A vitamin c intelligent delivery nano-assembly based on biomimetic imprinting technology and its anti-tumor application

Abstract

This invention discloses a vitamin C smart delivery nanocomponent based on biomimetic imprinting technology and its anti-tumor application. The core of the nanocomponent is a SiO2 nanoparticle containing cytochrome C, and the surface is directionally imprinted with a vitamin C-specific imprinted cavity using ascorbyl glucoside (AA2G), a stable derivative of vitamin C, as a molecular imprinting template. The preparation steps include: SiO2 nanoparticles containing cytochrome C. 2 The synthesis, amino-functionalization, boric acid-functionalization, template immobilization, directional imprinting, secondary amino-functionalization, template removal, and DMMA modification of NPs-CC led to the construction of the nano-component vMIPDcc containing cytochrome C in its core and having a vitamin C-specific imprinted cavity on its surface. This nanocomponent exhibits high selectivity (IF=8.4) and reliable recognition ability for vitamin C. vMIPDcc achieved an in vivo inhibition rate of 60.2% against colon cancer, providing new ideas and options for the development of anti-colon cancer drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a smart vitamin C delivery nanocomponent based on biomimetic imprinting technology and its anti-tumor application. Background Technology

[0002] Tumor immunotherapy, by activating the host's immune system to specifically recognize and kill tumor cells, has become a revolutionary breakthrough in cancer treatment following surgery, radiotherapy, and chemotherapy. However, the complex immunosuppressive network in the tumor microenvironment (TME), such as oxidative stress (manifested as high levels of reactive oxygen species, ROS) and metabolic abnormalities (such as high concentrations of glutathione, GSH), severely weakens the activity and infiltration capacity of immune cells, leading to poor treatment outcomes.

[0003] Currently, nano-strategies for regulating tumor metastasis (TME) largely rely on exogenous chemotherapeutic agents or scavengers (such as ROS scavengers and GSH depleting agents). Although these methods can improve the immunosuppressive state of TME to some extent, their exogenous components suffer from high biotoxicity, off-target effects, poor biocompatibility, and potential systemic side effects, limiting their clinical translation and application. In contrast, endogenous substances such as vitamin C (Vc) show great potential due to their naturally low toxicity, good biocompatibility, and unique bidirectional regulatory functions: in TME, Vc can act as an antioxidant to scavenge excess ROS and restore the function and vitality of immune cells (such as T cells and NK cells), and can also inhibit tumor metastasis through epigenetic regulatory mechanisms (such as promoting DNA demethylation). In addition, Vc can indirectly enhance the anti-tumor immune response by promoting ferroptosis and other pathways.

[0004] However, the clinical application of vitamin C faces many challenges: First, vitamin C is extremely unstable in vivo and is easily oxidized and degraded, resulting in low bioavailability; second, vitamin C lacks tumor targeting, is widely distributed in normal tissues, has a low therapeutic index, and high-dose systemic administration may cause adverse reactions such as kidney stones. Therefore, developing intelligent nanosystems that can efficiently deliver and target the release of vitamin C is key to overcoming its clinical application bottlenecks.

[0005] Molecularly imprinted polymers (MIPs), as polymeric materials with predefined recognition sites, possess advantages such as high specificity, high stability, and ease of preparation, making them ideal targeted delivery platforms. Furthermore, the "self-delivery" strategy utilizes the drug itself or a simply modified molecule to directly achieve delivery, avoiding the toxicity and biocompatibility issues of traditional carrier materials. However, no research has yet combined molecular imprinting technology with endogenous substances like vitamin C to construct intelligent self-delivery systems for anti-tumor therapy.

[0006] However, vitamin C is chemically reactive and unstable, easily affected by changes in heat, light, humidity, oxygen, and pH. Existing molecularly imprinted polymers that directly use vitamin C as a template are prone to template destruction during preparation, leading to a decrease in the accuracy of subsequent identification of imprinted molecules.

[0007] Therefore, developing intelligent delivery nanocomponents that can overcome the instability and non-targeting defects of vitamin C and reverse immunosuppression through precise regulation of the TME has significant scientific and clinical translational value. Summary of the Invention

[0008] To address the issue that existing molecularly imprinted polymers directly using Vitamin C as a template are prone to template destruction during preparation, leading to decreased accuracy in subsequent recognition of imprinted molecules, and the lack of research on intelligent Vitamin C delivery nanocomponents based on biomimetic imprinting technology for anti-tumor applications, this study selected ascorbate glucoside (AA2G), a stable derivative of Vitamin C, as a molecularly imprinted template. The steps included: synthesis of SiO2NPs-CC, amino functionalization, boric acid functionalization, template immobilization, directional imprinting, secondary amino functionalization, DMMA modification, and template removal. The constructed nanocomponent vMIPDcc contained a complete Vitamin C cavity, exhibiting high selectivity (IF=8.4) and reliable recognition ability for Vitamin C. Its core contained cytochrome C, achieving an in vivo inhibition rate of 60.2% against colon cancer. The specific technical solution is as follows: First aspect: Preparation of vMIPDcc

[0009] (I) Synthesis of SiO2NPs-CC

[0010] SiO2NPs-CC with a particle size of 40 nm was synthesized using the reverse microemulsion method, which is the core of vMIPDcc. (ii) Amin functionalization

