Implantable compositions for combination therapy of tumors

Abstract

To provide a novel compositions for tissue destruction in the treatment of abnormal biological tissue such as cancer.SOLUTION: In some aspects, the present disclosure pertains to methods of treating a tissue volume comprising (a) administering an implantable composition comprising a releasable membrane-active agent to a target site such that the membrane-active agent is locally released to the tissue volume and (b) performing irreversible, reversible and / or thermal treatment by application of a pulsed electric field to the tissue volume. In other aspects, the present disclosure pertains to embolic compositions that comprise releasable membrane-active agents.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] This disclosure relates to methods for treating tumors and compositions for use in treating tumors. [Background technology]

[0002] Various modalities are used to treat abnormal biological tissues such as cancer, including thermal ablation, cryoablation, ultrasound ablation, chemical ablation, RF electroablation, and irreversible electroporation.

[0003] Novel and different approaches to the disruption of biological tissues are desired. This disclosure describes novel compositions and combination approaches for tissue disruption using irreversible electroporation. [Overview of the project] [Means for solving the problem]

[0004] In some embodiments, the present invention discloses a method for treating a tissue volume, comprising (a) administering an implantable composition containing a releaseable membrane-active agent to a target site such that the membrane-active agent is locally released into the tissue volume, and (b) treating the tissue volume by applying a pulsed electric field to the tissue volume. For example, irreversible electroporation, reversible electroporation, and / or heat treatment may be carried out by applying a pulsed electric field.

[0005] As used herein, “membranolytic agent” is defined as a drug or molecule that affects the structure and dynamics of a cell membrane, such as cationic, amphiphilic, or anionic molecules. Cationic molecules bind to the cell membrane via charge-charge attraction. Amphiphilic molecules bind to the cell membrane via charged functional groups and bind to cell membrane lipids via hydrophobic interactions. Anionic molecules become hydrophobic when protonated and may interact with cell membrane lipids. As used herein, “membranolytic agent” is a subset of membrane activators and means a drug or molecule that disrupts the integrity of the membrane. Membranolytic agents often act as membrane solubilators when present in sufficiently high concentrations.

[0006] In some embodiments, the tissue volume corresponds to all or part of the healthy tissue surrounding the tumor and / or the tumor (for forming a margin via the vascular system). Examples of tumors include tumors of the pancreas, breast, lung, prostate, liver, kidney, brain, uterus, ovary, stomach, duodenum, or skin.

[0007] In some embodiments that may be used in combination with any of the above aspects and embodiments, the implantable composition is an embolic composition comprising a releaseable membrane activator, the embolic composition being administered to one or more blood vessels supplying blood to a tissue volume, or the embolic composition being administered percutaneously or endoscopically by injection.

[0008] In some embodiments that may be used in combination with any of the above aspects and embodiments, the implantable composition is a liquid composition that becomes a solid implant upon introduction into the body. For example, the implantable composition may be a gel having viscoelastic properties that is administered as a liquid and changes to a solid upon activation or contact with bodily fluids.

[0009] In some embodiments that may be used in combination with any of the above-described aspects and embodiments, the administered implantable composition is at least partially biostable or bioabsorbable.

[0010] In some embodiments that may be used in combination with any of the above aspects and embodiments, the implantable composition is a solid implantable composition. In some embodiments that can be used in combination with any of the above aspects and embodiments, the solid implantable composition is implantable particles. In some of these embodiments, the particles may have a maximum cross-sectional dimension in the range of 10 nm to 10 mm (for example, from 10 nm to 100 nm, to 1000 nm, to 10 microns (10 μm), to 100 microns (100 μm), to 1000 microns (1000 μm), and to 10 mm).

[0011] In some embodiments that may be used in combination with any of the above aspects and embodiments, the solid embolus composition may be a scaffold. Examples of scaffolds include, for example, discs, rods, stents, coils, fiducial markers, plates, tubes, or semipermeable woven meshes.

[0012] In some embodiments that may be used in combination with any of the above embodiments and models, the membrane activator is present throughout the solid implantable composition, or the membrane activator is present only in a portion of the solid implantable composition.

[0013] In some embodiments that can be used in combination with any of the above aspects and embodiments, the membrane activator is present on the surface of the solid transplantable composition but not in a bulk of the solid transplantable composition, or the membrane activator is present in a bulk of the solid transplantable composition but not on the surface of the solid transplantable composition, or the membrane activator is homogeneously present in the solid transplantable composition, or the membrane activator is present in the solid transplantable composition in a concentration gradient.

[0014] In some embodiments that may be used in combination with any of the above aspects and embodiments, the membrane activator is present in one or more layers arranged on an underlying substrate material, the membrane activator is encapsulated in an encapsulation material, or the membrane activator is dispersed in a matrix material.

[0015] In some embodiments that may be used in combination with any of the above-described aspects and embodiments, the membrane activator is coated onto the embolic beads, injected into the embolic beads, or conjugated to the embolic beads via covalent bonds.

[0016] In some embodiments that may be used in combination with any of the above aspects and embodiments, the membrane activator is selected from cationic molecules, amphiphilic molecules, and anionic molecules. For example, the membrane activator may be selected from cationic polymers, anionic polymers, and amphiphilic polymers.

[0017] In some embodiments that can be used in combination with any of the above aspects and embodiments, the membrane activator may be locally released into the tissue volume in a burst release profile, the membrane activator may be locally released into the tissue volume in a sustained release profile, or the membrane activator may be locally released into the tissue volume in a combination of a burst release profile and a sustained release profile.

[0018] In some embodiments that may be used in combination with any of the above aspects and embodiments, the release of the membrane activator is triggered by electrical pulses, ultrasound, magnetic field fluctuations, or thermal inputs.

[0019] In some embodiments that may be used in combination with any of the above-described aspects and embodiments, the treatment is carried out in the same procedure as the administration of the implantable composition.

[0020] In some embodiments that can be used in combination with any of the above aspects and embodiments, the treatment is performed in a procedure following the administration of the transplantable composition. In some embodiments that can be used in combination with any of the above aspects and embodiments, the treatment is performed in a procedure at a time ranging from the same day, the day after, or the second day, up to one week, up to two weeks, up to 30 days after the administration of the transplantable composition.

[0021] In some embodiments that can be used in combination with any of the above aspects and embodiments, one or more electrodes for treatment are disposed within or adjacent to the tissue volume.

[0022] In some embodiments that can be used in combination with any of the above aspects and embodiments, (a) one or more treatment electrodes are disposed adjacent to the tissue volume at one or more positions on the skin, (b) one or more treatment electrodes are disposed percutaneously within or adjacent to the tissue volume, (c) the treatment electrode is disposed within or adjacent to the tissue volume via a device inserted into one or more natural body lumens, or (d) a combination of any two or all three of the foregoing placement techniques.

[0023] Another aspect of the present disclosure relates to an embolization composition comprising a releasable membrane activator. In some of these embodiments, the embolization composition is a solid embolization composition. In some of these embodiments, the embolization composition is a liquid composition that becomes a solid embolization composition upon introduction into the body. For example, the embolization composition may be a gel having viscoelastic properties that are administered as a liquid and change to a solid upon activation or contact with body fluids.

[0024] In some embodiments that can be used in combination with any of the above aspects and embodiments, the solid embolization composition is at least partially bio-stable or bio-absorbable.

[0025] In some embodiments that can be used in combination with any of the above aspects and embodiments, the solid plug composition is transplantable particles. In some of these embodiments, the particles may have a maximum cross-sectional dimension in the range of 10 nm to 10 mm.

