Biodegradable alginate microspheres

Abstract

In some embodiments, the present specification discloses a method and composition for preparing biodegradable alginate microspheres for use in treating at least one of the following diseases: hepatocellular carcinoma, colorectal cancer liver metastasis, paraganglioma liver metastasis, neuroendocrine tumor liver metastasis, gastrointestinal cancer liver metastasis, breast cancer liver metastasis, melanoma liver metastasis, pancreatic cancer liver metastasis, cholangiocarcinoma liver metastasis, colorectal cancer lung metastasis, renal cancer lung metastasis, cirrhosis-associated thrombocytopenia, metastatic extrahepatic bile duct cancer, glioblastoma, renal cell carcinoma, prostate cancer, and uterine fibroids. In some embodiments, the method for preparing biodegradable alginate microspheres can utilize a microfluidic cassette. TIFF2026502140000003.tif113170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 433,889, filed December 20, 2023.

[0002] Technical Field The present disclosure relates generally to the production of microspheres, and more particularly to the production of loaded alginate microspheres. [Background technology]

[0003] Background of the Invention Alginate microspheres are widely used for drug delivery. Alginate is an attractive option because it is biocompatible and biodegradable. Alginate microspheres can be used to protect the encapsulated drug from conditions within the body.

[0004] Microparticles such as alginate microspheres produced by standard production methods often have broad size distributions, lack uniformity, fail to provide suitable release kinetics or other characteristics, and are difficult and expensive to produce. In addition, the microparticles can be large and prone to forming aggregates, necessitating a size selection process to remove particles deemed too large for administration to patients by injection or inhalation. This requires sieving, resulting in product loss.

[0005] It would be beneficial to have a biodegradable alginate microsphere production process that provides predictable particle size distribution and uniformity in addition to ease of production. Summary of the Invention

[0006] An embodiment of the present disclosure is a method for making a polymer matrix encapsulating liposomes, comprising the steps of: adding at least one liposome to a first channel on a microfluidic cassette; adding at least one crosslinkable polymer to a second channel on the microfluidic cassette; combining the at least one liposome and the at least one crosslinkable polymer to form a solution; pushing the solution through a third channel on the microfluidic cassette to shear the solution to create individual spheres; and adding an aqueous solution containing a crosslinker to a fourth channel on the microfluidic cassette, wherein the crosslinker and the crosslinkable polymer form a polymer matrix encapsulating the liposomes. In some embodiments, the at least one crosslinkable polymer is alginic acid. In some embodiments, the alginic acid is composed of stereomonomers of mannuronic acid and guluronic acid. In some embodiments, the alginic acid is composed of a mannuronic acid to guluronic acid ratio less than or equal to 1. In some embodiments, the alginic acid is composed of a mannuronic acid to guluronic acid ratio greater than or equal to 1.5. In some embodiments, the crosslinker is a divalent cation. In some embodiments, the crosslinker is a calcium salt. In some embodiments, the crosslinker is a barium salt. In some embodiments, the method further comprises washing the polymer matrix encapsulating the liposomes to remove excess crosslinker. In some embodiments, prior to adding at least one liposome to a first channel on a microcassette, the liposomes are loaded with at least one active agent. In some embodiments, the at least one active agent is radioactive. In some embodiments, the at least one active agent is a beta-emitting radionucleotide chelate. In some embodiments, the beta-emitting radionucleotide chelate comprises Re188. In some embodiments, the beta-emitting radionucleotide chelate comprises BMEDA. In some embodiments, the diameters of the first channel, the second channel, the third channel, and the fourth channel are each 20 to 120 μm. In some embodiments, the method further comprises a microsphere aspect ratio of 0.7 to 1.3 μm.In some embodiments, the method further comprises a flow rate differential between the dispersed and continuous phases of 0 to 100 μL / min. In some embodiments, the method further comprises a stream temperature of 5° C. to 55° C. In some embodiments, the method further comprises a stream pressure of 0 to 1500 mbar.

[0007] An embodiment of the present disclosure is a composition made by the method of claim 1, comprising at least one liposome, at least one active agent encapsulated in the intraliposomal aqueous compartment of the liposome, and a crosslinked polymer matrix, the polymer matrix being water-insoluble and hydrophilic, wherein the at least one liposome has a diameter of about 1 nm to 200 nm and the at least one liposome is embedded in the crosslinked polymer matrix. In some embodiments, the crosslinked polymer matrix comprises at least one active agent in free form. In some embodiments, the composition further comprises an aqueous medium surrounding the crosslinked polymer matrix in which the at least one liposome is embedded. In some embodiments, the aqueous medium comprises at least one active agent in free form. In some embodiments, the at least one active agent is radioactive. In some embodiments, the at least one radioactive agent is a beta-emitting nucleotide chelate. In some embodiments, the crosslinked polymer matrix comprises alginate. In some embodiments, at least one liposome contains at least one ionic salt in the intraliposomal aqueous compartment to create an ion gradient. In some embodiments, at least one liposome contains at least one buffer substance in the intraliposomal aqueous compartment to create a pH gradient. In some embodiments, at least one liposome contains at least one radiopaque material. In some embodiments, the crosslinked polymer matrix has a diameter of about 10-90 μm. In some embodiments, the crosslinked polymer is alginate.

