Oxidative stress response nano preparation as well as preparation method and application thereof

Abstract

The invention discloses an oxidative stress response nano preparation as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) mixing sodium cocoyl glutamate and 2-amino-2-methyl-1-propanol with water, slowly dropwise adding isopropyl triisopropoxy silane, adding a mixed solution of N-(3-trimethoxysilylpropyl) urea, sodium selenide and isopropyl triisopropoxy silane, and stirring to form nanoparticles; (2) dispersing the nano-particles in a buffer solution containing lysozyme and esterase, and oscillating and incubating to obtain organic silicon nano-particles with hollow structures; and (3) dispersing the organic silicon nanoparticles in a dihydroquercetin solution, filtering, washing uncombined substances, and carrying out vacuum drying to obtain the oxidative stress response nano preparation. The preparation disclosed by the invention can trigger drug release through ROS in an oxidative stress environment, so that the targeting property of the drug in pathological tissues such as tumors and the treatment effect of the drug are improved, the influence on normal tissues is reduced, and the treatment safety of the drug is enhanced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanocomposites, in particular to an oxidative stress-responsive nano-preparation and a preparation method and application thereof. BACKGROUND

[0002] Chemotherapy is one of the main means for treating malignant tumors in clinical practice. However, traditional anti-tumor chemotherapy drugs are often accompanied by serious side effects during treatment, mainly manifested as toxic damage to normal tissue cells, inhibition of immune system function, and occurrence of various complications, thereby seriously affecting the quality of life and treatment compliance of patients. The occurrence of these side effects is mainly attributed to the non-selective distribution of anti-cancer drugs in the body, which not only acts on tumor tissues, but also damages healthy tissues, resulting in low drug utilization rate, unsatisfactory treatment effect and even delayed treatment.

[0003] In order to solve the above problems, drug delivery systems (DDSs) have emerged. Among them, mesoporous silica nanoparticles (MSN) are widely studied as potential drug carriers due to their unique physicochemical properties, such as high specific surface area, large pore volume, adjustable pore size, and easy surface functionalization modification. MSN can achieve drug loading, slow release and targeted release of anti-tumor drugs, and has shown significant advantages in nanomedicine delivery systems.

[0004] However, as an inorganic material, MSN still has certain problems in long-term metabolic safety in the body. Studies have shown that MSN is prone to accumulate in the reticuloendothelial system (such as the liver and spleen), which may cause immune response and chronic toxicity problems, thereby limiting its further conversion and application in clinical practice. In addition, traditional MSN has strong structural rigidity and is difficult to achieve responsive degradation in pathological environments, which limits the controllability of drug release and the precision of treatment.

[0005] Therefore, it is urgent to develop a nano-drug carrier with high biocompatibility and high drug loading efficiency to improve the utilization efficiency of anti-tumor drugs and reduce their toxic side effects. SUMMARY

[0006] In view of the defects in the prior art, the present application provides an oxidative stress-responsive nano-preparation and a preparation method and application thereof.

[0007] The present application provides a preparation method of an oxidative stress-responsive nano-preparation, comprising the following steps:

[0008] (1) Sodium cocoyl glutamate and 2-amino-2-methyl-1-propanol were mixed with water, and isopropyltriisopropoxysilane was slowly added dropwise at 60-70°C. Then, a mixed solution of N-(3-trimethoxysilylpropyl)urea, sodium selenide and isopropyltriisopropoxysilane was added and stirred for 4-6 hours to form core-shell structured nanoparticles with an organosilicon layer on the surface.

[0009] (2) The nanoparticles obtained in step (1) are dispersed in a buffer solution containing lysozyme and esterase, and incubated with shaking at 37°C for 12-24 h. The core is selectively removed by enzyme etching to obtain organosilicon nanoparticles with hollow structure.

[0010] (3) Disperse the organosilicon nanoparticles obtained in step (2) in a dihydroquercetin solution, stir for 7-24 hours, filter, wash the unbound material, and vacuum dry to obtain the oxidative stress-responsive nano-formulation.

