Nano material for treating uveal melanoma and preparation method, application and drug thereof

Abstract

The application belongs to the field of medicine, and particularly relates to a nanomaterial for treating uveal melanoma, a preparation method, application and medicine of the nanomaterial, wherein the nanomaterial for treating uveal melanoma comprises a core and a hyaluronic acid coating layer covering the core; the core comprises a zinc / copper bimetallic organic framework and a compound of formula 1, wherein formula 1 is. In view of the pathogenesis, characteristics and treatment problems of ophthalmology UM, the application provides a brand-new nanomaterial which is based on the combined synergy of zinc / copper bimetallic organic framework, compound of formula 1 and HA component and structure, so as to realize synergy, to inhibit cancer cells of uveal melanoma in a high-selectivity manner based on multi-mechanism combined synergy, to be able to exert excellent treatment activity at a low drug dosage, and to be able to effectively inhibit metastasis. In addition, the nanomaterial has excellent biological safety.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, specifically relating to nanomaterials for treating uveal melanoma, their preparation methods, applications, and pharmaceutical applications. Background Technology

[0002] Uveal melanoma (UM) is a highly aggressive intraocular malignant tumor, differing significantly from cutaneous melanoma in its tumor-originating cells, driver mutations, and sites of occurrence and metastasis. This unique biological characteristic leads to poor response to conventional chemotherapy and most targeted or immunotherapies used for cutaneous melanoma. Once distant metastasis occurs, the median survival is only 6–12 months, resulting in a very poor clinical prognosis. Therefore, there is an urgent clinical need for new therapies that can effectively inhibit UM progression and distant metastasis.

[0003] For primary uveal melanoma, current local treatments mainly include surgical resection, radiotherapy, and laser therapy. While these can achieve good local control, they cannot effectively reduce the risk of long-term metastasis. For metastatic UM, since 90% of the metastatic lesions are located in the liver, current treatments primarily target liver metastases with local therapies (hepatic artery infusion chemotherapy, local radiotherapy, and surgical resection), combined with systemic chemotherapy, immunotherapy, and targeted therapy. However, traditional chemotherapy has limited efficacy; the objective response rate of immune checkpoint inhibitors (such as PD-1 / CTLA-4 inhibitors) is less than 10%; and the survival benefit of targeted inhibitors is also very limited. In recent years, the bispecific T-cell connector Tebentafusp became the first drug to improve the survival of metastatic patients, but it is only applicable to... For positive patients, the objective response rate is only 30% to 40%, which is insufficient to meet clinical needs.

[0004] With the continuous evolution of technology, some treatment methods for uveal melanoma have been developed. For example, patent document HK40128336A discloses a treatment method using a protein kinase C (PKC) inhibitor. Patent document CN121154782A discloses the application of a small molecule compound, DRP-104, in the preparation of a drug for treating uveal melanoma. Patent document US20260053905A1 discloses a uveal melanoma vaccine. Patent document WO2026013071A1 discloses a SETDB1 inhibitor for treating uveal melanoma. Patent document CN120131738A discloses the application of licorice extract in the treatment of uveal melanoma. However, none of the above technologies have fully verified the therapeutic effect of this treatment method in metastatic uveal melanoma.

[0005] In summary, although existing technologies have disclosed some treatment methods for uveal melanoma, there is still a lack of highly effective, universally applicable, and fully validated treatment options for metastatic uveal melanoma. Summary of the Invention

[0006] In view of the problems existing in the prior art, the first objective of the present invention is to provide a nanomaterial for treating uveal melanoma, aiming to provide a novel nanomaterial that can selectively target and inhibit uveal melanoma and help inhibit metastasis.

[0007] The second objective of this invention is to provide a method for preparing the aforementioned nanomaterial and its application in the preparation of drugs for treating uveal melanoma.

[0008] A third objective of this invention is to provide a medicament for treating uveal melanoma comprising the nanomaterials described above.

[0009] Uveal melanoma (UM) and cutaneous melanoma (CM), though both belonging to the melanoma family, are fundamentally two distinct diseases. The core difference lies in their origins: UM occurs in the uvea of ​​the eye, originating from neural crest cells, while CM occurs in the skin and is closely related to ultraviolet (UV) exposure. This difference in origin and location determines significant differences in their pathogenic genes: UM is primarily driven by mutations in the GNAQ / GNA11 genes, while CM commonly involves mutations in the BRAF gene. Consequently, their biological behaviors are also vastly different: UM primarily metastasizes hematogenously, with nearly 90% of metastases concentrated in the liver, and progresses rapidly after metastasis; CM, on the other hand, frequently metastasizes via the lymphatic system, with a wider range of metastatic sites. Notably, due to different molecular mechanisms, CM responds well to immunotherapy and BRAF / MEK targeted therapy, while UM is generally insensitive to these therapies, and once metastasized, the prognosis is extremely poor, with very limited treatment options. Addressing the characteristics of UM and the challenges of treating its tendency to metastasize, this invention provides the following solution:

[0010] Nanomaterials for treating uveal melanoma, comprising a core and a hyaluronic acid coating (also known as a HA layer) encapsulating the core; said core comprises a zinc / copper bimetallic organic framework and a compound of formula 1;

[0011] Formula 1;

[0012] In Formula 1, R1 is a halogen; R2 is H, a halogen, a C1-C4 alkyl group, or a C1-C4 alkoxy group; R3 is OH, ONa, OK, or NH2.