[0008] An aspect of the disclosure is a method of using a liposome-encapsulating polymer matrix made using the above method, where the active agent loaded into the polymer matrix actively destroys tumor cells.

[0009] An aspect of the disclosure is a method of using a polymer matrix encapsulating liposomes made using the above method to treat at least one of the following diseases using radioembolization: hepatocellular carcinoma, colorectal cancer liver metastasis, paraganglioma liver metastasis, neuroendocrine tumor liver metastasis, gastrointestinal cancer liver metastasis, breast cancer liver metastasis, melanoma liver metastasis, pancreatic cancer liver metastasis, cholangiocarcinoma liver metastasis, colorectal cancer lung metastasis, renal cancer lung metastasis, cirrhosis-associated thrombocytopenia, metastatic extrahepatic cholangiocarcinoma, glioblastoma, renal cell carcinoma, prostate cancer, and uterine fibroids.

[0010] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0011] The present technology will be better understood by reading the following detailed description of non-limiting embodiments thereof and examining the accompanying drawings.

[0012] [Figure 1] 1 shows a flow chart for the preparation of microspheres. [Figure 2] 1 illustrates an embodiment of a microfluidic cassette. [Figure 3A] Figures 3A, 3B, 3C, and 3D show particle size data comparing 188RNL BAM intermediate (PTN12-066), KI liposomes (PTN13-008), BAM KI liposomes 1:1 (PTN13-014), and BAM KI liposomes 2:1 (PTN13-015). [Figure 3B] See legend to Figure 3A. [Figure 3C]See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 4] Figures 4A and 4B show fluoroscopy images of the radiopaque prototype 188RNL-BAM-KI (4A) and the non-radiopaque prototype 188RNL-BAM (4B). Settings were 43 kV, 3.7 mA, 84.55 mGy, and 4 minutes. DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0014] When introducing elements of various embodiments of the present invention, the articles “a,” “an,” “the,” and “said” are intended to mean there is one or more of the element. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that additional elements may be present in addition to the listed elements. Any examples of operating parameters and / or environmental conditions do not exclude other parameters / conditions of the disclosed embodiments. In addition, it should be understood that references to “one embodiment,” “an embodiment,” “specific embodiment,” or “other embodiments” of the present invention are not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the recited features. Furthermore, references to terms such as “upper,” “lower,” “upper,” “lower,” “side,” “front,” “rear,” or other directional terms are made with reference to the illustrated embodiment and are not intended to be limiting or exclude other orientations.

[0015] Disclosed herein are novel fabrication methods for producing spheres using liposome-alginate sphere compositions and microfluidic chips, cartridges, or cassettes. In some embodiments, the microfluidic cassette is made of at least one of polydimethylsiloxane (PDMS), glass, silicon, polymethylmethacrylate (PMMA), polycarbonate, and cyclic olefin. In some embodiments, the diameter of the microparticle channel ranges from 1 μm to 1000 μm. In some embodiments, the size of the microparticle chip ranges from 1.5 inches to 4 inches or about 1 centimeter to 10 centimeters.

[0016] Certain embodiments are directed to compositions comprising liposome-containing alginate microspheres and methods for producing liposome-containing alginate microspheres. In some embodiments, the liposomes encapsulate one or more substances. Substances that can be encapsulated in liposomes and loaded into alginate microspheres include radiotherapeutic agents (including but not limited to rhenium-188), radiolabels (including but not limited to technetium-99m), chemotherapeutic agents (doxorubicin), magnetic particles (including but not limited to 10 μm ionic nanoparticles), and radiotransparent materials (including but not limited to iodinated contrast agents).

[0017] In some embodiments, the imaging agent is a radiolabel selected from the group comprising a radioisotope element selected from the group consisting of astatine, bismuth, carbon, copper, fluorine, gallium, indium, iodine, lutetium, nitrogen, oxygen, phosphorus, rhenium, rubidium, samarium, technetium, thallium, yttrium, and zirconium.

[0018] In some embodiments, the radiolabel is zirconium-89 ( 89 Zr), iodine-124( 124 I), iodine-131( 131 I), iodine-125( 125 I), iodine-123( 123 I), Bismuth-212(212 Bi), Bismuth-213( 213 Bi), astatine-211( 211 At), Copper-67( 67 Cu), Copper-64( 64 Cu), Rhenium-186( 186 Re), rhenium-188( 188 Re), Phosphorus-32 ( 32 P), samarium-153( 153 Sm), lutetium-177( 177 Lu), technetium-99m ( 99m Tc), gallium-67( 67 Ga), Indium-111( 111 In), thallium-201( 201 Tl), carbon-11( 11 C), nitrogen-13( 13 N), oxygen-15( 15 O), Fluorine-18( 18 F), and rubidium-82 ( 82 Ru).

