CN116712566A - Nanometer system for resisting coronavirus infection and preparation method and application thereof - Google Patents

Nanometer system for resisting coronavirus infection and preparation method and application thereof Download PDF

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CN116712566A
CN116712566A CN202310783988.2A CN202310783988A CN116712566A CN 116712566 A CN116712566 A CN 116712566A CN 202310783988 A CN202310783988 A CN 202310783988A CN 116712566 A CN116712566 A CN 116712566A
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sirna
protein
mesoporous silica
dendritic mesoporous
coronavirus
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朱钰方
陈佳杰
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Shanghai Institute of Ceramics of CAS
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Shanghai Institute of Ceramics of CAS
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    • 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
    • A61K47/6921—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 the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—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 the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • 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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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

Abstract

The invention relates to a nano system for resisting coronavirus infection, a preparation method and application thereof. The nanosystems against coronavirus infection comprise: the nano-porous silicon dioxide nanoparticle comprises dendritic mesoporous silicon dioxide nanoparticles, siRNA molecules loaded on the dendritic mesoporous silicon dioxide nanoparticles and S protein modified on the dendritic mesoporous silicon dioxide nanoparticles.

Description

Nanometer system for resisting coronavirus infection and preparation method and application thereof
Technical Field
The invention relates to a nano system for resisting coronavirus infection, a preparation method and application thereof, belonging to the field of biological materials.
Background
Viral infections have been threatening to human life and health, and in particular, new coronapneumonic diseases are caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although global scientists have been working on developing reliable strategies to combat new coronatine diseases such as vaccines and antibodies, there is still a significant impact on the life of people with various underlying diseases. Up to now, there are no drugs with high specificity for SARS-CoV-2 on the market, but general combination therapies such as symptomatic treatment, supportive treatment and antibiotic treatment, the effect of which depends more on the individual. Furthermore, abuse of existing drugs against SARS-CoV-2 infection may have some adverse effects on the body. Thus, there is an urgent need to develop new safe, effective therapies against such coronaviruses.
RNA interference (RNAi) is a method to achieve silencing of target gene expression by degradation of the corresponding messenger RNA (mRNA) based on specific double-stranded RNA (dsRNA), which is considered to be a powerful technique for treating different diseases at the genetic level, such as against cancer and viral infectious diseases. As an RNAi tool, synthetic short interfering RNAs (sirnas) can be used to specifically silence viral genes with high efficiency in the cytoplasm to protect the cell genome from attack by viral genetic material. Notably, siRNA therapy has several advantages in combating virus-induced epidemics: 1) siRNA can be used to target almost all virus-related genes and inhibit them by gene interference mechanisms, which is more advantageous than the use of chemical or small molecule compounds; 2) With the identification of the target gene sequence, the siRNA can be synthesized and evaluated in a short time before application; 3) The introduction of siRNA does not alter the host cell genome and has low cytotoxicity, showing high therapeutic safety. Thus, siRNA has shown great potential as an anti-coronavirus drug.
However, the use of siRNA is subject to some inherent limitations, such as: low stability, short biological half-life, easy degradation by endogenous nuclease, low cell membrane penetrating ability, poor targeting specificity, etc. In fact, some efforts have been made to design effective vectors to overcome biological barriers and optimize in vivo delivery of siRNA to treat viral infections, principally including viral vectors and non-viral vectors. Although viral vectors can successfully deliver siRNA to target host cells, their use is still limited by potential cytotoxicity and the potential threat of stimulating an innate immune response. Non-viral vectors are typically liposomes, polymeric nanoparticles, and dendrimers, which exhibit low siRNA loading, limited solubility and stability, and premature leakage problems that loaded drugs may present.
Disclosure of Invention
Aiming at the technical problems of poor anti-coronavirus infection effect and low safety in the prior art, the invention provides a nano system for resisting coronavirus infection, and a preparation method and application thereof.
In one aspect, the invention provides a nanosystem for combating coronavirus infection, comprising: the nano-porous silicon dioxide nanoparticle comprises dendritic mesoporous silicon dioxide nanoparticles, siRNA molecules loaded on the dendritic mesoporous silicon dioxide nanoparticles and S protein modified on the dendritic mesoporous silicon dioxide nanoparticles.
In the present disclosure, dendritic mesoporous silica nanoparticles are utilized as useful carriers for drug delivery due to their many advantages, such as regular uniform mesoporous channels, adjustable particles and pore sizes, controllable morphology, high thermal and chemical stability, large specific surface area, easily modified surfaces, high drug loading capacity, and good biocompatibility. The invention designs mesoporous silica nano particles with large aperture and high specific surface area, effectively delivers specific siRNA and has important significance for realizing the efficient silencing of coronavirus related genes.
Furthermore, coronaviruses generally consist of four structural proteins, namely spike protein (S), membrane protein (M), envelope protein (e) and nucleocapsid protein (N). In particular, the S protein is a glycoprotein present on the surface of coronaviruses and is capable of recognizing and entering the host cell by coronaviruses based on interaction with the host cell' S angiotensin converting enzyme 2 (ACE 2) receptor. Therefore, the inventor utilizes the characteristic to modify S protein on the nano delivery carrier, can simulate the process of infecting host cells with ACE2 high expression by coronavirus to effectively target and deliver antiviral siRNA into the host cells, improves the treatment efficiency and presents great attraction.
Therefore, the invention designs and prepares the mesoporous silica-based nano system with good biocompatibility, high siRNA load and S protein modification, and realizes the silencing of virus related genes by targeting the specific siRNA to host cells infected by coronaviruses, thereby being hopeful to realize safe and efficient anti-coronavirus treatment.