[0011] In some embodiments, the concentration of sodium cocoyl glutamate is 0.1 to 0.5 wt%, and the concentration of 2-amino-2-methyl-1-propanol is 1 to 3.0 wt%.

[0012] In some embodiments, the preparation method further includes any one of the following steps:

[0013] (1) The nano-formulation obtained in step (3) is uniformly dispersed in hyaluronic acid solution to obtain the oxidative stress-responsive nano-formulation HMON-Se@DHQ@HA;

[0014] (2) The nano-formulation obtained in step (3) is uniformly dispersed in an aqueous solution of sodium alginate to obtain the oxidative stress-responsive nano-formulation HMON-Se@DHQ@Alginate.

[0015] In some embodiments, the preparation method further includes the following steps:

[0016] The HMON-Se@DHQ@HA or HMON-Se@DHQ@Alginate was dispersed in a MES buffer containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS. After stirring, unreacted EDC and NHS were removed, and 1-3 OD aptamers were added and stirred to obtain an oxidative stress-responsive nanoformulation.

[0017] The nucleotide sequence of the aptamer is shown in SEQ ID NO.1.

[0018] In some embodiments, the mixed solution described in step (1)

[0019] The mass ratio of N-(3-trimethoxysilylpropyl)urea, sodium selenide, and isopropyltriisopropoxysilane (IPS) is 1:1:(2–10). IPS is the silicon source used to form the organosilicon layer, while N-(3-trimethoxysilylpropyl)urea and sodium selenide are reactants involved in forming the core-shell structure. By controlling the mass ratio of the three components, a proper reaction balance can be ensured, thereby obtaining a stable organosilicon core-shell structure. Too high a proportion of IPS will result in excessive organosilicon layer coverage, affecting the structure and size of the nanoparticles; too low a proportion may lead to insufficiently uniform or complete organosilicon layer coverage, thus affecting the stability and biocompatibility of the particles.

[0020] In some embodiments, in step (2), the lysozyme concentration is 0.2–1.0 mg / mL and the esterase concentration is 0.5–1.5 U / mL.

[0021] In some embodiments, in step (3), the dihydroquercetin solution is a methanol solution containing dihydroquercetin; the efficiency of enzyme etching directly affects the morphology, size, and surface properties of nanoparticles. Excessively high enzyme concentrations can lead to particle breakage or irregular morphology, while excessively low concentrations cannot form the desired hollow structure. This invention, by controlling the concentrations of lysozyme and esterase, can adjust the size and morphology of the particles to achieve optimal drug loading, release characteristics, and biocompatibility.

[0022] In some embodiments, the mass ratio of dihydroquercetin to organosilicon nanoparticles is 1:(5-20). As a drug molecule, dihydroquercetin must be loaded into the nanoparticles in an appropriate proportion. If the mass ratio of dihydroquercetin to organosilicon nanoparticles is too low, insufficient drug loading may occur, affecting the therapeutic effect; while an excessively high ratio may lead to excessive accumulation of drug molecules, affecting the stability of the nanoparticles and their drug release characteristics in vivo. This invention ensures effective drug loading by setting an appropriate mass ratio, while avoiding adverse effects on particle stability or release behavior.

[0023] The present invention also provides oxidative stress-responsive nanoformulations obtained by the preparation method described above.

[0024] In some embodiments, the particle size of the nano-formulation is 90–120 nm.

[0025] The present invention also provides the application of the oxidative stress-responsive nanoformulation obtained by the preparation method described above in the preparation of drugs for treating oxidative stress-related diseases.

[0026] In some embodiments, the oxidative stress-related diseases include inflammatory bowel disease, rheumatoid arthritis, cancer, atherosclerosis, and ischemia-reperfusion injury.