[0019] In certain aspects, ruthenium-188( 188 Re) liposomes (Rhe-LAM) can be used to treat liver tumors, particularly hepatocellular carcinoma (HCC). In some embodiments, HCC treatment can be achieved through radioembolization, in which ruthenium-188 delivers high doses of radiation targeted primarily to cancer cells, while the microspheres block the arterial blood supply to the tumor.

[0020] In one embodiment, a microfluidic channel is used to produce alginate microspheres (a polymer matrix with liposomes embedded) containing liposomes. A microfluidic cassette mixes two fluid streams, one containing liposomes and one containing alginate, by crossing them within the microfluidic channel. The mixed solution is then continuously pushed into the other microfluidic channel, where the solution is sheared to form spheres. The spheres will have an average diameter of 10-90 μm. The spheres then interact with a solution containing divalent ions, such as calcium or barium, whereupon the spheres gel with the liposomes encapsulated in the alginate matrix. In one embodiment, the solution is comprised of 1%-50% calcium or barium salt. In a more preferred embodiment, the solution is comprised of 1%-40% calcium or barium salt. In an even more preferred embodiment, the solution is comprised of 20%-30% calcium or barium salt. The properties of alginate-liposome microspheres are determined by variables in this process, such as the angle between the interacting channels, channel diameter, stream flow rate, stream temperature, stream pressure, and the ratio of mannuronic acid to guluronic acid in the alginate. Microfluidic methods yield alginate microspheres (including liposomes) with a narrow size distribution and uniform shape.

[0021] The influence of particle shape on flow dynamics can be significant. A direct numerical simulation (DNS) study observed that increasing the aspect ratio of spheroids reduced the local accumulation of spheres near the wall (7). This implies that particle distribution in a flow is influenced by their shape. Furthermore, it was found that spheroidal particles rotate slower near the wall, and their major axes tend to align with the flow direction (8). This indicates that particle orientation in a flow is also influenced by their shape. Furthermore, despite their slower rotation speeds, a higher degree of intermittency in the rotation rate was observed for spheroids, which increased with the aspect ratio. This suggests that particle motion is influenced by their shape, with spheroidal particles with higher aspect ratios exhibiting more erratic behavior (7). Overall, these results demonstrate that particle shape plays an important role in regulating flow dynamics.

[0022] The flow dynamics of spheres and droplets can be very different (7). For example, spheres typically move in a more predictable and symmetrical manner compared to droplets, which can exhibit more erratic and unpredictable motion (7). Furthermore, droplets have surface tension, which can cause them to deform and change shape as they move, which can affect their trajectory and interaction with the surrounding fluid. Overall, the differences in shape between spheres and droplets can result in different flow dynamics and behavior in fluids (8, 9).

[0023] The aspect ratio is the ratio of an object's longest axis to its shortest axis. From a perfect sphere to a slightly elongated particle, the aspect ratio varies depending on the degree of elongation. For a perfect sphere, the longest and shortest axes are the same, so the aspect ratio is 1. If the particle is somewhat elongated, the aspect ratio will be higher.

[0024] In one embodiment, the aspect ratio of the microsphere is 0.7 to 1.3. In a preferred embodiment, the aspect ratio of the microsphere is 0.8 to 1.2. In a more preferred embodiment, the aspect ratio of the microsphere is 0.9 to 1.1.

[0025] In one embodiment, the channel diameter is 20 to 120 μm. In a preferred embodiment, the channel diameter is 30 to 100 μm. In a more preferred embodiment, the channel diameter is 40 to 80 μm. In an even more preferred embodiment, the channel diameter is 60 to 80 μm. In one embodiment, the channel diameters of the first channel, the second channel, the third channel, and the fourth channel are each 20 to 120 μm. In one embodiment, any one of the channels can have a diameter of 20 to 120 μm. The diameter may be the same as or different from the diameter of any of the other channels.

[0026] In one embodiment, the difference in flow rate between the dispersed phase and the continuous phase is 0 to 100 μL / min. In a preferred embodiment, the difference in flow rate between the dispersed phase and the continuous phase is 0 to 30 μL / min. In a more preferred embodiment, the difference in flow rate between the dispersed phase and the continuous phase is 0 to 15 μL / min. In an even more preferred embodiment, the difference in flow rate between the dispersed phase and the continuous phase is 5 to 10 μL / min.

[0027] In one embodiment, the stream temperature is 5°C to 55°C. In a preferred embodiment, the stream temperature is 15°C to 40°C. In a more preferred embodiment, the stream temperature is 10°C to 40°C. In an even more preferred embodiment, the stream temperature is 20°C to 30°C.

[0028] In one embodiment, the stream pressure is 0 to 1500 mbar. In a preferred embodiment, the stream pressure is 50 to 100 mbar. In a more preferred embodiment, the stream pressure is 300 to 800 mbar. In an even more preferred embodiment, the stream pressure is 600 to 700 mbar.

