DNA hydrogel drug release system, and preparation method and application thereof

Abstract

The application discloses a DNA hydrogel drug release system, which is composed of a DNA hydrogel, small molecule ECM modulators encapsulated in the DNA hydrogel, and platelets loaded with cytotoxic drugs. The DNA hydrogel drug release system provided by the application can not only realize the time-space sequential release of the ECM modulators and the cytotoxic drugs, but also can significantly increase the accumulation of the cytotoxic drugs at a tumor site, thereby effectively enhancing the treatment effect of the tumor, especially the treatment effect of a solid tumor with dense ECM.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a DNA hydrogel drug release system, a preparation method and application thereof. BACKGROUND

[0002] The therapeutic effect of current solid tumor treatment is severely limited by the dense and abnormal extracellular matrix (ECM) of tumor cells. This complex network, characterized by excessive deposition of collagen, high interstitial fluid pressure and dysfunction of the vasculature, acts as a physical and biochemical barrier, severely hindering the penetration and accumulation of anti-tumor drugs at the tumor site. Therefore, even potent cytotoxic drugs often fail to reach their target cells at effective concentrations, resulting in poor treatment effect and frequent recurrence. Therefore, destroying the ECM barrier of tumor to improve drug delivery has become a key prerequisite for enhancing the treatment of solid tumors. However, in order to improve the therapeutic effect of tumor, it is not enough to rely on ECM remodeling alone, but also to deliver therapeutic drugs after the barrier is destroyed to maximize their accumulation at the target tumor site, which highlights the need for a coordinated strategy that can regulate ECM and drug delivery in a spatiotemporal manner.

[0003] However, conventional drug delivery systems often fail to meet the demand for spatiotemporal sequential release of ECM modulators and cytotoxic drugs. Traditional carriers are often affected by "non-selective synchronous release" or poor release sequence control, and cannot achieve spatiotemporal sequential release. Specifically, they either release ECM modulators and cytotoxic drugs simultaneously, resulting in premature drug clearance before ECM remodeling; or release cytotoxic drugs that are blocked by intact ECM in an uncoordinated sequential release, for example, first release cytotoxic drugs that are blocked by intact ECM. This inability to achieve precise spatiotemporal control severely limits the therapeutic effect of these drug delivery systems, highlighting the urgent need for new drug release systems that can program the release sequence of drugs. SUMMARY

[0004] The present application aims to provide a DNA hydrogel drug release system to solve at least one of the above technical problems.

[0005] According to one aspect of the present application, a DNA hydrogel drug release system is provided, which consists of a DNA hydrogel and small molecule ECM modulators and cytotoxic drug-loaded platelets encapsulated in the DNA hydrogel.

[0006] After the cytotoxic drug is loaded on the platelet, the overall size of the drug increases significantly, and the present application encapsulates it together with the small molecule ECM modulator in the DNA hydrogel, thereby preparing a DNA hydrogel drug release system based on size gating that can achieve spatiotemporal sequential release of ECM modulators and cytotoxic drugs.

[0007] The DNA hydrogel drug release system provided by the present application can not only realize the spatiotemporal sequential release of the ECM modulator and the cytotoxic drug, but also significantly increase the accumulation of the cytotoxic drug at the tumor site, thereby effectively enhancing the treatment effect of the tumor, especially the treatment effect of the solid tumor with dense ECM.

[0008] The DNA hydrogel drug release system provided by the present application can not only realize the spatiotemporal sequential release of the ECM modulator and the cytotoxic drug, but also significantly increase the accumulation of the cytotoxic drug at the tumor site, thereby effectively enhancing the treatment effect of the tumor, especially the treatment effect of the solid tumor with dense ECM.

[0009] In some embodiments, the molecular weight of the small molecule ECM modulator is not greater than 1000 Da, has high tissue penetration and cell permeability, and can efficiently act on the ECM synthesis, degradation or related signal transduction pathway. Preferably, the small molecule ECM modulator can be selected from at least one of the following drugs: losartan, resveratrol, MMP-2 / MMP-9 Inhibitor III (CAS: 244082-19-7), etc.

[0010] In some embodiments, the cytotoxic drug can be selected from at least one of the following: indocyanine green, azithromycin, paclitaxel, platinum-based chemotherapy drugs, etc. Among them, the platinum-based chemotherapy drugs include but are not limited to carboplatin, cisplatin, oxaliplatin, irinotecan, nedaplatin, and lobaplatin, etc.

[0011] In some embodiments, the cytotoxic drug can be loaded on the platelet by any method disclosed in the prior art to significantly increase the size of the drug. For example, the cytotoxic drug can be loaded on the platelet membrane by amide coupling reaction, or the method described in DOI: 10.1016 / j.cej.2023.142258, DOI: 10.1002 / adma.202109517, etc.

[0012] In some embodiments, the small molecule ECM modulator can be Losartan (Los), and the cytotoxic drug can be Indocyanine green (ICG). The Indocyanine green can be loaded on the platelet membrane through an amide coupling reaction to obtain the Indocyanine green loaded platelets (ICG / PLT) with a hydrodynamic diameter of about 1100-1400 nm, which is significantly larger than the diameter of Losartan and the pore diameter of the DNA hydrogel. The Indocyanine green and Losartan are delivered and released in cooperation and through the DNA hydrogel drug release system provided by the present application, which can significantly enhance the accumulation of ICG at the tumor site, thereby producing excellent light-activated photodynamic therapy effect in anti-tumor.

[0013] In some embodiments, the DNA hydrogel is formed by self-assembly of Y-type DNA monomers and L-type DNA monomers.

[0014] In some embodiments, the Y-type DNA monomers are synthesized by hybridization of DNA single strands with nucleotide sequences shown in SEQ ID NOs: 1-3, respectively; and the L-type DNA monomers are synthesized by hybridization of DNA single strands with nucleotide sequences shown in SEQ ID NOs: 4-5, respectively. Thus, the DNA hydrogel formed by self-assembly of the Y-type DNA monomers and the L-type DNA monomers has a clear reticular structure with an average pore diameter of about 50 nm, which is smaller than the diameter of ICG / PLT and large enough to allow the diffusion of the small molecule ECM modulator Losartan.

[0015] According to a second aspect of the present application, a method for preparing a DNA hydrogel drug release system is provided, comprising the following steps:

[0016] The DNA single strands for synthesizing the Y-type DNA monomers are added and dissolved in a buffer containing MgCl2, heated to 95℃ for 5-10 min, and annealed to 4℃ to obtain the Y-type DNA monomers;

[0017] The DNA single strands for synthesizing the L-type DNA monomers are added and dissolved in a buffer containing MgCl2, heated to 95℃ for 5-10 min, and annealed to 4℃ to obtain the L-type DNA monomers;

[0018] The small molecule ECM modulator and the platelets loaded with the cytotoxic drug are respectively dissolved or dispersed in a buffer;

[0019] The buffer containing the small molecule ECM modulator, the buffer containing the platelets loaded with the cytotoxic drug, and the Y-type DNA monomers are mixed, and then mixed with the L-type DNA monomers, and the mixture is self-assembled into the DNA hydrogel drug release system.

[0020] In some embodiments, the buffer can be a PBS buffer.

