A method for preparing a thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles.
By combining Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles with thermosensitive hydrogels, precise targeted delivery and sustained release of drugs in liver cancer treatment were achieved, solving the problem of poor targeting of existing embolization materials, improving the efficacy of liver cancer treatment and reducing side effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- AFFILIATED HOSPITAL OF NANTONG UNIV
- Filing Date
- 2024-12-30
- Publication Date
- 2026-06-30
AI Technical Summary
Existing embolization materials have poor targeting in tumor tissue, making it difficult to release drugs efficiently and continuously, resulting in limited therapeutic effects and significant side effects in liver cancer treatment.
A method was adopted to combine Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles with thermosensitive hydrogels. Annexin V recognizes phosphatidylserine on the surface of tumor cells to achieve precise targeted drug delivery, and the thermosensitive hydrogel forms a gel state at body temperature to achieve sustained drug release.
It improves drug accumulation at the tumor site, reduces the impact on normal tissues, enhances anti-cancer effects, reduces side effects, and achieves highly efficient and sustained therapeutic effects in interventional embolization therapy for liver cancer.
Smart Images
Figure CN119745836B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, and in particular to a method for preparing a thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles. Background Technology
[0002] Liver cancer is a common and highly lethal malignant tumor. Its early diagnosis rate is low, and most patients are diagnosed at an advanced stage, losing the opportunity for surgical resection. Traditional treatments for liver cancer, such as surgery, radiotherapy, and chemotherapy, while able to slow the progression of the disease to some extent, often suffer from limited efficacy, high recurrence rates, and significant side effects. Therefore, interventional embolization therapy has gradually become one of the main treatment methods for liver cancer.
[0003] Interventional embolization therapy for liver cancer involves directly injecting embolic agents into tumor blood vessels to block tumor blood supply and thus inhibit tumor growth. However, existing embolic materials have poor targeting properties in tumor tissue, making it difficult to release drugs efficiently and sustainably. Therefore, there is a need to develop new drug delivery systems to improve therapeutic efficacy. Summary of the Invention
[0004] The purpose of this invention is to solve the technical problem that embolization materials in the prior art have poor targeting in tumor tissues and are difficult to release drugs efficiently and continuously.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] A method for preparing Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles includes the following steps: using mPDA as a carrier, doxorubicin (DOX) is loaded onto the hydrogel system to obtain DOX@mPDA nanoparticles, and then Annexin V is modified onto the surface of DOX@mPDA to obtain DOX@mPDA-AV.
[0007] Preferably, the specific steps are as follows:
[0008] S1: Dissolve dopamine hydrochloride and Pluronic F127 in a mixture of water and ethanol, stir the solution at 600 rpm for 10 minutes, then slowly add TMB dropwise while continuing to stir at 500 rpm;
[0009] Next, ammonia was added dropwise, and the reaction system was stirred to promote the polymerization reaction. After the reaction was completed, the product was collected by centrifugation, then washed once with ethanol and centrifuged again, and then washed once with a washing solution of water and ethanol in a volume ratio of 1:1. The obtained mPDA particles were redispersed in HEPBS solution.
[0010] S2: Slowly add ammonia water to the mPDA solution, then add doxorubicin, and react for 30 minutes under stirring.
[0011] After the reaction was completed, the mixture was centrifuged to obtain DOX@mPDA particles. Finally, the obtained DOX@mPDA particles were redispersed in deionized water.
[0012] S3: The DOX@mPDA solution prepared in S2 was added to Annexin V-mCherry reagent, mixed, and incubated under light-protected conditions. After incubation, the solution was centrifuged and washed with water to obtain Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles.
[0013] Preferably, the mass ratio of dopamine to Pluronic F127 in S1 is 1:2.
[0014] This application also provides Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles, which are prepared using the above-described preparation method.
[0015] This application also provides a method for preparing a thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles. First, a PNIPMAm hydrogel is prepared, and then the PNIPMAm hydrogel is mixed with the aforementioned Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles to obtain the thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles.
[0016] Preferably, the PNIPMAm hydrogel is prepared by the following method:
[0017] Weigh 0.5 g NIPMAm and dissolve it in 3.5 mL of deionized water, then pour the solution into a glass bottle;
[0018] Then, 35 mg of APS was added as an initiator; the mixed solution was heated at 60°C for 12 hours to carry out thermally initiated polymerization; after the reaction, the product was a white solid, namely PNIPMAm hydrogel.
