Implantable compositions for combined tumor treatment - Patents.com

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

[0002] A variety of modalities are used to treat abnormal biological tissue, such as cancer, including thermal ablation, cryoablation, ultrasound ablation, chemical ablation, RF electrical ablation and irreversible electroporation.

[0003] New and different approaches to the destruction of biological tissue are desirable. This disclosure describes novel compositions and combination approaches for tissue destruction using irreversible electroporation. Summary of the Invention [Means for solving the problem]

[0004] In some embodiments, the present invention discloses a method of treating a tissue volume comprising: (a) administering to a target site an implantable composition comprising a releasable membrane-active agent such that the membrane-active agent is released locally to the tissue volume; and (b) effecting treatment of the tissue volume by application of a pulsed electric field to the tissue volume. For example, irreversible electroporation, reversible electroporation and / or heat treatment may be effected by application of a pulsed electric field.

[0005] As used herein, "membrane active agent" is defined as an agent or molecule that affects the structure and dynamics of cell membranes, such as cationic or amphipathic or anionic molecules. Cationic molecules bind to cell membranes through charge-charge attraction. Amphipathic molecules bind to cell membranes through charged functional groups and bind to cell membrane lipids through hydrophobic interactions. Anionic molecules become hydrophobic when protonated and can interact with cell membrane lipids. As used herein, "membranolytic agent" is a subset of membrane active agents and refers to agents or molecules that disrupt membrane integrity. Membrane active agents often become membrane lytic agents when present at high enough concentrations.

[0006] In some embodiments, the tissue volume corresponds to all or a portion of the tumor and / or healthy tissue surrounding the tumor (to form a margin via the vasculature). 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 that includes a releasable membrane active agent, and the embolic composition is administered to one or more blood vessels that supply blood to a tissue volume, or the embolic composition is administered percutaneously or endoscopically by injection.

[0008] In some embodiments, which 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 that is administered as a liquid and has viscoelastic properties that transform into a solid upon activation or contact with bodily fluids.

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

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

[0011] In some embodiments, which may be used in combination with any of the above aspects and embodiments, the solid embolic composition may be a scaffold, such as a disk, rod, stent, coil, fiducial marker, plate, tube, or semi-permeable woven mesh.

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

[0013] In some embodiments, which may be used in combination with any of the above aspects and embodiments, the membrane active agent is present on the surface of the solid implantable composition but not in a bulk of the solid implantable composition, or the membrane active agent is present in a bulk of the solid implantable composition but not on the surface of the solid implantable composition, or the membrane active agent is present homogeneously in the solid implantable composition, or the membrane active agent is present in the solid implantable composition in a concentration gradient.

[0014] In some embodiments, which may 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 an underlying substrate material, the membrane active agent is encapsulated within an encapsulating material, or the membrane active agent is dispersed in a matrix material.

[0015] In some embodiments that may be used in combination with any of the above aspects and embodiments, the membrane active agent is coated onto the embolic bead, injected into the embolic bead, or conjugated to the embolic bead via a covalent bond.

[0016] In some embodiments that may be used in combination with any of the above aspects and embodiments, the membrane active agent is selected from a cationic molecule, an amphiphilic molecule, and an anionic molecule. For example, the membrane active agent may be selected from a cationic polymer, an anionic polymer, and an amphiphilic polymer.

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

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

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

[0020] In some embodiments, which may be used in combination with any of the above aspects and embodiments, treatment is performed in a procedure subsequent to administration of the implantable composition. In some embodiments that may be used in combination with any of the above aspects and embodiments, treatment is performed procedurally at times ranging from the same day, the next day, or the second day, up to one week, up to two weeks, up to 30 days after administration of the implantable composition.

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

[0022] In some embodiments that may be used in combination with any of the above aspects and embodiments, (a) one or more therapeutic electrodes are placed adjacent to the tissue volume at one or more locations on the skin, (b) one or more therapeutic electrodes are placed percutaneously within or adjacent to the tissue volume, (c) therapeutic electrodes are placed within or adjacent to the tissue volume via a device that inserts 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 embolic composition that includes a releasable membrane active agent. In some of these embodiments, the embolic composition is a solid embolic composition. In some of these embodiments, the embolic composition is a liquid composition that becomes a solid embolic composition upon introduction into the body, for example, the embolic composition may be a gel that is administered as a liquid and has viscoelastic properties that transform into a solid upon activation or contact with bodily fluids.

[0024] In some embodiments, which may be used in combination with any of the above aspects and embodiments, the solid embolic composition is at least partially biostable or bioabsorbable.

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

[0026] In some embodiments, which may be used in combination with any of the above aspects and embodiments, the solid embolic composition is a scaffold. In some embodiments, which may be used in combination with any of the above aspects and embodiments, the membrane active agent is present throughout the solid embolic composition, or the membrane active agent is present in only a portion of the solid embolic composition.

