Silicon dioxide-loaded resveratrol double-ligand targeted nano-drug for breast cancer and preparation
By using mesoporous silica nanoparticles to load resveratrol in breast cancer treatment and improving targeting through dual ligand modification technology, the problems of insufficient targeting and major side effects in the existing technology are solved, and more efficient breast cancer treatment effects and lower side effects are achieved.
Patent Information
- Application Number
- CN202510056044.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-14
- Publication Date
- 2025-04-11
AI Technical Summary
The prior art has problems such as insufficient targeting, difficulty in controlling drug release and major side effects in the treatment of breast cancer, resulting in unsatisfactory treatment results and greater pain in patients.
Mesoporous silica nanoparticles are used as carriers to load resveratrol and modified by biligands, including a circular polypeptide (cRGD) and folic acid (FA) ligand containing two disulfide bonds, to construct targeted nanoformula to improve the cellular targeting of the drug and tumor penetration ability.
It significantly improves the bioavailability and therapeutic effect of resveratrol in breast cancer cells, reduces damage to normal cells, reduces the occurrence of side effects, and prolongs the patient's survival.
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Figure CN119792575A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomedicine, in particular to a silicon dioxide-loaded resveratrol dual-ligand targeted nano drug for breast cancer and its preparation. Background Art
[0002] Breast cancer is one of the most common malignant tumors in women. According to statistics, the incidence rate accounts for 7-10% of all malignant tumors in the body. Due to the limitations of current surgical treatments and the serious toxic side effects of anti-tumor drugs, the development of a new anti-breast cancer targeted delivery system has become inevitable.
[0003] Mesoporous silica drug-loaded nanoparticles have attracted extensive attention in tumor treatment in recent years, but there are still great challenges in key issues such as active targeting, blood circulation stability, and responsive controlled drug release.
[0004] Therefore, this technology will select resveratrol, a new green anti-cancer substance after "paclitaxel" with definite efficacy on breast cancer, as a model drug from a new perspective, and construct a silica-loaded resveratrol-containing two disulfide bonds cyclic polypeptide (cRGD)-folate (FA) dual ligand modified targeted recognition composite nanoformulation cRGD-FA-MSN (cRFM for short), so that it can obtain superior cell targeting, cell uptake or tumor penetration ability, and thus shows great advantages in tumor targeted therapy. This is undoubtedly of great clinical significance for solving the problem of "large toxic side effects and low bioavailability" of current clinical anti-breast cancer drugs and alleviating the suffering of breast cancer patients. It can also provide new targets for breast cancer treatment, and will also provide experimental basis and new ideas for the development of resveratrol preparations. Summary of the invention
[0005] In order to solve the problems of the prior art, the present invention provides a silicon dioxide-loaded resveratrol dual-ligand targeted nano drug for breast cancer and its preparation.
[0006] In order to solve the above technical problems, the present invention is achieved through the following technical solutions: In the first aspect, a silicon dioxide-loaded resveratrol dual-ligand targeted nanomedicine for breast cancer comprises:
[0007] Mesoporous silica nanoparticles are used as carriers, and resveratrol is loaded in the surface mesopores;
[0008] The surface of mesoporous silica nanoparticles was modified with a cyclic peptide (cRGD) ligand containing two disulfide bonds;
[0009] The mesoporous silica nanoparticles were further modified with folic acid (FA) ligands, which together with the cyclic peptide (cRGD) ligands formed a dual-ligand targeting system;
[0010] Nanoformulations have superior cell targeting, cellular uptake, and tumor penetration capabilities, and are used to enhance the bioavailability and therapeutic efficacy of resveratrol in breast cancer cells.
[0011] In this aspect, mesoporous silica nanoparticles are used as carriers to efficiently load resveratrol, a natural compound with anti-cancer activity, and modified with dual ligands (a cyclic polypeptide cRGD containing two disulfide bonds and folic acid FA) to achieve precise targeting and efficient delivery of breast cancer cells.
