CN110693851B - Mesoporous silica drug-loaded nanoparticle and preparation method and application thereof - Google Patents

Mesoporous silica drug-loaded nanoparticle and preparation method and application thereof Download PDF

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CN110693851B
CN110693851B CN201911016670.1A CN201911016670A CN110693851B CN 110693851 B CN110693851 B CN 110693851B CN 201911016670 A CN201911016670 A CN 201911016670A CN 110693851 B CN110693851 B CN 110693851B
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msn
pda
drug
peoz
deionized water
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CN110693851A (en
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王小宁
梁晓燕
闫梦茹
马远涛
高迎春
赵宁
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Xian Medical University
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    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The invention discloses mesoporous silica drug-loaded nanoparticles, which are prepared by taking mesoporous silica as a drug-loaded carrier, taking the mesoporous silica as the drug-loaded carrier, wrapping a polydopamine layer on the outer surface of the mesoporous silica carrier, and connecting tumor-oriented penetrating peptide iRGD and poly (2-ethyl-2-oxazoline) through Schiff base addition reaction.

Description

Mesoporous silica drug-loaded nanoparticle and preparation method and application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to mesoporous silica drug-loaded nanoparticles, and a preparation method and application thereof.
Background
Malignant tumors seriously threaten the health and life safety of human beings. Chemotherapy is one of the currently clinically important means for treating cancer. The nano delivery system of the antitumor drug has the advantages of small size, capability of obviously improving the solubility of the chemotherapeutic drug, improving the stability of the drug and prolonging the blood circulation time of the drug, thereby becoming a hotspot of the research of the current chemotherapeutic drug preparation. Among them, mesoporous silica has been proven to be one of the most potential drug delivery nanocarriers. The mesoporous silica has the pore diameter of 2-50nm, has the advantages of large specific surface area, high drug loading, good biocompatibility and easy surface modification of functional groups, and has great potential in the aspects of improving the water solubility of the antitumor drug, enhancing the retention of tumor parts (EPR effect) and the like. However, passive targeting to tumors via the EPR effect is of limited effectiveness, and nonspecific distribution of the drug may still lead to toxicity in healthy organs. Therefore, active recognition and uptake of the delivery system by tumor cells is a problem to be solved.
Modification of the carrier surface with various ligands has been shown to facilitate targeted drug delivery to tumor sites and increase cellular uptake. These ligands are recognized by specific receptors/antigens on the surface of tumor cells through specific interactions. A great deal of research proves that the tumor targeting of the carrier can be remarkably enhanced by the specific recognition effect of the ligand-receptor and the antibody-antigen. Meanwhile, the tumor tissue has high heterogeneity, compact extracellular matrix (ECM) and lack of lymphatic drainage, so that the pressure difference across capillaries is reduced, the hydraulic pressure between tissues is increased, and the penetration of nanoparticles into cells is prevented. Therefore, although the nanocarrier can be concentrated at the tumor tissue site due to active targeting, whether the nanocarrier can successfully enter the tumor cells by overcoming the obstruction of high interstitial fluid pressure of the tumor tissue is a critical issue.
The iRGD is a cyclic nonapeptide with the infiltration activity of targeting tumor new vessels and tumor cells, and the amino acid sequence of the cyclic nonapeptide is CRGDKGPDC. The RGD sequence of iRGD is firstly combined with an alpha v beta 3 integrin receptor specifically expressed in tumor vascular endothelium, then proteolytic cleavage is carried out, a CendR motif (RGDK/R) is exposed, and the CendR polypeptide sequence can be specifically combined with NRP-1 receptors on tumor neovascularization and cell surfaces, so that the permeability of tumor tissues to nanoparticles is remarkably improved, and the tumor targeting property and the retention time are enhanced. However, the slow release of the antitumor drug from the mesoporous silica may cause the concentration of the intracellular free drug to be maintained at a low level for a long time, thereby limiting the antitumor effect, and may even cause the tumor cells to develop drug resistance. Therefore, in order to ensure effective delivery of the antitumor drug to the tumor site and to achieve a sufficient drug concentration, the vehicle is required to be stable in the blood circulation and to rapidly release the drug to the cytoplasm of tumor cells. This can be achieved by a targeting vector with a trigger release mechanism. Since the pH of typical tumor tissues is about 6.5, and the pH of lysosomes and endosomes is 5.0-5.5, pH-triggered release targeting vehicles can be designed. The pKa value of poly (2-ethyl-2-oxazoline), PEOz is 4-6, if PEOz is used to modify mesoporous silicon dioxide, under the condition of physiological pH, the long chain distribution of PEOz and external side of mesoporous silicon channel are in "closed" state, and after the PEOz is come into tumor cell, under the condition of acid medium, the PEOz is protonated, and the electrostatic repulsion force is increased, so that the mesoporous silicon channel is in "open" state, and the medicine can be quickly released. Meanwhile, the PEOz has similar action with PEG (polyethylene glycol), can be used for modifying the surface of a carrier, can increase the surface hydrophilicity of the carrier, forms steric hindrance, enables nanoparticles to avoid recognition of a reticuloendothelial system, and avoids being absorbed and removed, thereby achieving the purposes of prolonging the internal circulation time of the medicament and improving the concentration of the medicament on tumor tissue parts.
The Polydopamine (PDA) is a main component of protein secreted by marine mussel organisms, has strong adhesion, can be adhered to the substrate surfaces of various organic matters and inorganic matters, and has good stability. The PDA surface is rich in catechol, amino and other active groups, is easy to generate Michael addition and Schiff base reaction with carbon-carbon double bonds, aldehyde groups and other functional groups, is easy to generate condensation reaction with carboxyl, can combine with a targeting ligand or adsorb organic molecules and the like through the actions of static electricity, conjugation and the like, and therefore can be used as a reaction platform for carrier modification. Meanwhile, polydopamine has strong absorption in a near-infrared light region in the range of 700-1100 nm, so that the polydopamine has high photo-thermal conversion capability and good photo-stability, and the photo-thermal conversion efficiency of the polydopamine is far higher than that of a gold nanorod commonly used for photo-thermal treatment.
