Glutathione-responsive virus-like mesoporous silica drug delivery system and construction and application thereof

Abstract

The application discloses a construction method of a glutathione-responsive virus-like mesoporous silica drug delivery system, and comprises the following steps: preparing a virus-like mesoporous silica VSCA-HA with hyaluronic acid modified disulfide bond grafted dipeptide; taking an anticancer drug DOX as a model drug, taking the VSCA-HA as a carrier, and loading the DOX by solvent evaporation to obtain a virus-like mesoporous silica drug delivery system VSCA-DOX-HA. The virus-like mesoporous silica nanoparticle is successfully prepared. On the basis of the virus-like mesoporous silica, the dipeptide is grafted through the disulfide bond, and the hyaluronic acid is used, so that the virus-like mesoporous silica has the glutathione reduction response, good biocompatibility and targeting property. The constructed drug delivery system has remarkable advantages, and provides a reference for the design of a future drug delivery system.

Description

Technical Field

[0001] This invention relates to the field of drug delivery technology, and more particularly to a glutathione-responsive viral mesoporous silica drug delivery system and its construction and application. Background Technology

[0002] According to the World Health Organization database, cancer is the second leading cause of death worldwide, after cardiovascular disease. Traditional cancer treatments typically include radiation therapy, surgery, and chemotherapy. However, the first two are limited to treating locally located cancers in a specific area of ​​the body. While chemotherapy allows drugs to enter the bloodstream or lymphatic system, most anticancer drugs have poor penetration into tumor tissue, insufficient drug specificity, and can cause side effects on normal cells. Therefore, developing new targeted drug delivery strategies to precisely release chemotherapy drugs at the tumor site, thereby overcoming the shortcomings of traditional therapies, has significant practical implications and research value.

[0003] Doxorubicin (DOX) is an anthracycline antibiotic that interferes with macromolecular biosynthesis and inhibits topoisomerase II by interfering with DNA insertion. It can be used to treat various cancers, including breast cancer, ovarian cancer, lymphoma, bladder cancer, acute lymphoblastic leukemia, Kaposi's sarcoma, gastric cancer, and lung cancer. As a commonly used first-line treatment, although doxorubicin possesses broad-spectrum antitumor activity, its nephrotoxicity, bone marrow suppression, symptomatic effects, and cardiotoxicity limit its routine use.

[0004] Mesoporous silica exhibits a stable structure with adjustable morphology and pore size, resulting in a uniform and ordered structure. Furthermore, its preparation is simple, cost-effective, and suitable for large-scale production. As a drug delivery system, it possesses a large surface area and pore volume, along with abundant silanol groups, making it easy to modify the surface and enhance the functionality of the drug-loaded system. Mesoporous silica nanoparticles also demonstrate good biosafety, are physiologically non-toxic, and biodegradable. Uniformly structured viral mesoporous silica nanoparticles can enter living cells in large quantities, exhibiting significantly higher uptake levels than solid silica nanoparticles and conventional mesoporous silica nanoparticles.

[0005] In summary, constructing a carrier for doxorubicin (DOX) using mesoporous silica as a substrate to further improve the drug's efficiency, intelligence, and targeting is an urgent problem to be solved. Summary of the Invention

[0006] This invention discloses a glutathione-responsive viral mesoporous silica drug delivery system, its construction, and its application.

[0007] This invention provides a method for constructing a glutathione-responsive viral mesoporous silica drug delivery system, comprising:

[0008] Preparation of hyaluronic acid-modified disulfide bond-grafted dipeptide viral mesoporous silica VSCA-HA;

[0009] Using the anticancer drug DOX as a model drug and VSCA-HA as a carrier, the drug was loaded onto DOX by solvent evaporation, resulting in a virus-like mesoporous silica drug delivery system, VSCA-DOX-HA.

[0010] Further, the preparation of disulfide-grafted dipeptide-like viral mesoporous silica (VSCA) in step 1 includes:

[0011] 1) Disperse CTAB in deionized water, then add TEA; after stirring in a water bath, add a mixed solution of cyclohexane and tetraethyl orthosilicate dropwise, continue stirring in a water bath, then stir in an oil bath, centrifuge to collect the precipitate, wash with water and ethanol alternately several times, and dry under vacuum to obtain VSN without template removal.

[0012] 2) Disperse the untemplated VSN in ethanol, add MPTMS dropwise, react under nitrogen protection, collect the product by centrifugation, wash with water and ethanol several times; then vacuum dry, remove the template by acid extraction, the same method as 1), to obtain VSN-SH;

[0013] 3) Disperse VSN-SH in methanol, add 2,2'-dithiopyridine, react at room temperature, centrifuge to collect the precipitate, wash repeatedly with water and alcohol, and dry to obtain VSPY; dissolve cysteine ​​in PBS solution, then add VSPY, stir at room temperature, centrifuge to collect the precipitate, wash repeatedly with water and alcohol, and dry to obtain VSC; disperse arginine in MES buffer in a flask, then add EDCI and SULFO-NHS, and stir at room temperature; disperse VSC in PBS buffer, then add the dispersion to the flask, react at room temperature overnight, centrifuge to collect the precipitate, wash with deionized water, and dry to obtain dipeptide-modified mesoporous silica VSCA;

[0014] 4) Sodium hyaluronate was added to deionized water and reacted overnight. EDCI and SULFO-NHS were added. After the reaction, the pH of the reaction solution was adjusted to 8-9. The dipeptide-modified mesoporous silica VSCA solution was added dropwise. After the reaction continued, the mixture was centrifuged, washed multiple times with deionized water, and freeze-dried to obtain VSCA-HA. The dipeptide-modified mesoporous silica VSCA solution was obtained by dispersing the dipeptide-modified mesoporous silica VSCA in 10 ml of PBS solution with pH=7.4.