[0011] APTES was added to a 3 mg / mL SiO2NPs-CC ethanol solution, shaken at room temperature, centrifuged, and the precipitate was washed once by ethanol centrifugation to obtain amino-functionalized SiO2NPs-CC (NH2-SiO2NPs-CC). (III) Boric acid functionalization

[0012] NH2-SiO2NPs-CC was added to a methanol solution containing 5 mg / mL FPBA and 1 mg / mL NaBH3CN. The mixture was shaken at room temperature for 24 h, centrifuged at 8000 rpm for 10 min, washed with ethanol and centrifuged three times. The precipitate was then placed in a vacuum desiccator at 45 °C overnight to obtain FPBA@SiO2NPs-CC. (iv) Template fixing

[0013] Mix 0.5 mol / mL AA2G in PBS with FPBA@SiO2NPs-CC, sonicate to disperse, shake at room temperature for 30 minutes, collect the precipitate, wash the precipitate 3 times with PBS, and collect the precipitate as template-anchored FPBA@SiO2NPs-CC (AA2G-FPBA@SiO2NPs-CC). (v) Oriented Imprint

[0014] AA2G-FPBA@SiO2NPs-CC was dispersed in 30 mL of anhydrous ethanol-ammonia-water solution, and 8 mL of ethanol solution containing functional monomers was added to react. After centrifugation, the precipitate was washed three times with ethanol and dried under vacuum at 50 °C overnight to obtain the directionally imprinted nanomaterial (AA2G@SiO2NPs-CC-core-shell). (vi) Secondary amino functionalization

[0015] AA2G@SiO2NPs-CC-core-shell was dispersed in 1 mL of ethanol. 20 μL of APTES was added to the ethanol solution of AA2G@SiO2NPs-CC-core-shell. The mixture was shaken at room temperature, centrifuged, and washed with ethanol before centrifugation twice to obtain the secondary amino-functionalized nanomaterial NH2@AA2G@SiO2NPs-CC-core-shell. (vii) Template Removal

[0016] NH2@AA2G@SiO2NPs-CC-core-shell was dispersed in 2 mL of eluent and shaken at room temperature for 1 hour to remove the template, yielding a nano-assembly with Vc-specific imprinted cavities on its surface. The nano-assembly was washed three times with ethanol and dried overnight under vacuum at 40°C to obtain the template-removed vMIP. CC . (viii) DMMA modification

[0017] Dissolve DMMA in double-distilled water and adjust the pH to 8 with 0.01M NaOH, then remove the template from the vMIP. CC Mix DMMA at a mass ratio of 1:2, place on a shaker and react at room temperature for 24 hours, centrifuge, and collect the centrifuged precipitate, which is vMIPDcc; (ix) Comparison materials

[0018] Control materials include: NIPD, NIPD CC vMIP, MIP CC vMIP CC vMIP+NIPD CC ; Preparation of NIPD: Cytochrome C was not added during the synthesis of the core, and template AA2G was not added during the directed blotting step. The remaining operation steps were exactly the same as those of vMIPDcc.

[0019] NIPD CC Preparation: In the directional imprinting step, template AA2G is not added, and the remaining operation steps are exactly the same as those of vMIPDcc.

[0020] Preparation of vMIP: Cytochrome C was not added during the synthesis of the core, and DMMA modification was not performed at the end. The remaining steps were the same as those for vMIPDcc.

[0021] MIP CC Preparation: No template AA2G was added in the directional imprinting step, and the remaining operation steps were the same as those for vMIPDcc.

[0022] vMIP CC Preparation: No DMMA modification is performed in the end, and the remaining operation steps are exactly the same as those for vMIPDcc.

[0023] Preparation of vMIP+NIPDCC: Equal masses of vMIP and NIPDCC are physically mixed to obtain the material. The second aspect is the characterization of vMIPDcc.

[0024] Transmission electron microscopy revealed that vMIPDcc consists of regular spherical nanoparticles with a particle size of approximately 50 nm; vMIPDcc exhibits GSH concentration-dependent degradation characteristics; and the theoretical template length of vMIPDcc is 0.91 nm. Third aspect: Optimization of imprint conditions

[0025] Based on the time dependence of imprint layer thickness growth and the theoretical length of epitope peptides, imprinting time and the ratio of functional monomers were systematically optimized using imprinting factor (IF) as the key evaluation index.

[0026] The optimal preparation conditions were determined by comparing experimental results: imprinting time: 10 minutes, and volume ratio of functional monomers: UPTES:BnTES:TEOS:BTEPDS: 5:15:40:40. This imprinting condition was used to prepare vMIPDcc. Fourth aspect: vMIPDcc performance evaluation (I) Performance Characterization of vMIPDcc

[0027] The vMIPDcc synthesized with the optimal monomer ratio of 5:15:40:40 and an blotting time of 10 minutes was characterized by TEM to investigate the particle size of vMIPDcc dynamic light scattering (DLS), the zeta potential of vMIPDcc, the functional group modification verification by Fourier transform infrared spectroscopy (FT-IR), and the encapsulation efficiency and encapsulation amount of cytochrome C protein by vMIPDcc. (II) Vc capture and verification