[0026] In some embodiments that can be used in combination with any of the above aspects and embodiments, the solid plug composition is a scaffold. In some embodiments that can be used in combination with any of the above aspects and embodiments, the membrane active agent is present throughout the solid plug composition, or the membrane active agent is present only in a part of the solid plug composition.

[0027] In some embodiments that can be used in combination with any of the above aspects and embodiments, the membrane active agent is present on the surface of the solid plug composition but not in most of the solid plug composition, the membrane active agent is present in most of the solid plug composition but not on the surface of the solid plug composition, the membrane active agent is homogeneously present in the solid plug composition, or the membrane active agent is present with a concentration gradient in the solid plug composition.

[0028] In some embodiments that can be used in combination with any of the above aspects and embodiments, the membrane active agent is present in one or more layers disposed on a lower substrate material, the membrane active agent is encapsulated within an encapsulating material, the membrane active agent is dispersed within a matrix material, or the membrane active agent is conjugated to a matrix material.

[0029] In some embodiments that can be used in combination with any of the above aspects and embodiments, the membrane active agent is selected from cationic molecules, amphiphilic molecules, and anionic molecules. For example, the membrane active agent may be selected from cationic polymers, anionic polymers, and amphiphilic polymers.

[0030] In some embodiments that may be used in conjunction with any of the above aspects and embodiments, the membrane activator is locally released in a burst-release profile, a sustained-release profile, or a combination of burst-release and sustained-release profiles when the embolic composition is implanted in the target.

[0031] Further aspects and embodiments of this disclosure will become apparent to those skilled in the art upon consideration of the detailed description that follows. [Brief explanation of the drawing]

[0032] [Figure 1] Figures 1A-1D show microspheres coated with fluorescently labeled PAHM. (Figure 1A) Representative fluorescence microscope images of microspheres coated with different amounts of PAHM (μg PAHM / mg PMMA). (Figure 1B) Fluorescence intensity of PAHM coating on individual microspheres. (Figure 1C) Representative fluorescence microscope images of microspheres coated with PAHM by different methods. (Figure 1D) Fluorescence intensity of PAHM on individual microspheres after coating by different methods. Data are shown as mean + / - SD (A) (n=16-63) (B) (n=165-186). Analysis of variance (ANOVA) using Tukey's HSD test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 2] Figures 2A and 2B show the quantification of PAHM in cell culture medium. (Figure 2A) UV-Vis absorption spectra at different PAHM concentrations. (Figure 2B) Calibration curve of absorbance versus PAHM concentration at 245 nm. [Figure 3] Figure 3 shows the release kinetics of PAHM from coated microspheres in cell culture medium at 37°C. Data are presented as mean + / - SD (n=4) and are fitted to a two-phase exponential association model. [Figure 4]Figures 4A and 4B show cell viability after treatment with IRE (Figure 4A) or PAHM (Figure 4B) applied separately. Data are presented as mean + / - SD (Figure 4A) (n=4-6) (Figure 4B) (n=9-13). Analysis of variance using Tukey's HSD test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 5] Figures 5A-5F show cell viability after IRE treatment combined with PAHM exposure for 15 minutes (Figure 5A), 4 hours (Figure 5B), and 24 hours (Figure 5C). Data are shown as mean + / - SD (n=3-12). Analysis of variance using Tukey's HSD test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). (Figure 5D) Combined index analysis (CI<1, =0.9-1 (dashed line), >1 indicates synergistic, near additive, and antagonistic effects, respectively). Dose reduction index analysis for IRE (Figure 5E) and PAHM (Figure 5F) (DRI<1, =1 (dashed line), >1 indicates unfavorable dose reduction, no dose reduction, and favorable dose reduction, respectively). [Figure 6] Figures 6A and 6B show cell viability after exposure to PAHM released from coated microspheres with average diameters of 71 μm (Figure 6A) and 100 μm (Figure 6B). Data are presented as mean + / - SD (n=6-12) and analyzed for variance using Tukey's HSD test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 7] Figure 7 shows cell viability after exposure to uncoated embolic microspheres. Data are presented as mean + / - SD (n=2). [Figure 8]Figures 8A-8F show cell viability after IRE treatment combined with exposure to PAHM released from coated microspheres: (Figure 8A) 15 minutes, (Figure 8B) 4 hours, (Figure 8C) 24 hours. Data are shown as mean + / - SD (n=3-9). Analysis of variance using Tukey's HSD test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). (Figure 8D) Combined index analysis (CI<1, =0.9-1 (dashed line), >1 indicates synergistic, near additive, and antagonistic effects, respectively). Dose-drop index analysis for IRE (Figure 8E) and microsphere-coated PAHM (Figure 8F) (DRI<1, =1 (dashed line), >1 indicates unfavorable dose-drop, no dose-drop, and favorable dose-drop, respectively). [Modes for carrying out the invention]

[0033] Electroporation is a phenomenon in which living cells respond to an electric field by opening pores in their cell membrane. At low electric field strengths, no pores are formed in the cell membrane. Beyond the electroporation threshold, pores begin to form reversibly. Reversible electroporation has long been used to introduce substances into living cells that would otherwise not be able to pass through the cell membrane. For example, it can be used to introduce genetic material or large molecules used in chemotherapy into the interior of cells. After the electric field is removed, the induced pores close through "transient" or "reversible" electroporation.

[0034] At relatively high amplitude electroporation doses, the pores formed in response to the applied electric field can be so extensive that the cell membrane cannot recover. If the cell membrane does not recover, the cell dies due to this electroporation, known as "irreversible" electroporation. The threshold for irreversible electroporation (IRE) varies depending on the size and shape of the cell, the overall tissue structure, and the characteristics of the interstitial fluid. The threshold itself can be defined by the local electric field strength and duration; for example, shorter durations require higher amplitude voltages, and longer durations require lower amplitude voltages.

[0035] In this disclosure, a method for treating tissue volume is described as comprising (a) administering an implantable composition containing a releaseable membrane activator to a target site such that the membrane activator is locally released into the tissue volume, and (b) performing IRE on the tissue volume.

[0036] During electroporation treatment according to this disclosure, a voltage is applied across two or more electrodes positioned inside or adjacent to the tissue volume being treated, thereby creating an electric field within the tissue volume. Parameters that can be adjusted with the IRE include the voltage magnitude, voltage duration (pulse length), number of pulses, number of electrodes, and electrode spacing.

[0037] The tissue volumes to which irreversible electroporation therapy pursuant to this disclosure may be applied include benign and malignant tumor volumes, as well as perfused or healthy tissue volumes containing viable and functional cells that constitute a bounded tumor volume. Examples of tumor volumes include pancreatic tumors, esophageal tumors, bladder tumors, bile duct tumors, liver tumors, kidney tumors, breast tumors, lung tumors, prostate tumors, brain tumors, uterine tumors including uterine fibroids, ovarian tumors, retroperitoneal tumors, limb tumors, pelvic tumors, and those present in neuroendocrine tumors or precancerous hyperplastic cell clusters or neoplastic structures.

[0038] A wide range of irreversible electroporation settings can be used in combination with the irreversible electroporation treatment of this disclosure, including voltages that provide electric field strengths in the range of 250–2500 V / cm, more typically 375–1750 V / cm; pulse counts in the range of 1–5000, more typically 5–300; pulse widths in the range of 0.5–1000 μs, more typically 1–250 μs; pulse frequencies in the range of 0.25–10 Hz; and electrode spacings in the range of 0.1–2 cm. The electrodes may be operated in monopolar, bipolar, or multiplexed configurations across a combination of multiple electrodes or probes.

