CN112535739B - Nanoparticle for improving gene transfection efficiency based on tumor self microenvironment and preparation method and application thereof - Google Patents

Nanoparticle for improving gene transfection efficiency based on tumor self microenvironment and preparation method and application thereof Download PDF

Info

Publication number
CN112535739B
CN112535739B CN202011425940.7A CN202011425940A CN112535739B CN 112535739 B CN112535739 B CN 112535739B CN 202011425940 A CN202011425940 A CN 202011425940A CN 112535739 B CN112535739 B CN 112535739B
Authority
CN
China
Prior art keywords
mesoporous silicon
mmsns
nano particles
silicon nano
metal ion
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202011425940.7A
Other languages
Chinese (zh)
Other versions
CN112535739A (en
Inventor
吴玫颖
梅林�
周韵
于冕
曾伟伟
陈婷
郝慧松
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sun Yat Sen University
Sun Yat Sen University Shenzhen Campus
Original Assignee
Sun Yat Sen University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sun Yat Sen University filed Critical Sun Yat Sen University
Priority to CN202011425940.7A priority Critical patent/CN112535739B/en
Publication of CN112535739A publication Critical patent/CN112535739A/en
Application granted granted Critical
Publication of CN112535739B publication Critical patent/CN112535739B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/32—Manganese; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/34—Copper; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B33/00—Silicon; Compounds thereof
    • C01B33/02—Silicon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03004—Glucose oxidase (1.1.3.4)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Inorganic Chemistry (AREA)
  • Nanotechnology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medical Informatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention discloses a nanoparticle for improving gene transfection efficiency based on tumor self microenvironment, which is prepared by firstly chemically synthesizing mesoporous silicon nanoparticle by a sol-gel method, then doping metal ions, carrying out surface modification on the metal ion mesoporous silicon nanoparticle by polyethyleneimine to obtain MMSNs-P, and finally mixing the MMSNs-P with glucose oxidase and nucleic acid medicine to obtain GR@MMSNs-P, namely the nanoparticle for improving gene transfection efficiency based on tumor self microenvironment. The nanoparticle overcomes the defect of shallow laser penetration depth in photochemical transfection technology, combines starvation/gene therapy, improves gene transfection efficiency by utilizing tumor microenvironment, and provides a new idea for tumor treatment.