Preferably, the grain diameter of the dendritic mesoporous silica nanoparticle is 20-100 nm, the diameter of the mesoporous pore canal is 3-10 nm, and the specific surface area is 200-1000 m 2 /g。
Preferably, the mass ratio of the siRNA molecules to the dendritic mesoporous silica nanoparticles is 1-200 mug/mg; the mass ratio of the S protein to the dendritic mesoporous silica nanoparticle is 1-200 mug/mg.
Preferably, the siRNA molecules are used to silence a corresponding gene of a coronavirus, the corresponding gene comprising one of a leader sequence, a replicase polyprotein 1a (pp 1 a) gene, an RdRp gene, an S protein gene, an N protein gene, an M protein gene, and an E protein gene.
Preferably, the particle size of the nano system for resisting coronavirus infection is 30-110 nm.
In another aspect, the present invention provides a method of preparing a nanosystem for combating coronavirus infection, comprising:
(1) Adding an aminosilane coupling agent into the dendritic mesoporous silica nanoparticle dispersion liquid, mixing, and washing to obtain amino-functionalized dendritic mesoporous silica nanoparticles;
(2) Dispersing amino-functionalized dendritic mesoporous silica nano particles in DEPC water, adding siRNA molecules, then carrying out ultrasonic mixing and centrifugation, then adding coronavirus S protein, carrying out secondary ultrasonic mixing and secondary centrifugation, and finally washing to obtain the coronavirus infected nano system.
Preferably, the concentration of the dendritic mesoporous silica nanoparticle dispersion liquid is 0.1-10 g/L; the solvent of the dendritic mesoporous silica nanoparticle dispersion liquid is at least one of ethanol, deionized water, PBS buffer solution and DMEM culture solution.
Preferably, the aminosilane coupling agent is at least one of gamma-aminopropyl trimethoxysilane, gamma-aminopropyl triethoxysilane, N-beta (aminoethyl) -gamma-aminopropyl trimethoxysilane, N-beta (aminoethyl) -gamma-aminopropyl triethoxysilane, aminoethylaminopropyl trimethoxysilane, and aminoethylaminopropyl triethoxysilane.
Preferably, the ratio of the aminosilane coupling agent to the dendritic mesoporous silica nanoparticle is 1-5.0 mL/1g.
Preferably, the mass ratio of the amino-functionalized dendritic mesoporous silica nanoparticle to the siRNA minute to the S protein is (50-2000): 10: (1-20).
In yet another aspect, the present invention provides the use of a nanosystem for combating coronavirus infection in the preparation of a material for combating coronavirus infection. By utilizing the characteristic that the S protein on the surface of the coronavirus is specifically combined with the ACE2 receptor on the surface of the host cell, the S protein modified nano system can simulate the path of coronavirus infection of the host cell to effectively and targetedly deliver and release siRNA into the host cell infected by the coronavirus and silence virus related genes, thereby achieving the aim of inhibiting coronavirus infection. This targeted delivery approach may improve the safety and efficiency of the anti-coronavirus process.
The beneficial effects are that:
the SARS-CoV-2S protein is modified on the nano-particles in the invention, and the aim is to simulate coronavirus to realize targeted delivery of the nano-particles to target cells which are easy to be infected by the coronavirus through specific recognition so as to realize treatment;
the invention is a dendritic mesoporous silica nanoparticle, the pore canal is different from the conventional mesoporous, is an open dendritic pore canal, has the pore diameter of 3-10 nm, and is more beneficial to loading and delivering macromolecules such as nucleic acid;
the siRNA loaded by the invention is a corresponding gene for silencing coronavirus (the corresponding gene comprises one of a leader sequence, replicase polyprotein 1a (pp 1 a) gene, rdRp gene, S protein gene, N protein gene, M protein gene and E protein gene) and aims at inhibiting the relevant life cycle of coronavirus in host cells, thereby inhibiting viral infection;
the invention takes dendritic mesoporous silica nano particles with good biocompatibility, large pore canal and high specific surface area as a matrix, integrates specific antiviral siRNA and virus S protein, and forms a nano system capable of resisting coronavirus infection. The nano system can efficiently load antiviral siRNA, improve the stability of the siRNA, protect the siRNA from nuclease degradation, effectively deliver the siRNA into host cells infected by coronaviruses by utilizing the specific targeting capability of S protein, realize the silencing of virus related genes and provide a new technology for resisting coronavirus infection. Compared with the prior art, the invention has obvious technical progress.
Drawings
FIGS. 1 (a) and (b) are a scanning electron micrograph and a transmission electron micrograph, respectively, of Dendritic Mesoporous Silica Nanoparticles (DMSN) prepared in the present invention;
in FIG. 2, (a), (b) and (c) are respectively amino-functionalized dendritic mesoporous silica nanoparticles (DMSN-NH) prepared in the present invention 2 ) Infrared absorption spectrum of (N) 2 Adsorption-desorption isotherms and corresponding pore size distribution curves;
FIGS. 3 (a) and (b) are, respectively, scanning electron micrographs and transmission electron micrographs of coronavirus infection-resistant nanosystems prepared in the present invention;
FIGS. 4 (a), (b), (c) and (d) are hydrodynamic dimensional changes, zeta potential changes, UV-visible absorbance changes, and BCA protein tests, respectively, of the process of preparing nanosystems for combating coronavirus infection in accordance with the present invention;
FIGS. 5 (a) and (b) are respectively siRNA and S protein loading profiles of the nanosystems prepared in the present invention against coronavirus infection;
FIG. 6 is a graph showing siRNA release profiles in a simulated physiological environment for coronavirus infection resistant nanosystems prepared in accordance with the present invention;
FIG. 7 is a graph showing the ability of the coronavirus infection resistant nanosystems prepared in the present invention to improve siRNA delivery stability using agarose gel electrophoresis;
fig. 8 (a), (b) and (c) are respectively a cytotoxicity test, a targeting verification test and a virus target gene silencing efficiency test of the nano-system for resisting coronavirus infection prepared in the present invention.