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

[0028] In some embodiments, which may 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 an underlying 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 may be used in combination with any of the above aspects and embodiments, the membrane active agent is selected from a cationic molecule, an amphiphilic molecule, and an anionic molecule. For example, the membrane active agent may be selected from a cationic polymer, an anionic polymer, and an amphiphilic polymer.

[0030] In some embodiments that may be used in conjunction with any of the above aspects and embodiments, upon implantation of the embolic composition in a subject, the membrane active agent is released locally in a burst release profile, a sustained release profile, or a combination of a burst release profile and a sustained release profile.

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

[0032] [Figure 1] Figures 1A-1D show microspheres coated with fluorescently labeled PAHM. (Figure 1A) Representative fluorescence microscopy 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 microscopy 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) with Tukey HSD test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Diagram 2] 2A and 2B show the quantification of PAHMs in cell culture media. (FIG. 2A) UV-Vis absorption spectra of different PAHM concentrations. (FIG. 2B) Calibration curve of absorbance at 245 nm versus PAHM concentration. [Diagram 3] Figure 3 shows the release kinetics of PAHMs from coated microspheres in cell culture medium at 37° C. Data are presented as mean + / - SD (n=4) and fit to a two-phase exponential association model. [Figure 4]Figures 4A-4B show cell viability after IRE (Figure 4A) or PAHM (Figure 4B) treatment applied separately. Data are shown as mean + / - SD (Figure 4A) (n=4-6) (Figure 4B) (n=9-13). Analysis of variance with Tukey HSD test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Diagram 5] Figures 5A-5F show cell viability after IRE treatment combined with PAHM exposure for 15 min (Figure 5A), 4 h (Figure 5B), and 24 h (Figure 5C). Data are shown as mean + / - SD (n=3-12). Analysis of variance with Tukey HSD test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). (Figure 5D) Combination index analysis (CI<1, =0.9-1 (dashed line), >1 indicates synergy, near-additive action, and antagonism, 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-6B show cell viability following exposure to PAHM released from coated microspheres with mean diameters of 71 µm (Figure 6A) and 100 µm (Figure 6B). Data are shown as mean + / - SD (n=6-12), analysis of variance with Tukey HSD test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 7] Figure 7 shows cell viability following 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 min (Figure 8B) 4 h (Figure 8C) 24 h. Data are shown as mean + / - SD (n=3-9). Analysis of variance with Tukey HSD test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). (Figure 8D) Combination index analysis (CI<1, =0.9-1 (dashed line), >1 indicates synergy, near-additive effect, and antagonism, respectively). Dose reduction index analysis for IRE (Figure 8E) and microsphere-coated PAHM (Figure 8F) (DRI<1, =1 (dashed line), >1 indicates unfavorable dose reduction, no dose reduction, and favorable dose reduction, respectively). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0034] At relatively high amplitude electroporation doses, the holes formed in response to the applied electric field can become so extensive that the cell membrane cannot recover. If the cell membrane does not recover, the cell will die from this electroporation, known as "irreversible" electroporation. The irreversible electroporation threshold (IRE) varies with cell size and shape, overall tissue structure, and intercellular fluid characteristics. The threshold itself can be defined by the local electric field strength and duration, e.g., higher amplitude voltages are required for shorter durations and lower amplitude voltages for longer durations.

[0035] In the present disclosure, a method of treating a tissue volume is described that includes the steps of: (a) administering to a target site an implantable composition comprising a releasable membrane active agent, such that the membrane active agent is released locally to the tissue volume; and (b) performing IRE on the tissue volume.

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

[0037] Tissue volumes to which irreversible electroporation therapy according to the present disclosure may be applied include benign and malignant tumor volumes, as well as perfused or healthy tissue volumes containing viable functioning cells that constitute a bounded tumor volume. Examples of tumor volumes include pancreatic, esophageal, bladder, bile duct, liver, kidney, breast, lung, prostate, brain, uterine tumors, including uterine fibroids, ovarian, retroperitoneal, limb, pelvic tumors, as well as 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 treatments of the present disclosure, including voltages providing field strengths ranging from 250-2500 V / cm, more typically 375-1750 V / cm, pulse numbers ranging from 1-5000, more typically 5-300, pulse widths ranging from 0.5-1000 μs, more typically 1-250 μs, pulse frequencies ranging from 0.25-10 Hz, and electrode spacing ranging from 0.1-2 cm. Electrodes can be operated in monopolar mode, bipolar mode, or multiplexed configurations across multiple electrode combinations or probes.

