A method for the preparation of microspheres for encapsulating radionuclides

Abstract

The present application relates to a kind of microsphere preparation methods for radionuclide, by the mixing self-assembly of metabolizable polymer and polyphenols, form stable microsphere structure in the presence of nuclide ion, realize the firm package of nuclide and daughter nuclide, form the drug carrier of stable structure, can be used for in vivo transport of nuclide ion.The microspheres formed by polymer and polyphenols are loaded with radionuclide, effectively intercept the recoil of nuclide during decay, inhibit the off-target of nuclide;Solve the problem of off-target of nuclide in radioactive targeted drug, improve treatment accuracy and safety, while ensuring that microsphere is metabolizable.

Description

Technical Field

[0001] This invention belongs to the field of tumor drug preparation technology, and in particular relates to a method for preparing microspheres for encapsulating radionuclides. Background Technology

[0002] Liposomes, as nanoscale drug carriers, are widely used in radiotargeted therapy to deliver radionuclides, such as alpha nuclides (e.g., α-nuclides). 225 Ac、 213 Liposomes, such as those containing alpha radionuclides (B), can simultaneously bind drug-loaded liposomes to targeting ligands, enabling precise tumor treatment. However, during the decay of radioactive alpha nuclides, daughter nuclides are prone to off-target effects due to recoil. Existing technologies, such as US Patent 20180243456A1, describe a method for delivering nuclides using liposomes combined with chelating agents; and the literature "Liposomal Delivery of Radionuclides for Cancer Therapy" (Journal of Nuclear Medicine, 2020) discusses the application of liposomes in radionuclide targeting. The most commonly used loading methods include nanoparticle drug delivery and liposome-based chelating agent capture of nuclides and their decay daughters. Existing technologies mainly employ chelating agents to chelate nuclides, specifically by pre-fixing the chelating agent (such as DOTA) onto or inside the liposome membrane, then introducing radionuclide ions, and achieving fixation of the radionuclide ions through a complexation reaction. Liposomes are aqueous cavities surrounded by a bilayer phospholipid membrane, with the chelating agent and nuclide linked by coordination bonds. The liposomes linked to the radionuclide then bind to the target ligand via a linker and are transported to the relevant tumor lesions via the bloodstream in vivo.

[0003] The drawbacks of the drug-loaded liposomes formed by the above loading methods are that the nuclides and their decay products are prone to off-target effects. Traditional chelating agents cannot effectively resist the decay recoil force, and the daughter nuclides easily escape from the liposomes; this is especially evident with α nuclides, represented by 225Ac and 212Pb, leading to reduced therapeutic precision and increased potential toxicity. Furthermore, it is difficult to precisely control the carrier size and distribution, resulting in non-uniform microsphere size, which affects in vivo distribution and targeting efficiency. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing microspheres for encapsulating radionuclides.

[0005] The technical solution adopted in this invention is: a method for preparing microspheres for encapsulating radionuclides, wherein polymers and polyphenolic substances are mixed with radionuclides and self-assembled to form microspheres encapsulating radionuclides.

[0006] Preferably, the polymer is polyethylene glycol (PEG) or chitosan, with a molecular weight range of 1000-20000 Da; the polyphenolic substance is tannic acid or catechin.

[0007] Preferably, the specific steps are as follows:

[0008] Step 1: Prepare polymer solution and polyphenol solution separately;

[0009] Step 2: Prepare a radionuclide ion solution by mixing the radionuclide ion solution with the polymer solution and performing a complexation reaction.

[0010] Step 3: The polyphenol solution is slowly added dropwise to the polymer solution containing radionuclides. Through the oxidative cross-linking of polyphenols and the hydrogen bonding / electrostatic interaction of polymers, microspheres loaded with radioactive nuclide ions are formed.

[0011] Preferably, the molar ratio of monomer polymer to nuclide is 10-100:1, and the weight ratio of polymer to polyphenol is 1:0.1-1.

[0012] Preferably, the same first solvent is used to prepare the polymer solution, the polyphenol solution, and the radionuclide ion solution; the concentration range of the polymer solution is 1-10 mg / mL, the concentration range of the polyphenol solution is 0.5-5 mg / mL, and the concentration of the radionuclide ion solution is 0.1-1 mCi / mL.

[0013] Preferably, the polymer solution and the polyphenol solution are adjusted to pH 5-7 using sodium hydroxide or hydrochloric acid.

[0014] Microspheres loaded with radionuclides are prepared by a method for preparing microspheres loaded with radionuclides.