[0039] The electrodes may be placed in or adjacent to a tissue volume to be treated by irreversible electroporation using a variety of methods, including (a) placement of one or more electrodes at one or more locations on the skin (including return electrode placement), (b) transcutaneous placement of one or more electrodes at one or more locations in tissue, (c) placement of one or more electrodes in or adjacent to one or more natural body lumens (e.g., blood vessels, gastrointestinal tract, bile ducts, lymphatic vessels, urinary tract, pulmonary airways, urethral lumen, bladder, or other body lumens), or (d) any combination of two or all three of these placement techniques.

[0040] With regard to placing one or more electrodes in or adjacent to a natural body lumen, a device such as a catheter, endoscope, bronchoscope, duodenoscope, or similar minimally invasive, single-use or reusable direct visualization probe may be advanced into the body lumen, in which case one or more electrodes may be placed on the device and in contact with the lumen wall, or one or more electrodes may be inserted from the device and inserted through the lumen wall adjacent to the device into the tissue surrounding the lumen wall.

[0041] As already stated, this disclosure describes a method for treating a tissue volume comprising (a) administering an implantable composition containing a releaseable membrane activator to a target site such that the membrane activator is locally released into the tissue volume, and (b) performing IRE on the tissue volume. In this regard, the membrane activator may be used to increase the number of cells killed at a given electric field strength and / or to decrease the electric field strength required to kill a given number of cells.

[0042] The membrane activator-releasing implantable composition may be selected from a membrane activator-releasing implantable composition that is at least partially biostable, and a membrane activator-releasing implantable composition that is bioabsorbable. In some embodiments, the membrane activator-releasing implantable composition may be an embolic composition containing a membrane activator, which may be administered to one or more blood vessels supplying blood to a tissue volume, thereby forming an embolus / occlusion. Thus, the method of the present disclosure can kill cells by blocking the blood supply to the cells, in addition to killing them with membrane activator-enhanced IRE.

[0043] Examples of membrane activators include cationic molecules, amphiphilic molecules, and anionic molecules. The membrane-active cation molecules can be selected from natural and synthetic cationic polymers having primary, secondary, tertiary, or quaternary amine groups.

[0044] Examples of natural cationic polymers include cationic polypeptides (polylysine, polyarginine, polyornithine, etc.), cationic polyamines (spermidine, spermine and their derivatives, etc.), cationic polysaccharides (chitosan, cationic gelatin, etc.), and cationic peptides rich in cationic amino acids including lysine and arginine (cecropin A, magainin, dermaceptin, bombinin, melittin, cathelicidin, human defensin, lactoferricin, histatin, indolicidin, tritriptycin, holotricin, coreoptericin, pyrhocolicin, etc.).

[0045] Synthetic cationic polymers are polymers containing primary, secondary, tertiary, or quaternary amine groups. These polymers can include molecular segments with linear, blocky, grafty, branched, dendritic, and network-like structures. These cationic polymers can be selected from the classes of polyacrylates, polymethacrylates, polyacrylamides, polymethacrylamides, polyamides, polyesters, polyorthoesters, poly(beta-aminoesters), polyethyleneimines, polypropyleneimines, polyamidoamines, polynorbornene, and their copolymers. These can be synthesized by free radical polymerization, ring-opening polymerization, polyaddition, and polycondensation reactions. Synthetic cationic polymers also include cationic peptides having unnatural sequences that can bind to cell membranes. Such cationic peptides are rich in lysine and arginine and can be synthesized using solid-phase methods well known in this field.

[0046] Membrane-soluble amphiphilic molecules include the above-mentioned natural and synthetic cationic polymers modified with hydrophobic groups and structures such as linear, branched, cyclic, and aromatic hydrocarbons containing saturated and unsaturated CC bonds. Membrane-active amphiphilic molecules also include the cationic peptides containing hydrophobic amino acids (such as alanine, glycine, leucine, isoleucine, phenoalanine, tyrosine, and tryptophan) or hydrophobic structures (such as hydrocarbone).

[0047] Membrane-active anionic molecules include certain carboxylic acid-containing synthetic polymers (such as polyalkylacrylic acids) and peptides ("GALA":WEAALAEALAEALAEHLAEALAEALEALAA, etc.). These polymers and peptides are protonated at acidic pH, becoming hydrophobic and adopting conformations that promote interaction with cellular lipid membranes.

[0048] Membrane dissolving agents are special membrane activators that cause cell membrane disruption. An example is amino-C2~C 10Alkyl acrylates and other aminoalkyl acrylates, amino-C2~C 10 Examples include aminoalkyl methacrylates such as alkyl methacrylates, copolymers of aminoalkyl acrylates and alkyl acrylates, and copolymers of aminoalkyl methacrylates and alkyl methacrylates. Examples of film-soluble aminoalkyl methacrylates include poly(6-aminohexyl methacrylate) (PAHM), poly(aminoethyl-co-butyl methacrylate), and poly(2-hexamethyleneimino)ethyl methacrylate.

[0049] In various embodiments, the membrane activator-releasing implantable composition may be a liquid composition that becomes a solid membrane activator-releasing implant when introduced into the body (for example, the composition may be in the form of a liquid embolization composition).

[0050] In various embodiments, the membrane activator-releasing implantable composition may be a solid composition. The membrane activator may be located in the entire solid composition or only in part of the solid composition. For example, the membrane activator may be present in one or more layers on an underlying substrate material (e.g., the layers may be formed entirely of the membrane activator or mixed with one or more further bioabsorbable or biostable matrix materials from which the membrane activator is released), the membrane activator may be encapsulated in an encapsulation material (e.g., a bioabsorbable encapsulation material or a biostable encapsulation material that is permeable to the membrane activator), or the membrane activator may be dispersed within the substrate material (e.g., by immersing the substrate material containing the membrane activator in an already existing substrate material, by forming the substrate as a matrix containing the membrane activator, etc.). The membrane activator may be bonded to one or more solid compositions via covalent bonds (such as amides, esters, ethers, thiol esters, dithiols, orthoesters, acetals, ketals, etc.) from which the membrane activator is released, or it may be bonded to one or more solid compositions via non-covalent bonds (such as biotin-avidin, metal chelates, charge-charge electrostatic interactions, polyethylene glycol / cyclodextrin, adamantane / cyclodextrin, etc., host-guest complexes), or it may be formed entirely from the membrane activator.

[0051] Accordingly, in addition to membrane activators, the implantable compositions of this disclosure may include a substrate material, a matrix material, and an encapsulation material that may be bioabsorbable and / or biostable. Examples of bioabsorbable and / or biostable materials include bioabsorbable and / or biostable polymers, bioabsorbable and / or biostable metals and metal alloys, and bioabsorbable and / or biostable ceramic and glass materials.

[0052] Bioabsorbable and biostable polymers, as well as polymer membrane activators, for use herein include homopolymers and copolymers. As used herein, “homopolymer” is a polymer containing multiple copies of a single constituent unit. “Copolymer” is a polymer containing multiple copies of at least two different constituent units, examples of which include random, statistical, gradient, periodic (e.g., alternating) and block (e.g., diblock, triblock, etc.) copolymers. Polymers for use in this disclosure may be linear or branched. Branched configurations include star configurations (e.g., configurations with three or more chains emanating from one branching point), comb configurations (e.g., configurations with a main chain and multiple side chains), dendritic configurations (e.g., dendritic and hyperbranched polymers), and the like.