[0039] Electrodes may be placed within or adjacent to the 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) percutaneous placement of one or more electrodes at one or more tissue locations; (c) placement of one or more electrodes within or adjacent to one or more natural body lumens (e.g., blood vessels, digestive tract, bile duct, lymphatic duct, urinary tract, pulmonary airway, urethral lumen, bladder, or other body lumen); or (d) a combination of any two or all three of these placement techniques.

[0040] With regard to placing one or more electrodes within 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 where one or more electrodes may be positioned on the device and in contact with the lumen wall, or one or more electrodes may be inserted from the device, through the lumen wall adjacent to the device, and into the tissue surrounding the lumen wall.

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

[0042] The membrane active agent-releasing implantable composition may be selected from membrane active agent-releasing implantable compositions that are at least partially biostable and membrane active agent-releasing implantable compositions that are bioabsorbable. In some embodiments, the membrane active agent releasing implantable composition may be an embolic composition comprising a membrane active agent, which may be administered to one or more blood vessels supplying blood to a tissue volume, thereby forming an embolism / occlusion. In this manner, the disclosed method may kill cells by cutting off their blood supply, in addition to killing cells by membrane active agent enhanced IRE.

[0043] Examples of membrane active agents include cationic, amphiphilic, and anionic molecules. Membrane active cationic molecules can be selected from natural and synthetic cationic polymers bearing primary, secondary, tertiary or quaternary amine groups.

[0044] Natural cationic polymers include cationic polypeptides (such as polylysine, polyarginine, polyornithine), cationic polyamines (such as spermidine, spermine and their derivatives), cationic polysaccharides (such as chitosan and cationic gelatin), cationic peptides rich in cationic amino acids including lysine and arginine (such as cecropin A, magainin, dermaseptin, bombinin, melittin, cathelicidin, human defensin, lactoferricin, histatin, indolicidin, trytrypticin, holothricin, coleopterisin, pyrphocolicin, etc.).

[0045] Synthetic cationic polymers are polymers that contain primary, secondary, tertiary or quaternary amine groups. These polymers can contain molecular segments of linear, block, graft, branched, dendritic and network structures. These cationic polymers can be selected from the classes of polyacrylates, polymethacrylates, polyacrylamides, polymethacrylamides, polyamides, polyesters, polyorthoesters, poly(beta-aminoesters), polyethyleneimines, polypropyleneimines, polyamidoamines, polynorbornenes, and copolymers thereof. They can be synthesized by free radical polymerization, ring-opening polymerization, polyaddition and polycondensation reactions. Synthetic cationic polymers also include cationic peptides with non-natural 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 the art.

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

[0047] Membrane active anionic molecules include certain carboxylic acid-containing synthetic polymers (such as polyalkylacrylic acids) and peptides (such as "GALA": WEAALAEALAEALAEHLAEALAEALEALAA). At acidic pH, these polymers and peptides become protonated and hydrophobic, adopting a conformation that facilitates interaction with cellular lipid membranes.

[0048] Membrane lytic agents are specific membrane active agents that cause cell membrane destruction. Examples include amino-C 2~C 10 Aminoalkyl acrylates such as alkyl acrylates, Amino-C 2 ~C 10 Aminoalkyl methacrylates such as alkyl methacrylates, and copolymers of aminoalkyl acrylates and alkyl acrylates, and copolymers of aminoalkyl methacrylates and alkyl methacrylates. Examples of membrane-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 active agent-releasing implantable composition can be a liquid composition that becomes a solid membrane active agent-releasing implant upon introduction into the body (e.g., the composition can be in the form of a liquid embolic composition).

[0050] In various embodiments, the membrane active agent-releasing implantable composition may be a solid composition. The membrane active agent may be located throughout the solid composition or only in a portion of the solid composition. For example, the membrane active agent may be present in one or more layers on an underlying substrate material (e.g., the layer may be formed entirely of the membrane active agent or may be mixed with one or more additional bioabsorbable or biostable matrix materials into which the membrane active agent is released), the membrane active agent may be encapsulated within an encapsulating material (e.g., a bioabsorbable encapsulating material or a biostable encapsulating material that is permeable to the membrane active agent), the membrane active agent may be dispersed within the substrate material (e.g., by immersing the substrate material in a substrate material already containing the membrane active agent, by forming the substrate as a matrix containing the membrane active agent, etc.). The membrane active agent may be attached to one or more solid compositions via a covalent bond (amide, ester, ether, thiol ester, dithiol, orthoester, acetal, ketal, etc.) from which the membrane active agent is released, the membrane active agent may be attached to one or more solid compositions via a non-covalent bond (biotin-avidin, metal chelates, charge-charge electrostatic interactions, host-guest complexes such as polyethylene glycol / cyclodextrin, adamantane / cyclodextrin, etc.) from which the membrane active agent is released, or by forming a solid composition entirely from the membrane active agent.

