Radionuclide-labeled nano delivery system and preparation method thereof

Abstract

The invention discloses a radionuclide-labeled nano delivery system and a preparation method thereof, and belongs to the technical field of nano drugs. The radionuclide-labeled nano delivery system comprises a radionuclide-labeled micelle, wherein a near-infrared fluorescent material and a targeted drug are wrapped in a hydrophobic core of the micelle; the surfaces of the micelles are modified with specific molecules capable of identifying tumor cells in a targeted manner; the radionuclide-labeled nano delivery system can specifically recognize the focus of iodine-refractory thyroid cancer, deliver molecular targeting drugs after precise targeting of tumors, reverse the dedifferentiation state of iodine-refractory thyroid cancer, increase and improve the number and quality of sodium-iodine transporters, and sensitize the absorption of radionuclides. Meanwhile, more radionuclides are delivered to the focus area, the treatment effect is synergistically enhanced, and in addition, the in-vivo delivery process of the bionic nano radiotherapy sensitizer can be traced based on the near-infrared fluorescent material.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomedicine, and in particular relates to a radionuclide-labeled nano delivery system and a preparation method thereof. Background Art

[0002] Differentiated thyroid cancer (DTC), which originates from thyroid follicular epithelial cells, accounts for the largest proportion of thyroid malignancies, reaching 90-95%. Despite the successful treatment with surgery, thyroid stimulating hormone (TSH) suppression therapy, 131 The prognosis is good after treatment with I and other methods, and the 10-year survival rate can reach 90%, but there are still 5% to 15% of DTC patients who need 131 Iodine-refractory differentiated thyroid cancer (RAIR-DTC) is a type of thyroid cancer that is insensitive to iodine therapy and develops into iodine-refractory differentiated thyroid cancer (RAIR-DTC). The average survival of RAIR-DTC patients is only 3 to 5 years, and the 10-year survival rate is less than 10%. Current treatment options for RAIR-DTC primarily include local therapies (including surgical resection, external beam radiotherapy, and ablation) and systemic therapies (including targeted therapies that inhibit tumor growth and / or angiogenesis, differentiation-inducing therapy, and chemotherapy). However, these treatments have not been effective. Therefore, developing advanced approaches to improve the treatment of iodine-refractory thyroid cancer is of great fundamental research and clinical significance.

[0003] RAIR-DTC pair 131 The main reason for insensitivity to I treatment is that various mechanisms lead to tumor dedifferentiation, resulting in downregulation, localization or dysfunction of the sodium / iodide symporter (NIS) of tumor cells. Among them, genetic changes play an important role, especially BRAFV600E mutation and TERT promoter mutation. BRAFV600E mutation can affect the proliferation and differentiation state of thyroid cancer cells by activating the mitogen-activated protein kinase (MAPK) pathway, and ultimately lead to "dedifferentiation" of thyroid cancer and reduced or even complete loss of expression of iodine metabolism-related proteins represented by NIS. Selective BRAF inhibitors vemurafenib and dabrafenib have shown a certain ability to induce redifferentiation in small sample studies (induction success rate of 40% to 69%), but in subsequent 131Treatment with BRAF has failed to improve the complete remission rate in RAIR-DTC patients. Furthermore, due to its systemic distribution, it increases the risk of adverse events such as cardiomyopathy, bleeding, and skin malignancies. Therefore, achieving targeted, controlled release of BRAF inhibitors to induce redifferentiation of dedifferentiated thyroid cancer is of great research significance for reversing the sensitivity of RAIR-DTC to radioiodine therapy and improving its efficacy.

[0004] In summary, the development of a nano-delivery platform that has radioactive therapeutic means and can deliver molecular targeted drugs in a directed and controllable manner, and has effective tracing capabilities, is of great significance for the treatment of iodine-refractory thyroid cancer. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a radionuclide-labeled nano-delivery system and a preparation method thereof, which solve the problems in the prior art.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A radionuclide-labeled nano-delivery system comprises: radionuclide-labeled micelles, wherein the hydrophobic core of the micelles is encapsulated with near-infrared fluorescent materials and targeted drugs; and the surface of the micelles is modified with specific molecules that can target and recognize tumor cells.

[0008] Furthermore, the radionuclide is: 131 I. 125 I. 188 Re、 89 Sr. 177 Any one of Lu.

[0009] Furthermore, the near-infrared fluorescent material is: Ag2S, Ag2Se, Ag2Te, Ag2S x Se 1-x 、Ag2Se x Te 1-x ,AgAuSe,AgAuTe,AgAuS,AgInS2,AgInSe 2、 Any one or more combinations of Au:Ag2Te, Ag2Te@Ag2S, Ag2Se@Ag2S, PbSe, PbS@CdS and PbS quantum dots.

[0010] Furthermore, the targeted drug is any one or more combinations of gefitinib, dabrafenib, PD-1 / PD-L1 inhibitors, sorafenib, sunitinib, imatinib, and olaparib.

[0011] Furthermore, the specific molecule capable of targeting and identifying tumor cells is: hyaluronic acid-tyramine.

[0012] Furthermore, the micelle is: DSPE-PEG 2000 -NH2 polymer micelles.

[0013] The preparation method of the above-mentioned radionuclide-labeled nano-delivery system comprises the following steps:

[0014] Micelle are prepared by emulsification-solvent evaporation method using amphiphilic block copolymers, and targeted drugs and near-infrared fluorescent materials are encapsulated in the hydrophobic core of micelle;

[0015] Modify the surface of micelles with specific molecules that can target and recognize tumor cells;

[0016] Radioactive nuclides were oxidized and labeled by the Lodogen method to obtain radioactive nuclide-labeled nanodelivery systems.