[0021] In some embodiments, the molar ratio of the DNA single strands (Y1, Y2, Y3) for synthesizing Y-type DNA monomers is 1:1.

[0022] In some embodiments, the molar ratio of the DNA single strands (L1, L2) for synthesizing L-type DNA monomers is 1:1.

[0023] In some embodiments, the molar ratio of Y-type DNA monomers and L-type DNA monomers can be (1.8-2.2):(2.8-3.2).

[0024] In some embodiments, the molar ratio of Y-type DNA monomers and L-type DNA monomers can be 2:3.

[0025] In some embodiments, the concentration of the small molecule ECM modulator in the buffer containing the small molecule ECM modulator can be 180-220 μM; the concentration of the platelets loaded with the cytotoxic drug in the buffer containing the platelets loaded with the cytotoxic drug can be 18-22 μM; the concentration of the Y-type DNA monomers can be 225-275 μM; and the concentration of the L-type DNA monomers can be 335-415 μM.

[0026] In some embodiments, the volume ratio of the buffer containing the small molecule ECM modulator, the buffer containing the platelets loaded with the cytotoxic drug, the Y-type DNA monomers, and the L-type DNA monomers can be (8-10):(8-10):(14-18):(14-18).

[0027] In some embodiments, the concentration of the buffer containing the small molecule ECM modulator is 200 μM; the concentration of the buffer containing the platelets loaded with the cytotoxic drug is 20 μM; the concentration of the Y-type DNA monomers is 250 μM; the concentration of the L-type DNA monomers is 375 μM; and the volume ratio of the buffer containing the small molecule ECM modulator, the buffer containing the platelets loaded with the cytotoxic drug, the Y-type DNA monomers, and the L-type DNA monomers is 9:9:16:16.

[0028] The DNA hydrogel drug release system provided by the present application has a simple preparation method, is easy to operate and control, and can overcome the disadvantages of the conventional drug delivery and / or release system, i.e., the drug release cannot be precisely controlled in time and space, thereby effectively enhancing the tumor treatment effect and being applicable to the preparation of an anti-tumor drug.

[0029] In some embodiments, the tumor is a solid tumor.

[0030] In some embodiments, the solid tumor can be at least one selected from the group consisting of hepatocellular carcinoma, breast cancer, neuroblastoma, melanoma, and the like. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Schematic diagram of the synthetic route of the DNA hydrogel drug release system (Los+ICG / PLT@DSH) of Example 2 of the present application;

[0032] Figure 2 Results related to the design, preparation and characterization of Los+ICG / PLT@DSH; wherein: (a) Schematic diagram of the synthetic route of ICG loading on PLT through amide coupling reaction; (b) Confocal fluorescence image showing the co-localization of ICG (green fluorescence) and janelled ring peptide labeled PLT membrane (blue fluorescence) in ICG / PLT (scale bar: 1 μm); (c) DLS analysis of ICG / PLT; (d) UV-Vis-NIR spectrum of ABTS confirming the generation of reactive oxygen species (characteristic peak 734 nm) under near-infrared irradiation of ICG / PLT; (e) Schematic diagram of the synthetic route of DSH; (f) PAGE analysis verifying the formation of Y / L type DNA monomer; (g) Actual photos of liquid Y / L type DNA monomer and gel-like DSH (stained with GelRed); (h) TEM image of the network structure of DSH (scale bar: 100 nm); (i) DSH pore size distribution analyzed by ImageJ (n = 750); (j) Rheological analysis of DSH, storage modulus G' > loss modulus G", confirming the hydrogel property; (k) Schematic diagram of the synthetic route of Los+ICG / PLT@DSH by mixing Y type DNA monomer, L type DNA monomer, Los and ICG / PLT; (l) Confocal image of Los+ICG / PLT@DSH showing the co-localization of Cy5 labeled Los (red fluorescence) and ICG / PLT (green fluorescence) (scale bar: 100 μm); (m) UV-Vis-NIR spectrum confirming the retention of Los (645 nm) and ICG / PLT (720 nm, 780 nm) absorption peaks in Los+ICG / PLT@DSH; (n) Rheological analysis of Los+ICG / PLT@DSH; (o) Injectability of Los+ICG / PLT@DSH through 29G needle;

[0033] Figure 3 Polyacrylamide gel electrophoresis (PAGE) diagram of Y type DNA monomer, L type DNA monomer and DSH;

[0034] Figure 4 SEM image of DSH, scale bar: 200 μm;

[0035] Figure 5Figure 1. In vitro validation of size-gated sequential release of Los+ICG / PLT@DSH. (a) Schematic diagram of the size-selective release mechanism and release kinetics of Los+ICG / PLT@DSH in PBS (neutral control) and tumor lysate (simulated tumor microenvironment); (b) TEM images of DSH in PBS for 14 days (scale bar: 200 nm); (c) Quantitative analysis of the change in pore size of DSH in PBS for 14 days (n≥100); (d) TEM images of DSH in tumor lysate for 14 days (scale bar: 200 nm); (e) Quantitative analysis of the change in pore size of DSH in tumor lysate for 14 days (n≥100); (f-g) Confocal images tracking the sequential release of Los+ICG / PLT@DSH in PBS (f) and tumor lysate (g), scale bar: 100 μm; (h) Schematic diagram of release quantification based on fluorescence signals and cumulative release curves of Los and ICG / PLT in PBS; (i) Schematic diagram of release quantification based on fluorescence signals and cumulative release curves of Los and ICG / PLT in tumor lysate;

[0036] Figure 6 Figure 2. Agarose gel electrophoresis (AGE) images showing the degradation of DSH in PBS and tumor lysate.

[0037] Figure 7 Figure 3. In vitro anti-tumor effect evaluation of Los+ICG / PLT@DSH. (a) Schematic diagram of the experimental procedure for evaluating the generation of reactive oxygen species and cell viability on day 3 (ICG / PLT retention period) and day 7 (ICG / PLT release period); (b-e) Flow cytometry quantitative analysis of intracellular reactive oxygen species in each treatment group on day 3 and day 7; (f-h) Flow cytometry analysis (f), CCK-8 detection (g), and Live / Dead staining (h) (scale bar: 200 μm) of 4T1 cells by each treatment group on day 3; (i-k) Flow cytometry analysis (i), CCK-8 detection (j), and Live / Dead staining (k) (scale bar: 200 μm) of 4T1 cells by each treatment group on day 7; NS indicates no statistical significance, **** P <0.0001;

[0038] Figure 8 Figure 4. Figure 7 (h) Quantitative data of the Live / Dead staining experiment in (h);

[0039] Figure 9 Figure 5. Figure 7 (k) Quantitative data of the Live / Dead staining experiment in (k);

