Iodine-131 protein-loaded nano-preparation, preparation method and application thereof

Abstract

The application discloses a kind of iodine-131 protein nano preparation and its preparation method and application.The preparation includes targeting carrier and radioactive iodine-131;The targeting carrier is formed by covalent connection of transferrin, polyethylene glycol and cyclodextrin;The radioactive iodine-131 is loaded in the hydrophobic cavity of the cyclodextrin by non-covalent physical inclusion effect.The application also discloses the preparation method of the preparation, and the targeting carrier is obtained by coupling of thiolated transferrin and maleimide group polyethylene glycol-cyclodextrin intermediate, then iodine-131 is loaded by saturated aqueous solution method.Cell experiment shows that the preparation can be efficiently taken up by refractory thyroid cancer cell with high expression of transferrin receptor, and is entered into cell by receptor-mediated active endocytosis pathway.The application bypasses inactivated sodium iodine symporter pathway, provides a new targeted radioactive strategy for the treatment of iodine-refractory differentiated thyroid cancer, thyroid undifferentiated cancer and thyroid medullary carcinoma.

Description

Technical Field

[0001] This invention relates to the fields of radiopharmaceuticals and nanomedicine, and in particular to an iodine-131 loaded protein nanoparticle formulation, its preparation method, and its application. Background Technology

[0002] Thyroid cancer is the most common endocrine malignancy, with differentiated thyroid cancer (DTC) accounting for approximately 90% and generally having a good prognosis. However, about 23% of DTC patients develop distant metastases, and about one-third of these metastatic DTC patients eventually develop tumor cell dedifferentiation, progressing to iodine-refractory differentiated thyroid cancer (RAIR-DTC). Furthermore, undifferentiated thyroid carcinoma (ATC) and medullary thyroid carcinoma (MTC) are also highly refractory. These three types of refractory thyroid cancer face a common and critical clinical challenge: the absence, abnormal localization, or impaired function of the sodium-iodine cotransporter (NIS) on the tumor cell membrane surface, rendering traditionally efficient and cost-effective radioactive iodine-131 (I-I-131) ineffective. 131 I) Internal irradiation therapy has completely failed.

[0003] Current treatment strategies for refractory thyroid cancer, such as multi-kinase inhibitors (sorafenib, lenvatinib, etc.), while prolonging progression-free survival to some extent, suffer from severe toxic side effects and unavoidable acquired resistance, requiring lifelong medication. Therefore, there is an urgent need to develop a novel targeted delivery strategy that does not rely on the NIS pathway to achieve [treatment]. 131 I is an effective treatment for refractory thyroid cancer. Summary of the Invention

[0004] The purpose of this invention is to provide an iodine-131 loaded protein nanoparticle formulation, its preparation method, and its application, in order to solve the problem of non-responsiveness of refractory thyroid cancer to radioactive iodine-131 treatment in the prior art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides an iodine-131-loaded protein nanoformulation comprising a targeting carrier and radioactive iodine-131 ( 131 I); The targeting carrier is composed of transferrin (Tf), polyethylene glycol (PEG), and cyclodextrin (CD) covalently linked together; The radioactive iodine-131 is in the form of triiodine anion ( 131 In the form of a non-covalent physical inclusion compound, the cyclodextrin is loaded into the hydrophobic cavity of the cyclodextrin.

[0006] Furthermore, the molecular weight of the polyethylene glycol is 2000 Da.

[0007] Furthermore, the cyclodextrin is selected from at least one of β-cyclodextrin and mono-(6-amino-6-deoxy)-β-cyclodextrin, preferably mono-(6-amino-6-deoxy)-β-cyclodextrin.

[0008] Furthermore, in the targeting carrier, transferrin is linked to polyethylene glycol via a thioether bond, and polyethylene glycol is linked to cyclodextrin via an amide bond.

[0009] Furthermore, the hydrated particle size of the nano-formulation in water is 180–260 nm, preferably 219.70 ± 4.52 nm; the Zeta potential is -10 mV to -20 mV, preferably -14.52 ± 0.85 mV.

[0010] Furthermore, in the nano-formulation, triiodine anion ( 131 It is encapsulated within the hydrophobic cavity of cyclodextrin, which is covalently linked to transferrin via polyethylene glycol chains.

[0011] In a second aspect, the present invention provides a method for preparing the iodine-131 loaded protein nanoparticle formulation according to the first aspect, comprising the following steps: (1) Transferrin was modified by thiolation to obtain thiolated transferrin (Tf-SH). (2) The cyclodextrin derivative was reacted with the heterodifunctional polyethylene glycol crosslinking agent NHS-PEG-MAL to obtain a polyethylene glycol-cyclodextrin intermediate (MAL-PEG-CD) with maleimide groups. (3) The intermediate of step (2) is coupled with the thiolated transferrin of step (1) via Michael addition reaction to obtain the targeting vector CD-PEG-Tf; (4) Using the saturated aqueous solution method, the targeting carrier from step (3) is mixed with a solution containing... 131 Mixing the triiodine anion solution of I, so that 131 The iodine-131 protein nanoparticles were encapsulated into the cyclodextrin cavity and purified to obtain the nanoparticles.