[0029] To encapsulate the active ingredient, a pH gradient is required between the fluid and the aqueous core of the liposome. In some embodiments, the fluid is continuous. Standard production methods often involve a washing procedure to remove the calcium chloride solution by centrifugation to create a pellet of alginate microspheres. The supernatant is discarded and the microspheres are resuspended in an appropriate ionic buffer. This process is repeated until an acceptable calcium chloride concentration is reached.

[0030] In some embodiments, tangential flow filtration (TFF) can be used to remove calcium chloride and for buffer exchange. TFF is a separation process in which fluid is passed parallel to the filter rather than perpendicular to the membrane. TFF can be used to concentrate solutions and remove salts for diafiltration. In some embodiments, the TFF membrane is a hydrophilic membrane. In some embodiments, the TFF membrane is polyethersulfone (PES). In some embodiments, Omega (商標) Minimate with membrane (商標) Tangential flow filtration capsules can be used to remove calcium chloride.

[0031] Certain embodiments described herein include pre-encapsulating a water-soluble, radiopaque material into pH-gradient liposomes. The water-soluble material can be incorporated into the liposomes via passive encapsulation. Liposomes spontaneously form when the dissolved liposome components are exposed to an aqueous solution. Once the liposomes are formed, the aqueous solution is encapsulated in the liposome lumen. In one embodiment, water-soluble potassium iodide is encapsulated in the pH-gradient liposomes. In some embodiments, ammonium sulfate liposomes can be prepared as described in U.S. Patent Application Publication No. 20220249374 (10).

[0032] An embodiment of the present disclosure is a composition comprising liposomes encapsulating at least one active agent in their intraliposomal aqueous compartments, the liposomes having a diameter of up to 200 nm and embedded in a water-insoluble, water-absorbing crosslinked polymer matrix. In some embodiments, the polymer matrix contains an amount of at least one active agent in free form. In some embodiments, an aqueous medium is added around the polymer matrix in which the liposomes are embedded. In some embodiments, the aqueous medium contains an amount of at least one active agent in free form. In some embodiments, the at least one active agent is radioactive to some extent. In some embodiments, the crosslinked polymer matrix is ​​alginate. In some embodiments, the liposomes contain at least one ionic salt in the intraliposomal aqueous compartment to create an ion gradient. In some embodiments, the liposomes contain at least one buffer substance in the intraliposomal aqueous compartment to create a pH gradient. In some embodiments, the liposomes contain at least one radiopaque material. In some embodiments, the at least one radioactive agent is a beta-emitting nucleotide chelate.

[0033] Polysaccharides such as alginate are widely used in various fields due to their structural properties, biocompatibility, and safety. When combined with divalent cations, alginate forms hydrogels that have been shown to be biocompatible and capable of encapsulating cells (1). However, previous studies have demonstrated that the purity of alginate can affect its biocompatibility in implants (2). Alginate is a copolymer composed of stereomonomers of mannuronic acid (M) and guluronic acid (G) (3, 4). The affinity of the G isomer for divalent cations, such as calcium, is stronger due to their atomic arrangement (4, 5). Two G monomers can form an "eggbox" structure, where calcium ions reside and crosslink the two polymer chains (4). Consequently, the M:G ratio can affect the crosslinking ability and physical properties of calcium alginate gels (6). A higher ratio of mannuronic acid can produce softer gels, while a higher ratio of guluronic acid can produce stiffer gels with narrower pores. This property has been exploited in the production of biodegradable implants, where different ratios of mannuronic acid to guluronic acid can be used to achieve the desired stiffness of the gel matrix.

[0034] In some embodiments, the average diameter of the polymer matrix in which the liposomes are embedded is less than 80 μm. In some embodiments, the cross-linked polymer is alginate. In some embodiments, the alginate is composed of a mannuronic acid to guluronic acid ratio less than or equal to 1. In some embodiments, the alginate is composed of a mannuronic acid to guluronic acid ratio greater than or equal to 1.5.

[0035] An embodiment of the present disclosure is a method comprising mixing (i) liposomes, (ii) at least one crosslinkable polymer, and (iii) an aqueous solution containing a crosslinker, such that the crosslinker and crosslinkable polymer form a water-insoluble, hydrophilic crosslinked polymer in which the liposomes are embedded. In some embodiments, the composition is washed to remove excess crosslinker. In some embodiments, a microfluidic channel is used to mix the liposomes, the aqueous crosslinker solution, and the crosslinkable polymer. An embodiment of the present disclosure is a method comprising shearing the crosslinked polymer into individual particles containing a microfluidic channel. In some embodiments, the liposomes within the microspheres are loaded with at least one active agent prior to mixing. In some embodiments, the at least one active agent is radioactive to some extent. In some embodiments, the at least one radioactive agent is a beta-emitting nucleotide chelate. In some embodiments, at least one of the chelators is BMEDA. In some embodiments, at least one of the chelators is DOTA.

[0036] An aspect of the present disclosure is a method for loading at least one chelating agent into a liposomal polymer matrix.