[0012] Traditional chemotherapy drugs often lack specificity and are prone to damage normal cells. However, this technology significantly improves the targeting ability of nanoformulations to breast cancer cells through dual ligand modification, achieves precise drug delivery, and reduces side effects; as a natural anticancer compound, resveratrol has poor water solubility and low bioavailability, which are the main problems limiting its clinical application. This technology effectively improves the stability and bioavailability of the drug by loading resveratrol on nanocarriers, enabling it to better play an anticancer role; because nanoformulations have superior cell uptake and tumor penetration capabilities, they can penetrate deep into tumor tissues and fully contact cancer cells, thereby enhancing the therapeutic effect; long-term use of traditional chemotherapy drugs can easily lead to drug resistance in cancer cells. However, this technology loads resveratrol on nanocarriers and combines it with a dual ligand targeting strategy, which is expected to reduce the drug resistance of cancer cells to drugs and prolong the survival of patients.
[0013] In the second aspect, the preparation of silica-loaded resveratrol dual-ligand targeted nanomedicine for breast cancer comprises the following steps:
[0014] S1: Resveratrol was loaded into the mesopores of mesoporous silica nanoparticles using a solvent-melt method to obtain drug-loaded nanoparticles;
[0015] S2: The circular peptide (cRGD) ligand containing two disulfide bonds is connected to the surface of drug-loaded nanoparticles through chemical bonding;
[0016] S3: Connecting the folic acid (FA) ligand to the surface of the drug-loaded nanoparticles treated in step b) through chemical bonding to obtain a dual-ligand targeted nanoformulation.
[0017] In some specific embodiments of the second aspect, step S1 further comprises removing the solvent using a rotary evaporator, and eluting with phosphate buffered saline (PBS) and ultrasonic dispersion treatment to obtain stable drug-loaded nanoparticles.
[0018] In some specific embodiments of the second aspect, step S2 also includes using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide as coupling agents to activate the carboxyl group on the cyclic polypeptide (cRGD) ligand, and then connecting it to the drug-loaded nanoparticles.
[0019] In some specific embodiments of the second aspect, step S3 also includes using a suitable chemical method to connect the folic acid (FA) ligand to the surface of the drug-loaded nanoparticles treated in step S2 to form a dual-ligand targeted nanoformulation.
[0020] In some specific embodiments of the second aspect, Pluronic F127 or other surfactants are introduced during the preparation process to enhance the stability and biocompatibility of the nanoformulation.
[0021] In some specific embodiments of the second aspect, the step of introducing Pluronic F127 comprises mixing Pluronic F127 with drug-loaded nanoparticles in a suitable solvent, and performing stirring and dialysis treatment to obtain a stable nanoformulation.
[0022] The beneficial effects of the present invention are:
[0023] 1. Through the precise targeting of the dual ligands (folic acid FA and a cyclic polypeptide cRGD containing two disulfide bonds), the present invention can ensure that resveratrol, an anti-cancer active ingredient, directly acts on breast cancer cells, thereby significantly improving the therapeutic effect. This targeted delivery method not only enhances the cellular uptake of the drug, but also increases the penetration depth of the drug in the tumor tissue, allowing the drug to kill cancer cells more effectively;
[0024] 2. Traditional chemotherapy drugs often lack specificity and are prone to damage normal cells, leading to a series of side effects. However, the present invention achieves precise drug delivery through the combination of nanocarriers and resveratrol, as well as the targeting effect of dual ligands, significantly reducing damage to normal cells and thus reducing the occurrence of side effects;
[0025] 3. As a natural anticancer compound, resveratrol has poor water solubility and low bioavailability, which are the main problems limiting its clinical application. The present invention uses nanocarriers to load resveratrol, which effectively improves the stability and bioavailability of the drug, allowing the drug to better exert its anticancer effect;
[0026] 4. Long-term chemotherapy can easily lead to drug resistance in cancer cells. However, the present invention is expected to reduce the drug resistance of cancer cells to drugs through the combination of nanocarriers and resveratrol, as well as the targeting effect of dual ligands, thereby prolonging the survival of patients. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 It is a schematic diagram of the changes in particle size and potential of each nanoparticle measured by using a laser particle size analyzer in an embodiment of the present invention.
[0028] Figure 2 It is a schematic diagram of measurement data of parameters such as charge and conductivity according to an embodiment of the present invention.
[0029] Figure 3 This is a schematic diagram of the present invention for detecting changes in main chemical bonds and functional groups.
[0030] Figure 4 It is a schematic diagram of the determination of the release degree of the present invention. DETAILED DESCRIPTION
[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.
