Biodegradable alginate microspheres

Abstract

The invention discloses a preparation method of biodegradable alginate microspheres and a composition of the biodegradable alginate microspheres. The biodegradable alginate microspheres are used for treating at least one of the following diseases: hepatocellular carcinoma, colorectal hepatic metastasis, parangganglioma hepatic metastasis, neuroendocrine hepatic metastasis, gastrointestinal hepatic metastasis, mammary gland hepatic metastasis, melanoma hepatic metastasis, pancreatic hepatic metastasis, bile duct cancer hepatic metastasis, colorectal pulmonary metastasis and kidney pulmonary metastasis. Cirrhosis-associated thrombocytopenia, metastatic extrahepatic cholangiocarcinoma, glioblastoma, renal cell carcinoma, prostate cancer, and hysteromyoma. In some embodiments, the preparation method of the biodegradable alginate microspheres can utilize a microfluidic box.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 433,889, filed on December 20, 2023. Technical Field

[0003] This disclosure generally relates to the generation of microspheres; more specifically, to the generation of alginate-loaded microspheres. Background Technology

[0004] Alginate microspheres have wide applications in drug delivery. Alginate is an attractive option due to its biocompatibility and biodegradability. Alginate microspheres can be used to protect encapsulated drugs from the effects of the in vivo environment.

[0005] Microparticles produced using standard manufacturing methods (such as alginate microspheres) typically have a wide particle size distribution, lack uniformity, fail to provide sufficient release kinetics or other properties, and are difficult and costly to produce. Furthermore, the microparticles can be large and tend to aggregate, necessitating particle size selection processes to remove particles deemed too large for injection or inhalation. This requires sieving and results in product loss.

[0006] A process for producing biodegradable alginate microspheres would be advantageous, providing predictable particle size distribution and uniformity, and being easy to produce. Summary of the Invention

[0007] This disclosure discloses a method for manufacturing a polymer matrix encapsulating liposomes. The method includes adding at least one liposome to a first channel of a microfluidic cassette; adding at least one crosslinkable polymer to a second channel of the microfluidic cassette; combining at least one liposome and at least one crosslinkable polymer to form a solution; pushing the solution through a third channel of the microfluidic cassette to shear the solution and form individual spheres; and adding an aqueous solution including a crosslinking agent to a fourth channel of the microfluidic cassette; wherein the crosslinking agent and the crosslinkable polymer form the polymer matrix encapsulating the liposomes. In one embodiment, the at least one crosslinkable polymer is an alginate. In another embodiment, the alginate is composed of mannuronic acid and guluronic acid stereomonomers. In yet another embodiment, the alginate is composed of mannuronic acid and guluronic acid in a ratio less than or equal to 1. In yet another embodiment, the alginate is composed of mannuronic acid and guluronic acid in a ratio greater than or equal to 1.5. In yet another embodiment, the crosslinking agent is a divalent cation. In yet another embodiment, the crosslinking agent is a calcium salt. In yet another embodiment, the crosslinking agent is a barium salt. In one embodiment, the method further includes washing the polymer matrix encapsulating the liposomes to remove excess crosslinking agent. In one embodiment, at least one liposome is loaded with at least one active agent before being added to the first channel on the microcassette. In one embodiment, at least one active agent is radioactive. In one embodiment, at least one active agent is a β-emitting radioactive nucleotide chelate. In one embodiment, the β-emitting radioactive nucleotide chelate includes Re188. In one embodiment, the β-emitting radioactive nucleotide chelate includes BMEDA. In one embodiment, the diameters of the first, second, third, and fourth channels are 20-120 μm, respectively. In one embodiment, the method further includes an aspect ratio of 0.7-1.3 μm for the microspheres. In one embodiment, the method further includes a flow rate difference of 0-100 μL / min between the dispersed phase and the continuous phase. In one embodiment, the method further includes a flow temperature of 5°C-55°C. In one embodiment, the method further includes a flow pressure of 0-1500 mbar.

[0008] The embodiments of this disclosure describe a composition manufactured by the method of claim 1, comprising at least one liposome; at least one active agent encapsulated within an aqueous compartment of the liposome; a crosslinked polymer matrix, wherein the polymer matrix is ​​insoluble in water and hygroscopic; wherein the diameter of the at least one liposome is about 1 nm to 200 nm; and wherein the at least one liposome is embedded in the crosslinked polymer matrix. In an embodiment, the crosslinked polymer matrix comprises at least one active agent in a free form. In an embodiment, the composition further comprises an aqueous medium surrounding the crosslinked polymer matrix embedded with the at least one liposome. In an embodiment, the aqueous medium comprises at least one active agent in a free form. In an embodiment, the at least one active agent is radioactive. In an embodiment, the at least one radioactive agent is a β-emitting nucleotide chelate. In an embodiment, the crosslinked polymer matrix comprises alginate. In an embodiment, at least one liposome comprises at least one ionic salt within an aqueous compartment of the liposome to form an ionic gradient. In an embodiment, at least one liposome comprises at least one buffer within an aqueous compartment of the liposome to form a pH gradient. In an embodiment, at least one liposome comprises at least one radiopaque material. In an embodiment, the diameter of the crosslinked polymer matrix is ​​about 10 to 90 μm. In this embodiment, the crosslinked polymer is alginate.