[0013] Addressing the pathogenesis, characteristics, and treatment challenges of ophthalmic melanoma (UM), this invention provides a novel nanomaterial based on the synergistic combination of a zinc / copper bimetallic organic framework, a compound of formula 1, and HA components and structures. This synergistic effect enables highly selective targeting and inhibition of uveal melanoma cancer cells through multiple mechanisms, exhibiting excellent therapeutic activity even at low doses. Furthermore, it effectively inhibits metastasis. Moreover, the nanomaterial described in this invention also demonstrates excellent biocompatibility.

[0014] In this invention, the zinc / copper bimetallic organic framework is a zinc / copper bimetallic organic framework assembled from a divalent copper source, a divalent zinc source, and a ligand of formula 2;

[0015] Formula 2;

[0016] In Formula 2, R4 and R5 are H or C1~C4 alkyl groups.

[0017] The research of this invention shows that the zinc / copper bimetallic organic framework, in conjunction with the overall synergistic components and structure described in this invention, can further synergize and enhance the specificity and targeting of nanomaterials for ophthalmic umbilicus, which can further benefit the treatment of umbilicus.

[0018] In this invention, the molar ratio of copper to zinc in the zinc / copper bimetallic organic framework is 1:10~40, and more preferably 1:20~30. This preferred ratio facilitates the combination of this organic framework with other components and structures, further synergistically improving the inhibitory activity and selectivity of UM.

[0019] This invention demonstrates that, under the optimal control of copper and zinc elements, the compatibility of nanomaterials with ophthalmic microscopic umbilicus (UM) can be further enhanced, which helps to further improve their therapeutic activity and selectivity at low doses.

[0020] In Formula 1 of this invention, the halogen can be Cl or Br.

[0021] In this invention, the compound of formula 1 includes at least one of formula 1A, formula 1B, and formula 1C;

[0022] Formula 1A;

[0023] Formula 1B;

[0024] Formula 1C.

[0025] Preferably, Formula 1A is used as Formula 1, which, together with other components and structures of the present invention, can further synergistically enhance the inhibitory activity and selectivity of UM.

[0026] As an optional scheme, the content of compound of formula 1 in the core is 5~15 μg / mg, and more preferably 8~10 μg / mg;

[0027] In the nanomaterial, the weight ratio of the core to hyaluronic acid is 1:0.2~0.3.

[0028] The present invention also provides a method for preparing the nanomaterial, wherein a zinc / copper bimetallic organic framework is obtained, and it is composite-loaded with a compound of formula 1 to obtain a core, and then the core is mixed with hyaluronic acid for coating to obtain the nanomaterial.

[0029] In this invention, the assembly method of the zinc / copper bimetallic organic framework can be well known, for example, it can be assembled based on the copper and zinc raw materials and the ligand of formula 2.

[0030] In this invention, the divalent copper source and the divalent zinc source can be water-soluble raw materials of each group of metals. The raw materials of the divalent copper source and the divalent zinc source are mixed in a Cu / Zn molar ratio of 1:1 to 20 (more preferably 1:1.5 to 2).

[0031] The total molar ratio of copper and zinc to that in Formula 2 is 1:1 to 8; more specifically, it can be a 1:3 to 5 mixture. Assembly can be carried out at room temperature, and the solvent can be a C1 to C3 alcohol. The assembly time can be 3 to 10 hours.

[0032] The present invention also provides the application of the nanomaterial in the preparation of a drug for treating uveal melanoma.

[0033] The present invention demonstrates that the nanomaterials described herein can be adapted to the disease characteristics of uveal melanoma, achieving excellent selectivity for ophthalmic targeted therapy, obtaining good therapeutic effects at low doses, and effectively inhibiting its metastasis.

[0034] In this invention, uveal melanoma is ocular uveal melanoma caused by cells whose genetic characteristics are similar to at least one of Mum-2B and C918 cells.

[0035] In this invention, the nanomaterials and pharmaceutically acceptable excipients are combined to prepare a pharmaceutically acceptable dosage form.