[0019] This application also provides a thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles, which is prepared using the preparation method described above.
[0020] This application also provides the application of a thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles in the preparation of drugs for treating liver cancer, wherein the thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles is prepared by the above-described preparation method.
[0021] Preferably, the drug is used in TACE treatment of liver cancer.
[0022] Compared with the prior art, this application has at least the following beneficial effects:
[0023] (1) Enhanced targeting: This invention achieves precise targeted delivery to liver cancer cells by modifying mPDA nanoparticles with Annexin V. Annexin V can specifically recognize phosphatidylserine (PS) on the surface of tumor cells, significantly improving the accumulation of the drug at the tumor site, thereby enhancing the anti-cancer effect of the drug and reducing non-specific effects on normal tissues.
[0024] (2) Lower toxicity: Compared with traditional chemotherapy drugs, the mPDA drug-loaded nanoparticles of the present invention significantly reduce the accumulation of drugs in normal cells through targeted delivery, thereby reducing systemic toxicity. In addition, the mPDA material has good biocompatibility, which reduces the toxic side effects of the nanoparticles themselves on the body and improves the safety and tolerability of treatment.
[0025] (3) Enhanced therapeutic effect by combining with hydrogel: This invention combines Annexin V-modified mPDA nanoparticles with thermosensitive hydrogel to form a stable embolization system. The hydrogel forms a gel state at body temperature, which can achieve sustained drug release, prolong the drug's residence time at the tumor site, enhance the anti-cancer effect, and simultaneously embolize the tumor's blood supply vessels, effectively inhibiting tumor growth and metastasis, further improving the efficacy of interventional treatment for liver cancer.
[0026] (4) Significant comprehensive therapeutic effect: Through the combined effects of targeted delivery, reduced toxicity and thermosensitive hydrogel, this invention achieves efficient drug release and sustained therapeutic effect in interventional embolization therapy for liver cancer. It not only improves the anti-tumor efficacy of the drug, but also reduces the side effects and adverse reactions of conventional treatment methods, and has broad clinical application prospects. Attached Figure Description
[0027] Figure 1 This is a transmission electron microscope image of mPDA nanoparticles in one embodiment of the present invention;
[0028] Figure 2 This is a transmission electron microscope image of DOX@mPDA nanoparticles in one embodiment of the present invention;
[0029] Figure 3 This is a transmission electron microscope image of DOX@mPDA-AV nanoparticles in one embodiment of the present invention;
[0030] Figure 4 This is a scanning electron microscope image of the hydrogel in one embodiment of the present invention;
[0031] Figure 5 This illustrates the curing effect of the hydrogel in one embodiment of the present invention.
[0032] Figure 6 The ultraviolet absorption spectra of different nanoparticles were used in the verification experiment of this invention;
[0033] Figure 7 This is a particle size distribution diagram of different nanoparticles used in the verification experiment of this invention;
[0034] Figure 8 This is a graph showing the zeta potential measurement results of different nanoparticles in the verification experiment of this invention;
[0035] Figure 9 This is a diagram showing the uptake of different nanoparticles by Huh7 cells in the verification experiment of this invention;
[0036] Figure 10 The diagram shows the effect of different nanoparticles on Huh7 cells in the CCK 8 method used in the verification experiment of this invention. Detailed Implementation
[0037] The present invention will be further described in detail below with reference to specific embodiments. Example
[0038] A method for preparing a thermosensitive hydrogel system of mesoporous polydopamine drug-loaded nanoparticles modified with Annexin V, wherein the hydrogel system uses mPDA as a carrier to load DOX to obtain DOX@mPDA nanoparticles, then Annexin V is modified onto the surface of DOX@mPDA to obtain DOX@mPDA-AV, and the DOX@mPDA-AV nanoparticles are dispersed in the thermosensitive hydrogel to form a composite system.
[0039] The specific steps of the preparation method are as follows:
[0040] S1: Preparation of mPDA NPs:
[0041] Dissolve 250 mg of dopamine hydrochloride and 500 mg of Pluronic F127 in a mixture of water and ethanol (50% v / v; 50 mL), and stir the solution at 600 rpm for 10 minutes to ensure complete dissolution. Then, slowly add 0.5 mL of trimethylbenzene (TMB) dropwise, and continue stirring at 500 rpm for 30 minutes.