[0051] Thus, in addition to the membrane active agent, the implantable compositions of the present disclosure may include substrate materials, matrix materials, and encapsulating materials 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 and polymeric membrane active agents for use herein include homopolymers and copolymers. As used herein, a "homopolymer" is a polymer that contains multiple copies of a single constitutional unit. A "copolymer" is a polymer that contains multiple copies of at least two different constitutional 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 can be linear or branched. Branched configurations include star configurations (e.g., configurations with three or more chains coming out of one branch 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 may be selected, for example, from the following: polycarboxylic acid homopolymers and copolymers, including polyacrylic acid; alkyl acrylate and alkyl methacrylate homopolymers and copolymers, including poly(methyl methacrylate-bn-butyl acrylate-b-methyl methacrylate) and poly(styrene-bn-butyl acrylate-b-styrene) triblock copolymers; polyamides, including nylon 6,6, nylon 12; polyether-block-polyamide copolymers (e.g., Pebax® resins); polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl halides, such as polyvinyl chloride; vinyl homopolymers and copolymers, such as ethylene-vinyl acetate copolymer (EVA); vinyl aromatic homopolymers and copolymers, such as polystyrene, styrene-maleic anhydride copolymer; styrene-butadiene copolymers; styrene-ethylene-butylene copolymers (e.g., Kraton® G series polymers). poly(styrene-b-ethylene / butylene-b-styrene (SEBS)) copolymers, available as styrene-isoprene copolymers (e.g., poly(styrene-b-isoprene-b-styrene), acrylonitrile-styrene copolymers, acrylonitrile-butadiene-styrene copolymers, styrene-butadiene copolymers, and styrene-isobutylene copolymers (e.g., polyisobutylene-polystyrene block copolymers, such as poly(styrene-b-isobutylene-b-styrene) or vinyl aromatic alkene copolymers, including SIBS, which are described, for example, in U.S. Pat. No. 6,545,097 to Pinchuk et al.; ionomers; polyesters, including aliphatic polyesters, such as homopolymers and copolymers of polyethylene terephthalate and lactide (including d-, l-, and meso-lactide) (poly(L-lactide) and poly(d,l-lactide)); glycolide (glycolic acid); and ε-caprolactone, poly(l-lactide-co-glycolide) and poly(d,polycarbonates, including trimethylene carbonate (and its alkyl derivatives), including polylactide-co-glycolides such as l-lactide-co-glycolide; polyanhydrides; polyorthoesters; polyether homopolymers and copolymers, including polyalkylene oxide polymers such as polyethylene oxide (PEO) and polyetheretherketone; polyolefin homopolymers and copolymers, including polyalkylenes such as polypropylene, polyethylene, polybutylene (such as polybut-1-ene and polyisobutylene); polyolefin elastomers (e.g., Santoprene) and ethylene propylene diene monomers. Fluorinated homopolymers and copolymers including (EPDM) rubber, polytetrafluoroethylene (PTFE), poly(tetrafluoroethylene-co-hexafluoropropene) (FEP), modified ethylene-tetrafluoroethylene copolymers (ETFE) and polyvinylidene fluoride (PVDF), silicone homopolymers and copolymers including polydimethylsiloxane, polyurethanes, polypeptides, proteins, polysaccharides, biopolymers such as 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 the present disclosure may also be selected from homopolymers and copolymers containing one or more of 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 nylons, polycarbonates, polyesters, poly(ether ketones) (PEEK) and poly(arylene ether ketones) PAEK.

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

[0056] Membrane active agents may be released from the implantable composition by any type of chemical reaction including diffusion, dissolution, biodegradation, hydrolysis (through covalent bonds), or by enzymatic cleavage of bonds, ionization and deionization, or any combination of the above.

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

[0058] The membrane active agent may be released from the implantable composition via a triggerable release mechanism, for example, as a result of an electrical pulse (e.g., to heat the implantable composition with or without a corresponding phase transition and drive the membrane active agent from the implantable composition by electromigration, etc.), ultrasound (e.g., to heat the implantable composition or to rupture capsules containing the membrane active agent thereby releasing the membrane active agent, etc.), magnetic field fluctuations (e.g., magnetic particles may be provided in the implantable composition and vibrated to release the membrane active agent, for example, by heating the implantable composition and rupturing capsules containing the membrane active agent), injection of a chemical agent (e.g., injection of an acid or base, low or high tension salt, breaking bonds between the membrane active agent and the implantable composition causing release of the membrane active agent, etc.), changes in diffusion rates or membrane transport due to thermal excitation, weakening of chemical bonds and alteration of release, or combinations of the above.

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

[0060] The membrane active agent-releasing implantable composition may be in the form of, for example, a membrane active agent-releasing scaffold. Such scaffolds may be in a variety of forms, such as disks, rods, stents, coils, fiducial markers, woven meshes and tubes. Such scaffolds may contain pores in the range of 0.1 nm to 1 mm, or may be completely non-porous.