[0015] Furthermore, the method of loading DOX with drugs by evaporating solvent includes: preparing a DOX solution using anhydrous ethanol as a solvent; preparing a mixed solution with a carrier in a certain proportion; stirring gently at room temperature in the dark for 24 hours; and then opening the bottle and evaporating the solvent to obtain the drug-containing carrier VSCA-DOX.

[0016] Sodium hyaluronate was added to deionized water and reacted overnight. EDCI and SULFO-NHS were then added. After the reaction, the pH of the reaction solution was adjusted to 8-9. The drug-containing carrier VSCA-DOX solution was added dropwise, and the reaction was continued. After centrifugation, the solution was washed multiple times with deionized water and then freeze-dried to obtain VSCA-DOX-HA. The drug-containing carrier VSCA-DOX solution was obtained by dispersing the drug-containing carrier VSCA-DOX in PBS solution at pH 7.4.

[0017] Secondly, the present invention also provides a glutathione-responsive viral mesoporous silica drug delivery system constructed by the method described above.

[0018] Finally, this invention provides the application of the glutathione-responsive viral mesoporous silica drug delivery system constructed by the method in anti-tumor applications.

[0019] This invention provides a glutathione-responsive viral mesoporous silica drug delivery system, its construction, and its application. First, this invention successfully synthesized viral mesoporous silica grafted with dipeptides via disulfide bonds, and modified it with hyaluronic acid, resulting in uniform particle size, good morphology, and redox responsiveness and targeting properties. Second, using the anticancer drug DOX as a model drug, a targeted intelligent responsive drug delivery system was successfully constructed, with injection as the chosen route of administration. Experimental results show that, compared to traditional active pharmaceutical ingredients, the targeted intelligent responsive drug delivery system exhibits stronger antitumor activity. This provides a reference for future research in the field of tumor treatment.

[0020] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit the disclosure of the present invention. Attached Figure Description

[0021] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1A schematic diagram illustrating the synthesis process of a series of modified mesoporous silica nanoparticles provided in the embodiments of the present invention;

[0024] Figure 2 Morphology characterization (A: TEM; B: SEM) and particle size analysis (C) of a series of mesoporous silica nanoparticles provided in the embodiments of the present invention;

[0025] Figure 3 The series of infrared spectra and thermogravimetric analysis curves of silicon nanoparticles disclosed in the embodiments of the present invention;

[0026] Figure 4 Nitrogen adsorption curves and pore size distribution curves of a series of mesoporous silica nanoparticles provided in the embodiments of the present invention;

[0027] Figure 5 SAXS diagrams of a series of mesoporous silica nanoparticles provided in the embodiments of the present invention;

[0028] Figure 6 Surface characteristic diagrams of a series of mesoporous silica nanoparticles provided in the embodiments of the present invention; A: Initial contact angle; B: Contact angle change trend over time; C: Zeta potential;

[0029] Figure 7 The color reactions of a series of mesoporous silica nanoparticles provided in the embodiments of the present invention are as follows: (a): VSN; (b): VSN-SH; (c): VSPY; (d): VSC; (e): VSCA; (f): VSCA-HA;

[0030] Figure 8 The UV-Vis spectrophotometer scan (range) results and standard curves provided for embodiments of the present invention are shown below (A: anhydrous ethanol; B: pH 7.4; C: pH 5.0; D: standard curve).

[0031] Figure 9 Characterization diagrams of the DOX drug delivery system provided in the embodiments of the present invention (A: BET diagram; B: pore size distribution diagram; C: infrared spectrum; D: XRD; E: SAXD; F: DSC);

[0032] Figure 10 The diagram shows the release of drug-loaded carriers in different media according to embodiments of the present invention (A and B: drug-loaded carrier VSCA-DOX; C: drug-loaded carrier VSCA-DOX-HA);

[0033] Figure 11 Cytotoxicity experiment diagrams of the drug-loaded carriers VSCA-DOX and VSCA-DOX-HA provided in the embodiments of the present invention;

[0034] Figure 12 The distribution of silicon in animals after intravenous injection of the carrier provided in the embodiments of the present invention (A: distribution of silicon in various organs after 24 hours; B: distribution of silicon in various organs after 7 days; C: change of silicon content in blood over time).