[0012] Further, in step (1), the thiolizing agent is 2-iminothiacyclopentane hydrochloride (Traut reagent).

[0013] Further, in step (2), the cyclodextrin derivative is mono-(6-amino-6-deoxy)-β-cyclodextrin (6-NH_2-β-CD), and the heterobifunctional polyethylene glycol crosslinking agent is NHS-PEG2000-MAL; the reaction is carried out in anhydrous dimethyl sulfoxide (DMSO) with triethylamine (TEA) added as a catalyst; after the reaction is completed, the reaction solution can be used directly in the next step, or it can be used after ultrafiltration purification.

[0014] Furthermore, in step (3), the Michael addition reaction is carried out under nitrogen protection and light-protected conditions.

[0015] Further, in step (4), the saturated aqueous solution method specifically includes: mixing elemental iodine (I2) and Na... 131 I mixes with water to form 131 The solution is then mixed with the targeting carrier; wherein the mass ratio of the targeting carrier to elemental iodine is 1:5 to 1:20, preferably 1:10; the reaction temperature is 20 to 30°C, preferably 25°C; the reaction time is 2 to 6 hours, preferably 4 hours; the reaction is carried out under light-protected conditions with stirring.

[0016] Further, in step (4), the purification is carried out by dialysis or ultrafiltration; the dialysis method uses a dialysis bag with a molecular weight cutoff of 10 kDa, and the dialysis time is 8 to 24 hours, preferably 12 hours.

[0017] Thirdly, the present invention provides the use of the iodine-131 loaded protein nanoformulation according to the first aspect in the preparation of a medicament for treating refractory thyroid cancer.

[0018] Furthermore, the refractory thyroid cancer includes at least one of iodine-refractory differentiated thyroid cancer (RAIR-DTC), undifferentiated thyroid carcinoma (ATC), and medullary thyroid carcinoma (MTC).

[0019] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. Overcoming the "iodine resistance" bottleneck: Utilizing TfR as an alternative target, through Tf-mediated active targeting and receptor-mediated endocytosis, bypassing the NIS inactivation barrier, providing a novel treatment strategy for refractory thyroid cancer; 2. Highly efficient nuclide loading and stable delivery system: Iodine is non-covalently physically encapsulated using β-CD hydrophobic cavities, achieving an encapsulation efficiency of over 91% and a radioactive encapsulation efficiency of up to 92.20%, while exhibiting good radioactive stability in both PBS and serum-containing culture media; 3. Excellent tumor targeting selectivity: Cell uptake experiments confirmed that this nano-formulation exhibited high uptake in TfR-highly expressed ATC and MTC cells, while almost no uptake was observed in normal thyroid cells; 4. A well-defined receptor-mediated endocytosis mechanism: Free Tf competitive blocking experiments confirmed that the cellular entry process depends on Tf-TfR specific recognition; 5. Good potential for clinical translation: The materials used have good biocompatibility, the preparation process is stable, and the safety is high. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the preparation of the iodine-131 loaded protein nanoparticles provided in Example 1 of the present invention and their uptake mechanism by refractory thyroid cancer cells. Figure 2 The target vector CD-PEG2000-Tf provided in Embodiment 1 of the present invention 1 H-NMR spectrum; Figure 3 The FTIR spectrum of the targeting vector CD-PEG2000-Tf provided in Embodiment 1 of the present invention; Figure 4 The SDS-PAGE spectrum of the targeting vector CD-PEG2000-Tf provided in Embodiment 1 of the present invention; Figure 5 Provided for Embodiment 1 of the present invention 131 UV-Vis comparison of CD-PEG2000-Tf and blank carrier CD-PEG2000-Tf; Figure 6 Provided for Embodiment 1 of the present invention 131 Zeta potential plot of CD-PEG2000-Tf; Figure 7 Provided for Embodiment 1 of the present invention 131 Particle size distribution curve of CD-PEG2000-Tf; Figure 8 Provided for Embodiment 1 of the present invention 131 Transmission electron microscopy (TEM) image of CD-PEG2000-Tf; Figure 9 Provided for Embodiment 1 of the present invention 131 The physical stability evaluation results of CD-PEG2000-Tf nano-formulation in aqueous system, where A is the trend of Zeta potential change and B is the change of average hydrodynamic particle size and polydispersity index (PDI). Figure 10 Provided for Embodiment 1 of the present invention 131 -UV-Vis spectrum of in vitro encapsulation stability of CD-PEG2000-Tf nanoformulation over 15 days; Figure 11 Provided for Embodiment 1 of the present invention 131 -Retention rate (%) of CD-PEG2000-Tf radioactivity in PBS and culture medium containing 10% FBS over 72 hours; Figure 12 The image shows a multichannel fluorescence imaging of the iodine-131 protein nanoparticles (FITC-labeled) provided in Example 1 of this invention in different thyroid cell lines, where (A) human undifferentiated thyroid cancer cells 8305c (abbreviated as 8305c cells), (B) CAL-62 thyroid cancer cells (abbreviated as CAL-62 cells), (C) C643 thyroid cancer cells (abbreviated as C643 cells), (D) TT thyroid cancer cells (abbreviated as TT cells), (E) BCPAP papillary thyroid cancer cells (abbreviated as BCPAP cells), and (F) Nthy-ori3-1 normal thyroid follicular epithelial cells (abbreviated as Nthy-ori3-1 normal thyroid cells). Figure 13 The results are semi-quantitative analysis of the uptake efficiency of iodine-131 protein nanoparticles by various cell lines provided in Example 1 of this invention, where A is the percentage of fluorescently positive area (%Area) and B is the average fluorescence intensity (MFI). Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in further detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0022] The main experimental materials and reagents in the specific embodiments of the present invention are shown in Table 1.