Therefore, a mesoporous silica drug-loading system (MSNs @ PDA-PEOz-iRGD) jointly modified by tumor-oriented penetrating peptide iRGD and pH response material PEOz is constructed by taking PDA as a reaction platform, the aims of tumor penetration, trigger release and photothermal therapy are expected to be achieved, and the MS @ Ns PDA-PEOz-iRGD system and the application thereof in the field of tumor therapy are not reported at present.
Disclosure of Invention
The invention aims to provide mesoporous silica drug-loaded nanoparticles which can play roles in tumor penetration, trigger release and photothermal therapy after being loaded with antitumor drugs.
The second purpose of the invention is to provide a preparation method of the drug-loaded nanoparticles.
The third purpose of the invention is to provide the application of the drug-loaded nanoparticles.
The first technical scheme adopted by the invention is as follows: mesoporous silica drug-loaded nanoparticles are prepared by taking mesoporous silica as a drug-loaded carrier, wrapping a polydopamine layer on the outer surface of the mesoporous silica drug-loaded nanoparticles, and connecting tumor-oriented penetrating peptide iRGD and poly (2-ethyl-2-oxazoline) through Schiff base addition reaction.
The second technical scheme adopted by the invention is as follows: a preparation method of mesoporous silica drug-loaded nanoparticles specifically comprises the following steps:
step 1, weighing CTAB, dissolving in deionized water, adding NaOH, stirring at 60-80 ℃ until CTAB is completely dissolved, slowly adding TEOS, stirring, cooling to room temperature, centrifuging, washing with deionized water and absolute ethyl alcohol, extracting and refluxing to obtain a sample, washing with deionized water and absolute ethyl alcohol, and vacuum drying to obtain a sample labeled as MSN;
step 2, respectively adding isopropanol and APTES into the MSN powder obtained in the step 1, extracting, refluxing, centrifuging, respectively cleaning for 3-5 times by using ethanol and deionized water, and vacuum drying to obtain a sample marked as MSN-NH2。
Step 3, adding Tris-HCl buffer solution and dopamine hydrochloride into MSN-NH obtained in step 22And stirring the mixture under the condition of keeping out of the sun, centrifuging the mixture to obtain a dark brown precipitate, dissolving the dark brown precipitate in deionized water, performing ultrasonic treatment, centrifuging the solution again, and performing freeze drying to obtain a sample labeled as MSN @ PDA.
Step 4, adding a Tris-HCl buffer solution containing HOOC-PEOz-OH into the MSN @ PDA powder obtained in the step 3, stirring at room temperature, centrifuging, washing with deionized water, and freeze-drying to obtain a sample which is marked as MSN @ PDA-PEOz;
and step 5, adding Tris-HCl buffer solution containing iRGD into the MSN @ PDA-PEOz powder obtained in the step 4, stirring at room temperature, centrifuging, washing with deionized water, and freeze-drying to obtain the mesoporous silica drug-loaded nanoparticle MSN @ PDA-PEOz-iRGD.
The invention adopting the second technical proposal is also characterized in that,
NaOH, CTAB, TEOS and H in step 12The molar ratio of O is 1-5: 1: 5-10: 5000-10000; stirring time after NaOH is added is 30-60 min, stirring time after TEOS is added is 2-4 h, centrifuging time is 5-10 min, centrifuging speed is 10000-15000 rpm, the times of deionized water and ethanol cleaning are 3-5 times, vacuum drying temperature is 60-85 ℃, vacuum drying time is 12-24 h, extracting agent for extraction backflow is ethanol solution containing ammonium nitrate, and the concentration of ammonium nitrate is 4-8 g/L.
In the step 2, the mass ratio of the isopropanol to the APTES to the MSN powder is 100-1000: 1: 100-500; the extraction reflux temperature is 60-85 ℃, the extraction reflux time is 12-24 h, the centrifugation time is 5-10 min, the centrifugation speed is 10000-15000 rpm, the vacuum drying temperature is 60-85 ℃, and the vacuum drying time is 12-24 h.
MSN-NH in step 32Powder, Tris-HCl buffer and dopamine hydrochlorideThe mass ratio is 1-10: 1-5: 1, the amount of Tris-HCl buffer solution is 10mmol, the pH value is 8.5, the stirring time is 12-24 hours, the stirring speed is 300-500 r/min, the speed of two times of centrifugation is 10000-15000 rpm, the time of two times of centrifugation is 10-15 minutes, the ultrasonic frequency is 20-40 kHz, the ultrasonic time is 10-20 minutes, and the freeze drying time is 24-48 hours.
In the step 4, the mass ratio of MSN @ PDA to Tris-HCl buffer solution containing HOOC-PEOz-OH is 1-10: 1, the concentration of HOOC-PEOz-OH in the Tris-HCl buffer solution is 1mg/mL, the stirring time at room temperature is 6-12 h, the centrifugation speed is 10000-15000 rpm, the centrifugation time is 10-15 min, the ultrasonic frequency is 20-40 kHz, deionized water is washed for 3-5 times, and the freeze drying time is 24-48 h.
The mass ratio of the Tris-HCl buffer solution containing iRGD to the MSN @ PDA-PEOz powder in the step 5 is 1: 1-10, and the concentration of the iRGD in the Tris-HCl buffer solution is 0.1 mg/mL; stirring time is 1-3 h, centrifugal speed is 10000-15000 rpm, centrifugal time is 10-15 min, deionized water washing is performed for 3-5 times, and freeze drying time is 24-48 h.
The third technical scheme adopted by the invention is the application of the mesoporous silica drug-loaded nanoparticles in the targeted drug delivery of anticancer drugs.
The third technical solution adopted by the present invention is further characterized in that,
the anticancer drug is any one of paclitaxel, adriamycin and docetaxel.