[0035] Figure 13 The time-drug concentration curves of DOX and VSCA-DOX-HA provided in the embodiments disclosed in this invention;

[0036] Figure 14 The following figures illustrate the results of co-incubating VSCA-DOX and VSCA-DOX-HA with 4T1 cells for 4 and 24 hours, respectively, according to embodiments of the present invention.

[0037] Figure 15 A diagram showing the apoptosis results of the DOX drug delivery system provided in the embodiments of this invention;

[0038] Figure 16 The fluorescence distribution results of tumor-bearing mice and the fluorescence intensity of different organs provided in the embodiments of the present invention (A: fluorescence distribution map at different times; B: fluorescence intensity map of isolated organs; C: relative MFI values ​​of different organs);

[0039] Figure 17 The pharmacodynamic evaluation indicators after injection administration provided in the embodiments of the present invention;

[0040] Figure 18 HE and TUNEL staining images of tumor tissue provided in the embodiments of the present invention (A: H&E staining image; B: TUNEL staining image);

[0041] Figure 19 This is an immunohistochemical image of Ki67 in tumor tissue provided in an embodiment of the present invention. Detailed Implementation

[0042] The present invention will be further explained below with reference to specific implementation schemes, but this is not intended to limit the scope of protection of the present invention.

[0043] 1. This implementation plan first provides a method for preparing carrier mesoporous silica. Different experimental conditions can be used to prepare mesoporous silica with different morphologies. Non-spherical particles can reduce macrophage phagocytosis and prolong in vivo circulation time, thus improving drug delivery efficiency. It has been confirmed that uniformly structured viral mesoporous silica nanoparticles can enter living cells in large quantities and exhibit significantly higher uptake levels than solid silica nanoparticles and conventional mesoporous silica nanoparticles. This implementation plan uses the surfactant cetyltrimethylammonium bromide (CTAB) as a template to construct virus-like mesoporous silica nanoparticles. The template is removed using calcination and acid-alcohol extraction. Disulfide bonds are used to graft dipeptides to construct a redox-responsive intelligent drug delivery system. Hyaluronic acid is then modified to endow it with tumor microenvironment targeting capabilities. The specific steps include: the synthesis process is as follows... Figure 1 As shown;

[0044] 1) Synthesis of VSN: 1 g CTAB was dispersed in 40 ml of deionized water, and then 0.18 g TEA was added. After stirring in a 60 °C water bath for 2 h, a mixed solution of 20 ml cyclohexane and tetraethyl orthosilicate (TEOS) (16 ml: 4 ml) was added dropwise. The mixture was stirred in a 60 °C water bath for 48 h, and then stirred in a 98 °C oil bath for 24 h. The precipitate was collected by centrifugation, washed repeatedly with water and ethanol alternately, and dried under vacuum at 45 °C for 8 h to obtain template-unremoved VSN. The template-unremoved VSN was dispersed in 200 ml of methanol, and 8 ml of concentrated hydrochloric acid was added. The mixture was refluxed at 70 °C to remove the template, thus obtaining sample VSN.

[0045] Synthesis of VSN-SH: Unremoved VSN was dispersed in 50 ml of ethanol, and 0.7 ml of MPTMS was added dropwise. The reaction was carried out at 70 °C for 12 h under nitrogen protection. The product was collected by centrifugation and washed several times with water and ethanol. Then, it was dried under vacuum at 45 °C for 8 h, and the template was removed by acid extraction as above to obtain the sample VSN-SH.

[0046] Synthesis of VSCA: The VSN-SH obtained in the previous step was dispersed in 200 ml of methanol, and 176 mg of 2,2'-dithiodipyridine was added. After reacting at room temperature for 8 h, the precipitate was collected by centrifugation, washed repeatedly with water and alcohol, and dried to obtain sample VSPY. 48 mg of cysteine ​​was dissolved in 200 ml of PBS solution, and then 200 mg of VSPY was added. After stirring at room temperature for 12 h, the precipitate was collected by centrifugation, washed repeatedly with water and alcohol, and dried to obtain sample VSC. 200 mg of arginine was dispersed in 20 ml of MES buffer in a flask, and then 200 mg of EDCI and 100 mg of SULFO-NHS were added. The mixture was stirred at room temperature for 30 min. The previously prepared VSC was dispersed in 10 ml of PBS buffer, and then the dispersion was added to the flask. The mixture was reacted overnight at room temperature, and the precipitate was collected by centrifugation. After washing three times with deionized water, the precipitate was dried to obtain dipeptide-modified mesoporous silica nanoparticles VSCA.

[0047] Synthesis of VSCA-HA: 200 mg of sodium hyaluronate was added to 100 ml of deionized water and reacted overnight. 200 mg of EDCI and 200 mg of SULFO-NHS were added, and the reaction was carried out for 1.5 h. The pH of the reaction solution was adjusted to 8-9, and the dipeptide-modified mesoporous silica VSCA solution was added dropwise. After the reaction was continued, the mixture was centrifuged, washed multiple times with deionized water, and freeze-dried to obtain VSCA-HA. The dipeptide-modified mesoporous silica VSCA solution was obtained by dispersing the dipeptide-modified mesoporous silica VSCA in 10 ml of PBS solution at pH 7.4.