[0023] Table 1. Main Experimental Materials and Reagents

[0024] The main experimental instruments in the specific embodiments of the present invention are shown in Table 2.

[0025] Table 2. Main Experimental Instruments

[0026] The English abbreviations and corresponding full names of relevant medical terms used in this invention are shown in Table 3.

[0027] Table 3. English abbreviations and corresponding full names of relevant medical terms

[0028] Example 1: Iodine-131 loaded protein nanoparticles ( 131 Preparation of -CD-PEG2000-Tf) 1. Synthesis of the targeting vector CD-PEG2000-Tf (1) Preparation of thiolized transferrin (Tf-SH) Accurately weigh 100 mg of lyophilized transferrin (Tf) powder and dissolve it in phosphate-buffered saline (PBS) at pH 8.0. Add 40 equivalents of 2-iminothiacyclopentane hydrochloride (Traut's reagent) and react magnetically for 2 hours under nitrogen protection, at room temperature, and in the dark. After the reaction is complete, centrifuge at 7000 rpm for 6 minutes at 4°C using an ultrafiltration tube with a molecular weight cutoff of 30 kDa. Wash and concentrate with PBS, and collect the Tf-SH solution for later use.

[0029] (2) Preparation of intermediate MAL-PEG2000-CD Accurately weigh 100 mg of mono-(6-amino-6-deoxy)-β-cyclodextrin (NH2-β-CD) and dissolve it in 2 mL of anhydrous DMSO. Add 1.0 equivalent of NHS-PEG2000-MAL and 2.0 equivalent of triethylamine (TEA), and stir magnetically at room temperature for 2 hours to obtain the MAL-PEG2000-CD reaction solution. This reaction solution can be used directly for the next step, or it can be lyophilized after removing small molecule impurities by ultrafiltration (10 kDa) for later use.

[0030] (3) Coupling and purification of CD-PEG2000-Tf The DMSO solution of MAL-PEG2000-CD obtained in the previous step was slowly added dropwise to the Tf-SH solution using a micro-injection pump. The reaction was continued for 8 hours under nitrogen protection, at room temperature, and in the dark. After the reaction, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed in ultrapure water for 24 hours (changing the water every 4-6 hours). The retained solution in the dialysis bag was collected, lyophilized, and the resulting white, loose powdered targeting carrier CD-PEG2000-Tf was dried at -20°C and stored in the dark. The structural characterization of the obtained targeting carrier is detailed in Experimental Example 1.

[0031] 2. Preparation of iodine-131 loaded nano-formulations It was prepared using the saturated aqueous solution method. The optimal conditions were set at a mass ratio of elemental iodine (I₂) to CD-PEG2000-Tf of 10:1: 50 mg of elemental iodine was accurately weighed and added to 1 mL of 1.0 mCi Na₂. 131 Solution I (containing 1M NaI as a carrier) and 4mL of ultrapure water were stirred at room temperature in the dark to form... 131 Complexation solution. Separately, dissolve 5 mg of lyophilized CD-PEG2000-Tf in 5 mL of ultrapure water. Slowly add the carrier solution dropwise to the above-mentioned complexing solution. 131 The solution was stirred at 25°C and 1500 rpm in the dark for 4 hours. After the reaction was completed, the mixture was transferred to a 10 kDa dialysis bag and dialyzed in ultrapure water at 4°C in the dark for 12 hours (with one solution change in between). The retentate in the bag was collected to obtain the final product. 131 -CD-PEG2000-Tf nanoformulation. The physicochemical properties of the obtained nanoformulation are detailed in Experimental Example 2, and the encapsulation efficiency and stability evaluation are detailed in Experimental Example 3.

[0032] Example 2: Preparation and Characterization of Iodine-131 Protein Nanoparticles under Different Feed Ratios 1. Preparation of nano-formulations with a feed ratio of 1:5 (carrier:iodine) The targeting vector CD-PEG2000-Tf was synthesized according to the method in Example 1. 10 mg of elemental iodine was accurately weighed and added to 1 mL of 1.0 mCi Na¹³¹I solution (containing 1 M NaI as a carrier) and 4 mL of ultrapure water. The mixture was stirred at room temperature in the dark to form... 131 Complexation solution. Separately, dissolve 50 mg of lyophilized CD-PEG2000-Tf in 5 mL of ultrapure water. Slowly add the carrier solution dropwise to the above-mentioned complexing solution. 131 The solution was stirred at 25°C and 1500 rpm in the dark for 4 hours. After the reaction was complete, the solution was transferred to a 10 kDa dialysis bag and dialyzed in ultrapure water at 4°C in the dark for 12 hours. The retentate was collected to obtain the product with a feed ratio of 1:5. 131 -CD-PEG2000-Tf nanoformulation.