[0017] Furthermore, the steps of encapsulating the targeted drug and the near-infrared fluorescent material in the hydrophobic core of the micelle are as follows:

[0018] Under ultrasonic conditions, the near-infrared fluorescent material, the gel layer, and the targeted drug system completely dissolved in dichloromethane are dropped into the aqueous phase system for ice-bath ultrasonic emulsification to obtain an oil-in-water emulsion;

[0019] The oil-in-water emulsion was then subjected to rotary evaporation to remove the organic solvent and finally concentrated by ultrafiltration.

[0020] Furthermore, the steps of modifying the micelle surface with specific molecules that can target and recognize tumor cells are as follows:

[0021] Dissolve the specific molecule, EDC, and NHS in MES buffer, and add EDC and NHS solutions successively to the specific molecule solution while stirring to perform activation reaction;

[0022] After the activation reaction, the pH value of the reaction solution is adjusted to 8.3, and micelles containing targeted drugs and near-infrared fluorescent materials are added for cross-linking; finally, after purification, specific molecules can be modified on the surface of the micelles.

[0023] Furthermore, the steps of oxidatively labeling radionuclides by the Lodogen method are:

[0024] Dissolve Lodogen in dichloromethane, add to a test tube and blow dry to obtain a Lodogen labeled test tube;

[0025] Then, the surface-modified micelles, PBS and strontium chloride solution were added to react with stirring, and then the radionuclide-labeled nanodelivery system was obtained by purification.

[0026] Beneficial effects of the present invention:

[0027] The radionuclide-labeled nano-delivery system of the present invention can specifically identify iodine-refractory thyroid cancer lesions, accurately target tumors and deliver molecular targeted drugs, reverse the "dedifferentiation" state of iodine-refractory thyroid cancer, increase and improve the number and quality of sodium-iodine transporters, enhance the absorption of radionuclides, and simultaneously deliver more radionuclides to the lesion area, synergistically enhancing the therapeutic effect. In addition, based on near-infrared fluorescent materials, the in vivo delivery process of biomimetic nano-radiotherapy sensitizers can be traced. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0029] Figure 1 Schematic diagram of the structure of the radionuclide-labeled nanodelivery system;

[0030] Figure 2 This is a transmission electron microscopy image of the radionuclide-labeled nanodelivery system prepared in Example 1;

[0031] Figure 3 Figure 1 shows the particle size distribution and surface charge results of the radionuclide-labeled nanodelivery system prepared in Example 1;

[0032] Figure 4 This is a graph showing the efficiency of the radionuclide-labeled nano-delivery system loaded with molecular targeted drugs prepared in Example 1;

[0033] Figure 5 This is a graph showing the efficiency of radionuclide loading in the radionuclide-labeled nanodelivery system prepared in Example 1;

[0034] Figure 6 This is a diagram showing the tumor tracking effect of the radionuclide-labeled nanodelivery system prepared in Example 1 in tumor-bearing mice;

[0035] Figure 7 This is a diagram showing the evaluation effect of the radionuclide-labeled nanodelivery system prepared in Example 1 in inducing redifferentiation of iodine-refractory thyroid cancer lesions in vivo. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0037] Example 1

[0038] In this embodiment, DSPE-PEG 2000 The hydrophobic inner cavity of the -NH2 polymer micelle is loaded with fluorescent probe Au:Ag2Te quantum dots and hydrophobic molecules targeting the small molecule drug dabrafenib. Through the amino sites on the surface of the micelle, hyaluronic acid molecules that specifically target dedifferentiated thyroid cancer are modified on it, and then the radionuclide drug is labeled using a nucleophilic substitution reaction. 131 I, thereby realizing a radionuclide-labeled nanodelivery system 131 The preparation of I-HA-DP@Da&AAT was carried out, and the in vivo tracking and sensitization to radiotherapy after inducing "redifferentiation" of iodine-refractory thyroid cancer in tumor-bearing mice were studied.

[0039] like Figure 1 As shown, the preparation process of a radionuclide-labeled nano-delivery system includes:

[0040] 1. Synthesis of Au:Ag2Te Quantum Dots in the NIR-IIb Region

[0041] 1) Synthesis of Ag2Te Quantum Dots: First, 0.0334 g of silver acetate (AgAc, 0.2 mmol) and 10 g of dichlorophenyltrichloroethane (DDT) were mixed and placed in a 100 mL three-necked flask and heated to 120°C under a nitrogen atmosphere to obtain a clear solution. Subsequently, 0.25 mL of tributyl phosphate-tellurium (TBP-Te, 0.05 mmol) solution was rapidly injected into the solution under vigorous stirring, and the reaction was continued at 120°C for 5 minutes. Another 0.25 mL of TBP-Te solution was injected and reacted for 5 minutes. After that, chloroform was added to the product, mixed thoroughly, and centrifuged at 10,000 g / min for 15 minutes to remove trace precipitates.

[0042] 2) Synthesis of Au:Ag2Te Quantum Dots: First, a gold precursor solution (Au-CFO) was prepared by dissolving 1 mmol of HAuCl4·3H2O in 10 mL of 20% oleylamine / chloroform solution (volume ratio 5:1). Subsequently, 0.01 mmol of Ag2Te quantum dots synthesized by the above method was dispersed in 20 mL of chloroform, and 0.1 mL of Au-CFO was added to react for 12 hours. Finally, the resulting Au:Ag2Te quantum dots were centrifuged and purified in acetone.