[0036] In this invention, the excipients can be components known in the pharmaceutical field, such as conventional stabilizers, dispersants, antioxidants, etc.

[0037] The present invention also provides a medicament for treating uveal melanoma, which comprises a pharmaceutically effective amount of nanomaterials.

[0038] The medicament for treating uveal melanoma according to the present invention comprises pharmaceutically acceptable excipients;

[0039] The drug is available in dosage forms for ocular or intravenous injection.

[0040] Beneficial effects

[0041] This invention provides a nanomedicine for treating uveal melanoma (also known as Formula 1A / CuZn-MOF@HA), which can selectively target and inhibit uveal melanoma cancer cells based on the synergistic combination of its components and multiple mechanisms. It can also exert excellent therapeutic activity at low doses and effectively inhibit metastasis. Moreover, the nanomaterials described in this invention also have excellent biocompatibility.

[0042] This invention incorporates copper ions into a zinc metal-organic framework (Zn-MOF) to form a uniform and stable nanoplatform (CuZnMOF), which is then loaded with compound formula 1 and encapsulated with hyaluronic acid (HA) to ensure targeted delivery to CD44-overexpressing UM cells. This design achieves orthogonal inhibition of the energy pathway: Zn 2+ Compound 1 and Cu synergistically block glycolysis, while Cu 2+ It inhibits the tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation (OXPHOS), comprehensively blocking energy acquisition in uveal melanoma cells. Furthermore, the inhibition of glycolysis amplifies the cytotoxicity of copper death and catalyzes the generation of mitochondrial superoxide, inducing oxidative stress in tumor cells and exacerbating mitochondrial dysfunction. Formula 1A / CuZn-MOF@HA exhibited potent antitumor and antimetastatic efficacy in two aggressive uveal melanoma cell lines and their corresponding animal models. More notably, transcriptomic analysis elucidated an additional key mechanism: Formula 1A / CuZn-MOF@HA significantly downregulated the MAPK signaling pathway, which is persistently activated in over 90% of uveal melanoma cases due to mutations in the GNAQ or GNA11 genes and is a crucial driver of tumor proliferation and metastasis.

[0043] In summary, the nanomedicine described in this invention combines a multi-mechanism strategy of targeted full-pathway energy blocking, copper death amplification, and inhibition of core oncogenic pathways, making it a highly promising therapeutic agent for primary and metastatic uveal melanoma. Attached Figure Description

[0044] Figure 1 SEM, TEM characterization, and elemental distribution map of Formula 1A / CuZn-MOF@HA prepared in Example 1 are shown; where a is the SEM image, b is the TEM image, and c is the elemental distribution map.

[0045] Figure 2 Fourier transform infrared spectra of CuZn-MOF, Formula 1A / CuZn-MOF, and Formula 1A / CuZn-MOF@HA prepared in Example 1.

[0046] Figure 3 The graph shows the killing effect of Formula 1A / CuZn-MOF@HA on invasive UM cells and normal cells at different concentrations in Example 3.

[0047] Figure 4 The diagram illustrates the inhibitory effect of the drug in Example 4 on the migration and invasion abilities of UM cells; where a is a graph showing the migration rate of MUM-2B cells in the scratch assay, b is a graph showing the number of invaded MUM-2B cells in the Transwell assay, c is a statistical graph showing the cell migration rates of MUM-2B and C918 cells in the scratch assay, d is a statistical graph showing the number of migrated cells of the two cell types in the Transwell migration assay, and e is a statistical graph showing the number of invaded cells of the two cell types in the Transwell invasion assay.

[0048] Figure 5 The image shows the drug distribution of Formula 1A / CuZn-MOF@HA in animal models of UM ocular tumors and subcutaneous tumors in Example 6; where a is the in vivo fluorescence image in the ocular tumor animal model, b is the fluorescence image of isolated organs and tumors in the subcutaneous tumor animal model, c is the total fluorescence intensity change curve of the tumor-bearing eye and the contralateral healthy eye in the ocular tumor animal model, and d is the average fluorescence intensity change curve of the tumor and major organs in the subcutaneous tumor animal model.

[0049] Figure 6 The graph shows the inhibitory effects of Formula 1A / CuZn-MOF@HA and different single-component or two-component drugs on UM subcutaneous tumors in Example 7; where a is a representative gross photograph of each group of subcutaneous tumors, b is a curve of tumor volume change, and c is a statistical graph of the weight of excised tumors after treatment.

[0050] Figure 7 This is a representative image showing the inhibitory effects of Formula 1A / CuZn-MOF@HA and different single-component or two-component drugs on the in situ proliferation of ocular UM tumors and invasion of adjacent tissues in a mouse model in Example 8; where a is a gross photograph of the tumor-bearing eye in each group, and b is an HE-stained section of the tumor-bearing eye in each group.