Detailed Description
The invention is further illustrated by the following embodiments, which are to be understood as merely illustrative of the invention and not limiting thereof.
In the present disclosure, the nanosystems against coronavirus infection consist of dendritic mesoporous silica nanoparticles, siRNA molecules loaded on the particles, and virus S proteins modified on the particles. The particle size of the nano system is 30-100 nanometers. The nano system for resisting coronavirus infection has good biocompatibility and higher siRNA loading capacity, can realize corresponding virus gene silencing by targeting delivery of siRNA to host cells which are easy to be infected by coronavirus while improving the stability of the siRNA, and has potential application value in resisting coronavirus infection.
In one embodiment of the invention, dendritic mesoporous silica nanoparticles are prepared by a sol-gel method, amino functionalization is performed on the nanoparticles, and finally siRNA molecules and S proteins are integrated sequentially by electrostatic interaction. The following illustrates exemplary methods for preparing nanosystems resistant to coronavirus infection.
And (3) preparing a dendritic mesoporous silica nanoparticle dispersion liquid. Dispersing a template surfactant and a mineralizer into deionized water, and adding a silicon source to react for a certain time to obtain white nano particles. Wherein the reaction temperature is 60-100 ℃ and the reaction time is 1-24 hours. And placing the obtained white nano particles in a hydrochloric acid/methanol mixed solution, and stirring and etching to obtain dendritic mesoporous silica nano particles (DMSN). Wherein the template surfactant may be at least one of cetyltrimethyl para-toluene sulfonamide (CTAT), cetyltrimethylammonium bromide (CTAB), and cetyltrimethylammonium chloride (CTAC). The mineralizer may be at least one of triethanolamine, ammonia water, diethylamine, ethylamino, propylamino and butylamino. The silicon source may be at least one of tetraethyl orthosilicate and tetramethyl orthosilicate. The mass ratio of the template surfactant to the silicon source may be 1: (1-10). The mass ratio of the mineralizer to the silicon source may be 1: (1-20). The solubility of hydrochloric acid in the mixed solution of hydrochloric acid and methanol can be 0.2-2 mol/L. The rotation speed of the stirring etching can be 100-1000 rpm, and the time can be 1-12 hours. As one example, it includes: cetyl trimethyl para-toluene sulfonamide (CTAT) and Triethanolamine (TEA) were added to deionized water and stirred rapidly in an oil bath at 60-100deg.C until completely dissolved. Then slowly dripping a certain amount of tetraethyl orthosilicate (TEOS), stirring for a period of time at a constant speed, centrifuging to collect white nano particles, and washing with deionized water and ethanol. The collected nanoparticles were added to a hydrochloric acid/methanol mixed solution and stirred and etched at 60 ℃. Finally, dendritic Mesoporous Silica Nanoparticles (DMSN) were collected by centrifugation and, after three washes with ethanol, redispersed in ethanol.
Taking a proper volume of nanoparticle ethanol solution, adding a certain amount of aminosilane coupling agent, stirring for 24 hours, centrifuging, and washing with ethanol and deionized water for several times to obtain amino-functionalized dendritic mesoporous silica nanoparticle (DMSN-NH) 2 ). Wherein the aminosilane coupling agent is any one of gamma-aminopropyl trimethoxysilane, gamma-aminopropyl triethoxysilane, N-beta (aminoethyl) -gamma-aminopropyl trimethoxysilane, N-beta (aminoethyl) -gamma-aminopropyl triethoxysilane, aminoethylaminoethyl aminopropyl trimethoxysilane and aminoethylaminoethyl aminopropyl triethoxysilane.
DMSN-NH 2 Dispersing in DEPC water, adding specific siRNA molecules according to a certain proportion, carrying out ultrasonic treatment for 10 minutes, centrifuging, adding a certain amount of coronavirus S protein, carrying out ultrasonic treatment for 10 minutes, centrifuging, and washing for several times by using DEPC water to obtain the nano system for resisting coronavirus infection, wherein the nano system is loaded with siRNA molecules and modified S proteins. Wherein the specific siRNA molecule can silence any one of corresponding genes of coronavirus, namely a leader sequence, replicase polyprotein 1a (pp 1 a) gene, rdRp gene, S protein gene, N protein gene, M protein gene and E protein gene. Further, the DMSN-NH in the third step 2 The mass ratio of the siRNA to the S protein is 50-2000:10:1-20.
Performance characterization of the prepared nano system shows that the nano system can efficiently load siRNA and modified virus S protein due to the fact that the dendritic mesoporous silica nanoparticle matrix has a large pore canal and a high specific surface area, and the load can be achieved byAnd (5) adjusting the mixing proportion. The nano system can obviously improve the stability of siRNA and protect the siRNA from nuclease degradation in the siRNA delivery process. By adjusting DMSN-NH 2 And the mixing proportion of the siRNA, the behavior of the nanometer system for releasing the siRNA in a physiological environment can be controlled. The nano system has high biocompatibility and is beneficial to application in biomedicine.
