Radiopharmaceutical-loaded hyaluronic acid gel microspheres as well as preparation method and application thereof

Abstract

The invention relates to a radiopharmaceutical-loaded hyaluronic acid gel microsphere as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: mixing a hyaluronic acid solution with methacrylic anhydride for reaction to obtain methacrylated hyaluronic acid; mixing the methacrylated hyaluronic acid solution with a compound solution containing a quaternary ammonium salt group or a sulfonate group for reaction to obtain methacrylated hyaluronic acid with positive electricity; the preparation method comprises the following steps: mixing positively charged methacrylated hyaluronic acid with a photoinitiator solution to prepare a water phase; dissolving a surfactant in an organic solvent to obtain an oil phase; curing the water phase and the oil phase through a microfluidic device to obtain hyaluronic acid gel microspheres; the hyaluronic acid gel microspheres are used as carriers to load radiopharmaceuticals, and the hyaluronic acid gel microspheres loaded with the radiopharmaceuticals are obtained. The preparation method of the hyaluronic acid gel microspheres loaded with the radiopharmaceuticals is simple, and the adsorption rate is gt; and after the treatment is finished, the radioactive gel microspheres can also be degraded.

Description

Hyaluronic acid gel microspheres loaded with radiopharmaceuticals, their preparation methods and applications Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to hyaluronic acid gel microspheres loaded with radiopharmaceuticals, their preparation methods, and applications. Background Technology

[0002] Currently, liver cancer ranks 6th in incidence and 4th in mortality worldwide. It is predicted that by 2025, more than one million people will be diagnosed with liver cancer annually, with hepatocellular carcinoma accounting for 90% of all liver cancer cases. Furthermore, the liver is also the most common site of metastasis for malignant tumors, accounting for approximately 25% of all malignant tumors, with colorectal cancer being the most common. Intra-arterial brachytherapy (IAB) or transarterial radioembolization (TARE) using radioactive microspheres is a promising form of intravascular brachytherapy for treating malignant solid tumors. The goal of IAB is to deliver a high dose of radionuclide to the tumor while avoiding radiation damage to surrounding normal tissues. The liver has a dual blood supply, with approximately 95% of the blood supply to liver cancer coming from the hepatic artery. Therefore, applying embolization therapy, blocking the hepatic artery supplying the liver tumor, causes ischemic necrosis of the tumor tissue. Combining embolization with radionuclide therapy can further kill tumor tissue. Transarterial radioembolization (TARE) involves injecting radiolabeled or radiolabeled microspheres into the liver lesion via the hepatic artery. The radiolabeled microspheres remain at the terminal blood vessels of the tumor, emitting a high dose of high-energy beta rays. Simultaneously, the loaded drug is slowly released through the microspheres. This achieves the goal of killing tumor tissue while reducing the toxicity of the loaded drug. Due to the limited tissue penetration depth of the rays, it can also effectively protect nearby healthy liver tissue, thereby improving the objective response rate. It has become an effective palliative therapy for liver cancer.

[0003] Currently, there are three commercially available radioactive microspheres in clinical use, namely those from the United States. 90 Y-glass microspheres TheraSphere™, Australia 90 Y-resin microspheres SIR-Spheres® and the Netherlands 166Ho-type polylactic acid microspheres (QuiremSpheres®) exhibit significantly different physical properties due to variations in the selected radionuclides and carriers. However, they generally suffer from non-biodegradability and can only be labeled with a single radionuclide. If the embolized vessel can be reopened after treatment, not only can blood flow be restored, but a second radioembolization treatment can also be performed if necessary. To address this, some radioactive microspheres utilize biodegradable biomaterials, such as polylactic-glycolic acid copolymer (PLGA) microspheres, polylactic acid (PLA) microspheres, and human serum albumin (HAS) microspheres. However, their degradation rate in vivo cannot be well controlled; they either remain undegraded for extended periods or degrade too rapidly, resulting in excessive radionuclide leaching.

[0004] Hyaluronic acid (HA), also known as hyaluronic acid, is a large-molecule acidic mucopolysaccharide and an important component of the intercellular matrix of skin cells, possessing a variety of important physiological functions. Hyaluronic acid can diffuse and degrade between tissues. Through chemical modification or cross-linking, its unique biocompatibility and degradability make it suitable for use as a drug sustained-release and controlled-release carrier. Current reports on HA radioactive microspheres all involve directly labeling the microspheres with radionuclides. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides hyaluronic acid gel microspheres loaded with radiopharmaceuticals, their preparation method, and applications. The purpose of this invention is to provide a method for preparing gel microspheres capable of loading targeted radiopharmaceuticals and their application in arterial embolization therapy for tumors. This allows for the reduction of the dosage of small molecule radionuclides, peptide radionuclides, antibodies, or protein radionuclides during treatment through the sustained-release function of the microspheres, thereby minimizing toxicity. Furthermore, the gel microspheres degrade after treatment.

[0006] This invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide a method for preparing hyaluronic acid gel microspheres loaded with radiopharmaceuticals, comprising the following steps:

[0008] (1) Hyaluronic acid solution was mixed with methacrylic anhydride and reacted to obtain methacrylated hyaluronic acid;

[0009] (2) The obtained methacrylamide hyaluronic acid solution is mixed and reacted with a compound containing quaternary ammonium salt group or sulfonate group to obtain positively charged methacrylamide hyaluronic acid;

[0010] (3) The positively charged methacrylamide hyaluronic acid obtained is mixed with a photoinitiator solution to prepare an aqueous phase;

[0011] (4) Dissolve the surfactant in an organic solvent to obtain the oil phase;

[0012] (5) The obtained aqueous phase and the obtained oil phase are passed through a microfluidic device to solidify the obtained microdroplets to obtain hyaluronic acid gel microspheres;

[0013] (6) Using the obtained hyaluronic acid gel microspheres as a carrier, a radiopharmaceutical is loaded to obtain the radiopharmaceutical loaded hyaluronic acid gel microspheres.