Adding the MSN @ PDA-PEOz-iRGD into a PBS solution of an anti-cancer drug, stirring at 37-60 ℃, washing the obtained solid for 3-5 times by distilled water, and drying in vacuum at 60-80 ℃ for 12-24 hours to obtain a drug-loaded preparation, wherein the mass ratio of the anti-cancer drug to the MSN @ PDA-PEOz-iRGD is 1: 2-10.
The preparation method has the beneficial effects that the prepared mesoporous silica drug-loaded nanoparticles (MSNs @ PDA-PEOz-iRGD) jointly modified by the tumor-oriented penetrating peptide iRGD and the pH response material PEOz, which are constructed by taking the PDA as a reaction platform, can achieve the purposes of tumor penetration, trigger release and photo-thermal treatment. The preparation method is feasible and reliable in operation, the obtained carrier has high drug loading, the drug has obvious pH responsive release, has obvious tumor cell targeting capability, has a better photothermal treatment effect, has a good in vivo and in vitro anti-tumor effect, and provides a theoretical basis for the research of a novel targeted drug delivery system.
Drawings
FIG. 1 is a transmission image/scanning electron microscope image of mesoporous silica drug-loaded nanoparticles of the invention;
FIG. 2 is a drug release curve chart of the drug-loaded MSN @ PDA-PEOz-iRGD of the present invention under different pH conditions;
FIG. 3 is a graph showing the results of the inhibition of the growth of Hela tumor cells by the drug-loaded MSN @ PDA-PEOz-iRGD of the present invention;
FIG. 4 is the quantitative analysis of the uptake of the drug-carrying system by the hepatoma carcinoma cell Bel-7402 of the present invention;
FIG. 5 is a schematic diagram of the evaluation result of the photo-thermal responsiveness of the hepatoma carcinoma cell Bel-7402 to a drug loading system.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
The invention relates to mesoporous silica drug-loaded nanoparticles, which are prepared by taking mesoporous silica as a drug-loaded carrier, wrapping a polydopamine layer on the outer surface of the mesoporous silica drug-loaded nanoparticles, and connecting tumor-oriented penetrating peptide iRGD and poly (2-ethyl-2-oxazoline) through Schiff base addition reaction.
As shown in fig. 1, which is a transmission diagram of the mesoporous silica drug-loaded nanoparticle prepared by the present invention, it can be known that the particle size of the mesoporous silica particle is about 100nm, the distribution is uniform, and clear mesoporous channels can be seen inside the mesoporous silica particle.
The invention provides a preparation method of mesoporous silica drug-loaded nanoparticles, which is implemented by the following steps:
step 1, weighing 250 mg-500 mg CTAB, dissolving in 120-200 mL deionized water solution, adding 875-1750 mu L2 mol NaOH, and violently stirring at 60-80 ℃ for 30-60 min at the stirring speed of 1000 r/min-3000 r/min. After CTAB is completely dissolved, slowly adding 1.25-2.5 mL TEOS at a rate of 0.5-1 drop/min, and continuously stirring for 2-4 h. And (3) standing and cooling the product after reaction to room temperature, centrifuging at 10000-15000 rpm for 5-10 min, and washing with deionized water and absolute ethyl alcohol for 3-5 times respectively. And finally, extracting and refluxing for 24-48 h by using an ethanol solution (4-8 g/L) containing ammonium nitrate to remove the template agent, respectively cleaning for 3-5 times by using deionized water and absolute ethyl alcohol after obtaining a sample, and drying for 12-24 h in a vacuum drying oven at the temperature of 60-85 ℃, wherein the obtained sample is marked as MSN.
Step 2, weighing 200-400 mg of MSN powder, putting the MSN powder into a 500-1000 mL round-bottom flask, adding 200-400 mL of isopropanol and 0.8-1.6 mL of APTES, stirring, extracting and refluxing for 12-24 h at 60-85 ℃, centrifuging for 5-10 min at 10000-15000 rpm, respectively washing for 3-5 times by using ethanol and deionized water, drying for 12-24 h in a vacuum drying oven at 60-85 ℃, and marking as MSN-NH2。
Step 3, weighing 100-200 mg of MSN-NH2Pouring the powder into a 150-250 mL round-bottom flask, sequentially adding 50-100 mL Tris-HCl buffer solution (10mmol, pH8.5) and 50-100 mg dopamine hydrochloride, stirring at room temperature under a dark condition for 12-24 h (300-500 r/min), centrifuging (10000-15000 rpm, 10-15 min) to obtain a dark brown precipitate, dissolving the precipitate in deionized water, performing ultrasonic treatment (20-40 kHz, 10-20 min), centrifuging again (10000-15000 rpm, 10-15 min), removing redundant PDA, and freeze-drying the obtained precipitate for 24-48 h to obtain the MSN @ PDA.
Step 4, weighing 100-200 mg of MSN @ PDA powder, adding the powder into a 100-250 mL round-bottom flask, adding 20-50 mL of Tris-HCl buffer solution (10mmol, pH8.5) containing 100-200 mg of HOOC-PEOz-OH (molecular weight 2000-10000), stirring at room temperature for 6-12 h, centrifuging (10000-15000 rpm, 10-15 min), washing with deionized water for 3-5 times, and freeze-drying the obtained precipitate for 24-48 h to obtain the MSN @ PDA-PEOz.
Step 5, weighing 100-200 mg of MSN @ PDA-PEOz powder, adding the powder into a 150-250 mL round-bottom flask, adding 50-100 mL of Tris-HCl buffer solution (8-10 mmol, pH 8.5-9.0) containing 50-100 mg of iRGD, stirring for 1-3 h at room temperature, centrifuging (10000-15000 rpm, 10-15 min), washing for 3-5 times with deionized water, and freeze-drying for 24-48 h to obtain the mesoporous silica drug-loaded nanoparticle MSN @ PDA-PEOz-iRGD.