The present invention will be further illustrated by the following examples. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations upon the scope of the invention, since numerous insubstantial modifications and variations will now occur to those skilled in the art in light of the foregoing disclosure. The specific process parameters and the like described below are also merely examples of suitable ranges, i.e., one skilled in the art can make a suitable selection from the description herein and are not intended to be limited to the specific values described below.
Example 1 preparation of nanosystems (DRS-1) against coronavirus infection
Step (1): 0.6836g of cetyltrimethyl-p-toluene-sulfonamide (CTAT) and 0.4g of Triethanolamine (TEA) were added to 36mL of deionized water and stirred rapidly in an oil bath at 80deg.C until completely dissolved. Then, 2mL of tetraethyl orthosilicate (TEOS) was slowly dropped, stirred at a constant speed for 2 hours, and after centrifugation, white nanoparticles were collected and washed three times with deionized water and ethanol, respectively. The collected nanoparticles were added to a hydrochloric acid/methanol (1:15) mixed solution and placed in a 60 ℃ stirring and etching. Finally, centrifugally collecting dendritic mesoporous silica nano particles (DMSN), washing with ethanol three times, and then redispersing in ethanol;
step (2): 60mL of nanoparticle ethanol solution with the concentration of 5g/L is taken, 0.9mL of gamma-aminopropyl triethoxysilane is added, the mixture is stirred for 24 hours and centrifuged, and the mixture is washed three times by ethanol and deionized water respectively, so as to obtain amino-functionalized dendritic mesoporous silica nanoparticle (DMSN-NH) 2 )；
Step (3): DMSN-NH 2 Dispersing in DEPC water, adding siRNA molecule against RdRp gene of coronavirus, centrifuging after 10 min, and adding coronavirusS protein sonicated for 10 min followed by centrifugation (wherein DMSN-NH 2 The mass ratio of siRNA to S protein is 160:1:1), and the nano system for resisting coronavirus infection, which loads siRNA molecules and modifies S protein, is obtained by washing the nano system with DEPC water for three times. The mass ratio of the obtained siRNA molecules to the dendritic mesoporous silica nanoparticles is 6.1 mug/mg; the mass ratio of the S protein to the dendritic mesoporous silica nanoparticle is 8.3 mug/mg.
Fig. 1 is a scanning electron microscope photograph and a transmission electron microscope photograph of DMSN prepared according to the above method, which prove that silica nanoparticles having good dispersibility, uniform size and dendritic macropores are successfully prepared.
FIG. 2 is a schematic diagram of DMSN-NH prepared according to the present invention prepared as described above 2 Infrared absorption spectrum of (N) 2 Adsorption-desorption isotherms and corresponding pore size distribution curves prove the success of amino functionalization, and the pore canal is still large enough after the amino functionalization, thereby being beneficial to the loading of siRNA macromolecules.
Example 2 preparation of nanosystems (DRS-2) against coronavirus infection
Step (1): 0.6836g of cetyltrimethyl-p-toluene-sulfonamide (CTAT) and 0.4g of Triethanolamine (TEA) were added to 36mL of deionized water and stirred rapidly in an oil bath at 80deg.C until completely dissolved. Then, 2mL of tetraethyl orthosilicate (TEOS) was slowly dropped, stirred at a constant speed for 2 hours, and after centrifugation, white nanoparticles were collected and washed three times with deionized water and ethanol, respectively. The collected nanoparticles were added to a hydrochloric acid/methanol (1:15) mixed solution and placed in a 60 ℃ stirring and etching. Finally, centrifugally collecting dendritic mesoporous silica nano particles (DMSN), washing with ethanol three times, and then redispersing in ethanol;
step (2): 60mL of nanoparticle ethanol solution with the concentration of 5g/L is taken, 0.9mL of gamma-aminopropyl triethoxysilane is added, the mixture is stirred for 24 hours and centrifuged, and the mixture is washed three times by ethanol and deionized water respectively, so as to obtain amino-functionalized dendritic mesoporous silica nanoparticle (DMSN-NH) 2 )；
Step (3): DMSN-NH 2 Dispersing in DEPC water, and adding siRNA molecule ultrasound aiming at RdRp gene of coronavirusCentrifuging after 10 min, adding coronavirus S protein, and centrifuging after 10 min (wherein DMSN-NH 2 The mass ratio of siRNA to S protein is 80:1:1), and the nanometer system for resisting coronavirus infection, which loads siRNA molecules and modifies S protein, is obtained by washing the nanometer system with DEPC water for three times. The mass ratio of the obtained siRNA molecules to the dendritic mesoporous silica nanoparticles is 12.0 mug/mg; the mass ratio of the S protein to the dendritic mesoporous silica nanoparticle is 16.4 mug/mg.