[0014] In one embodiment of the present invention, in step (1), the mass concentration of the hyaluronic acid solution is 0.8~1.2%;

[0015] And / or, the molecular weight of the hyaluronic acid is 200,000 to 2,500,000;

[0016] And / or, the volume ratio of the hyaluronic acid solution to the methacrylic anhydride is 80~120:2~3;

[0017] And / or, the pH of the reaction is 8-10;

[0018] And / or, the reaction is carried out at a temperature of -4 to 0°C for a time of 24 to 72 h.

[0019] In one embodiment of the present invention, in step (2), the mass concentration of the methacrylated hyaluronic acid solution is 4-6%;

[0020] And / or, the compound containing quaternary ammonium salt or sulfonate groups is diallyl dimethyl ammonium chloride (DADMAC), polydiallyl dimethyl ammonium chloride (Poly(DADM AC) / PDMDAAC), methacryloyloxyethyl trimethyl ammonium chloride (DMC), polymethacryloyloxyethyl trimethyl ammonium chloride (Poly(AET AC) / PQ-6), dimethyl diallyl ammonium chloride, polydimethyl diallyl ammonium chloride, acryloyloxyethyl trimethyl ammonium chloride, polyacryloyloxyethyl trimethyl ammonium chloride (Poly(AET AC) / PQ-5), acrylamide and acryloyloxyethyl trimethyl ammonium chloride copolymer (P(AM- AETAC) / CPAM), acrylamide and methacryloyloxyethyltrimethylammonium chloride copolymer (P(AM- ME TAC), polyquaternium-10 (PQ-1), polyquaternium-6,-11 (PQ-6,-11), poly(4-vinylpyridine) quaternium, polyquaternium-10 (PQ-10 / JR400), polyquaternium-7 (PQ-7), polyquaternium-22 (PQ-22), polyquaternium-47 (PQ-47 / Merquat), cationic guar gum (C-Guar / Guar Hydroxypropyltrimonium Chloride), cationic precipitate powder (C-Starch), chitosan quaternary ammonium salt (Quaternary) Chitosan, poly(4-vinylpyridine) quaternary ammonium salt (Q-PVP), polyvinylbenzyltrimethylammonium chloride, epichlorohydrin-dimethylamine copolymer, quaternized polyethyleneimine (PEI), quaternized siloxane polymer, quaternized chitosan, quaternized starch, sodium polystyrene sulfonate (PSS or PSSNa), poly(2-acrylamide-2-methylpropanesulfonic acid) (PAMPS), polypropylene sulfonic acid (PAPS), perfluorosulfonic acid membrane (nafion), sulfonated polyether ether ketone (SPEEK), sulfonated polyphenylene sulfide (SPPS), sodium dodecylbenzene sulfonate (SDBS or LAS), sodium dodecyl sulfate (SDS or SLES), sodium α-olefin sulfonate (ASO), methyl orange, Congo red, celecoxib, p-toluenesulfonic acid (PTSA), heparin, 3,4-ethylenedioxythiophene, poly(3,4-ethylenedioxythiophene), polystyrene sulfonate, styrene sulfonate, or one or more of the following:

[0021] In one embodiment of the present invention, in step (2), the molar ratio of the methacrylamide hyaluronic acid to the compound containing quaternary ammonium groups or sulfonate groups is 1:5-50, preferably 1:10;

[0022] And / or, the reaction is carried out at a temperature of 37-40°C for a time of 12-72 h.

[0023] In one embodiment of the present invention, after the reaction in steps (1) and (2) is completed, dialysis and freeze-drying are performed; the molecular weight of the dialysis is 8000~14000; the dialysis time is 2~4 days; the freeze-drying temperature is -45~-60℃ and the time is 5~9 days.

[0024] In one embodiment of the present invention, in step (3), the volume concentration of the photoinitiator solution is 0.25~1%;

[0025] And / or, the photoinitiator is 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), ethyl 2,4,6-trimethylbenzoylphosphonate (TPO-L), bis(2,4,6-trimethylbenzoyl)diphenylphosphine oxide (BAPO), a mixture of 2,4,6-trimethylbenzophenone and methylbenzophenone (TZT), 4-phenylbenzophenone (PBZ), phenyl dimethoxyacetophenone (BDK), isooctyl p-dimethylaminobenzoate (EHA), methyl o-benzoylbenzoate (OMBB), 4-benzoyl-4'-methyl-diphenyl sulfide (BMS), benzophenone (BP), polybutylene glycol 250 di-(2-carboxymethoxybenzophenone) ester (OmnipolBP), polybutylene glycol 250 di-(2-carboxymethoxythioxanthone) ester (OmnipolBP). TX), 1-hydroxycyclohexylphenyl ketone (photoinitiator 184), 2-benzyl-2-dimethylamino-1-(4-morpholinophenyl)butanone (photoinitiator 369), 2-dimethylamino-2-(4-methyl)benzyl-1-[4-(4-morpholino)phenyl]-1-butanone (photoinitiator 379), 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (photoinitiator 659), bis(2,6-difluoro-3-pyrrolephenyl)dicenoctanetane (photoinitiator 784), bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide (photoinitiator 819), 2-methyl-1-[4-(methylthio)phenyl]-2 One or more of the following: morpholinyl-1-propanone (photoinitiator 907), 2-dimethylamino-2-benzyl-1-[4-(4-morpholinyl)phenyl]-1-butanone (photoinitiator 910), 2-hydroxy-2-methyl-1-phenylpropanone (photoinitiator 1173), 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (photoinitiator 2959), lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP), thioxanthone (TX), 2-isopropylthioxanthone (ITX), 2,4-diethylthiazolinone (DETX), 4-methylbenzophenone (MBF), methyl benzoylformate (Darocur MBF), bifunctional ketone sulfone (photoinitiator 1001M), ethyl 4-dimethylaminobenzoate (EDB), and 2,2-dimethoxy-2-phenylacetophenone;

[0026] And / or, the mass concentration of positively charged methacrylamide hyaluronic acid in the aqueous phase is 5-10%.