[0051] Figure 8 The figures shown are representative of the inhibitory effects of Formula 1A / CuZn-MOF@HA and different single-component or two-component drugs on UM liver metastases in a mouse model in Example 9; where a is a gross photograph of the tumor-bearing liver in each group, and b is an HE-stained section of the tumor-bearing liver in each group.

[0052] Figure 9The figures show the proportions of Ki67 and TUNEL-positive cells in mouse models of UM subcutaneous tumors, orthotopic ocular tumors, and liver metastases after treatment with Formula 1A / CuZn-MOF@HA and different single-component or two-component drugs in Example 10; where a is the proportion of Ki67-positive cells and b is the proportion of TUNEL-positive cells. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] Part 1: Material Preparation

[0055] Preparation Example 1

[0056] The bimetallic drug-loaded nanoplatform of this invention is a zinc / copper bimetallic organic framework (MOF) with a supported 1A structure and hyaluronic acid coated on its surface. Its preparation steps include:

[0057] Step 1: Dissolve copper nitrate and zinc nitrate in methanol at a Cu / Zn molar ratio of 4:6, and add formula 2A ( A methanol solution containing copper and zinc in a molar ratio of 1:4 (total molar amount of copper and zinc to that of formula 2A) was magnetically stirred at 450 rpm for 6 hours at room temperature. The precipitate was separated by ultrafiltration, washed twice with ethanol, and then dried under vacuum at 60°C to obtain CuZn-MOF (the assembled bimetallic MOF is Cu1Zn). 24 -MOF). SEM, TEM, and elemental distribution maps are shown in […]. Figure 1 The Fourier transform infrared spectra of CuZn-MOF, Formula 1A / CuZn-MOF, and Formula 1A / CuZn-MOF@HA obtained in steps 1 to 3 are shown below. Figure 2 ;

[0058] Step 2: Dissolve CuZn-MOF in ethanol, sonicate with 1 mg / ml of Formula 1A ethanol solution for 2 hours, centrifuge at 10,000 rpm for 10 minutes, and then wash twice with ethanol to obtain Formula 1A / CuZn-MOF (the content of Formula 1A is 9 μg / mg).

[0059] Step 3: Disperse Formula 1A / CuZn-MOF in a 5 mg / ml hyaluronic acid solution (the weight ratio of Formula 1A / CuZn-MOF to hyaluronic acid is 1:0.25), and sonicate until completely dissolved. Centrifuge the mixture at 10,000 rpm for 10 minutes, wash twice with deionized water, and then freeze-dry to obtain Formula 1A / Cu1Zn. 24 -MOF@HA (also labeled as Formula 1A / CuZn-MOF@HA, nanomaterials or nanomedicines).

[0060] Preparation Example 2

[0061] Compared to Preparation Example 1, the only difference was the change in the Cu / Zn molar ratio in step 1, which was 1:19, 1:9, and 2:8. The total amount of Cu / Zn metal elements and other operations and conditions were the same as in Preparation Example 1, and the resulting nanomaterials were labeled as Formula 1A / Cu1Zn. 199 -MOF@HA, Formula 1A / Cu1Zn 99 -MOF@HA and formula 1A / Cu1Zn 49 -MOF@HA.

[0062] Preparation Example 3

[0063] Compared to Preparation Example 1, the only difference is that Formula 1A in step 2 is replaced with Formula 1B or Formula 1C, respectively. All other operations and conditions are the same as in Preparation Example 1. The resulting nanomaterials are labeled as Formula 1B / CuZn-MOF@HA (also labeled as Formula 1B / Cu1Zn). 24 -MOF@HA) and formula 1C / CuZn-MOF@HA (also denoted as formula 1C / Cu1Zn) 24 -MOF@HA).

[0064] Comparative Preparation Example 1

[0065] Compared with Preparation Example 1, the only difference is that in step 1, copper is missing, and the missing copper is supplemented by zinc by weight. All other operations and parameters are the same as in Preparation Example 1. The final material is labeled as Formula 1A / Zn-MOF@HA.

[0066] Comparative Preparation Example 2

[0067] Compared with Preparation Example 1, the only difference is that step 2 is removed. All other operations and parameters are the same as in Preparation Example 1. The final material is labeled CuZn-MOF@HA.

[0068] Comparative preparation example 3

[0069] Compared with Preparation Example 1, the only difference is that in step 1, copper is missing and the missing copper is made up by zinc in equimolar amounts, and step 2 is removed. All other operations and parameters are the same as in Preparation Example 1. The final material is labeled as Zn-MOF@HA.

[0070] Comparative preparation example 4

[0071] Compared with Preparation Example 1, the only difference is that in step 1, copper is missing and the missing copper is made up by zinc in equimolar amounts, and steps 2 and 3 are removed. All other operations and parameters are the same as in Preparation Example 1. The final material is labeled as Zn-MOF.