[0042] Next, 2.5 mL of ammonia water was added dropwise, and the reaction system was stirred at 500 rpm for 2 hours to promote the polymerization reaction. The entire process was carried out at around 15°C.
[0043] After the reaction was complete, the product was collected by centrifugation at 14,000 rpm for 15 minutes. Subsequently, it was washed once with ethanol and centrifuged again, and then washed once with a mixture of water and ethanol (50% v / v) to remove unreacted residues.
[0044] Finally, the obtained mPDA particles were redispersed in HEPBS solution.
[0045] S2: Preparation of DOX@mPDA:
[0046] 1 mL of 1 mg / mL mPDA solution was slowly added dropwise to 50 μL of ammonia water, followed by the addition of 0.2 mg of doxorubicin, and the mixture was reacted for 30 minutes with stirring.
[0047] After the reaction was completed, the mixture was centrifuged at 14,000 rpm for 8 minutes. Finally, the obtained DOX@mPDA particles were redispersed in 1 mL of deionized water.
[0048] S3: Preparation of DOX@mPDA-Annexin V:
[0049] Take 0.25 mL of the DOX@mPDA solution prepared in S2 and add 100 μL of Annexin V-mCherry reagent. After mixing, stir and incubate for 2 hours in the dark.
[0050] After incubation, the solution was centrifuged and washed with water to obtain DOX@mPDA-AV nanoparticles. The particles were dissolved in deionized water for subsequent experiments.
[0051] S4: Preparation of poly(N-isopropylmethacrylamide) hydrogel (PNIPMAm):
[0052] Weigh 0.5 g NIPMAm and dissolve it in 3.5 mL of deionized water, then pour the solution into a glass bottle.
[0053] Then, 35 mg of ammonium persulfate (APS) was added as an initiator. The mixed solution was heated at 60°C for 12 hours to carry out a thermally initiated polymerization reaction. After the reaction, the product was a white solid.
[0054] When the solid is stored in a 4°C refrigerator, it transforms into a homogeneous solution. This solution will be mixed with drug-loaded nanoparticles in subsequent experiments.
[0055] S5: Preparation of PNIPMAM / DOX@mPDA-AV:
[0056] The PNIPMAm solution obtained in S4 and the DOX@mPDA-AV particles obtained in S3 were gently stirred and mixed evenly at room temperature to prepare a thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles. The volume ratio of the PNIPMAm solution to the DOX@mPDA-AV solution was 3.5:1.
[0057] The DOX loading in the DOX@mPDA-AV nanoparticle solution of the thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles was approximately 8%.
[0058] The thermosensitive hydrogel system with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles prepared in the above embodiments can be applied to TACE treatment of liver cancer. By combining Annexin V-modified mPDA drug-loaded nanoparticles with thermosensitive hydrogel, efficient targeted drug delivery and sustained anti-tumor effect can be achieved in interventional embolization treatment of liver cancer.
[0059] Verification Experiment 1: TEM observation of the structural morphology of mPDA, DOX@mPDA, and DOX@mPDA-AV:
[0060] Experimental procedure: Under light-protected conditions, dissolve nanoparticles in ultrapure water, take an appropriate amount of diluted nano-therapeutic agent and drop it onto a copper grid for electron microscopy sample preparation. After 30 minutes, use absorbent paper to wipe off the excess liquid, let it dry, and then take pictures for observation.
[0061] Test results: such as Figure 1 As shown, the obtained mPDA has a clear mesoporous structure and relatively uniform pore size; Figure 2 As shown, compared to the unloaded pure mPDA particles, the surface of the drug-loaded DOX@mPDA particles appears slightly rough, indicating that doxorubicin (DOX) has been successfully loaded onto the surface and pores of the nanoparticles.
[0062] like Figure 3 As shown, DOX@mPDA-AV particles further modified with Annexin V also exhibit similar surface characteristics, demonstrating the loading of drugs and targeting molecules and ensuring that the particles have good structure and function for use in tumor-targeted therapy.
[0063] The scanning electron microscopy results of the PNIPMAm hydrogel are as follows: Figure 4 As shown, it exhibits a regular grid structure with uniform pore size. And as... Figure 5 As shown, the liquid PNIPMam hydrogel at room temperature will solidify significantly after standing in a 37°C water bath for about 50 seconds.
