1.3 and 1.5 times lower, and for T24, 1.4 and 1.9 times lower at 2 mM and 5 mM Ca, respectively. ++ In cases of elevated Ca++ -Concentration achieved the same ablation efficiency with 2 times fewer pulses and 3 times fewer pulses in ISEA with 2 mM and 5 mM Ca respectively. ++ can be achieved. Compared to ISEA without Ca ++ In addition to the diagrams, representative fluorescence images of urothelial cells after exposure to 200, 300 ns PEFs at 2 kV are shown. Two hours after exposure, the cells were stained with 2.25 pM Hoechst-33342 (blue dye), a membrane-permeable nucleic acid dye that stains all cells, and propidium iodide (red dye), a membrane-impermeable dye that selectively labels cells with impaired plasma membranes, indicating Cell death is indicated by staining. Yellow circles in the images show the electrode imprints. LD50 of electric fields for PEF pulses. The bar chart compares the LD50 values ​​of different cell lines at varying extracellular calcium concentrations. 2+-Concentrations. In an environment with 0 Ca 2+ There is a significant difference in sensitivity to electroporation between cell lines, with some cancer cell lines exhibiting increased sensitivity to pulsed electric fields (PEFs) while others are less sensitive. The addition of Ca 2+ It reduces sensitivity differences and more closely aligns the response of the most resistant cancer cell line with that of healthy cells. This leads to more selective killing of cancer cells compared to healthy cells, thereby increasing the effectiveness of PEF treatment against cancer cells. Fig. 2 For 3D spheroidal electroporation, the inventors designed an electrode arrangement with two parallel plate electrodes. The homogeneous electric field between these electrodes reduces measurement inaccuracies caused by an inhomogeneous field distribution. Experiments with 3D cultures showed increased ablation efficiency when performed in ISEA, which was enriched with 2 mM and 5 mM Ca ++ was enriched. Four hours after exposure, the proportion of cells stained with propidium iodide (indicating cell death) relative to cells stained with Hoechst (indicating total cell count) was [missing information] after treatments with 2-5 mM Ca ++ ISEA was 22-25% higher compared to the control conditions. (Not shown: Immediately after resection, the bladder was emptied and electroporated in PS with 200–300 ns PEF at a frequency of 10 Hz. (Mean ± SE, n = 10 separate experiments for each condition). After 2 hours, the bladders were stained with TTC to assess the ablation field. White areas indicate lesions, while red areas indicate viable bladder tissue. The ablation area was determined using algorithms. The lesion was detected using machine learning. After lesion detection, profiling was performed to determine the lesion area, perimeter, major and minor axes, and form factor. The ablation area was significantly larger at the same electric field when electroporation was performed in 5 mM Ca ++ -PS compared to a Ca ++-free PS was performed. Consequently, a significantly longer perimeter as well as a significantly longer major and minor axis of the lesion were observed. The unchanged shape factor suggests that the shape irregularities were similar regardless of the electroporation conditions. Fig. 3 Monitoring of cell membrane electropermeabilization using YO-PRO-1 dye uptake. The inventors used a custom-made 3D-printed micromanipulator with tungsten electrodes positioned at a 45-degree angle to the cell layer, allowing for precise targeting of the cells. The setup panel shows the electroporation electrodes and the selected group of target cells located between them. The fluorescence fields below indicate YO-PRO-1 (YP) uptake within the defined area at various time points relative to the electroporation load. To quantify YO-PRO-1 uptake, the inventors employed machine learning software trained to identify cells in bright-field images and monitor fluorescence changes over time. The fluorescence time course (mean ± se)) was recorded in both normal urothelial cells and urothelial cancer cells after electroporation with 200 pulses of 300 ns at 10 kV / cm, with the cells suspended in an ISEA solution containing Ca. ++ The uptake dynamics were fitted to a single exponential curve. Notably, YO-PRO-1 uptake in SV-HUC cells was 2.1 to 2.3 times lower, while uptake in HABLAK cancer cells was 4.48 to 4.49 times lower than uptake in cancerous T24 and UC3 cells. These results suggest that T24 and UC3 cancer cells experience greater membrane disruption than SV-HUC and HABLAK cells when in the Ca 2+ -enriched ISEA solution are electroporated. Fig. 4: The study used 3D models derived from primary cell lines of Cells were obtained from patients with muscle-invasive bladder cancer who had undergone radical cystectomy. To ensure phenotypic stability, only primary cell lines exhibiting stable proliferation over six or more passages were selected. The three selected primary cell lines displayed characteristics comparable to commercially available cell lines, such as the cancerous RT4 and the healthy urothelial SV-HUC-1 cell line. The spheroids were electroporated using nanosecond pulses between two tungsten rods mounted on a micromanipulator, with YO-PRO uptake monitored 10 minutes post-exposure. The mean fluorescence intensity of the spheroids (± se) was recorded for further analysis.The results showed that patient-derived cancer organoids exhibited a 2- to 3-fold higher uptake of YO-PRO1 dye than spheroids from healthy SV-HUC-1 cells, confirming the results of previous monolayer studies. DETAILED DESCRIPTION 1. Materials and methods Cell line culture