[0040] Figure 10 Related results graphs for in vivo verification experiment of spatiotemporal remodeling of tumor microenvironment (TME) by Los+ICG / PLT@DSH; Wherein: (a) Schematic diagram of in vivo TME remodeling experiment process; (b) Real-time fluorescence tracking image of Los-Cy5 and ICG / PLT fluorescence; (c) Quantitative analysis of Los-Cy5 and ICG / PLT fluorescence in tumor area; (d) Masson staining images of tumor sections in each group (scale: 50 μm); (e) Quantitative analysis of Masson staining results; (f) Immunohistochemical (IHC) staining images of α-SMA of tumor sections in each group after treatment with different treatment regimens (scale: 50 μm); (g) Quantitative analysis of α-SMA immunohistochemical staining results; (h) Immunohistochemical staining images of COL-1 of tumor sections in each group after treatment with different treatment regimens (scale: 50 μm); (i) Quantitative analysis of COL-1 immunohistochemical staining results; (j) Immunofluorescence (IF) staining images of CD31 of tumor sections in each group after treatment with different treatment regimens (scale: 50 μm); (k) Quantitative analysis of CD31 immunofluorescence staining results; (l) Immunofluorescence staining images of HIF-1α of tumor sections in each group after treatment with different treatment regimens (scale: 50 μm); (m) Quantitative analysis of HIF-1α immunofluorescence staining results; (n) Schematic diagram of mechanism of action of Los+ICG / PLT@DSH in remodeling TME; NS represents no statistical significance, **** P <0.0001;

[0041] Figure 11 Masson staining, α-SMA staining, COL-1 staining, HIF-1α staining and CD31 staining images of 4T1 tumor sections on day 1, day 3 and day 7 after treatment with Los+ICG / PLT@DSH (scale: 100 μm);

[0042] Figure 12 Quantitative analysis results of Masson staining, α-SMA staining, COL-1 staining, HIF-1α staining and CD31 staining of 4T1 tumor sections on day 1, day 3 and day 7 after treatment with Los+ICG / PLT@DSH (n=3);

[0043] Figure 13Figure 6 is a series of graphs showing the results of in vivo anti-4T1 tumor effect evaluation experiments of Los+ICG / PLT@DSH; wherein: (a) is a schematic diagram of the experimental procedure of the in vivo anti-4T1 tumor effect evaluation experiment; (b-c) are graphs showing the changes in tumor volume of 4T1 tumor-bearing mice in each group over time after treatment with different treatment regimens; (d) is a graph showing the monitoring of tumor weight of 4T1 tumor-bearing mice in each group after treatment with different treatment regimens; (e) is a graph showing the cumulative survival rate of mice in each group after treatment with different treatment regimens; (f) is a graph showing the H&E staining images of 4T1 tumor sections in each group after treatment with different treatment regimens (scale bar: 100 µm); (g) is a graph showing the TUNEL immunohistochemical staining images of 4T1 tumor sections in each group after treatment with different treatment regimens (scale bar: 100 µm); (h) is a graph showing the Ki67 immunohistochemical staining images of 4T1 tumor sections in each group after treatment with different treatment regimens (scale bar: 100 µm); (i-j) are graphs showing the quantitative analysis of TUNEL (i) and Ki67 (j) immunohistochemical staining results by Image J software; * P <0.05, **** P <0.0001;

[0044] Figure 14 Figure 7 is a Violin plot showing the tumor volume of 4T1 tumor-bearing mice in each group on day 13 after treatment with different treatment regimens; * P <0.05, **** P <0.0001;

[0045] Figure 15Figure (a) is a schematic diagram of the experimental procedure for the in vivo anti-SK-N-BE(2) tumor efficacy evaluation experiment of Los+ICG / PLT@DSH; (b) is a graph showing the change in tumor volume over time in SK-N-BE(2) tumor-bearing mice treated with different treatment regimens; (c) is a graph showing the tumor weight monitoring of SK-N-BE(2) tumor-bearing mice treated with different treatment regimens; (d) is a graph showing the cumulative survival rate of mice treated with different treatment regimens; (e) is a graph showing the H&E staining images of SK-N-BE(2) tumor sections of SK-N-BE(2) tumor-bearing mice treated with different treatment regimens (scale bar: 100 µm); (f-g) are graphs showing the TUNEL (f) and Ki67 (g) immunohistochemical staining images of SK-N-BE(2) tumor sections of SK-N-BE(2) tumor-bearing mice treated with different treatment regimens (scale bar: 100 µm); (h-i) are graphs showing the quantitative analysis of TUNEL (h) and Ki67 (i) immunohistochemical staining results by Image J software; (j) is a graph showing the H&E staining images of the main organs (heart, liver, spleen, lung, kidney) of mice treated with different treatment regimens (scale bar: 50 µm); (k) is a graph showing the blood routine analysis of mice treated with different treatment regimens; (l) is a graph showing the biochemical analysis of liver function and kidney function-related markers of mice treated with different treatment regimens. * P <0.05, **** P <0.0001;

[0046] Figure 16 Figure is a graph showing the growth curve of SK-N-BE(2) tumor volume in mice treated with different treatment regimens.

[0047] Figure 17 Figure is a Violin plot of SK-N-BE(2) tumor volume in mice treated with different treatment regimens on day 13. * P <0.05, **** P <0.0001;

[0048] Figure 18 Figure is a graph showing the body weight monitoring of 4T1 tumor-bearing mice treated with different treatment regimens.

[0049] Figure 19 Figure is a graph showing the body weight monitoring of SK-N-BE(2) tumor-bearing mice treated with different treatment regimens. DETAILED DESCRIPTION

[0050] The present invention will be further described in detail below with reference to the embodiments. The embodiments are for illustrative purposes only and do not limit the invention in any way. Unless otherwise specified, the raw materials and reagents used in the embodiments are conventional products that can be obtained commercially; experimental methods that do not specify specific conditions in the embodiments are generally performed under conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0051] In this invention, all DNA oligonucleotide sequences were synthesized and supplied by Sangon Biotech (Shanghai) Co., Ltd., and used directly without further purification. Losartan (Los) was purchased from Macklin (Shanghai, China). Indocyanine green N-hydroxysuccinimide ester (ICG-NHS) was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Anhydrous magnesium chloride (MgCl2) was supplied by China National Medicines Corp. Ltd. (Beijing, China). Cy5-NHS and AlexaFluor TM The 488 dye was supplied by Sigma-Aldrich (St. Louis, USA). DyLight TM Phalloidin 488 was purchased from CST Bioreactor Co., Ltd. (Shanghai, China). Gelred was sourced from Suzhou Everbright Inc. (Suzhou, China). RPMI-1640 medium, fetal bovine serum (FBS), and penicillin-streptomycin were supplied by Gibco (Waltham, USA). Bio-Rad (Shanghai, China) provided gel loading buffer and agarose for experimental use. Cell Counting Kit-8 (CCK-8), Annexin V-FITC / PI apoptosis assay kit, and Live / Dead cell viability assay kit were supplied by Aladdin Reagent (Shanghai, China). Reactive oxygen species assay kit (DCFH-DA) was purchased from Meilunbio (Dalian, China). Primary antibodies (anti-CD31, anti-HIF-1α, and anti-Ki67) and secondary antibodies were purchased from Abcam (Cambridge, USA). Other primary antibodies (anti-COL-1, anti-α-SMA) were supplied by Proteintech (Wuhan, China). The fluorescent and colorimetric TUNEL apoptosis detection kit was provided by Beyotime Biotechnology (Shanghai, China). Bovine serum albumin (BSA) and Triton X-100 were purchased from Thermo Fisher Scientific (Shanghai, China).