[0032] The silica-loaded resveratrol dual-ligand targeted nanomedicine for breast cancer comprises: mesoporous silica nanoparticles as carriers, with resveratrol loaded in the surface mesopores; the surface of the mesoporous silica nanoparticles is modified with a cyclic polypeptide (cRGD) ligand containing two disulfide bonds; the mesoporous silica nanoparticles are further modified with a folic acid (FA) ligand, which together with the cyclic polypeptide (cRGD) ligand constitutes a dual-ligand targeting system; the nanoformulation has excellent cell targeting, cell uptake ability and tumor penetration ability, and is used to improve the bioavailability and therapeutic effect of resveratrol in breast cancer cells.
[0033] The preparation of silica-loaded resveratrol dual-ligand targeted nanomedicine for breast cancer comprises the following steps:
[0034] S1: Resveratrol was loaded into the mesopores of mesoporous silica nanoparticles using a solvent-melt method to obtain drug-loaded nanoparticles;
[0035] S2: The circular peptide (cRGD) ligand containing two disulfide bonds is connected to the surface of drug-loaded nanoparticles through chemical bonding;
[0036] S3: Connecting the folic acid (FA) ligand to the surface of the drug-loaded nanoparticles treated in step b) through chemical bonding to obtain a dual-ligand targeted nanoformulation.
[0037] Step S1 also includes removing the solvent using a rotary evaporator, eluting with phosphate buffered saline (PBS) and ultrasonic dispersion treatment to obtain stable drug-loaded nanoparticles.
[0038] Step S2 also includes using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide as coupling agents to activate the carboxyl group on the cyclic polypeptide (cRGD) ligand, and then connecting it to the drug-loaded nanoparticles.
[0039] Step S3 also includes using a suitable chemical method to connect the folic acid (FA) ligand to the surface of the drug-loaded nanoparticles treated in step S2 to form a dual-ligand targeted nanoformulation.
[0040] The method also includes introducing Pluronic F127 or other surfactants during the preparation process to enhance the stability and biocompatibility of the nanoformulation. The step of introducing Pluronic F127 includes mixing Pluronic F127 with drug-loaded nanoparticles in a suitable solvent, and performing stirring and dialysis treatment to obtain a stable nanoformulation.
[0041] Example: Reference Figure 1 , Figure 2 , Figure 3 and Figure 4 Shown: Resveratrol and mesoporous silica nanoparticles (RES@DMSN) were prepared by solvent-melt method. (Two other methods were also performed)
[0042] (1) Weigh 100 mg of mesoporous silica nanoparticles, 60 mg of resveratrol, and 4 mg of lysine, add them into a 100 ml round-bottom flask, then add 50 ml of methanol solution, and shake thoroughly to dissolve them.
[0043] (2) Fix the round-bottom flask on a rotary evaporator and set the parameters: 50°C, negative pressure -0.2 kPa, 50 min. Rotary evaporation is used to remove methanol and load resveratrol into the mesopores on the surface of silica to obtain white drug-loaded nanoparticles.
[0044] (3) PBS elution and cooling: Add 4 ml of phosphate buffered saline (PBS) to the removed round-bottom flask, shake gently, fully emulsify, and elute.
[0045] (4) Ultrasonic dispersion: Use a 5 ml pipette to transfer the emulsified liquid into a 50 ml centrifuge tube. After cooling in an ice bath, use an ultrasonic emulsification sonicator with a frequency set to 150 W and a working mode of 5 min (5 s / 5 s, on / off) to evenly disperse the formed drug-loaded nanoparticles by ultrasound.
[0046] (5) The obtained nanoparticles were centrifuged in a low-temperature centrifuge (4°C, 12000 rpm / min) for 10 minutes, and then the supernatant was taken and centrifuged again, and the supernatant was taken as a sample, and finally stored at 4°C in the dark for later use. The same method was used to prepare mesoporous titanium dioxide (TiO2) nanoparticles (RES@DMSN).
[0047] (6) cRGD, 1 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloride (EDC), and 4 mg N-hydroxysuccinimide were dissolved in deionized water and stirred for 30 min to activate the carboxyl groups on cRGD. RES@DMSN was then added to obtain RES@DMSN-cRGD. The mixture was stirred at 4°C overnight, the unreacted cRGD was removed by centrifugation, and the mixture was resuspended in deionized water.