[0064] Verification Experiment 2: Measurement of Ultraviolet Absorption Spectrum:
[0065] Please see Figure 6 DOX@mPDA has a distinct characteristic peak at 485 nm, which is consistent with the characteristic peak of DOX, indicating that doxorubicin (DOX) has been successfully loaded onto mPDA.
[0066] Verification Experiment 3: Measurement of particle size, distribution, and surface potential of mPDA and DOX@mPDA:
[0067] Nanoparticles were dissolved in ultrapure water and placed in a dish (ensuring no air bubbles were generated). Their particle size, dispersibility, and potential were measured using a Malvern particle size analyzer. The results show that the prepared nanoparticles have uniform particle size and strong dispersibility. Figure 7 As shown, the particle sizes of mPDA and DOX@mPDA are 146 nm and 164 nm, respectively. Furthermore, DLS analysis reveals that these nanoparticles have a narrow particle size distribution, indicating good dispersibility in ultrapure water.
[0068] Depend on Figure 8 The Zeta potential results for mPDA, DOX@mPDA, and DOX@mPDA-AV nanoparticles are shown. The changes in Zeta potential reflect the effects of nanoparticle surface modification and drug loading on surface charge. Loading DOX increases the surface charge, while Annexin V modification neutralizes the charge, indicating that Annexin V modification was successful.
[0069] Verification Experiment 4: Targeted uptake effect of DOX@mPDA-AV on Huh7 cells:
[0070] Experimental procedure: Huh7 cells were seeded in a 35 mm confocal dish (1.5 × 10⁻⁶ cells / mL). 5 Cells were cultured at 100 cells / dish for 24 hours. Then, doxorubicin (DOX) was added to a final concentration of 5 μg / mL and the cells were treated for 24 hours to induce apoptosis, ensuring an apoptosis rate of 70%.
[0071] Cells were then washed three times with PBS buffer to remove residual drug. After washing, DOX@PDA and DOX@PDA-AV nanoparticles were added, adjusting the final PDA concentration to 0.2 mg / mL. The PDA nanoparticles were labeled with FITC fluorescence to monitor cellular uptake. Drug treatment lasted for 1 hour, after which cells were immediately fixed with 2.5% glutaraldehyde fixative at 4°C for 15 minutes. After fixation, nuclear DNA was stained with DAPI, and cells were washed twice with PBS to remove excess dye. Finally, the cells were imaged using a confocal laser scanning microscope (CLSM) to observe drug uptake.
[0072] Experimental results: such as Figure 9 As shown, the amount of mPDA taken up by the Annexin V-modified group was significantly greater than that of the unmodified group, indicating that Annexin V modification effectively improved the targeting and uptake of drug-loaded nanoparticles. Furthermore, the presence of red Annexin V and green mPDA at the same position indicates that Annexin V successfully modified the DOX@mPDA nanoparticles.
[0073] Verification Experiment 5: In vitro cytotoxicity experiment:
[0074] Experimental procedure: Prepare Huh7 cell suspension (10... 5 The culture medium was filled into 96-well plates (200 μL per well) and incubated for 24 h. The original medium was then replaced with different concentrations of DOX, DOX@mPDA, and DOX@mPDA-AV (1, 2, 4, 8, 16, and 20 μg / mL), and incubated again for 24 h. Then, 110 μL of 10% CCK8 solution prepared with fresh medium was added to each well, and the plates were incubated for 4 h. The optical density (OD) at 450 nm was measured using a microplate reader. Three independent experiments were performed, with five replicates for each experiment.
[0075] Experimental results: such as Figure 10 As shown, compared with free DOX and DOX@mPDA, the relative cell viability of Huh7 cells decreased significantly after incubation with DOX@mPDA-AV for 24 h in a concentration-dependent manner, indicating that DOX@mPDA-AV has significant cytotoxic effects.
[0076] The materials and their sources involved in the embodiments and verification experiments of this application are as follows:
[0077] Material source Dopamine hydrochloride, Pluronic F127, NIPMAm, APS Sigma Annexin V-mCherry, CCK-8, DAPI Azure Sky Doxorubicin Hydrochloride Aladdin
[0078] This application provides a thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles. By modifying mPDA nanoparticles with Annexin V, precise targeted delivery to liver cancer cells is achieved. Annexin V can specifically recognize phosphatidylserine (PS) on the surface of tumor cells, significantly improving drug accumulation at the tumor site, thereby enhancing the anti-cancer effect and reducing non-specific effects on normal tissues.