[0072] Part Two: Cell Research

[0073] Example 1

[0074] Tests on the killing effect of all formulations in Examples 1-3 on UM cells:

[0075] Human invasive uveal melanoma cell lines Mum-2B and C918 were seeded in 96-well plates and incubated overnight in an incubator (5% CO2, 37°C). The culture medium consisted of 99% RPMI-1640 medium and 1% penicillin-streptomycin solution. Different concentrations of Formula 1A / Cu1Zn were used for further analysis. 199 -MOF@HA, Formula 1A / Cu1Zn 99 -MOF@HA, Formula 1A / Cu1Zn 49 -MOF@HA, Formula 1A / Cu1Zn 24 -MOF@HA, Formula 1B / Cu1Zn 24 -MOF@HA, or formula 1C / Cu1Zn 24 -MOF@HA treatment for 24 hours. Subsequently, cells were washed with phosphate-buffered saline (PBS), incubated in CCK-8 solution at 37°C for 2 hours, and the optical density (OD) value was measured at 450 nm using a microplate reader to calculate the survival rate of UM cells after different drug treatments. IC50 50 The values ​​are shown in Table 1:

[0076] Table 1 shows the IC50 of all preparations used in Examples 1-3 for two types of UM cells. 50 Value (μg / ml)

[0077] As shown in Table 1, Equation 1A / Cu1Zn 24 -MOF@HA exhibited the strongest killing effect against both types of invasive UM cells, therefore, formula 1A / Cu1Zn was used in subsequent experiments. 24 -MOF@HA is represented by Equation 1A / CuZn-MOF@HA.

[0078] Example 2

[0079] The killing effects of Formula 1A / CuZn-MOF@HA and different single or two-component drugs on UM cells: Mum-2B and C918 cells were seeded in 96-well plates and incubated overnight. They were then treated for 24 hours with different concentrations of CuCl2, Formula 1A, Zn-MOF@HA, Formula 1A / Zn-MOF@HA, CuZn-MOF@HA, Formula 1A / CuZn-MOF@HA, or a mixture of CuCl2+Zn-MOF+Formula 1+HA (with the same content of each component in Formula 1A / CuZn-MOF@HA). Subsequently, the cells were washed with PBS and incubated in CCK-8 solution at 37°C for 2 hours. OD values ​​were then measured, and the half-maximal inhibitory concentrations (IC50) of Formula 1A / CuZn-MOF@HA, different single-component or two-component drugs (CuCl2, Formula 1A, Zn-MOF@HA, Formula 1A / Zn-MOF@HA, CuZn-MOF@HA), and single-component mixtures with equivalent component content to Formula 1A / CuZn-MOF@HA were calculated and compared against the two types of invasive UM cells. 50 IC 50 The results are shown in Table 2:

[0080] Table 2. Effects of different drugs on the IC50 of two types of UM cells. 50 Value (μg / ml)

[0081] Note: The data for Formula 1A / CuZn-MOF@HA are experimental data from the same batch as those in Example 2.

[0082] As shown in Table 2, compared with other drugs or mixtures of components, the Formula 1A / CuZn-MOF@HA prepared in Preparation Example 1 has the strongest antitumor effect.

[0083] This invention physically mixes the corresponding active components in the same ratio, collects complete dose-response curves, and uses the Chou-Talalay equation to quantitatively calculate the synergistic index CI of the combined use of the components (Table 3) to analyze the interaction mode between the active components.

[0084] The Chou-Talalay equation calculates the CI value based on dose-response curves of single drugs and combination drugs.

[0085] Where: CI=(D1 / D 1m )+(D2 / D 2m );

[0086] D1 and D2 are the doses of each component in the combined administration group.

[0087] D1m and D 2m This is the dose required to achieve the same effect when drug A and drug B are administered alone. If there are three or more drugs, their synergy is calculated in a similar manner.

[0088] The CI values ​​of different drug components are shown in Table 3.

[0089] Table 3. Synergistic effects (combination index, CI) between different drug combinations in two types of UM cells.

[0090] As shown in Table 3, the present invention formula 1A / CuZn-MOF@HA can achieve better synergy.