The MSN @ PDA-PEOz-iRGD is applied to the targeted drug delivery of the anti-cancer drugs, and the specific operation is as follows: the preparation method comprises the steps of weighing an anti-cancer drug and MSN @ PDA-PEOz-iRGD according to the mass ratio of 1: 2-10, adding the MSN @ PDA-PEOz-iRGD into a PBS (phosphate buffer solution) solution (pH 7.4) of the anti-cancer drug, stirring at 37-60 ℃, washing obtained solids with distilled water for 3-5 times, and drying at 60-80 ℃ in vacuum for 12-24 hours to obtain the drug-carrying preparation.
Wherein the anticancer drug is one of paclitaxel, adriamycin and docetaxel.
Compared with the prior art, the invention has the following advantages:
(1) the invention modifies PEOz on the surface of mesoporous silicon dioxide, so that the drug release has pH responsiveness, can effectively respond to the pH value of a tumor part, and improves the effective drug concentration in a tumor cell.
(2) The invention modifies PEOz on the surface of mesoporous silicon dioxide, which can achieve hydrophilic modification and prolong the in vivo circulation time of the drug;
(3) the iRGD is modified on the drug delivery system, so that the targeting and the penetration of tumor tissues can be realized simultaneously, and the anti-tumor effect is enhanced;
(4) the method takes the PDA as a reaction platform, has simple preparation method operation, easy product acquisition and simple post-treatment, and is suitable for industrial production;
(5) the invention takes PDA as a reaction platform, and the prepared carrier has the photo-thermal treatment effect at the same time.
Example 1
A preparation method of mesoporous silica drug-loaded nanoparticles is specifically implemented according to the following steps:
step 1, Synthesis of MSN
CTAB 250mg was weighed and dissolved in 120mL deionized water, 875 μ L NaOH 2M was added, and the mixture was stirred vigorously at 60 ℃ for 30 min. After CTAB was completely dissolved, 1.25mL TEOS was slowly added and stirring was continued for 2 h. And (3) standing and cooling the product after reaction to room temperature, centrifuging for 5min at 10000rpm, washing with deionized water and ethanol for 3 times respectively, extracting and refluxing for 24 hours by using an ethanol solution (4g/L) containing ammonium nitrate to remove the template agent to obtain a sample, washing with deionized water and absolute ethanol for 3 times respectively, drying in a vacuum drying oven at 60 ℃ for 12 hours, and marking as MSN.
Step 2, MSN-NH2Synthesis of (2)
Weighing 200mg MSN powder, placing into 500mL round bottom flask, adding 200mL isopropanol and 0.8mL APTES, stirring at 60 deg.C, extracting under reflux for 12h, centrifuging at 10000rpm for 5min, washing with ethanol and deionized water for 3 times, vacuum drying at 60 deg.C for 12h, and labeling as MSN-NH2。
Step 3, synthesis of MSN @ PDA
Weighing 100mg MSN-NH2Pouring the powder into a 150mL round-bottom flask, adding 50mL Tris-HCl buffer solution (10mmol, pH8.5), adding 50mg dopamine hydrochloride, stirring at room temperature (300r/min) for 12h in a dark condition, centrifuging (10000rpm, 10min), dissolving the precipitate in deionized water, performing ultrasonic treatment (20kHz, 10min), centrifuging again (10000rpm, 10min), removing redundant PDA, and freeze-drying the obtained precipitate for 24h to obtain the MSN @ PDA.
Step 4, synthesis of MSN @ PDA-PEOz
Weighing 100mg of MSN @ PDA powder, adding the powder into a 100mL round-bottom flask, adding 20mL Tris-HCl buffer solution (10mmol, pH8.5) containing 100mg of HOOC-PEOz-OH (molecular weight 2000), stirring at room temperature for 6h, centrifuging (10000rpm, 10min), washing with deionized water for 3 times, and freeze-drying the obtained precipitate for 24h to obtain the MSN @ PDA-PEOz.
Step 5, synthesizing MSN @ PDA-PEOz-iRGD
Weighing 100mg of MSN @ PDA-PEOz powder, adding the powder into a 150mL round-bottom flask, adding 50mL of Tris-HCl buffer solution (8mmol, pH8.5) containing 50mg of iRGD, stirring at room temperature for 1h, centrifuging (10000rpm, 10min), washing with deionized water for 3 times, and freeze-drying for 24h to obtain the MSN @ PDA-PEOz-iRGD.
Example 2
A preparation method of mesoporous silica drug-loaded nanoparticles is specifically implemented according to the following steps:
step 1, Synthesis of MSN
CTAB 300mg was weighed into a solution containing 150mL deionized water, 1000. mu.L NaOH 2M was added, and the mixture was stirred vigorously at 70 ℃ for 40 min. After CTAB was completely dissolved, 1.5mL TEOS was slowly added and stirring was continued for 3 h. After the reaction, the product is kept stand and cooled to room temperature, is centrifuged at 12000rpm for 8min, and is washed 4 times by deionized water and ethanol respectively. And finally, extracting and refluxing for 36h by using an ethanol solution (5g/L) containing ammonium nitrate to remove the template agent, respectively washing the sample by using deionized water and absolute ethyl alcohol for 4 times, and drying the sample in a vacuum drying oven at 70 ℃ for 12h, wherein the sample is marked as MSN.
Step 2, MSN-NH2Synthesis of (2)
Weighing 300mg MSN powder, placing into 1000mL round bottom flask, adding 300mL isopropanol and 1.2mL APTES, stirring at 80 deg.C, extracting under reflux for 24h, centrifuging at 12000rpm for 10min, washing with ethanol and deionized water for 4 times, vacuum drying at 80 deg.C for 24h, and labeling as MSN-NH2。
Step 3, synthesis of MSN @ PDA
Weighing 120mg of MSN-NH2And pouring the powder into a 250mL round-bottom flask, adding 100mL Tris-HCl buffer (10mmol, pH8.5), adding 100mg dopamine hydrochloride, stirring at room temperature (400r/min) in a dark condition for 24h, centrifuging (12000rpm, 15min), dissolving the precipitate in deionized water, performing ultrasonic treatment (25kHz, 15min), centrifuging again (12000rpm, 12min), removing redundant PDA, and freeze-drying the obtained precipitate for 24h to obtain the MSN @ PDA.