[0015] Preferably, the nuclide is an α-nuclide or a β-nuclide;

[0016] Preferred 227 Th、 225 Ac、 223 Ra、 213 Bi、 212 Pb, 188 Re、 177 Lu、 161 Tb, 90 Y、 89 Sr and 67 One or more of Cu.

[0017] Application of microspheres loaded with radionuclides in the preparation of radiotargeted drugs.

[0018] A liposome loaded with a radionuclide, comprising microspheres encapsulating a radionuclide.

[0019] Preferably, phospholipids and cholesterol are weighed in a round-bottom flask, an organic solvent is added, and the mixture is stirred until completely dissolved; the organic solvent is evaporated until a uniform transparent / semi-transparent lipid film is formed; the film is vacuum dried overnight in an oven to remove residual solvent; a buffer solution containing microspheres is added to the dried lipid film, the temperature is controlled at 40-55°C, and the film is dispersed by shaking / stirring to allow the lipid film to hydrate and peel off to form multilayer liposomes (MLVs); after hydration, the suspension is placed at 4°C overnight (to promote membrane stability); the material is extruded through a polycarbonate membrane using a liposome extruder to obtain liposome-loaded microspheres.

[0020] The advantages and positive effects of this invention are as follows: the microspheres formed by polymers and polyphenols effectively intercept radionuclides that recoil during decay, thus inhibiting off-target effects. The polymers provide a metabolizable matrix, while the polyphenols provide reducing and cross-linking functions. The self-assembly of the radionuclides avoids the need for additional chelating agents. Furthermore, the microspheres can be used alone as carriers for radionuclides or for lipid construction, significantly improving the stability of the connection between the radionuclides and liposomes. This solves the problem of off-target effects of radionuclides in radiotargeted drugs, improves treatment precision and safety, and ensures that the microspheres are metabolizable. Attached Figure Description

[0021] Figure 1 Schematic diagram of the preparation process of microspheres loaded with radionuclides;

[0022] Figure 2 Example 1: Scanning electron microscope image of microsphere morphology. Detailed Implementation

[0023] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0024] This invention relates to a method for preparing microspheres for encapsulating radionuclides. Through the self-assembly of a metabolizable polymer and polyphenolic substances, a stable microsphere structure is formed in the presence of radionuclide ions, achieving robust encapsulation of the radionuclide and its daughter nuclides, thus forming a structurally stable drug carrier capable of in vivo transport of radionuclide ions. These microspheres can be used as inclusions in liposomal radiopharmaceuticals or as carriers of radionuclides independently. This method solves the problem of radionuclide off-target effects in radiotargeted drugs, improving therapeutic precision and safety while ensuring the metabolizability of the microspheres.

[0025] The polymers used to prepare the microspheres can be natural or synthetically produced metabolizable polymers, such as polyethylene glycol (PEG) or chitosan, with a molecular weight range of 1000-20000 Da; the polyphenols are tannic acid or catechins; the polymer and polyphenols can form a cavity structure through non-covalent bonds such as van der Waals forces and hydrogen bonds, facilitating the chelation of nuclides. The cavity size can be controlled according to the different polymer monomers, resulting in cavity structures of different sizes suitable for loading different types of nuclides. This microsphere structure can be used for encapsulating various nuclides, such as... 225 Ac and 212 α nuclides, represented by Pb, and 177 Lu, 188 Re and 90 Beta-nuclides, such as Y, are included. Adding nuclides during microsphere formation creates numerous cavities, resulting in a high throughput of nuclides. This encapsulation method offers significant advantages over traditional chelating agents. Furthermore, the multi-layered molecular cross-linking of the microspheres effectively intercepts recoiled nuclides during decay, preventing off-target effects.

[0026] Methods for preparing microspheres loaded with radionuclides, such as Figure 1 As shown, the specific steps include the following:

[0027] Step 1: Prepare polymer solution and polyphenol solution separately; dissolve the selected polymer component in the first solvent at a concentration range of 1-10 mg / mL; dissolve the polyphenol component in the same first solvent at a concentration range of 0.5-5 mg / mL; the first solvent can be deionized water, and use sodium hydroxide or hydrochloric acid as pH adjuster to adjust the pH value of the solution to 5-7, and stir at room temperature (20-30℃) for 30-60 min until completely dissolved.