[0037] The experiments were performed on T24, UC3, and RT4 urothelial cells from human tumors and on normal human urothelial cells HABLAK and SV-HUC1. The cells were cultured at 37 °C in a 5% CO2 atmosphere in their recommended media and expanded to 80–90% confluence. All media were supplemented with 2 mM L-glutamine (Gibco, Paisley, UK), 10% FBS (Gibco, New York, USA), and 1X penicillin-streptomycin (Gibco, St. Louis, USA). Culture of organoids from patient material

[0038] After obtaining written informed consent, urothelial carcinoma samples were taken from patients undergoing surgery. The tissue was then fragmented and collected by centrifugation (480 g, 10 min, ambient temperature). The sediment was resuspended in a buffer containing collagenase (3000 U / ml) and hyaluronidase (1000 U / ml) and incubated with moderate stirring (37 °C, 30 min). Proteolytic degradation was continued by adding fresh collagenase for 30 minutes at 37 °C. The residues were removed through a cell sieve (70 pm mesh size), and the filtrate was sedimented by centrifugation (150 g, 7 min, ambient temperature). The cells were counted and stratified at 4 × 10 6 / ml resuspended to 4*10 4Cells were obtained in 10 pl increments. These cells were mixed with 30 pl of Matrigel on ice, immersed in a 24-well plate, and inverted to create hanging droplets. After a short incubation at 37 °C, the plates were inverted again, replenished with 500 pl of BTM culture medium per well, and incubated in a cell culture incubator (37 °C, 5% CC>2, humid atmosphere). To ensure the genotypic and phenotypic stability of the primary cell lines, the cells were passaged at least six times. Only cells that maintained their morphology and division rate were selected for subsequent organoid formation. The cell plates containing primary cell spheroids in Matrigel were placed on ice to liquefy the Matrigel. The cell suspension in Matrigel was then centrifuged and resuspended in BTM culture medium.The organoids were transferred to 96-well plates coated with 1% agarose, creating a low-adhesion environment. A 1% v / w agarose solution (Sigma Aldrich) was prepared in deionized water and autoclaved. After one minute of microwave heating, 75 pl were added to each well to form a meniscus on the well bottom. After 10 minutes at room temperature, the agarose cooled and gelled to form a non-adherent round-bottom well. The desired number of cells was added to each well, with a total volume of 150 pl per well. The medium was replaced by half every 2–3 days. This method for generating spheroids is reproducible, cost-effective, and allows for high throughput. The cultures were maintained for four weeks to reach the desired size for experimental use.After the incubation period required to obtain a homogeneous spheroidal shape. The spheroids were collected and placed on the glass using a standard 1000 pL pipette. Production of the ISE A Table 1: Example of an "ISEA" (Intravesical Solution for Pulsed Electric Field Ablation)

[0039] The inventors prepared an "ISEA" with the compounds listed above (see Table 1). The pH of both solutions was adjusted to a physiological pH of 7.2–7.4 using 5 M NaOH. The liquids underwent vacuum-controlled filtration using a Steriflip (Millipore, Darmstadt, Germany). Osmolality and conductivity of the solutions were measured using the Advanced Micro Osmometer Moldel 3300 (Advanced Instruments, Norwood, MA, USA) and the Oakton ECTestr High+ (Cole-Parmer, Vernon Hills, IL, USA). Setup for the electroporation of cell monolayers