[0033] 2. Preparation of nano-formulations with a feed ratio of 1:20 (carrier: iodine) Accurately weigh 100 mg of elemental iodine, add 1 mL of 1.0 mCi Na¹³¹I solution and 4 mL of ultrapure water to prepare... 131 Complexing solution. Separately, dissolve 5 mg of lyophilized CD-PEG2000-Tf in 5 mL of ultrapure water. Perform the remaining procedures as above to obtain a feed ratio of 1:20. 131 -CD-PEG2000-Tf nanoformulation.

[0034] 3. Physicochemical property characterization The characterization was performed using the same method as in Experiment 2. The results are shown in Table 4. Table 4. Results obtained in Example 2 131 Physicochemical properties characterization results of CD-PEG2000-Tf nano-formulation

[0035] TEM observation showed that both groups of nanoparticles were regularly spherical and well dispersed, with particle sizes of 30–55 nm and 40–70 nm, respectively, in the dry state. These results indicate that stable nanoparticle formulations with particle sizes of 180–260 nm and zeta potentials in the range of -10 mV to -20 mV can be prepared within the claimed feed ratio range (1:5 to 1:20), and the feed ratio of 1:10 (Example 1) exhibits the optimal encapsulation efficiency.

[0036] Example 3: Preparation and characterization of iodine-131 protein nanoparticles under different reaction conditions 1. Reaction temperature 20℃, reaction time 6 hours The targeting vector CD-PEG2000-Tf was synthesized according to the method in Example 1. 50 mg of elemental iodine was accurately weighed, added to 1 mL of 1.0 mCi Na¹³¹I solution and 4 mL of ultrapure water, and stirred at room temperature in the dark to form... 131 Complexation solution. Separately, dissolve 5 mg of lyophilized CD-PEG2000-Tf in 5 mL of ultrapure water. Slowly add the carrier solution dropwise to the above-mentioned complexing solution. 131 The solution was stirred at 20°C and 1500 rpm in the dark for 6 hours. After the reaction was completed, the solution was transferred to a 10 kDa dialysis bag and dialyzed in ultrapure water at 4°C in the dark for 12 hours. The retentate was collected to obtain the nano-formulation.

[0037] 2. Reaction temperature 30℃, reaction time 2 hours carrier solution and 131 After mixing the complexing solutions, stir at 30°C and 1500 rpm in the dark for 2 hours. The remaining operations are the same as above, and the nano-formulation is obtained.

[0038] 3. Physicochemical property characterization The characterization was performed using the same method as in Experimental Example 2. The results are shown in Table 5. Table 5. Results obtained in Example 3 131 Physicochemical properties characterization results of CD-PEG2000-Tf nano-formulation

[0039] DLS measurements showed that the average hydrated particle sizes of the two nano-formulations in aqueous phase were 215.80±4.90 nm and 225.40±5.30 nm, respectively; the PDI values ​​were 0.210±0.014 and 0.219±0.016, respectively; and the zeta potentials were -15.20±0.85 mV and -13.80±0.90 mV, respectively. These results confirm that uniformly sized and well-dispersed iodine-131 protein nano-formulations can be obtained within a temperature range of 20–30 °C and a reaction time range of 2–6 hours.

[0040] Example 4: Preparation and Characterization of Iodine-131 Loaded Protein Nanoparticles Based on β-Cyclodextrin 1. Preparation of intermediate MAL-PEG2000-β-CD Accurately weigh 100 mg of β-cyclodextrin (β-CD) and dissolve it in 2 mL of anhydrous DMSO. Add 1.2 equivalents of N,N'-carbonyldiimidazole (CDI), activate at room temperature for 1 hour, then add 1.0 equivalent of ethylenediamine, and continue the reaction for 2 hours to obtain amino-modified β-cyclodextrin (NH2-β-CD). After purification, dissolve the obtained NH2-β-CD in 2 mL of anhydrous DMSO, add 1.0 equivalent of NHS-PEG2000-MAL and 2.0 equivalents of triethylamine (TEA), and react magnetically at room temperature for 2 hours to obtain MAL-PEG2000-β-CD reaction solution.

[0041] Note: If amino-modified β-CD is used directly as the starting material, the operation can be simplified as follows: replace mono-(6-amino-6-deoxy)-β-cyclodextrin with an equal molar amount of amino-modified β-CD, and the rest is the same as in Example 1.

[0042] 2. Synthesis of the targeting vector β-CD-PEG2000-Tf Following the method in Example 1, MAL-PEG2000-β-CD was coupled with thiolated transferrin (Tf-SH) via Michael addition reaction, purified by dialysis, and lyophilized to obtain a white, loose powder-like targeting carrier β-CD-PEG2000-Tf.