[0028] vMIPD prepared under optimal blotting conditions can specifically recognize Vc molecules, and it also recognizes structurally similar small molecules (Vc, V...). B1 V B6 The cross-reactivity of DA, 4met-Glu, and 4met-Man was <18.5%, indicating that the imprinted layer was successfully formed on the SiO2NPs-CC surface. (III) Free radical scavenging test

[0029] ABTS+• cationic free radicals can be reduced to ABTS under the action of reducing agent Vc. (iv) Based on ABTS + • Scavenging method for evaluating the antioxidant activity of nanomaterials in serum

[0030] Compared with the control group (NIPD), only the vMIPD treatment group caused a significant color change from blue-green to colorless in the ABTS+• solution. (v) In vitro cell targeting

[0031] Compared with the control group (NIPD), the vMIPcc treatment group significantly reduced the characteristic absorption peak of ABTS+• at 734 nm, indicating that vMIPcc has excellent ability to capture Vc and use it for ROS scavenging. (vi) Cellular uptake and intracellular localization of nanomaterials

[0032] vMIPcc has a large capture capacity for Vc, with a maximum capture capacity of 0.69 μmol / g. Fifth aspect: Evaluation of the in vitro antitumor effect of vMIPDcc

[0033] Cell viability is assessed using a live / dead cell staining assay.

[0034] Compared with other treatment groups, CT26 cells in the vMIPDcc treatment group showed significantly enhanced red fluorescence, indicating that the targeted delivery system can effectively promote tumor cell apoptosis. Conversely, the free Vc and CC treatment groups showed only weak fluorescence signals, indicating that the free drugs had almost no cell-killing effect due to their difficulty in penetrating the cell membrane. Meanwhile, no significant fluorescence signals were observed in the NIPD group, vMIP group, MIPcc group, and vMIPcc group.

[0035] It is noteworthy that although vMIPD alone did not show a direct tumor-suppressing effect in delivering vitamin C, it significantly enhanced the anti-tumor effect of cytochrome c, suggesting that vitamin C plays a key synergistic role in this delivery system. Sixth aspect: Evaluation of the in vivo antitumor effect of vMIPDcc

[0036] Mice carrying CT26 tumors were randomly assigned to seven experimental groups and intravenously injected with PBS, NIPD, and NIPD, respectively. CC vMIP, vMIP CC vMIP+NIPD CC 、vMIPDcc.

[0037] Compared with other groups, the vMIPDcc group showed a significant tumor growth inhibition effect within 15 days, with a final tumor inhibition rate of 60.2%, which was significantly better than the other groups.

[0038] Compared with the prior art, the beneficial effects of this application are as follows: 1. Highly efficient biomimetic capture By utilizing molecular imprinting technology, the system can not only deliver drugs, but also actively capture and enrich vitamin C in vivo, realizing a new drug utilization model that combines "open source" and "cost reduction".

[0039] 2. High security The component (Vc) used is an endogenous substance in the human body, with good biocompatibility, avoiding the potential toxicity and side effects of exogenous drug carriers. Attached Figure Description

[0040] Figure 1 Diagram illustrating the in vivo antitumor mechanism of vMIPDcc nanomodules; Figure 2 A schematic diagram of the synthesis process of vMIPDcc nanomodules; Figure 3 Time-dependent degradation of vMIPDcc nanomodules; Figure 4 AA2G's 3D structure; Figure 5 The imprinting time optimization of epitope imprints vMIPDcc under different monomer ratios, among which, 'a' represents UPTES:BnTES:TEOS:BTEPDS = 5:15:40:40. b represents UPTES:BnTES:TEOS:BTEPDS = 10:10:40:40. c is UPTES:BnTES:TEOS:BTEPDS=5:5:45:45; Figure 6 Performance characterization of vMIPDcc nanomodules, among which, vMIPDcc synthesized with the optimal monomer ratio of 5:15:40:40 and an imprinting time of 10 minutes was characterized by TEM. b is the particle size distribution of dynamic light scattering (DLS). c represents the Zeta potential plot of vMIPDcc. d is the Fourier transform infrared (FT-IR) verification diagram for functional group modification. e represents the encapsulation efficiency and encapsulation amount of the CC protein in the vMIPDcc pair; Figure 7 ,VC capture and verification, among which, vMIPDcc, prepared with the optimal ratio of functional monomers, recognizes Vc and V. B1 V B6 Comparison of DA, 4met-Glu, and 4met-Man b represents the reaction equation where the ABTS+• cationic free radical is reduced to ABTS under the action of reducing agent Vc. c represents the vMIPDcc treatment group, which caused a color change in the ABTS+• solution. d represents the effect of the vMIPDcc treatment group on the characteristic absorption peak of ABTS+• at 734 nm. e represents the adsorption isotherm of Vc by the Vc-imprinted vMIPDcc and the non-imprinted NIPD. f represents the amount of Vc captured by vMIPDcc; Figure 8 vMIPDcc in vitro antitumor activity; Figure 9 The in vivo anti-tumor effects of vMIPDcc, among which, a represents the weight changes in the seven groups of mice. b represents the changes in tumor volume in the seven groups of mice. c is a statistical graph showing the changes in tumor volume in seven groups of mice.