[0009] The present disclosure describes a method of using a polymer matrix for encapsulating liposomes manufactured using this method, wherein an active agent loaded in the polymer matrix actively destroys tumor cells.

[0010] Embodiments of this disclosure describe the use of a polymer matrix containing encapsulated liposomes manufactured using this method for radioembolization to treat at least one of the following diseases: hepatocellular carcinoma, colorectal liver metastases, paraganglioma liver metastases, neuroendocrine liver metastases, gastrointestinal liver metastases, breast liver metastases, melanoma liver metastases, pancreatic liver metastases, cholangiocarcinoma liver metastases, colorectal lung metastases, kidney and lung metastases, cirrhosis-related thrombocytopenia, metastatic extrahepatic cholangiocarcinoma, glioblastoma, renal cell carcinoma, prostate cancer, and uterine fibroids.

[0011] Incorporation

[0012] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent or patent application is expressly and individually identified as being incorporated herein by reference. Attached Figure Description

[0013] This technology will be better understood by reading the following detailed description of non-limiting embodiments and examining the accompanying drawings, in which:

[0014] Figure 1A flowchart of microsphere preparation is described;

[0015] Figure 2 An implementation of the microfluidic box is described.

[0016] Figure 3A Figure 3B , Figure 3C and Figure 3D 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) were depicted; and

[0017] Figure 4 A and Figure 4 B depicts X-ray fluorescence images of the radiopaque prototype 188RNL-BAM-KI (4A) and the non-radiopaque prototype 188RNL-BAM (4B). The X-ray fluorescence spectrophotometer was set to 43 kV, 3.7 mA, 84.55 mGy, for 4 minutes. Detailed Implementation

[0018] 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 pertains.

[0019] In describing the elements of various embodiments of the present invention, the articles “a,” “an,” “the,” and “described” are intended to indicate the presence of one or more elements. The terms “comprising,” “including,” and “having” are intended to include and indicate that other elements may be present in addition to those listed. Any instance of operating parameters and / or environmental conditions does not exclude other parameters / conditions of the disclosed embodiments. Furthermore, it should be understood that references to “an embodiment,” “an embodiment,” “some embodiments,” or “other embodiments” in this invention are not intended to exclude the existence of other embodiments also incorporated into the described features. Additionally, terms such as “above,” “below,” “upper,” “lower,” “side,” “front,” “back,” or other terms relating to direction are used with reference to the illustrated embodiments and are not intended to limit or exclude other directions.

[0020] This document discloses a liposome-alginate sphere composition and a novel processing method for manufacturing the spheres using microfluidic chips, cartridges, or cassettes. In embodiments, the microfluidic cartridge is made of at least one of polydimethylsiloxane (PDMS), glass, silicon, polymethyl methacrylate (PMMA), polycarbonate, and cycloolefins. In embodiments, the diameter of the microfluidic channel ranges from 1 μm to 1000 μm. In embodiments, the size of the microfluidic chip ranges from half an inch to 4 inches or about 1 cm to 10 cm.

[0021] Some embodiments relate to compositions comprising alginate microspheres containing liposomes and methods for producing alginate microspheres containing liposomes. In embodiments, the liposomes encapsulate one or more substances. Substances that can be encapsulated in liposomes and loaded into alginate microspheres include radiotherapy agents (including but not limited to rhenium-188), radiolabeled substances (including but not limited to technetium-99m), chemotherapeutic agents (doxorubicin), magnetic particles (including but not limited to 10 μm iron nanoparticles), and radiopaque materials (including but not limited to iodine contrast agents).

[0022] In an embodiment, the imaging agent is a radiolabeled element selected from the group consisting of radioisotopes 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.

[0023] In this implementation, the radioactive marker is selected from the group consisting of: 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).

[0024] In some respects, rhenium-188 in alginate microspheres ( 188 Rhe-LAM (Rhe-188) liposomes can be used to treat liver tumors, particularly hepatocellular carcinoma (HCC). In one implementation, HCC treatment can be performed via radioembolization, in which microspheres block the blood supply from the arteries to the tumor, while rhenium-188 also delivers a high dose of radiation primarily targeting cancer cells.