[0043] 2. Synthesis of Hyaluronic Acid-Tyramine (HA-TA)

[0044] 1) Using an electronic analytical balance, accurately weigh 350 mg of HA (monomer molecular weight 379, average molecular weight 3500, approximately 10 monomers per polymer chain, i.e., 10 carboxyl groups) and 700 mg of TA (molecular weight 137, each monomer of this small molecule contains one amino group). After optimizing the coupling efficiency through multiple feed ratios, the current feed ratio was selected for the reaction (at this ratio, the molar ratio of amino to carboxyl groups was 5:1). Prior to the reaction, 350 mg each of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, molecular weight 155) and N-hydroxysuccinimide (NHS, molecular weight 115) were weighed, with their molar ratios to the carboxyl groups in HA being approximately 2.2:1 and 3:1, respectively.

[0045] 2) Take an appropriate amount of pre-prepared MES buffer solution (pH = 5.5) to dissolve HA, EDC, and NHS respectively. Under ice bath and magnetic stirring at 400 rpm, EDC and NHS are successively added dropwise to the reaction bottle containing HA solution using a small-scale pipette. After activating the carboxyl groups in the dark for 30 minutes, TA dissolved in an appropriate amount of deionized water is added dropwise. Finally, the volume is adjusted to an appropriate volume with deionized water according to the actual size of the reaction bottle and stirrer and the actual stirring effect brought by the stirring speed. Try to keep the reaction under ice bath conditions throughout the reaction for 24 hours before stopping the reaction (if ice bathing cannot be guaranteed throughout the reaction, it is recommended to keep ice bathing for at least the first 4 hours to reduce the degradation of the active ester in the reaction and avoid affecting the coupling efficiency).

[0046] 3) The reaction product was carefully transferred to a pre-treated dialysis bag (molecular cutoff, 1000 Da) and dialyzed in deionized water for 48 h (deionized water was replaced every 4 h for the first 12 h, and every 8 h thereafter) to purify the reaction product to remove free TA and excess EDC and NHS to obtain a pure hyaluronic acid-tyramine (HA-TA) aqueous solution. After dialysis, the solution was poured into a large open container such as a cell culture dish or beaker, pre-frozen, and then transferred to a freeze dryer for lyophilization for 2-3 days to obtain a white, flocculent hyaluronic acid-tyramine compound. After collection, the container was sealed and stored in a -20°C refrigerator for later use.

[0047] 3. 131 Synthesis of I-HA-DP@Da&AAT

[0048] 1) Raw material preparation: 0.6 mg of Au:Ag2Te quantum dots were dissolved in chloroform, most of the solvent was dried with nitrogen, and then an appropriate amount of anhydrous ethanol was added and centrifuged (10000 rpm, 3 min) to wash the supernatant. The excess ligand in the supernatant was removed to obtain the required Au:Ag2Te quantum dot solid raw material; 6 mg of amphiphilic block copolymer DSPE-PEG was weighed using an electronic analytical balance. 2000 -NH2, 0.75 mg of the hydrophobic small molecule drug dabrafenib, 6 mg of the prepared hyaluronic acid-tyramine conjugate HA-TA, 6 mg of the cross-linker EDC, and 4 mg of the cross-linking stabilizer NHS; prepare 10 mL of deionized water in a 20 mL glass reaction bottle; prepare two small ice boxes and an appropriate amount of dichloromethane.

[0049] 2) Preparation process 1: Using a cell ultrasonic disruptor and a rotary evaporator as the main preparation instruments. The first step is to complete emulsification using a cell ultrasonic disruptor, that is, under ultrasonic conditions (3mm probe, 30% ultrasonic power, 3s / 2s ultrasonic interval, 4min ultrasonic time), the Au:Ag2Te quantum dots and polymer DSPE-PEG completely dissolved in dichloromethane are emulsified. 2000 A total of 200 μL of a water-immiscible oil phase containing NH2 and the small molecule dabrafenib was slowly added dropwise to a 10 mL aqueous phase in a reaction flask for ultrasonic emulsification in an ice bath. After ultrasonication, a milky oil-in-water (O / W) emulsion was obtained. During the rearrangement of the polymer, micelles were formed to encapsulate the drug and quantum dots. The size of the micelles was related to the ultrasonic conditions. The prepared emulsion was quickly transferred to a round-bottom flask and rotary evaporation (40°C, 10 minutes) was performed to remove the organic solvent, resulting in a clear nanomicelle suspension. The suspension was then concentrated by ultrafiltration to obtain the micelle main body DP@Da&AAT.

[0050] 3) Preparation process 2: The second step is to carry out surface functionalization modification of the micelle host DP@Da&AAT. The prepared HA-TA is modified on the surface of DP@Da&AAT through an aminocarboxyl reaction to obtain HT-DP@Da&AAT.

[0051] First, HA-TA, EDC, and NHS were dissolved in MES buffer (adjusted to pH = 5.5), and EDC and NHS solutions were added to the HA-TA solution successively while stirring. The reaction was allowed to react at room temperature for 30 minutes to activate the carboxyl groups on HA-TA. After activation, the pH of the reaction solution was adjusted to 8.3, and DP@Da&AAT was added dropwise. After cross-linking at room temperature for 24 hours, the mixture was purified using a 100KDa ultrafiltration tube to obtain functionally modified HT-DP@Da&AAT micelles.

[0052] 4) Preparation process three: The third step is to use the Lodogen method to functionalize the micelle main body HT-DP@Da&AAT 131 I / I mark.