[0048] (7) Synthesis of F127: First, pre-treat the reagents: take an appropriate amount of solid calcium hydride and place it in toluene and stir it continuously for 24 hours to remove the water in the toluene, then distill it under normal pressure to obtain anhydrous toluene; obtain anhydrous DMSO by distillation under reduced pressure; take an appropriate amount of F127 and dissolve it in the pre-treated dry toluene, and use a rotary evaporator to completely evaporate the toluene and water to obtain dry F127. L-Lactide (L-LA) is placed in ethyl acetate (which has been dehydrated with activated molecular sieves) (T = 90°C) that is continuously heated and stirred to dissolve to form a supersaturated solution, which is cooled until crystals precipitate, filtered under reduced pressure, and vacuum dried for 48 hours to obtain recrystallized L-LA. All raw materials before synthesis are dried in a vacuum oven for 48 hours in advance, and the synthesis instrument is kept dry by preheating and dehumidification.
[0049] (8) Synthesis of RES@DMSN-cRGD-F127: Accurately weigh RES@DMSN-cRGD (180 mg) and DCC (82.52 mg) into a round-bottom flask, add 20 ml of anhydrous DMSO to dissolve and stir at room temperature for 24 h. Pluronic F127 (5 g, 0.4 mmol) and dry DMAP (4.89 mg, 0.04 mmol) dried by azeotropic dehydration with anhydrous toluene were added to the above round-bottom flask and stirred at room temperature for 48 h. The reaction product was centrifuged (8000r / min, 5min), the precipitate was discarded, and the supernatant was placed in 500ml DMSO (analytical grade) for 5h (dialysis bag molecular cutoff 3500Da), and DMSO was replaced at intervals of 0.5h, 1h, and 2h to remove biotin and catalysts that did not participate in the reaction and other small molecule byproducts; then dialyzed with distilled water for 48h (changing water every 3h), and the dialysis was stopped after the dialysis bag had no DMSO smell. The reaction product was centrifuged, the precipitate was discarded, and the supernatant was taken and dried with a rotary evaporator (the maximum temperature of the water bath was <50℃, and the supernatant was continued to be rotated for 6h until no bubbles were generated). After being completely dissolved with CH2CL2, it was dispersed dropwise into 8-10 times the volume of ice methanol (-20℃ refrigerated) for precipitation, and then centrifuged at 10000rpm and the supernatant was discarded and dried to obtain purified RES@DMSN-cRGD-F127.
[0050] 2. Characterization Experiment
[0051] refer to Figure 1 and Figure 2 As shown, ① Particle size and potential: A laser particle size analyzer is used to measure the changes in particle size and potential of each nanoparticle.
[0052] refer to Figure 3 As shown, ②Infrared spectrum: detect the changes of main chemical bonds and functional groups. (Compared)
[0053] Determination of stability
[0054] Determination of targeting
[0055] 4.1 Grouping and dosing method
[0056] 48 SD rats were randomly divided into a control group and an experimental group, with 24 rats in each group. They were fed 24 hours before administration, and each group was injected with resveratrol and resveratrol FA-BT nanoparticles at a dose of 50 mg.kg-1 through the tail vein. Three rats were randomly killed at 5, 10, 20, 40, 80, 160, and 320 minutes, and plasma, heart, liver, spleen, lung, and kidney tissues were collected and centrifuged for later use.
[0057] 4..2 Determination
[0058] Sample treatment: Take the standby sample and add 0.9% saline, place it in -18 freezer for standby. Take 150μL of plasma and tissue homogenate in a 1.5ml centrifuge tube, add 500μL of ethyl acetate, and shake it with a vortex oscillator for 10min, let it stand for 5min, and dilute it to 200μL with methanol for HPLC determination. Chromatographic conditions: C18, mobile phase: acetonitrile: water = 30:70
[0059] Injection volume: 5 μL, column temperature: 25 degrees, flow rate: 1 ml min-1. The results are shown in Table 1.
[0060] Table 1 Recovery rate, intra-day precision and inter-day precision of RES@DMSN-cRGD-F127 in different tissues (n=7)
[0061]
[0062] The results showed that the recovery rates were all above 90%, and the intra-day precision and inter-day precision were both below 5%, which met the requirements for sample analysis.
[0063] 4.3 Calculation
[0064] Relative uptake rate (Re) = (AUCi)T / (AUCi)R, total targeting coefficient (Te) = (AUCi)T / Σ(AUCi)R, distribution efficiency Rte = (Te)T / (Te). If the Re and Rte of a certain organ in the calculation results are both greater than 1, it can be determined that the organ is targeted.