[0091] Example 3

[0092] The killing effects of different concentrations of Formula 1A / CuZn-MOF@HA on UM cells and normal cells:

[0093] Mum-2B, C918, normal rat retinal cells R28, and human umbilical vein endothelial cells (HUVECs) were seeded in 96-well plates and incubated overnight. They were then treated with different concentrations of Formula 1A / CuZn-MOF@HA for 24 hours. Subsequently, the cells were washed with PBS and incubated in CCK-8 solution at 37°C for 2 hours. OD values ​​were then measured, and the cytotoxic effects on different cell types were calculated and compared based on the results. Figure 3 As shown, after treatment with 20 μg / mL Formula 1A / CuZn-MOF@HA for 24 hours, the relative survival rates of MUM-2B and C918 cells decreased to approximately 11.5% and 0.8%, respectively, while R28 and HUVECs maintained high survival rates of 92.2% and 92.4%, respectively. These results indicate that Formula 1A / CuZn-MOF@HA possesses a broad therapeutic window, enabling it to minimize damage to normal ocular and vascular cells at doses that effectively eradicate tumor tissue.

[0094] Example 4

[0095] Inhibitory effects of Formula 1A / CuZn-MOF@HA and different single-component drugs on UM cell migration and invasion:

[0096] Scratch assay: MuM-2B and C918 cells were seeded in 6-well plates and cultured until approximately 90% confluence. A vertical line was drawn in the center of each well using a sterile pipette tip (1 mL). Cells were then incubated for 24 h with PBS or different drugs (CuCl2 0.10 µg / mL, LND 0.15 µg / mL, nanomedicine 10 µg / mL). Cell images were taken at 0 h and 24 h, and the images were processed using ImageJ software to compare the scratch healing rate after different drug treatments and to compare their anti-tumor metastasis effects. Figure 4 As shown in Figures a and c, compared with the 86.1% scratch healing rate of MUM-2B cells in the PBS group and the effects of other single-component or two-component drugs, Formula 1A / CuZn-MOF@HA had the strongest anti-migration effect, reducing the healing rate to 41.4%.

[0097] Transwell migration / invasion assay: MuM-2B and C918 cells were resuspended in serum-free medium and mixed with drug solutions (final concentrations: CuCl2 0.2 µg / mL, LND 0.3 µg / mL, nanomedicine 20 µg / mL). This mixture was added to the upper chamber of a Transwell chamber (coated or uncoated with Matrigel). The lower chamber was filled with complete medium containing 10% serum and the corresponding drug concentration to establish a chemotactic gradient. After 4 hours of incubation, cells were fixed with paraformaldehyde and stained with crystal violet. Cells that did not migrate / invade were wiped from the surface of the upper chamber, leaving only cells that had penetrated to the surface of the lower chamber. Image J was used to quantitatively analyze the number of cells that had penetrated the membrane and compare their anti-tumor metastasis effects. Figure 4 As shown in b, d, and e, compared with the PBS group and other single-component or two-component drugs, Formula 1A / CuZn-MOF@HA showed the strongest effect against UM cell migration and invasion, with inhibition rates exceeding 80% compared to the PBS control group.

[0098] pass Figure 4 It is known that the present invention, Formula 1A / CuZn-MOF@HA, has the strongest ability to resist UM cell metastasis compared with other single-component or two-component drugs.

[0099] Part Three: Animal Model Research

[0100] All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of Xiangya Hospital, Central South University.

[0101] Example 5

[0102] Safety assessment of Formula 1A / CuZn-MOF@HA:

[0103] Healthy 6-8 week old male BALB / c mice were randomly divided into a control group and an experimental group (n=3). The control group was injected with saline via the tail vein, while the experimental group was injected with 1A / CuZn-MOF@HA (15mg / kg) once. The general condition and weight of the mice were observed and monitored. The mice were euthanized after 14 days, and blood was collected for complete blood count and liver and kidney function tests. The results are shown in Table 4.

[0104] Table 4. Results of blood routine tests and liver and kidney function tests in mice during the safety assessment experiment.

[0105] As shown in Table 4, no significant abnormalities were observed in either group of mice within 14 days. There was no difference in weight gain, and no statistically significant differences in blood routine tests or liver and kidney function. The experimental group mice showed a slight decreasing trend in white blood cell count, but the change was small and statistically insignificant, and no signs of infection were observed.

[0106] Example 6

[0107] Drug distribution of Formula 1A / CuZn-MOF@HA in animal models of umbilical ocular tumour and subcutaneous tumor:

[0108] A subcutaneous tumor model was established using BALB / c nude mice (6-8 weeks old, half male and half female). MUM-2B cell suspension was subcutaneously injected into the right axilla of each mouse. Tumor growth was monitored, and when the tumor volume reached approximately 800 mm², the tumor was considered closed. 3 Mice were euthanized. The tumor was aseptically removed, and after removing necrotic tissue, the remaining viable tumor tissue was cut into small pieces approximately 3 mm in diameter. These pieces were subcutaneously implanted into the right axilla of new recipient mice through a small surgical incision to establish a passage model. Tumor volume was monitored regularly, and the tumor volume was expressed as length × (width × length)... 2 The result is calculated as () × 0.523.