[0041] in Figures 1-9 middle, *, **, ***, and **** represent P<0.05, P<0.01, P<0.001, and P<0.0001, respectively, indicating that the difference between the two groups is statistically significant. Detailed Implementation

[0042] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present application in any way.

[0043] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0044] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0045] Main reagents and materials

[0046]

[0047] instrument Example 1: Fabrication of vMIPDcc nanomodules

[0048] 1. Preparation of SiO2NPs-CC

[0049] SiO2NPs-CC was synthesized using a reverse microemulsion method.

[0050] 1.77 g Triton X-100, 1.6 mL n-hexanol, and 6.5 mL cyclohexane were added sequentially to a 25 mL pear-shaped flask and mixed thoroughly. Then, 480 μL of 2 mg / mL cytochrome C aqueous solution was slowly added dropwise. After the system became clear, 50 μL of 28% ammonia solution was added. After magnetic stirring for 10 minutes, 1 mL of mixed precursor solution was added to the system. The reaction mixture was stirred at 25 °C and 700 rpm for 24 h. After the reaction was completed, 10 mL of acetone was added to the system to break the emulsion. The mixture was centrifuged at 10,000 rpm for 15 minutes, and the precipitate was collected. The precipitate was washed three times with ethanol. The precipitate was SiO2NPs-CC with a particle size of about 40 nm. It was stored at 4 °C for later use. SiO2NPs-CC is cytochrome C encapsulated by silica nanoparticles, i.e., the core of vMIPDcc.

[0051] 1 mL Mixed precursor solution: Take 30 μL of TEOS and 70 μL of BTEPDS, add them to 1 mL of anhydrous ethanol to prepare a mixed precursor solution (total volume approximately 1.1 mL). Accurately measure 1 mL from this mixture for later use; TEOS: Tetraethyl orthosilicate; BTEPDS: Bis-[3-(triethoxysilyl)propyl]-disulfide. 2. Amine functionalization

[0052] Add 200 mL of APTES to 20 mL of 3 mg / mL SiO2NPs-CC ethanol solution, shake at room temperature for 2 h, centrifuge at 8000 rpm for 10 min, wash with ethanol and centrifuge once to obtain the precipitate. The precipitate is NH2-SiO2NPs-CC, which is amino-functionalized SiO2NPs-CC.

[0053] APTES: 3-Aminopropyltriethoxysilane. 3. Boric acid functionalization

[0054] 60 mg of NH2-SiO2NPs-CC was added to 20 mL of methanol solution containing 5 mg / mL of FPBA and 1 mg / mL of NaBH3CN. The mixture was shaken at room temperature for 24 h, centrifuged at 8000 rpm for 10 min, washed with ethanol and centrifuged three times. The precipitate was then placed in a vacuum desiccator at 45 °C overnight to obtain FPBA@SiO2NPs-CC.

[0055] FPBA stands for 4-formylphenylboronic acid. 4. Template fixing

[0056] Mix 2 mL of 0.5 mol / mL AA2G in PBS solution with 8 mg of FPBA@SiO2NPs-CC, sonicate to disperse, shake at room temperature for 30 minutes, collect the precipitate, wash the precipitate 3 times with PBS solution, and collect the precipitate as AA2G-FPBA@SiO2NPs-CC. AA2G-FPBA@SiO2NPs-CC is template-anchored FPBA@SiO2NPs-CC.

[0057] AA2G is ascorbate glucoside, PBS solution: concentration 0.01M, pH 7.4. 5. Oriented Imprint

[0058] The AA2G-FPBA@SiO2NPs-CC obtained in step 4 was dispersed in 30 mL of anhydrous ethanol-ammonia-water solution [0.9 mL ammonia (approximately 25%), 2 mL double-distilled water (ddH2O), and anhydrous ethanol added to 30 mL]. 8 mL of ethanol solution containing the functional monomer was added, and the mixture was shaken at room temperature for 10 min. The mixture was then centrifuged (10,000 rpm, 10 min). The precipitate was washed three times with ethanol and then vacuum-dried overnight at 50 °C to obtain the directionally imprinted nanomaterial (AA2G@SiO2NPs-CC-core-shell).

[0059] The volume ratio of functional monomers is 5:15:40:40:UPTES:BnTES:TEOS:BTEPDS.

[0060] UPTES: 3-Urea-propyltriethoxysilane BnTES: Benzyltriethoxysilane TEOS: Tetraethyl orthosilicate BTEPDS: bis-[3-(triethoxysilyl)propyl]-disulfide. 6. Secondary amino functionalization

[0061] The AA2G@SiO2NPs-CC-core-shell obtained in step 5 was dispersed in 1 mL of ethanol. 20 μL of APTES was added to the ethanol solution of AA2G@SiO2NPs-CC-core-shell, and the mixture was shaken at room temperature for 1 h. The precipitate was centrifuged twice, followed by washing with ethanol, to obtain the secondary amino-functionalized nanomaterial (NH2@AA2G@SiO2NPs-CC-core-shell). 7. Template Removal

[0062] The NH2@AA2G@SiO2NPs-CC-core-shell obtained in step 6 was dispersed in 2 mL of elution buffer (acetonitrile:water:acetic acid = 50:49:1), and shaken at room temperature for 1 hour to remove the template, finally obtaining vMIPD nanodevices with Vc-specific imprinted cavities on the surface. After washing with ethanol 3 times, i.e., the vMIPcc with template removed, it was dried overnight under vacuum at 40°C for later use. 8. Modification with dimethylmaleic anhydride (DMMA)

[0063] 8 mg of DMMA was dissolved in 1 mL of double-distilled water (ddH2O), and the pH was adjusted to 8.0 with 0.01 M NaOH. This solution was then mixed thoroughly with 4 mg of dried vMIPcc nanomaterials at a mass ratio of 1:2 (vMIPcc:DMMA). The mixture was placed in a shaker and reacted at room temperature for 24 h. Finally, the mixture was centrifuged at 8000 rpm for 10 min, and the precipitate was collected. This precipitate is vMIPDcc.