[0061] The membrane active agent-releasing implantable compositions may be implanted into the 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 native body lumens (e.g., blood vessels, gastrointestinal tract, bile duct, lymphatic duct, urinary tract, or other body lumens that contain distal capillaries or hypervascular lesions to occlude blood flow), or a combination thereof. Placement of one or more implantable compositions within or adjacent to a native body lumen may be performed using devices such as catheters, endoscopes, microcatheters, needles, or other flexible probes that hold the body lumen open.

[0062] As already noted, in various aspects of the present disclosure, a method of treating a tissue volume is described that includes: (a) administering a membrane active agent-releasing implantable composition to a target site such that the membrane active agent is released locally in the tissue volume; and (b) performing IRE in the tissue volume.

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

[0064] In some embodiments, IRE may be performed on a tissue volume in one or more procedures subsequent to the procedure in which the implantable composition is administered to the target site. For example, (e.g., when the implantable composition is a sustained release composition), the one or more subsequent procedures may be performed at a time ranging from 0.1 days to 6 weeks after administration of the implantable composition to the target site.

[0065] As can be seen from the above, an advantage of the compositions and methods of the present disclosure is that essentially any desired time course for membrane active agent release and essentially any desired IRE schedule can be established, 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), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), ethanol (200 proof), Alexa Fluor® 488 NHS Ester were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Hydroxylamine hydrochloride and PD-10 desalting columns (Sephadex® G-25 medium; exclusion limit: M r5000) were purchased from Millipore Sigma (Burlington, MA, USA). Electroporation cuvettes (4 mm gap) were obtained from Harvard Apparatus (Holliston, MA, USA). UV-transparent cuvettes (semi-micro, 1.5 mL) were purchased from USA Scientific (Ocala, FL, USA).

[0067] COMPUSYN Modeling of Drug Synergy. Combination index (CI) and dose reduction index (DRI) for each therapeutic combination were calculated by the Chou-Talalay method using the CompuSyn software program (ComboSyn, Paramus, NJ, 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)). CI values ​​are a dimensionless numerical representation of drug interactions, with CI=1 indicating additive effects, CI<1 indicating synergistic effects, and CI>1 indicating antagonism. DRI is an index that indicates how much the dosage of each drug can be reduced when used in combination at a certain Fa compared to when each drug is administered alone; DRI=1 means no reduction in dosage, DRI>1 means a favorable reduction in dosage, and DRI<1 means an unfavorable reduction in dosage.

[0068] Statistical analysis. The significance of differences in cell viability between different treatment groups was determined by analysis of variance (ANOVA) and Tukey HSD test for multiple comparisons. The significance of differences in the fluorescence intensity of PAHMs from coated microspheres was determined by analysis of variance and Tukey HSD test for multiple comparisons. PAHM release profiles were fitted with a two-phase exponential association model. All analyses were performed using GraphPad prism, version 9.0.0 (GraphPad software, San Diego, CA, 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. PAHM was then 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 synthesized as described in Ji, W., "Ji, W., Synthetic polymers with well-defined structures for DNA vaccine delivery and cancer therapy," PhD dissertation, University of Minnesota, November 2013. 1 The PAHMs were characterized using H NMR and gel permeation chromatography (GPC). 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 Non-hydrolyzed poly(methyl methacrylate) (PMMA) beads (diameter 90–106 μm) were treated with a membrane active agent, specifically poly(6-aminohexyl methacrylate) (PAHM),

[0071] [ka]

[0072] (where n is an integer) and were coated with PAHM by incubating the beads with PAHM in ethanol followed by evaporation of the ethanol, as further detailed below.

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

[0074] Example 4: Optimized method for coating embolic microspheres with PAHM PMMA microspheres with an average diameter of 71 μm or 100 μm were provided by Boston Scientific Corporation (Maple Grove, MN, USA). PMMA microspheres were coated with PAHMs by a solvent evaporation method. A mixture of fluorescently labeled and unlabeled PAHMs (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 PAHM solution was added to 50 mg of PMMA microspheres (100 μm diameter). The vials were exposed on an orbital shaker (Lab-Line Instruments, Melrose Park, IL, USA) at room temperature for 3 days to evaporate the ethanol. After drying, the microspheres were coated with PAHM at a concentration of 2 μg / mg or 10 μg / mg (μg PAHM / mg PMMA), depending on the feed ratio. The coated microspheres were stored in a vacuum desiccator at room temperature (protected from light) until use.