[0027] In one embodiment of the present invention, in step (4), the oil phase is an oily solution of surfactant Span 80 with a volume concentration of 8-12%;

[0028] And / or, the organic solvent is n-octanol or liquid paraffin.

[0029] In one embodiment of the present invention, in step (5), the chip through which the aqueous phase passes in the microfluidic device is PDMS-50 or PDMS-100;

[0030] And / or, the flow rate of the aqueous phase is 150-9000 μL / h;

[0031] And / or, the flow rate of the oil phase is 200~45000 μL / h.

[0032] In one embodiment of the present invention, in step (6), the particle size of the hyaluronic acid gel microspheres is 38~60 μm;

[0033] And / or, the radiopharmaceutical is a small molecule radiopharmaceutical, a peptide radiopharmaceutical, an antibody or a protein radiopharmaceutical; specifically, the radiopharmaceutical includes one or more of the following: small molecules targeting PSMA, small molecules targeting FAP, small molecule analogs targeting SSTR, small molecule analogs containing RGD sequences, PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, HER2 antibodies, CD20 antibodies, VEGF, EGFR antibodies, TNF-α antibodies, IL-17A antibodies, IL-12 / 23 antibodies, IL-4 / 13 antibodies, CD80 / 86 antibodies, IgE antibodies, and small molecule or peptide drugs containing proteins (such as tyrosine);

[0034] And / or, the radionuclides in the radiopharmaceutical include 18 F, 47 Sc、 66 Ga、 67 Ga、 68 Ga、 99m Tc, 64 Cu、 67 Gu、 89 Zr、 86 Y、 90 Y、 109 Pd, 111 In、 131 I, 125 I, 153 Sm、 166 Ho、 177 Lu、 186 Re、 188 Re、 89 Sr、 149 Tb, 161 Tb, 212 Pb, 212 Bi、 213 Bi、 225 Ac、 227 Ac、 223Ra、 226 Th and 227 One or more of Th.

[0035] A second objective of this invention is to provide hyaluronic acid gel microspheres loaded with radiopharmaceuticals obtained by the aforementioned preparation method.

[0036] A third objective of this invention is to provide the application of the aforementioned hyaluronic acid gel microspheres loaded with radiopharmaceuticals in the preparation of tumor embolization therapeutic drugs;

[0037] The tumors include lung cancer, stomach cancer, esophageal cancer, liver cancer, colorectal cancer, breast cancer, cervical cancer, pancreatic cancer, thyroid cancer, lymphoma, bladder cancer, kidney cancer, endometrial cancer, prostate cancer, ovarian cancer, skin cancer, nasopharyngeal cancer, gallbladder cancer, lip and oral cancer, laryngeal cancer, testicular cancer, osteosarcoma, or chondrosarcoma.

[0038] Existing technologies often employ mechanical stirring to prepare microspheres, which makes size control difficult and results in inconsistent microsphere sizes. This invention utilizes microfluidic microsphere preparation, setting the aqueous and oil phases to a fixed flow rate, then shearing them through a chip channel of fixed size to obtain uniform spherical droplets, which are then cured using a UV lamp to obtain microspheres. Because the flow rates of the aqueous and oil phases and the chip size are fixed values, the microspheres are less susceptible to fluctuations from other factors during the microsphere planting process, thus resulting in microspheres with more uniform particle size and morphology.

[0039] Most commercially available radiopharmaceutical-loaded microspheres chelate radionuclides using chelating agents, which couple the radionuclides to the radionuclide. This invention, however, uses physical adsorption to adsorb targeted small-molecule radiopharmaceuticals, peptide radiopharmaceuticals, and antibody radiopharmaceuticals. This invention uses compounds containing quaternary ammonium salts or sulfonate groups, such as methacryloyloxyethyltrimethylammonium chloride, as materials introduced into the microsphere synthesis. The introduction of quaternary ammonium salt or sulfonate groups allows for the synthesis of a positively charged microsphere, enabling the loading of radiopharmaceuticals via potentiometric adsorption. This loading method simplifies and facilitates drug delivery, and normal in vivo conditions promote the release of the loaded drug, resulting in higher biosafety. Furthermore, the gel microspheres of this invention can load a variety of radiopharmaceuticals compared to other microspheres. The microspheres of this invention can directly adsorb radiopharmaceuticals for tumor treatment and can release them within the tumor, resulting in better tumor cell killing.

[0040] The gel microspheres of this invention can deliver targeted radiopharmaceuticals into tumors to kill tumor cells, achieving better therapeutic effects. Gel microspheres loaded with small-molecule radiopharmaceuticals, peptide radiopharmaceuticals, and antibody radiopharmaceuticals are used to embolize blood vessels near the tumor. While the radionuclides kill cells through radiation, the radiopharmaceuticals released from the microspheres target antigens on the tumor surface, killing tumor cells at closer range. This treatment method reduces damage to normal tissues from radionuclides while maintaining the efficiency of targeted tumor therapy.

[0041] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0042] This invention provides hyaluronic acid gel microspheres loaded with radiopharmaceuticals, their preparation method, and applications. The radiotargeting gel microspheres of this invention can degrade after treatment, thereby restoring embolized blood vessels. Furthermore, the gel microspheres are prepared using biocompatible and biodegradable materials such as hyaluronic acid, exhibiting excellent safety. The preparation method of the radiotargeting gel microspheres of this invention is simple, and the conditions for loading small molecule radionuclides, polypeptide radionuclides, antibodies, and protein radionuclides are also easy to implement, achieving a drug loading rate >85%. Attached Figure Description

[0043] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0044] Figure 1 is the Fourier transform infrared spectrum of methacrylamide hyaluronic acid with quaternary ammonium salt groups prepared in Example 1; where a is the characteristic peak of the quaternary ammonium salt group;

[0045] Figure 2 is an optical microscope image of the hyaluronic acid gel microspheres prepared in Example 1;