[0040] Two days before exposure to the pulsed electric field (PEF), the cells were placed in 24-well plates with a density of 0.15 x 10 6 Cells were seeded per well to ensure homogeneous monolayer formation. One day prior to each experiment, the specific growth medium for each cell line was replaced with DMEM containing 10% fetal bovine serum (FBS) to guarantee consistent cell viability and uniform conductivity during electroporation. The DMEM medium was replaced with ISEA solution 10 seconds before exposure to PEFs. Electrode arrays

[0041] The inventors have developed three specially designed electrode arrays for PEF administration: • For / nv / fro experiments on cell monolayers, the inventors used Steinles Stell rods with an outer diameter of 0.9 mm. The electrodes were insulated and connected center-to-center at a distance of 1.9 mm. The contact electrodes were designed to generate an electric field that gradually decreases with distance, allowing for the comparison of cell killing over a range of electric field strengths within a single sample. • For Ex-v / vo studies and YO-PRO-1 uptake experiments on cell monolayers and patient-derived spheroids, an additional array of tungsten rods (0.5 mm diameter, 1 mm spacing between centers) was used. • For the viability test of RT4 after electroporation, we used plate electrodes consisting of two stainless steel plates, each 11 mm long, 4 mm wide, and 0.6 mm thick, spaced 4 mm apart. The electrode was designed to fit into a 94-well plate. Electroporation process

[0042] The parameters of the electric field - pulse count, frequency and intensity - were optimized in initial experiments to generate a visible and compact lesion in cell monolayers using 300 nanosecond pulses.

[0043] A modified Anet A8 3D printer (Shenzhen Anet Technology Co., China) with a custom electrode holder was used for the precise positioning of the electrodes. The electrodes were used perpendicular to the cell monolayers. A 24-well plate of cell monolayers or a 94-well plate of spheroids was securely fixed in a frame attached to the printer bed. The printer was programmed to position the electrodes precisely in the center of the first well, where they remained for 25 seconds for PEF exposure before moving on to the subsequent wells. This scheme allowed for standardized intervals between treatments and imaging. Pulse generation and monitoring

[0044] A pulse generator built at the Institute for High Magnetic Fields (VGTII, Vilnius, Lithuania) delivered pulses from 100 ns to 1 ms. For nsPEF, trains of 200 pulses at 1.4 kV with a frequency of 10 Hz were delivered. Pulse shape and amplitude were monitored using a TBS1052C oscilloscope (Tektronix, Beaverton, OR, US). In vitro electroporation of cell monolayers protocol

[0045] For fluorescence imaging, cells were incubated with 2.25 pM Hoechst-33342 (Thermo Fisher Scientific), a membrane-permeable nucleic acid dye that stains all cells, and 75 pM propidium iodide (Thermo Fisher Scientific), a membrane-permeable dye that selectively labels cells with damaged plasma membranes, thus indicating cell death. After the addition of the dye, the cells were incubated for 20 minutes prior to imaging to allow the dye to diffuse. Once the cell membrane is permeable, propidium enters the cell, and its emission increases through binding to intracellular nucleic acids. The delayed uptake of PI is a recognized marker for cell death.Given that most membrane repairs occur within 10–15 minutes of exposure, we expected that introducing PI 2 hours after electroporation or later would exclusively label permanently permeabilized (i.e., dead) cells. PI-positive cells also exhibited Hoechst positivity and appeared pink when the channels were merged.