Description

Nanoparticle for improving gene transfection efficiency based on tumor self microenvironment and preparation method and application thereof
Technical Field
The invention relates to the field of biological medicine, in particular to a nanoparticle for improving gene transfection efficiency based on tumor self microenvironment, and a preparation method and application thereof.
Background
The RNA interference (RNAi) technology can inhibit the expression of specific genes to exert gene therapy effect, has the advantages of high specificity, high safety, high silencing efficiency, stable action and the like, and has attractive application prospect in tumor therapy. Research shows that the nano delivery system (DDS) can protect genes from degradation, improve the internal circulation time, enhance the uptake of cancer cells to genes and improve the gene transfection efficiency. However, exogenous RNA is easy to be trapped in lysosomes after entering cells, and is difficult to enter cytoplasm to play a role to influence transfection effect, so that the escape of the lysosomes of RNA is a great difficulty to be solved by research.
The photochemical transfection technology provides a new idea for realizing lysosome escape of RNA, and the action mechanism is as follows: under the excitation of laser, the photosensitizer generates Reactive Oxygen Species (ROS), damages the lysosome membrane and promotes the lysosome escape of genes. For example, patent CN108524946a discloses that the included photosensitizer can generate Reactive Oxygen Species (ROS) under the control of exogenous red light, so as to realize photochemical internalization (PCI) -mediated lysosome escape and photo-promoted polymer degradation, and finally achieve the purposes of reducing toxic and side effects of materials, improving transfection efficiency and enhancing tumor gene therapy effect. However, since the laser penetration depth is shallow, it is difficult to treat deep tumors, the technique is greatly limited, and additional photosensitizers are required. Emerging chemokinetic therapies are the use of Fenton (Fenton) or Fenton-like reactions to catalyze the overexpression of H in tumor tissue 2 O 2 In situ generation of OH (a type of ROS), which in turn causes mitochondrial destruction, DNA strand breaks, protein or lipid damage, and apoptosis of tumor cells to treat tumors. The OH produced by the therapy is expected to play a role similar to PCI, promote the lysosome escape of genes, and overcome the defect of shallow penetration depth of PCI-stimulated light tissues. However, this technique for promoting RNA transfection using OH generated by Fenton or Fenton-like reaction has not been reported yet. Furthermore, H in tumor microenvironment 2 O 2 Limited, it is difficult to generate enough OH by Fenton reaction, and also limitedIts use in gene transfection.
Disclosure of Invention
The first object of the present invention is to overcome the disadvantages of the prior art, such as low transfection efficiency in gene therapy, low penetration depth of PCI-excited light tissue and H in tumor microenvironment 2 O 2 The limited defect is that the nanoparticle for improving the gene transfection efficiency based on the tumor self microenvironment is provided.
The second object of the present invention is to provide a method for preparing the nanoparticle for improving gene transfection efficiency based on the tumor self microenvironment.
The third object of the present invention is to provide the application of the nanoparticle for improving gene transfection efficiency based on the tumor self microenvironment.
The above object of the present invention is achieved by the following technical solutions:
a nanoparticle for improving gene transfection efficiency based on tumor self microenvironment comprises mesoporous silicon nanoparticle (MMSNs-P) with surface modified with polyethylenimine doped with metal ion, nucleic acid medicine entrapped in the mesoporous silicon nanoparticle, and glucose oxidase (GOx).