[0109] For the in situ intraocular tumor model, 8-week-old BALB / c nude mice (half male and half female) were anesthetized. The pupils were dilated with 0.5% tropicamide, and the corneal surface of the right eye was gently rinsed with sterile saline and disinfected with povidone-iodine solution to reduce the risk of infection. Subsequently, a small incision was carefully made behind the limbus using a sterile 30G needle. Then, a suspension of MUM-2B cells was carefully injected into the suprachoroidal space or vitreous cavity using a microinjector (33G). The needle was held in place for 60 seconds before being withdrawn to prevent backflow. Immediately after injection, tobramycin eye ointment was applied to the corneal surface to prevent postoperative infection. The formation and progression of the intraocular tumor were monitored through regular ophthalmic examinations.

[0110] Cy5-labeled formula 1A / CuZn-MOF@HA (15 mg / kg) was injected via tail vein into tumor-bearing mice of the above two models, and fluorescence imaging was performed using an in vivo imaging system (IVIS). Non-invasive real-time imaging data of the orthotopic MUM-2B tumor in the right eye of BALB / c nude mice were collected at 3, 8, 24, 1, 2, 3, 5, and 7 days after injection. Mice in the subcutaneous model were euthanized, and the major organs (heart, liver, spleen, lung, and kidney) and tumors were collected for immediate in vitro fluorescence imaging to quantify the accumulation of formula 1A / CuZn-MOF@HA in different organs. The detection time points were 8 hours, 24 hours, 1 day, 2 days, 3 days, 5 days, and 7 days.

[0111] like Figure 5 As shown, except for a slightly lower fluorescence intensity than the liver during peak metabolic period, Formula 1A / CuZn-MOF@HA maintained the highest fluorescence signal within the tumor and persisted for up to 7 days post-injection, demonstrating excellent tumor targeting and long-term retention capabilities. In normal organs of tumor-bearing mice, the liver showed higher fluorescence intensity, suggesting that Formula 1A / CuZn-MOF@HA is primarily metabolized in the liver. Furthermore, as the site of over 90% of metastatic umbilical melanoma (UM), drug accumulation in the liver offers significant therapeutic advantages for metastatic uveal melanoma.

[0112] Example 7

[0113] Antitumor effect of Formula 1A / CuZn-MOF@HA in a UM subcutaneous tumor animal model:

[0114] After the UM model was established, tumor-bearing mice were randomly divided into control group (NS), Formula 1A, Zn-MOF@HA, Formula 1A / Zn-MOF@HA, CuZn-MOF@HA and Formula 1A / CuZn-MOF@HA groups. Due to the burning of normal tissue caused by acidic CuCl2 solution, the CuCl2 group was excluded from in vivo verification.

[0115] For the subcutaneous tumor model, treatment began 7 days after tumor implantation. Tumor size and body weight were measured every other day, followed by intratumoral injections (nanomedicine group 15 mg / kg, clonidine 0.22 mg / kg) for a total of 5 times. Figure 6 As shown in the figures, the tumor volume growth curves and the weight of ex vivo tumors after treatment indicate that Formula 1A / CuZn-MOF@HA exhibits the strongest in vivo antitumor effect compared to other single-component or two-component drugs. Compared to the NS group, the tumor growth inhibition rate of the Formula 1A / CuZn-MOF@HA group was 85.3%.

[0116] Example 8

[0117] Anti-extraocular invasion effect of Formula 1A / CuZn-MOF@HA in an animal model of umbilical ocular tumor:

[0118] Intraocular tumor models were treated 3 days after tumor cell inoculation. Tumor-bearing mice received intravitreal injections of 0.5 µL every three days (50 µg nanomedicine or 0.73 µg clonidine), for a total of three times. Body weight was measured and ophthalmic observation was performed before each treatment to monitor tumor progression. Mice were euthanized three days after the last injection. Tumor-bearing eyeballs were collected, photographed, weighed, and fixed with ocular fixative. After standard dehydration, the tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE).

[0119] like Figure 7 As shown, due to the highly invasive nature of MUM-2B cells, the tumors in the NS-treated group rapidly occupied the intraocular space, leading to ocular expansion. With scleral dissolution and ocular rupture, tumor cells spread into the orbit and proliferated rapidly, ultimately resulting in significant proptosis. While Formula 1A and Zn-MOF@HA treatments inhibited tumor growth and scleral invasion to some extent, they were insufficient to prevent orbital infiltration. The Formula 1A / Zn-MOF@HA and CuZn-MOF@HA groups showed better control of UM proliferation and greater preservation of scleral integrity; however, extraocular invasion still occurred. Among all six treatment groups, the tumor-bearing eye in the Formula 1A / CuZn-MOF@HA group exhibited the most normal and intact ocular tissue structure, the lowest ocular weight, and the smallest extraocular tumor invasion, demonstrating the strongest anti-tumor proliferation and anti-invasive effects.