[0065] 1.4 Statistical analysis
[0066] Statistical analysis was performed using spss17.0 statistical software, and pharmacokinetic parameters were analyzed using DAS software. The test results were expressed as x±s.
[0067] 1.5.1 The blood drug concentration-time curve of RES@DMSN-cRGD-F127 nanoparticles was 1.8 times higher than that of the control group, the elimination rate was reduced from (2.82±0.60) to (0.72±0.08), and t1 / 2z(h) was extended from (0.43±0.18) to (0.96±0.16). The results showed that resveratrol FA-BT nanoparticles can change the elimination rate and prolong the duration of action in vivo. See Table 2.
[0068] 1.5.2 Pharmacokinetic parameters of RES@DMSN-cRGD-F127 nanoparticles
[0069] Table 2 Pharmacokinetic parameters of RES@DMSN-cRGD-F127 nanoparticles in rats (n=3, x±s)
[0070]
[0071] 1.2 Distribution of RES@DMSN-cRGD-F127 nanoparticles in vivo
[0072] The results showed that the Rte and Re of RES@DMSN-cRGD-F127 nanoparticles in the liver, spleen, and kidney were greater than 1, reaching 2.367, 2.128, and 2846, respectively, indicating that they have a certain degree of targeting to the liver, spleen, and kidney. See Table 3.
[0073] Table 3 Rte, Re, Te of RES@DMSN-cRGD-F127 nanoparticles in plasma and tissues
[0074]
[0075] Determination of release rate.
[0076] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A silicon dioxide-loaded resveratrol dual-ligand targeted nanomedicine for breast cancer, characterized in that: include: Mesoporous silica nanoparticles are used as carriers, and resveratrol is loaded in the surface mesopores; The surface of the mesoporous silica nanoparticles is modified with a cyclic polypeptide (cRGD) ligand containing two disulfide bonds; The mesoporous silica nanoparticles are further modified with folic acid (FA) ligands, which together with the cyclic polypeptide (cRGD) ligands form a dual ligand targeting system; The nanoformulation has excellent cell targeting, cell uptake ability and tumor penetration ability, and is used to improve the bioavailability and therapeutic effect of resveratrol in breast cancer cells.
2. Preparation of silica-loaded resveratrol dual-ligand targeted nanomedicine for breast cancer, characterized in that: The following steps are involved: S1: Resveratrol was loaded into the mesopores of mesoporous silica nanoparticles using a solvent-melt method to obtain drug-loaded nanoparticles; S2: The circular peptide (cRGD) ligand containing two disulfide bonds is connected to the surface of drug-loaded nanoparticles through chemical bonding; S3: Connecting the folic acid (FA) ligand to the surface of the drug-loaded nanoparticles treated in step b) through chemical bonding to obtain a dual-ligand targeted nanoformulation.
3. The preparation of the silicon dioxide-loaded resveratrol dual-ligand targeted nanomedicine for breast cancer according to claim 2, characterized in that: The step S1 also includes removing the solvent using a rotary evaporator, eluting with phosphate buffered saline (PBS) and performing ultrasonic dispersion treatment to obtain stable drug-loaded nanoparticles.
4. The preparation of the silicon dioxide-loaded resveratrol dual-ligand targeted nanomedicine for breast cancer according to claim 2, characterized in that: The S2 step also includes using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide as coupling agents to activate the carboxyl group on the cyclic polypeptide (cRGD) ligand, and then connecting it with the drug-loaded nanoparticles.
5. The preparation of the silicon dioxide-loaded resveratrol dual-ligand targeted nanomedicine for breast cancer according to claim 2, characterized in that: The step S3 also includes using a suitable chemical method to connect the folic acid (FA) ligand to the surface of the drug-loaded nanoparticles treated in step S2 to form a dual-ligand targeted nanoformulation.
6. The preparation of the silicon dioxide-loaded resveratrol dual-ligand targeted nanomedicine for breast cancer according to claim 2, characterized in that: The method also includes introducing Pluronic F127 or other surfactants during the preparation process to enhance the stability and biocompatibility of the nanoformulation.
7. The preparation of the silicon dioxide-loaded resveratrol dual-ligand targeted nanomedicine for breast cancer according to claim 6, characterized in that: The step of introducing Pluronic F127 includes mixing Pluronic F127 with drug-loaded nanoparticles in a suitable solvent, and performing stirring and dialysis treatment to obtain a stable nanoformulation.