In the nano-particle for improving gene transfection efficiency based on tumor self microenvironment, the mesoporous silicon nano-particle with the surface modified with the metal ion doped with polyethyleneimine is used as a carrier of a nucleic acid drug and GOx, the metal ion is used for participating in Fenton reaction, PEI is a transfection reagent, and GOx is used for improving H in tumors 2 O 2 Concentration of reagent. The nano particles can fully utilize tumor microenvironment to perform in-situ catalysis to improve gene transfection efficiency, and particularly, after the nano particles enter a body through intravenous injection and are accumulated at tumor positions, the nano particles can be degraded in an acidic tumor microenvironment without laser irradiation, so that GOx is released to consume glucose, the acidity is enhanced, and intracellular H is improved 2 O 2 Concentration, utilization of metal ions with Fenton catalytic activity H 2 O 2 In situ activation of Fenton reaction to generate OH promotes transfection of nucleic acid medicine, and realizes 'starvation'/gene cooperative treatment of tumor.
Preferably, the particle size of the nanoparticle is 50 to 400nm.
Preferably, the metal is one or more of Fe, cu, or Mn.
Preferably, the nucleic acid agent is DNA, RNA, more preferably small interfering RNA (siRNA).
Preferably, the mass ratio of the mesoporous silicon nanoparticle with the surface modified by the polyethyleneimine and doped with metal ions to GOx to the nucleic acid medicine is 15-200: 1-40: 1 to 4 percent of the total weight of the composite,
more preferably, the mass ratio of the mesoporous silicon nanoparticle with the surface modified by the polyethyleneimine and doped with metal ions to GOx to the nucleic acid medicine is 15-120: 1-30: 1 to 4.
The invention also provides a preparation method of the nano-particles, which comprises the following steps:
s1, preparing mesoporous silicon nano particles by a sol-gel method, then reacting with a metal ion compound under an alkaline condition, and obtaining metal ion doped mesoporous silicon nano particles (MMSNs) by a high-temperature hydrothermal method;
s2, performing amination modification on the MMSNs obtained in the step S1, performing carboxylation modification to obtain carboxylation metal ion doped mesoporous silicon nanoparticles, dropwise adding PEI ethanol solution, and performing coupling reaction on PEI and the surface of the carboxylation metal ion doped mesoporous silicon nanoparticles to obtain MMSNs-P;
s3, uniformly stirring GOx and MMSNs-P obtained in the step S2 to obtain GOx-loaded nano particles, then adding nucleic acid medicines, uniformly mixing, and standing to obtain the nano-particles.
Preferably, the mass ratio of the mesoporous silicon nano particles, the metal ion compound and the alkaline substance in the step S1 is 5-50:8-30:10-30.
More preferably, the metal ion compound in the step S1 is Fe-containing 2+ 、Cu 2+ 、Mn 2+ The alkaline substance is disodium maleate or urea.
Preferably, the amination-modifying reagent of step S2 is 3-aminopropyl triethoxysilane (APTES).
Preferably, the agent for carboxylation modification in step S2 is succinic acid.
Preferably, in the step S2, the mass ratio of the MMSNs, the amination modification reagent to the carboxylation modification reagent is 1 to 3:8 ～ 30 ～ 2 ～ 7.
As a preferred embodiment, the method for preparing the nanoparticle comprises the steps of:
(1) Mixing and stirring hexadecyl trimethyl ammonium chloride, ultrapure water and triethanolamine, preheating to 45-95 ℃, slowly adding tetraethyl orthosilicate, stirring at 45-95 ℃ for reaction for 3-6 hours to obtain mesoporous silicon nano particles, and extracting with a methanol solution of sodium chloride to remove a surfactant; and mixing mesoporous silicon nano particles with metal ions and alkaline substances in an aqueous solution, and performing hydrothermal reaction to obtain the MMSNs.
(2) Adding APTES into the ethanol mixture of MMSNs, and heating and refluxing to obtain aminated MMSNs (MMSNs-NH) 2 ) The method comprises the steps of carrying out a first treatment on the surface of the MMSNs-NH 2 Mixing and reacting the ethanol solution of (2) with succinic acid, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (EDCI) and 1-Hydroxybenzotriazole (HOBT) to obtain carboxylated MMSNs (MMSNs-COOH); EDCI and HOBT are added into ethanol solution of MMSNs-COOH, then the mixture is dripped into PEI ethanol solution, and the mixture is stirred for reaction to obtain MMSNs-P.