[0046] Fluorescence microscopy was performed using a Leica DM18 inverted microscope (Leica, Wetzlar, Germany) with a 10x, 0.38 NA objective. Samples were imaged with DAPI and Cy3 filter sets for Hoechst and PI signals, respectively. With the 10x, 0.38 NA objective, the camera acquired nine images of adjacent regions (3x3 square), which were then automatically stitched together to create a high-resolution image of the sample. Cell monolayers were imaged with DAPI and Cy3 filter sets for Hoechst and Pr signals, respectively. The LAS X software (Leica, Wetzlar, Germany) enabled automatic repositioning of the stage, selection of filter cubes, and image acquisition synchronized with the illumination and camera operation. Image analysis monolayer

[0047] For image analysis after PEF exposure, a rectangular region of interest (ROI) (0.5 mm x 2.5 mm) was defined between the electrode centers. Using CellProfiler software, the primary ROI was subdivided into 26 smaller ROIs. In each ROI, the number of cells stained with PI and Hoechst was counted. The percentage of dead cells was calculated as the ratio of PI-positive cells to the total number. The ROIs furthest from the ablation site, with negligible electric field exposure, served as sham controls. Image analysis of 3D cell viability experiments

[0048] 3D spheroids of RT4 urothelial carcinoma cells were cultured on 96-well low-adhesion plates coated with 1% agarose as described above. After the incubation period required to achieve a homogeneous spheroidal shape, the spheroids were collected and placed onto the glass using a 1000-pl standard pipette. Simulations of the electric field

[0049] The distribution of the electric field was simulated using the software Sim4Lifelite v7.3.0 (Zurich Med Tech, Switzerland), which was combined with the experimental electrode positions. The values ​​of the electric field were calculated in a plane 5 pm above the bottom of the depression for 1 V between the electrodes and then scaled to the applied voltage. Ex-vivo protocol

[0050] Mice were euthanized under isoflurane anesthesia (5% v / v) and killed by cervical dislocation; subsequently, the bladders of the mice were removed. The freshly resected bladders were emptied of urine and placed in ISEA solution. Electrodes were inserted into the bladder wall at 1 mm intervals. Ablation was performed with 300 ns pulses, 200 nsPEF pulses at 10 Hz, creating lesions 3–4 mm in length. The lesion in the bladder was imaged with the ViewPix 700 (Biobtep) 2 hours after TTC staining (30 mM / 20 min) and formaldehyde fixation. Image analysis of lesions

[0051] The TTC-stained lesions were detected and segmented using the Ilastik software. Initial manual annotations of lesion and non-lesion areas were made to three randomly selected blisters to train the software and enable accurate segmentation based on intensity and texture features. Once segmentation was complete, the data were imported into Cell Profiler, where thresholding was performed to more accurately define and refine the lesion boundaries. This integrated approach, combining machine learning-based segmentation with subsequent thresholding, ensured precise and consistent quantification of electroporation-induced lesions in the mouse blister model and provided robust data for further analysis. YOPRO-1 Imaging

[0052] For the experiments to track YOPRO-1 fluorescence changes after PEF, the cell monolayers were placed on the glass coverslip on the microscope stage. Positioned. A specially designed electrode holder positioned the tungsten electrodes, which contacted the coverslip, at a stable 45-degree angle between the coverslip and the electrodes. In an experiment, groups of more than 100 cells in a monolayer were simultaneously exposed to PEF across a broad spectrum of electric fields. Fluorescence was recorded over a period of 3 minutes, and the baseline fluorescence (F0) was subtracted from the fluorescence after PEF exposure. YOPRO-1 Fluorescence changes in cell monolayers and cell spheroids

[0053] For the analysis of the Ca ++For the image acquisition, a rectangular area of ​​800 x 800 pm was selected, extending from an edge of the connecting line between the tips of the two electrodes. Subsequent analyses involved segmenting the cell regions in bright-field images using the software llastic, which employs machine learning-based pixel classification. First, the regions of interest, including the cell regions and background, were manually labeled to create a training dataset. These labels were then used by llastic to apply supervised machine learning algorithms for pixel classification based on intensity and texture features, enabling accurate cell segmentation even in cases of low contrast or overlap.The resulting probability maps were then processed in CellProfiler to define thresholds and cell boundaries, ensuring consistent and reliable quantification. Fluorescence intensity in each region of interest (ROI) was monitored for 180 seconds, with an image acquired every 3 seconds. Cells were categorized based on their location within the ROI and correlated with the corresponding electric field distributions to enable observations across a broader range of electric field intensities within a single experiment. Statistical analysis