[0064] The mechanism of action of vMIPDcc nanomodules against tumors in vivo is as follows: Figure 1 As shown; The Vc surface molecular imprinted layer was constructed using a borate affinity-controlled directional surface imprinting method, as follows: Figure 2 As shown, the process includes: core material synthesis → amino functionalization of material surface → boric acid functionalization of material surface → template fixation and directional imprinting → secondary amino functionalization → template removal → DMMA modification. Example 2: Preparation of control materials

[0065] Control materials include: NIPD, NIPD CC vMIP, MIPCC vMIP CC vMIP+NIPD CC ; NIPD preparation: No template fixation and orientation imprinting were performed; the remaining steps were the same as in Example 1. NIPD CC Preparation: Except for the absence of AA2G during template fixation, the other steps are the same as in Example 1; vMIP preparation: Except for the absence of cytochrome C and DMMA modification, the synthesis of SiO2NPs was the same as in Example 1. MIP CC Preparation: No template AA2G was added in the directional imprinting step, and the remaining steps were the same as in Example 1.

[0066] vMIP CC Preparation: No DMMA modification was performed in the end, and the remaining steps were the same as in Example 1.

[0067] vMIPcc preparation: No DMMA modification was performed, and the remaining steps were the same as in Example 1.

[0068] vMIP+NIPD CC Preparation: vMIP and NIPD CC Mix at a mass ratio of 1:1. Example 3: Characterization of vMIPDcc

[0069] To avoid the influence of DMMA eluted during CH3COOH elution on absorbance values ​​during characterization, the products vMIP and NIP of vMIPDcc and NIPD particles treated with PBS at pH 6.5 were used for subsequent detection of relevant OD values.

[0070] GSH responsiveness: In a buffer solution containing 10 mM GSH (simulating the intracellular environment), transmission electron microscopy showed that vMIPDcc was completely degraded after 48 h of treatment with 10 mM GSH, while its morphology did not change significantly in the absence of GSH, demonstrating its GSH concentration-dependent degradation characteristic. Figure 3 ).

[0071] AA2G's 3D structure, such as Figure 4 As shown, the theoretical template length is 0.91 nm. Example 4: Optimization of Imprint Conditions

[0072] Based on the time dependence of imprint layer thickness growth and the theoretical length of epitope peptides, imprinting time and the ratio of functional monomers were systematically optimized using imprinting factor (IF) as the key evaluation index.

[0073] We selected the following three functional monomer ratios to prepare vMIPDcc. The functional unit ratio of UPTES:BnTES:TEOS:BTEPDS is 5:15:40:40. (Volume ratio) The functional monomer ratio of UPTES:BnTES:TEOS:BTEPDS is 10:10:40:40 (volume ratio). The functional monomer ratio of UPTES:BnTES:TEOS:BTEPDS is 5:5:45:45 (volume ratio). vMIPD was examined at 5, 10, 15, 20, and 25 minutes respectively. CC NIPD for V C The adsorption amount and IF value are determined as follows: vMIPcc and NIPD particles were treated with PBS at pH 6.5. Then, 1 mL of Vitamin C PBS solution (0.5 μmol / mL) was added to vMIPcc and NIPD (3 mg) prepared at different blotting ratios and times. The mixtures were ultrasonically dispersed and shaken at room temperature for 30 min, followed by centrifugation (4000 rpm, 30 min). The vMIPcc and NIPD particles were collected separately. The materials were eluted with 30 μL of acetonitrile:water:acetic acid = 50:49:1 for 1 hour. The eluent was collected, and its OD value at 260 nm was measured.

[0074] IF = Absorbance (vMIPDcc) / Absorbance (NIPD) The results are as follows Figure 5 As shown, the blotting time was 10 minutes, the volume ratio of functional monomers was UPTES:BnTES:TEOS:BTEPDS of 5:15:40:40, and the highest IF value of vMIPDcc for Vc was 8.4. Subsequent experiments used this optimal functional monomer ratio to prepare vMIPDcc. Example 5: vMIPDcc Performance Evaluation 1. vMIPDcc performance characterization

[0075] vMIPDcc synthesized with the optimal monomer ratio of 5:15:40:40 and an imprinting time of 10 minutes was characterized by TEM, and the results are as follows: Figure 6 As shown in a; The particle size of dynamic light scattering (DLS) is as follows Figure 6 As shown in b; The zeta potential of vMIPDcc is as follows Figure 6 As shown in c; Fourier transform infrared spectroscopy (FT-IR) verification of functional group modification verification, such as Figure 6 As shown in d; vMIPDcc's encapsulation efficiency and encapsulation amount of cytochrome C protein are as follows: Figure 6 As shown in e. 2. Vc capture and verification