(3) Dispersing MMSNs-P in water, mixing the water with GOx, stirring uniformly to obtain G@MMSNs-P, adding siRNA under vortex condition to uniformly mix, and standing to obtain GR@MMSNs-P, namely the nano-particles for improving gene transfection efficiency based on tumor self microenvironment.
Preferably, the amount of cetyltrimethylammonium chloride in step (1) is 1 to 6g, the amount of triethanolamine is 0.01 to 0.10g, and the volume of water is 10 to 30mL.
Preferably, in the step (1), the volume of the tetraethoxysilane is 0.5-5 mL, the concentration of the methanol solution of the extractant sodium chloride is 5-15 mg/mL, and the dosage is 100-200 mL.
Preferably, the high temperature hydrothermal reaction condition in the step (1) is 150-200 ℃ and the reaction time is 24-72 h.
Preferably, in the step (2), the MMSNs are used in an amount of 50-150 mg, the APTES is used in an amount of 0.2-5 mL, the reflux temperature is 60-85 ℃ and the time is 8-15 h.
Preferably, the MMSNs-NH of step (2) 2 The mass ratio of succinic acid, EDCI and HOBT is 1: 2-3: 2 to 4: 2-4, and the stirring time is 10-15 h.
Preferably, in the step (2), the mass ratio of the MMSNs-COOH, PEI, EDCI to the HOBT is 1.0-2.5: 1.0 to 2.5:1.0 to 3:1.0 to 3, and the stirring time is 24 to 48 hours.
Preferably, in the step (3), the mass ratio of the MMSNs-P, GOx to the siRNA is 15-120: 1-30: 1 to 4, stirring for 12 to 24 hours, swirling for 20 to 50 seconds, and standing for 0.5 to 2 hours.
The invention also provides application of the nanoparticle for improving gene transfection efficiency based on the tumor self microenvironment in preparation of a medicament for treating tumors.
Compared with the prior art, the invention has the following advantages:
the invention provides a nanoparticle for improving gene transfection efficiency based on tumor self microenvironment, which has controllable particle size, good biocompatibility and biodegradability, can utilize tumor microenvironment to perform in-situ catalysis, consume glucose in the tumor microenvironment and promote H in tumor 2 O 2 Concentration, in-situ generation of ROS, promotion of lysosome escape of RNA, and overcoming of H in laser penetration depth and tumor microenvironment in PCI technology 2 O 2 The limited problems are solved, the gene transfection efficiency is improved to achieve the effect of treating tumors, and the systemic toxic and side effects can be reduced at the same time, so that the 'hunger'/gene cooperative treatment of tumors is realized.
Drawings
Fig. 1a and b are projection electron microscope (TEM) images of MMSNs obtained in example 1 of the present invention. Fig. 1c is an element mapping diagram of MMSNs.
FIG. 2 is an X-ray photoelectron spectroscopy (XPS) chart of the MMSNs obtained in example 1 of the present invention.
Fig. 3 is a TEM image corresponding to the degradability experiment of mmss in example 2 of the present invention.
FIG. 4 shows the compositions MMSNs, MMSNs-NH according to example 3 of the present invention 2 Particle size potential of MMSNs-COOH, MMSNs-PA drawing.
FIG. 5 shows the gel blocking assay results of example 4 of the present invention, lanes 1-7, lane 1: naked siRNA; lanes 2-7: the mass ratio (G@MMSNs-P/siRNA) of GR@MMSNs-P is 1, 5, 10, 15, 20 and 30.
FIG. 6 shows the results of RNase protection assay according to example 5 of the present invention, lanes 1-8, lane 1: naked siRNA; lane 2: bare sirna+rnase a enzyme; lanes 3-4: GR@MMSNs-P with mass ratio of 15 and 30; lanes 5-6: gr@mmsns-P (mass ratio 15, 30) +heparin sodium; lanes 7-8: GR@MMSNs-P (mass ratio 15, 30) +RNase A enzyme+heparin sodium.
FIG. 7 shows the toxicity results of GR@MMSNs-P on NP69 cells at various concentrations.
FIG. 8 shows the proliferative effects of MSNs, MMSNs, G@MMSNs-P, R@MMSNs-P, and GR@MMSNs-P on 4T1 cells.
FIG. 9 is the effect of MSNs, MMSNs, and G@MMSNs-P on intracellular ROS production.
FIG. 10 is the effect of GR@MSNs-P, GR@MMSNs-P on lysosomal escape.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 preparation method of manganese-doped mesoporous silicon nanoparticles (MMSNs)