[0043] 3. Preparation of iodine-131 loaded nano-formulations Using the same saturated aqueous solution method as in Example 1, with elemental iodine and β-CD-PEG2000-Tf at a mass ratio of 10:1, the mixture was stirred at 25°C in the dark for 4 hours, and then purified through a 10kDa dialysis bag to obtain... 131 -β-CD-PEG2000-Tf nanoformulation.

[0044] 4. Physicochemical property characterization DLS measurement results showed that 131 The average hydrated particle size of the -β-CD-PEG2000-Tf nanoparticles in aqueous phase was 206.80±4.70 nm, the PDI was 0.205±0.013, and the Zeta potential was -15.60±0.82 mV. UV-Vis spectra were observed at 350 nm. 131 Characteristic absorption peaks were observed. TEM observation showed that the particles were regularly spherical and well dispersed.

[0045] The above results show that iodine-131 protein nanoparticles can also be successfully constructed by replacing mono-(6-amino-6-deoxy)-β-cyclodextrin with β-cyclodextrin, further verifying the universality of the technical solution of the present invention for different types of cyclodextrin.

[0046] Example 5: In vivo pharmacodynamic study of iodine-131 loaded protein nanoformulation in mice with refractory thyroid cancer. 1. Establishment of animal models Female BALB / c nude mice (weighing 18–22 g) aged 4–6 weeks were subcutaneously inoculated with a suspension of human undifferentiated thyroid cancer cells CAL-62 (or 8305c, C643) (1 × 10⁻⁶ g) in the axilla of the right forelimb. 7 (cells / mouse). When the tumor volume grew to approximately 100–150 mm³, the tumor-bearing mice were randomly divided into the following 4 groups (6 mice in each group): Group A (saline control group): 100 μL of saline was injected via the tail vein; Group B (Free Na¹³¹I group): 100 μL Na¹³¹I solution (containing 200 μCi¹³¹I) was injected into the tail vein. Group C (blank vector group): 100 μL of CD-PEG2000-Tf vector solution without ¹³¹I was injected via tail vein (dose equivalent to the amount of vector in Group D). Group D 131 -CD-PEG2000-Tf nano-formulation group): 100μL injected via tail vein. 131 -CD-PEG2000-Tf nanoformulation (containing 200μCi¹³¹I, carrier amount 5mg / kg).

[0047] 2. Pharmacodynamic evaluation Tumor volume (V = length × width² / 2) was measured every 3 days after drug administration, and mouse weight was also measured. The observation was continued for 21 days. On day 21 after drug administration, mice were sacrificed, tumor tissue was dissected and weighed, and the tumor inhibition rate (TGI%) was calculated as (1 - average tumor weight of the drug group / average tumor weight of the control group) × 100%.

[0048] 3. Biological distribution research Three tumor-bearing mice were taken and injected with the corresponding preparation via the tail vein. They were then sacrificed at 2h, 6h, 12h, 24h, 48h and 72h after administration. Blood, tumors and major organs (heart, liver, spleen, lungs, kidneys, thyroid, stomach and bones) were collected, weighed and radioactivity counted using a gamma counter. The percentage of injected dose per gram of tissue was calculated (%ID / g).

[0049] 4. Expected Results Based on the in vitro targeted uptake characteristics of the nano-formulation of this invention (Experimental Example 4), it is expected that the nano-formulation group D will have the highest radioactive enrichment at the tumor site (tumor %ID / g significantly higher than group B), and the TGI% will be significantly better than the free Na¹³¹I group (P<0.01), and the radioactive damage to major organs (especially the thyroid gland) will be significantly lower than that of the free Na¹³¹I group. In vivo efficacy results will further confirm the targeted therapeutic effect of the iodine-131 loaded protein nano-formulation of this invention on refractory thyroid cancer.

[0050] In summary, Examples 1 to 4 provide preparation schemes for iodine-131 protein-loaded nanoparticles under different feed ratios (1:5, 1:10, 1:20), different reaction conditions (20℃ / 6h, 25℃ / 4h, 30℃ / 2h), and different types of cyclodextrin (β-cyclodextrin, mono-(6-amino-6-deoxy)-β-cyclodextrin). The hydrated particle size of the preparations obtained by each scheme is in the range of 180–260 nm, and the Zeta potential is in the range of -10 mV to -20 mV, indicating that the technical solution of the present invention has broad process adaptability and good reproducibility. Example 5 further verifies the in vivo antitumor efficacy of the nanoparticles.

[0051] Example 6: Application of Iodine-131 Loaded Protein Nanoparticles 1. Preparation of fluorescently labeled iodine-131 protein nanoparticles (for tumor imaging and tracing) 100 mg of the CD-PEG2000-Tf carrier prepared in Example 1 was weighed and dissolved in PBS (pH=8.0). 10 mg of fluorescein isothiocyanate (FITC) was added, and the mixture was stirred at room temperature in the dark for 3 hours. The reaction solution was purified by dialysis in ultrapure water using a dialysis bag with a molecular weight cutoff of 10 kDa, and then lyophilized to obtain the fluorescently labeled carrier CD-PEG2000-Tf-FITC. Following the saturated aqueous solution method in Example 1, the carrier was loaded at a feed ratio of 10:1. 131 I, to obtain ¹³¹I₃ - - Cyclodextrin - PEG2000 - Transferrin - FITC probe ( 131 (-CD-PEG2000-Tf-FITC), store at 4°C protected from light. This fluorescently labeled preparation can be used for targeted uptake tracking of tumor cells (see Experimental Example 4).