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

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

[0077] Embolization microspheres (100 μm diameter) were coated with fluorescently labeled PAHM by the solvent evaporation method. Representative fluorescence micrographs (Figure 1A) show that increasing concentrations of PAHM resulted in thicker coatings, 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 by three different solvent evaporation methods. Representative fluorescent micrographs of microspheres coated by the three methods are shown in Figure 1C, and quantification of the fluorescence intensity is shown in Figure 1D. In method 3, the ethanol was evaporated quickly within 10 min, yielding a bright, relatively uniform coating (Figure 1C). In method 2, slow evaporation of ethanol over 12 h appeared to slightly increase the amount of PAHM coated onto the microspheres, as indicated by the 1.2-fold brighter fluorescence compared to method 3 (Figure 1D). However, slowing the evaporation further to 3 days in method 3 resulted in a much dimmer coating (Figure 1C,D). We chose to coat the microspheres using method 3 in all subsequent experiments, as it was faster while still 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. 1 mL of PAHM solution was added to 100 mg of microspheres (71 μm or 100 μm) in a glass vial. Ethanol was evaporated using method 1 above.

[0080] Example 5: In vitro release kinetics of coated microspheres In a 12-well plate, 25 mg of coated microspheres (71 µm or 100 µm diameter) were suspended in 1 mL of cell culture medium (without phenol red) and incubated at 37 °C for 3 h at 5% CO. 2 The cells were incubated for 1 week in a humidified environment at 37 °C containing 100 µL of ethanol. At specific time points, 750 µL of the supernatant was sampled and replaced with fresh medium. The sampled supernatant was then diluted with 0.75 mL release medium (final volume: 1.5 mL) in a transparent UV cuvette (semi-micro, 1.5 mL) (USA Scientific, Ocala, FL, USA). The amount of PAHMs in the supernatant was measured by measuring the absorbance at 245 nm using a Cary100 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Figures 2A and 2B show the quantification of PAHMs in the cell culture medium. Figure 2A shows the UV-Vis absorption spectra of different PAHM concentrations. Figure 2B shows the calibration curve of absorbance at 245 nm against PAHM concentrations.

[0081] The release kinetics profiles, expressed as the cumulative percentage released over time, are shown in Figure 3. After an initial burst release within the first 4 hours, sustained release of PAHM continued over a period of 1 week. PAHM was released slightly faster from the 71 μm PMMA microspheres than from the 100 μm. Within the first hour, 51% and 38% of the PAHM were released from the 71 μm and 100 μm PMMA microspheres. After 4 hours, 69% and 59% of the PAHM were released from the 71 μm and 100 μm PMMA microspheres. More than 20% of the 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 a medium consisting of Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 2 g / L glucose, 2 mM L-glutamine, 2 g / L sodium bicarbonate, 10% heat-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 at 4 °C for 2 h at 5% CO. 2 The cells were incubated in a humidified environment at 37 °C for 1 h. When the cells reached approximately 80% confluency, the medium was removed, the cells were harvested, and either split 1:3–1:6 to continue culturing or used immediately for cytotoxicity experiments.

[0083] Example 7: Cytotoxicity of IRE in combination with free PAHM or PAHM released from coated microspheres The harvested 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 diluted to various concentrations. To 1 mL of cell suspension, 0.5 mL of PAHM dilution was added to obtain final concentrations ranging from 0 to 50 μg / mL. Coated microspheres (71 μm or 100 μm diameter) and uncoated microspheres were suspended in cell culture medium to an initial concentration of 25 mg / mL and diluted to various concentrations. To 1 mL of cell suspension, 0.5 mL of coated microsphere dilution was added to obtain final concentrations ranging from 0 to 5 mg / mL (corresponding to 0 to 50 μg / mL of microsphere-coated PAHM).

[0085] After 15 min of incubation, 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 prepared cell suspension was pipetted into an electroporation cuvette (BTX45-0126, Harvard Apparatus, Holliston, MA) between two aluminum plate electrodes (4 mm apart). The cuvette was placed in an external electric field generated by an electric pulse generator (BTX ECM Square Wave Electroporation System, BTX Model No. 830, Harvard Apparatus, Holliston, MA, USA) delivering 50 electric 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 15 min exposure time, 400 μL of treated cell suspension was transferred to a microcentrifuge tube, centrifuged, and the supernatant was discarded. PAHM was washed off 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 in 12-well plates (180,000 cells / well). For samples with 4 and 24 h exposure time, 400 μL of treated cell suspension was plated in 12-well plates (180,000 cells / well) and 600 μL of free PAHM or coated microsphere dilutions were added to maintain the appropriate PAHM dose. Incubate in 5% CO. 2After 4 or 24 h of incubation in a humidified environment at 37° C., cells were washed with 1 mL of DPBS (twice) and fed with 1 mL of fresh cell culture medium.