[0053] Weigh 10 μg of Lodogen and dissolve it in 10 μL of dichloromethane. Add it to the bottom of a 1.5 mL hard plastic test tube and blow dry it gently with N2. Dotted crystals can be seen at the bottom of the test tube. This is the Lodogen-labeled test tube. Add 200 μL of HT-DP@Da&AAT (1 mg / mL), 200 μL of PBS and Na 131 I solution 30 μL (0.898 mCi) / NaI solution, stirred at room temperature for 7 min, the reaction solution was removed, and free Na was removed using a 10 KDa ultrafiltration tube. 131 I, after purification, the final product is obtained 131 I-HA-DP@Da&AAT drug.

[0054] 4. 131 I-HA-DP@Da&AAT in vivo tracking

[0055] Tail vein injection 200 μL 131 After I-DP@Da&AAT 1mg / kg (calculated based on Au:Ag2Te concentration) was injected into tumor-bearing mice (Suzhou Jingweiyu Biotechnology Co., Ltd.), the tumors were observed under near-infrared in vivo imaging, and near-infrared fluorescence signals from the tumor sites of the tumor-bearing mice were collected for real-time dynamic tracking. 131 Migration of I-DP@Da&AAT in tumor-bearing mice.

[0056] 5. 131 Evaluation of I-HA-DP@Da&AAT-induced redifferentiation of iodine-refractory thyroid cancer lesions in vivo

[0057] Tail vein injection 200 μL 131After I-DP@Da&AAT 1 mg / kg (calculated based on Au:Ag2Te concentration) was injected into tumor-bearing mice (Suzhou Jingweiyu Biotechnology Co., Ltd.), the mice were fed for 1 week, and the mice were killed. The tumor tissues were removed for tumor tissue protein extraction and tissue protein immunoblotting WB to verify the changes in the expression of NIS, an indicator of tumor iodine uptake.

[0058] Among them, the transmission electron microscopy image of the delivery system prepared in this example is as follows Figure 2 As shown, it can be seen that: nano micelles 131 I-HA-DP@Da&AAT presents a regular spherical morphology on the copper mesh with uniform size. The particle size is about 80nm. The Au:Ag2Te quantum dots loaded inside the micelles can be clearly seen. The results prove that we have successfully synthesized nanomicelles with uniform particle size, regular morphology, and good encapsulation of functional drugs. 131 I-HA-DP@Da&AAT.

[0059] The particle size distribution and surface charge results of the delivery system are shown in Figures 2 and 3, respectively. Figure 3 As shown in A and B in the figure, it can be seen that the hyaluronic acid HA used for surface modification of nanomicelles has a charge of about -21.5mV. The charge of the hyaluronic acid-tyramine conjugate HT obtained after modification is reduced to -11.2mV. The change in charge can also indirectly prove the success of the coupling. Using the amphiphilic block copolymer DSPE-PEG2 000 -NH2 synthesized micelles have a surface charge of about +11.5mV. 131 I-HA was further coupled and modified to the surface of micelles for functional modification. The surface charge of micelles changed from the initial +11.5mV to about -9.32mV after modification. This proves that iodine-labeled hyaluronic acid HA was successfully modified on the micelle body, and nanomicelles loaded with multiple functional components were successfully obtained. 131 I-HA-DP@Da&AAT. The hydrated particle size of the unmodified micelle DP@Da&AAT is about 90nm. 131 The actual hydrated particle size of I-HA-DP@Da&AAT is about 100 nm.

[0060] The efficiency of the nano-delivery system loading targeted drugs is shown in the figure Figure 4 As shown in the figure, the absorption spectrum of the molecular targeted inhibitor Dabrafenib was first measured by UV-visible absorption spectrometer, and the characteristic absorbance at its characteristic absorption peak was determined by setting a concentration gradient. According to the Lambert-Beer law, the standard curve of Dabrafenib was fitted ( Figure 4 A), so that the drug concentration of Dabrafenib can be calculated by measuring the size of the characteristic absorbance.131 After I-HA-DP@Da&AAT, its absorption spectrum was measured. As shown in the figure, a significant characteristic absorption peak appeared at 348nm, the characteristic absorption peak position of Dabrafenib, indicating that the hydrophobic small molecule drug Dabrafenib was successfully coated on 131 The hydrophobic core of I-HA-DP@Da&AAT was then obtained. The absorbance of free Dabrafenib in the supernatant after ultrafiltration purification was then measured and the free Dabrafenib after a batch of synthesis was quantified using a standard curve to obtain the actual Dabrafenib loading amount. Under the final optimized synthesis process, the encapsulation efficiency of Dabrafenib was approximately 87% and the drug loading was approximately 8.3% ( Figure 4 B).

[0061] The efficiency of radionuclide loading in the nano-delivery system is shown in the figure Figure 5 As shown in the figure, it can be seen that under the labeling process used in this study, significant 131 I-HA-DP@Da&AAT signal, but only very low free state 131 I signal is quantified by integration method. 131 The radiochemical purity of I-HA-DP@Da&AAT was 98%, that is, the labeling efficiency was 98%.

[0062] The tumor tracking effect of the nano-delivery system in tumor-bearing mice is shown in the figure. Figure 6 As shown, the active targeted drug group was systematically evaluated using the NIR-II fluorescence imaging platform. 131 I-HA-DP@Da&AAT and non-active targeted drug group 131 The in vivo targeting level of I-DP@Da&AAT. Figure 4-7 Shown, indicating 131 I-HA-DP@Da&AAT drug has good tumor targeting ability. The drug can be observed to be significantly enriched in the tumor site 1 hour after administration, and after 24 hours of administration, the active targeted drug group 131 I-HA-DP@Da&AAT tumor site signal-to-noise ratio can reach 6.9, rather than active targeted drug group 131 I-DP@Da&AAT is only 1.8. Through semi-quantitative fluorescence comparison of isolated tumor tissue, active targeted drug 131 The drug enrichment of I-HA-DP@Da&AAT is a non-active targeted drug 131 I-DP@Da&AAT is approximately 4.2 times that of I-DP@Da&AAT.