[0052] 4T1 and SK-N-BE(2) cell lines were purchased from Procell (Wuhan, China). These cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin solution at 37℃ in a humidified atmosphere of 5% CO2.

[0053] 4-6-week-old female BALB / c mice were purchased from Guangdong Medical Laboratory Animal Center. To establish tumor models, 5×10 6 4T1 and SK-N-BE(2) cells were subcutaneously injected into the unilateral inguinal region of the recipient mice, respectively. All animals were given careful treatment and care according to the guidelines of the Southern Medical University Institutional Animal Care and Use Committee.

[0054] In the present application, all experiments were repeated at least three times. The data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, Inc., USA). The differences between two groups were compared using unpaired two-tailed Student's t-test. The differences between multiple groups were compared using one-way analysis of variance (ANOVA). * P <0.05 was considered statistically significant.

[0055] Example 1 Synthesis of DNA hydrogel

[0056] The nucleotide sequences of the DNA single strands (ssDNA) Y1~Y3 for synthesizing Y-type DNA monomers and the DNA single strands L1~L2 for synthesizing L-type DNA monomers are shown in Table 1.

[0057] Table 1 Nucleotide sequences of Y1~Y3 and L1~L2

[0058]

[0059] The synthesis of the DNA hydrogel includes the following steps:

[0060] (1) Dissolve the DNA single strands Y1~Y3 in a PBS buffer solution containing MgCl2 (Mg 2+ concentration of 2 mM) and perform a thermal annealing process, heat to 95℃, incubate for 5~10 min, then gradually reduce the temperature from 95℃ to 4℃ at a controlled rate of 1℃ per min, to obtain Y-type DNA monomers; wherein the molar ratio of Y1, Y2 and Y3 is 1:1:1.

[0061] (2) Dissolve the DNA single strands L1~L2 in a PBS buffer solution containing MgCl2 (Mg 2+The L-type DNA monomer is obtained by mixing the Y-type DNA monomer (250 μM, 16 μL) and the L-type DNA monomer (375 μM, 16 μL) with PBS containing MgCl2 (Mg2+concentration is 2 mM, 18 μL) and performing a thermal annealing process, heating to 95℃, incubating for 5-10 min, and then gradually reducing the temperature from 95℃ to 4℃ at a controlled rate of 1℃ per minute; wherein the molar ratio of L1 to L2 is 1:1.

[0062] (3) The Y-type DNA monomer (250 μM, 16 μL) and the L-type DNA monomer (375 μM, 16 μL) are mixed with PBS containing MgCl2 (Mg2+concentration is 2 mM, 18 μL) to rapidly form a gel within 10 seconds, and a DNA hydrogel is obtained. 2+ The DNA hydrogel is obtained by mixing the Y-type DNA monomer (250 μM, 16 μL) and the L-type DNA monomer (375 μM, 16 μL) with PBS containing MgCl2 (Mg2+concentration is 2 mM, 18 μL) to rapidly form a gel within 10 seconds.

[0063] Example 2 Synthesis of a DNA hydrogel drug release system (Los+ICG / PLT@DSH)

[0064] The preparation methods of the Y-type DNA monomer and the L-type DNA monomer are the same as in Example 1.

[0065] The synthesis route of Los+ICG / PLT@DSH is shown in Figure 1 and includes the following steps:

[0066] (1) Preparation of ICG-loaded platelets (ICG / PLT):

[0067] A whole blood sample is collected, and platelets are separated by differential centrifugation, and the platelets are resuspended in PBS containing 1 µM PGE1 to obtain a PLT suspension;

[0068] ICG-NHS is dissolved in ddH2O to prepare a 40 μM ICG-NHS solution; then the PLT suspension and the ICG-NHS solution are mixed, and a large number of amino acids in the amino acid residues of the platelet membrane protein can directly react with ICG-NHS to form an amide coupling reaction under the condition of pH=7.8 at room temperature, and ICG is loaded on the PLT; after the reaction is completed, the ICG / PLT complex is removed by centrifugation at 1000×g for 10 min to remove impurities, and finally the ICG / PLT complex is resuspended in PBS and stored at 4℃ for standby use. g The reaction process of the amide coupling reaction is shown below:

[0069]

[0070]

[0071] (2) The Los powder is directly dissolved in PBS to a final concentration of 2 mM; and a Los solution with a desired concentration is obtained by dilution before use.

[0072] ​(3) The Los-containing PBS (200 μM, 9 μL), ICG / PLT-containing PBS (20 μM, 9 μL) and Y-type DNA monomer (250 μM, 16 μL) were mixed uniformly, and then mixed with L-type DNA monomer (375 μM, 16 μL); the mixed solution was converted into a gel-like substance within 10 seconds at ambient temperature, which was Los+ICG / PLT@DSH.

[0073] Test Example 1, Characterization of ICG / PLT

[0074] To construct Los+ICG / PLT@DSH, the inventors loaded the cytotoxic therapeutic agent ICG onto the PLT membrane through amide coupling reaction, and synthesized the larger-sized therapeutic component ICG / PLT Figure 2 a) in the formula.

[0075] To confirm whether ICG / PLT was successfully prepared, the inventors observed the PLT by phalloidin staining. The PLT cultured in a confocal dish was fixed with 4% paraformaldehyde for 15 minutes, and then permeabilized with 0.5% Triton X-100. Subsequently, it was blocked with 5% BSA for one hour. Then, the PLT was labeled with DyLight 488 Phalloidin and incubated in the dark for 15 minutes. A confocal laser scanning microscope (Nikon, Japan) was used to acquire images. TM

[0076] In addition, 2,2’-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) was also used as a probe to evaluate the generation of reactive oxygen species (ROS) to study the potential of ICG / PLT for photodynamic therapy under near-infrared (NIR) irradiation. ABTS reacts with ROS to form cationic ABTS ●+ , which shows a clear absorption peak at 736 nm. The samples were prepared and divided into five groups: PBS (control), ICG alone, ICG / PLT, ICG+NIR and ICG / PLT+NIR. After exposure to NIR irradiation (980 nm, 0.5 W / cm 2 , Microenerg CME-L980, Beijing, China) for 10 minutes, each group was mixed with 100 μL of 5 mM ABTS solution (ICG concentration was 20 μM in all groups except the PBS group). After incubation for 5 minutes, the absorption spectrum of the solution was recorded in the wavelength range of 400-900 nm using a UV-visible spectrophotometer (Hitachi-UV-3600 Plus, Japan).

[0077] ​A particle size analyzer (Litesizer TM 500, Anton Paar) was used to evaluate the particle size distribution of ICG / PLT.

[0078] The results are shown in b~d of Figure 2 .

[0079] Confocal fluorescence microscopy showed that ICG (green fluorescence) co-localized with the platelet membrane stained by phalloidin (blue fluorescence), confirming that ICG had been successfully loaded onto PLT (b of Figure 2 ). Dynamic light scattering (DLS) analysis showed that the hydrodynamic diameter of the synthesized ICG / PLT particles was about 1221 nm (c of Figure 2 ), which was significantly larger than that of the small molecule Losartan (Los). The significant size difference between ICG / PLT and Los provided a key structural basis for subsequent size-gated sequential release of Los and ICG / PLT, allowing the selective diffusion of small molecule Los while retaining larger particle ICG / PLT within the hydrogel matrix. In addition, ICG / PLT particles retained the ability to generate reactive oxygen species (ROS) after photoactivation (d of Figure 2 ), which is a basic property of efficient photodynamic therapy (PDT).