[0048] 2) The VSN, VSN-SH, VSCA obtained in the above preparation process, and the final VSCA-HATEM, SEM, and particle size distribution are as follows: Figure 2 As shown, VSN exhibits a monodisperse, virus-like morphology with numerous uniformly distributed protruding nanotubes on its surface. Simultaneously, the modified mesoporous silica carriers also retain virus-like morphology characteristics, with uniform nanoparticle size distribution. The particle size distribution of VSN, VSCA, and VSCA-HA is concentrated between 50-60 nm using imaging techniques. However, due to the influence of hydration, the hydrated particle size measured by the Malvern laser particle size analyzer is larger than the true value, ranging from 110-120 nm. The modified mesoporous silica nanoparticles are slightly larger than the previous carrier, but the VSN-SH nanoparticles do not follow this pattern. This may be because the hydrophobic interactions between nanoparticles are enhanced after thiol modification, resulting in a significant increase in hydrated particle size, thus verifying the success of thiol grafting.

[0049] 3) such as Figure 3 As shown, the infrared spectrum was analyzed, and the 477cm² value in the VSN spectrum was... -1 The peak of Si-O-Si bending vibration is shown at 1096 cm⁻¹. -1The presence of Si-O-Si asymmetric stretching vibration peaks at 2935 and 3854 cm⁻¹ confirms the presence of silicon dioxide. The VSN-SH spectrum shows higher peaks compared to the VSN spectrum at 2935 and 3854 cm⁻¹. -1 A new peak appeared at 2935 cm⁻¹, which can be attributed to the stretching vibration of hydrocarbons. -1 The peak position indicates the presence of a methylene group, proving that the thiol group grafted along with the methylene group was successful during the reaction of VSN and MPTMS. (VSCA at 1670 cm⁻¹) -1 The peak at cm⁻¹ represents the stretching vibration of the carbonyl group, confirming the presence of the amide bond. The carbonyl stretching vibration peak of VSCA-HA is lower than that of VSCA, decreasing from 1670 cm⁻¹. -1 Move to 1646cm -1 A red shift occurred, indicating successful hyaluronic acid modification. Observing the thermogravimetric analysis (TGA) plots, VSN, VSN-SH, VSC, VSCA, and VSCA-HA showed weight losses of 0.87%, 9.62%, 12.17%, 19.40%, and 23.86% respectively within the temperature range of 25–700℃. The increasing weight loss of the series of nanoparticles demonstrates the increase in organic groups in the products, indicating successful nanoparticle modification. Compared to the spectrum of VSN, the VSN-SH spectrum showed an enhanced S2p peak at 164 eV and an S2s peak at 230 eV, as well as an enhanced C1 s peak, proving successful grafting of thiol groups.

[0050] 4) The nitrogen adsorption curves and pore size distribution curves of the above series of mesoporous silica nanoparticles are as follows: Figure 4 As shown, by Figure 4 As shown in Figure A, VSN, VSN-SH, VSPY, VSC, VSCA, and VSCA-HA all exhibit distinct type IV adsorption / desorption isotherms, indicating the presence of a uniform mesoporous structure. The pore size distribution diagrams show that they all possess mesoporous channels; specific values ​​are shown in Table 1. The pore sizes are 2.86, 2.10, 2.30, 2.05, 2.05, and 2.20 nm, respectively. (Observation...) Figure 5 The discovery that a series of mesoporous silica nanoparticles have similar peak shapes proves that the mesoscopic structure remains unchanged and ordered after modification.

[0051] Table 1. Specific surface area, pore volume, and pore size of a series of mesoporous silica nanoparticles.

[0052]

[0053] 4) Surface properties of a series of mesoporous silica nanoparticles, such as Figure 6As shown, the initial contact angle of VSN after thiol modification significantly increased, indicating the presence of hydrophobic thiol functional groups during grafting, based on the contact angle trend and surface Zeta potential of the mesoporous silica nanoparticles. Furthermore, the initial contact angles of all carriers were less than 45°, demonstrating hydrophilicity and facilitating the free movement of water molecules. The materials are hydrophilic and possess drug-carrying potential. The Zeta potentials of VSN, VSN-SH, VSC, VSCA, and VSCA-HA were -21.90 (±1.84), -28.05 (±3.75), -29.91 (±2.26), -24.89 (±0.44), and -35.09 (±2.39) mV, respectively. VSN is electronegative, and its electronegativity increased after thiol modification. Hyaluronic acid contains a negative charge, and the electronegativity of VSCA significantly increased after hyaluronic acid modification, indicating successful hyaluronic acid modification.

[0054] 5) such as Figure 7 As shown, thiol-containing compounds can react with DTNB, breaking the disulfide bonds of DTNB to produce 2-nitro-5-thiobenzoic acid (NTB). - If it can be ionized in water under neutral or alkaline pH conditions, it will generate NTB. 2- Divalent anion. This NTB 2- The ions appear yellow. A yellow solution indicates successful grafting of the thiol group. Once the disulfide bond is formed, the thiol group transforms into a disulfide bond, and the color reaction disappears. For example... Figure 7 As shown, the thiol-grafted VSN-SH undergoes a color reaction. Upon reaction with 2,2'-dithiodipyridine, a disulfide bond is formed, and the VSPY solution becomes clear and colorless. This demonstrates the successful grafting of the thiol group and the formation of the disulfide bond.