[0120] Example 9

[0121] The role of Formula 1A / CuZn-MOF@HA in an animal model of UM liver metastases:

[0122] To establish a liver metastasis model, anesthetized BALB / c nude mice (6-8 weeks old, equal number of males and females) were placed in the right lateral decubitus position. A small incision was made in the left upper abdominal wall, and the peritoneum was dissected to expose the spleen. MUM-2B cells were gently injected into the spleen using an insulin injector. The needle was held in place for one minute, followed by cauterization and firm pressure with a sterile cotton swab to stop bleeding. Five minutes later, to ensure that metastatic lesions formed in the liver only through the portal vein circulation and to prevent the growth of the primary tumor at the injection site, a total splenectomy was performed, and the abdominal incision was sutured.

[0123] Treatment began 7 days after modeling, with intraperitoneal injections of the following drugs: nanomedicine group 15 mg / kg, clonidine 0.22 mg / kg, every two days for a total of 10 doses. Body weight was monitored before each administration. Two days after the last administration, mice were euthanized. Tumor-bearing livers were collected, photographed, weighed, fixed in 4% paraformaldehyde solution, embedded in paraffin, and sectioned for HE staining.

[0124] like Figure 8 As shown, numerous metastatic nodules of varying sizes were observed in the liver of the NS group. Compared to the control group, different drugs exhibited varying therapeutic effects on metastatic nodules. The Formula 1A / CuZn-MOF@HA group produced the fewest and smallest metastatic nodules, with the lowest liver weight increase and liver / body weight ratio. HE-stained sections of liver tissue revealed dense clusters of tumor cells and scattered satellite lesions in the surrounding liver tissue. The Formula 1A / CuZn-MOF@HA group showed the mildest lesions, with the most significant necrosis in the metastatic lesions. These results suggest that Formula 1A / CuZn-MOF@HA has a significant inhibitory effect on liver metastasis of uveal melanoma.

[0125] Example 10

[0126] Tumor cell proliferation and death in three UM animal models after treatment with Formula 1A / CuZn-MOF@HA: After euthanizing the animals, tumor tissues (subcutaneous tumors, tumor-bearing eyeballs, and tumor-bearing livers from Examples 6-9) were collected and fixed with 4% paraformaldehyde or ophthalmic fixative. After standard dehydration, the tissues were embedded in paraffin and cut into 5-micrometer-thick sections. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was used to detect dead cells within the tumor, and immunohistochemistry or immunofluorescence staining was performed using a specific antibody against the proliferation marker Ki67. Figure 9 As shown, in the tumor tissue of the Formula 1A / CuZn-MOF@HA treatment group, proliferating cells (Ki67) + The proportion of ) was the lowest, while that of dead cells (TUNEL) + The highest proportion of these cells confirmed their powerful ability to induce tumor cell death and inhibit proliferation.

Claims

1. A nanomaterial for treating uveal melanoma, characterized in that, It includes a core and a hyaluronic acid coating layer that encapsulates the core; the core includes a zinc / copper bimetallic organic framework and a compound of formula 1; Compound of Formula 1 is of Formula 1A, Formula 1B or Formula 1C; Formula 1A; Formula 1B; Formula 1C; The zinc / copper bimetallic organic framework is a zinc / copper bimetallic organic framework assembled from a divalent copper source, a divalent zinc source and a formula 2 ligand; Formula 2; In Formula 2, R4 and R5 are H or C1~C4 alkyl groups; In zinc / copper bimetallic organic frameworks, the molar ratio of copper to zinc is 1:1 to 20.

2. The nanomaterial for treating uveal melanoma as described in claim 1, characterized in that, In the nucleus, the content of compound of formula 1 is 5~15 μg / mg; In the nanomaterial, the weight ratio of the core to hyaluronic acid is 1:0.2~0.

3.

3. A method for preparing the nanomaterial according to any one of claims 1 to 2, characterized in that, A zinc / copper bimetallic organic framework was obtained and composited with a compound of formula 1 to obtain a core. The core was then mixed with hyaluronic acid for coating to prepare the nanomaterial.

4. The use of the nanomaterial according to any one of claims 1 to 2 in the preparation of a medicament for treating uveal melanoma.

5. The application as described in claim 4, characterized in that, The nanomaterials and pharmaceutically acceptable excipients are combined to prepare a pharmaceutically acceptable dosage form.

6. A drug for treating uveal melanoma, characterized in that, The nanomaterial comprising a pharmaceutically effective amount as described in any one of claims 1 to 2.

7. The medicament for treating uveal melanoma as described in claim 6, characterized in that, It contains pharmaceutically acceptable excipients; It has dosage forms for ocular or intravenous injection.

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