[0037] An embodiment of the present disclosure is a method for treating a disease using the composition herein, wherein a polymer matrix embedded with liposomes occludes blood vessels in unresectable tumors. In some embodiments, the blood vessels in unresectable tumors are occluded using at least one substance known to be biodegradable. In some embodiments, the active substance loaded into the polymer matrix actively destroys tumor cells.

[0038] An embodiment of the present disclosure is a method of treating at least one of the following diseases using radioembolization: (i) hepatocellular carcinoma, (ii) colorectal cancer liver metastasis, (iii) paraganglioma liver metastasis, (iv) neuroendocrine tumor liver metastasis, (v) gastrointestinal cancer liver metastasis, (vi) breast cancer liver metastasis, (vii) melanoma liver metastasis, (viii) pancreatic cancer liver metastasis, (ix) cholangiocarcinoma liver metastasis, (x) colorectal cancer lung metastasis, (xi) renal cancer lung metastasis, (xii) cirrhosis-associated thrombocytopenia, (xiii) metastatic extrahepatic cholangiocarcinoma, (xiv) glioblastoma, (xv) renal cell carcinoma, (xvi) prostate cancer, and (xvii) uterine fibroids.

[0039] To fabricate the microspheres, 2 ml of sterile gradient liposomes are combined with 2 ml of 3% sterile alginate solution to form a 1:1 alginate mixture (Figure 1). The mixture is mixed until homogeneous. The alginate mixture is combined with a sterile continuous stream of water for injection (WFI) and ethanol using a focused laminar flow microchannel. This is collected in a 2% CaCl2 crosslinking solution and mixed for 10-90 minutes. After mixing, the microspheres are dispensed into sterile centrifuge tubes. The tubes are centrifuged at 10K rpm for 15-20 minutes to produce a pellet. The supernatant is discarded, and saline is added to the pelleted tube. This centrifugation, supernatant removal, and saline addition are repeated three times to obtain the microsphere intermediate.

[0040] Labeled microspheres are obtained by adding 2 ml of the microsphere intermediate to a vial of Re-BMEDA chelate and incubating at 37° C. for 60 minutes.

[0041] The activity of the labeled microspheres before purification is measured. A Sephadex column is prepared with Dulbecco's phosphate buffered saline (DPBS). The labeled liposomes are dispensed onto the Sephadex column. The labeled liposomes are eluted with DPBS buffer and collected in a sterile glass vial. The specific activity is measured and the percent efficiency is calculated. A test is performed to ensure the absence of endotoxin contamination.

[0042] A method for producing alginate microspheres involves mixing a 3% alginate solution with ammonium sulfate liposomes in solution using a microfluidic device 200 that provides continuous, focused, laminar flow. In one embodiment, the microfluidic device is composed of at least one central fluid inlet channel 202 and 204 and at least one outer fluid inlet channel 212 (Figure 2). The main principle of a microfluidic mixer is to reduce the diffusion distance and time between different fluid components. In one embodiment, the microfluidic device is designed to provide either chaotic micro-mixing or microfluidic hydrodynamic focusing.

[0043] In one embodiment, solutions are mixed by having two streams, one containing liposomes in inlet channel 202 and the other containing alginate in inlet channel 204, intersecting in microfluidic channel 206. The mixed solution is then continuously pushed into another microfluidic channel 208, where the solution is sheared to create spheres. The spheres will have an average diameter of 10 to 80 μm. The spheres then interact with a stream of 30% calcium chloride solution from channel 210, where they gel, with the liposomes encapsulated in the alginate matrix. In this process, the properties of the alginate-liposome microspheres are determined by variables such as the angle between the interacting channels, channel diameter, stream flow rate, stream temperature, and stream pressure. The microfluidic technology is performed in a microfluidic cassette or chip, and the process variables are controlled by an associated controller. In one embodiment, the angle between the interacting channels ranges from θ = 1° to θ = 85°.

[0044] The alginate liposome microspheres were then loaded with 188Re-BMEDA (rhenium-188 BMEDA) by incubating the microspheres with 188Re-BMEDA at elevated temperature for 1 hour. The BMEDA solution was prepared by interacting rhenium-188, derived from tungsten-188, with BMEDA and sodium glucoheptonate solution at 80°C for 1 hour. The 188Re-BMEDA was then encapsulated within the ammonium sulfate liposomes in the alginate microspheres. [Example]

[0045] Example 1 The 188RNL-BAM intermediate was produced using the atomizer nozzle method and can be used for applications including, but not limited to, characterization and preclinical studies (Figure 3C, PTN12-066).

[0046] The materials used included DPBS, 3% alginate, liposomes, and 20 gL CaCl2.

[0047] The nozzle and tubing were cleaned by flushing with 10 ml of water, followed by 10 ml of 70% IPA, and finally rinsing with 10 ml of water. After cleaning, the nozzle and tubing were air purged with a 10 mL syringe.

[0048] Two mL of 3% alginate solution was combined with 2 ml of ammonium sulfate (pH) gradient liposomes in a centrifuge tube.