Taking 2g of cetyl trimethyl ammonium chloride and 0.02g of triethanolamine in 18mL of water, stirring for 20min at 80 ℃, slowly and dropwise adding 1.5mL of tetraethyl orthosilicate, continuously stirring at 80 ℃ for reaction for 4h, centrifugally collecting precipitate, washing twice with water and ethanol, adding 100mL of sodium chloride in methanol (8 mg/mL) for extraction three times to remove surfactant, and centrifugally washing with water to collect a product; 320mg of the product was dissolved in 10mL of water, to which 160mg of MnSO was added 4 ·H 2 O and 200mg of disodium maleate are stirred uniformly at room temperature and then transferred into a hydrothermal kettle at 160 DEG CAnd reacting for 48 hours to obtain the MMSNs. After MMSNs were diluted by a certain multiple, the morphology was observed with a transmission electron microscope and elemental analysis was performed. From figures 1a and b, it can be seen that the nanoparticle has obvious spherical and porous structures, uniform particle size and about 100 nm. The mapping graph of the element c indicates that the MMSNs contain Si, O and Mn elements. As shown in fig. 2, the XPS map of MMSNs indicates that Mn element exists, and three valence states (Mn 2+ 、Mn 3+ 、Mn 4+ )。
EXAMPLE 2 degradation experiments of manganese-doped mesoporous silicon nanoparticles (MMSNs)
10mg of MMSNs were dispersed in Simulated Body Fluids (SBF) treated under different conditions (pH 7.4 or 5.5, glutathione, i.e. GSH concentration 0 or 10 mM) with stirring in a water bath at 37 ℃. At 12h,24h,72h, 1mL of the pellet was centrifuged and the pellet was resuspended in ethanol and observed by TEM. As shown in fig. 3, MMSNs degraded well when mimicking tumor microenvironment (acidic and high glutathione concentrations), substantially completely degrading in 72 hours.
Example 3 preparation of nanoparticles (GR@MMSNs-P)
(1) Dissolving 50mg of MMSNs in 100mL of ethanol, heating to 78 ℃, adding 0.5mL of APTES, and refluxing for 10 hours to obtain MMSNs-NH 2 . 100mg of succinic acid, 130mg of EDCI and 130mg of HOBT are dissolved in 10mL of absolute ethanol, and 50mg of MMSNs-NH are added 2 After 30min of ultrasonic treatment, stirring for 12h at room temperature to obtain MMSNs-COOH. 100mg of MMSNs-COOH were dissolved in 5mL of absolute ethanol, 130mg of EDCI and 130mg of HOBT were added, and the mixture was sonicated for 30min. 120mg PEI was weighed and 2mL absolute ethanol was added. And (3) dropwise adding the nanoparticle solution into the PEI solution under the stirring condition, and stirring at room temperature for 2d after ultrasonic treatment to obtain the MMSNs-P. As shown in FIG. 4, the MMSNs, MMSNs-NH during synthesis are determined 2 The successful synthesis of MMSNs-P was evident from the variation of the particle size (FIG. 4 a) and the potential (FIG. 4 b) of MMSNs-COOH and MMSNs-P.
(2) 10mg of MMSNs-P is dispersed in 8mL of water, GOx aqueous solution (1 mg/mL,2 mL) is added, stirred and incubated for 24h at room temperature, and the mixture is centrifuged and washed with water to obtain G@MMSNs-P. G@MMSNs-P (1 mg/mL, 200. Mu.L) was vortexed on a vortexing apparatus, siRNA (0.2 mg/mL, 50. Mu.L) was slowly added, and after vortexing for 30s, the mixture was allowed to stand at room temperature for 30min to obtain GR@MMSNs-P.
EXAMPLE 4 gel blocking experiment
50 mu LsiRNA (0.2 mg/mL) aqueous solution is taken, slowly dripped into 200 mu L G@MMSNs-P solution (0.05, 0.25, 0.50, 0.75, 1.0 and 1.5 mg/mL) with different concentrations under vortex condition, and after vortex for 30 seconds, GR@MMSNs-P with different mass ratios is obtained after room temperature incubation for 30 minutes. Bare siRNAs were used as control groups, and after mixing with a loading buffer, they were subjected to electrophoresis on a 1% agarose gel at 80V for 20 minutes in Tris-Acetate-EDTA (TAE) buffer. As shown in FIG. 5, the carrier can encapsulate siRNA when the mass ratio of G@MMSNs-P to siRNA is more than 15.
EXAMPLE 5 RNase protection assay