[0055] In summary, the mesoporous silica drug delivery carrier provided in this embodiment exhibits superior performance compared to other materials: (1) its large specific surface area and suitable pore size enable efficient loading of drug molecules; (2) its uniform morphology can be precisely controlled by changing experimental conditions; (3) its surface is rich in silanol groups, making it easy to modify with different groups; and (4) its stable and rigid framework gives mesoporous silica ideal chemical, thermal, and mechanical stability. Based on these advantages, the construction of a mesoporous silica drug delivery system with intelligent response capabilities holds great promise for research and development in the field of disease diagnosis and treatment.

[0056] 2. This implementation plan investigated the biosafety of the aforementioned mesoporous silica. Biosafety-related experiments were conducted on a series of modified virus-like mesoporous silica nanoparticles. The in vitro toxicity of the carrier was evaluated through in vitro degradation simulation experiments, blood compatibility experiments, and cellular-level cytotoxicity studies. Additionally, the in vivo toxicity of the carrier was systematically evaluated by observing the clinical manifestations, weight changes, complete blood count and blood biochemical indicators, and changes in tissue sections of various organs after administration via tail vein injection to mice.

[0057] Specifically, the series of mesoporous silica nanoparticles exhibited a "fast initially, slow later" degradation trend in four simulated environments (A: simulated body fluid; B: simulated body fluid + GSH; C: simulated body fluid + H2O2; D: pH 5.0 environment). Furthermore, the degradation rates of the modified mesoporous silica nanoparticles were all higher than those of the unmodified carriers, demonstrating good biocompatibility. Among them, the carriers containing disulfide bonds (VSC, VSCA, VSCA-HA) showed higher degradation rates than VSN in the simulated solution containing GSH and H2O2, proving that the modified carriers have a dual redox response, indirectly proving the successful grafting of disulfide bonds. Due to the higher degree of ionization of the silanol groups on the surface of the mesoporous silica nanoparticles under neutral conditions, the degradation rate of the series of carriers was higher at pH 7.4 than at pH 5.0.

[0058] The results of the cytotoxicity assay showed that the VSCA and VSCA-HA vectors had no significant effect on the survival rate of 4T1 cells within the experimental concentration range, which was much higher than the concentration in the clinical trial. The vectors VSCA and VSCA-HA did not produce significant cytotoxicity to 4T1 cells and had good cell safety.

[0059] During the in vivo toxicity experiments, mice exhibited no abnormal clinical behavior and showed no significant weight changes, demonstrating that injection of mesoporous silica nanoparticles had no significant effect on body weight and exhibited good in vivo safety. Blood routine test indicators in blood samples were all within the normal reference range for ICR mice, indicating that the VSCA-HA carrier did not cause abnormalities in blood components within the tested range. The experimental results show that the VSCA-HA carrier has good biosafety and low toxicity, demonstrating safety as a drug carrier both in vivo and in vitro, and possessing the potential to construct drug delivery systems.

[0060] 3. This implementation plan provides the construction of a mesoporous silica drug delivery system; the model drug DOX is loaded into a series of mesoporous silica substrates to construct a highly efficient DOX drug delivery system. A series of characterizations were performed on the drug delivery carrier, measuring its specific surface area, TGA, DSC, infrared spectroscopy, and other physicochemical properties to obtain the drug distribution before and after loading. The in vivo distribution and cytotoxicity of the drug delivery system were investigated, and its pharmacokinetics in vivo were studied, providing a reference for subsequent research on the antitumor activity of intelligent drug delivery systems.

[0061] 1) The detection wavelength, standard curve, and precision of DOX; the UV-Vis spectrophotometer scan (range) results and the standard curve are as follows: Figure 8As shown in the figure, the maximum absorption peaks of DOX in anhydrous ethanol and PBS solutions at pH 7.4 and 5.0 appear at 480 nm and 481 nm. Therefore, 480 nm was chosen as the optimal detection wavelength for DOX in anhydrous ethanol, and 481 nm as the optimal detection wavelength for DOX in PBS solutions at pH 7.4 and 5.0. A maximum absorption peak also appears at 230 nm, but the nearby wavelengths have more interference compared to 480 nm, so this was not chosen as the optimal detection wavelength. As shown in Figure D, the R² values ​​of all three standard curves are above 0.999, indicating that the results are reliable.

[0062] The absorbance of each DOX in low, medium and high concentration solutions in three media was measured at the optimal detection wavelength, and the precision results are shown in Table 2.

[0063] Observe the data in Table 2, and the correlation coefficient (R) of the linear standard curve of DOX in different solutions. 2 The values ​​were all above 0.999, indicating a good linear relationship between DOX concentration and absorbance. In different solutions, the relative standard deviations of DOX intra-day and inter-day precision were all less than 2.5%, demonstrating that the in vitro analytical methodology met the requirements.