[0054] In the experiments to assess the electric field strength, 26 measurements were carried out in a single experiment, with one measurement per region. Areas of interest (ROI) were measured. Data from two ROIs positioned symmetrically to the ablation center (e.g., the two most peripheral ROIs) were averaged to obtain 13 distinct data points. For each cell line, various experimental conditions (e.g., solution types and pulse counts) were randomized and tested. The electric field response data were fitted with a sigmoidal function derived from the Hill equation. The fits and their 95% confidence interval were calculated using GraphPad software (Boston, MA, US). The LD50 values ​​between the different conditions were compared using the additional F-test (sum of squares): A significant F-test result indicates that the separate models fit the data significantly better, suggesting that the LD50 values ​​differ between the two conditions.For the comparison of more than two dose-response curves, a multi-test correction with Bonferroni fitting was performed.

[0055] Fluorescence measurements of the YOPRO-1 uptake were averaged at each time point under identical electric field strengths (typically 100 cells per experiment and 6 technical replicates in one condition, 4 biological replicates of the experiments). All trials included sham-exposed controls (no EP). Data were plotted as means ± s. Data fitting and plotting were performed using GraphPad software.

[0056] To analyze the significance of the differences, a two-sided Student's t-test was performed; p < 0.05 was considered statistically significant. We restricted the statistical comparison to the fluorescence intensity at the end of the observation period. Statistical significance at earlier time points can be estimated based on the distance between the error bars of the compared groups: A distance exceeding the length of the error bar indicates a significant difference at p < 0.05 or better. 2. Results Experimental workflow and data analysis workflow

[0057] The experimental workflow is illustrated in Fig. 1. Urothelial carcinoma cells were treated with nsPEFs using an automated high-throughput system with multiple electrode arrays. The system was programmed to position the electrodes precisely in the center of the first well, where they remained for PEF exposure before moving to subsequent wells. 25 pM Hoechst-33342 (Thermo Fisher Scientific), a membrane-permeable nucleic acid dye, was added 1 hour prior to exposure. To assess cell death, 75 pM propidium iodide (Pl) solution was added several hours after exposure to permanently label permeabilized (dead) cells (the time interval was determined in preliminary experiments). After labeling the living cells, images of the samples were examined using epifluorescence microscopy.

[0058] Data analysis workflow: For the acquired images of cell monolayers, the area of ​​interest (ROI) was positioned in the center of the ablation, perpendicular to the line connecting the centers of the electrode imprints. Using a pixel-based approach for cell detection, we calculated the ratio of cells stained with PI / YO-PRO-1 to those stained with Hoechst. The data points from each ROI were plotted with sigmoidal curves generated using the Hill equation, producing dose-response curves that facilitate the comparison of ED50 under different conditions. Electroporation in ISEA leads to increased killing of cancer cells:

[0059] The addition of Ca 2+The ISEA solution shifted the dose-response curves towards lower electric field strengths, indicating a more effective treatment. LD50 (electric field dose that kills 50% of the cells) after exposure to 100, 200, and 300 PEF was significantly lower for all cell lines than LD50 in Ca ++ depleted ISEA (P < 0.05 for all data points). The LD50 (electric field dose at which 50% (which kills cells) for the cell death of SV-HUC cells was 2 mM Ca in ISEA. ++ 1.2 times lower and in ISEA with 5 mM Ca ++ 1.5 times lower than in Ca ++ -free ISEA (p<0.001). For the cancer cell line UC3, the LD50 was 1.3 and 1.5 times lower, and for T24, 1.4 and 1.9 times lower in ISEA with 2 mM and 5 mM Ca, respectively. ++ In cases of elevated Ca ++ -Concentration achieved the same ablation efficiency with 2 times fewer pulses and 3 times fewer pulses in ISEA with 2 mM and 5 mM Ca respectively. ++ compared to ISEA without Ca++ In addition to the diagrams, representative fluorescence images of urothelial cells are shown after exposure to 200 pulses of 300-ns PEFs at 2 kV. Two hours after exposure, the cells were stained with 2.25 pM Hoechst-33342 (blue), a membrane-permeable nucleic acid dye that stains all cells, and propidium iodide (red), a membrane-impermeable dye that selectively labels cells with impaired plasma membranes, indicating cell death. The images clearly show an expanded ablation field with increased Ca ++ -Concentration in the ISEA solution, despite the constant electric field applied between the electrodes. In a 0 Ca 2+ISEA reveals a significant difference in sensitivity to electroporation between cell lines, with some cancer cell lines exhibiting increased sensitivity to pulsed electric fields while others are less sensitive. The addition of Ca ++ The sensitivity difference is reduced, so that the response of the most resistant cancer cell line matches that of healthy urothelial cells. This results in an overall increased killing selectivity for cancer cells compared to healthy cells. Electroporation in ISEA increases cell death in 3D cell cultures and animal experiments.