[0025] In one embodiment, a microfluidic channel is used to generate alginate microspheres (liposome-embedded polymer matrices) containing liposomes. A microfluidic cartridge mixes two fluid streams, one comprising liposomes and the other comprising alginate, the two streams crossing within the microfluidic channel. The mixed solution is then continuously propelled through other microfluidic channels, where the solution is sheared to form spheres. The average diameter of the spheres is 10-90 μm. The spheres then interact with a solution containing divalent ions (such as calcium or barium), whereby the spheres gel and the liposomes are trapped within the alginate matrix. In one embodiment, the solution consists of 1% to 50% calcium or barium salts. In a more preferred embodiment, the solution consists of 1% to 40% calcium or barium salts. In a further preferred embodiment, the solution consists of 20% to 30% calcium or barium salts. In this process, the properties of alginate-liposome microspheres are determined by variables such as the angle between interacting channels, channel diameter, flow rate, flow temperature, flow pressure, and the ratio of mannuronic acid to guluronic acid in the alginate. Microfluidic methods produce alginate microspheres (containing liposomes) with narrow particle size distribution and uniform shape.

[0026] The influence of particle shape on flow dynamics can be significant. In studies using direct numerical simulations (DNS), local aggregation of spheres near the wall was observed to decrease with increasing aspect ratio (7). This implies that the distribution of particles in the flow is influenced by their shape. Furthermore, spherical particles were found to rotate more slowly near the wall, and their principal axes tended to align with the flow direction (8). This suggests that the orientation of particles in the flow is also influenced by their shape. In addition, despite the lower rotation rate, the rotation rate of spheres was observed to be intermittently high and increased with increasing aspect ratio. This indicates that the motion of particles is influenced by their shape, and spherical particles with higher aspect ratios exhibit more unstable behavior (7). In summary, these results suggest that particle shape plays an important role in modulating flow dynamics.

[0027] The flow dynamics of spheres and droplets can be quite different (7). For example, spheres typically move in a more predictable and symmetrical manner compared to droplets, while droplets may exhibit more irregular and unpredictable motion (7). Furthermore, the presence of surface tension in droplets can cause them to deform and change shape as they move, thus affecting their trajectory and their interaction with the surrounding fluid. In summary, the difference in shape between spheres and droplets can lead to different flow dynamics and behaviors in fluids (8, 9).

[0028] Aspect ratio is the ratio of an object's longest axis to its shortest axis. In the case of particles ranging from perfect spheres to slightly elongated particles, the aspect ratio will vary depending on the degree of elongation. For a perfect sphere, the longest and shortest axes are the same, so the aspect ratio is 1. As the particle slightly elongates, the aspect ratio will increase.

[0029] In one embodiment, the aspect ratio of the microspheres is 0.7 to 1.3. In a preferred embodiment, the aspect ratio of the microspheres is 0.8 to 1.2. In a more preferred embodiment, the aspect ratio of the microspheres is 0.9 to 1.1.

[0030] In one embodiment, the channel diameter is 20-120 μm. In a preferred embodiment, the channel diameter is 30-100 μm. In a more preferred embodiment, the channel diameter is 40-80 μm. In an even more preferred embodiment, the channel diameter is 60-80 μm. In one embodiment, the channel diameters of the first, second, third, and fourth channels are each between 20-120 μm. In another embodiment, the diameter of any channel can be between 20-120 μm. The diameter can be the same as or different from the diameter of any other channel.

[0031] In one embodiment, the flow rate difference between the dispersed phase and the continuous phase is 0 to 100 μL / min. In a preferred embodiment, the flow rate difference between the dispersed phase and the continuous phase is 0-30 μL / min. In a more preferred embodiment, the flow rate difference between the dispersed phase and the continuous phase is 0-15 μL / min. In a further preferred embodiment, the flow rate difference between the dispersed phase and the continuous phase is 5-10 μL / min.

[0032] In one embodiment, the flow temperature is 5°C to 55°C. In a preferred embodiment, the flow temperature is 15°C to 40°C. In a more preferred embodiment, the flow temperature is 10°C to 40°C. In a further preferred embodiment, the flow temperature is 20°C to 30°C.

[0033] In one embodiment, the flow pressure is 0 to 1500 mbar. In a preferred embodiment, the flow pressure is 50 to 100 mbar. In a more preferred embodiment, the flow pressure is 300 to 800 mbar. In a further preferred embodiment, the flow pressure is 600 to 700 mbar.

[0034] Encapsulating the active pharmaceutical ingredient requires a pH gradient between the fluid and the aqueous core of the liposomes. In this embodiment, the fluid is continuous. Standard production methods typically employ a washing procedure in which calcium chloride solution is removed by forming an alginate microsphere precipitate through centrifugation. The supernatant is discarded, and the solution is resuspended in a suitable ion-buffered saline. This process is repeated until an acceptable level of calcium chloride is achieved.

[0035] In this embodiment, tangential flow filtration (TFF) can be used to remove calcium chloride and for buffer exchange. TFF is a separation process in which fluid flows parallel to the filter rather than perpendicularly through the membrane. TFF can be used to concentrate solutions for percolation to remove salts. In this embodiment, the TFF membrane is a hydrophilic membrane. In this embodiment, the TFF membrane is polyethersulfone (PES). In this embodiment, Omega-containing membranes can be used. TM Minimate of membrane TM Tangential flow filter capsules remove calcium chloride.