Step 4, synthesis of MSN @ PDA-PEOz
Weighing 120mg of MSN @ PDA powder, adding the powder into a 150mL round-bottom flask, adding 30mL of Tris-HCl buffer solution (10mmol, pH8.5) containing 120mg of HOOC-PEOz-OH (molecular weight is 3000), stirring at room temperature for 8h, centrifuging (12000rpm, 12min), washing with deionized water for 4 times, and freeze-drying the obtained precipitate for 24h to obtain the MSN @ PDA-PEOz.
Step 5, synthesizing MSN @ PDA-PEOz-iRGD
Weighing 120mg of MSN @ PDA-PEOz powder, adding the powder into a 150mL round-bottom flask, adding 60mL of Tris-HCl buffer solution (10mmol, pH8.5) containing 60mg of iRGD, stirring at room temperature for 2h, centrifuging (12000rpm, 12min), washing with deionized water for 4 times, and freeze-drying for 24h to obtain the MSN @ PDA-PEOz-iRGD.
Example 3
A preparation method of mesoporous silica drug-loaded nanoparticles is specifically implemented according to the following steps:
step 1, Synthesis of MSN
CTAB 400mg was weighed into a solution containing 200mL of deionized water, 1200. mu.L of 2M NaOH was added, and the mixture was stirred vigorously at 80 ℃ for 45 min. After CTAB was completely dissolved, 2.0mL TEOS was slowly added and stirring was continued for 3 h. After the reaction, the product was left to stand and cooled to room temperature, centrifuged at 15000rpm for 10min, and washed 5 times with deionized water and ethanol, respectively. And finally, extracting and refluxing for 48 hours by using an ethanol solution (6g/L) containing ammonium nitrate to remove the template agent, respectively washing the sample by using deionized water and absolute ethyl alcohol for 5 times, and drying the sample in a vacuum drying oven at the temperature of 80 ℃ for 24 hours, wherein the mark is MSN.
Step 2, MSN-NH2Synthesis of (2)
Weighing 250mg MSN powder, placing into 500mL round bottom flask, adding 250mL isopropanol and 1.0mL APTES, stirring at 70 deg.C, extracting under reflux for 24 hr, centrifuging at 12000rpm for 8min, washing with ethanol and deionized water for 4 times, vacuum drying at 70 deg.C for 12 hr, and labeling as MSN-NH2。
Step 3, synthesis of MSN @ PDA
Weighing 150mg of MSN-NH2And pouring the powder into a 250mL round-bottom flask, adding 80mL Tris-HCl buffer (10mmol, pH8.5), adding 80mg dopamine hydrochloride, stirring at room temperature (500r/min) for 24h in a dark condition, centrifuging (12000rpm, 12min), dissolving the precipitate in deionized water, performing ultrasonic treatment (30kHz, 15min), centrifuging again (12000rpm, 15min), removing redundant PDA, and freeze-drying the obtained precipitate for 24h to obtain the MSN @ PDA.
Step 4, synthesis of MSN @ PDA-PEOz
Weighing 150mg of MSN @ PDA powder, adding the powder into a 250mL round-bottom flask, adding 30mL Tris-HCl buffer solution (10mmol, pH8.5) containing 150mg of HOOC-PEOz-OH (molecular weight is 5000), stirring at room temperature for 10h, centrifuging (12000rpm, 15min), washing with deionized water for 5 times, and freeze-drying the obtained precipitate for 36h to obtain the MSN @ PDA-PEOz.
Step 5, synthesizing MSN @ PDA-PEOz-iRGD
Weighing 150mg of MSN @ PDA-PEOz powder, adding the powder into a 250mL round-bottom flask, adding 80mL of Tris-HCl buffer (10mmol, pH8.5) containing 80mg of iRGD, stirring at room temperature for 2h, centrifuging (12000rpm, 15min), washing with deionized water for 4 times, and freeze-drying for 36h to obtain the MSN @ PDA-PEOz-iRGD.
Example 4
A preparation method of mesoporous silica drug-loaded nanoparticles is specifically implemented according to the following steps:
step 1, Synthesis of MSN
CTAB 450mg was weighed into a solution containing 200mL of deionized water, 1500. mu.L of 2M NaOH was added, and the mixture was stirred vigorously at 80 ℃ for 50 min. After CTAB was completely dissolved, 2.0mL TEOS was slowly added and stirring was continued for 4 h. After the reaction, the product was left to stand and cooled to room temperature, centrifuged at 15000rpm for 5min, and washed with deionized water and ethanol for 4 times, respectively. And finally, extracting and refluxing for 48 hours by using an ethanol solution (6g/L) containing ammonium nitrate to remove the template agent, respectively washing the sample by using deionized water and absolute ethyl alcohol for 5 times, and drying the sample in a vacuum drying oven at the temperature of 80 ℃ for 12 hours, wherein the mark is MSN.
Step 2, MSN-NH2Synthesis of (2)
Weighing 300mg MSN powder, placing into 1000mL round bottom flask, adding 300mL isopropanol and 1.2mL APTES, stirring at 80 deg.C, extracting under reflux for 24h, centrifuging at 14000rpm for 8min, washing with ethanol and deionized water for 4 times, vacuum drying at 80 deg.C for 12h, and labeling as MSN-NH2。
Step 3, synthesis of MSN @ PDA
Weighing 180mg of MSN-NH2And pouring the powder into a 250mL round-bottom flask, adding 100mL Tris-HCl buffer (10mmol, pH8.5), adding 80mg dopamine hydrochloride, stirring at room temperature (500r/min) in a dark condition for 24h, centrifuging (15000rpm, 12min), dissolving the precipitate in deionized water, performing ultrasonic treatment (40kHz, 15min), centrifuging again (12000rpm, 12min), removing redundant PDA, and freeze-drying the obtained precipitate for 24h to obtain the MSN @ PDA.