[0080] Test Example 2, Characterization of DSH and Los+ICG / PLT@DSH

[0081] The migration behavior of ssDNA, Y-type DNA monomer, L-type DNA monomer, and DSH was analyzed by gel electrophoresis. Scanning electron microscopy (SEM, Hitachi S-3000N, Japan) and transmission electron microscopy (TEM, Hitachi HT7800, Japan) were used to observe the structural characteristics of DSH. To confirm the encapsulation of Los and ICG / PLT into DSH, Los was labeled with Cy5-NHS fluorescent dye, and then confocal microscopy was used for imaging to capture the visual features of DSH and Los+ICG / PLT@DSH. In addition, the rheological properties of DSH and Los+ICG / PLT@DSH were analyzed using a MCR 302e rheometer (Anton Paar, Austria).

[0082] The present application synthesized Y-type DNA monomer and L-type DNA monomer by thermal annealing partially complementary DNA single strands (synthesis schematic as shown in e of Figure 2 ). Polyacrylamide gel electrophoresis (PAGE) analysis confirmed the successful synthesis of these two monomers (e of Figure 2(f in the text). Compared to isolated, partially complementary ssDNA, Y and L-type DNA monomers exhibited slower migration rates in PAGE, attributed to their increased molecular weight. The slower migration rate in PAGE is evidenced by the self-assembly of the two monomers into a DNA hydrogel (DSH). Figure 3 This indicates the formation of a larger supramolecular structure. For example... Figure 2 As shown in g, DNA monomers remain in a fluid state at ambient temperature, while DSH forms a gel-like substance that settles at the bottom of the microcentrifuge tube, visually confirming gelation. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) reveal that DSH has a distinct network structure. Figure 2 h and Figure 4 The average pore size is approximately 50 nm. Figure 2 (i) This well-defined porous structure, with a pore size smaller than the diameter of ICG / PLT (approximately 1221 nm), but large enough to allow for small molecule Los diffusion, provides a structural basis for size-gated sequential drug release. Furthermore, rheological analysis indicates that the shear storage modulus (G') is higher than the shear loss modulus (G''). Figure 2 (j) is a marker of hydrogel behavior, confirming the successful synthesis of DSH with stable mechanical properties.

[0083] The Los+ICG / PLT@DSH system was prepared by mixing Y-type DNA monomers, L-type DNA monomers, Los, and ICG / PLT. Figure 2 (k in the text). To verify the co-encapsulation of Los and ICG / PLT within the DSH, Los was pre-labeled with Cy5-NHS before assembly. Laser scanning confocal microscopy images showed clear co-localization of Cy5 fluorescence (Ex / Em = 640 / 664 nm, corresponding to Los) and ICG fluorescence (Ex / Em = 785 / 810 nm) within the DSH structure, confirming the effective co-encapsulation of the two components. Figure 2 The UV-Vis-NIR spectroscopy further validated the integrity of the encapsulated drug: the characteristic absorption peaks of Los (645 nm) and ICG / PLT (720 nm and 780 nm) were well preserved in the final Los+ICG / PLT@DSH. Figure 2 The presence of 'm' indicates that encapsulation did not alter the chemical properties of any component. Rheological characterization of Los+ICG / PLT@DSH showed that it retained typical hydrogel properties comparable to pure DSH. Figure 2 The presence of n in the formula (G'>G'') confirms stable gel formation. Furthermore, the DNA hydrogel provided by this invention can be easily aspirated and injected using a 29G needle. Figure 2The injectability and flowability of Los+ICG / PLT@DSH were confirmed by measuring the viscosity of the suspension in o), which is a key feature for minimally invasive drug delivery in subsequent in vivo anti-tumor applications.

[0084] In vitro release characteristics of Los+ICG / PLT@DSH in Example 3

[0085] The ability of Los+ICG / PLT@DSH to achieve spatiotemporal sequential drug release mainly relies on the size-gated sequential release: small molecule Los (diameter less than DSH pore size) diffuses rapidly, while larger size ICG / PLT (about 1100-1400 nm) is retained until DSH degrades and the pore size expands to allow its release. This size-selective behavior is further regulated by the tumor microenvironment, where high DNase activity accelerates DSH degradation, ensuring spatiotemporal control. To verify the release characteristics of Los+ICG / PLT@DSH, the present application evaluated the drug release curves under two environments: PBS (as a neutral control) and tumor lysate (simulating the tumor microenvironment) (a) in Example 3. Figure 5

[0086] The preparation method of the tumor lysate comprises the following steps: subcutaneously injecting 4T1 cells into BALB / c mice to construct a 4T1 tumor model, when the tumor volume reaches 300-500 mm 3 , collect the tumor tissue; cut the tissue into fine pieces; take 30 mg of the tissue, wash it with PBS, and then absorb the water with a water-absorbing paper, put it into a 1.5 mL centrifuge tube, place it on ice, put 2 grinding steel balls into each tube, add RIPA lysis buffer, and grind it in a grinder until no tissue is visible; after precooling the centrifuge, centrifuge at 15000 rpm and 4°C for 10-15 min, take the supernatant (containing enzyme protein), and obtain the tumor lysate, which is stored at low temperature.

[0087] In this test example, Los+ICG / PLT@DSH contains Los 36 μM and ICG 3.6 μM.

[0088] To study the sequential release characteristics of Los+ICG / PLT@DSH, Los+ICG / PLT@DSH was incubated with PBS and tumor lysate in transwell chambers, respectively. At 1, 3, 5, 7, and 14 days after incubation, the remaining Los+ICG / PLT@DSH in the upper layer was collected for TEM analysis to evaluate DSH degradation and pore size changes.

[0089] To evaluate the release behavior of the two drugs, Los+ICG / PLT@DSH was incubated with PBS and tumor lysate, respectively. At 1, 3, and 7 days after incubation, the remaining samples were transferred to glass slides and monitored for drug release by confocal laser scanning microscopy.

[0090] ​To assess the stability of DSH, agarose gel electrophoresis (AGE) was used to detect the change of DNA content of Los+ICG / PLT@DSH in PBS and tumor lysate.

[0091] To further verify the sequential release of the two drugs, Los+ICG / PLT@DSH was placed in dialysis bags and incubated with PBS and tumor lysate, respectively. The medium outside the dialysis bag was collected at predetermined time intervals, and the change of fluorescence intensity was measured.

[0092] All the above experiments were carried out in a light-proof environment to minimize fluorescence quenching.

[0093] The results are shown in Figure 5 .

[0094] The structural changes of DSH were monitored by TEM, and the results showed that in PBS, the hydrogel degraded slowly, and the pore size moderately increased from about 50 nm to about 430 nm at day 14 (b~c in Figure 5 ). In contrast, the tumor lysate triggered rapid enzymatic degradation: the pore size expanded to more than 1200 nm at day 7, and to about 1560 nm at day 14 (d~e in Figure 5 ), exceeding the diameter of ICG / PLT, enabling its release.