[0064] Table 2. Standard Curve Formulas and Precision of DOX in Different Media

[0065]

[0066] 2) Drug loading capacity of DOX drug delivery system: The drug loading capacity and encapsulation efficiency of the drug delivery carrier are shown in Table 3.

[0067] Table 3. Drug loading capacity of different drug delivery carriers

[0068]

[0069] 3) Characterization of the DOX drug delivery system; the characterization results of the DOX drug delivery system are as follows: Figure 9 As shown, when the active pharmaceutical ingredient (DOX) is loaded into the carrier, some of the material's physicochemical properties change. Specific surface area, pore volume, and pore size all change. Analysis of the specific surface area measurement results shows that the specific surface area and pore size of the drug-loaded carrier are significantly smaller than those of the original blank carrier, which fully demonstrates that the DOX active pharmaceutical ingredient is effectively loaded into the carrier's pores.

[0070] Observation of the infrared spectrum revealed that the DOX characteristic peak of the drug carrier was masked, indicating that the active pharmaceutical ingredient was successfully loaded into the pore structure of mesoporous silica.

[0071] Observation of X-ray diffraction results showed that the active pharmaceutical ingredient DOX exhibited a large number of obvious drug crystallization diffraction peaks, indicating that it exists in a highly crystalline state. The spectrum of the drug-loaded carrier showed that it exists in an amorphous state, indicating that the highly porous structure of the carrier improved the distribution of the drug and inhibited drug crystallization.

[0072] The active pharmaceutical ingredient (DOX) has an exothermic peak at 200-220℃, but the exothermic peak disappears after the drug is loaded into the carrier, indicating successful drug loading.

[0073] 4) In vitro drug release from the DOX drug delivery system, such as the in vitro release of the DOX drug delivery carrier. Figure 10 As shown, the release results of VSCA-DOX in different concentrations of GSH are: 10mM GSH > 2mM GSH > 1mM GSH > 0mM GSH. The release results of VSCA-DOX in different concentrations of H2O2 are: 10mM H2O2 > 2mM H2O2 > 1mM H2O2 > 0mM H2O2. This indicates that the release of the modified viral mesoporous silica nanoparticle drug delivery system containing disulfide bonds is concentration-dependent in redox environments, exhibiting redox responsiveness. This can be attributed to the breaking of disulfide bonds in a reducing environment, promoting DOX release; and the increased hydrophilicity and hydrolysis of disulfide bonds after oxidation to sulfoxides or sulfones in an oxidizing environment. Furthermore, the drug delivery carrier modified with hyaluronic acid exhibits stronger selective response under acidic conditions, which is beneficial for the drug delivery system to exert a stronger effect at the tumor site.

[0074] 5) Cytotoxicity test results of the DOX drug delivery system, including cytotoxicity tests of the drug delivery carriers VSCA-DOX and VSCA-DOX-HA. Figure 11 As shown, the results indicate that the cytotoxicity of the drug-loaded carrier is concentration-dependent; the higher the concentration of the drug-loaded carrier in the culture medium, the lower the cell viability. Cell viability decreases with increasing carrier concentration, and the trend of cell viability at all concentrations is: DOX > VSCA-DOX > VSCA-DOX-HA. This indicates that the drug-loaded carrier enhances the killing effect on tumor cells, demonstrating the feasibility of constructing a DOX drug delivery system.

[0075] 6) In vivo distribution results of mesoporous silica carriers: The distribution of silicon in the animal body 24 hours and 7 days after intravenous injection is shown below. Figure 12 As shown in the figure. Experimental results indicate that, 24 hours and 7 days after tail vein injection of mesoporous silica nanoparticles, silicon was most abundant in the liver, with similar distribution in other tissues, suggesting that the carrier is primarily metabolized by the liver after tail vein injection. Within 24 hours after administration, the silicon content in plasma samples decreased slightly, then gradually increased, and subsequently decreased slowly after 24 hours.

[0076] 7) Pharmacokinetic standard curve, precision, and recovery rate of DOX: The standard curve, precision, and recovery rate of DOX in plasma samples are shown in Table 4. The results in the table show that the concentration of the model drug DOX in plasma samples is related to the absorbance R-value. 2 The linearity is greater than 0.999, indicating a good linear relationship, which can be used for subsequent experiments. The intra-day and inter-day precision RSDs for low, medium, and high concentrations are all less than 10%, proving that the precision and extraction recovery rate of this test method meet the requirements for biological sample determination.

[0077] Table 4. Standard curve, precision, and extraction recovery rate of DOX in plasma samples.

[0078]

[0079]

[0080] 8) Pharmacokinetics of the DOX drug delivery system, blood concentration-time curves as shown in the figure. Figure 13 As shown in Table 5, the pharmacokinetic parameters obtained using DAS software are presented.

[0081] Table 5. Pharmacokinetic parameters of the active pharmaceutical ingredient and carrier formulation after injection administration.