Example 3 preparation of nanosystems against coronavirus infection (DRS-3):
step (1): 0.6836g of cetyltrimethyl-p-toluene-sulfonamide (CTAT) and 0.4g of Triethanolamine (TEA) were added to 36mL of deionized water and stirred rapidly in an oil bath at 80deg.C until completely dissolved. Then, 2mL of tetraethyl orthosilicate (TEOS) was slowly dropped, stirred at a constant speed for 2 hours, and after centrifugation, white nanoparticles were collected and washed three times with deionized water and ethanol, respectively. The collected nanoparticles were added to a hydrochloric acid/methanol (1:15) mixed solution and placed in a 60 ℃ stirring and etching. Finally, centrifugally collecting dendritic mesoporous silica nano particles (DMSN), washing with ethanol three times, and then redispersing in ethanol;
step (2): 60mL of nanoparticle ethanol solution with the concentration of 5g/L is taken, 0.9mL of gamma-aminopropyl triethoxysilane is added, the mixture is stirred for 24 hours and centrifuged, and the mixture is washed three times by ethanol and deionized water respectively, so as to obtain amino-functionalized dendritic mesoporous silica nanoparticle (DMSN-NH) 2 )；
Step (3): DMSN-NH 2 Dispersing in DEPC water, adding siRNA molecule against RdRp gene of coronavirus, centrifuging after ultrasonic treatment for 10 min, adding S protein of coronavirus, centrifuging after ultrasonic treatment for 10 min (wherein DMSN-NH 2 The mass ratio of siRNA to S protein is 40:1:1), and the nanometer system for resisting coronavirus infection, which loads siRNA molecules and modifies S protein, is obtained by washing the nanometer system with DEPC water for three times. The mass ratio of the obtained siRNA molecules to the dendritic mesoporous silica nanoparticles is 22.6 mug/mg; the mass ratio of the S protein to the dendritic mesoporous silica nanoparticle is 30.8 mug/mg.
FIG. 3 is a scanning electron micrograph and a transmission electron micrograph of a nanosystem against coronavirus infection prepared according to the above method. From the results, the loading of siRNA and modification of S protein did not significantly change the structure and morphology of the dendritic mesoporous silica nanoparticle itself, and the blurring of the nanoparticle pore channels suggests the loading of siRNA and S protein on the nanoparticle.
FIG. 4 shows hydrodynamic size change, zeta potential change, UV visible absorbance change, and BCA protein test of the nanosystems process for preparing anti-coronavirus infection according to the above method. From the results, it can be seen that the nano system indeed integrates the siRNA and the viral S protein, the process of loading the siRNA and modifying the S protein increases the hydrodynamic size of the dendritic mesoporous silica nanoparticle, changing the zeta potential of the dendritic mesoporous silica nanoparticle.
Example 4 preparation of nanosystems (DRS-4) against coronavirus infection
Step (1): 0.6836g of cetyltrimethyl-p-toluene-sulfonamide (CTAT) and 0.4g of Triethanolamine (TEA) were added to 36mL of deionized water and stirred rapidly in an oil bath at 80deg.C until completely dissolved. Then, 2mL of tetraethyl orthosilicate (TEOS) was slowly dropped, stirred at a constant speed for 2 hours, and after centrifugation, white nanoparticles were collected and washed three times with deionized water and ethanol, respectively. The collected nanoparticles were added to a hydrochloric acid/methanol (1:15) mixed solution and placed in a 60 ℃ stirring and etching. Finally, centrifugally collecting dendritic mesoporous silica nano particles (DMSN), washing with ethanol three times, and then redispersing in ethanol;
step (2): 60mL of nanoparticle ethanol solution with the concentration of 5g/L is taken, 0.9mL of gamma-aminopropyl triethoxysilane is added, the mixture is stirred for 24 hours and centrifuged, and the mixture is washed three times by ethanol and deionized water respectively, so as to obtain amino-functionalized dendritic mesoporous silica nanoparticle (DMSN-NH) 2 )；
Step (3): DMSN-NH 2 Dispersing in DEPC water, adding siRNA molecule against RdRp gene of coronavirus, centrifuging after ultrasonic treatment for 10 min, adding S protein of coronavirus, centrifuging after ultrasonic treatment for 10 min (wherein DMSN-NH 2 The mass ratio of siRNA to S protein is 20:1:1), and the siRNA molecules are loaded and the S protein is modified by washing the siRNA with DEPC water three timesIs resistant to coronavirus infection. The mass ratio of the obtained siRNA molecules to the dendritic mesoporous silica nanoparticles is 42.6 mug/mg; the mass ratio of the S protein to the dendritic mesoporous silica nanoparticle is 58.0 mug/mg.
Example 5 preparation of nanosystems against coronavirus infection (DRS-5):
step (1): 0.6836g of cetyltrimethyl-p-toluene-sulfonamide (CTAT) and 0.4g of Triethanolamine (TEA) were added to 36mL of deionized water and stirred rapidly in an oil bath at 80deg.C until completely dissolved. Then, 2mL of tetraethyl orthosilicate (TEOS) was slowly dropped, stirred at a constant speed for 2 hours, and after centrifugation, white nanoparticles were collected and washed three times with deionized water and ethanol, respectively. The collected nanoparticles were added to a hydrochloric acid/methanol (1:15) mixed solution and placed in a 60 ℃ stirring and etching. Finally, centrifugally collecting dendritic mesoporous silica nano particles (DMSN), washing with ethanol three times, and then redispersing in ethanol;
step (2): 60mL of nanoparticle ethanol solution with the concentration of 5g/L is taken, 0.9mL of gamma-aminopropyl triethoxysilane is added, the mixture is stirred for 24 hours and centrifuged, and the mixture is washed three times by ethanol and deionized water respectively, so as to obtain amino-functionalized dendritic mesoporous silica nanoparticle (DMSN-NH) 2 )；
Step (3): DMSN-NH 2 Dispersing in DEPC water, adding siRNA molecule against RdRp gene of coronavirus, centrifuging after ultrasonic treatment for 10 min, adding S protein of coronavirus, centrifuging after ultrasonic treatment for 10 min (wherein DMSN-NH 2 The mass ratio of siRNA to S protein is 10:1:1), and the nanometer system for resisting coronavirus infection, which loads siRNA molecules and modifies S protein, is obtained by washing the nanometer system with DEPC water for three times. The mass ratio of the obtained siRNA molecules to the dendritic mesoporous silica nanoparticles is 66.0 mug/mg; the mass ratio of the S protein to the dendritic mesoporous silica nanoparticle is 89.8 mug/mg.