[0079] Furthermore, compared to traditional chemotherapy drugs, the mPDA drug-loaded nanoparticles of this application significantly reduce drug accumulation in normal cells and lower systemic toxicity through targeted delivery. In addition, the mPDA material exhibits good biocompatibility, reducing the toxic side effects of the nanoparticles themselves and improving the safety and tolerability of treatment. Moreover, this application combines Annexin V-modified mPDA nanoparticles with a thermosensitive hydrogel to form a stable embolization system. The hydrogel forms a gel state at body temperature, enabling sustained drug release, prolonging the drug's residence time at the tumor site, enhancing the anti-cancer effect, and simultaneously embolizing tumor-supplying blood vessels, effectively inhibiting tumor growth and metastasis, further improving the efficacy of interventional treatment for liver cancer. Through the combined effects of targeted delivery, reduced toxicity, and the thermosensitive hydrogel, this invention achieves efficient drug release and sustained therapeutic effects in interventional embolization treatment of liver cancer, not only improving the anti-tumor efficacy of the drug but also reducing the side effects and adverse reactions of conventional treatment methods, showing broad clinical application prospects.
Claims
1. A method for preparing Annexin V modified mesoporous polydopamine drug-loaded nanoparticles, characterized in that: The process includes the following steps: The Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles are loaded with doxorubicin using mPDA as a carrier to obtain DOX@mPDA nanoparticles. Then, Annexin V is modified onto the surface of DOX@mPDA to obtain DOX@mPDA-AV.
2. The method of claim 1, wherein the preparation of the modified mesoporous polydopamine nanoparticles loaded with Annexin V is characterized by: The specific steps are as follows: S1: Dissolve dopamine hydrochloride and Pluronic F127 in a mixture of water and ethanol, stir the solution at 600 rpm for 10 minutes, then slowly add TMB dropwise while continuing to stir at 500 rpm; Next, ammonia was added dropwise, and the reaction system was stirred to promote the polymerization reaction. After the reaction was completed, the product was collected by centrifugation, then washed once with ethanol and centrifuged again, and then washed once with a washing solution of water and ethanol in a volume ratio of 1:
1. The obtained mPDA particles were redispersed in HEPBS solution. S2: Slowly add ammonia water to the mPDA solution, then add doxorubicin, and react for 30 minutes under stirring. After the reaction was completed, the mixture was centrifuged to obtain DOX@mPDA particles. Finally, the obtained DOX@mPDA particles were redispersed in deionized water. S3: The DOX@mPDA solution prepared in S2 was added to Annexin V-mCherry reagent, mixed, and incubated under light-protected conditions. After incubation, the solution was centrifuged and washed with water to obtain Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles.
3. The method of claim 2, wherein the preparation of the modified mesoporous polydopamine nanoparticles loaded with Annexin V is characterized by: The mass ratio of dopamine to Pluronic F127 in S1 is 1:
2.
4. A mesoporous polydopamine-loaded nanoparticle loaded with Annexin V, characterized in that: It is prepared using any one of the preparation methods of claims 1-3.
5. A method for preparing a thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles, characterized in that: First, PNIPMAm hydrogel was prepared. Then, PNIPMAm hydrogel was mixed with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles as described in claim 4 to obtain a thermosensitive hydrogel system of Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles.
6. The method for preparing a thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles according to claim 5, characterized in that: The PNIPMAm hydrogel was prepared by the following method: Weigh 0.5 g NIPMAm and dissolve it in 3.5 mL of deionized water, then pour the solution into a glass bottle; Then, 35 mg of APS was added as an initiator; the mixed solution was heated at 60°C for 12 hours to carry out thermally initiated polymerization; after the reaction, the product was a white solid, namely PNIPMAm hydrogel.
7. A thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles, characterized in that: It is prepared using the preparation method described in claim 5 or 6.
8. The application of a thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles in the preparation of drugs for treating liver cancer, characterized in that: The thermosensitive hydrogel system loaded with Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles is prepared by the preparation method in claim 5 or 6.
9. The application of the thermosensitive hydrogel system of Annexin V-modified mesoporous polydopamine drug-loaded nanoparticles according to claim 8 in the preparation of drugs for treating liver cancer, characterized in that: The drug is used in TACE treatment of liver cancer.