[0082]

[0083] As shown in the above pharmacokinetic parameters and concentration-time curves, under the same dosage conditions, the area under the pharmacokinetic curve (AUC(0-∞)) of the VSCA-DOX-HA group was significantly larger than that of the DOX group (p<0.05). In addition, the apparent volume of distribution (Vz) decreased (p<0.05). The half-life was prolonged, indicating that the PK / PD properties of the drug were optimized after being incorporated into VSCA-HA, thus enhancing the therapeutic effect.

[0084] In summary, the hydrophobic drug DOX was loaded into the carrier pores via solvent evaporation. Compared to the original blank carrier, the specific surface area and pore size of the drug-loaded carrier were significantly reduced, indicating that the drug was effectively loaded into the pores of the mesoporous silica, thus leading to the reduction in specific surface area and pore size. The absence of characteristic infrared peaks for DOX-containing VSCA-DOX and VSCA-DOX-HA in the infrared spectrum indicates that the active pharmaceutical ingredient (DOX) was successfully loaded into the pore structure of the mesoporous silica rather than simply adsorbed on the surface. X-ray diffraction results showed that the drug-loaded carrier exhibited typical broad peaks, rather than drug crystallization diffraction peaks, indicating that it was loaded into the mesoporous channels of the mesoporous silica in an amorphous state. DSC analysis revealed that the exothermic peak disappeared after the drug was loaded into the carrier, indicating successful drug loading.

[0085] In vitro release experiments showed that the drug delivery vehicle selectively releases DOX under redox conditions. This is because disulfide bonds break or hydrolyze under redox conditions, increasing the release rate of DOX. In cytotoxicity experiments, the trend of cell viability was: DOX > VSCA-DOX > VSCA-DOX-HA. This indicates that the drug delivery vehicle has enhanced killing power against tumor cells. VSCA-DOX-HA, due to its modification with hyaluronic acid, exhibits enhanced targeting and thus stronger killing effect. This demonstrates the feasibility of constructing a DOX drug delivery system.

[0086] In vivo pharmacokinetic studies have shown that VSCA-DOX-HA has a long-circulation effect, which can effectively prolong the circulation time of the drug in the body.

[0087] 4. The antitumor activity of the mesoporous silica drug delivery system provided in this implementation plan;

[0088] 1) Cellular uptake results of the DOX drug delivery system, and the results of co-incubation of VSCA-DOX and VSCA-DOX-HA with 4T1 cells for 4 and 24 hours are as follows: Figure 14 As shown.

[0089] observe Figure 14 Flow cytometry analysis showed that the cellular uptake capacity, from highest to lowest, was VSCA-DOX-HA > VSCA-DOX > DOX, indicating that both VSCA-DOX and VSCA-DOX-HA possess good anti-tumor potential, with VSCA-DOX-HA being superior to VSCA-DOX. Confocal analysis confirmed this finding. Cellular uptake experiments demonstrated that the VSCA-DOX-HA drug delivery system effectively delivered drugs to cells.

[0090] 2) Cell apoptosis results of the DOX drug delivery system, as follows: Figure 15 As shown in the results, both VSCA-DOX and VSCA-DOX-HA showed stronger apoptosis rates than the DOX group, especially the VSCA-DOX-HA experimental group, which had a higher apoptosis rate. This indicates that VSCA-DOX-HA has a strong inhibitory effect on tumor cell proliferation and has the potential to construct an excellent drug delivery system.

[0091] 3) Distribution of VSCA and VSCA-HA-loaded fluorescent Dir in tumor-bearing mice as follows: Figure 16As shown in the fluorescence distribution map, the fluorescence intensity of VSCA loaded with Dir, injected into mice via the tail vein, gradually increased, peaked at 8 hours, and then gradually decreased. For VSCA-HA loaded with Dir, fluorescence was observed at the tumor site 2 hours after tail vein injection, and the fluorescence intensity increased over time, reaching its peak at 4 hours. The fluorescence intensity remained significant at 12 hours, and a small amount of fluorescence traces were observed at the tumor site at 24 hours. The fluorescence intensity map of ex vivo organs indicates that at 24 hours, VSCA accumulated most abundantly in the lungs, while VSCA-HA accumulated more abundantly in the spleen.

[0092] 4) In vivo pharmacodynamic evaluation of the DOX drug delivery system, including pharmacodynamic parameters of the saline group, DOX group, VSCA-DOX group, and VSCA-DOX-HA group after injection administration. Figure 17 As shown in the figure, the observation results indicate that during the experimental period, the tumors in mice injected with saline via the tail vein grew rapidly, while the mice injected with the active pharmaceutical ingredient DOX showed a certain tumor-inhibiting effect. The VSCA group, modified with disulfide bonds, exhibited redox-responsive function, further enhancing its tumor-inhibiting effect. Among the experimental groups, the VSCA-DOX-HA group showed the best tumor-inhibiting effect, which can be attributed to its combination of redox-responsiveness and the presence of hyaluronic acid, improving its targeting to tumor sites—a point previously confirmed. The mouse body weight change curve over time during the experimental period shows that the experimental group mice had a slightly lower body weight compared to the control group, but the trend was not significantly different, indicating a relatively safe outcome.