[0095] Agarose gel electrophoresis (AGE) of residual DNA in DSH also confirmed the above trend: DNA loss was minimal in PBS (structurally stable), while it was significantly degraded in tumor lysate, with a clear decrease in undigested DNA at day 7 ( Figure 6 ); showing the environment-dependent degradation kinetics essential for targeted release.

[0096] Confocal imaging of Cy5-labeled Los (Los-Cy5) and ICG / PLT further visualized the release kinetics. In PBS, Los-Cy5 stably diffused from day 1 to day 7, while ICG / PLT was still trapped due to incomplete pore expansion (f in Figure 5 ). However, in tumor lysate, pore expansion was triggered by DSH degradation at day 1, allowing rapid release of Los-Cy5; from day 3 to day 7, the pore size was sufficiently enlarged to release ICG / PLT, resulting in the sequence of Los release followed by ICG / PLT release (g in Figure 5 ). This is consistent with the pore size changes observed by TEM, confirming the size-gated control.

[0097] Quantitative fluorescent analysis of the supernatant further confirmed the release rates. Los-Cy5 showed rapid, near-complete release in both environments: 89.7% in PBS and 85.7% in tumor lysate by day 14, reaching a plateau by day 7 (79.4% and 73.5%, respectively). However, for ICG / PLT, release was strictly dependent on the environment: in PBS, only 12.1% was released by day 7 and 41.9% by day 14 due to less DSH degradation; in tumor lysate, 69.3% was released by day 7 and 78.6% by day 14. Figure 5 This stark contrast demonstrates that Los releases rapidly regardless of the environment (driven by size), while ICG / PLT release is delayed and only accelerated in tumor lysate (driven by both size and enzymatic degradation), directly validating the release profile of the drug size-gated sequential release in Los+ICG / PLT@DSH.

[0098] The above results demonstrate that Los+ICG / PLT@DSH achieves programmable spatiotemporal release: small molecule Los first remodels the ECM (through early, size-driven release), while larger-sized ICG / PLT is retained and released later in the tumor microenvironment (through degradation-driven pore size expansion), laying the foundation for synergistic anti-tumor efficacy.

[0099] Test Example 4, Evaluation of Anti-tumor Effect in Vitro of Los+ICG / PLT@DSH

[0100] The in vitro anti-tumor effect of Los+ICG / PLT@DSH was verified according to the experimental method outlined in a of Figure 7

[0101] Tumor cells (4T1) were divided into four groups (PBS, ICG, ICG / PLT@DSH, and Los+ICG / PLT@DSH), and each treatment formulation was administered at an ICG dose of 3.6 μM, and evaluated at two time points (3 days and 7 days).

[0102] 5 x 10 5 Tumor cells were seeded in the lower chamber of a transwell. Subsequently, the cells were cultured with complete medium and incubated at 37°C for 24 hours. The drugs were placed in the upper chamber of the transwell. For the 3-day group, the cells were divided into four groups: (I) PBS group, treated with PBS for 3 days, then irradiated with a 980 nm laser (0.5 W / cm 2 2 ​​irradiation; (III) ICG / PLT@DSH group, treated with ICG / PLT@DSH for 3 days, followed by 980 nm laser (0.5 W / cm 2 irradiation; (IV) Los+ICG / PLT@DSH group, treated with Los+ICG / PLT@DSH for 3 days, followed by 980 nm laser (0.5 W / cm 2 irradiation. For the 7-day group, the treatment procedure was the same as the 3-day group, except that the drug exposure time was extended to 7 days. Cell-free wells were used as blank controls.

[0103] (1) In vitro ROS detection

[0104] ICG is the core of the Los+ICG / PLT@DSH cytotoxic phase, and its PDT efficacy mainly depends on its ability to release and generate ROS around tumor cells under near-infrared (NIR) light irradiation. Therefore, according to the drug release curve determined in Test Example 3 (at day 3, ICG / PLT mainly remained within the DSH, while by day 7, most had been released), the intracellular ROS level was evaluated using flow cytometry at two selected time points, day 3 and day 7, respectively.

[0105] After drug treatment, the culture supernatant was removed from all experimental groups, and the cell monolayer was gently rinsed with PBS. Then, freshly prepared DCFH-DA probe solution (2’,7’-dichlorodihydrofluorescein diacetate) was added to each well, after which the culture plate was transferred to a humidified 37 °C incubator for 20 minutes for probe loading. After incubation, the dye remaining outside the cells was eliminated by three consecutive PBS washes, and then the cells were resuspended in serum-free medium. The CytoFLEX flow cytometry system (Beckman Coulter, USA) was used for quantitative evaluation of intracellular ROS, and instrument calibration and data acquisition were performed according to the standardized protocol. The data were analyzed using FlowJo software (TreeStar, Inc., Ashland, USA).

[0106] As shown in b~c of FIG. 3, at day 3, both the ICG / PLT@DSH and Los+ICG / PLT@DSH groups showed significantly lower ROS generation than the ICG group. This is attributed to the fact that the DSH had not degraded by day 3, and its pore size was still relatively small; at this stage, the larger-sized ICG / PLT could not escape from the hydrogel. In contrast, as shown in d~f of FIG. 3, by day 7, the ROS level in the ICG / PLT@DSH group was significantly higher than that in the ICG group, which is attributed to the fact that the DSH had degraded by day 7, and its pore size had increased; at this stage, the larger-sized ICG / PLT could escape from the hydrogel. Figure 7 Figure 7 ​As shown in Figure 6D, by day 7, the ROS levels generated under NIR irradiation in the ICG / PLT@DSH and Los+ICG / PLT@DSH groups were comparable to that of the ICG group. This indicates that after the effective enzymatic degradation of DSH and subsequent pore size expansion, ICG / PLT can be released into the extracellular environment to exert its photodynamic activity, verifying the timed activation of PDT by Los+ICG / PLT@DSH.

[0107] (2) Evaluation of the in vitro anti-tumor effect of Los+ICG / PLT@DSH

[0108] The in vitro anti-tumor effect of Los+ICG / PLT@DSH was evaluated using flow cytometry with Annexin V / PI staining, CCK-8, and Live / Dead cell staining experiments.

[0109] CCK-8 assay: Performed according to the manufacturer's instructions. After drug treatment, cells were collected and transferred to a 96-well plate, then the cells were incubated with 4 μL CCK-8 solution for 4 hours. The optical density (OD value) was determined at 450 nm using an Absorbance Microplate Reader (Winooski, USA). The cell viability (%) was calculated as follows:

[0110] Cell viability (%) = (OD 处理 - OD 空白 ) / (OD PBS - OD 空白 ) × 100%.

[0111] Live / Dead cell assay: Performed according to the manufacturer's instructions. After drug treatment, cells were collected and washed thoroughly with PBS, then incubated with Calcein-AM / PI co-staining reagent at 37°C for 20-40 minutes. After washing twice with PBS, the cells were analyzed using a confocal microscope.