[0027] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0028] 1. The hollow organosilicon nanoparticles prepared in this invention combine the mesoporous properties of inorganic materials with the biodegradability of organic structures, significantly improving drug loading efficiency and the metabolic safety of the carrier. They can efficiently encapsulate DHQ, achieving a DHQ loading capacity as high as 146.7 μg / mg, enhancing its stability and prolonging its duration of action in vivo. The formulation can trigger drug release via ROS under oxidative stress, thereby improving drug targeting in tumors and other diseased tissues, enhancing therapeutic efficacy, reducing impact on normal tissues, and improving therapeutic safety.

[0029] 2. The HMON-Se nanoparticles of the present invention exhibit good stability and sustained-release properties in the absence of H2O2, enabling continuous release of drugs in vivo, prolonging the duration of drug efficacy, and avoiding the side effects caused by frequent administration.

[0030] 3. This invention significantly alters the surface electrical properties of nanoparticles through surface modification with materials such as hyaluronic acid (HA) and sodium alginate, thereby enhancing their stability and dispersibility in vivo. Simultaneously, through electrostatic interactions and biocompatibility modifications, it improves their ability to target and deliver to tumor cells.

[0031] 4. This invention uses hollow organosilicon nanoparticles as a carrier, which can effectively load drugs and improve the precision and effectiveness of treatment by controlling the drug release rate.

[0032] 5. By introducing a targeting aptamer, this invention further improves the targeting and delivery efficiency of drug formulations to tumor cells, providing a new strategy for personalized targeted therapy, enhancing therapeutic effects, and reducing side effects. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a scanning electron microscope image of HMON in Embodiment 1 of the present invention.

[0035] Figure 2 This is a transmission electron microscope image of HMON from Embodiment 1 of the present invention.

[0036] Figure 3This is a particle size distribution diagram of HMON-Se@DHQ in Example 1 of the present invention.

[0037] Figure 4 This is a Zeta potential diagram from Embodiment 3 of the present invention.

[0038] Figure 5 This is a Zeta potential diagram from Embodiment 4 of the present invention. Detailed Implementation

[0039] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0040] This invention successfully achieved the loading and controlled release of the natural antioxidant drug dihydroquercetin (DHQ) by constructing a hollow nanoplatform with organosilicon as a carrier and possessing oxidative stress response characteristics.

[0041] This invention designs and prepares oxidative stress-responsive hollow organosilicon nanoparticles (HMON-Se@DHQ), and enhances their targeting and drug release capabilities against tumor cells through various modifications (such as hyaluronic acid, sodium alginate, and aptamers). First, dihydroquercetin (DHQ) loading was successfully achieved by synthesizing core-shell nanoparticles and forming hollow structures using enzymatic etching. The nanoparticles exhibit significant ROS-triggered release characteristics under oxidative stress, with the cumulative release rate of DHQ significantly increasing with increasing H₂O₂ concentration. Modification with HA and alginate enhances the cytotoxicity of the formulation, especially with the aid of the targeting aptamer, greatly enhancing the formulation's targeted delivery capability to tumor cells. In summary, the nanoformulation developed in this invention not only possesses excellent drug release control characteristics but also effectively targets tumor cells, demonstrating potential for anti-tumor therapeutic applications.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0043] Example 1: Preparation of nano-formulations responding to oxidative stress

[0044] (1) Synthesis of core-shell nanoparticles:

[0045] Add 0.2 g of sodium cocoyl glutamate and 0.5 mL of 2-amino-2-methyl-1-propanol (AMP) to 20 mL of deionized water, and heat and stir in a 60 °C water bath (magnetic stirring, 600 rpm). Under constant temperature conditions, slowly add 2.0 mL of isopropyltriisopropoxysilane (IPS) dropwise over approximately 30 minutes.

[0046] After stirring for another 15 minutes, a precursor mixture obtained by thoroughly mixing N-(3-trimethoxysilylpropyl)urea (0.4 g), sodium selenide (0.4 g), and isopropyltriisopropoxysilane (1.0 mL) was added. The reaction temperature was maintained at 65 °C, and the reaction was continued for 46 hours to form core-shell structured nanoparticles with an organosilicon layer on the surface. After the reaction was completed, the precipitate was collected by centrifugation (12000 rpm, 10 min), washed three times with deionized water, and set aside for later use.