[0093] 5) Histopathological analysis of the DOX drug delivery system: After the pharmacodynamic experiment, the excised tumor tissue was sectioned, and the H&E staining and TUNEL staining results were as follows: Figure 18 As shown in the figure. Experimental results showed that in the untreated saline group, tumor cells in the tumor tissue sections were densely packed with intact nuclei. Compared to the saline group, tumor cells in the DOX, VSCA-DOX, and VSCA-DOX-HA groups were sparsely packed, with the VSCA-DOX-HA group showing the sparsest arrangement and nuclear atrophy and deformation, indicating that VSCA-DOX-HA has a strong inhibitory effect. TUNEL staining results also confirmed that VSCA-DOX-HA induced more severe apoptosis than in the other groups.

[0094] 6) Immunohistochemical results of isolated tumor tissue, such as Figure 19As shown in the figure, the Ki67 antigen nucleus is closely related to cell proliferation. Immunohistochemistry can directly observe the expression of Ki67 in tumor tissues of the Saline, DOX, VSCA-DOX, and VSCA-DOX-HA groups. Brown in the figure represents the expression of the Ki67 antigen nucleus. Observation of the experimental results revealed that Ki67 expression in the control group (saline) was significantly higher than that in the experimental group, indicating active tumor cell proliferation. In the experimental groups, the expression order was: DOX > VSCA-DOX > VSCA-DOX-HA, indicating that the VSCA-DOX-HA group has a good ability to inhibit tumor proliferation, consistent with the pharmacodynamic experimental results, demonstrating a good anti-tumor effect. This effect can be attributed to the combined effects of multiple factors, including the intelligent response of the drug delivery system, good biocompatibility, and targeting ability.

[0095] In summary, the antitumor activity study of the DOX drug delivery system constructed in this implementation scheme showed that VSCA-DOX-HA with tumor targeting function had the highest cell entry efficiency, stronger ability to promote cell apoptosis, and stronger killing effect on tumor cells.

[0096] The modified virus-like mesoporous silica nanoparticles prepared in this invention, by grafting dipeptides and hyaluronic acid onto the virus-like mesoporous silica base via disulfide bonds, exhibit glutathione reduction response, good biocompatibility, and targeting ability. The constructed drug delivery system demonstrates significant advantages and provides a reference for the design of future drug delivery systems. The VSCA-DOX-HA system provided in this embodiment can accumulate at the tumor site, persist for a longer period, and exhibit stronger targeting capabilities. In vivo and in vitro experiments jointly demonstrate that the intelligent drug delivery system has a stronger tumor cell killing rate and better tumor suppression effect. Furthermore, it demonstrates good biocompatibility, providing a reference for the subsequent application of DOX drugs.

[0097] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the following claims.

[0098] It should be understood that the present invention is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The scope of the invention is limited only by the appended claims.

Claims

1. A method for constructing a glutathione-responsive viral mesoporous silica drug delivery system, characterized in that, include: Preparation of hyaluronic acid-modified disulfide bond-grafted dipeptide viral mesoporous silica VSCA-HA; Using the anticancer drug DOX as a model drug and VSCA-HA as a carrier, DOX was loaded with the drug using a solvent evaporation dry method, resulting in a virus-like mesoporous silica drug delivery system, VSCA-DOX-HA.

2. The construction method according to claim 1, characterized in that, Step 1, which involves preparing virus-like mesoporous silica (VSCA) with disulfide-grafted dipeptides, includes: 1) Disperse CTAB in deionized water and add TEA; after stirring in a water bath, add a mixed solution of cyclohexane and tetraethyl orthosilicate dropwise, continue stirring and react in an oil bath, collect the precipitate by centrifugation, wash and dry to obtain VSN without template removal; 2) Disperse the unremoved VSN in ethanol, add MPTMS, react under nitrogen protection, centrifuge, wash and dry, and then remove the template by acid extraction to obtain VSN-SH; 3) Disperse VSN-SH in methanol, add 2,2'-dithiopyridine, react at room temperature, centrifuge, wash and dry to obtain VSPY; dissolve cysteine ​​in PBS, add VSPY, react at room temperature, centrifuge, wash and dry to obtain VSC; disperse arginine in MES buffer, add EDCI and SULFO-NHS, stir at room temperature, add PBS dispersion of VSC, react at room temperature overnight, centrifuge, wash and dry to obtain VSCA; 4) Dissolve sodium hyaluronate in water, add EDCI and SULFO-NHS after the reaction, adjust the pH to 8-9, add VSCA in PBS dispersion, continue the reaction, centrifuge and wash, freeze dry to obtain VSCA-HA.

3. The construction method according to claim 1, characterized in that, The solvent evaporation dry loading of DOX includes: dissolving DOX in anhydrous ethanol, mixing it with VSCA-HA, stirring at room temperature in the dark for 24 h, and then evaporating the solvent by opening the bottle to obtain VSCA-DOX-HA.

4. A glutathione-responsive viral mesoporous silica drug delivery system constructed according to any one of claims 1-3.

5. The application of the glutathione-responsive viral mesoporous silica drug delivery system constructed according to any one of claims 1-3 in the preparation of antitumor drugs.

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