[0063] The evaluation effect of this nano-delivery system on inducing redifferentiation of iodine-refractory thyroid cancer lesions in vivo is shown in the figure below. Figure 7As shown, it can be seen that at a dose of 6 mg / kg, 1 week of administration 131 A single administration cycle of I-HA-DP@Da&AAT drug can achieve BRAF at the mouse level. V600E Redifferentiation of iodine-refractory thyroid cancer tissues is characterized by increased differentiation level, characterized by increased NIS protein expression.

[0064] Example 2

[0065] This example uses DSPE-PEG 2000 The hydrophobic inner cavity of the -NH2 polymer micelles is loaded with fluorescent probes Au:Ag2Te quantum dots and the drug zoledronic acid. Through the amino sites on the surface of the micelles, hyaluronic acid molecules that specifically target dedifferentiated thyroid cancer are modified on them, and then the radionuclide drug is labeled using a nucleophilic substitution reaction. 89 Sr, thereby realizing the preparation of radionuclide-labeled nanodelivery system, and conducting in vivo tracing and sensitized radiotherapy research in tumor-bearing mice.

[0066] 1. Synthesis of Au:Ag2Te Quantum Dots in the NIR-IIb Region

[0067] 1) Synthesis of Ag2Te Quantum Dots: First, 0.0334 g of silver acetate (AgAc, 0.2 mmol) and 10 g of dichlorophenyltrichloroethane (DDT) were mixed and placed in a 100 mL three-necked flask and heated to 120°C under a nitrogen atmosphere to obtain a clear solution. Subsequently, 0.25 mL of tributyl phosphate-tellurium (TBP-Te, 0.05 mmol) solution was rapidly injected into the solution under vigorous stirring, and the reaction was continued at 120°C for 5 minutes. Another 0.25 mL of TBP-Te solution was injected and reacted for 5 minutes. After that, chloroform was added to the product, mixed thoroughly, and centrifuged at 10,000 g / min for 15 minutes to remove trace precipitates.

[0068] 2) Synthesis of Au:Ag2Te Quantum Dots: First, a gold precursor solution (Au-CFO) was prepared by dissolving 1 mmol of HAuCl4·3H2O in 10 mL of 20% oleylamine / chloroform solution (volume ratio 5:1). Subsequently, 0.01 mmol of Ag2Te quantum dots synthesized by the above method was dispersed in 20 mL of chloroform, and 0.1 mL of Au-CFO was added to react for 12 hours. Finally, the resulting Au:Ag2Te quantum dots were centrifuged and purified in acetone.

[0069] 2. Synthesis of Hyaluronic Acid-Tyramine (HA-TA)

[0070] 1) Using an electronic analytical balance, accurately weigh 350 mg of HA (monomer molecular weight 379, average molecular weight 3500, approximately 10 monomers per polymer chain, i.e., 10 carboxyl groups) and 700 mg of TA (molecular weight 137, each monomer of this small molecule contains one amino group). After optimizing the coupling efficiency through multiple feed ratios, the current feed ratio was selected for the reaction (at this ratio, the molar ratio of amino to carboxyl groups was 5:1). Prior to the reaction, 350 mg each of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, molecular weight 155) and N-hydroxysuccinimide (NHS, molecular weight 115) were weighed, with their molar ratios to the carboxyl groups in HA being approximately 2.2:1 and 3:1, respectively.

[0071] 2) Take an appropriate amount of pre-prepared MES buffer solution (pH = 5.5) to dissolve HA, EDC, and NHS respectively. Under ice bath and magnetic stirring at 400 rpm, EDC and NHS are successively added dropwise to the reaction bottle containing HA solution using a small-scale pipette. After activating the carboxyl groups in the dark for 30 minutes, TA dissolved in an appropriate amount of deionized water is added dropwise. Finally, the volume is adjusted to an appropriate volume with deionized water according to the actual size of the reaction bottle and stirrer and the actual stirring effect brought by the stirring speed. Try to keep the reaction under ice bath conditions throughout the reaction for 24 hours before stopping the reaction (if ice bathing cannot be guaranteed throughout the reaction, it is recommended to keep ice bathing for at least the first 4 hours to reduce the degradation of the active ester in the reaction and avoid affecting the coupling efficiency).

[0072] 3) The reaction product was carefully transferred to a pre-treated dialysis bag (molecular cutoff, 1000 Da) and dialyzed in deionized water for 48 h (deionized water was replaced every 4 h for the first 12 h, and every 8 h thereafter) to purify the reaction product to remove free TA and excess EDC and NHS to obtain a pure hyaluronic acid-tyramine (HA-TA) aqueous solution. After dialysis, the solution was poured into a large open container such as a cell culture dish or beaker, pre-frozen, and then transferred to a freeze dryer for lyophilization for 2-3 days to obtain a white, flocculent hyaluronic acid-tyramine compound. After collection, the container was sealed and stored in a -20°C refrigerator for later use.