[0047] (2) Enzymatic etching to form hollow nanoparticles:

[0048] The obtained nanoparticles were dispersed in 20 mL of Tris-HCl buffer (pH 7.4) containing 0.75 mg / mL lysozyme and 1 mg / mL esterase, and incubated at 37°C in a constant temperature shaker (150 rpm) for 18 h. After incubation, the nanoparticles were washed by centrifugation (12000 rpm, 10 min) to remove enzyme residues, yielding hollow organosilicon nanoparticles (denoted as HMON-Se).

[0049] The morphological features and size of HMON were analyzed using a SU8010 scanning electron microscope, such as... Figure 1 As shown, HMONs are regular spherical in shape, with uniform size, approximately 90–110 nm. The hollow structure of HMONs was analyzed using a JEOL JEM-2100F transmission electron microscope, as shown... Figure 2 As shown.

[0050] (3) Preparation of nano-formulations by drug loading:

[0051] 10 mg of the obtained hollow organosilicon nanoparticles (HMON-Se) were dispersed in 10 mL of a 0.5 mg / mL dihydroquercetin (DHQ) ethanol solution and reacted under magnetic stirring in the dark for 12 h to achieve drug molecule loading. After the reaction was completed, the nanoparticles were collected by centrifugation (12000 rpm, 10 min), washed three times with anhydrous ethanol and deionized water, and finally dried under vacuum at 40 °C for 24 h to obtain an oxidative stress-responsive hollow organosilicon nanoparticle formulation (denoted as HMON-Se@DHQ). The particle size distribution of HMON-Se@DHQ was tested using an equivalent dynamic light scattering (DLS) instrument. Figure 3As shown, the HMON-Se@DHQ nanoparticles have a concentrated particle size distribution in the range of 135–165 nm, with an average particle size of 149 ± 8 nm and a PDI value of 0.20, indicating that the formulation has good stability and dispersibility in aqueous solution. This particle size is suitable for passively targeting tumor tissue through the EPR effect and can also effectively avoid rapid renal clearance, providing favorable conditions for subsequent in vivo delivery.

[0052] After centrifuging HMON-Se@DHQ, the supernatant was collected, and the absorbance of the supernatant at around 280 nm was detected using Nanodrop. The amount of DHQ loaded into the cavity was calculated based on the corresponding standard curve, and the DHQ loading amount was found to be 146.7 μg / mg.

[0053] Example 2 Intracellular ROS-induced release experiment

[0054] This embodiment verifies that the prepared HMON-Se@DHQ nanoformulation exhibits oxidative stress-responsive release behavior under different concentrations of H2O2 induction, meaning that the release rate of drug DHQ can be accelerated in a high ROS environment, indicating that it possesses ROS-triggered release capability. The experimental steps are as follows:

[0055] (1) Disperse an equal amount of HMON-Se@DHQ nanoparticles (1 mg as DHQ) in 5 mL of PBS;

[0056] (2) Add H2O2 to final concentrations of 0 μM, 100 μM, 500 μM, and 1 mM respectively;

[0057] (3) Put the solution into a dialysis bag, seal it, and place it in a centrifuge tube containing 20 mL of PBS. Shake it at a constant temperature of 37 °C in a shaker.

[0058] (4) Take 1 mL of the external solution every 1, 2, 4, 8, 12 and 24 hours and add an equal amount of PBS;

[0059] (5) The content of DHQ released in the external liquid was detected by HPLC, and the cumulative release rate (%) was calculated.

[0060] The results are shown in Table 1:

[0061] Table 1. Statistics on the cumulative release rate of DHQ under different H2O2 concentrations (%)

[0062]

[0063]

[0064] As shown in Table 1, under simulated physiological conditions, the DHQ release from HMON-Se@DHQ nanoparticles exhibits significant oxidative stress response characteristics. In the absence of H2O2, the DHQ release rate was only 21.1% within 24 hours, demonstrating good stability and sustained-release performance. However, under 1mM H2O2 conditions, the cumulative release reached 78.4% within 24 hours, significantly higher than the low ROS group, indicating that the nano-formulation of this application can achieve accelerated release under oxidative stress.