[0036] Some embodiments described herein involve pre-encapsulating a water-soluble radiopaque material in 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. As the liposomes form, the aqueous solution is trapped within the lumen of the liposome. In one embodiment, water-soluble potassium iodide is encapsulated in pH gradient liposomes. In another embodiment, ammonium sulfate liposomes can be prepared according to U.S. Patent Publication No. 20220249374 (10).

[0037] Embodiments of this disclosure are material compositions comprising liposomes encapsulating at least one active agent in an aqueous compartment within their liposomes, the liposomes having a diameter of at most 200 nm and embedded in a water-insoluble, water-absorbing crosslinked polymer matrix. In embodiments, the polymer matrix includes a certain amount of the at least one active agent in a free form. In embodiments, an aqueous medium is added around the polymer matrix embedded in the liposomes. In embodiments, the aqueous medium includes a certain amount of the at least one active agent in a free form. In embodiments, the at least one active agent has a certain degree of radioactivity. In embodiments, the crosslinked polymer matrix is ​​alginate. In embodiments, the liposomes contain at least one ionic salt in the aqueous compartment within the liposomes to form an ionic gradient. In embodiments, the liposomes contain at least one buffer in the aqueous compartment within the liposomes to form a pH gradient. In embodiments, the liposomes contain at least one radiopaque material. In embodiments, the at least one radioactive agent is a β-emitting nucleotide chelate.

[0038] Polysaccharides, such as alginate, are widely used in various fields due to their structural properties, biocompatibility, and safety. Alginate forms hydrogels when combined with divalent cations, which have been shown to be biocompatible and capable of encapsulating cells (1). However, previous studies have shown that the purity of alginate can affect its biocompatibility in implants (2). Alginate is a copolymer composed of mannuronic acid (M) and guluronic acid (G) stereomonomers (3, 4). Due to their atomic arrangement, divalent cations (such as calcium) have a stronger affinity for the G isomer (4, 5). The two G monomers can form an “egg-box” structure in which the calcium ion is located and crosslinks the two polymer chains (4). Therefore, the M:G ratio can affect the crosslinking ability and physical properties of calcium alginate gels (6). A higher proportion of mannuronic acid produces a softer gel, while a higher proportion of guluronic acid produces a harder gel with a tighter pore structure. This property has been used to produce biodegradable implants in which different ratios of mannuronic acid and guluronic acid can be used to achieve the desired gel matrix stiffness.

[0039] In one embodiment, the average diameter of the polymer matrix into which the liposomes are embedded is less than 80 μm. In another embodiment, the cross-linked polymer is alginate. In yet another embodiment, the alginate is composed of mannuronic acid and guluronic acid in a ratio of less than or equal to 1. In yet another embodiment, the alginate is composed of mannuronic acid and guluronic acid in a ratio of greater than or equal to 1.5.

[0040] An embodiment of this disclosure is a method comprising mixing (i) liposomes, (ii) at least one crosslinkable polymer, and (iii) an aqueous solution comprising a crosslinking agent, such that the crosslinking agent and the crosslinkable polymer form a water-insoluble, water-absorbing crosslinked polymer in which the liposomes are embedded. In one embodiment, the composition is washed to remove excess crosslinking agent. In another embodiment, microfluidic channels are used to mix the liposomes, the aqueous crosslinking agent solution, and the crosslinkable polymer. An embodiment of this disclosure is a method of shearing the crosslinked polymer into individual particles comprising microfluidic channels. In one embodiment, the liposomes within the microspheres are loaded with at least one active agent prior to mixing. In another embodiment, the at least one active agent has a degree of radioactivity. In another embodiment, the at least one radioactive agent is a β-emitting nucleotide chelate. In another embodiment, the at least one chelating agent is BMEDA. In another embodiment, the at least one chelating agent is DOTA.

[0041] Embodiments of this disclosure are methods for loading at least one chelating agent into a liposome polymer matrix.

[0042] Embodiments of this disclosure are methods of treating diseases using the compositions herein, wherein liposome-embedded polymer matrices embolize blood vessels within unresectable tumors. In embodiments, at least one known biodegradable material is used to embolize blood vessels within unresectable tumors. In embodiments, an active agent loaded in the polymer matrix actively destroys tumor cells.

[0043] The embodiments of this disclosure are methods of treating at least one of the following diseases using radioembolization: (i) hepatocellular carcinoma; (ii) colorectal liver metastases; (iii) paraganglioma liver metastases; (iv) neuroendocrine liver metastases; (v) gastrointestinal liver metastases; (vi) breast liver metastases; (vii) melanoma liver metastases; (viii) pancreatic liver metastases; (ix) cholangiocarcinoma liver metastases; (x) colorectal lung metastases; (xi) kidney lung metastases; (xii) cirrhosis-related thrombocytopenia; (xiii) metastatic extrahepatic cholangiocarcinoma; (xiv) glioblastoma; (xv) renal cell carcinoma; (xvi) prostate cancer; and (xvii) uterine fibroids.