[0046] Figure 3 shows the particle size distribution of the hyaluronic acid gel microspheres prepared in Example 1;

[0047] Figure 4 shows the microsphere loading in Example 1. 177 Drug loading rate of Lu-labeled PSMA-617;

[0048] Figure 5 shows the microsphere loading in Example 1. 177 Release rate of Lu-labeled PSMA-617;

[0049] Figure 6 shows an optical microscope image of the hyaluronic acid gel microspheres prepared in Comparative Example 1;

[0050] Figure 7 shows the microsphere loading in Example 2. 177 Drug loading rate of Lu-labeled SSTRs;

[0051] Figure 8 shows the microsphere loading in Example 2. 177 Release rate of Lu-labeled SSTRs;

[0052] Figure 9 shows the microsphere loading in Example 3. 131 Drug loading rate of I-labeled PD-L1;

[0053] Figure 10 shows the small molecule radiopharmaceutical loaded in Example 4. 177 Tumor change curves in mice treated with Lu-PSMA-617 microspheres;

[0054] Figure 11 shows the small molecule radiopharmaceutical loaded in Example 4 ( 177 Photographs of tumors in mice treated with Lu-PSMA-617 microspheres. Detailed Implementation

[0055] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0056] This invention provides a method for preparing radioactive gel microspheres, comprising the following preparation steps:

[0057] (1) Aqueous solution of hyaluronic acid is mixed with methacrylic anhydride and reacted to obtain methacrylated hyaluronic acid;

[0058] (2) The aqueous solution of the obtained methacrylamide hyaluronic acid is mixed with an aqueous solution of a compound containing quaternary ammonium salt groups or sulfonate groups to obtain positively charged methacrylamide hyaluronic acid;

[0059] (3) The positively charged methacrylamide hyaluronic acid obtained is mixed with a phosphate solution of photoinitiator to prepare an aqueous phase;

[0060] (4) Dissolve the surfactant in an organic solvent to obtain the oil phase;

[0061] (5) The obtained aqueous phase and the obtained oil phase are passed through a microfluidic device to solidify the obtained microdroplets to obtain hyaluronic acid gel microspheres;

[0062] (6) Using the obtained hyaluronic acid gel microspheres as a carrier, a radiopharmaceutical is loaded onto the microspheres to obtain the radiopharmaceutical-loaded hyaluronic acid gel microspheres. The radiopharmaceutical is a small molecule radiopharmaceutical, a polypeptide radiopharmaceutical, an antibody radiopharmaceutical, or a protein radiopharmaceutical.

[0063] In this invention, the mass concentration of the hyaluronic acid solution is preferably 0.8-1.2%, and more preferably 1%.

[0064] In this invention, the molecular weight of hyaluronic acid is preferably 200,000 to 2,500,000.

[0065] In this invention, the volume ratio of the hyaluronic acid solution to the methacrylic anhydride mixture is preferably 80-120:2-3, and more preferably 100:2.5.

[0066] In this invention, the pH of the reaction is preferably 8 to 10, and more preferably 9.

[0067] In this invention, the pH is preferably adjusted using a 5M NaOH solution.

[0068] In this invention, the reaction temperature is preferably -4 to 0°C, and more preferably 0°C.

[0069] In this invention, the temperature required for the reaction is preferably provided by an ice bath.

[0070] In this invention, the reaction time is preferably 24-72 h, more preferably 36-60 h, and even more preferably 48 h.

[0071] In this invention, after the reaction is complete, it is preferable to dialyze and freeze-dry the solution containing methacrylamide hyaluronic acid.

[0072] In this invention, the permeable molecular weight of the dialysis is preferably 8,000 to 14,000, more preferably 9,000 to 12,000, and even more preferably 10,000.

[0073] In this invention, the dialysis time is preferably 2 to 4 days, and more preferably 3 days.

[0074] In this invention, the freeze-drying temperature is preferably -45 to -60°C, and more preferably -55°C.

[0075] In this invention, the freeze-drying time is preferably 5 to 9 days, and more preferably 7 days.

[0076] In this invention, the methacrylamide hyaluronic acid after freeze-drying is a white, spongy substance.

[0077] After preparing methacrylamide hyaluronic acid, a solution is prepared. The methacrylamide hyaluronic acid solution is mixed with a solution containing quaternary ammonium salt or sulfonate, and a positively charged methacrylamide hyaluronic acid is generated by the reaction.

[0078] In this invention, the mass concentration of the methacrylamide hyaluronic acid solution is preferably 4-6%, and more preferably 5%.

[0079] In this invention, the mass concentration of the solution containing quaternary ammonium salt or sulfonate groups is preferably 0.3-1.2%, more preferably 0.3-0.8%, and even more preferably 0.6%.

[0080] In this invention, the compound containing quaternary ammonium salt or sulfonate group is one or more of acryloyloxyethyltrimethylammonium chloride, dimethyl diallyl ammonium chloride, acrylamidopropyltrimethylammonium chloride, 2-(N,N-dimethylamino)ethyl methacrylate, acrylamide, ammonium acryloyldimethyl taurate, sodium 2-acrylamido-2-methylpropanesulfonate, methacrylamide, acrylic acid, methacrylic acid, N,N-dimethylacrylamide, 2-acrylamido-2-ethylpropanesulfonic acid, and 2-methacrylamido-2-methylpropanesulfonic acid, and is more preferably methacryloyloxyethyltrimethylammonium chloride.

[0081] In this invention, methacryloyloxyethyltrimethylammonium chloride can also be replaced with other cationic compounds that have the same effect as methacryloyloxyethyltrimethylammonium chloride.

[0082] In this invention, the steps further include mixing the methacrylamide hyaluronic acid solution with the solution containing quaternary ammonium salt or sulfonate.

[0083] In this invention, when the methacrylamide hyaluronic acid solution is mixed with a compound solution containing quaternary ammonium salt or sulfonate groups, it is preferable to add the compound solution containing quaternary ammonium salt or sulfonate groups dropwise to the methacrylamide hyaluronic acid solution for mixing.