[0053] Specific examples of bioabsorbable and biostable polymers can be selected from, for example, the following: polycarboxylic acid homopolymers and copolymers containing polyacrylic acid; alkyl acrylate and alkyl methacrylate homopolymers and copolymers containing poly(methyl methacrylate-bn-butylacrylate-b-methyl methacrylate) and poly(styrene-bn-butylacrylate-b-styrene) triblock copolymers; polyamides containing nylon 6,6 and nylon 12; polyether-block-polyamide copolymers (e.g., Pebax® resin); polyvinyl halides such as polyvinyl alcohol, polyvinylpyrrolidone, and polyvinyl chloride; vinyl homopolymers and copolymers such as ethylene-vinyl acetate copolymer (EVA); vinyl aromatic homopolymers and copolymers such as polystyrene and styrene-maleic anhydride copolymer; styrene-butadiene copolymer; styrene-ethylene-butylene copolymer (e.g., Kraton® G series polymers) - Available as poly(styrene-b-ethylene / butylene-b-styrene (SEBS)) copolymer, styrene-isoprene copolymer (e.g., poly(styrene-b-isoprene-b-styrene)), acrylonitrile-styrene copolymer, acrylonitrile-butadiene-styrene copolymer, styrene-butadiene copolymer and styrene-isobutylene copolymer (e.g., polyisobutylene-polystyrene block copolymer, e.g., poly(styrene-b-isobutylene-b-styrene) or Polyesters, glycolides (glycolic acid), and ε-caprolactone, poly(l-lactide-co-glycolide) and poly(d,l-lactide) homopolymers and copolymers of vinyl aromatic alkene copolymers, ionomers, polyethylene terephthalate and lactide (including d-,l- and meso-lactide) (poly(L-lactide) and poly(d,l-lactide)), including poly(L-lactide) and poly(d,l-lactide) (as described in U.S. Patent No. 6,545,097, for example, by Pinchuk et al., including poly(L-lactide-co-glycolide) and poly(d,Polycarbonates containing polylactide co-glycolides such as l-lactide co-glycolides, polymethylene carbonates (and their alkyl derivatives), polyanhydrides, polyoltoesters, polyalkylene oxide polymers such as polyethylene oxide (PEO) and polyether ether ketones, polyolefin homopolymers and copolymers containing polyalkylenes such as polypropylene, polyethylene, and polybutylene (polybuta-1-ene and polyisobutylene), polyolefin elastomers (e.g., Santoprene) and ethylene propylene diene monomers. Biopolymers such as (EPDM) rubber, polytetrafluoroethylene (PTFE), poly(tetrafluoroethylene-co-hexafluoropropene) (FEP), modified ethylene-tetrafluoroethylene copolymer (ETFE), and polyvinylidene fluoride (PVDF), as well as fluoride homopolymers and copolymers containing polydimethylsiloxane, silicone homopolymers and copolymers containing polydimethylsiloxane, polyurethanes, polypeptides, proteins, polysaccharides, fibrin, fibrinogen, collagen, elastin, chitosan, gelatin, starch, glycosaminoglycans such as hyaluronic acid, and blends and further copolymers of the above.

[0054] Examples of biostable polymers for use in this disclosure may also be selected from homopolymers and copolymers containing, for example, one or more olefin monomers including fluorinated olefin monomers, acrylate monomers, methacrylate monomers, vinyl monomers, dialkylsiloxane monomers and combinations thereof, as well as various other polymers such as polyurethanes, polyureas, polyamides including nylon, polycarbonates, polyesters, poly(etherketone) (PEEK) and poly(arylene etherketone) PAEK.

[0055] Examples of bioabsorbable polymers for use in this disclosure may also be selected from biodegradable polyester homopolymers and copolymers, poly(orthoester) homopolymers and copolymers, polyanhydride homopolymers and copolymers, amino acid homopolymers and copolymers including tyrosine-based polymers, and polyethylene glycol homopolymers and copolymers.

[0056] Membrane activators can be released from a transplantable composition by any type of chemical reaction, including diffusion, dissolution, biodegradation, hydrolysis (via covalent bonds), or by enzymatic cleavage of bonds, ionization and deionization, or any combination thereof.

[0057] The membrane activator may be released from an implantable composition in the following manner: burst release profile (e.g., within a period of 0 to 6 hours), sustained release profile (e.g., over a period of 6 hours to 3 months), pulsatile release profile, biphasic or multiphasic release profile, or a combination of the foregoing.

[0058] The membrane activator may be released from the implantable composition via a triggerable release mechanism, for example, as a result of: electrical pulses (e.g., to heat the implantable composition with or without a corresponding phase transition and drive the membrane activator out of the implantable composition by electrical transfer, etc.); ultrasound (e.g., to heat the implantable composition or to rupture a capsule containing the membrane activator and thereby release the membrane activator, etc.); magnetic field fluctuations (e.g., magnetic particles may be provided in the implantable composition and vibrated to release the membrane activator, for example, by heating the implantable composition or by rupturing a capsule containing the membrane activator); injection of chemicals (e.g., by injecting an acid or base, a low-tension or high-tension salt to break the bond between the membrane activator and the implantable composition and cause the release of the membrane activator, etc.); changes in diffusion rate or membrane transport due to thermal excitation, weakening of chemical bonds and modification of release, or a combination of the above.

[0059] The membrane activator-releasing implantable composition may, for example, be in the form of membrane activator-releasing particles. Such particles can take on a variety of shapes, including spherical and elongated shapes. Such particles may have a maximum cross-sectional dimension (e.g., diameter in a sphere) in the range of 10 nm to 10 mm.

[0060] The membrane activator-releasing implantable composition may, for example, be in the form of a membrane activator-releasing scaffold. Such scaffolds may take various forms such as disks, rods, stents, coils, reference markers, woven meshes, and tubes. Such scaffolds may contain pores ranging from 0.1 nm to 1 mm, or they may not be porous at all.

[0061] The membrane-releasing implantable composition may be implanted into target tissue using any suitable implantation method, including percutaneous placement of the implantable composition, placement of the implantable composition within or adjacent to one or more natural body lumens (e.g., blood vessels including distal capillary or hypervascular lesions to block blood flow, gastrointestinal tract, bile duct, lymphatic vessel, urinary tract, or other body lumens), or a combination thereof. Placement of one or more implantable compositions within or adjacent to natural body lumens may be performed using devices such as catheters, endoscopes, microcatheters, needles, or other flexible probes that maintain an open body lumen.

[0062] As already stated, various embodiments of this disclosure describe methods for treating tissue volume, comprising (a) administering a membrane activator-releasing implantable composition to a target site such that a membrane activator is locally released into the tissue volume, and (b) performing IRE on the tissue volume.

[0063] In some embodiments, the same procedure can be used to administer the implantable composition to a target site and perform IRE on the tissue volume. For example, the implantable composition may be administered to the target site, and IRE may be performed on the tissue volume after sufficient time (e.g., in the range of 30 seconds to 180 minutes) has been allowed for a burst release from the implantable composition into the tissue volume.

[0064] In some embodiments, IRE may be performed on tissue volume in one or more steps following the procedure in which the implantable composition is administered to the target site. For example (for example, if the implantable composition is a sustained-release composition), one or more subsequent steps may be performed at a time in the range of 0.1 days to 6 weeks after the administration of the implantable composition to the target site.