[0049] The tube was vortexed until the solution was well mixed.

[0050] The liposome-alginate solution was drawn up into a 10 ml syringe.

[0051] The volume of liposome-alginate solution in the syringe was 35 mL.

[0052] The syringe was placed in a syringe pump and tubing was attached from the nozzle to the outlet of the syringe.

[0053] Another syringe was used to draw up 3 ml of DI water and set aside until needed.

[0054] The BAM collection bowl was filled with 20 g / L CaCl2 solution until the liquid level reached 1 cm.

[0055] The nozzle was opened and set to 5.0 W. The collection dish was stirred at setting 2 with a 1 inch stir bar.

[0056] The syringe pump was set to 0.5 mL / min.

[0057] Once the syringe was empty, it was replaced with the syringe that had been set aside earlier. The pump was set to 0.5 mL / min.

[0058] After the syringe was emptied, the BAM was incubated for 10 minutes at setting 2. After incubation, 137 mL of CaCl2 and BAM solution was collected.

[0059] The contents of the dish were collected into a 50 ml centrifuge tube. The tube was labeled with the notebook number, date, and description.

[0060] The tubes were centrifuged at 2500 RPM for 10 minutes. The supernatant from each tube was aspirated and replaced with 20 ml of DPBS.

[0061] The tubes were resuspended and centrifuged at 2500 RPM for 10 minutes. The supernatant in each tube was aspirated and replaced with 20 ml of DPBS.

[0062] The tubes were stored at 2–8°C.

[0063] Example 2 Potassium iodide liposomes were prepared for evaluation in determining radiopacity (FIG. 3C, PTN13-008).

[0064] Materials used included potassium iodide, distearoylphosphatidylcholine (DSPC), cholesterol, ammonium sulfate, ethanol, hydrochloric acid, pH paper, and Dulbecco's phosphate buffered saline (DPBS).

[0065] A 480 mg / mL potassium iodide I solution was prepared by dispensing 156.9 g of potassium iodide into a beaker and diluting the solution with 250 g of deionized (DI) water.

[0066] A 300 mM ammonium sulfate solution was prepared by dispensing 29.6 g of ammonium sulfate into a beaker and diluting with 733.2 g of DI water.

[0067] The pH of the potassium iodide was adjusted to 5.5 by dispensing 0.625 mL of 0.12 M hydrochloric acid solution into the potassium iodide solution.

[0068] 175 mL of potassium iodide solution was dispensed into a separate 250 mL beaker, which was heated to 65-66°C.

[0069] 13.1 g of DSPC was added to a 1 L flask, and 5.1 g of cholesterol was added to the same flask. 40.2 g of ethanol was dispensed into the flask.

[0070] A water bath attached to a rotary evaporator was set to 75° C. Once this temperature was reached, the flask was attached to the rotary evaporator and the flask was rotated for 15 minutes.

[0071] After dissolving the lipid in the ethanol, the potassium iodide solution was poured into the flask when the solution was at 65-66°C.

[0072] The contents of the flask were homogenized with a homogenizer at speed 4 for 2 minutes.

[0073] The water bath was set to 45°C and once this temperature was reached the flask was returned to the water bath.

[0074] The flask was cooled to 45°C for 15 minutes.

[0075] While the flask was cooling, the Microfluidizer refrigerator was turned on and set to 20°C. The Microfluidizer was primed by passing 90-100g of ammonium sulfate solution through the machine. The pressure was set to 22 kpsi. After at least five passes through the machine, the remaining ammonium sulfate was removed.

[0076] After the flask had cooled, the contents were fed into a microfluidizer, which was turned on and the output collected once the contents began to turn yellow-white.

[0077] When the liquid in the hopper began to reduce by a quarter, the collected output was returned to the hopper for four passes.

[0078] The ethanol in the solution was evaporated by setting the water bath to 28° C. and the vacuum to 28 torr. The solution was rotated in a rotary evaporator under these conditions for 1 hour.

[0079] The solution was stored at 2-8°C.

[0080] Example 3 Potassium iodide liposome BAM was prepared using the ultrasonic nozzle method and undiluted liposomes (Figure 3C, PTN13-014).

[0081] The materials used included 3% alginate, 20 g / L calcium chloride, DPBS, and liposomes.

[0082] The nozzle assembly was cleaned by flushing the tubing and nozzle with 10 mL of IPA, followed by 10 mL of water, followed by a 10 mL purge with air. Waste was collected in an appropriately named and labeled container.

[0083] 2 mL of 3% alginate solution was combined with 2 mL of potassium iodide liposomes in a centrifuge tube, which was vortexed until the solution was homogenous.

[0084] The solution was drawn up into a 10 mL syringe, and the volume of the alginate liposome solution was 3.6 mL.

[0085] The syringe was placed in a syringe pump, and a rubber tube was attached from the nozzle to the syringe.

[0086] Another syringe was used to draw up 4 mL of DI water and set aside until needed.