Step 4, synthesis of MSN @ PDA-PEOz
Weighing 180mg of MSN @ PDA powder, adding the powder into a 250mL round-bottom flask, adding 30mL Tris-HCl buffer solution (10mmol, pH8.5) containing 200mg of HOOC-PEOz-OH (molecular weight 8000), stirring at room temperature for 10h, centrifuging (15000rpm, 15min), washing with deionized water for 4 times, and freeze-drying the obtained precipitate for 36h to obtain the MSN @ PDA-PEOz.
Step 5, synthesizing MSN @ PDA-PEOz-iRGD
Weighing 180mg of MSN @ PDA-PEOz powder, adding the powder into a 250mL round-bottom flask, adding 100mL of Tris-HCl buffer solution (9mmol, pH8.5) containing 80mg of iRGD, stirring at room temperature for 2h, centrifuging (15000rpm, 12min), washing with deionized water for 4 times, and freeze-drying for 48h to obtain the MSN @ PDA-PEOz-iRGD.
Example 5
A preparation method of mesoporous silica drug-loaded nanoparticles is specifically implemented according to the following steps:
step 1, Synthesis of MSN
CTAB 500mg was weighed and dissolved in 200mL deionized water, 1750. mu.L NaOH 2M was added, and the mixture was stirred vigorously at 80 ℃ for 60 min. After CTAB was completely dissolved, 2.5mL TEOS was slowly added and stirring was continued for 4 h. After the reaction, the product was left to stand and cooled to room temperature, centrifuged at 15000rpm for 10min, and washed 5 times with deionized water and ethanol, respectively. And finally, extracting and refluxing for 48 hours by using an ethanol solution (8g/L) containing ammonium nitrate to remove the template agent, respectively washing the sample by using deionized water and absolute ethyl alcohol for 5 times, and drying the sample in a vacuum drying oven at the temperature of 85 ℃ for 24 hours, wherein the mark is MSN.
Step 2, MSN-NH2Synthesis of (2)
Weighing 400mg MSN powder, placing into 1000mL round bottom flask, adding 400mL isopropanol and 1.6mL APTES, stirring at 85 deg.C, extracting under reflux for 24h, centrifuging at 15000rpm for 10min, washing with ethanol and deionized water for 5 times, vacuum drying at 85 deg.C for 24h, and labeling as MSN-NH2。
Step 3, synthesis of MSN @ PDA
Weighing 200mg of MSN-NH2And pouring the powder into a 250mL round-bottom flask, adding 100mL Tris-HCl buffer (10mmol, pH8.5), adding 100mg dopamine hydrochloride, stirring at room temperature (500r/min) in a dark condition for 24h, centrifuging (15000rpm, 15min), dissolving the precipitate in deionized water, performing ultrasonic treatment (40kHz, 20min), centrifuging again (15000rpm, 15min), removing redundant PDA, and freeze-drying the obtained precipitate for 48h to obtain the MSN @ PDA.
Step 4, synthesis of MSN @ PDA-PEOz
Weighing 200mg of MSN @ PDA powder, adding the powder into a 250mL round-bottom flask, adding 50mL of Tris-HCl buffer solution (10mmol, pH8.5) containing 200mg of HOOC-PEOz-OH (molecular weight is 10000), stirring at room temperature for 12h, centrifuging (15000rpm, 15min), washing with deionized water for 5 times, and freeze-drying the obtained precipitate for 48h to obtain the MSN @ PDA-PEOz.
Step 5, synthesizing MSN @ PDA-PEOz-iRGD
Weighing 200mg of MSN @ PDA-PEOz powder, adding the powder into a 250mL round-bottom flask, adding 100mL of Tris-HCl buffer solution (10mmol, pH 9.0) containing 100mg of iRGD, stirring for 3h at room temperature, centrifuging (15000rpm, 15min), washing for 5 times by deionized water, and freeze-drying for 48h to obtain the MSN @ PDA-PEOz-iRGD.
Detecting the drug release condition of the drug-loaded DOX @ MSN-PDA-PEOz-iRGD under different pH conditions:
weighing 100mg of DOX @ MSN-PDA-PEOz-iRGD, pouring into a 100mL round bottom flask, adding 20mL of deionized water, carrying out ultrasonic treatment for 20min, adding 50mg of doxorubicin hydrochloride, stirring to dissolve the doxorubicin hydrochloride, and stirring at room temperature for 24h at 600r/min in the dark. And (3) centrifuging (14000r/min, 30min) the obtained solution, separating, washing the drug-loaded nano-composite at the lower layer with deionized water for 4 times, removing redundant doxorubicin hydrochloride, and freeze-drying the obtained precipitate to obtain drug-loaded powder.
50mg of the drug-loaded powder is precisely weighed and placed in a dialysis bag (MWCO:3500), 1mL of PBS buffer solution (pH 7.4) is added, two ends of the dialysis bag are tied, the dialysis bag is placed in 50mL centrifuge tubes respectively filled with 20mL of PBS buffer solution with pH of 5.0 and 7.4, the dialysis bag is fixed and placed in a constant temperature culture oscillator in a flat mode, the temperature is set to be 37 ℃, and the rotating speed is 150 r/min. At predetermined time points (20min, 40min, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 36h, 48h), 1mL was sampled while an equal volume of equal temperature PBS solution was added. And (3) measuring the absorbance of the sample solution at the wavelength of 480nm, calculating the DOX content, and calculating the cumulative release degree. Fig. 2 is a drug release profile of the drug-loaded formulation under different pH conditions. It can be seen that the drug release is fastest in the release medium with the pH value of 5.0, the cumulative release degrees at 2h, 4h and 12h are respectively 27.7%, 34.3%, 56.8% and 69.2% at 24h, which shows that the carrier material of the invention has pH sensitivity and the drug release is accelerated under the condition of the pH value of 5.0.