[0084] In this invention, the reaction temperature is preferably 37~40℃, and more preferably 38℃.

[0085] In this invention, the reaction time is preferably 12-72 h, more preferably 24-60 h, and even more preferably 48 h.

[0086] In this invention, after the reaction is complete, it is preferable to dialyze and freeze-dry the solution of positively charged methacrylated hyaluronic acid.

[0087] In this invention, the permeable molecular weight of the dialysis is preferably 8,000 to 14,000, more preferably 9,000 to 12,000, and even more preferably 10,000.

[0088] In this invention, the dialysis time is preferably 2 to 4 days, and more preferably 3 days.

[0089] In this invention, the freeze-drying temperature is preferably -45 to -60°C, and more preferably -55°C.

[0090] In this invention, the freeze-drying time is preferably 5 to 9 days, and more preferably 7 days.

[0091] In this invention, the positively charged methacrylamide hyaluronic acid after freeze-drying is a white, spongy substance.

[0092] After obtaining positively charged methacrylamide hyaluronic acid, it was mixed with a photoinitiator solution to form an aqueous phase.

[0093] In this invention, the volume concentration of the photoinitiator solution is preferably 0.25-1%, and more preferably 0.5%.

[0094] In this invention, the photoinitiator is preferably lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) or photoinitiator 2959, and more preferably lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP).

[0095] In this invention, the temperature at which positively charged methacrylamide hyaluronic acid is mixed with the photoinitiator solution is preferably 40-50°C, and more preferably 45°C.

[0096] In this invention, the mass concentration of positively charged methacrylamide hyaluronic acid in the aqueous phase is preferably 5-10%, more preferably 10%.

[0097] After obtaining the aqueous phase, the aqueous phase and the oil phase are processed through a microfluidic device to obtain hyaluronic acid gel microspheres.

[0098] In this invention, the oil phase is preferably an oily solution of surfactant Span 80 with a volume concentration of 8-12%, and more preferably an oily solution of surfactant Span 80 with a volume concentration of 10%.

[0099] In this invention, the solvent of the oily solution is preferably n-octanol or liquid paraffin.

[0100] In this invention, the microfluidic chip used is PDMS-50 to 200 in size, with an aqueous phase flow rate of 150-9000 μL / h and an oil phase flow rate of 200-45000 μL / h. More preferably, the chip size is PDMS-100, with an aqueous phase flow rate of 250 μL / h and an oil phase flow rate of 340 μL / h.

[0101] In this invention, after the aqueous phase is sheared into microdroplets by the oil phase, it is preferably irradiated with ultraviolet light for 1 to 60 minutes to solidify the droplets into microspheres, more preferably for 5 to 30 minutes, and even more preferably for 6 minutes.

[0102] In this invention, after irradiation, a cleaning step is also included. During cleaning, if n-butanol is chosen as the solvent for the oily solution, anhydrous ethanol is preferred; if liquid paraffin is chosen as the solvent for the oily solution, isopropanol is preferred. Then, all components are rinsed with clean water.

[0103] In this invention, after the cleaning process, the steps of sieving, dehydration, and drying are also included.

[0104] In this invention, the particle size of the sieve is preferably 38~60 μm; the dehydration is preferably carried out with anhydrous ethanol; and the drying is preferably carried out with nitrogen until powdered hyaluronic acid gel microspheres are obtained.

[0105] The prepared hyaluronic acid gel microspheres were used as carriers to load small molecule radionuclides, polypeptide radionuclides, antibodies and protein radionuclides to obtain radioactive gel microspheres.

[0106] In this invention, the radionuclide is preferably... 18 F, 47 Sc、 66 Ga、 67 Ga、 68 Ga、 99m Tc, 64 Cu、 67 Gu、 89 Zr、 86 Y、 90 Y、 109 Pd, 111 In、 131 I, 125 I, 153 Sm、 166 Ho、 177 Lu、 186 Re、 188 Re、 89 Sr、 149 Tb, 161 Tb, 212 Pb, 212 Bi、 213 Bi、 225 Ac、 227 Ac、 223 Ra、 226 Th and 227 One or more of Th.

[0107] In this invention, the drug loaded with hyaluronic acid is preferably one or more of the following: small molecules targeting PSMA, small molecules targeting FAP, small molecule analogs targeting SSTR, small molecule analogs containing RGD sequences, PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, HER2 antibodies, CD20 antibodies, VEGF, EGFR antibodies, TNF-α antibodies, IL-17A antibodies, IL-12 / 23 antibodies, IL-4 / 13 antibodies, CD80 / 86 antibodies, IgE antibodies, and small molecules or peptides containing proteins (such as tyrosine).

[0108] In this invention, the activity of the radionuclide is preferably 1 × 10⁻⁶. -6 ~10 Ci.

[0109] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0110] The CAS numbers for the following products are: Hyaluronic acid: 37326-33-3; Methacrylamide: 760-93-0; Lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP): 85073-19-4; Surfactant Span 80: 1338-43-8; Methacryloxyethyltrimethylammonium chloride: 5039-78-1. All products were purchased from Shanghai Titan Technology Co., Ltd.; Black rat C57 was purchased from Changzhou Cavens Laboratory Animal Co., Ltd.

[0111] Example 1

[0112] This embodiment provides a method for preparing hyaluronic acid gel microspheres loaded with radiopharmaceuticals, as detailed below:

[0113] The hyaluronic acid aqueous solution was first purified using a cation exchange resin, i.e., the hyaluronic acid raw material was purified. 1 g of purified hyaluronic acid (MW 20~2.5 million) was weighed and added to 100 mL of ultrapure water, and stirred thoroughly to dissolve, obtaining a hyaluronic acid solution. The hyaluronic acid solution was placed on a magnetic stirrer at 150 rpm in an ice bath at 0°C, and 2.5 mL of methacrylic anhydride (MA) was added dropwise. The pH was adjusted to 9 with 5 M NaOH, and the reaction was carried out in an ice bath at 0°C for 48 hours. Dialysis was performed in ultrapure water for 3 days using a dialysis bag (MW 10000), followed by freeze-drying at -55°C for one week to obtain white, spongy methacryloyl hyaluronic acid (HAMA).