[0060] For 3D spheroidal electroporation, the inventors developed an electrode array with two parallel plate electrodes. The homogeneous electric field between these electrodes reduces measurement inaccuracies caused by an inhomogeneous field distribution. Experiments with 3D cultures showed increased ablation efficiency when performed in ISEA, which was enriched with 2 mM and 5 mM Ca ++ was enriched. Four hours after exposure, the proportion of cells stained with propidium iodide (indicating cell death) relative to cells stained with Hoechst (indicating total cell count) was [missing information] after treatments with 2-5 mM Ca ++ ISEA was 22–25% higher than under control conditions (p < 0.05) (Fig. 2). The ionic The composition of ISEA allows ions to penetrate the extracellular matrix of the tumor, thereby enabling the ablation of deeper tumor structures that may be inaccessible to standard intravesical chemotherapy due to their larger molecular size. This improved ablation efficiency through electroporation with ISEA promises a more precise and effective treatment approach, potentially improving patient outcomes and minimizing the risk of residual cancer cells after treatment.

[0061] The efficacy of electroporation with ISEA was demonstrated in a mouse model. Immediately after resection, the bladder was emptied and electroporated in ISEA with a peak electric field (PEF) of 200–300 ns at a frequency of 10 Hz (mean ± SE, n = 10 separate experiments for each condition). After 2 hours, the bladders were stained with TTC to assess the ablation field. White areas indicate lesions, while red areas indicate viable bladder tissue. The ablation area was determined using machine learning algorithms. The ablation area was significantly larger at the same electric field when electroporation was performed in 5 mM Ca ++ -ISEA compared to Ca ++ ISEA was performed without electroporation (p<0.01). Consequently, a significantly longer circumference as well as a main and secondary axis of the lesion were observed (p<0.05). The unchanged shape factor suggests that the shape irregularities were similar regardless of the electroporation conditions. Exposure to the ISEA solution enables selective permeabilization of cancer cells while minimizing the effects on normal urothelial cells.

[0062] The inventors used a custom-made 3D-printed micromanipulator with tungsten electrodes positioned at a 45-degree angle to the cell layer, allowing for precise targeting of the cells. The fluorescence time course (mean ± se) was recorded in both normal urothelial cells and urothelial cancer cells after electroporation with 200 pulses of 300 ns at 10 kV / cm, with the cells in ISEA solution containing Ca 2+ were suspended. The uptake dynamics followed a simple exponential trend. In particular, YO-PRO-1 uptake in SV-HUC cells was reduced by a factor of 2.1 and 2.3, while HABLAK cancer cells showed a reduction of 11.5. At kV / cm, SV-HUC cells showed uptake that was 4.48 and 4.49 times lower, respectively, compared to the cancerous T24 and UC3 cell lines. Similar results were observed at a lower electric field strength of 10 kV / cm, where SV-HUC cells showed uptake that was 1.6 and 1.7 times lower, respectively, and HABLAK cells showed uptake that was 4.76 and 4.58 times lower, respectively, compared to the uptake values ​​of T24 and UC3 cancer cells. These results suggest that T24 and UC3 cancer cells undergo greater membrane disruption than SV-HUC and HABLAK cells when electroporated in the ISEA solution (Fig. 3). To assess whether the selectivity of PEFs in the permeabilization of urothelial carcinoma cells persists in more complex models, we used bladder cancer organoids (BCOs) derived from primary cell lines of patients with muscle-invasive bladder cancer who had undergone radical cystectomy.Electroporation was performed on these spheroids using nanosecond pulses applied between two tungsten rods mounted on a micromanipulator, with YO-PRO-1 uptake monitored for 10 minutes post-exposure. The mean fluorescence intensity of the spheroids (± se) was recorded for analysis. The results showed that patient-derived cancer organoids exhibited up to 2.3-fold higher YO-PRO-1 uptake compared to spheroids derived from healthy SV-HUC-1 cells, confirming the results of previous monolayer studies. Ten minutes post-exposure in ISEA, YO-PRO-1 fluorescence increased 1.6-fold (p = 0.0856) in BCO 154 spheroids and 2.3-fold (p = 0.0021) in BCO 319 spheroids. In the most resistant cell line, BCO T270, YO-PRO-1 fluorescence was only slightly increased (1.01-fold) compared to spheroids from healthy cells (p = 0.0856).Spheroids derived from the RT4 urothelial carcinoma cell line showed a 1.2-fold increase in fluorescence compared to healthy urothelial cell spheroids (p = 0.4274). Despite variations in the sensitivity of the cancer cells within the organoids, exposure to ISEA enabled selective permeabilization of the cancer cells (Fig. 4).