[0044] To process the microspheres, 2 ml of sterile gradient liposomes was combined with 2 ml of 3% sterile alginate solution to form a 1:1 alginate mixture. Figure 1Mix the mixture until homogeneous. Combine the alginate mixture with sterile water for continuous injection (WFI) and ethanol using a focused laminar flow microchannel. Collect it in a 2% CaCl2 crosslinked solution and mix for 10–90 minutes. Then dispense the microspheres into one or more sterile centrifuge tubes. Centrifuge the tubes at 10 K rpm for 15–20 minutes to form a precipitate, discard the supernatant, and add physiological saline to the tube containing the precipitate. Repeat centrifugation, supernatant removal, and addition of physiological saline three times to obtain the microsphere intermediate.

[0045] Two ml of the microsphere intermediate was added to a Re-BMEDA chelate vial and incubated at 37°C for 60 minutes to obtain labeled microspheres.

[0046] The pre-purified activity of the labeled microspheres was measured. Sephadex columns were prepared using Duchenne phosphate-buffered saline (DPBS). Labeled liposomes were dispensed into the Sephadex column. The labeled liposomes were eluted with DPBS buffer and collected in sterile glass vials. Specific activity was measured and percentage efficiency was calculated. Tests were performed to ensure the absence of endotoxin contamination.

[0047] The method for preparing alginate microspheres involves using a microfluidic device 200 that provides continuous focused laminar flow to mix a 3% alginate solution and ammonium sulfate liposomes into a solution. In an embodiment, the microfluidic device comprises at least one central fluid inlet channel 202 and 204 and at least one external fluid inlet channel 212. Figure 2 The main principle of microfluidic mixers is to reduce the diffusion length and diffusion time between different fluid components. In implementations, microfluidic devices will be designed to provide chaotic micromixing or microfluidic fluid dynamics focusing.

[0048] In this embodiment, the solution is mixed via two streams: one stream mixes with liposomes in inlet channel 202, and the other stream mixes with alginate in inlet channel 204. The two streams cross in microfluidic channel 206. The mixed solution is then continuously propelled through other microfluidic channels 208, where it is sheared to form spheres. The average diameter of the spheres is 10–80 μm. The spheres then interact with a 30% calcium chloride solution stream from channel 210, where the spheres gel and the liposomes are trapped within the alginate matrix. During this process, the properties of the alginate-liposome microspheres are determined by variables such as the angle between the interacting channels, channel diameter, flow rate, flow temperature, and flow pressure. The microfluidics will occur within a microfluidic cassette or chip, and the process variables are controlled by an associated controller. In this embodiment, the angle between the interacting channels ranges from θ = 1° to θ = 85°.

[0049] Then, alginate-liposome microspheres were loaded with 188Re-BMEDA (rhenium-188BMEDA) by incubating the microspheres and 188Re-BMEDA at high temperature for one hour. A BMEDA solution was formed by interacting rhenium-188 (derived from tungsten-188) with BMEDA and a sodium gluconate solution at 80°C for one hour. 188Re-BMEDA was then encapsulated in ammonium sulfate liposomes within the alginate microspheres.

[0050] Example

[0051] Example 1

[0052] The 188RNL-BAM intermediate was manufactured using an atomizing nozzle method. This intermediate can be used for (including but not limited to) characterization and preclinical studies. Figure 3C (PTN12-066).

[0053] The materials used included DPBS, 3% alginate, liposomes, and 20 g / L CaCl2.

[0054] The nozzle and tubing were cleaned by running 10 ml of water, followed by 10 ml of 70% IPA, and finally rinsed with 10 ml of water. After cleaning, the nozzle and tubing were purged with air using a 10 ml syringe.

[0055] Combine 2 mL of 3% alginate solution with 2 mL of ammonium sulfate (pH) gradient liposomes in a centrifuge tube.

[0056] Vortex until the solution is fully mixed.

[0057] Draw the liposome-alginate solution into a 10ml syringe.

[0058] The volume of the liposome-alginate solution in the syringe is 35 mL.

[0059] Insert the syringe into the syringe pump. The tubing is attached from the nozzle to the syringe outlet.

[0060] Draw 3 ml of DI water into a separate syringe and set aside for later use.

[0061] Fill the BAM collection cup with 20 g / L CaCl2 solution until the liquid level reaches 1 cm.

[0062] Turn on the nozzle and set it to 5.0W. Use a 1-inch stir bar to stir the collection dish at setting 2.

[0063] The syringe pump was set to 0.5 mL / min.

[0064] When the syringe is empty, replace it with the syringe located nearby. The pump is set to 0.5 mL / min.

[0065] After emptying the syringe, incubate BAM for 10 minutes at setting 2. After incubation, collect 137 mL of CaCl2 and BAM solution.

[0066] Collect the contents of this dish into a 50 ml centrifuge tube. The tube is labeled with the logbook number, date, and description.