[0114] Weigh 1 g of HAMA and add it to 100 mL of ultrapure water. Stir thoroughly to dissolve and obtain HAMA solution. Add 6 mL of methacryloyloxyethyltrimethylammonium chloride to the solution and react for 48 hours. Dialyze the solution in ultrapure water for 3 days using a dialysis bag (MW 10000). Then freeze-dry the solution at -55℃ for one week to obtain white, spongy, positively charged methacryloyl hyaluronic acid (HAMA-DMC) (Fourier transform infrared spectrum shown in Figure 1).

[0115] Weigh 50 mg of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) and dissolve it in 10 mL of phosphate solution to prepare a 0.5% (w / v) photoinitiator LAP solution. Weigh 200 mg of HAMA-DMC and add it to 2 mL of 0.5% (w / v) LAP solution. Place the solution in a 45°C water bath and shake intermittently until completely dissolved to obtain a 10% (w / v) HAMA-DMC gel solution. This solution should be used as the aqueous phase and stored in the dark.

[0116] Add 4 mL of surfactant Span 80 to 40 mL of n-octanol and mix thoroughly to obtain the oil phase. Use a 10 mL syringe to draw 5 mL of the oil phase and a 5 mL syringe to draw 2 mL of the aqueous phase. Place both syringes on a dual-channel syringe pump using a PDMS chip (PDMS-50). Set the flow rate of the aqueous phase to 250 μL / h and the flow rate of the oil phase to 340 μL / h. After the aqueous phase is sheared into microdroplets by the oil phase, irradiate the reaction solution with UV light for 6 min to solidify the droplets into microspheres. Collect the microspheres after solid-liquid separation, and repeatedly wash them with anhydrous ethanol to remove the oil phase from the surface. Then wash them again with ultrapure water and collect the microspheres again to obtain gel microspheres with a particle size range of 38 µm to 60 µm. Dehydrate the microspheres with anhydrous ethanol and then dry them with nitrogen to obtain powdered dried microspheres, which are the hyaluronic acid gel microspheres. The optical microscope image is shown in Figure 2, and the particle size distribution is shown in Figure 3.

[0117] After preparing hyaluronic acid gel microspheres, the positive charge on the microspheres was used to adsorb radiolabeled small molecules, peptides, antibodies, and proteins, thus obtaining radiotargeting gel microspheres.

[0118] Preparation of adsorption 177 Lu-labeled PSMA-617 gel microspheres were used to investigate the effect of time on adsorption rate.

[0119] Weigh 1 mg of hyaluronic acid gel microspheres and dissolve them in 20 µL of ultrapure water to prepare a hyaluronic acid gel microsphere solution for later use; add 0.2 mCi 177Lu solution was placed in a 1.5 mL EP tube, followed by 2 μL of 1 mg / mL PSMA-617 solution, and then 0.4 mol sodium acetate buffer solution. The total reaction volume was 50 μL. The entire reaction system was placed in a metal bath and reacted at 95 °C, 800 rpm for 30 min to obtain... 177 Lu-PSMA-617, using EDTA as the developing solvent, was analyzed by TLC. 177 The labeling efficiency of Lu-PSMA-617. The above hyaluronic acid gel microspheres were labeled with a efficiency greater than 90%. 177 The Lu-PSMA-617 solution was divided into four equal parts. Four parts of hyaluronic acid gel microspheres were added to four parts of the solution respectively. 177 In Lu-PSMA-617 solution, four portions of the mixed solution were shaken for 0 min, 20 min, 40 min, and 60 min, respectively. After the reaction was completed, centrifugation and solid-liquid separation were performed. The activity of the hyaluronic acid microspheres and the supernatant was measured to obtain positively charged hyaluronic acid gel microspheres. 177 The adsorption rate of Lu-PSMA-617 is shown in Figure 4. The change in adsorption rate with reaction time is illustrated in Figure 4. As can be seen from Figure 4, the adsorption rate does not change with increasing reaction time, and the highest adsorption rate reaches 88% in the first reaction time.

[0120] Radioactive-targeting gel microspheres were taken at any of the above times and soaked in phosphate-buffered saline (PBS, pH 7.2). Solid-liquid separation was performed at 1 h, 2 h, 4 h, 8 h, and 24 h, respectively. The radioactivity of the microspheres was measured using a gamma radioimmunoassay counter, as shown in Figure 5. The release rate of the radioactive-targeting gel microspheres within 48 hours was approximately 30%.

[0121] Comparative Example 1

[0122] This comparative example provides a method for preparing hyaluronic acid gel microspheres, as detailed below:

[0123] The aqueous and oil phases were prepared according to the method in Example 1. The aqueous phase was drawn up using a 1 mL syringe, and 40 mL of the oil phase was placed in a round-bottom flask. The flask was then placed on a magnetic stirrer (the speed of the oil phase was controlled by adjusting the stirrer's rotation speed). Microspheres were prepared by mechanical stirring, with the aqueous phase flowing at a rate of 250 μL / min and the stirrer rotating at 750 rpm, pushing the aqueous phase into the oil phase. After the aqueous phase was complete, polymerization was carried out under UV light to obtain hyaluronic acid gel microspheres.

[0124] The optical microscope image of the obtained hyaluronic acid gel microspheres is shown in Figure 6. As can be seen from Figures 2 and 6, compared with mechanical stirring, the hyaluronic acid microspheres prepared by microfluidic control are better controlled in size, more uniform, and have more complete morphology.

[0125] Example 2

[0126] This embodiment provides a method for preparing hyaluronic acid gel microspheres loaded with radiopharmaceuticals. Similar to Example 1, the only difference is that the oil phase solvent, n-butanol, is replaced with liquid paraffin, and the microspheres are washed with isopropanol to obtain hyaluronic acid gel microspheres. These hyaluronic acid gel microspheres are then used as a carrier for loading... 177 Lu-labeled octreotide.