[0065] As can be seen from the above, the advantage of the compositions and methods of this disclosure is that an essentially arbitrary time course and an essentially arbitrary IRE schedule can be established for the release of the desired membrane activator, which can be adjusted based on tumor biology. [Examples]

[0066] Materials: Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin-streptomycin (10,000 U / mL; 10,000 μg / mL), Dulbecco's phosphate-buffered saline (DPBS), MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), dimethyl sulfoxide (DMSO), ethanol (200 proof), Alexa Fluor® 488 NHS Ester (purchased from Thermo Fisher Scientific, Waltham, Massachusetts, USA). Hydroxylamine hydrochloride and PD-10 desalting column (Sephadex® G-25 medium; exclusion limit: M rThe 5000ml tubes were purchased from Millipore Sigma (Burlington, Massachusetts, USA). The electroporation cuvettes (4mm gap) were obtained from Harvard Apparatus (Holliston, Massachusetts, USA). The UV clear cuvettes (semi-micro, 1.5mL) were purchased from USA Scientific (Ocala, Florida, USA).

[0067] Compusyn modeling of drug synergistic effects. The combination index (CI) and dose reduction index (DRI) for each treatment combination were calculated using the Chou-Talalay method with the CompuSyn software program (ComboSyn, Inc., Paramus, New Jersey, USA) (Chou, T.-C., "Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies," Pharmacol. Rev., 58, 621-681 (2006)). The CI value is a dimensionless numerical representation of drug interactions, where CI=1 indicates additive effect, CI<1 indicates synergistic effect, and CI>1 indicates antagonistic effect. DRI is an index that shows how much the dosage of each drug can be reduced when used in combination with a given Fa compared to when each drug is administered alone. DRI=1 means no reduction in dosage, DRI>1 means a desirable reduction in dosage, and DRI<1 means an undesirable reduction in dosage.

[0068] Statistical analysis. The significance of differences in cell viability between different treatment groups was determined by multiple comparisons using analysis of variance (ANOVA) and Tukey's HSD test. The significance of differences in PAHM fluorescence intensity between coated microspheres was determined by multiple comparisons using ANOVA and Tukey's HSD test. PAHM emission profiles were fitted using a two-phase exponential association model. All analyses were performed using GraphPad prism, version 9.0.0 (GraphPad software, San Diego, California, USA).

[0069] Example 1: Synthesis of poly(6-aminohexyl methacrylate) (PAHM) Poly(6-aminohexyl methacrylate) (PAHM) was synthesized as follows: N-(tert-butoxycarbonyl)aminohexyl methacrylate (tBocAHM) was first synthesized as described in Zhu, C. et al., "Cationic methacrylate copolymers containing primary and tertiary amino side groups: Controlled synthesis via RAFT polymerization, DNA condensation, and in vitro gene transfection," J. Polym. Sci. Part A Polym. Chem., (2010). doi:10.1002 / pola.24064. Next, PAHM was synthesized via atom transfer radical polymerization (ATRP) of tBocAHM, followed by deprotection of the tBoc side chain, based on the method reported by Ji, W. et al., "Poly(2-aminoethyl methacrylate) with Well-Defined Chain Length for DNA Vaccine Delivery to Dendritic Cells," Biomacromolecules, 12, 4373-4385 (2011). This polymer was then synthesized as described in Ji, W.'s "Ji, W., Synthetic polymers with well-defined structures for DNA vaccine delivery and cancer therapy" (PhD dissertation, University of Minnesota, November 2013), 1 Characterization was performed using 1H NMR and gel permeation chromatography (GPC). Number average molecular weight (M) of PAHM. n ) is 2.08 × 10 4The dispersity (D) was 1.26 and the average degree of polymerization (DP) was 100.

[0070] Example 2: Bead formation Unhydrolyzed poly(methyl methacrylate) (PMMA) beads (90 - 106 μm in diameter) were incubated with the membrane surfactant, specifically poly(6 - aminohexyl methacrylate) (PAHM),

[0071]

Chemical formula

[0072] (where n is an integer), and as will be detailed further below, the beads were incubated with PAHM in ethanol and then coated by evaporating the ethanol.

[0073] Example 3: Fluorescent labeling of PAHM PAHM was fluorescently labeled with the NHS ester of Alexa Fluor® 488 dye (Thermo Fisher Scientific) (λ ex / λ em : 494 / 517 nm; absorption coefficient = 71,000 cm -1 M -1 ). The theoretical labeling degree was 1% based on the feed ratio, and the labeling efficiency was 25 - 33% (per manufacturer). PD - 10 desalting column (Sephadex® G - 25 medium; exclusion limit: M rUnreacted dyes were removed by gel filtration using a Millipore Sigma 5000 gel. The purified fluorescently labeled PAHM solution was frozen overnight at -80°C and freeze-dried for 4 days at 0.020 mBar (0.002 kPa) in a FreeZone® freeze-drying system (Labconco, Kansas City, Missouri) equipped with a Maxima® C Plus vacuum pump (Model M8c, Thermo Fisher Scientific, Waltham, Massachusetts). The dried polymer was stored in a vacuum desiccator (protected from light) at room temperature until use.

[0074] Example 4: Optimization method for coating embolic microspheres with PAHM PMMA microspheres with an average diameter of 71 μm or 100 μm were supplied by Boston Scientific Corporation (Maple Grove, Minnesota, USA). The PMMA microspheres were coated with PAHM by solvent evaporation. A mixture of fluorescently labeled PAHM and unlabeled PAHM (labeled:unlabeled ratio 1:8) was dissolved in ethanol (Thermo Fisher Scientific) at a total concentration of 0.1 mg / mL or 0.5 mg / mL. In a glass vial, 1 mL of the PAHM solution was added to 50 mg of PMMA microspheres (100 μm in diameter). The vial was exposed to an orbital shaker (Lab-Line Instruments, Melrose Park, Illinois, USA) at room temperature for 3 days to evaporate the ethanol. Depending on the supply ratio, after drying, the microspheres were coated with PAHM at a concentration of 2 μg / mg or 10 μg / mg (μg PAHM / mg PMMA). The coated microspheres were stored in a vacuum desiccator at room temperature (protected from light) until use.

[0075] To optimize and accelerate the coating process, PMMA microspheres were coated with PAHM using three different solvent evaporation methods. A mixture of fluorescently labeled PAHM and unlabeled PAHM (labeled:unlabeled ratio 1:8) was dissolved in ethanol at a total concentration of 0.8 mg / mL. 0.5 mL of the PAHM solution was added to 100 mg of PMMA microspheres (100 μm in diameter) in a glass vial. In coating method 1, the ethanol was evaporated as described above (on a slowly rotating orbital shaker for 3 days). In coating method 2, the uncovered vial was placed in a vacuum desiccator on a slowly rotating orbital shaker for 12 hours. In coating method 3, the ethanol was evaporated with compressed air for 10 minutes. The coated microspheres were stored in a vacuum desiccator (protected from light) at room temperature until use.

[0076] To visualize the PAHM coating, fluorescence and bright-field images of coated microspheres were acquired using an Olympus® IX70 inverted fluorescence microscope equipped with an Olympus® DP72 camera and an X-Cite 120 wide-field fluorescence microscope excitation light source (Excelitas Technologies, Waltham, Massachusetts). Fluorescently labeled PAHM was visualized using an excitation wavelength of 480±50 nm and an emission wavelength of 535±50 nm. The fluorescence intensity of PAHM in individual coated microspheres was quantified using ImageJ, an open-source Java®-based image processing program developed by the National Institutes of Health and the Optical Computational Instruments Laboratory (University of Wisconsin-LOCI).