[0073] 3. Synthesis of Radionuclide Nanodelivery System

[0074] 1) Raw material preparation: 0.6 mg of Au:Ag2Te quantum dots were dissolved in chloroform, most of the solvent was dried with nitrogen, and then an appropriate amount of anhydrous ethanol was added and centrifuged (10000 rpm, 3 min) to wash the supernatant. The excess ligand in the supernatant was removed to obtain the required Au:Ag2Te quantum dot solid raw material; 6 mg of amphiphilic block copolymer DSPE-PEG was weighed using an electronic analytical balance.2000 -NH2, 0.75 mg of zoledronic acid, 6 mg of prepared hyaluronic acid-tyramine conjugate HA-TA, 6 mg of cross-linker EDC, and 4 mg of cross-linking stabilizer NHS; prepare 10 mL of deionized water in a 20 mL glass reaction bottle; prepare two small ice boxes and an appropriate amount of dichloromethane.

[0075] 2) Preparation process 1: Using a cell ultrasonic disruptor and a rotary evaporator as the main preparation instruments. The first step is to complete emulsification using a cell ultrasonic disruptor, that is, under ultrasonic conditions (3mm probe, 30% ultrasonic power, 3s / 2s ultrasonic interval, 4min ultrasonic time), the Au:Ag2Te quantum dots and polymer DSPE-PEG completely dissolved in dichloromethane are emulsified. 2000 A water-immiscible oil phase system containing 200 μL of NH₂ and zoledronic acid was slowly added dropwise to a 10 mL aqueous phase in a reaction flask for ultrasonic emulsification in an ice bath. After ultrasonication, a milky oil-in-water (O / W) emulsion was obtained. During the rearrangement of the polymer, micelles were formed, encapsulating the drug and quantum dots. The size of the micelles was related to the ultrasonic conditions. The prepared emulsion was quickly transferred to a round-bottom flask and rotary evaporated (40°C, 10 minutes) to remove the organic solvent, resulting in a clear nanomicelle suspension. This suspension was then concentrated by ultrafiltration to obtain the micelle bulk.

[0076] 3) Preparation process 2: The second step is to carry out surface functional modification of the micelle body, and the prepared HA-TA is modified on the micelle surface through aminocarboxyl reaction.

[0077] First, HA-TA, EDC, and NHS were dissolved in MES buffer (adjusted to pH = 5.5), and EDC and NHS solutions were added to the HA-TA solution successively while stirring. The reaction was carried out at room temperature for 30 minutes to activate the carboxyl groups on HA-TA. After activation, the pH of the reaction solution was adjusted to 8.3, and micelles were added dropwise. After cross-linking at room temperature for 24 hours, the micelles were purified using a 100KDa ultrafiltration tube to obtain functionally modified micelles.

[0078] 4) Preparation process three: In the third step, the functionalized micelle body is labeled with radionuclides using the Lodogen method.

[0079] Weigh 10 μg of Lodogen and dissolve it in 10 μL of dichloromethane. Pour the mixture into the bottom of a 1.5 mL rigid plastic test tube and gently blow dry with N2. Dotted crystals will be visible at the bottom of the test tube, thus obtaining a Lodogen-labeled test tube. Add 200 μL of HT-DP@Da&AAT (1 mg / mL), 200 μL of PBS, and 30 μL of strontium chloride solution. Stir at room temperature for 7 minutes, then remove the reaction solution and use a 10 kDa ultrafiltration tube to remove free nuclides. Purify the final product.

[0080] 4. In vivo tracking of radionuclide nanodelivery systems

[0081] After tail vein injection of 200 μL of 1 mg / kg (calculated as Au:Ag2Te concentration) radionuclide nanodelivery system into tumor-bearing mice (Suzhou Jingweiyu Biotechnology Co., Ltd.), the tumor sites of the tumor-bearing mice were observed under near-infrared in vivo imaging, and the near-infrared fluorescence signals of the tumor sites of the tumor-bearing mice were collected to track the migration of the radionuclide nanodelivery system in the tumor-bearing mice in real time.

[0082] Example 3

[0083] This example uses DSPE-PEG 2000 The hydrophobic inner cavity of the -NH2 polymer micelle is loaded with fluorescent probe Au:Ag2Te quantum dots and the drug bevacizumab. Through the amino sites on the surface of the micelle, hyaluronic acid molecules that specifically target dedifferentiated thyroid cancer are modified on it, and then the radionuclide drug is labeled using a nucleophilic substitution reaction. 90 Y, thereby realizing the preparation of radionuclide-labeled nanodelivery system, and conducting in vivo tracing and sensitized radiotherapy research in tumor-bearing mice.

[0084] 1. Synthesis of Au:Ag2Te Quantum Dots in the NIR-IIb Region

[0085] 1) Synthesis of Ag2Te Quantum Dots: First, 0.0334 g of silver acetate (AgAc, 0.2 mmol) and 10 g of dichlorophenyltrichloroethane (DDT) were mixed and placed in a 100 mL three-necked flask and heated to 120°C under a nitrogen atmosphere to obtain a clear solution. Subsequently, 0.25 mL of tributyl phosphate-tellurium (TBP-Te, 0.05 mmol) solution was rapidly injected into the solution under vigorous stirring, and the reaction was continued at 120°C for 5 minutes. Another 0.25 mL of TBP-Te solution was injected and reacted for 5 minutes. After that, chloroform was added to the product, mixed thoroughly, and centrifuged at 10,000 g / min for 15 minutes to remove trace precipitates.