[0127] Investigating the effect of reaction time on the effect of hyaluronic acid gel microspheres 177 The effect of Lu-SSRT adsorption rate. 1 mg of hyaluronic acid gel microspheres was dissolved in 20 µL of ultrapure water to prepare a hyaluronic acid gel microsphere solution for later use; 0.2 mCi of... 177 Lu-SSRT. The above-mentioned hyaluronic acid gel microspheres were labeled with a labeling efficiency greater than 90%. 177 The Lu-SSRT solution was divided into four equal portions. Four portions of hyaluronic acid gel microspheres were added to four portions of... 177 In Lu-SSRT solution, four portions of the mixed solution were shaken for 0 min, 20 min, 40 min, and 60 min, respectively. After the reaction, centrifugation and solid-liquid separation were performed, and the activity of the hyaluronic acid microspheres and supernatant was measured to obtain positively charged hyaluronic acid gel microspheres. 177 The adsorption rate of Lu-SSRT is shown in Figure 7. The change in adsorption rate with reaction time is illustrated in Figure 7. As can be seen from Figure 7, the adsorption rate does not change with increasing reaction time, and the adsorption rate reaches a maximum of 93% in the first reaction time.

[0128] In vitro release of hyaluronic acid gel microspheres: The radiotargeting gel microspheres prepared in the above steps at a reaction temperature of 37℃ and a reaction time of 10 min were soaked in phosphate-buffered saline (PBS, pH 7.2) and 10% fetal bovine serum (FBS), respectively. Solid-liquid separation was performed at 1 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h, respectively. The radioactivity of the microspheres was measured using a gamma radioimmunoassay counter, as shown in Figure 8 (the two curves in Figure 8 overlap). The release rate reached 90% after 7 days.

[0129] Example 3

[0130] Preparation of load 125 I-PD-L1 radioactive gel microspheres: Weigh 1 mg of the hyaluronic acid gel microspheres prepared in Example 1 to prepare a hyaluronic acid gel microsphere solution for later use; take 0.2 mCi 131 I-PD-L1 solution was added to a hyaluronic acid gel microsphere solution, and the mixture was stirred and reacted at 37°C for 10 min. After the reaction, the solid and liquid phases were separated and washed with water to obtain...131 I-PD-L1 radioactive gel microspheres, as shown in Figure 9, were found to have an adsorption rate of 83%.

[0131] Example 4

[0132] The hyaluronic acid gel microspheres prepared in Example 1 were used in a mouse tumor treatment experiment.

[0133] A prostate cancer tumor model with high PSMA expression was constructed subcutaneously in black mice. The mice were divided into four groups: a control group, a group receiving 150 μC, and a group receiving a control group. 177 Lu-PSMA-617 treatment group, hyaluronic acid gel microsphere treatment group (MS), loaded with 150 μCi 177 The treatment group of hyaluronic acid gel microspheres with Lu-PSMA-617 ( 177 Lu-PSMA-617 MS); where 150 μCi 177 Lu-PSMA-617 treatment group, hyaluronic acid gel microsphere treatment group, and group loaded with 150 μCi 177 The Lu-PSMA-617 hyaluronic acid gel microsphere treatment group involved applying 150 μCi of hyaluronic acid gel microspheres. 177 Lu-PSMA-617, hyaluronic acid gel microspheres, loaded with 150 μCi 177 Lu-PSMA-617 hyaluronic acid gel microspheres were injected into mouse tumors. Tumor growth was recorded, and tumors were recorded when any group reached a volume of 1500 mm². 3 All mice in each group were sacrificed, and tumors were removed and photographed. Tumor growth curves were plotted to compare changes in tumor volume and the effectiveness of tumor treatment in each group. The results are shown in Figures 10 and 11. A tumor growth curve of 150 μCi was found to be effective. 177 The Lu-PSMA-617 hyaluronic acid gel microsphere group showed the best therapeutic effect on tumors with high PSMA expression, with significantly smaller tumor volume changes compared to other groups, a phenomenon also confirmed by solid tumor photographs.

[0134] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing hyaluronic acid gel microspheres loaded with radiopharmaceuticals, characterized in that, The process includes the following steps: (1) mixing hyaluronic acid solution with methacrylic anhydride to obtain methacrylated hyaluronic acid; (2) mixing the obtained methacrylated hyaluronic acid solution with a compound containing quaternary ammonium salt groups or sulfonate groups to obtain positively charged methacrylated hyaluronic acid; (3) mixing the obtained positively charged methacrylated hyaluronic acid with a photoinitiator solution to form an aqueous phase; (4) dissolving a surfactant in an organic solvent to obtain an oil phase; (5) using a microfluidic device to solidify the obtained droplets between the obtained aqueous phase and the obtained oil phase to obtain hyaluronic acid gel microspheres; (6) using the obtained hyaluronic acid gel microspheres as a carrier to load a radiopharmaceutical to obtain the radiopharmaceutical-loaded hyaluronic acid gel microspheres.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass concentration of the hyaluronic acid solution is 0.8-1.2%; and / or, the molecular weight of the hyaluronic acid is 200,000-2,500,000; and / or, the volume ratio of the hyaluronic acid solution to methacrylic anhydride is 80-120:2-3; and / or, the pH of the reaction is 8-10; and / or, the temperature of the reaction is -4-0℃ and the time is 24-72 h.