[0077] Embolistic microspheres (100 μm in diameter) were coated with fluorescently labeled PAHM by solvent evaporation. A representative fluorescence micrograph (Figure 1A) shows that increasing the concentration of PAHM results in a thicker coating, as microspheres coated with 10 μg / mg (μg PAHM / mg PMMA) exhibited 10.5 times brighter fluorescence than microspheres coated with 2 μg / mg (μg PAHM / mg PMMA) (Figure 1B).

[0078] To optimize the coating process, embolic microspheres (100 μm in diameter) were coated with 4 μg / mg (μg PAHM / mg PMMA) of fluorescently labeled PAHM using three different solvent evaporation methods. Representative fluorescence micrographs of microspheres coated by the three methods are shown in Figure 1C, and the quantification of fluorescence intensity is shown in Figure 1D. In Method 3, ethanol evaporated rapidly within 10 minutes, resulting in a bright, relatively uniform coating (Figure 1C). In Method 2, the slow evaporation of ethanol over 12 hours appeared to slightly increase the amount of PAHM coated on the microspheres, as indicated by fluorescence 1.2 times brighter compared to Method 3 (Figure 1D). However, further delaying the evaporation in Method 3 to 3 days resulted in a much dimmer coating (Figure 1C, D). In all subsequent experiments, we chose to coat the microspheres using Method 3 because it was fast while producing a good coating.

[0079] All subsequent experiments (PAHM release kinetics and cytotoxicity assays) used microspheres coated with unlabeled PAHM at a concentration of 10 μg / mg (μg PAHM / mg PMMA). PAHM was dissolved in ethanol at a concentration of 1 mg / mL. In a glass vial, 1 mL of PAHM solution was added to 100 mg of microspheres (71 μm or 100 μm). The ethanol was evaporated using Method 1 described above.

[0080] Example 5: In vitro emission kinetics of coated microspheres In a 12-well plate, 25 mg of coated microspheres (71 μm or 100 μm in diameter) were suspended in 1 mL of cell culture medium (without phenol red) and incubated for 1 week in a humidified environment at 37°C with 5% CO2. At specific time points, 750 μL of supernatant was sampled and replaced with fresh medium. The sampled supernatant was then diluted with 0.75 mL of release medium (final volume: 1.5 mL) in a clear UV cuvette (semi-micro, 1.5 mL) (USA Scientific, Ocala, Florida, USA). The amount of PAHM in the supernatant was determined by measuring the absorbance at 245 nm using a Cary100 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, California, USA). Figures 2A and 2B show the quantification of PAHM in cell culture medium. Figure 2A shows the UV-Vis absorption spectra at different PAHM concentrations. Figure 2B shows the calibration curve of absorbance at 245 nm with respect to PAHM concentration.

[0081] The release kinetic profile is expressed as the cumulative percentage released over time and is shown in Figure 3. Following the initial burst release within the first four hours, sustained PAHM release continued for one week. PAHM was released slightly faster from 71 μm PMMA microspheres than from 100 μm PMMA microspheres. Within the first hour, 51% and 38% of PAHM were released from 71 μm and 100 μm PMMA microspheres. After four hours, 69% and 59% of PAHM were released from 71 μm and 100 μm PMMA microspheres. More than 20% of PAHM was released from both sizes of microspheres between days 2 and 7.

[0082] Example 6: Cell Culture The human pancreatic adenocarcinoma cell line AsPC-1 was obtained from ATCC. AsPC-1 cells were cultured in Roswell Park Memorial Laboratory (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, Massachusetts, USA), containing 2 g / L glucose, 2 mM L-glutamine, 2 g / L sodium bicarbonate, 10% thermo-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific), 100 U / mL penicillin (Thermo Fisher Scientific), and 100 μg / mL streptomycin (Thermo Fisher Scientific). AsPC-1 cells were cultured in tissue culture flasks and incubated in a humidified environment of 37°C with 5% CO2. When the cells reached approximately 80% confluence, the culture medium was removed, the cells were harvested, and divided into 1:3 to 1:6 groups for continued cultivation or immediate use for cytotoxicity experiments.

[0083] Example 7: Cytotoxicity of IRE in combination with free PAHM or PAHM released from coated microspheres The collected cells were centrifuged, and the supernatant was discarded. The cell pellet was resuspended in cell culture medium (without phenol red), diluted to a density of 675,000 cells / mL, and 1 mL aliquots were dispensed into 2 mL microcentrifuge tubes.

[0084] Free PAHM was dissolved in cell culture medium to an initial concentration of 250 μg / mL and then diluted to various concentrations. 0.5 mL of PAHM diluent was added to 1 mL of cell suspension to obtain final concentrations of 0–50 μg / mL. Coated microspheres (71 μm or 100 μm in diameter) and uncoated microspheres were suspended in cell culture medium to an initial concentration of 25 mg / mL and then diluted to various concentrations. 0.5 mL of coated microsphere diluent was added to 1 mL of cell suspension to obtain final concentrations of 0–5 mg / mL (corresponding to 0–50 μg / mL of microsphere-coated PAHM).

[0085] After incubation for 15 minutes, the cells were treated with IRE as described in Shao, Q. et al., “Physical and Chemical Enhancement of and Adaptive Resistance to Irreversible Electroporation of Pancreatic Cancer,” Ann. Biomed. Eng., 46, 25-36 (2018). Briefly, 400 μL of the prepared cell suspension was pipettered into an electroporation cuvette (BTX45-0126, Harvard Apparatus, Holliston, Massachusetts) between two aluminum plate electrodes (4 mm apart). The cuvette was placed in an external electric field generated by an electrical pulse generator (BTX ECM square wave electroporation system, BTX Model No. 830, Herbert Apparatus, Holliston, Massachusetts, USA) that delivered 50 electrical pulses (pulse duration 100 μs, frequency 1 Hz) at 150, 225, 300, 375, or 450 V (corresponding to electric field strengths of 375, 562.5, 750, 937.5, or 1125 V / cm).

[0086] For samples with a 15-minute exposure time, 400 μL of treated cell suspension was transferred to a microcentrifuge tube, centrifuged, and the supernatant was discarded. PAHM was washed from the cells twice with 1 mL of Dulbecco's phosphate-buffered saline (DPBS) (Thermo Fisher Scientific). The cell pellet was resuspended in 1 mL of cell culture medium and plated into a 12-well plate (180,000 cells / well). For samples with 4 and 24-hour exposure times, 400 μL of treated cell suspension was plated into a 12-well plate (180,000 cells / well), and 600 μL of free PAHM or coated microsphere diluent was added to maintain an appropriate PAHM dose. After incubation for 4 or 24 hours in a humidified environment of 37°C with 5% CO2, the cells were washed twice with 1 mL of DPBS and given 1 mL of fresh cell culture medium.

[0087] Cell viability was evaluated using the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) (Thermo Fisher Scientific) assay (Mosmann, T., "Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays," J.Immunol.Methods, 65, 55-63 (1983)). 100 μL of MTT solution (5 mg / mL in DPBS) was added to each well. After incubation for 4 hours in a humidified environment at 37°C with 5% CO2, 850 μL of medium was removed from each well. Formazan crystals were dissolved in 1 mL of DMSO (Thermo Fisher Scientific), and the absorbance was measured at 540 nm using a BioTek Cytation® Cell Imaging Multi-Mode Reader (BioTek Instruments, Winooski, Vermont). Cell viability was determined by normalizing with the absorbance of untreated cells (using the same exposure time).