To demonstrate that negatively charged heparin sodium can promote siRNA release by electrostatic interaction, 10. Mu.L of GR@MMSNs-P complex (containing 0.4. Mu.g siRNA) with a mass ratio of 15, 30 was added to 5. Mu.L of 150mg/mL heparin sodium solution and incubated at 50℃for 4h. 10. Mu.LsiRNA (0.4. Mu.g) and GR@MMSNs-P complex (containing 0.4. Mu.g siRNA) with a mass ratio of 15 and 30 were incubated with 40ng RNase A enzyme at 37℃for 10 minutes, respectively. The RNase A enzyme is inhibited by adding an RNase enzyme inhibitor. Then, heparin sodium solution was added for the same incubation operation. The eight components of bare siRNA, bare siRNA+RNase A enzyme, GR@MMSNs-P (mass ratio 15, 30) +heparin sodium, GR@MMSNs-P (mass ratio 15, 30) +RNase A enzyme+heparin sodium were subjected to 1% agarose gel electrophoresis experiments. As shown in FIG. 6, heparin sodium can promote the release of siRNA in the complex ( lanes 5, 6 have bands run out), bare siRNA is degraded by RNase A enzyme, no band (lane 2), G@MMSNs-P can protect siRNA from RNase A enzyme degradation, and siRNA can be released by negatively charged heparin sodium (lanes 7, 8).
EXAMPLE 6 cytotoxicity assay
(1) Toxicity to Normal cells (human nasopharyngeal epithelial cells)
The cell model adopts human nasopharyngeal epithelial cells (NP 69 cells), and the nucleic acid medicine adopts Twist siRNA capable of inhibiting cancer cell metastasis. Different concentrations of GR@MMSNs-P were incubated with NP69 cells for 48h, the control group was non-medicated cells, and CCK8 was assayed for cytotoxicity, as seen in FIG. 7, the nanoparticles GR@MMSNs-P were essentially non-toxic to normal cells.
(2) Toxicity to 4T1 breast cancer cells
The cell model adopts 4T1 breast cancer cells, and the nucleic acid medicine adopts Twist siRNA capable of inhibiting cancer cell metastasis. Different concentrations of MSNs (without manganese-doped mesoporous silicon), MMSNs, G@MMSNs-P, R@MMSNs-P, GR@MMSNs-P and 4T1 cells were incubated for 48h, the control group was a non-medicated cell, and cytotoxicity was measured by CCK8, as shown in FIG. 8, the nanoparticles GR@MMSNs-P were able to effectively inhibit proliferation of 4T1 breast cancer cells due to "starvation"/gene cooperative therapy effect as the dosing concentration was increased.
Example 7 intracellular ROS production assay
4T1 cells were inoculated into a laser confocal dish and cultured for 24h, with fresh complete culture broth being replaced. To which MSNs, MMSNs, MMSNs +H is added 2 O 2 (H 2 O 2 Concentration 100. Mu.M), G@MMSNs-P, which were set at 40. Mu.g/mL based on MMSNs concentration, a blank control group was additionally set, G@MMSNs-P group incubated with cells in low sugar medium was set, after 6h incubation, the medium containing the drug was removed, washed twice with PBS, 0.5mL 10. Mu.M 2, 7-dichlorofluorescein diacetate (DCFH-DA) working solution was added, and incubated in 37℃cell incubator for 30min in the absence of light, and the probe was loaded, and the greater the intracellular ROS production and the more pronounced the fluorescent signal were observed by CLSM. As can be seen from FIG. 9, manganese-doped mesoporous silica MMSNs are capable of generating ROS and H within cells 2 O 2 The addition of (2) increases the amount of ROS produced in the cell; glucose oxidase-loaded G@MMSNs-P has a stronger intracellular ROS-producing capacity than MMSNs, and its ROS-producing capacity is dependent on glucose concentration, and ROS production is greatly reduced in the absence of glucose.
Example 8 evaluation of lysosomal escape ability (lysosomal co-localization experiment)
The fluorescence labeled siRNA (FAM-siRNA) is used for replacing the siRNA, and the co-delivery nanoparticles GR@MSNs-P and GR@MMSNs-P (the former is not doped with manganese, and the latter is doped with manganese) loaded with the FAM-siRNA are prepared. 4T1 cells were inoculated into a laser confocal dish and cultured for 24h, with fresh complete culture broth being replaced. GR@MSNs-P and GR@MMSNs-P were added thereto to give a final FAM-siRNA concentration of 200pmol/mL, and incubated for 3 and 12 hours. Cell nuclei and lysosomes were labeled with Hoechst33342 and Lyso Tracker Red dyes, respectively, and CLSM was observed for photographs. As can be seen from fig. 10, in the same case, the manganese-doped gr@mmsns-P had a stronger lysosomal escape ability (small overlap of FAM-labeled siRNA and lysosome fluorescence) over time compared to the manganese-free system (gr@msns-P).