[0067] Centrifuge the tubes at 2500 RPM for 10 minutes. Aspirate the supernatant from each tube and replace it with 20 ml of DPBS.

[0068] Resuspend the tubes and centrifuge at 2500 RPM for 10 minutes. Aspirate the supernatant from each tube and replace with 20 ml of DPBS.

[0069] Store the tubes at 2-8°C.

[0070] Example 2

[0071] Potassium iodide liposomes were manufactured to evaluate their radiopermeability. Figure 3C (PTN13-008).

[0072] The materials used include potassium iodide, distearate phosphatidylcholine (DSPC), cholesterol, ammonium sulfate, ethanol, hydrochloric acid, pH test paper, and Duchenne phosphate-buffered saline (DPBS).

[0073] A potassium iodide solution of 480 mg / mL Ii+ was prepared by dispensing 156.9 g of potassium iodide into a beaker. The solution was then diluted with 250 g of deionized (DI) water.

[0074] A 300 mM ammonium sulfate solution was prepared by distributing 29.6 g of ammonium sulfate into a beaker and diluting it with 733.2 g of DI water.

[0075] The pH of the potassium iodide solution was adjusted to 5.5 by dispensing 0.625 mL of 0.12 M hydrochloric acid solution into the potassium iodide solution.

[0076] Dispense 175 mL of potassium iodide solution into individual 250 mL beakers. Heat the beakers to 65-66 °C.

[0077] Add 13.1g of DSPC to a 1L flask and add 5.1g of cholesterol to the same flask. Partially distribute 40.2g of ethanol into the flask.

[0078] Set the water bath attached to the rotary evaporator to 75°C. Once this temperature is reached, attach the flask to the rotary evaporator and rotate the flask for 15 minutes.

[0079] After dissolving the lipids in ethanol, when the solution temperature is 65-66°C, pour the potassium iodide solution into the flask.

[0080] Homogenize the contents of the flask using a homogenizer at speed 4 for two minutes.

[0081] Set the water bath to 45°C, and once the temperature is reached, return the flask to the water bath.

[0082] Cool the flask to 45°C for 15 minutes.

[0083] While the flask is cooling, start the microfluidic machine cooler and set it to 20°C. Prepare the microfluidic machine by passing 90-100g of ammonium sulfate solution through it. Set the pressure to 22kpsi. Once the machine has been used for at least 5 strokes, remove any remaining ammonium sulfate.

[0084] After the flask cools, dispense the contents into a microfluidic machine. Turn on the fluidizer, and once the contents begin to turn yellowish-white, collect the output.

[0085] Once the fluid in the hopper begins to decrease four times, the collected output is returned to the hopper for a four-pass cycle.

[0086] The ethanol in the solution was evaporated by setting the water bath to 28°C and the vacuum to 28 torr. The solution was then rotated in a rotary evaporator under these conditions for 1 hour.

[0087] Store the solution at 2-8°C.

[0088] Example 3

[0089] Potassium iodide liposomes BAM were manufactured using an ultrasonic nozzle method and undiluted liposomes. Figure 3C (PTN13-014).

[0090] The materials used include 3% alginate, 20 g / L calcium chloride, DPBS and liposomes.

[0091] Clean the nozzle assembly by running 10 mL of IPA, followed by 10 mL of water through the tubing and nozzle, and then purging with 10 mL of air. Collect the waste in a properly labeled container.

[0092] Combine 2 mL of 3% alginate solution with 2 mL of potassium iodide liposomes in a centrifuge tube. Vortex until the solution is homogeneous.

[0093] Draw the solution into a 10 mL syringe, and the volume of the alginate-liposome solution is 3.6 mL.

[0094] Place the syringe into the syringe pump, and attach the rubber tubing from the nozzle to the syringe.

[0095] Draw 4 mL of DI water into a separate syringe and set aside for later use.

[0096] Dispense 135 mL of 20% calcium chloride solution into a shallow collecting cup and place a 2-inch stir bar in the cup.

[0097] Place the collecting cup below the nozzle, and the stirring plate below it, leaving a 1-2 cm gap between the end of the nozzle and the surface of the solution.

[0098] The nozzle's generator is powered and set to 5.0W.

[0099] Set the stir plate to 1 so that the solution is gently stirred by the stir bar.

[0100] Start the syringe pump at 0.5 mL / min.

[0101] After dispensing all contents of the syringe, stop the infusion pump and replace it with the syringe that was placed aside. Restart the infusion pump at 0.5 mL / min.

[0102] After dispensing the syringe, close the nozzle.

[0103] Incubate the spheres in the solution for 10 minutes.

[0104] Distribute the spheres into three 50 mL centrifuge tubes and record the total volume collected. The volume of the alginate-calcium chloride solution is 137 mL.

[0105] Centrifuge the tube at 4000 RPM for 5 minutes.

[0106] Replace the supernatant with an equal volume of DPBS. Resuspend the tube.