[0088] Figures 4A-4B show the viability of AsPC-1 cells after exposure to IRE or PAHM. Higher electric field strengths reduced cell viability after IRE alone, but the decline was relatively gradual (Figure 4A). More than 70% of cells exposed to 750 V / cm or less survived. Even at the highest electric field strength (1125 V / cm), 46%, 50%, and 68% of cells were killed at 15 minutes, 4 hours, and 24 hours. In contrast, PAHM alone showed a much more significant cell death effect (Figure 4B). In cells treated with PAHM at doses of 25 μg / mL or higher, more than 45%, 90%, and 85% of cells were killed after continuous exposure to PAHM for 15 minutes, 4 hours, and 24 hours. 100% of cells were killed after 24 hours of treatment with 40 μg / mL or 50 μg / mL PAHM.

[0089] Incubating cells for 4 or 24 hours after IRE treatment did not appear to affect cell viability, indicating that the cell-killing effect of IRE is largely time-dependent (Figure 4A). Only when exposed to 1125 V / cm did 24-hour incubation reduce cell viability. Unlike IRE, PAHM-induced cell death was time-dependent. Interestingly, treating cells with PAHM for 4 hours seemed to reduce cell viability more than treating them for 24 hours. At PAHM doses ≤20 μg / mL, a significant decrease in cell viability was observed after 4 hours of exposure, but the cells showed some recovery after 24 hours. At PAHM doses of 25 μg / mL or higher, no statistically significant difference in cell viability was observed between 4-hour and 24-hour exposures, and therefore this recovery was not observed.

[0090] To evaluate the ability of PAHM to enhance the cell-killing effect of IRE, two electric field intensities were combined with three PAHM doses (Figures 5A-5C). For all exposure times, the combination of PAHM and IRE resulted in significantly more cell death. When cells were treated with PAHM for only 15 minutes, the viability of cells exposed to 562.5 V / cm decreased from 95% to 64%, 48%, and 33% with PAHM concentrations of 5 μg / mL, 15 μg / mL, and 25 μg / mL, respectively (Figure 5A). After 4 hours of exposure to PAHM, the viability of cells exposed to 562.5 V / cm decreased from 86% to 37%, 10%, and 2% with PAHM concentrations of 5 μg / mL, 15 μg / mL, and 25 μg / mL, respectively (Figure 5B). Similar to PAHM treatment alone, cells treated with IRE and PAHM for 24 hours showed some recovery in cell viability; however, the viability of cells exposed to 562.5 V / cm decreased from 91% to 72%, 17%, and 3% with PAHM concentrations of 5 μg / mL, 15 μg / mL, and 25 μg / mL, respectively (Figure 5C).

[0091] To investigate the ability of PAHMs to enhance the efficacy of IRE, the combination index (CI) and dose-reduction index (DRI) for each combination of IRE and PAHM were calculated using the Chou-Talalay method with the CompuSyn software program (CompuSyn, Paramus, New Jersey). The CI is plotted against the fractional effect (Fa), which is the fraction of cell proliferation inhibited by specific combinations of IRE and free PAHM (Figure 5D). For 15 minutes of PAHM exposure, most CIs were greater than 1.12, suggesting that the combination of IRE and free PAHM is slightly antagonistic. For longer PAHM exposures, the CI tended to decrease with higher Fa levels. For 4 hours of PAHM exposure, the combinations with the three highest Fa levels appeared synergistic. For 24 hours of PAHM exposure, combinations with low Fa levels showed mild to moderate antagonism; however, combinations with higher Fa levels were mostly additive or showed moderate synergistic effects. The DRIs for IRE field strength and PAHM dose are shown in Figures 5E and 5F. The DRI for IRE tended to increase with Fa at all three exposure times, and all DRIs were greater than 1. This indicates that, for any combination with PAHM, lower field strengths can kill a similar percentage of cells as higher field strengths alone. The DRI for PAHM was greater than 1 for all combinations except one, indicating that the PAHM dose can also be reduced when used in combination. For 15-minute and 24-hour exposures, the DRI tended to decrease with Fa; however, the DRI tended to increase with Fa at 4-hour exposure to PAHM.

[0092] Figures 6A-6B show the viability of AsPC-1 cells after exposure to microspheres (71 μm or 100 μm in diameter) coated with 10 μg / mg (μg PAHM / mg PMMA). Since PMMA microspheres alone (uncoated) showed little to no toxicity (Figure 7), all cell death was attributable to PAHM released from the coated microspheres. Similar to free PAHM, the exposure time to PAHM released from 71 μm coated microspheres had a significant effect on cell viability (Figure 6A). Only moderate cell death occurred after 15 minutes of exposure to 71 μm coated microspheres, with even the highest dose (50 μg / mL microsphere-coated PAHM) killing only 47% of cells. Four hours of exposure to 71 μm coated microspheres significantly reduced cell viability. With microsphere-coated PAHM at concentrations of 20 μg / mL or higher, the viability was less than 13%. After 24 hours of exposure to 71 μm coated microspheres, cells recovered much of their viability, similar to that observed after 15 minutes of exposure. Only with 50 μg / mL microsphere-coated PAHMs did 24-hour exposure result in statistically significantly more cell death than 15 minutes of exposure. Similar results were observed for cells treated with 100 μm coated microspheres for 15 minutes or 24 hours; however, cytotoxicity was minimal after 4 hours of exposure (Figure 6B). Even with 50 μg / mL microsphere-coated PAHMs, 19% of cells remained viable after 4 hours of exposure to 100 μm coated microspheres. Only with microsphere-coated PAHMs of 25 μg / mL or higher did longer exposure times result in significantly lower cell viability compared to 15 minutes of exposure.

[0093] Example 8: Cytotoxicity of IRE in combination with PAHM released from coated microspheres Due to the slower release of PAHM and the much smaller difference in cell viability between 4-hour and 24-hour exposure times, 100 μm coated microspheres were selected to investigate the ability of PAHM released from coated microspheres to enhance IRE (Figures 8A-8F). Released PAHM enhanced the IRE cell-killing effect after 15 minutes, particularly after 4 hours; however, a 24-hour exposure time did not significantly reduce cell viability. After 15 minutes of exposure, the viability of cells exposed to 912.5 V / cm decreased from 95% to 40-54% when coated microspheres were added; however, there was no statistically significant difference between the three microsphere doses (Figure 8A). Cell viability decreased from 86% to 65%, 56%, and 19% with 5 μg / mL, 15 μg / mL, and 25 μg / mL microsphere-coated PAHMs in addition to IRE (Figure 8B), with the most substantial IRE enhancement observed with 4 hours of exposure to 100 μm coated microspheres. This enhancement almost completely disappeared after 24 hours of exposure. Compared to IRE alone, the only combination that significantly reduced cell viability was 562.5 V / cm electric field strength and 25 μg / mL microsphere-coated PAHM (Figure 8C). This reduction was also relatively gradual, from 91% with IRE alone to 75% with the combination.