[0052] 2. Radiopharmaceutical preparations for the treatment of refractory thyroid cancer Take the preparation of Example 1 131 The CD-PEG2000-Tf nanoparticle formulation, dispersed in injectable saline, is sterilized by filtration through a 0.22μm microporous membrane to obtain an injectable radiopharmaceutical formulation. This formulation, through transferrin-mediated active targeting, is specifically taken up by refractory thyroid cancer cells with high transferrin receptor expression, and can be used to prepare drugs for treating iodine-resistant differentiated thyroid cancer, undifferentiated thyroid carcinoma, medullary thyroid carcinoma, and other refractory thyroid cancers.

[0053] Experimental Example 1: Structural Characterization of the Targeting Vector CD-PEG2000-Tf The target carriers obtained by freeze-drying in Example 1 were characterized as follows: 1H NMR spectrum ( 1 H-NMR: The lyophilized powder was dissolved in heavy water (D). 2O The results were obtained using a 400MHz nuclear magnetic resonance spectrometer. Figure 2 As shown, the resonance peak at 5.01 ppm is attributed to the characteristic absorption of the C1-H (terminal proton) of the glucose unit in the β-cyclodextrin molecule; the sharp, strong peak at 3.63 ppm is the repetitive methylene (-CH2-CH2-O-) proton peak on the PEG2000 chain; and the multiple broad peaks appearing in the 2.5–3.5 ppm region are attributed to the alkyl proton signals of various amino acid residues on the Tf polypeptide chain. These results suggest that the final product simultaneously contains β-CD, PEG2000, and Tf-related structural information, preliminarily confirming that β-cyclodextrin, PEG molecular chains, and transferrin have successfully undergone covalent cross-linking.

[0054] Fourier transform infrared spectroscopy (FTIR): Detection was performed using the KBr pellet method. Results are as follows: Figure 3 As shown, at 3358cm -1 The broad and strong absorption peak at 1107 cm⁻¹ is caused by the stretching vibrations of numerous hydroxyl groups (-OH) along the outer edge of the β-cyclodextrin cavity; -1 The strong absorption peak at 1656 cm⁻¹ is attributed to the typical asymmetric stretching vibration of the ether bond (COC) in the PEG chain; simultaneously, at 1656 cm⁻¹... -1 A distinct absorption peak characteristic of transferrin, the amide I band (C=O stretching vibration), was observed. These results further indicate that the sample simultaneously possesses β-CD, PEG2000, and protein-related functional group characteristics.

[0055] Polyacrylamide gel electrophoresis (SDS-PAGE): Results are as follows Figure 4 As shown, compared to the unmodified Tf (approximately 77 kDa), the band of the CD-PEG2000-Tf sample shifted upwards to approximately 180 kDa and exhibited a diffuse characteristic. Due to the hydrophilicity and spatial conformation of the PEG2000 chain, its apparent molecular weight in gel electrophoresis is often greater than the theoretical value (the theoretical conjugate molecular weight is approximately 80 kDa), and a single Tf may couple multiple PEG-CD chains. Therefore, the observed increase in molecular weight is as expected, indicating that Tf successfully coupled high molecular weight PEG and CD.

[0056] Experimental Example 2: 131 Physicochemical property characterization of -CD-PEG2000-Tf The nano-formulation prepared under the optimal feed ratio (10:1) in Example 1 was subjected to the following tests: Ultraviolet-Vis spectrophotometry (UV-Vis): such as Figure 5 As shown, compared to the blank carrier CD-PEG2000-Tf, the iodine-loaded nanoparticles, after dialysis to remove free elemental iodine and iodide ions from the system, exhibit a distinct and specific absorption peak at 350 nm in their UV absorption curve. This peak is... 131 The typical absorption characteristics confirmed that iodine had been successfully encapsulated as a guest molecule into the hydrophobic lumen of β-cyclodextrin.

[0057] Particle size and zeta potential: determined using dynamic light scattering (DLS). Results are as follows. Figure 6 and Figure 7 As shown, 131 The average hydrodynamic particle size of the CD-PEG2000-Tf nanoparticles in aqueous phase was 219.70±4.52 nm, and the polydispersity index (PDI) was 0.214±0.012. The distribution curve showed a single peak, indicating that the system had good dispersibility. The zeta potential was -14.52±0.85 mV, indicating that the particle surface had a certain degree of negative charge.

[0058] Transmission electron microscopy (TEM): such as Figure 8As shown, the nanoparticles exhibit a regular spherical or elliptical appearance, with clear boundaries between particles and good dispersion, showing no obvious adhesion or cross-linking. Their physical particle size distribution in the dry state ranges from 33 to 66 nm, with an average particle size of approximately 46.8 nm. The size observed by TEM is smaller than the hydrodynamic particle size measured by DLS. This is because DLS measures the hydration layer and soft layer structure of the particles in aqueous solution, while TEM observes the geometric size after dehydration and negative staining. This difference is consistent with the general rules of nanocolloid systems.