[0112] Flow cytometry: Performed using the Annexin V-FITC / PI apoptosis detection kit according to the manufacturer's instructions. After drug treatment, cells were collected by centrifugation and resuspended in binding buffer (400 μL), then labeled with Annexin V-FITC and PI. After incubation for 20 minutes, data acquisition was performed using a CytoFLEX flow cytometry system (Beckman Coulter, USA).

[0113] As Figure 7As shown in f, on day 3, the cell viability of the ICG / PLT@DSH group (88.5%) and the Los+ICG / PLT@DSH group (89.5%), as measured by flow cytometry, was higher than that of the ICG group (63.3%). This result indicates that by day 3, unreleased ICG / PLT was ineffective in generating photodynamic activity and failed to induce significant cytotoxicity, which is consistent with the purpose of the Los+ICG / PLT@DSH design provided in this invention to delay PDT until after ECM remodeling. By day 7, the cell viability of the ICG group, ICG / PLT@DSH group, and Los+ICG / PLT@DSH group were 63.0%, 60.0%, and 59.4%, respectively, significantly lower than that of the PBS group (…). Figure 7 (i). This indicates that the successful release of ICG / PLT and the subsequent photodynamic effect led to enhanced cytotoxicity, which is the expected result of the Los+ICG / PLT@DSH sequential release provided by this invention. CCK-8 ( Figure 7 (g and j in the sample) and Calcein-AM / PI co-staining experiment ( Figure 7 h and k in Figure 8 , Figure 9 The results all demonstrated tumor cell apoptosis under different treatment regimens, with Los+ICG / PLT@DSH showing the most significant anti-apoptotic effect.

[0114] The above results further validate the controlled sequential release of two drugs with different molecular sizes in Los+ICG / PLT@DSH and demonstrate that the drug release system does not impair the PDT efficacy of ICG.

[0115] Example 5: In vivo validation of Los+ICG / PLT@DSH achieving spatiotemporal remodeling of the tumor microenvironment through sequential drug release.

[0116] Using 4T1 tumor-bearing mice, according to Figure 10 The experimental method outlined in section a verifies whether Los+ICG / PLT@DSH can achieve spatiotemporal control of the tumor microenvironment (TME).

[0117] A 4T1 tumor model was established by subcutaneous injection of 4T1 cells into BALB / c mice. When the tumor volume reached 100 mm... 3Mice were randomly divided into six experimental groups: PBS group, ICG group, ICG@DSH group, ICG / PLT@DSH group, Los+ICG@DSH group, and Los+ICG / PLT@DSH treatment group (groups I-VI), with 3 mice in each group. Los was labeled with Cy5-NHS fluorescent dye, and each treatment agent was administered at a dose of 36 μM Los and 3.6 μM ICG, respectively. All treatment agents were injected circumferentially along the tumor margin (1-2 cm) in a 50 μL volume.

[0118] (1) In vivo release characteristics of Los+ICG / PLT@DSH

[0119] To assess the in vivo release kinetics of Los+ICG / PLT@DSH, the distribution dynamics of Cy5-labeled Los and ICG / PLT in tumor tissues were longitudinally tracked at predetermined time points after administration (days 0, 1, 2, 3, 4, 5, 6, 10, and 13) using the IVIS Spectrum imaging platform. Fluorescence signals were quantitatively evaluated using Living Image 4.3.1 analysis software (PerkinElmer, USA).

[0120] like Figure 10 As shown in b~c, the fluorescence intensity of Los-Cy5 decreased significantly from day 0 to day 3 and disappeared completely on day 3, indicating rapid release and metabolism of Los-Cy5, consistent with the early ECM remodeling process expected by Los+ICG / PLT@DSH. In contrast, the fluorescence intensity of ICG / PLT remained almost unchanged for the first three days and gradually decreased from day 3 to day 10, reflecting its delayed release to tumor cells.

[0121] (2) Verification of ECM reshaping effect of Los+ICG / PLT@DSH

[0122] Tumor tissues were collected from different treatment groups (PBS, ICG, ICG@DSH, ICG / PLT@DSH, Los+ICG@DSH and Los+ICG / PLT@DSH) at different time points (days 1, 3 or 7 after treatment) for histological analysis to verify the ECM remodeling effect of Los+ICG / PLT@DSH.

[0123] The excised tumors were fixed with 4% paraformaldehyde for 24 h, followed by ethanol dehydration and paraffin embedding. Four-micrometer-thick tissue sections were prepared using a microtome, followed by deparaffinization, hydration, and antigen retrieval treatment. After blocking with 5% BSA for 30 min, the specimens were incubated with CD31 and HIF-1a primary antibodies at 4 °C overnight, followed by incubation with fluorescently labeled secondary antibodies at room temperature for 1 h. Masson trichrome staining and immunohistochemical staining of COL-1 and a-SMA were also performed. Finally, the immunofluorescence / immunohistochemical treated samples were observed by a digital slide scanner to evaluate the extracellular matrix remodeling and hypoxia improvement.

[0124] Masson staining results showed that the fibrosis was significantly reduced in the Los+ICG@DSH and Los+ICG / PLT@DSH groups (Fig. 2d~e), which was a key evidence of ECM remodeling. Immunohistochemical (IHC) analysis of a-smooth muscle actin (a-SMA, a specific marker of myofibroblast activation) and collagen I (COL-1) expression further confirmed that Los+ICG@DSH and Los+ICG / PLT@DSH exhibited significant tumor ECM remodeling effects (Fig. 2f~i). Figure 10 Figure 10

[0125] Immunofluorescence (IF) analysis of the angiogenesis marker CD31 showed that the expression level of CD31 was significantly increased in the Los+ICG@DSH and Los+ICG / PLT@DSH groups (Fig. 2j~k), which provided evidence for the improvement of vasculature after ECM remodeling. This modulation explained the reason for the decreased expression of hypoxia-inducible factor-1a (HIF-1a, a marker of tumor hypoxia) (Fig. 2l~m) in the same treatment groups. This alleviation of hypoxia provided a prerequisite for ICG to subsequently exert better PDT effects. As shown in Fig. 2, during this period, Masson staining, IHC staining of a-SMA and COL-1, and IF staining of HIF-1a all showed statistically significant reductions in tumor tissues in the Los+ICG / PLT@DSH group, while CD31 staining showed a significant increase. Quantitative analysis further indicated that the most significant ECM remodeling occurred between day 1 and day 3 (Fig. 2n), creating favorable conditions for subsequent ICG / PLT adhesion to tumor cells and exerting enhanced therapeutic effects. Figure 10 Figure 10 Figure 11 Figure 12

[0126] ​​​​​​The above results show that the Los+ICG / PLT@DSH provided by the application can achieve sequential drug delivery and spatiotemporal control of TME. Specifically, intratumoral injection of Los+ICG / PLT@DSH leads to rapid release of Los, thereby destroying ECM, remodeling TME, relieving hypoxia, and promoting the adhesion, penetration and activation of ICG / PLT in the tumor, which is the basic principle of the DNA hydrogel drug release system of the application Figure 10 wherein n is as defined in the above.

[0127] Test Example 6: Evaluation of the in vivo anti-tumor effect of Los+ICG / PLT@DSH

[0128] In order to systematically verify the in vivo anti-tumor effect of Los+ICG / PLT@DSH and its universality in different tumor types, the application conducted experiments in two different solid tumor models: triple-negative breast cancer (4T1) and neuroblastoma (SK-N-BE(2)).