[0065] Example 3: Preparation of hyaluronic acid encapsulated with EGCG loaded onto hollow mesoporous organosilicone nanoparticles

[0066] The HMON-Se@DHQ prepared in Example 1 was uniformly dispersed in a hyaluronic acid (HA) solution and stirred at room temperature for 24 hours. HA was adsorbed onto the surface of HMON-Se@DHQ through electrostatic interaction to obtain HMON-Se@DHQ@HA.

[0067] The change in zeta potential after encapsulating hyaluronic acid was measured using an SZ-100Z particle size analyzer.

[0068] like Figure 4 As shown, since the HA on the surface of the nanoparticles carries a negatively charged carboxyl group, the Zeta potential drops to -21.5mV after HA modification. This is because the negative charge formed after the dissociation of the carboxyl group (-COOH) in the HA molecule covers the surface of the nanoparticles, thereby significantly changing its surface charge, further confirming the successful modification of HA.

[0069] Example 4: Preparation of sodium alginate supported on hollow mesoporous organosilica nanoparticles

[0070] A 1 mg / mL sodium alginate aqueous solution was prepared and magnetically stirred at room temperature for 30 minutes to ensure complete dissolution. HMON-Se@DHQ nanoparticles were dispersed in the sodium alginate solution at a concentration of 0.2 mg / mL. The mixture was magnetically stirred at room temperature for 24 hours to allow negatively charged sodium alginate to be adsorbed onto the particle surface via electrostatic attraction. Free sodium alginate was removed by ultracentrifugation (12,000 rpm, 10 min), and the nanoparticles were washed twice with PBS. Resuspension yielded surface-modified HMON-Se@DHQ@Alginate.

[0071] The change in zeta potential after encapsulating hyaluronic acid was measured using an SZ-100Z particle size analyzer.

[0072] like Figure 5 As shown, the Zeta potential decreased to approximately -23.4 mV after modification with sodium alginate, and the significant decrease in surface potential indicates that the modification was successful.

[0073] In Example 5, hollow mesoporous organosilicon nanoparticles loaded with EGCG encapsulate hyaluronic acid linker aptamers

[0074] The HMON-Se@DHQ@HA prepared in Example 2 and the HMON-Se@DHQ@Alginate prepared in Example 3 were dispersed in a pH 6.0 MES buffer containing 400 mM EDC and 100 mM NHS and stirred for 15 min. After removing unreacted EDC and NHS, the 2OD aptamer (nucleotide sequence as shown in SEQ ID NO.1) was added to the nano suspension and stirred for 12 hours to obtain HMON-Se@DHQ@HA@aptamer and HMON-Se@DHQ@Alginate@aptamer.

[0075] Example 6: Effects of Oxidative Stress-Response Nanoparticles on Tumor Cell Proliferation

[0076] This embodiment provides the application of oxidative stress-degradable nano-formulations that can inhibit the proliferation of tumor cells, specifically including the following steps:

[0077] 100 μL of HuH-7 cell (human hepatocellular carcinoma cell) suspension was prepared in a 96-well plate with a density of 1 × 10⁻⁶ mcg. 5 Each well was repeated 6 times, and the culture plate was then pre-cultured in an incubator for 24 hours to allow cells to fully adhere. After 24 hours, the old culture medium was removed, and different concentration gradients of HMON-Se (group a), DHQ (group b), HMON-Se@DHQ (group c), HMON-Se@DHQ@HA (group d), HMON-Se@DHQ@Alginate (group e), HMON-Se@DHQ@HA@aptamer (group f) and HMON-Se@DHQ@Alginate@aptamer (group g) were added to the culture wells. The cells were incubated for another 24 hours. Finally, 100 μL of a mixture of CCK-8 and culture medium (Vcck-8:V culture medium = 1:9) was added, and the absorbance at 450 nm was measured using a microplate reader to calculate the cell viability.