[0087] Cell viability was assessed using the MTT (3-(4,5-dimethyl-thiazol-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)). To each well, 100 μL of MTT solution (5 mg / mL in DPBS) was added. The plates were incubated in 5% CO. 2 After 4 h of incubation in a humidified environment at 37 °C containing 100 mM NaCl, 850 μL of medium was removed from each well. Formazan crystals were dissolved in 1 mL of DMSO (Thermo Fisher Scientific) and absorbance was measured at 540 nm using a BioTek Cytation™ 3 Cell Imaging Multi-Mode Reader (BioTek Instruments, Winooski, VT). Cell viability was determined by normalizing by the absorbance of untreated cells (using the same exposure time).

[0088] The viability of AsPC-1 cells after exposure to IRE or PAHM is shown in Figure 4A-4B. Although higher electric field strengths reduced cell viability after IRE alone, the decrease in cell viability was relatively gradual (Figure 4A). For cells exposed to 750 V / cm or less, more than 70% of cells survived. The highest electric field strength (1125 V / cm) also killed 46%, 50%, and 68% of cells at 15 min, 4 h, and 24 h. In contrast, PAHM alone showed a much more significant cell killing effect (Figure 4B). For cells treated with PAHM at doses of 25 μg / mL or more, more than 45%, 90%, and 85% of cells were killed when exposed to PAHM for 15 min, 4 h, and 24 h consecutively. Treatment with 40 μg / mL or 50 μg / mL PAHM for 24 h killed 100% of cells.

[0089] The cell killing effect of IRE was found to be largely time-dependent, as incubation of cells for 4 or 24 hours after IRE treatment did not seem to affect cell viability (Figure 4A). Only when exposed to 1125 V / cm did cell viability decrease when cells were incubated for 24 hours. Unlike IRE, PAHM-induced cell death was dependent on exposure time. Interestingly, treating cells with PAHM for 4 hours seems to decrease cell viability more than treating cells for 24 hours. At PAHM doses ≤ 20 μg / mL, a significant decrease in cell viability was observed at 4 hours of exposure, but cells showed some recovery after 24 hours. This recovery was not observed at PAHM doses ≥ 25 μg / mL, as no statistically significant difference was observed in cell viability at 4 and 24 hours of exposure.

[0090] To evaluate the ability of PAHM to enhance the cell killing effect by IRE, two electric field strengths were combined with three PAHM doses (Figure 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 min, the viability of cells exposed to 562.5 V / cm decreased from 95% to 64%, 48%, and 33% for 5 μg / mL, 15 μg / mL, and 25 μg / mL of PAHM (Figure 5A). With 4 h of exposure to PAHM, the viability of cells exposed to 562.5 V / cm decreased from 86% to 37%, 10%, and 2% for 5 μg / mL, 15 μg / mL, and 25 μg / mL of PAHM (Figure 5B). Similar to PAHM treatment alone, cells treated with IRE and PAHM for 24 h show some recovery of cell viability; however, viability of cells exposed to 562.5 V / cm decreased from 91% to 72%, 17% and 3% at 5 μg / mL, 15 μg / mL and 25 μg / mL of PAHM (Figure 5C).

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

[0092] The viability of AsPC-1 cells after exposure to microspheres (71 μm or 100 μm diameter) coated at 10 μg / mg (μg PAHM / mg PMMA) is shown in Figure 6A-6B. As PMMA microspheres alone (uncoated) showed little to no toxicity (Figure 7), all cell death is attributed to PAHM released from the coated microspheres. Similar to free PAHM, exposure time to PAHM released from 71 μm coated microspheres showed a significant effect on cell viability (Figure 6A). Only moderate cell death occurred after 15 min exposure to 71 μm coated microspheres, as even the highest dose (50 μg / mL microsphere-coated PAHM) killed only 47% of the cells. A 4-h exposure to 71 μm coated microspheres greatly reduced cell viability. At 20 μg / mL or higher microsphere-coated PAHM, less than 13% of cells survived. After 24 hours of exposure to the 71 μm coated microspheres, cells regained much of their viability, similar to that seen after a 15 min exposure. Only with 50 μg / mL microsphere-coated PAHM did a statistically significant number of cells die after 24 hours of exposure compared with 15 min. Similar results were seen for cells treated with 100 μm coated microspheres for 15 min or 24 h; however, after 4 hours of exposure, little cytotoxicity was observed (Figure 6B). Even with 50 μg / mL microsphere-coated PAHM, 19% cell viability remained after 4 hours of exposure to the 100 μm coated microspheres. Only for ≥25 μg / mL microsphere-coated PAHM did longer exposure times result in significantly lower cell viability than the 15 min 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 and 24 h exposure times, 100 μm coated microspheres were selected to examine the ability of released PAHM from coated microspheres to enhance IRE (Figure 8A-8F). Released PAHM enhanced the cell killing effect of IRE after 15 min, especially after 4 h; however, 24 h exposure time did not significantly decrease cell viability. At 15 min 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). The most substantial IRE enhancement was seen after 4 h of exposure to 100 μm coated microspheres, as cell viability exposed to 562.5 V / cm decreased from 86% to 65%, 56%, and 19% with 5 μg / mL, 15 μg / mL, and 25 μg / mL microsphere-coated PAHM (Figure 8B). This enhancement was nearly gone after 24 h of exposure. The only combination that significantly decreased cell viability when compared to IRE alone was a field strength of 562.5 V / cm and 25 μg / mL microsphere-coated PAHM (Figure 8C). This decrease was also relatively gradual, from 91% with IRE alone to 75% with the combination.