Detecting the growth inhibition rate of the drug-loaded preparation on the hepatoma carcinoma cell Bel-7402:
MTT assayThe drug-loaded preparation has the inhibition rate on hepatoma carcinoma cell Bel-7402. Bel-7402 cells 1X 104One cell/well is inoculated on a 96-well culture plate, the culture plate is placed in an incubator, after cells adhere to the wall, DOX @ MSN-PDA-PEOz and DOX @ MSN-PDA-PEOz with the final concentration of paclitaxel of 0.5, 1, 2, 4, 8 and 16 mu g/mL are respectively added, an equal volume of serum culture medium is added into a control group, the final volume of each well is 200 mu l, and each group is provided with 6 multiple wells. And after further culturing for 24h, adding 150 mu L of MTT (methyl thiazolyl tetrazolium) with the concentration of 10 mu g/mL in dark, incubating for 4h at 37 ℃, discarding the supernatant, adding 100 mu LDMSO into each hole, detecting the absorbance (A) value at the 490nm wavelength by using a microplate reader, and calculating the cell growth inhibition rate. FIG. 3 is a graph showing the results of the inhibition rate of tumor growth. It can be seen that when the drug concentration is more than 0.05 mug/mL, the inhibition rate of the DOX @ MSN-PDA-PEOz-iRGD group on cells is significantly greater than that of the DOX @ MSN-PDA-PEOz group and the DOX @ MSN group, and when the drug concentration is 0.5 mug/mL, the inhibition rate of the DOX @ MSN-PDA-PEOz group on the growth of tumor cells reaches 80%.
Detecting the uptake of Bel-7402 cells to a drug loading system:
3X 10 of Bel-7402 cells5Inoculating to 6-well culture plate at 37 deg.C and 5% CO2Culturing for 24h in a constant temperature incubator until the cells adhere to the wall. After the cells are attached to the wall, the original cell culture solution is poured out, 1mL of the drug-containing carrier is added to the experimental group, and 1mL of DMEM culture solution containing 10% fetal calf serum and 1% double antibody in volume fraction is added to the blank group. Standing at 37 deg.C for 5% CO2The incubation was continued in the incubator for 24 h. After 24h of administration and culture, pouring out the culture solution in a 12-hole plate in a clean bench, washing the cells for 3-5 times by PBS (phosphate buffer solution) to absorb the medicine which does not enter the cells, then adding 4% paraformaldehyde solution into each hole by 1mL, then putting the cells into an incubator, culturing for 30min, then sucking out the paraformaldehyde solution in the clean bench, washing the cells for 3-5 times by PBS, adding 0.4mL of 0.25% trypsin digestion solution into each hole, incubating for 5min in the incubator at 37 ℃, adding 0.6mL of serum-containing culture solution into each hole after the cells are digested into a spherical shape, stopping digestion, blowing into cell suspension, and transferring into a 1.5mL centrifuge tube by a pipette. The cell suspension was centrifuged for 5min at 1500 rpm. Removing supernatant after centrifugation, adding 1ml of precooled PBS, blowing to obtain cell suspension, centrifuging, and rinsingThe process was repeated three times. Blowing and resuspending the finally obtained cell mass by 0.5ml precooled PBS, transferring the cell mass into a flow tube through a 300-mesh cell sieve, measuring the fluorescence intensity of intracellular DOX by using a flow cytometer, comparing the uptake condition of the cells to each drug-loaded preparation, and as shown in figure 4, the cell mass is a schematic diagram of the quantitative analysis result of the liver cancer cell Bel-7402 on the uptake of the drug-loaded system, wherein the number of the cells used in each analysis is not less than 1 multiplied by 105The number of collected cells was 10000. As can be seen from FIG. 3, the DOX @ MSN-PDA-PEOz-iRGD group had the highest cellular uptake, with fluorescence intensities of 1.27 and 1.73 times that of DOX @ MSN and DOX @ MSN-PDA-PEOz, respectively.
Evaluation of photo-thermal responsiveness:
bel-7402 cells were incubated with DOX @ MSN, DOX @ MSN-PDA-PEOz-iRGD for 4 hours, washed with PBS, and then incubated at 1.5W/cm2The solution is exposed to 808nm laser for 5 minutes, the temperature of the solution is recorded by an infrared thermal imager, the recording result is shown in figure 5, and the temperature of DOX @ MSN-PDA-PEOz and DOX @ MSN-PDA-PEOz-iRGD is obviously higher than that of the DOX @ MSN group from figure 5, which indicates that the preparation has obvious photothermal effect.

Claims (10)

1. A mesoporous silica drug-loaded nanoparticle is characterized in that mesoporous silica is used as a drug-loaded carrier, a poly-dopamine layer is wrapped outside the mesoporous silica, and a tumor-oriented penetration peptide iRGD and poly (2-ethyl-2-oxazoline) are connected through Schiff base addition reaction to obtain a mesoporous silica drug-loaded nanoparticle MSN @ PDA-PEOz-iRGD;
the preparation method of the mesoporous silica drug-loaded nanoparticles is specifically implemented by the following steps:
step 1, weighing CTAB, dissolving in deionized water, adding NaOH, violently stirring at 60-80 ℃ until CTAB is completely dissolved, slowly adding TEOS, stirring, cooling to room temperature, centrifuging, washing with deionized water and absolute ethyl alcohol, extracting and refluxing to obtain a sample, washing with deionized water and absolute ethyl alcohol, and vacuum drying to obtain a sample labeled as MSN;
step 2, respectively adding isopropanol and APTES into the MSN powder obtained in step 1, extracting, refluxing, centrifuging, and extracting with ethanolAnd respectively cleaning with deionized water for 3-5 times, and vacuum drying to obtain a sample marked as MSN-NH2；
Step 3, adding Tris-HCl buffer solution and dopamine hydrochloride into MSN-NH obtained in step 22Stirring in a dark condition, centrifuging to obtain a dark brown precipitate, dissolving the dark brown precipitate in deionized water, performing ultrasonic treatment, centrifuging again, and freeze-drying to obtain a sample labeled as MSN @ PDA;
step 4, adding a Tris-HCl buffer solution containing HOOC-PEOz-OH into the MSN @ PDA powder obtained in the step 3, stirring at room temperature, centrifuging, washing with deionized water, and freeze-drying to obtain a sample which is marked as MSN @ PDA-PEOz;
and 5, adding an iRGD-containing Tris-HCl buffer solution into the MSN @ PDA-PEOz powder obtained in the step 4, stirring at room temperature, centrifuging, washing with deionized water, and freeze-drying to obtain the mesoporous silica drug-loaded nanoparticle MSN @ PDA-PEOz-iRGD.