[0087] 135 mL of 20% calcium chloride solution was dispensed into a shallow collection bowl and a 2-inch stir bar was placed in the bowl.

[0088] A collection bowl was placed under the nozzle, and a stirring plate was placed underneath it, leaving a gap of 1–2 cm between the tip of the nozzle and the surface of the solution.

[0089] A nozzle generator was created and set to 5.0W.

[0090] The stir plate was set to 1 so that the stir bar was slightly stirring the solution.

[0091] The syringe pump was operated at 0.5 mL / min.

[0092] After the entire contents of the syringe had been dispensed, the syringe pump was stopped and replaced with the syringe that had been set aside. The syringe pump was returned to 0.5 mL / min.

[0093] After dispensing from the syringe, the nozzle was tightened.

[0094] The spheres were incubated in the solution for 10 minutes.

[0095] The spheres were dispensed into three 50 mL centrifuge tubes and the total volume collected was recorded. The volume of the alginate-calcium chloride solution was 137 mL.

[0096] The tube was centrifuged at 4000 RPM for 5 minutes.

[0097] The supernatant was replaced with an equal volume of DPBS and the tube was resuspended.

[0098] The tube was centrifuged again at 4000 RPM for 5 minutes.

[0099] The supernatant was removed and replaced with 10 mL of DPBS per tube.

[0100] The contents of the tubes were resuspended and transferred to one tube, resulting in a BAM volume of 33 mL.

[0101] The tubes were labeled with the lab notebook number, description, and the current date and stored at 2–8°C.

[0102] Example 4 Potassium iodide liposome BAM was made using the ultrasonic nozzle method and a 2:1 ratio of undiluted liposomes (Figure 3C, PTN13-015).

[0103] The materials used included 3% alginate, 20 g / L calcium chloride, DPBS, and liposomes.

[0104] The nozzle assembly was cleaned by flushing the tubing and nozzle with 70 mL of IPA, followed by 10 mL of water, followed by a 10 mL purge with air. Waste liquid was collected in an appropriately named and labeled container.

[0105] 1.2 mL of 3% alginate solution was combined with 2.8 mL of potassium iodide liposomes in a centrifuge tube, which was vortexed until the solution was homogenous.

[0106] The solution was drawn up into a 10 mL syringe, and the volume of the alginate liposome solution was 3.6 mL.

[0107] The syringe was placed in a syringe pump, and a rubber tube was attached from the nozzle to the syringe.

[0108] Another syringe was filled with 4 mL of DI water and set aside until needed for use.

[0109] 135 mL of 20% calcium chloride solution was dispensed into a shallow collection bowl and a 2-inch stir bar was placed in the bowl.

[0110] A collection bowl was placed under the nozzle, and a stirring plate was placed underneath it, leaving a gap of 1–2 cm between the tip of the nozzle and the surface of the solution.

[0111] The nozzle generator was turned on and set to 5.0W.

[0112] The stir plate was set to 1 so that the stir bar was slightly stirring the solution.

[0113] The syringe pump was operated at 0.5 mL / min.

[0114] After the entire contents of the syringe had been dispensed, the syringe pump was stopped and the syringe was replaced with the syringe that had been set aside. The syringe pump was returned to 0.5 mL / min.

[0115] After dispensing from the syringe, the nozzle was tightened.

[0116] The spheres were incubated in the solution for 10 minutes.

[0117] The spheres were dispensed into three 50 mL centrifuge tubes, with a total volume of alginate-calcium chloride solution of 138 mL.

[0118] The three tubes were centrifuged at 4000 RPM for 5 minutes.

[0119] The supernatant was replaced with an equal volume of DPBS and resuspended.

[0120] The tube was centrifuged at 4000 RPM for 5 minutes.

[0121] The supernatant was removed and replaced with 10 mL of DPBS per tube.

[0122] The tubes were resuspended and the contents were transferred to one tube for a total volume of 37 mL of BAM.

[0123] The tubes were labeled with the lab notebook number, description, and the current date and stored at 2–8°C.

[0124] Example 5 The Mastersizer is a laser diffraction particle size analyzer capable of measuring particle sizes from 0.1 μm to 3 mm. Particle size measurements were performed using the Mastersizer. Figures 3A, 3B, 3C, and 3D show particle size data comparing 188RNL BAM intermediate (PTN12-066), KI liposomes (PTN13-008), BAM KI liposomes 1:1 (PTN13-014), and BAM KI liposomes 2:1 (PTN13-015).

[0125] Example 6 Fluoroscopic images of the radiopaque prototype (KI) versus the non-radiopaque prototypes (RNL-BAM-KI and RNL-BAM) at settings of 43 kV, 3.7 mA, 84.55 mGy, and 4 minutes. Figures 4A and 4B.

[0126] Although the present technology has been described herein with reference to embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the technology. It is therefore to be understood that numerous modifications can be made to the exemplary embodiments and other arrangements can be devised without departing from the spirit and scope of the present technology as defined by the appended claims.