[0078] Cell viability = (A 实验组 -A 空白 ) / (A 对照 -A 空白 )×100%.

[0079] The results are shown in Table 2:

[0080] Table 2. Statistical results of cell viability (%)

[0081]

[0082] Table 2 shows that HMON-Se exhibits good biocompatibility and negligible cytotoxicity. In in vitro cell experiments, the cell viability of the DHQ, HMON-Se@DHQ, HMON-Se@DHQ@HA, HMON-Se@DHQ@Alginate, HMON-Se@DHQ@HA@aptamer, and HMON-Se@DHQ@Alginate@aptamer groups showed a gradual decreasing trend, indicating that the cytotoxicity of the formulation was more pronounced after modification with HA or Alginate. Especially after the addition of the targeting aptamer, cell viability further decreased, suggesting a significant enhancement in its targeting recognition and delivery capabilities.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0084]

Claims

1. A method for preparing an oxidative stress-responsive nano-formulation, characterized in that, Includes the following steps: (1) Sodium cocoyl glutamate and 2-amino-2-methyl-1-propanol were mixed with water, and isopropyltriisopropoxysilane was slowly added dropwise at 60-70°C. Then, a mixed solution of N-(3-trimethoxysilylpropyl)urea, sodium selenide and isopropyltriisopropoxysilane was added and stirred for 4-6 hours to form core-shell structured nanoparticles with an organosilicon layer on the surface. (2) The nanoparticles obtained in step (1) are dispersed in a buffer solution containing lysozyme and esterase, and incubated with shaking at 37°C for 12-24 h. The core is selectively removed by enzyme etching to obtain organosilicon nanoparticles with hollow structure. (3) Disperse the organosilicon nanoparticles obtained in step (2) in a dihydroquercetin solution, stir for 7-24 hours, filter, wash the unbound material, and vacuum dry to obtain the oxidative stress-responsive nano-formulation.

2. The preparation method according to claim 1, characterized in that, It also includes any of the following steps: (1) The nano-formulation obtained in step (3) is uniformly dispersed in hyaluronic acid solution to obtain the oxidative stress-responsive nano-formulation HMON-Se@DHQ@HA; (2) The nano-formulation obtained in step (3) is uniformly dispersed in an aqueous solution of sodium alginate to obtain the oxidative stress-responsive nano-formulation HMON-Se@DHQ@Alginate.

3. The preparation method according to claim 2, characterized in that, It also includes the following steps: The HMON-Se@DHQ@HA or HMON-Se@DHQ@Alginate was dispersed in a MES buffer containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS. After stirring, unreacted EDC and NHS were removed, and 1-3 OD aptamers were added and stirred to obtain an oxidative stress-responsive nanoformulation. The nucleotide sequence of the aptamer is shown in SEQ ID NO.

1.

4. The preparation method according to claim 1, characterized in that, In the mixed solution described in step (1), the mass ratio of N-(3-trimethoxysilylpropyl)urea, sodium selenide and isopropyltriisopropoxysilane is 1:1:(2-10).

5. The preparation method according to claim 1, characterized in that, In step (2), the concentration of lysozyme is 0.2–1.0 mg / mL and the concentration of esterase is 0.5–1.5 U / mL.

6. The preparation method according to claim 1, characterized in that, The mass ratio of dihydroquercetin to organosilicon nanoparticles is 1:(5-20).

7. The oxidative stress-responsive nanoformulation obtained by the preparation method according to any one of claims 1 to 6.

8. The oxidative stress-responsive nanoformulation according to claim 7, characterized in that, The particle size of the nano-formulation is 90–180 nm.

9. The application of the oxidative stress-responsive nanoformulation obtained by the preparation method of claim 1 in the preparation of drugs for treating oxidative stress-related diseases.

10. The application according to claim 9, characterized in that, The oxidative stress-related diseases include inflammatory bowel disease, rheumatoid arthritis, cancer, atherosclerosis, and ischemia-reperfusion injury.

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