[0028] Step 2: Prepare a radionuclide ion solution using the first solvent, with a radionuclide ion concentration of 0.1-1 mCi / mL; add the radionuclide ion solution to the polymer solution, with a polymer (based on monomers) to radionuclide molar ratio of 10:1 to 100:1, and stir at room temperature for 10-30 minutes to promote preliminary complexation.

[0029] Step 3: Slowly add the polyphenol solution dropwise to the polymer solution containing the radionuclide, with a polymer to polyphenol weight ratio of 1:0.1 to 1:1. React at room temperature for 0.1-4 hours at a stirring speed of 500-1000 rpm. Microspheres loaded with radioactive nuclide ions are formed through self-assembly via oxidative cross-linking of the polyphenol and hydrogen bonding / electrostatic interactions of the polymer.

[0030] Step 4: After the reaction is complete, centrifuge at 10,000-20,000 g for 10-20 min, or purify the microspheres by ultrafiltration (molecular weight cutoff 100 kDa) to remove unreacted components and obtain microspheres loaded with radionuclides.

[0031] The size of microspheres can be controlled by adjusting factors such as polymer concentration, the ratio of polymer to polyphenols, and polymer molecular weight. This allows for the control of the particle size of nuclide-loaded microspheres within the range of 50-500 nm, ensuring a polydispersity index (PDI) of less than 0.2. Experiments have shown that increasing the polymer solution concentration increases the microsphere size, increasing the polyphenol ratio enhances the crosslinking density, and using higher molecular weight polymers results in microspheres with even larger particle sizes.

[0032] In the microsphere structure, the polymer provides a metabolizable matrix, while the polyphenols provide reducing and cross-linking functions, achieving radionuclide encapsulation through self-assembly and avoiding additional chelating agents. The prepared radionuclide-loaded microspheres exhibit high stability, with in vitro decay experiments showing a daughter radionuclide retention rate >95%. These microspheres possess numerous active phenolic hydroxyl groups, which can be synthesized using Williamson ethers or through haloalkanes to introduce structures such as PEG (polyethylene glycol) or alkyl chains, thereby connecting with targeting ligands and enabling selective binding to tumor cells. Furthermore, the microsphere size can be flexibly controlled by using polymers of different molecular weights, allowing for compatibility with different types of radionuclides and further facilitating liposome loading. Smaller microspheres are beneficial for liposome encapsulation and exhibit higher radionuclide retention rates.

[0033] Microspheres can be combined with liposomes, and the loading of radionuclides can be achieved by loading microspheres with liposomes. Liposomes loaded with microspheres can be prepared by thin-film hydration method. During the hydration process, buffer solution containing microspheres is added, and the loading of microspheres is achieved during the liposome forming process.

[0034] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0035] Example 1:

[0036] Dissolve PEG with a molecular weight of 5000 Da in deionized water to prepare 10 ml of a 5 mg / mL solution, and adjust the pH to 6; add [the following ingredient is missing from the original text] 2250.5 ml of Ac ion 0.5 mCi / mL solution was stirred for 20 minutes; then 10 ml of 2 mg / mL tannic acid solution (PEG:tannic acid = 1:0.4) was added dropwise, and the mixture was stirred at room temperature for 1 hour to form microspheres; after purification, the microspheres were obtained and tested. Dynamic light scattering (DLS) showed that the average particle size was 150 nm and the PDI was 0.15.

[0037] Example 2:

[0038] Dissolve PEG with a molecular weight of 400 Da in deionized water to prepare 10 ml of a 3 mg / mL solution, and adjust the pH to 6; add 225 Acetyl ions 0.3 mCi / mL 0.5 ml, stirred for 15 minutes; then 1 mg / mL tannic acid solution (PEG:tannic acid = 1:0.3) 10 ml was added dropwise, stirred at room temperature for 1.5 hours to form microspheres; after purification, the microspheres were obtained and detected. Dynamic light scattering (DLS) showed an average particle size of 80 nm and a PDI of 0.12.

[0039] Using PEG with a smaller molecular weight can reduce the size of microspheres, making it easier to encapsulate them into liposome carriers and improving the overall nanoscale compatibility and in vivo delivery efficiency of the drug system.