FIG. 5 is an analysis of the loading of siRNA and S protein of the nanosystems against coronavirus infection prepared in examples 1-5 of the present invention. It can be found that as DMSN-NH is synthesized 2 And the proportion of siRNA is reduced, the loading capacity of siRNA is increased, the loading efficiency is reduced, and when DMSN-NH 2 And the mass ratio of siRNA is below 40:1 (namely DRS-3), the load of siRNA is close to saturation. When DMSN-NH 2 And the saturated loading amount of siRNA is up to 66.0 mug/mg when the mass ratio of siRNA is 10:1 (namely DRS-5). Similarly, protein S is loaded in a corresponding manner, and the saturated loading amount is as high as 89.8 mug/mg. In fig. 5 (a), it has been demonstrated that the loading of siRNA molecules can be adjusted by varying the mass ratio of DMSN-NH2 to siRNA when DRS is synthesized. As can be seen from fig. 5 (b), regarding the S protein, the loading of the S protein can be characterized according to the BSA detection method, and can also be adjusted by changing the mass ratio of DMSN-NH2 to the S protein when synthesizing DRS.
EXAMPLE 6 preparation of nanosystems (DR) against coronavirus infection
Step (1): 0.6836g of cetyltrimethyl-p-toluene-sulfonamide (CTAT) and 0.4g of Triethanolamine (TEA) were added to 36mL of deionized water and stirred rapidly in an oil bath at 80deg.C until completely dissolved. Then, 2mL of tetraethyl orthosilicate (TEOS) was slowly dropped, stirred at a constant speed for 2 hours, and after centrifugation, white nanoparticles were collected and washed three times with deionized water and ethanol, respectively. The collected nanoparticles were added to a hydrochloric acid/methanol (1:15) mixed solution and placed in a 60 ℃ stirring and etching. Finally, centrifugally collecting dendritic mesoporous silica nano particles (DMSN), washing with ethanol three times, and then redispersing in ethanol;
step (2): 60mL of nanoparticle ethanol solution with the concentration of 5g/L is taken, 0.9mL of gamma-aminopropyl triethoxysilane is added, the mixture is stirred for 24 hours and centrifuged, and the mixture is washed three times by ethanol and deionized water respectively, so as to obtain amino-functionalized dendritic mesoporous silica nanoparticle (DMSN-NH) 2 )；
Step (3): DMSN-NH 2 Dispersing in DEPC water, adding siRNA molecule against RdRp gene of coronavirus, ultrasonic treating for 10 min, and centrifuging (wherein DMSN-NH 2 And siRNA mass ratio of 40:1), and washed three times with DEPC water, resulting in a nanosystem against coronavirus infection loaded with siRNA molecules only. The mass ratio of the obtained siRNA molecules to the dendritic mesoporous silica nanoparticles is 22.8 mug/mg. Relative to example 3, siRNA molecule loadings on DR nanosystemsThere was no significant difference because the loading of the S protein at the latter step had relatively little effect on the loading of the siRNA molecule at the former step.
Comparative example 1 preparation of nanosystems (DS) against coronavirus infection
Step (1): 0.6836g of cetyltrimethyl-p-toluene-sulfonamide (CTAT) and 0.4g of Triethanolamine (TEA) were added to 36mL of deionized water and stirred rapidly in an oil bath at 80deg.C until completely dissolved. Then, 2mL of tetraethyl orthosilicate (TEOS) was slowly dropped, stirred at a constant speed for 2 hours, and after centrifugation, white nanoparticles were collected and washed three times with deionized water and ethanol, respectively. The collected nanoparticles were added to a hydrochloric acid/methanol (1:15) mixed solution and placed in a 60 ℃ stirring and etching. Finally, centrifugally collecting dendritic mesoporous silica nano particles (DMSN), washing with ethanol three times, and then redispersing in ethanol;
step (2): 60mL of nanoparticle ethanol solution with the concentration of 5g/L is taken, 0.9mL of gamma-aminopropyl triethoxysilane is added, the mixture is stirred for 24 hours and centrifuged, and the mixture is washed three times by ethanol and deionized water respectively, so as to obtain amino-functionalized dendritic mesoporous silica nanoparticle (DMSN-NH) 2 )；
Step (3): DMSN-NH 2 Dispersing in DEPC water, adding coronavirus S protein, sonicating for 10 min, and centrifuging (wherein DMSN-NH 2 And a mass ratio of S protein of 40:1), and washed three times with DEPC water, to obtain a nanosystem against coronavirus infection that modifies only S protein. The mass ratio of the S protein to the dendritic mesoporous silica nanoparticle is 47.4 mug/mg. The S protein loading on the DS nanosystems was significantly increased relative to example 3, since there was no previous step of loading of siRNA molecules, allowing for an increase in the particle sites capable of loading S protein.
Example 7
First, standard curves of siRNA at different concentrations were plotted according to the absorbance of siRNA at 260 nm. Next, the coronavirus infection-resistant nanosystems prepared in examples 1 to 5 were dispersed in PBS buffer, and then placed in a constant temperature shaker at 37℃for continuous shaking. Wherein, at regular time intervals, the micro supernatant is obtained by centrifugation to test absorbance. And calculating the siRNA release conditions of different time periods according to the standard curve.