[0059] Experimental Example 3: Encapsulation Efficiency and Stability Evaluation 1. Encapsulation efficiency and radioactive encapsulation efficiency like Figure 9-13 As shown, the specific data indicates that the chemical encapsulation efficiency (UV-Vis standard curve method) is 85.14±2.6%, 91.02±4.5%, and 82.36±3.1% at feed ratios of 5:1, 10:1, and 20:1, respectively, and 10:1 is determined to be the optimal feed ratio.

[0060] Radioactive encapsulation efficiency (ultrafiltration centrifugation method, 10:1 group): 92.20±0.30%, which is highly consistent with the chemical encapsulation efficiency.

[0061] 2. Physical and colloidal stability The nano-formulation of Example 1 (10:1 group) was stored at 4°C for 15 days, and samples were taken for testing on days 1, 7, and 15. Figure 9 As shown, the average hydrated particle size of the freshly prepared nanoparticles was 219.70±4.52 nm, and the Zeta potential was -14.52±0.85 mV. After one day of storage, the particle size was 220.15±3.80 nm, the Zeta potential was -14.38±0.92 mV, and the PDI stabilized at 0.218±0.015. On days 7 and 15, the particle sizes were 222.48±4.65 nm and 226.35±5.12 nm, respectively, and the Zeta potentials were -13.95±1.10 mV and -13.42±1.25 mV, respectively. Within 15 days, the PDI remained below 0.25, and the solution was clear and transparent, without precipitation or aggregation, indicating good physical stability.

[0062] 3. Encapsulation stability The evaluation was conducted by tracking the absorbance value at 350 nm. For example... Figure 10 As shown, the absorbance was 0.254±0.011 on day 1, 0.244±0.010 on day 7, and 0.233±0.010 on day 15. One-way ANOVA showed no statistically significant difference in the peak absorbance values ​​over the 15 days (P>0.05), indicating that the degree of dissociation of iodine from the cyclodextrin cavity was very low, and the encapsulation stability was good.

[0063] 4. Radioactive stability The nano-formulation was placed in PBS and culture medium containing 10% fetal bovine serum (FBS) and incubated at 37°C for 72 hours. The radioactivity retention rate was determined by ultrafiltration centrifugation. Results are as follows: Figure 11 As shown, immediately after dialysis purification (0h), the radioactivity retention rates in both media were close to 99%. After incubation in PBS at 37°C for 72h, the radioactivity retention rate remained at (93.45±0.88)%. In a medium containing 10% FBS, the retention rates at 12h, 24h, and 72h were (95.12±0.86)%, (92.38±1.05)%, and (86.55±1.32)%, respectively. Maintaining a high radioactivity encapsulation level of over 92% in a simulated blood environment for 24h indicates that the formulation can effectively resist competitive substitution in the blood circulation environment, and will not cause large-scale radionuclide leakage before reaching the target lesion, possessing the radioactivity stability required for clinical application.

[0064] Experiment Example 4: Study on in vitro cellular uptake and targeting mechanisms To verify the targeted uptake mechanism of the iodine-131 loaded protein nanoparticles described in this invention (see...), Figure 1 Using the material prepared in Example 2 131 Experiments were conducted using CD-PEG2000-Tf-FITC.

[0065] Cell lines: human undifferentiated thyroid carcinoma cells (CAL-62, 8305c, C643), medullary carcinoma cells (TT), papillary carcinoma cells (BCPAP), and normal thyroid follicular epithelial cells (Nthy-ori3-1).

[0066] Two groups were set up: a "Target" group and a "Block" group (pre-incubated with 2 mg / mL free Tf for 30 minutes). Cells in each group were then incubated with 0.05 mg / mL of Tf. 131 After co-incubating with CD-PEG2000-Tf-FITC for 4 hours, the nuclei were stained with Hoechst33342, observed under a fluorescence microscope, and semi-quantitatively analyzed using ImageJ software.

[0067] The results are as follows Figure 12 As shown: In the targeted group, high-intensity green fluorescence was observed in all three types of ATC cells and TT cells, moderate-intensity fluorescence was observed in BCPAP cells, while normal thyroid cells showed almost no fluorescence. In the receptor competition blocking group, the fluorescence intensity of all tumor cells significantly decreased to near-normal cell levels.

[0068] Semi-quantitative analysis results ( Figure 13The results showed that the percentage of fluorescently positive area (%Area) and mean fluorescence intensity (MFI) of ATC and MTC cells in the targeted group were both at a high level, with statistically significant differences compared to the blocking group (P<0.0001). This confirms that the uptake of this nano-formulation is an active endocytosis process mediated by transferrin receptor, and that it has high targeting selectivity for highly malignant and refractory thyroid cancer cells.