[0094] CI and DRI for the combination of IRE and coated PMMA microspheres were also calculated. As shown by the CI in Figure 8D, the combination of IRE with 15 min exposure to PAHM released from coated microspheres is strongly antagonistic at low Fa but becomes synergistic at intermediate Fa. At 4 h exposure, most CIs were in the range of 0.9–1.1 (or close to it), indicating a nearly additive effect. All 24 h exposure time combinations showed CIs of 1.1 or higher. At low Fa, the combination showed strong antagonism, but the antagonism decreased with Fa. The DRI 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 for all three exposure times, and at Fa>0.1, the DRI of IRE is all greater than 1. The DRI of microsphere-coated PAHM tended to increase with Fa for the 15 min and 24 h exposure times, but decreased with Fa for the 4 h exposure time. When Fa>0.2, the DRI of microsphere-coated PAHM was greater than 1.

Claims

1. An implantable composition for use in treating a tissue volume, comprising: the implantable composition is an embolic composition comprising a releasable membrane active agent; the releasable membrane active agent is selected from aminoalkyl acrylates, aminoalkyl methacrylates, copolymers of aminoalkyl acrylates and alkyl acrylates, and copolymers of aminoalkyl methacrylates and alkyl methacrylates; An implantable composition, comprising: (a) an embolic composition comprising the releasable membrane active agent, which is administered to a target site such that the membrane active agent is released locally in the tissue volume; and (b) treatment of the tissue volume is achieved by application of a pulsed electric field to the tissue volume.

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

3. The transplantable composition of claim 1, wherein the tissue volume corresponds to all or a portion of a tumor, or to a tumor and all or a portion of healthy tissue surrounding the tumor.

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

5. The implantable composition of claim 1, wherein the embolic composition is administered to one or more blood vessels supplying blood to the tissue volume, or the embolic composition is administered percutaneously or endoscopically by injection.

6. The implantable composition of claim 1, wherein the implantable composition is at least partially biostable or bioabsorbable.

7. The implantable composition of claim 1, wherein the implantable composition is a solid implantable composition when administered to the target site, or wherein the implantable composition is a liquid composition that becomes a solid implant upon administration to the target site.

8. The implantable composition of claim 7, wherein the solid implantable composition is an implantable particle or wherein the solid implantable composition is a scaffold.

9. The implantable composition of claim 7, wherein the solid implantable composition is an implantable particle 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 disc, rod, stent, coil, fiducial marker, plate, tube, or semi-permeable woven mesh.

10. The implantable composition of claim 7, wherein the membrane active agent is present throughout the solid implantable composition; the membrane active agent is present in only a portion of the solid implantable composition; the membrane active agent is present on the surface of the solid implantable composition but not in a majority of the solid implantable composition; the membrane active agent is present in a majority of the solid implantable composition but not on the surface of the solid implantable composition; the membrane active agent is present homogeneously in the solid implantable composition; the membrane active agent is present in a concentration gradient in the solid implantable composition; the membrane active agent is present in one or more layers disposed on an underlying substrate material; the membrane active agent is encapsulated in an encapsulating material; or the membrane active agent is dispersed within a matrix material.

11. The implantable composition of claim 7, wherein the solid implantable composition is an embolic particle and the membrane active agent is coated on, injected into, or conjugated to the embolic particle via a covalent bond.

12. The implantable composition of claim 1, wherein release of the membrane active agent is triggered by an electrical pulse, ultrasound, a magnetic field fluctuation, or thermal input.

13. The implantable composition of claim 1, wherein the treatment is performed in the same procedure as administration of the implantable composition.

14. The implantable composition of claim 1, wherein the treatment is performed in a procedure following administration of the implantable composition.

15. The implantable composition of claim 1, wherein the treatment is performed procedurally at a time ranging from the same day to 30 days after administration of the implantable composition.

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

17. The implantable composition of claim 1, wherein: (a) one or more therapeutic electrodes are positioned adjacent to the tissue volume at one or more locations on the skin; (b) one or more therapeutic electrodes are positioned percutaneously within or adjacent to the tissue volume; (c) one or more therapeutic electrodes are positioned 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.

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