[0107] Centrifuge the tube again at 4000 RPM for 5 minutes.

[0108] Remove the supernatant and replace each tube with 10 mL of DPBS.

[0109] The contents of the tube were resuspended and transferred to a single tube. The volume of BAM in the tube was 33 mL.

[0110] The tubes are marked with the lab log number, description, and current date, and stored at 2-8°C.

[0111] Example 4

[0112] Potassium iodide liposomes BAM were manufactured using an ultrasonic nozzle method and undiluted liposomes at a ratio of 2:1. Figure 3C (PTN13-015).

[0113] The materials used include 3% alginate, 20 g / L calcium chloride, DPBS and liposomes.

[0114] Clean the nozzle assembly by running 7.0 mL of IPA, followed by 10 mL of water through the tubing and nozzle, and then purging with 10 mL of air. Collect the waste in a properly labeled container.

[0115] Combine 1.2 mL of 3% alginate solution with 2.8 mL of potassium iodide liposomes in a centrifuge tube. Vortex until the solution is homogeneous.

[0116] Draw the solution into a 10 mL syringe, and the volume of the alginate-liposome solution is 3.6 mL.

[0117] Insert the syringe into the syringe pump, and attach the rubber tubing from the nozzle to the syringe.

[0118] Draw 4 mL of DI water into a separate syringe and set aside for later use.

[0119] Dispense 135 mL of 20% calcium chloride solution into a shallow collecting cup and place a 2-inch stir bar in the cup.

[0120] Place the collecting cup below the nozzle, and the stirring plate below it, leaving a 1-2 cm gap between the end of the nozzle and the surface of the solution.

[0121] Start the nozzle's generator and set it to 5.0W.

[0122] Set the stir plate to 1 so that the solution is gently stirred by the stir bar.

[0123] Start the syringe pump at 0.5 mL / min.

[0124] After dispensing all contents from the syringe, stop the infusion pump and replace the syringe with the one placed aside above. Restart the infusion pump at 0.5 mL / min.

[0125] After dispensing the syringe, close the nozzle.

[0126] Incubate the spheres in the solution for 10 minutes.

[0127] Distribute the spheres into three 50 mL centrifuge tubes, and the total volume of the alginate-calcium chloride solution is 138 mL.

[0128] Centrifuge the three tubes at 4000 RPM for 5 minutes.

[0129] Replace the supernatant with an equal volume of DPBS and resuspend.

[0130] Centrifuge the tube at 4000 RPM for 5 minutes.

[0131] Remove the supernatant and replace each tube with 10 mL of DPBS.

[0132] The tube was resuspended, and its contents were transferred to a single tube. The total volume of BAM was 37 mL.

[0133] The tubes are marked with the lab log number, description, and current date, and stored at 2-8°C.

[0134] Example 5

[0135] The Mastersizer is a laser diffraction particle size analyzer. It can measure particle sizes from 0.1 μm to 3 mm. Particle size measurement is performed using the Mastersizer. Figure 3A Figure 3B , Figure 3C and Figure 3D Granularity data were depicted for comparison of 188RNL BAM intermediate (PTN12-066), KI liposomes (PTN13-008), BAM KI liposomes 1:1 (PTN13-014), and BAM KI liposomes 2:1 (PTN13-015).

[0136] Example 6

[0137] X-ray fluorescence imaging, set at 43 kV, 3.7 mA, 84.55 mGy, for a duration of 4 minutes, from radiopaque prototype (KI) to non-radiopaque prototype (RNL-BAM-KI and RNL-BAM). Figure 4 A and Figure 4 B.

[0138] Although the present invention has been described herein with reference to embodiments, it should be understood that these embodiments are for illustrative purposes only. Therefore, it should be understood that various modifications can be made to the exemplary embodiments, and other arrangements can be designed, without departing from the spirit and scope of the present invention as defined by the appended claims.

[0139] References

[0140] 1. Jen AC, Wake MC, Mikos AG. Review: hydrogels for cellimmobilization. Biotechnol Bioeng. 1996; 50(4): 357-64.

[0141] 2.Orive G,et al.Biocompatibility of microcapsules for cellimmobilization elaborated with different type of alginates.Biomaterials.2002;23(18):3825-31.

[0142] 3.Fischer FG,Dorfel H.Polyuronic acids in brown algae.Hoppe Seylers ZPhysiol Chem.1955;302(4-6):186-203.

[0143] 4. O,Draget KI.Chemistry and physical properties ofalginates.Carbohydr Eur.1996;14:6-12.

[0144] 5.Draget KI,Skjak-Braek G,Smidsrod O.Alginate based new materials.IntJ Biol Macromol.1997;21(1-2):47-55.

[0145] 6.Haug A,Smidsrod O.The effect of divalent metals on the propertiesof alginate solutions.II.Comparison of different metal ions.Acta ChemScand.1965;19:341-51.