[0127] References TIFF2026502140000002.tif138162

Claims

1. adding at least one liposome to a first channel on a microfluidic cassette; adding at least one crosslinkable polymer to a second channel on the microfluidic cassette; combining the at least one liposome and at least one crosslinkable polymer to form a solution; forcing the solution into a third channel on the microfluidic cassette to shear the solution to create individual spheres; and adding an aqueous solution containing a crosslinker to a fourth channel on the microfluidic cassette, wherein the crosslinker and crosslinkable polymer form a polymer matrix encapsulating liposomes.

1. A method of making a polymer matrix encapsulating liposomes, comprising:

2. The method of claim 1 , wherein the at least one crosslinkable polymer is alginate.

3. The method of claim 2, wherein the alginic acid is composed of stereomonomers of mannuronic acid and guluronic acid.

4. 4. The method of claim 3, wherein the alginate is composed of a mannuronic acid to guluronic acid ratio of less than or equal to 1.

5. 4. The method of claim 3, wherein the alginate is composed of a mannuronic acid to guluronic acid ratio greater than or equal to 1.

5.

6. The method of claim 1 , wherein the cross-linking agent is a divalent cation.

7. The method of claim 1 , wherein the cross-linking agent is a calcium salt.

8. The method of claim 1 , wherein the cross-linking agent is a barium salt.

9. 10. The method of claim 1, further comprising washing the polymer matrix encapsulating the liposomes to remove excess cross-linking agent.

10. 10. The method of claim 1, wherein the at least one liposome is loaded with at least one active agent prior to adding the at least one liposome to the first channel on the microcassette.

11. 11. The method of claim 10, wherein the at least one active agent is radioactive.

12. 11. The method of claim 10, wherein the at least one active agent is a beta-emitting radionucleotide chelate.

13. 13. The method of claim 12, wherein the beta-emitting radionucleotide chelate comprises Rhe188.

14. 13. The method of claim 12, wherein the beta-emitting radionucleotide chelate comprises BMEDA.

15. 2. The method of claim 1, wherein the first channel, the second channel, the third channel, and the fourth channel each have a diameter of 20 to 120 μm.

16. 10. The method of claim 1, further comprising a microsphere aspect ratio of 0.7 to 1.

3.

17. 10. The method of claim 1, further comprising a flow rate difference between the dispersed phase and the continuous phase of 0 to 100 μL / min.

18. 10. The method of claim 1, further comprising a stream temperature of 5°C to 55°C.

19. 10. The method of claim 1, further comprising a stream pressure of 0 to 1500 mbar.

20. 10. A composition made by the method of claim 1, comprising: at least one liposome, at least one active agent encapsulated in the intraliposomal aqueous compartment of said liposome; A crosslinked polymer matrix, the polymer matrix being water insoluble and absorbing water. Including, the at least one liposome has a diameter of about 1 nm to 200 nm; and the at least one liposome is embedded in the crosslinked polymer matrix; The composition.

21. 21. The composition of claim 20, wherein the crosslinked polymer matrix comprises at least one active agent in free form.

22. 21. The composition of claim 20, further comprising an aqueous medium surrounding the crosslinked polymer matrix in which the at least one liposome is embedded.

23. 23. The composition of claim 22, wherein the aqueous medium comprises at least one active substance in free form.

24. 21. The composition of claim 20, wherein the at least one active agent is radioactive.

25. 25. The composition of claim 24, wherein the at least one radioactive substance is a beta-emitting nucleotide chelate.

26. 21. The composition of claim 20, wherein the crosslinked polymer matrix comprises alginate.

27. 21. The composition of claim 20, wherein the at least one liposome comprises at least one ionic salt within the intraliposomal aqueous compartment to create an ion gradient.

28. 21. The composition of claim 20, wherein the at least one liposome comprises at least one buffering substance within the intraliposomal aqueous compartment to create a pH gradient.

29. 21. The composition of claim 20, wherein the at least one liposome comprises at least one radiopaque material.

30. 21. The composition of claim 20, wherein the crosslinked polymer matrix has a diameter of about 10 to 90 μm.

31. 21. The composition of claim 20, wherein the cross-linked polymer is alginate.

32. 10. A method of using the polymer matrix encapsulating liposomes produced using the method of claim 1, wherein the active agent loaded into the polymer matrix actively destroys tumor cells.

33. The polymer matrix encapsulating liposomes produced using the method of claim 1 is used to treat the following diseases: Hepatocellular carcinoma, colorectal cancer liver metastasis, paraganglioma liver metastasis, neuroendocrine tumor liver metastasis, gastrointestinal cancer liver metastasis, breast cancer liver metastasis, melanoma liver metastasis, pancreatic cancer liver metastasis, cholangiocarcinoma liver metastasis, colorectal cancer lung metastasis, renal cancer lung metastasis, cirrhosis-associated thrombocytopenia, metastatic extrahepatic cholangiocarcinoma, glioblastoma, renal cell carcinoma, prostate cancer, and uterine fibroids and (c) treating at least one of the above conditions with radioembolization therapy.

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