[0040] Example 3:

[0041] Dissolve chitosan with a molecular weight of 10000 Da in deionized water to prepare 10 mL of an 8 mg / mL solution. Adjust the pH to 5.5 with hydrochloric acid and stir at room temperature for 40 min until completely dissolved. 177 A Lu ion solution with a concentration of 0.8 mCi / mL was added to a chitosan solution at a molar ratio of chitosan monomer to radionuclide of 60:1. The mixture was stirred at room temperature for 25 min to complete the initial complexation. A 10 mL solution of catechin (4 mg / mL) was prepared and adjusted to pH 5.5. This solution was then slowly added dropwise to the polymer solution containing the radionuclide at a chitosan:catechin weight ratio of 1:0.8. The mixture was reacted at room temperature with stirring at 800 rpm for 2.5 h. The mixture was then purified by centrifugation at 15000 g for 15 min to obtain the encapsulated... 177 Lu microspheres. Average particle size 280 nm, PDI 0.16, nuclide release rate <4.5% after 48 h.

[0042] Example 4: Assembly of microsphere-loaded liposomes

[0043] The microspheres prepared in Examples 1 and 2 were used to form liposomes. The specific steps are as follows:

[0044] Weigh phospholipids and cholesterol (dispalmitoylphosphatidylcholine (DPPC) + cholesterol, molar ratio 7:3) into a round-bottom flask, add organic solvent (5 mL chloroform), and magnetically stir for 15–30 min at room temperature or slightly above the phospholipid phase transition temperature (40–50℃) until completely dissolved. Stirring should be done in a dark, nitrogen atmosphere at a speed of 300–500 rpm. Transfer the solution to a rotary evaporator and evaporate the organic solvent under reduced pressure at 35–38℃ and 100–150 rpm until a uniform, transparent / semi-transparent lipid film is formed. Continue vacuum drying overnight in an oven to remove residual solvent. Add PBS buffer containing microspheres to the dried lipid film, maintain a temperature of 40–55℃, and intermittently shake / magnetically stir / vortex for 30–120 min, manually shaking or stirring at low speed (300–500 rpm) every 5–10 min to hydrate and peel off the lipid film to form multilayer liposomes (MLVs). After hydration, the suspension was placed at 4°C overnight (to promote membrane stability). Using a liposome extruder, the liposomes were passed through a polycarbonate membrane (100 nm initially, then 50-200 nm) at 40-55°C, 7-21 times per membrane (14-42 times in total); liposome microspheres were obtained.

[0045] Comparative Example 1:

[0046] Preparation of carriers using traditional DOTA chelation method 225 Ac liposomes. The specific steps are as follows:

[0047] Weigh phospholipids and cholesterol (dispalmitoylphosphatidylcholine (DPPC) + cholesterol, molar ratio 7:3) into a round-bottom flask, add organic solvent (5 mL chloroform), and magnetically stir for 15–30 min at room temperature or slightly above the phospholipid phase transition temperature (40–50℃) until completely dissolved. Stirring should be done in a dark, nitrogen-filled atmosphere at a speed of 300–500 rpm. Transfer the solution to a rotary evaporator and evaporate the organic solvent under reduced pressure at 35–38℃ and 100–150 rpm until a uniform, transparent / semi-transparent lipid film is formed. Add PBS buffer to the dried lipid film, maintain the temperature at 40–55℃, and intermittently shake / magnetically stir / vortex for 30–120 min, manually shaking or stirring at low speed (300–500 rpm) every 5–10 min to hydrate and peel off the lipid film to form multilayer liposomes (MLVs). After hydration, incubate the suspension overnight at 4℃ (to promote membrane stability). Blank liposome microspheres (80–150 nm) were obtained by passing them through a polycarbonate membrane using a liposome extruder at 40–55 °C. DOTA was covalently grafted onto the surface of the blank liposomes using DOTA-NHS ester. The reaction was incubated in a buffer solution at pH 7–8 in the dark for 2–4 h. Unreacted DOTA reagent was removed by ultrafiltration or dialysis to obtain the DOTA-liposome precursor.225 Ac nitrate was dissolved in 0.1 M HCl and neutralized to pH 6. DOTA-liposome suspension was then added, and the mixture was heated at 60°C for 30 min. After the reaction, unchelated nitrate was removed by ultrafiltration. 225 Ac, obtained carrying 225 Ac liposomes.

[0048] Comparative Example 2:

[0049] The loading was prepared according to the method in Example 2. 225 The Ac microspheres differ in that they are not treated with tannic acid solution. Liposomes loaded with the microspheres are then prepared according to the method in Example 4.

[0050] Example 5: In vitro stability test

[0051] The liposomes containing microspheres from Example 1 or Example 2 prepared in Example 4, and the liposomes prepared in Comparative Examples 1 or 2, were subjected to in vitro stability tests.