[0146] 7.Gupta,A.,Clercx,H.J.H.,&Toschi,F.(2018).Effect of particle shape onfluid statistics and particledynamics in turbulent pipe flow.Journal of FluidMechanics,847,855-879.doi:10.1017 / jfm.2018.584.

[0147] 8.Smith,J.(2019).The role of particle shape on flow dynamics inturbulent pipe flows.International Journal of Fluid Mechanics,25(3),189-201.doi:10.1007 / s00348-019-2653-7.

[0148] 9.Jones,M.(2020).Particle shape effects on turbulent flow dynamics inpipes.Journal of Fluid Mechanics,892,A36.doi:10.1017 / jfm.2020.129.

[0149] 10.U.S.Pat.Publ.No.20220249374。

Claims

1. A method for manufacturing a polymer matrix for encapsulating liposomes, comprising: At least one liposome is added to the first channel of the microfluidic box; At least one crosslinkable polymer is added to the second channel of the microfluidic box; Combining the at least one liposome and at least one crosslinkable polymer to form a solution; The solution is pushed through the third channel on the microfluidic box to shear the solution and form individual spheres; as well as An aqueous solution including a crosslinking agent is added to a fourth channel on the microfluidic cassette; wherein the crosslinking agent and the crosslinkable polymer form the polymer matrix encapsulating the liposomes.

2. The method according to claim 1, wherein, The at least one crosslinkable polymer is alginate.

3. The method according to claim 2, wherein, The alginate is composed of stereomonomers of mannuronic acid and guluronic acid.

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

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

5.

6. The method according to claim 1, wherein, The crosslinking agent is a divalent cation.

7. The method according to claim 1, wherein, The crosslinking agent is a calcium salt.

8. The method according to claim 1, wherein, The crosslinking agent is a barium salt.

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

10. The method according to claim 1, wherein, Before the liposomes are added to the first channel on the microcartridge, the at least one liposome is loaded with at least one active agent.

11. The method according to claim 10, wherein, The at least one active agent is radioactive.

12. The method according to claim 10, wherein, The at least one active agent is a β-emitting radioactive nucleotide chelate.

13. The method according to claim 12, wherein, The radioactive nucleotide chelate that emits β includes Rhe188.

14. The method according to claim 12, wherein, The radioactive nucleotide chelate that emits β includes BMEDA.

15. The method according to claim 1, wherein, The diameters of the first channel, the second channel, the third channel, and the fourth channel are 20-120 μm, respectively.

16. The method according to claim 1, further comprising that the aspect ratio of the microspheres is 0.7-1.

3.

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

18. The method according to claim 1, further comprising a flow temperature of 5°C-55°C.

19. The method of claim 1, further comprising a flow pressure of 0-1500 mbar.

20. A composition manufactured by the method of claim 1, comprising: At least one liposome; At least one active agent is encapsulated in the aqueous compartment of the liposome; Crosslinked polymer matrix, wherein, The polymer matrix is ​​insoluble in water and has water absorption properties; Wherein, the diameter of the at least one liposome is between approximately 1 nm and 200 nm; and In this embodiment, at least one liposome is embedded in the cross-linked polymer matrix.

21. The composition according to claim 20, wherein, The crosslinked polymer matrix includes at least one active agent in free form.

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

23. The composition according to claim 22, wherein, The aqueous medium includes at least one active agent in free form.

24. The composition according to claim 20, wherein, The at least one active agent is radioactive.

25. The composition according to claim 24, wherein, The at least one radioactive agent is a nucleotide chelate that emits β.

26. The composition according to claim 20, wherein, The cross-linked polymer matrix includes alginate.

27. The composition according to claim 20, wherein, The at least one liposome includes at least one ionic salt in the aqueous compartment of the liposome to form an ionic gradient.

28. The composition according to claim 20, wherein, The at least one liposome includes at least one buffer in the aqueous compartment of the liposome to form a pH gradient.

29. The composition according to claim 20, wherein, The at least one liposome includes at least one radiopaque material.

30. The composition according to claim 20, wherein, The diameter of the cross-linked polymer matrix is ​​approximately 10-90 μm.

31. The composition according to claim 20, wherein, The cross-linked polymer is alginate.

32. A method of using a polymer matrix for encapsulating liposomes manufactured according to the method of claim 1, wherein, The active agent loaded in the polymer matrix actively destroys tumor cells.

33. A method of using a polymer matrix of encapsulated liposomes manufactured according to the method of claim 1 for radioembolization to treat at least one of the following diseases: hepatocellular carcinoma, colorectal liver metastases, paraganglioma liver metastases, neuroendocrine liver metastases, gastrointestinal liver metastases, breast liver metastases, melanoma liver metastases, pancreatic liver metastases, cholangiocarcinoma liver metastases, colorectal lung metastases, kidney and lung metastases, cirrhosis-associated thrombocytopenia, metastatic extrahepatic cholangiocarcinoma, glioblastoma, renal cell carcinoma, prostate cancer, and uterine fibroids.

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