FIG. 6 shows siRNA release profiles of coronavirus infection resistant nanosystems prepared in examples 1-5 of the present invention in PBS buffer. The results show that with DMSN-NH 2 And the proportion of siRNA is increased, the speed of the nanometer system for releasing the siRNA is reduced, and the release amount is also reduced. Wherein, when DMSN-NH 2 And when the mass ratio of the siRNA is 160:1 (namely DRS-1), the final release of the siRNA by the nano system is only about 10%, and most of the siRNA is not released; when DMSN-NH 2 And when the mass ratio of the siRNA is 10:1 (namely DRS-5), the speed of the nanometer system for releasing the siRNA is fastest, and the highest point of release is 52% after 6 hours, and then the siRNA is not released any more; when DMSN-NH 2 And a mass ratio of siRNA of 40:1 (i.e., DRS-3), the nanosystems exhibit sustained, slow siRNA release behavior, which is beneficial for sustained intracellular gene silencing. As can be seen from the analysis, the above phenomenon may be due to DMSN-NH 2 The higher the duty ratio is, the larger the adsorption force of the siRNA after being uniformly loaded on the nano particles is, which is unfavorable for the release of guest molecules; and DMSN-NH 2 The lower the duty ratio, the more siRNA is loaded on each nanoparticle, the smaller the siRNA adsorption force of the outermost layer of the nanoparticle is, and the easier the siRNA is released. Thus, by modulating DMSN-NH 2 And the mixing proportion of the siRNA can control the behavior of the nanometer system for releasing the siRNA in a physiological environment, thereby meeting the treatment requirements of different antiviral infections.
Example 8
First, free siRNA and nanosystem DRS-3 loaded with siRNA were dispersed in an aqueous solution containing ribonuclease a (RNase a), and then placed in a constant temperature shaker at 37 ℃ for 30 minutes with shaking. After the treatment was completed, 2. Mu.L of ethylenediamine tetraacetic acid sodium salt (EDTA) (250 mM) solution was added to the solution to terminate degradation. Finally, 1% agarose gel electrophoresis (90V, 15 min) was used to electrophoretically analyze nucleic acid molecules in different liquids.
FIG. 7 shows the ability of the nanosystem DRS-3 prepared in example 3 of the present invention to improve siRNA delivery stability against coronavirus infection using agarose gel electrophoresis. As a result, it was found that the free siRNA was hardly seen in the electrophoresis band after RNase A treatment, while the electrophoresis band was still present after RNase A treatment of the siRNA-loaded nanosystem DRS-3, and there was no significant change compared with the nanosystem without RNase A treatment. This suggests that free siRNA would be rapidly degraded by RNase a, whereas nanosystem delivery of siRNA could significantly improve siRNA stability, protecting it from nuclease degradation.
Example 9
1) Cytotoxicity assays were performed using CCK-8. First, DMSN-NH 2 And the DRS-3 nanosystems were dispersed in DMEM medium to prepare suspensions at concentrations of 0, 12.5, 25, 50, 100 and 200. Mu.g/mL. Next, 293T cells (human embryonic kidney cells) overexpressed by purchased ACE2 were seeded overnight in 96-well plates, and then the prepared nanoparticle-containing suspension was added to the 96-well plates, 100 μl of each well. After 24 hours of co-culture with the cells, CCK-8 test solution was added to each well, and after 2 hours of further culture, absorbance at 450nm was measured with a microplate reader, and the cell viability was determined by calculation. The results show that DMSN-NH 2 The nano particles per se show better biocompatibility, and when the concentration is up to 200 mug/mL, the cell survival rate still keeps more than 90%; moreover, the nano system DRS-3 loaded with siRNA and modified S protein also has lower toxicity, which is beneficial to biomedical application;
2) Cell targeting validation experiments were performed by fluorescent labeling. First, FAM fluorescent dye is labeled on siRNA so that DR and DRS-3 nano system can excite green fluorescence. Next, 293T cells (human embryonic kidney cells) overexpressing ACE2 at a concentration were seeded overnight in 48-well plates; removing the culture solution, adding DMEM culture solution containing single siRNA, DR and DRS-3 nanometer system (100 μg/mL) into a 48-well plate for 5 hours; removing the culture solution, and fixing with 4% paraformaldehyde for 15 min; DAPI (blue fluorescence after staining nuclei) and LysoBrite Red fluorescent stain (Red fluorescence after staining intracellular lysosomes) were then added for staining, followed by three washes with PBS, and finally observation with a fluorescence microscope. The results indicate that siRNA is rarely present in siRNA treated cells alone, on the one hand because nucleic acid molecules are easily degraded, and on the other hand that siRNA has poor membrane permeability, making it difficult to enter cells. In addition, green fluorescence of siRNA was present in DR nanosystem treated cells, indicating that the use of nanosystems facilitates siRNA delivery into cells. More obviously, more obvious green fluorescence appears in cells cultured by the DRS-3 nanosystem modified by the S protein, which indicates that the specific binding characteristics of the virus S protein and ACE2 are utilized, the targeting to host cells is more favorable, the uptake of the nanosystem by the host cells is promoted, and the active targeting is favorable for delivering siRNA into coronavirus infected cells to realize the therapeutic effect. Moreover, it can be seen that the green fluorescence and the red fluorescence are more recombined, which also indicates that the nano system is successfully phagocytized by cells and enters lysosomes;
3) Viral target gene silencing efficacy test experiments were performed by q-PCR. First, an ACE2 overexpressing 293T stable transformant containing the RdRp gene of coronavirus was constructed to mimic host cells after coronavirus infection: firstly, constructing a selected virus RdRp gene on a lenti-CMV-MCS-3 xFlag-PGK-Puro vector; packaging the corresponding slow viruses and measuring the virus titer; finally, the packaged RdRp lentivirus is used for infecting 293T cells over-expressed by ACE2, and RdRp-293T stable transgenic strain is obtained. Secondly, verification of the efficiency of siRNA silencing RdRp gene is carried out: rdRp-293T cells at a concentration were seeded overnight in 6-well plates; removing culture solution, adding siRNA and DMSN-NH into 6-hole plate 2 The DR, DRS-3 nanosystems (100. Mu.g/mL) DMEM broth was incubated for 48 hours; removing the culture solution, adding Trizol into each hole, and extracting mRNA of different groups; after mRNA extraction, cDNA was obtained by reverse transcription, and q-PCR was performed.