[0086] 2) Synthesis of Au:Ag2Te Quantum Dots: First, a gold precursor solution (Au-CFO) was prepared by dissolving 1 mmol of HAuCl4·3H2O in 10 mL of 20% oleylamine / chloroform solution (volume ratio 5:1). Subsequently, 0.01 mmol of Ag2Te quantum dots synthesized by the above method was dispersed in 20 mL of chloroform, and 0.1 mL of Au-CFO was added to react for 12 hours. Finally, the resulting Au:Ag2Te quantum dots were centrifuged and purified in acetone.

[0087] 2. Synthesis of Hyaluronic Acid-Tyramine (HA-TA)

[0088] 1) Using an electronic analytical balance, accurately weigh 350 mg of HA (monomer molecular weight 379, average molecular weight 3500, approximately 10 monomers per polymer chain, i.e., 10 carboxyl groups) and 700 mg of TA (molecular weight 137, each monomer of this small molecule contains one amino group). After optimizing the coupling efficiency through multiple feed ratios, the current feed ratio was selected for the reaction (at this ratio, the molar ratio of amino to carboxyl groups was 5:1). Prior to the reaction, 350 mg each of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, molecular weight 155) and N-hydroxysuccinimide (NHS, molecular weight 115) were weighed, with their molar ratios to the carboxyl groups in HA being approximately 2.2:1 and 3:1, respectively.

[0089] 2) Take an appropriate amount of pre-prepared MES buffer solution (pH = 5.5) to dissolve HA, EDC, and NHS respectively. Under ice bath and magnetic stirring at 400 rpm, EDC and NHS are successively added dropwise to the reaction bottle containing HA solution using a small-scale pipette. After activating the carboxyl groups in the dark for 30 minutes, TA dissolved in an appropriate amount of deionized water is added dropwise. Finally, the volume is adjusted to an appropriate volume with deionized water according to the actual size of the reaction bottle and stirrer and the actual stirring effect brought by the stirring speed. Try to keep the reaction under ice bath conditions throughout the reaction for 24 hours before stopping the reaction (if ice bathing cannot be guaranteed throughout the reaction, it is recommended to keep ice bathing for at least the first 4 hours to reduce the degradation of the active ester in the reaction and avoid affecting the coupling efficiency).

[0090] 3) The reaction product was carefully transferred to a pre-treated dialysis bag (molecular cutoff, 1000 Da) and dialyzed in deionized water for 48 h (deionized water was replaced every 4 h for the first 12 h, and every 8 h thereafter) to purify the reaction product to remove free TA and excess EDC and NHS to obtain a pure hyaluronic acid-tyramine (HA-TA) aqueous solution. After dialysis, the solution was poured into a large open container such as a cell culture dish or beaker, pre-frozen, and then transferred to a freeze dryer for lyophilization for 2-3 days to obtain a white, flocculent hyaluronic acid-tyramine compound. After collection, the container was sealed and stored in a -20°C refrigerator for later use.

[0091] 3. Synthesis of Radionuclide Nanodelivery System

[0092] 1) Raw material preparation: 0.6 mg of Au:Ag2Te quantum dots were dissolved in chloroform, most of the solvent was dried with nitrogen, and then an appropriate amount of anhydrous ethanol was added and centrifuged (10000 rpm, 3 min) to wash the supernatant. The excess ligand in the supernatant was removed to obtain the required Au:Ag2Te quantum dot solid raw material; 6 mg of amphiphilic block copolymer DSPE-PEG was weighed using an electronic analytical balance.2000 -NH2, 0.75 mg bevacizumab and 6 mg prepared hyaluronic acid-tyramine conjugate HA-TA, 6 mg cross-linker EDC, 4 mg cross-linking stabilizer NHS; prepare 10 mL of deionized water in a 20 mL glass reaction bottle; prepare two small ice boxes and an appropriate amount of dichloromethane.

[0093] 2) Preparation process 1: Using a cell ultrasonic disruptor and a rotary evaporator as the main preparation instruments. The first step is to complete emulsification using a cell ultrasonic disruptor, that is, under ultrasonic conditions (3mm probe, 30% ultrasonic power, 3s / 2s ultrasonic interval, 4min ultrasonic time), the Au:Ag2Te quantum dots and polymer DSPE-PEG completely dissolved in dichloromethane are emulsified. 2000 A total of 200 μL of a water-immiscible oil phase system containing NH2 and bevacizumab was slowly added dropwise to a 10 mL aqueous phase system in a reaction flask for ice-bath ultrasonic emulsification. After the ultrasonic treatment, a milky oil-in-water (O / W) emulsion was obtained. During the rearrangement of the polymer, micelles were formed to encapsulate the drug and quantum dots. The size of the micelles was related to the ultrasonic conditions. The prepared emulsion was quickly transferred to a round-bottom flask and rotary evaporated (40°C, 10 minutes) to remove the organic solvent to obtain a clear nanomicelle suspension. The micelle body was finally obtained by ultrafiltration concentration.

[0094] 3) Preparation process 2: The second step is to carry out surface functional modification of the micelle body, and the prepared HA-TA is modified on the micelle surface through aminocarboxyl reaction.

[0095] First, HA-TA, EDC, and NHS were dissolved in MES buffer (adjusted to pH = 5.5), and EDC and NHS solutions were added to the HA-TA solution successively while stirring. The reaction was carried out at room temperature for 30 minutes to activate the carboxyl groups on HA-TA. After activation, the pH of the reaction solution was adjusted to 8.3, and micelles were added dropwise. After cross-linking at room temperature for 24 hours, the micelles were purified using a 100KDa ultrafiltration tube to obtain functionally modified micelles.

[0096] 4) Preparation process three: In the third step, the functionalized micelle body is labeled with radionuclides using the Lodogen method.