Claims (4)

1. The nano particle for improving gene transfection efficiency based on the tumor self microenvironment is characterized by comprising mesoporous silicon nano particles with the surfaces modified with polyethyleneimine and doped with metal ions, glucose oxidase entrapped in the mesoporous silicon nano particles and nucleic acid drugs;
the mass ratio of the mesoporous silicon nano particles with the surfaces modified with the polyethyleneimine and doped with metal ions, glucose oxidase and nucleic acid medicine is 15-200: 1-40: 1-4, wherein the mass ratio of the mesoporous silicon nano particles with the surfaces modified with polyethyleneimine, doped metal ions and coated with glucose oxidase to the nucleic acid medicine is more than 15;
the metal is Mn;
the nucleic acid drug is small interfering RNA.
2. The nanoparticle according to claim 1, wherein the nanoparticle has a particle size of 50 to 400nm.
3. A method of preparing nanoparticles according to claim 1 or 2, comprising the steps of:
s1, preparing mesoporous silicon nano particles by a sol-gel method, then reacting with a metal ion compound under an alkaline condition, and obtaining the metal ion doped mesoporous silicon nano particles by a high-temperature hydrothermal method; the metal ion compound contains Mn 2+ A compound;
s2, carrying out amination modification on the metal ion doped mesoporous silicon nano particles obtained in the step S1, then carrying out carboxylation modification to obtain carboxylated metal ion doped mesoporous silicon nano particles, then dropwise adding the carboxylated metal ion doped mesoporous silicon nano particles into a polyethyleneimine ethanol solution, and carrying out coupling reaction on polyethyleneimine and the surface of the carboxylated metal ion doped mesoporous silicon nano particles to obtain polyethyleneimine modified metal ion doped mesoporous silicon nano particles;
s3, uniformly stirring glucose oxidase and the polyethyleneimine modified metal ion doped mesoporous silicon nano particles obtained in the step S2, and then adding nucleic acid medicines and uniformly mixing to obtain the nano-particles;
in the step S1, the mass ratio of the mesoporous silicon nano particles, the metal ion compound and the alkaline substance is 5-50: 8-30: 10 to 30 percent; the alkaline substance in the step S1 is disodium maleate or urea; the mass ratio of the carboxylated metal ion doped mesoporous silicon nano particles to the polyethyleneimine in the step S2 is 3-20: 10 to 20.
4. Use of the nanoparticle for improving gene transfection efficiency based on tumor self microenvironment according to claim 1 or 2 in the preparation of a medicament for treating tumor.
CN202011425940.7A 2020-12-08 2020-12-08 Nanoparticle for improving gene transfection efficiency based on tumor self microenvironment and preparation method and application thereof Active CN112535739B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202011425940.7A CN112535739B (en) 2020-12-08 2020-12-08 Nanoparticle for improving gene transfection efficiency based on tumor self microenvironment and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011425940.7A CN112535739B (en) 2020-12-08 2020-12-08 Nanoparticle for improving gene transfection efficiency based on tumor self microenvironment and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN112535739A CN112535739A (en) 2021-03-23
CN112535739B true CN112535739B (en) 2023-05-05