3. The preparation method according to claim 1, characterized in that, In step (2), the mass concentration of the methacrylated hyaluronic acid solution is 4-6%; and / or, the compound containing quaternary ammonium salt or sulfonate group is diallyl dimethyl ammonium chloride, polydiallyl dimethyl ammonium chloride, methacryloyloxyethyl trimethyl ammonium chloride, polymethacryloyloxyethyl trimethyl ammonium chloride, dimethyl diallyl ammonium chloride, polydiallyl dimethyl ammonium chloride, acryloyloxyethyl trimethyl ammonium chloride, polyacryloyloxyethyl trimethyl ammonium chloride, acrylamide and acryloyloxyethyl trimethyl ammonium chloride copolymer, acrylamide and methacryloyloxyethyl trimethyl ammonium chloride copolymer, polyquaternium salt-10, polyquaternium salt-6,-11, poly(4-vinylpyridine) quaternary ammonium salt, polyquaternium salt-10, polyquaternium salt-7, poly Quaternary ammonium salt-22, polyquaternary ammonium salt-47, cationic guar gum, cationic precipitate powder, chitosan quaternary ammonium salt, poly(4-vinylpyridine) quaternary ammonium salt, polyvinylbenzyltrimethylammonium chloride, epichlorohydrin-dimethylamine copolymer, quaternized polyethyleneimine, quaternized siloxane polymer, quaternized chitosan, quaternary ammonium starch, sodium polystyrene sulfonate, poly2-acrylamide-2-methylpropanesulfonic acid, polypropylene sulfonic acid, perfluorosulfonic acid membrane, sulfonated polyether ether ketone, sulfonated polyphenylene sulfide, sodium dodecylbenzene sulfonate, sodium dodecyl sulfate, sodium α-olefin sulfonate, methyl orange, Congo red, celecoxib, p-toluenesulfonic acid, heparin, 3,4-ethylenedioxythiophene, poly3,4-ethylenedioxythiophene, polystyrene sulfonate and styrene sulfonate, one or more of these.

4. The preparation method according to claim 1, characterized in that, In step (2), the molar ratio of the methacrylated hyaluronic acid to the compound containing quaternary ammonium groups or sulfonate groups is 1:5-50; and / or, the reaction temperature is 37-40°C and the time is 12-72 h.

5. The preparation method according to claim 1, characterized in that, In step (3), the volume concentration of the photoinitiator solution is 0.25~1%; and / or, the photoinitiator is 2,4,6-trimethylbenzoyl-diphenylphosphine oxide, ethyl 2,4,6-trimethylbenzoylphosphonate, bis(2,4,6-trimethylbenzoyl)diphenylphosphine oxide, a mixture of 2,4,6-trimethylbenzophenone and methyl benzophenone, 4-phenylbenzophenone, phenyl dimethoxyacetophenone, isooctyl p-dimethylaminobenzoate, methyl o-benzoylbenzoate, 4-benzoyl-4'-methyl-diphenyl sulfide, benzophenone, polybutylene glycol 250 di-(2-carboxymethoxybenzophenone) ester, polybutylene glycol 250 di-(2-carboxymethoxybenzophenone) ester, etc. 2-Carboxymethoxythioxanthone ester, 1-hydroxycyclohexylphenyl ketone, 2-benzyl-2-dimethylamino-1-(4-morpholinophenyl)butanone, 2-dimethylamino-2-(4-methyl)benzyl-1-[4-(4-morpholino)phenyl]-1-butanone, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, bis(2,6-difluoro-3-pyrrolophenyldicyclopentadiene, One or more of bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide, 2-methyl-1-[4-(methylthio)phenyl]-2-morpholinyl-1-propanone, 2-dimethylamino-2-benzyl-1-[4-(4-morpholinyl)phenyl]-1-butanone, 2-hydroxy-2-methyl-1-phenylpropanone, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, thioxanthone, 2-isopropylthioxanthone, 2,4-diethylthiazolinone, 4-methylbenzophenone, methyl benzoylformate, difunctional ketone sulfone, ethyl 4-dimethylaminobenzoate, and 2,2-dimethoxy-2-phenylacetophenone; and / or, the mass concentration of positively charged methacrylamide hyaluronic acid in the aqueous phase is 5-10%.

6. The preparation method according to claim 1, characterized in that, In step (4), the oil phase is an oily solution of surfactant Span 80 with a volume concentration of 8-12%; and / or, the organic solvent is n-octanol or liquid paraffin.

7. The preparation method according to claim 1, characterized in that, In step (5), the chip through which the aqueous phase passes in the microfluidic device is PDMS-50 or PDMS-100; and / or, the flow rate of the aqueous phase is 150-9000 μL / h; and / or, the flow rate of the oil phase is 200~45000 μL / h.

8. The preparation method according to claim 1, characterized in that, In step (6), the hyaluronic acid gel microspheres have a particle size of 38-60 μm; and / or, the radiopharmaceutical is a small molecule radiopharmaceutical, a peptide radiopharmaceutical, an antibody radiopharmaceutical, or a protein radiopharmaceutical; and / or, the radionuclide in the radiopharmaceutical includes 18 F, 47 Sc、 66 Ga、 67 Ga、 68 Ga、 99m Tc, 64 Cu、 67 Gu、 89 Zr、 86 Y、 90 Y、 109 Pd, 111 In、 131 I, 125 I, 153 Sm、 166 Ho、 177 Lu、 186 Re、 188 Re、 89 Sr、 149 Tb, 161 Tb, 212 Pb, 212 Bi、 213 Bi、 225 Ac、 227 Ac、 223 Ra、 226 Th and 227 One or more of Th.

9. Hyaluronic acid gel microspheres loaded with radiopharmaceuticals obtained by the preparation method according to any one of claims 1 to 8.

10. The use of the hyaluronic acid gel microspheres loaded with radiopharmaceuticals as described in claim 9 in the preparation of tumor embolization therapeutic drugs; wherein the tumors include lung cancer, gastric cancer, esophageal cancer, liver cancer, colorectal cancer, breast cancer, cervical cancer, pancreatic cancer, thyroid cancer, lymphoma, bladder cancer, kidney cancer, endometrial cancer, prostate cancer, ovarian cancer, skin cancer, nasopharyngeal carcinoma, gallbladder cancer, lip and oral cancer, laryngeal cancer, testicular cancer, osteosarcoma, or chondrosarcoma.

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