FIG. 8 shows DMSN-NH prepared in examples 3 and 6 of the present invention 2 In vitro cell experiments with DR and DRs-3 nanosystems. The result shows that the single siRNA has no obvious silencing effect on RdRp genes in RdRp-293T cells, which is caused by the reasons of low stability, easy degradation by nuclease, low cell membrane penetrating capacity and the like of the siRNA; in addition, the RdRp gene in the cells treated by the DR nanometer system has oneThe definite down-regulation means that the nanometer system is favorable for delivering siRNA into cells and releasing the siRNA, thus realizing a certain silencing effect of target genes; more importantly, rdRp genes in cells treated by the DRS-3 nanosystem show the lowest expression level, which indicates that the specific combination of S protein and ACE2 receptor endows the nanosystem with targeting property, can target and deliver siRNA to host cells and promote the siRNA to be transported into the cells, and finally improves the silencing efficiency of coronavirus target genes. Therefore, the prepared nano system has outstanding effect on antiviral infection, and provides a new reference for efficient and safe antiviral treatment.
The nanosystems against viral infection and the method of preparing the same according to the present invention are not limited to the above-described embodiments 1 to 9. The foregoing is merely exemplary of the principles of the invention, and several simple deductions or substitutions may be made without departing from the principles of the invention, which should be regarded as belonging to the scope of the invention.

Claims (10)

1. A nanosystem for combating coronavirus infection, comprising: the nano-porous silicon dioxide nanoparticle comprises dendritic mesoporous silicon dioxide nanoparticles, siRNA molecules loaded on the dendritic mesoporous silicon dioxide nanoparticles and S protein modified on the dendritic mesoporous silicon dioxide nanoparticles.
2. The nano system for resisting coronavirus infection according to claim 1, wherein the particle diameter of the dendritic mesoporous silica nano particles is 20-100 nm, the diameter of mesoporous pore canal is 3-10 nm, and the specific surface area is 200-1000 m 2 /g。
3. The nanosystem against coronavirus infection as claimed in claim 1, wherein the mass ratio of siRNA molecules to dendritic mesoporous silica nanoparticles is 1-200 μg/mg; the mass ratio of the S protein to the dendritic mesoporous silica nanoparticle is 1-200 mug/mg.
4. The nanosystem of claim 1, wherein the siRNA molecule is used to silence a corresponding gene of a coronavirus, the corresponding gene comprising one of a leader sequence, a replicase polyprotein 1a (pp 1 a) gene, an RdRp gene, an S protein gene, an N protein gene, an M protein gene, and an E protein gene.
5. The nanosystem resistant to coronavirus infection as claimed in any one of claims 1-4, wherein the particle size of the nanosystem resistant to coronavirus infection is 30-110 nm.
6. A method of preparing a nanosystem resistant to coronavirus infection as claimed in any one of claims 1 to 5, comprising:
(1) Adding an aminosilane coupling agent into the dendritic mesoporous silica nanoparticle dispersion liquid, mixing, and washing to obtain amino-functionalized dendritic mesoporous silica nanoparticles;
(2) Dispersing amino-functionalized dendritic mesoporous silica nano particles in DEPC water, adding siRNA molecules, then carrying out ultrasonic mixing and centrifugation, then adding coronavirus S protein, carrying out secondary ultrasonic mixing and secondary centrifugation, and finally washing to obtain the coronavirus infected nano system.
7. The method of claim 6, wherein the dendritic mesoporous silica nanoparticle dispersion has a concentration of 0.1 to 10g/L; the solvent of the dendritic mesoporous silica nanoparticle dispersion liquid is at least one of ethanol, deionized water, PBS buffer solution and DMEM culture solution.
8. The method according to claim 6, wherein the aminosilane coupling agent is at least one of γ -aminopropyl trimethoxysilane, γ -aminopropyl triethoxysilane, N- β (aminoethyl) - γ -aminopropyl trimethoxysilane, N- β (aminoethyl) - γ -aminopropyl triethoxysilane, aminoethylaminopropyl trimethoxysilane, and aminoethylaminopropyl triethoxysilane;
the ratio of the aminosilane coupling agent to the dendritic mesoporous silica nanoparticle is 1-5.0 mL/1g.
9. The preparation method according to any one of claims 6 to 8, wherein the mass ratio of the amino-functionalized dendritic mesoporous silica nanoparticle, siRNA minute and S protein is (50 to 2000): 10: (1-20).
10. Use of a nanosystem of any one of claims 1-5 for the preparation of a material against coronavirus infection.
CN202310783988.2A 2023-06-29 2023-06-29 Nanometer system for resisting coronavirus infection and preparation method and application thereof Pending CN116712566A (en)

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