[0094] CI and DRI were also calculated for the combination of IRE and coated PMMA microspheres. As shown by the CI in Figure 8D, the combination of 15-minute exposure to PAHM released from coated microspheres and IRE is strongly antagonistic at low Fa but synergistic at intermediate Fa. At 4-hour exposure, most CIs were in the range of 0.9–1.1 (or close to it), showing a nearly additive effect. All 24-hour exposure time combinations showed a CI of 1.1 or greater. At low Fa, this combination showed a strong antagonistic effect, but the antagonistic effect decreased with increasing Fa. The DRIs of IRE field strength and microsphere-coated PAHM dose are shown in Figures 11E and 11F. The DRI of IRE tended to increase with Fa at all three exposure times, and at Fa > 0.1, the DRI of IRE was greater than 1 in all cases. The DRI of microsphere-coated PAHMs tended to increase with Fa for 15-minute and 24-hour exposure times, but decreased with Fa for 4-hour exposure times. When Fa > 0.2, the DRI of microsphere-coated PAHMs was greater than 1. The technical concepts that can be understood from the above embodiments are described below as an addendum. [Note 1] An embolic composition containing a releaseable membrane activator. [Note 2] The embolic composition according to Appendix 1, wherein the membrane activator is selected from cationic molecules, amphiphilic molecules, and anionic molecules. [Note 3] The embolic composition according to Appendix 1, wherein the membrane activator is selected from cationic polymers, anionic polymers, and amphiphilic polymers. [Note 4] The embolic composition according to Appendix 1, wherein the membrane activator is a natural or synthetic cationic polymer having primary, secondary, tertiary, and / or quaternary amine groups. [Note 5] The embolic composition according to Appendix 1, wherein the membrane activator is selected from aminoalkyl acrylate homopolymer, aminoalkyl acrylate copolymer, aminoalkyl methacrylate homopolymer, and aminoalkyl methacrylate copolymer. [Note 6] The embolic composition according to Appendix 1, wherein the membrane activator is selected from poly(6-aminohexyl methacrylate), poly(aminoethyl-co-butyl methacrylate), and poly(2-hexamethyleneimino)ethyl methacrylate. [Note 7] The embolic composition according to any one of the appendices 1 to 6, wherein the embolic composition is a liquid composition that becomes a solid embolic composition when introduced into the body. [Note 8] The embolic composition according to any one of the appendices 1 to 6, wherein the embolic composition is a solid embolic composition. [Note 9] The embolic composition according to Appendix 8, wherein the solid embolic composition is at least partially biostable or bioabsorbable. [Note 10] The embolic composition according to any one of appendices 8 to 9, wherein the solid embolic composition is implantable particles. [Note 11] The embolic composition according to any one of the appendices 8 to 9, wherein the solid embolic composition is a scaffold. [Note 12] The embolic composition according to Appendix 11, wherein the scaffold is selected from a disc, rod, stent, coil, reference marker, plate, tube, or semi-permeable woven mesh. [Note 13] The embolic composition according to any one of the appendices 8 to 12, wherein the membrane activator is present throughout the solid embolic composition or only in a part of the solid embolic composition. [Note 14] The embolic composition according to any one of the appendices 8 to 13, wherein the membrane activator is present on the surface of the solid embolic composition but not in the majority of the solid embolic composition, the membrane activator is present in the majority of the solid embolic composition but not on the surface of the solid embolic composition, the membrane activator is present homogeneously in the solid embolic composition, the membrane activator is present in the solid embolic composition with a concentration gradient, the membrane activator is present in one or more layers arranged on a lower substrate material, the membrane activator is encapsulated in an encapsulation material, the membrane activator is dispersed in a matrix material, or the membrane activator is conjugated in a matrix material. [Note 15] The embolic composition according to any one of Appendix 1 to 14, wherein, upon implantation of the embolic composition in a subject, the membrane activator is locally released in a burst release profile, a sustained release profile, or a combination of a burst release profile and a sustained release profile.

Claims

1. An implantable composition for use in the treatment of human body tissue, The implantable composition is an embolic composition comprising a releaseable membrane activator, The releaseable membrane activator is selected from aminoalkyl acrylate, aminoalkyl methacrylate, copolymer of aminoalkyl acrylate and alkyl acrylate, and copolymer of aminoalkyl methacrylate and alkyl methacrylate. (a) The embolic composition containing the releaseable membrane activator is administered to a target site such that the membrane activator is locally released into the tissue, and (b) treatment of the tissue is carried out by applying a pulsed electric field to the tissue. The aforementioned tissue is a transplantable composition that corresponds to all or part of a tumor, or all or part of the tumor and the healthy tissue surrounding it.

2. The implantable composition according to claim 1, wherein the treatment results in irreversible electroporation, reversible electroporation, or heat treatment.

3. The transplantable composition according to claim 1, wherein the tumor is selected from tumors of the pancreas, breast, lung, prostate, liver, kidney, brain, uterus, ovary, stomach, duodenum, or skin.

4. The implantable composition according to claim 1, wherein the embolic composition is administered to one or more blood vessels that supply blood to the tissue, or the embolic composition is administered percutaneously or endoscopically by injection.

5. The transplantable composition according to claim 1, wherein the transplantable composition is at least partially biostable or bioabsorbable.

6. The implantable composition according to claim 1, wherein the implantable composition is a solid implantable composition when administered to the target site, or a liquid composition that becomes a solid implant upon introduction when administered to the target site.

7. The transplantable composition according to claim 6, wherein the transplantable composition of the solid is transplantable particles, or the transplantable composition of the solid is a scaffold.

8. The implantable composition according to claim 6, wherein the solid implantable composition is implantable particles having a maximum cross-sectional dimension in the range of 10 nm to 10 mm, or the solid implantable composition is a scaffold selected from a disk, rod, stent, coil, reference marker, plate, tube, or semipermeable woven mesh.

9. The implantable composition according to claim 6, wherein the membrane activator is present throughout the solid implantable composition, the membrane activator is present only in a portion of the solid implantable composition, the membrane activator is present on the surface of the solid implantable composition but not in the majority of the solid implantable composition, the membrane activator is present in the majority of the solid implantable composition but not on the surface of the solid implantable composition, the membrane activator is homogeneously present in the solid implantable composition, the membrane activator is present in the solid implantable composition with a concentration gradient, the membrane activator is present in one or more layers arranged on a lower substrate material, the membrane activator is encapsulated in an encapsulation material, or the membrane activator is dispersed in a matrix material.

10. The implantable composition according to claim 6, wherein the solid implantable composition is an embolic particle, and the membrane activator is coated on the embolic particle, injected into the embolic particle, or conjugated to the embolic particle via covalent bonds.

11. The implantable composition according to claim 1, wherein the release of the membrane activator is triggered by an electrical pulse, ultrasound, magnetic field fluctuation, or thermal input.

12. The transplantable composition according to claim 1, wherein the treatment is carried out in the same procedure as the administration of the transplantable composition.

13. The transplantable composition according to claim 1, wherein the treatment is carried out in a step following the administration of the transplantable composition.

14. The transplantable composition according to claim 1, wherein the treatment is carried out by procedure at a time within the range from the same day to 30 days after administration of the transplantable composition.

15. The implantable composition according to claim 1, wherein one or more electrodes for the treatment are positioned within or adjacent to the tissue.

16. The implantable composition according to claim 1, wherein (a) one or more therapeutic electrodes are positioned adjacent to the tissue at one or more locations on the skin; (b) one or more therapeutic electrodes are positioned percutaneously within or adjacent to the tissue; (c) one or more therapeutic electrodes are positioned within or adjacent to the tissue via a device inserted into one or more natural body tubules; or (d) a combination of any two or all three of the aforementioned positioning techniques.

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