Claims

Patent claims 1. Solution for use in electrical field ablation of the bladder wall for electroporation of urothelial carcinoma, comprising the following components: - approx. 50 - approx. 200 mM sodium ions (Na + ); - approx. 1 - approx. 10 mM potassium ions (K + ); - approx. 0.5 - approx. 7 mM magnesium ions (Mg ++ ); - approx. 0.5 - approx. 20 mM calcium ions (approx ++ ); - approx. 5 - approx. 50 mM buffer with a pH of approx. 7 - approx. 8; - Solvents.

2. Solution according to claim 1, comprising the following components: - approx. 50 - approx. 200 mM sodium chloride (NaCl), preferably approx. 100 - approx. 150 mM NaCl.

3. Solution according to claim 1 or 2, comprising the following components: - approx. 1 - approx. 10 mM potassium chloride (KCl), preferably approx. 4 - 6 mM KCl.

4. Solution according to any of the preceding claims, comprising the following components: - approx. 0.5 - approx. 7 mM magnesium chloride (MgCh), preferably approx. 2 - 4 mM MgCh.

5. Solution according to any of the preceding claims, comprising the following components: - approx. 0.5 - approx. 20 mM calcium chloride (CaCh), preferably approx. 2 - 10 mM CaCh, further preferably approx. 5 mM CaCh.

6. Solution according to any of the preceding claims, comprising the following component: - Approximately 10 - 30 mM buffer with a pH of approximately 7 - 8.

7. Solution according to one of the preceding claims, characterized in that the buffer is a Tris buffer, preferably a Tris-HCl buffer.

8. Solution according to one of the preceding claims, characterized in that the buffer is a HEPES buffer.

9. Solution according to one of the preceding claims, characterized in that the buffer has a pH value of approximately 7.2 - approximately 7.

6.

10. Solution according to one of the preceding claims, characterized in that it has an osmolarity of approximately 200 - approximately 400 mOsm / kg, preferably approximately 250 - approximately 350 mOsm / kg, further preferably approximately 290 - approximately 300 mOsm / kg.

11. Solution according to one of the preceding claims, characterized in that it has a conductivity of approximately 1 - approximately 2 S / m, preferably approximately 1.2 - approximately 1.6 S / m.

12. Solution according to one of the preceding claims, characterized in that it has a pH value of approximately 7 - approximately 8, preferably of approximately 7.2 - approximately 7.

6.

13. Solution according to one of the preceding claims, characterized in that the solvent is water (H2O).

14. Solution according to one of the preceding claims, characterized in that it has a temperature of approximately 4 - approximately 50°C, preferably approximately 20 - approximately 40°C, more preferably approximately 36 - approximately 37°C, and most preferably approximately 36.6°C.

15. Method for electrical field ablation of the bladder wall for electroporation of a urothelial carcinoma in a mammal, comprising introducing the solution according to one of claims 1 to 14 into the bladder, preferably the emptied bladder, and administering electrical impulses to the bladder wall, preferably the urothelial carcinoma.

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