[0076] First, vMIPDcc and NIPD particles were treated with PBS at pH 6.5. Then, a solution of vitamin C and structurally similar interfering agents (0.5 μmol / mL, interfering agents being VB1, VB6, DA, 4-met-Glu, and 4-met-Man) was prepared using PBS. Next, vMIPDcc and NIPD (3 mg) were added to the above peptide solution (200 μL), sonicated, and shaken on a shaker for 30 min (at room temperature). Subsequently, the particles were washed three times with 0.01 M PBS solution at pH 7.4, and collected by centrifugation at 4000 rpm for 30 min. Then, 30 μL of eluent (acetonitrile:water:acetic acid, v / v / v = 50:49:1) was added to the vMIPDcc or NIPD and reacted for 1 h. Finally, the mixture was centrifuged at 4000 rpm (30 min), the supernatant was collected, and its OD260 nm was measured (average of three measurements).

[0077] like Figure 7 As shown in Figure a, the vMIPDcc prepared with the optimal ratio of functional monomers can specifically recognize Vc molecules, and it also recognizes structurally similar small molecules (Vc, V...). B1 V B6 The cross-reactivity of DA, 4met-Glu, and 4met-Man was <18.5%, indicating that the imprinted layer was successfully formed on the SiO2NPs surface. 3. Free radical scavenging test

[0078] When evaluating the radical scavenging ability of vMINPD using the ABTS method, ABTS is first prepared. ＋ Free radical solution: Dissolve 3.0 mg ABTS in 1 mL of 0.5 mM H2O2, add HRP (final concentration 0.1 μg / mL), and incubate in the dark for 6 hours to generate ABTS. ＋ • During the test, 0.9 mL of the nanomaterial solution to be tested (containing 5 mg NPs, incubated with Vc) was mixed with 100 μL of fresh ABTS. ＋ • Mix the working solution, sonicate for 5 minutes, centrifuge at 10,000 rpm for 5 minutes, and immediately measure the absorbance of the supernatant at 734 nm.

[0079] like Figure 7 As shown in b, the blue-green ABTS+• cationic free radical can be reduced to colorless ABTS under the action of reducing agent Vc.

[0080] 4. Based on ABTS ＋• Scavenging method for evaluating the antioxidant activity of nanomaterials in serum

[0081] Whole blood was collected from Balb / c mice (CT26 cell xenograft model) and placed in a coagulation-promoting tube. After centrifugation at 3000 rpm for 5 min, serum was separated and set aside. 5 mg of NPs was mixed with 1 mL of fresh serum by sonication and incubated for 30 min, after which the serum was discarded. 0.9 mL of the above-treated nanomaterial solution was then added to 100 μL of fresh ABTS. ＋ • Disperse the working solution by sonication for 5 min, then centrifuge at 10000 rpm for 5 min. Take the supernatant and immediately measure the absorbance at 734 nm.

[0082] like Figure 7 As shown in c, compared with the control group, both the vMIPDcc and vMIPD treatment groups could cause a significant color change from blue-green to colorless in the ABTS+• solution. 5. In vitro cell targeting

[0083] CT26 cells were cultured in RPMI 1640 medium containing 10% FBS. When using sterile materials, serum-free medium was prepared according to the required concentration.

[0084] CT26 cells were seeded in 35 mm culture dishes and cultured for 24 h. Afterward, the cells were incubated with the nanomaterials for a period of time. The cells were then washed three times with PBS, and 100 μL of DAPI staining solution was added to each culture dish. After incubation for 10 min, the cells were washed three times with PBS buffer. Finally, the cells were observed using a confocal laser scanning microscope (CLSM). For flow cytometry analysis, the cell processing method was similar to the above steps (DAPI staining step omitted), followed by trypsin digestion, washing twice with PBS, and redispersing in PBS solution for flow cytometry analysis.

[0085] The results are as follows Figure 7 As shown in d, UV spectroscopy further confirmed that, compared with the control group, the vMIPDcc treatment group significantly reduced the characteristic absorption peak of ABTS+• at 734 nm, indicating that vMIPDcc has excellent ability to capture Vc and be used for ROS scavenging. 6. Cellular uptake and intracellular localization of nanomaterials

[0086] To investigate the cellular uptake of nanoparticles, CT26 cells were seeded in culture dishes (confocal microscopy) and cultured for 24 hours. Subsequently, 200 μL of nanoparticles or an equivalent concentration of controls (200 μg / mL) were co-incubated with CT26 cells for different durations (4 / 8 h). After incubation, the cells were washed three times with PBS and then fixed with 4% paraformaldehyde for 10 minutes. Afterward, the cells were washed twice with PBS and stained with DAPI (100 μL, 10 min) for confocal microscopy detection.

[0087] The results are as follows Figure 7 As shown in Figure e, the adsorption isotherms of Vc by the Vc-imprinted vMIPDcc and the non-imprinted NIPD show that the Kd value of Vc by vMIPDcc is (1.61±0.12)×10⁻⁶. -4 M.