[0069] Transferrin receptor (TfR) is highly expressed on the surface of various malignant tumor cells due to high metabolic demands, and its expression level is positively correlated with tumor malignancy. Studies have found that TfR is significantly highly expressed in follicular thyroid carcinoma (FTC) and atopic thyroid carcinoma (ATC). Utilizing the specific recognition between transferrin (Tf) and TfR, active targeted drug delivery can be achieved. Meanwhile, cyclodextrin (CD) possesses a unique cavity structure that is hydrophobic internally and hydrophilic externally, allowing for efficient loading of iodine molecules through non-covalent physical inclusion. Polyethylene glycol (PEG), as a linker arm, can improve the water solubility and stability of the carrier.

[0070] Based on this, the present invention proposes an innovative entry strategy that bypasses the NIS pathway, constructing an iodine-131-loaded protein nanoparticle formulation that is not NIS-dependent. By utilizing transferrin to actively target refractory thyroid cancer cells with high TfR expression, radioactive iodine is precisely delivered into the tumor cells through receptor-mediated endocytosis, thereby breaking through the treatment bottleneck of "iodine loss" in refractory thyroid cancer.

[0071] In summary, this invention successfully constructed a non-NIS-dependent iodine-131-loaded protein nanoparticle formulation. 131 The invention, using CD-PEG2000-Tf, exhibits stable preparation process, high radionuclide loading rate, strong targeting, and good biocompatibility. It demonstrates excellent targeted uptake ability against refractory thyroid cancer cells in vitro. This invention provides a novel strategy and reliable experimental basis for overcoming the clinical treatment bottleneck of refractory thyroid cancer, possessing extremely high scientific research value and clinical translational potential.

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nano-formulation loaded with iodine-131 protein, characterized in that, Includes a target carrier and radioactive iodine-131; The targeting carrier is composed of transferrin, polyethylene glycol, and cyclodextrin covalently linked together. The radioactive iodine-131, in the form of triiodine anions, is loaded into the hydrophobic cavity of the cyclodextrin through non-covalent physical inclusion.

2. The iodine-131 loaded protein nanoparticle formulation according to claim 1, characterized in that, The molecular weight of the polyethylene glycol is 2000 Da; And / or, the cyclodextrin is selected from at least one of β-cyclodextrin and mono-(6-amino-6-deoxy)-β-cyclodextrin; And / or, in the target carrier, transferrin is linked to polyethylene glycol via a thioether bond, and polyethylene glycol is linked to cyclodextrin via an amide bond; And / or, the hydrated particle size of the nano-formulation in water is 180–260 nm, and the zeta potential is -10 mV to -20 mV.

3. The iodine-131 loaded protein nanoparticle formulation according to claim 1, characterized in that, In the nano-formulation, triiodide anions are encapsulated within the hydrophobic cavity of cyclodextrin, and cyclodextrin is covalently linked to transferrin via polyethylene glycol chains.

4. A method for preparing the iodine-131 loaded protein nanoparticle formulation according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Transferrin was modified by thiolation to obtain thiolated transferrin; (2) The cyclodextrin derivative was reacted with a heterodifunctional polyethylene glycol crosslinking agent to obtain a polyethylene glycol-cyclodextrin intermediate with maleimide groups; (3) The intermediate of step (2) is coupled with the thiolated transferrin of step (1) via Michael addition reaction to obtain the targeting vector CD-PEG-Tf; (4) Using the saturated aqueous solution method, the targeting carrier from step (3) is mixed with a solution containing... 131 Mixing the triiodine anion solution of I, so that 131 The iodine-131 protein nanoparticles were encapsulated into the cyclodextrin cavity and purified to obtain the nanoparticles.

5. The method for preparing the iodine-131 loaded protein nanoparticles according to claim 4, characterized in that, In step (1), the thiolizing agent is 2-iminothiacyclopentane hydrochloride.

6. The method for preparing the iodine-131 loaded protein nanoparticles according to claim 4, characterized in that, In step (2), the cyclodextrin derivative is mono-(6-amino-6-deoxy)-β-cyclodextrin, and the heterobifunctional polyethylene glycol crosslinking agent is NHS-PEG2000-MAL; the reaction is carried out in anhydrous dimethyl sulfoxide, and triethylamine is added as a catalyst.

7. The method for preparing the iodine-131 loaded protein nanoparticle formulation according to claim 4, characterized in that, In step (4), the saturated aqueous solution method includes: mixing elemental iodine and Na... 131 I mixes with water to form 131 The solution is then mixed with the targeting carrier; The mass ratio of the target carrier to elemental iodine is 1:5 to 1:20, the reaction temperature is 20 to 30°C, the reaction time is 2 to 6 hours, and the mixture is stirred in the dark.

8. The method for preparing the iodine-131 loaded protein nanoparticles according to claim 4, characterized in that, In step (4), the purification is carried out by dialysis or ultrafiltration; the molecular weight cutoff for dialysis is 10 kDa, and the dialysis time is 8 to 24 hours.

9. The use of the iodine-131 loaded protein nanoformulation according to any one of claims 1-3 in the preparation of a medicament for treating refractory thyroid cancer.

10. The application of the iodine-131 loaded protein nanoparticles according to claim 9 in the preparation of a drug for treating refractory thyroid cancer, characterized in that, The refractory thyroid cancer includes at least one of iodine-resistant differentiated thyroid cancer, undifferentiated thyroid carcinoma, and medullary thyroid carcinoma.

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