[0097] Weigh 10 μg of Lodogen and dissolve it in 10 μL of dichloromethane. Pour the mixture into the bottom of a 1.5 mL rigid plastic test tube and gently blow dry with N2. Dotted crystals will be visible at the bottom of the test tube, thus obtaining a Lodogen-labeled test tube. Add 200 μL of HT-DP@Da&AAT (1 mg / mL), 200 μL of PBS, and 30 μL of yttrium chloride solution. Stir at room temperature for 7 minutes, then remove the reaction solution and use a 10 kDa ultrafiltration tube to remove free nuclides. Purify the final product.

[0098] 4. In vivo tracking of radionuclide nanodelivery systems

[0099] After tail vein injection of 200 μL of 1 mg / kg (calculated as Au:Ag2Te concentration) radionuclide nanodelivery system into tumor-bearing mice (Suzhou Jingweiyu Biotechnology Co., Ltd.), the tumor sites of the tumor-bearing mice were observed under near-infrared in vivo imaging, and the near-infrared fluorescence signals of the tumor sites of the tumor-bearing mice were collected to track the migration of the radionuclide nanodelivery system in the tumor-bearing mice in real time.

[0100] In summary, the radionuclide nanodelivery system of the present invention can specifically identify tumors, avoiding collateral damage to surrounding normal tissues; increase radionuclide accumulation at the lesion site, enhancing radionuclide therapy sensitivity; and deliver tumor-targeted drugs, synergistically enhancing the therapeutic effect. Furthermore, the nanodelivery system's in vivo delivery process can be tracked using near-infrared second-zone fluorescence imaging technology. This strategy enables on-demand, precise drug delivery to cancer patients, enhances radionuclide therapy efficacy, reduces radiotherapy side effects, improves the quality of life of cancer patients, and prolongs their survival. This strategy is of great significance for improving patients' quality of life and reducing the economic costs of disease treatment.

[0101] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0102] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

1. A radionuclide-labeled nano-delivery system, characterized in that: include: Radioactive nuclide-labeled micelles, the hydrophobic core of which is encapsulated with near-infrared fluorescent materials and targeted drugs; the surface of the micelles is modified with specific molecules that can target and recognize tumor cells.

2. A radionuclide-labeled nano-delivery system according to claim 1, characterized in that: The radionuclides are: 131 I. 125 I. 188 Re、 89 Sr. 177 Any one of Lu.

3. The radionuclide-labeled nano-delivery system according to claim 1, characterized in that: The near-infrared fluorescent material is: Ag2S, Ag2Se, Ag2Te, Ag2S x Se 1-x 、Ag2Se x Te 1-x , any one or more combinations of AgAuSe, AgAuTe, AgAuS, AgInS2, AgInSe2, Au:Ag2Te, Ag2Te@Ag2S, Ag2Se@Ag2S, PbSe, PbS@CdS and PbS quantum dots.

4. The radionuclide-labeled nano-delivery system according to claim 1, characterized in that: The targeted drug is any one or more combinations of gefitinib, dabrafenib, PD-1 / PD-L1 inhibitors, sorafenib, sunitinib, imatinib, and olaparib.

5. The radionuclide-labeled nano-delivery system according to claim 1, characterized in that: The specific molecule capable of targeting and identifying tumor cells is: hyaluronic acid-tyramine.

6. The radionuclide-labeled nano-delivery system according to claim 1, characterized in that: The micelle is: DSPE-PEG 2000 -NH2 polymer micelles.

7. The method for preparing a radionuclide-labeled nano-delivery system according to any one of claims 1 to 6, characterized in that: The following steps are involved: Micelle are prepared by emulsification-solvent evaporation method using amphiphilic block copolymers, and targeted drugs and near-infrared fluorescent materials are encapsulated in the hydrophobic core of micelle; Modify the surface of micelles with specific molecules that can target and recognize tumor cells; Radioactive nuclides were oxidized and labeled by the Lodogen method to obtain radioactive nuclide-labeled nanodelivery systems.

8. The method for preparing a radionuclide-labeled nano-delivery system according to claim 7, characterized in that: The steps of encapsulating the targeted drug and near-infrared fluorescent material in the hydrophobic core of the micelle are as follows: Under ultrasonic conditions, the near-infrared fluorescent material, the gel layer, and the targeted drug system completely dissolved in dichloromethane are dropped into the aqueous phase system for ice-bath ultrasonic emulsification to obtain an oil-in-water emulsion; The oil-in-water emulsion was then subjected to rotary evaporation to remove the organic solvent and finally concentrated by ultrafiltration.

9. The method for preparing a radionuclide-labeled nano-delivery system according to claim 7, characterized in that: The steps for micelle surface modification to target specific molecules that can recognize tumor cells are as follows: Dissolve the specific molecule, EDC, and NHS in MES buffer, and add EDC and NHS solutions successively to the specific molecule solution while stirring to perform activation reaction; After the activation reaction, the pH value of the reaction solution is adjusted to 8.3, and micelles containing targeted drugs and near-infrared fluorescent materials are added for cross-linking; finally, after purification, specific molecules can be modified on the surface of the micelles.

10. The method for preparing a radionuclide-labeled nano-delivery system according to claim 7, characterized in that: The steps for oxidative labeling of radionuclides by the Lodogen method are: Dissolve Lodogen in dichloromethane, add to a test tube and blow dry to obtain a Lodogen labeled test tube; Then, the surface-modified micelles, PBS and strontium chloride solution were added to react with stirring, and then the radionuclide-labeled nanodelivery system was obtained by purification.

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