[0129] BALB / c mice were subcutaneously injected with 4T1 or SK-N-BE(2) cells to construct tumor models, and when the tumor volume reached 100 mm 3 When the tumor volume reached 100 mm 2 , the mice were randomly divided into six experimental groups: PBS group, ICG group, ICG@DSH group, ICG / PLT@DSH group, Los+ICG@DSH group and Los+ICG / PLT@DSH treatment group (groups I-VI), with 4-6 mice in each group. Each treatment formulation was administered at a dose of Los 36 μM and ICG 3.6 μM. All drugs were administered by peritumoral injection (circular injection 1-2 cm from the tumor edge) to avoid intratumoral leakage, and each group maintained a uniform injection volume of 50 μL to ensure consistency of administration.

[0130] After injection, light irradiation was performed on days 1, 3, 5 and 7 (980 nm, 0.5 W / cm 2 for 10 minutes). Tumor growth and mouse body weight changes were monitored for 13 consecutive days. At the end of the experimental period, representative mice were selected to obtain tumor tissue for histopathological processing, including H&E staining, immunohistochemical analysis (TUNEL-based apoptosis detection and Ki67 proliferation assessment). The remaining experimental subjects were subjected to longitudinal survival monitoring for 60 days.

[0131] The formula for calculating the volume of the tumor ellipsoid is V =½(W 2 ×L), where L is the maximum length (mm) and W is the perpendicular width (mm).

[0132] Biosafety assessment: To assess the biosafety of Los+ICG / PLT@DSH, mice were sacrificed 24 hours after the last administration. Major organs (heart, liver, spleen, lung, and kidney) were collected for H&E staining. Simultaneously, blood samples were collected for hematological and biochemical analysis.

[0133] In a 4T1 tumor-bearing BALB / c nude mouse model, mice were randomly divided into six groups (PBS, ICG, ICG@DSH, ICG / PLT@DSH, Los+ICG@DSH, and Los+ICG / PLT@DSH), and each drug was administered 50 μL via intratumoral injection, followed by NIR irradiation (980 nm, 0.5 W / cm²) on days 1, 3, 5, and 7. 2 ), to ensure the PDT efficacy of the released ICG / PLT ( Figure 13 (a) Dynamic monitoring results showed that the tumors in the Los+ICG / PLT@DSH group exhibited the most significant growth inhibition, in stark contrast to the rapid progression in the PBS, ICG, ICG@DSH, ICG / PLT@DSH, and Los+ICG@DSH groups. Figure 13 (b~c) 13 days after treatment, the Los+ICG / PLT@DSH group showed the smallest tumor burden ( Figure 13 d and Figure 14 ) and longest survival time ( Figure 13 The histopathological analysis (H&E, TUNEL, and Ki67 staining) further confirmed the presence of extensive tumor cell necrosis, apoptosis, and reduced proliferation in the Los+ICG / PLT@DSH group, demonstrating its potent cytotoxicity. Figure 13 f~j in the middle.

[0134] Consistent results were observed in the SK-N-BE(2) neuroblastoma model. A similar experimental design was used ( Figure 15 In the SK-N-BE(2) neuroblastoma model, Los+ICG / PLT@DSH also showed excellent tumor growth inhibition. Figure 15 b~c and Figures 16-17 Survival analysis showed that all mice in this group survived for 40 days, and 25% of the mice in this group were still alive at the 60-day endpoint, while mice in the control group (PBS, ICG, ICG@DSH, ICG / PLT@DSH) died within 20-40 days. Figure 15 (d). Histological evaluation was similar to that of the 4T1 tumor model, with the Los+ICG / PLT@DSH group showing the highest levels of coagulative necrosis and apoptosis (d). Figure 15 (e~i in the middle).

[0135] No significant weight loss, acute toxicity, or organ damage was observed in either tumor model. Figures 18-19 The biosafety assessment results further confirmed that Los+ICG / PLT@DSH did not cause pathological changes in major organs. Figure 15 (j) or abnormal blood routine / biochemical parameters ( Figure 15 The data (k~l) verified that the Los+ICG / PLT@DSH provided by this invention has excellent biocompatibility and biosafety.

[0136] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A DNA hydrogel drug release system characterized by, The composition comprises a DNA hydrogel, and a small molecule ECM regulator and a platelet loaded with a cytotoxic drug encapsulated in the DNA hydrogel; the small molecule ECM regulator is losartan; the cytotoxic drug is indocyanine green; the DNA hydrogel is formed by self-assembly of Y-type DNA monomers and L-type DNA monomers, wherein the molar ratio of the Y-type DNA monomers to the L-type DNA monomers is (1.8-2.2):(2.8-3.2); the Y-type DNA monomers are synthesized by hybridization of DNA single strands with nucleotide sequences shown in SEQ ID NOs: 1-3, and the molar ratio of any two DNA single strands is 1:1; the L-type DNA monomers are synthesized by hybridization of DNA single strands with nucleotide sequences shown in SEQ ID NOs: 4-5, and the molar ratio of any two DNA single strands is 1:

1.

2. The method of claim 1, wherein the DNA hydrogel drug release system is prepared by the steps of: The method comprises the following steps: DNA single strands of the Y-type DNA monomers are added and dissolved in a buffer containing MgCl2, heated to 95℃, and incubated for 5-10 min; annealed to 4℃ to obtain the Y-type DNA monomers; the molar ratio of any two DNA single strands of the DNA single strands of the Y-type DNA monomers is 1:1; DNA single strands of the L-type DNA monomers are added and dissolved in a buffer containing MgCl2, heated to 95℃, and incubated for 5-10 min; annealed to 4℃ to obtain the L-type DNA monomers; the molar ratio of any two DNA single strands of the DNA single strands of the L-type DNA monomers is 1:1 The small molecule ECM regulator and the platelet loaded with the cytotoxic drug are respectively dissolved or dispersed in a buffer; The buffer containing the small molecule ECM regulator, the buffer containing the platelet loaded with the cytotoxic drug, and the Y-type DNA monomers are mixed, and then mixed with the L-type DNA monomers, and the mixture is self-assembled into a DNA hydrogel drug release system; wherein the molar ratio of the Y-type DNA monomers to the L-type DNA monomers is (1.8-2.2):(2.8-3.2).

3. The method of claim 2, wherein the DNA hydrogel drug release system is prepared by the steps of: The concentration of the buffer containing the small molecule ECM regulator is 180-220 μM; the concentration of the buffer containing the platelet loaded with the cytotoxic drug is 18-22 μM; the concentration of the Y-type DNA monomers is 225-275 μM; the concentration of the L-type DNA monomers is 335-415 μM; and the volume ratio of the buffer containing the small molecule ECM regulator, the buffer containing the platelet loaded with the cytotoxic drug, the Y-type DNA monomers, and the L-type DNA monomers is (8-10):(8-10):(14-18):(14-18).

4. Use of the DNA hydrogel drug release system according to claim 1 in the preparation of an antitumor drug, characterized by, The tumor is selected from at least one of breast cancer and neuroblastoma.

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