2. The preparation method of the mesoporous silica drug-loaded nanoparticle of claim 1, which is specifically implemented by the following steps:
step 1, weighing CTAB, dissolving in deionized water, adding NaOH, violently stirring at 60-80 ℃ until CTAB is completely dissolved, slowly adding TEOS, stirring, cooling to room temperature, centrifuging, washing with deionized water and absolute ethyl alcohol, extracting and refluxing to obtain a sample, washing with deionized water and absolute ethyl alcohol, and vacuum drying to obtain a sample labeled as MSN;
step 2, respectively adding isopropanol and APTES into the MSN powder obtained in the step 1, extracting, refluxing, centrifuging, respectively cleaning for 3-5 times by using ethanol and deionized water, and vacuum drying to obtain a sample marked as MSN-NH2；
Step 3, adding Tris-HCl buffer solution and dopamine hydrochloride into MSN-NH obtained in step 22Stirring in a dark condition, centrifuging to obtain a dark brown precipitate, dissolving the dark brown precipitate in deionized water, performing ultrasonic treatment, centrifuging again, and freeze-drying to obtain a sample labeled as MSN @ PDA;
step 4, adding a Tris-HCl buffer solution containing HOOC-PEOz-OH into the MSN @ PDA powder obtained in the step 3, stirring at room temperature, centrifuging, washing with deionized water, and freeze-drying to obtain a sample which is marked as MSN @ PDA-PEOz;
and 5, adding an iRGD-containing Tris-HCl buffer solution into the MSN @ PDA-PEOz powder obtained in the step 4, stirring at room temperature, centrifuging, washing with deionized water, and freeze-drying to obtain the mesoporous silica drug-loaded nanoparticle MSN @ PDA-PEOz-iRGD.
3. The preparation method of the mesoporous silica drug-loaded nanoparticles as claimed in claim 2, wherein NaOH, CTAB, TEOS and H are added in step 12The molar ratio of O is 1-5: 1: 5-10: 5000-10000; stirring time after NaOH is added is 30-60 min, stirring speed is 1000-3000 r/min, TEOS adding speed is 0.5-1 drop/min, stirring time after TEOS is added is 2-4 h, centrifuging time is 5-10 min, centrifuging speed is 10000-15000 rpm, the times of washing with deionized water and ethanol for two times are 3-5 times, vacuum drying temperature is 60-85 ℃, vacuum drying time is 12-24 h, extracting and refluxing extractant is ethanol solution containing ammonium nitrate, time is 24-48 h along with removal, and concentration of ammonium nitrate in the ethanol solution is 4-8 g/L.
4. The preparation method of the mesoporous silica drug-loaded nanoparticle as claimed in claim 2, wherein the mass ratio of the isopropanol, the APTES and the MSN powder in the step 2 is 100-1000: 1: 100-500; the extraction reflux temperature is 60-85 ℃, the extraction reflux time is 12-24 h, the centrifugation time is 5-10 min, the centrifugation speed is 10000-15000 rpm, the vacuum drying temperature is 60-85 ℃, and the vacuum drying time is 12-24 h.
5. The preparation method of the mesoporous silica drug-loaded nanoparticles of claim 2, wherein the MSN-NH in the step 32The mass ratio of the powder to the Tris-HCl buffer solution to the dopamine hydrochloride is 1-10: 1-5: 1, the stirring time is 12-24 hours, the stirring speed is 300-500 r/min, and the two centrifugation speeds are both10000-15000 rpm, 10-15 min of two-time centrifugation, 20-40 kHz of ultrasonic frequency, 10-20 min of ultrasonic time and 24-48 h of freeze drying time.
6. The preparation method of the mesoporous silica drug-loaded nanoparticle as claimed in claim 2, wherein in step 4, the mass ratio of MSN @ PDA to Tris-HCl buffer solution containing HOOC-PEOz-OH is 1-10: 1, the concentration of HOOC-PEOz-OH in Tris-HCl buffer solution is 1mg/mL, the stirring time at room temperature is 6-12 h, the centrifugation rate is 10000-15000 rpm, the centrifugation time is 10-15 min, the ultrasound frequency is 20-40 kHz, deionized water is washed for 3-5 times, and the freeze-drying time is 24-48 h.
7. The preparation method of the mesoporous silica drug-loaded nanoparticles as claimed in claim 2, wherein the mass ratio of Tris-HCl buffer solution containing iRGD to MSN @ PDA-PEOz powder in the step 5 is 1: 1-10, and the concentration of iRGD in Tris-HCl buffer solution is 0.1 mg/mL; stirring time is 1-3 h, centrifugal speed is 10000-15000 rpm, centrifugal time is 10-15 min, deionized water washing is performed for 3-5 times, and freeze drying time is 24-48 h.
8. The application of the mesoporous silica drug-loaded nanoparticle of claim 1 in preparing targeted drug delivery anticancer drugs.
9. The application of the mesoporous silica drug-loaded nanoparticle in preparing targeted anticancer drugs according to claim 8, wherein the anticancer drug is any one of paclitaxel, doxorubicin and docetaxel.
10. The application of the mesoporous silica drug-loaded nanoparticles in the preparation of targeted anticancer drugs according to claim 9, wherein MSN @ PDA-PEOz-iRGD is added into PBS solution of the anticancer drugs, stirring is carried out at 37-60 ℃, the obtained solid is washed by distilled water for 3-5 times, vacuum drying is carried out at 60-80 ℃ for 12-24 hours, and a drug-loaded preparation is obtained, wherein the mass ratio of the anticancer drugs to the MSN @ PDA-PEOz-iRGD is 1: 2-10.
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