Family

ID=75019555

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202011425940.7A Active CN112535739B (en) 2020-12-08 2020-12-08 Nanoparticle for improving gene transfection efficiency based on tumor self microenvironment and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN112535739B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114377149B (en) * 2021-12-14 2023-11-10 西北工业大学 Mn-based degradable MOF nano-reactor and preparation method and application thereof

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012009448A2 (en) * 2010-07-13 2012-01-19 The Regents Of The University Of California Cationic polymer coated mesoporous silica nanoparticles and uses thereof
EP2648764A4 (en) * 2010-12-07 2015-03-25 Univ Western Australia Multifunctional nanoparticles
CN102327622B (en) * 2011-09-08 2014-08-06 上海交通大学 Method for loading siRNA (small interfering Ribonucleic Acid) by using mesoporous silicon dioxide nanoparticles
CN108210931A (en) * 2016-12-15 2018-06-29 深圳大学 Nanometer diagnosis and treatment agent, preparation method and application
WO2018162676A1 (en) * 2017-03-08 2018-09-13 Université De Strasbourg DISINTEGRATABLE POROUS ORGANOSILICA OR ORGANOMETALOXIDE NANOPARTICLES AND USES THEREOF AS VEHICLE FOR CONTROLLED DELIVERY OF siRNA
CN109078176B (en) * 2018-08-14 2020-09-22 武汉大学 Tumor cell membrane coated nano material and preparation method and application thereof
CN111388451B (en) * 2020-04-29 2023-05-23 南京工业大学 Protein self-assembled iron-based nanoparticle, preparation method thereof and application thereof in antitumor drug delivery system

Also Published As

Publication number Publication date
CN112535739A (en) 2021-03-23

Similar Documents

Publication Publication Date Title
Ding et al. Novel engineered bacterium/black phosphorus quantum dot hybrid system for hypoxic tumor targeting and efficient photodynamic therapy
CN110384806B (en) Preparation and application of drug-loaded polydopamine/dendrimer-gold nanoparticles
CN106456454B (en) Disintegrable core/shell silica particles for encapsulating and releasing biologically active macromolecules
CN109395087A (en) A kind of nanometer delivering NO donor and Nano medication altogether is total to delivery system
CN107242996B (en) A kind of gel rubber material and preparation method thereof for oncotherapy
Yu et al. Chemodynamic therapy combined with multifunctional nanomaterials and their applications in tumor treatment
CN109513000A (en) It is a kind of deliver melittin photoactive nanoparticles support preparation method and application
CN112535739B (en) Nanoparticle for improving gene transfection efficiency based on tumor self microenvironment and preparation method and application thereof
Chen et al. Tumor-specific nanomedicine via sequential catalytic reactions for accurate tumor therapy
CN112741903B (en) DNA/nano compound and preparation method and application thereof
CN113350524B (en) Iron-blocking magnetic nano-drug responding to tumor microenvironment as well as preparation method and application thereof
Chen et al. An overview of recent advancements on manganese-based nanostructures and their application for ROS-mediated tumor therapy
Zhang et al. Tumor microenvironment-responsive degradable silica nanoparticles: design principles and precision theranostic applications
Liu et al. Glucose oxidase-modified metal–organic framework for starving-enhanced chemodynamic therapy
CN112089836A (en) Preparation method and application of copper sulfide/hyperbranched macromolecular nano-bionic enzyme
CN109550050B (en) Melanin-loaded molybdenum dioxide drug-loaded compound and preparation and application thereof
Yang et al. Targeted intelligent mesoporous polydopamine nanosystems for multimodal synergistic tumor treatment
CN114949186A (en) POD-GOD (peroxidase-GOD) synergistically modified mesoporous silicon nanomaterial, preparation method and application
CN110917172B (en) Molybdenum oxide nanosheet plugging hollow mesoporous silicon nanomaterial and preparation and application thereof
Li et al. pH-responsive degradable mesoporous organosilica nanoparticle for tumor targeting and phototherapy combined with chemotherapy
CN115337397A (en) PDA nano-drug loaded with siRNA/PEI, preparation method and application thereof
CN110665005B (en) Iron-doped polymer nanoparticle and preparation method and application thereof
Wang et al. DNA-based nanosystems to generate reactive oxygen species for nanomedicine
CN117383616A (en) Hollow drug-loaded nano-enzyme, preparation method and anti-tumor drug
CN115300632B (en) Nanocapsule composition, preparation method and application

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right

Effective date of registration: 20240605

Address after: 518107 Room 501, building 3, Herun Jiayuan, Huaxia Road, Guangming Street, Guangming New District, Shenzhen City, Guangdong Province

Patentee after: Shenzhen, Zhongshan University

Country or region after: China

Patentee after: SUN YAT-SEN University

Address before: No.135, Xingang West Road, Haizhu District, Guangzhou City, Guangdong Province

Patentee before: SUN YAT-SEN University

Country or region before: China

TR01 Transfer of patent right