[0088] Furthermore, we examined the amount of Vc captured by vMIPDcc, such as Figure 7 As shown in f, the maximum capture capacity of vMIPDcc for Vc is 0.69 μmol / g. Example 6: Evaluation of the in vitro antitumor effect of vMIPDcc

[0089] Cell viability is assessed using a live / dead cell staining assay.

[0090] Construct 3D tumor spheroids using CT26 cells. Place CT26 cells (5 × 10⁶ cells per well) into wells. 3 (Numbers) were seeded onto Absin Bioscience 96-well plates. After 3 days, individual tumor spheroids were treated with NPs and incubated for half an hour.

[0091] CT26 cells were stained with a Calcein / PI mixture for 30 minutes before CLSM imaging was performed. Green fluorescence represents live cells, and red fluorescence represents apoptotic cells.

[0092] The results are as follows Figure 8 As shown, compared with other treatment groups, CT26 cells in the vMIPDcc treatment group exhibited significantly enhanced red fluorescence signals, indicating that this targeted delivery system can effectively promote tumor cell apoptosis. Conversely, the free Vc and CC treatment groups showed only weak fluorescence signals, indicating that the free drugs had almost no cell-killing effect due to their difficulty in penetrating the cell membrane. Meanwhile, no significant fluorescence signals were observed in the NIPD group, vMIP group, MIPcc group, and vMIPcc group.

[0093] Notably, although vMIPD alone did not exhibit a direct tumor-suppressing effect on vitamin C, it significantly enhanced the antitumor activity of cytochrome c, suggesting that vitamin C plays a crucial synergistic role in this delivery system. These results demonstrate that we have successfully constructed a vitamin C-based vMIPDcc delivery system that not only efficiently captures and delivers vitamin C but also synergistically enhances the antitumor activity of cytochrome c, thereby significantly improving the killing effect on tumor cells. Example 7: Evaluation of the in vivo antitumor effect of vMIPDcc

[0094] To evaluate the synergistic antitumor effects of different nanoparticles in vivo, mice carrying CT26 tumors were randomly assigned to seven experimental groups, with six mice in each group. Each group received a tail vein injection of PBS, NIPD, or NIPD every three days. CC vMIP, vMIP CC vMIP+NIPD CC The mice were injected with vMIPDcc (10 mg NPs / kg, in 0.01 M PBS solution at pH 7.4) five times. Mouse weight and tumor volume were recorded every two days.

[0095] The results of weight changes over time are as follows Figure 9 As shown in figure a, the body weight of mice in each group did not change significantly, indicating that the treatment regimen has good biosafety.

[0096] Compared with other groups, the vMIPDcc group showed a significant tumor growth inhibition effect within 15 days. Figure 9 (b and 9c) achieved a final tumor inhibition rate of 60.2%, significantly superior to the other groups. This result is consistent with the conclusions of the in vitro tumor inhibition experiment, further confirming that the vitamin C smart delivery nanocomponent based on biomimetic imprinting technology has a significant in vivo anti-tumor effect.

[0097] The present invention has been described in detail above with general descriptions and specific embodiments. However, modifications or improvements can be made to the present invention, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of vMIPDcc in the preparation of anti-colon cancer agents, characterized in that, The vMIPDcc core is a SiO2 NPs-CC containing cytochrome C, and the surface has a Vc-specific imprinted cavity as a nano-component. The preparation steps of the vMIPDcc are as follows: (1) Preparation of SiO2 NPs-CC SiO2 NPs-CC was synthesized using a reverse microemulsion method, as follows: Triton X-100, n-hexanol, and cyclohexane were added sequentially to a pear-shaped flask and mixed thoroughly. A 2 mg / mL aqueous solution of cytochrome C was slowly added dropwise, followed by 28% ammonia. After magnetic stirring for 10 minutes, a mixed precursor solution was added to the system. The reaction mixture was stirred at 25°C and 700 rpm for 24 hours. After the reaction was complete, acetone was added to the system to break the emulsion. The mixture was centrifuged at 10,000 rpm for 15 minutes, and the precipitate was collected. The precipitate was washed three times with ethanol. The precipitate was SiO2 NPs-CC with a particle size of approximately 40 nm and stored at 4°C for later use. The ratio of Triton X-100, n-hexanol, cyclohexane, 2 mg / mL cytochrome C aqueous solution, 28% ammonia solution, mixed precursor solution, and acetone was 1.77 g: 1.6 mL: 6.5 mL: 480 μL: 50 μL: 1 mL: 10 mL. Mixed precursor solution: Take TEOS and BTEPDS, add them to anhydrous ethanol, and mix well to obtain the solution. The volume ratio of TEOS, BTEPDS and anhydrous ethanol is 3:7:

100. The TEOS is tetraethyl orthosilicate, the BTEPDS is bis-[3-(triethoxysilyl)propyl]-disulfide, and the SiO2 NPs-CC is cytochrome C encapsulated by silica nanoparticles, i.e., the core of vMIPDcc. (2) Amin functionalization APTES was added to a 3 mg / mL SiO2 NPs-CC ethanol solution, shaken at room temperature for 2 h, centrifuged at 8000 rpm for 10 min, and the precipitate was washed with ethanol and centrifuged once. The precipitate was NH2-SiO2 NPs-CC. The volume ratio of APTES to the 3 mg / mL SiO2 NPs-CC ethanol solution is 1:

100. The APTES is 3-aminopropyltriethoxysilane, and the NH2-SiO2 NPs-CC is amino-functionalized SiO2 NPs-CC. (3) Boric acid functionalization The NH2-SiO2 NPs-CC was added to a methanol solution containing 5 mg / mL FPBA and 1 mg / mL NaBH3CN, shaken at room temperature for 24 h, centrifuged at 8000 rpm for 10 min, washed with ethanol and centrifuged three times, and then the centrifuged precipitate was placed in a vacuum desiccator at 45 °C overnight to obtain FPBA@SiO2 NPs-CC. The ratio of the NH2-SiO2 NPs-CC to the methanol solution containing 5 mg / mL FPBA and 1 mg / mL NaBH3CN is 3 mg: 1 mL; The FPBA mentioned is 4-formylphenylboronic acid; (4) Template fixing Mix 0.5 μmol / mL AA2G in PBS solution with the FPBA@SiO2 NPs-CC, disperse by ultrasonication, shake at room temperature for 30 minutes, collect the precipitate, wash the precipitate 3 times with PBS solution, and collect the precipitate to obtain AA2G-FPBA@SiO2 NPs-CC; The ratio of the 0.5 μmol / mL AA2G PBS solution to the FPBA@SiO2 NPs-CC is 1 mL: 4 mg; The AA2G is ascorbate glucoside; the PBS solution has a concentration of 0.01M and a pH of 7.

4. The AA2G-FPBA@SiO2 NPs-CC is a template-anchored FPBA@SiO2 NPs-CC; (5) Oriented imprints The AA2G-FPBA@SiO2 NPs-CC was dispersed in 30 mL of anhydrous ethanol-ammonia-water solution, and 8 mL of ethanol solution containing functional monomers was added. The mixture was reacted for 10 min, centrifuged at 10,000 rpm for 10 min, and the precipitate was washed three times with ethanol and dried under vacuum at 50 °C overnight to obtain the oriented imprinted nanomaterial, namely AA2G@SiO2 NPs-CC-core-shell. The 30 mL anhydrous ethanol-ammonia-water solution contains approximately 0.9 mL of ammonia (25%) and 2 mL of double-distilled water. The functional unit is composed of UPTES, BnTES, TEOS, and BTEPDS, with a volume ratio of UPTES:BnTES:TEOS:BTEPDS of 5:15:40:

40. The UPTES is 3-ureidopropyltriethoxysilane, and the BnTES is benzyltriethoxysilane; (6) Secondary amino functionalization The AA2G@SiO2 NPs-CC-core-shell was dispersed in 1 mL of ethanol, and 20 μL of APTES was added to the ethanol solution of the AA2G@SiO2 NPs-CC-core-shell. The mixture was shaken at room temperature for 1 h, and the precipitate was centrifuged and washed with ethanol twice to obtain the secondary amino-functionalized nanomaterial NH2@AA2G@SiO2 NPs-CC-core-shell. (7) Template Removal The NH2@AA2G@SiO2 NPs-CC-core-shell was dispersed in 2 mL of elution buffer and shaken at room temperature for 1 hour to remove the template, resulting in a nano-assembly with a Vc-specific imprinted cavity on the surface. It was washed three times with ethanol and dried overnight under vacuum at 40°C to obtain the template-removed vMIPcc. The eluent is composed of acetonitrile, water, and acetic acid, with a volume ratio of acetonitrile:water:acetic acid of 50:49:

1. (8) DMMA modification A DMMA aqueous solution with pH 8.0 and 8 mg / mL was mixed with dry vMIPcc. The mass ratio of vMIPcc to DMMA was 1:

2. The mixture was placed in a shaker and reacted at room temperature for 24 h. The mixture was then centrifuged at 8000 rpm for 10 min. The centrifuged precipitate was collected and was vMIPDcc. The DMMA is dimethylmaleic anhydride.

2. The application as described in claim 1, characterized in that, In step (1) the preparation of SiO2 NPs-CC, the Triton X-100 is 1.77 g, the n-hexanol is 1.6 mL, the cyclohexane is 6.5 mL, the 2 mg / mL cytochrome C aqueous solution is 480 μL, the 28% ammonia solution is 50 μL, the mixed precursor solution is 1 mL, and the acetone is 10 mL.

3. The application as described in claim 1, characterized in that, In step (2) amino functionalization, the APTES is 200 μL; the 3 mg / mL SiO2NPs-CC ethanol solution is 20 mL.

4. The application as described in claim 1, characterized in that, In step (3) boric acid functionalization, the NH2-SiO2 NPs-CC is 60 mg, and the methanol solution containing 5 mg / mL FPBA and 1 mg / mL NaBH3CN is 20 mL.

5. The application as described in claim 1, characterized in that, In step (4) template fixation, the PBS solution of 0.5 μmol / mL AA2G is 2 mL, and the FPBA@SiO2 NPs-CC is 8 mg.

6. The application as described in claim 1, characterized in that, In step (8) DMMA modification, the 8 mg / mL DMMA aqueous solution at pH 8.0 is 1 mL; the dried vMIPcc is 4 mg.

7. The application as described in any one of claims 1-6, characterized in that, The vMIPDcc showed an inhibition rate of 60.2% against colon cancer.

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