[0052] First, the particle size and colloidal stability of each group of liposomes were tested. Liposomes loaded with radionuclides were placed in PBS (pH 7.4, 37℃) buffer solution, and the particle size and PDI were measured at 0 h and 72 h. The results are shown in Table 1. The liposomes prepared by this method exhibited excellent stability, with almost no change in particle size and good dispersibility. The comparative liposomes, after prolonged soaking, became loose and aggregated, indicating poor stability.

[0053] Table 1

[0054] Further testing was conducted on the radionuclide encapsulation efficiency and drug loading in the examples and comparative groups. The results are shown in Table 2, with Examples 1 and 2 exhibiting better encapsulation efficiency. The liposomes from the examples and comparative groups were placed in PBS (pH 7.4, 37°C) solution, and the cumulative release rate and daughter radionuclide retention rate were measured over 48 hours. The results are shown in Table 2, demonstrating that the examples effectively maintained radionuclide loading and exhibited good stability.

[0055] Table 2

[0056] In addition, in vivo decay was simulated, and measurements were taken in both the example group and the comparative group. 225 Ac decay chain progeny escape rate. Specifically, the total escape rate of progeny in Example 1 and Example 2 groups was <4%, the escape rate in Comparative Example 1 group was >23%, and the escape rate in Comparative Example 2 group was >32%. This demonstrates that the liposomes in the Example groups have better resistance to decay recoil.

[0057] Example 6: In vitro cytotoxicity test

[0058] Using HeLa cells as a model, the cytotoxicity of liposomes in the Example Group and the Comparative Group was detected by the CCK-8 assay, and cell viability was examined under conditions of 0.1-0.5 mCi / mL. The results showed that the cell viability of Example 1 and Example 2 groups was approximately 87%, demonstrating the better biocompatibility of the Example Group; the cell viability of Comparative Group 1 was 68%, exhibiting increased free radionuclide toxicity; the cell viability of Comparative Group 2 was 59%, which was attributed to the unstable structure of the radionuclide-loaded lipids, leading to higher toxicity.

[0059] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A method for preparing microspheres for encapsulating radionuclides, characterized in that: Polymers, polyphenols, and radionuclides are mixed and self-assembled to form microspheres encapsulating radionuclides.

2. The method for preparing microspheres for encapsulating radionuclides according to claim 1, characterized in that: The polymer is polyethylene glycol (PEG) or chitosan, with a molecular weight range of 1000-20000 Da; the polyphenols are tannic acid or catechins.

3. The method for preparing microspheres for encapsulating radionuclides according to claim 1 or 2, characterized in that: The specific steps are as follows: Step 1: Prepare polymer solution and polyphenol solution separately; Step 2: Prepare a radionuclide ion solution by mixing the radionuclide ion solution with the polymer solution and performing a complexation reaction. Step 3: The polyphenol solution is slowly added dropwise to the polymer solution containing radionuclides. Through the oxidative cross-linking of polyphenols and the hydrogen bonding / electrostatic interaction of polymers, microspheres loaded with radioactive nuclide ions are formed.

4. The method for preparing microspheres for loading radionuclides according to claim 3, characterized in that: The molar ratio of monomer polymer to nuclide is 10-100:1, and the weight ratio of polymer to polyphenol is 1:0.1-1.

5. The method for preparing microspheres for encapsulating radionuclides according to claim 4, characterized in that: Polymer solutions, polyphenol solutions, and radionuclide ion solutions were prepared using the same primary solvent; the concentration range of the polymer solution was 1–10 mg / mL, the concentration range of the polyphenol solution was 0.5–5 mg / mL, and the concentration range of the radionuclide ion solution was 0.1–1 mCi / mL.

6. The method for preparing microspheres for encapsulating radionuclides according to claim 3, characterized in that: The pH of polymer solutions and polyphenol solutions is adjusted to 5-7 using sodium hydroxide or hydrochloric acid.

7. Microspheres loaded with radionuclides prepared by the method for preparing microspheres loaded with radionuclides according to any one of claims 1-6.

8. The microspheres containing radionuclides according to claim 7, characterized in that: The nuclide is α 227 Th、 225 Ac、 223 Ra、 213 Bi、 212 Pb, 188 Re、 177 Lu、 161 Tb, 90 Y、 89 Sr and 67 One or more of Cu.

9. The use of the microspheres containing radionuclides as described in claim 7 or 8 in the preparation of radiotargeted drugs.

10. A liposome loaded with a radionuclide, characterized in that: Including the microspheres containing radionuclides as described in claim 7 or 8.

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