CN105056239A - Composite material of functional mesoporous silica loaded drug and siRNA, preparation and application thereof in preparation of anticancer drugs - Google Patents
Composite material of functional mesoporous silica loaded drug and siRNA, preparation and application thereof in preparation of anticancer drugs Download PDFInfo
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Abstract
Belonging to the fields of functional material loaded drugs and gene technologies, the invention discloses a composite material of a functional mesoporous silica loaded drug and siRNA, a preparation method and application thereof in preparation of anticancer drugs. The composite material is prepared by the method comprising the steps of: adding mesoporous silica into a 3-mercaptopropyl trimethoxysilane solution, conducting heating reaction and separation to obtain thiolated mesoporous silica, dispersing the thiolated mesoporous silica into a 2, 2-dithiodipyridine solution, and carrying out heating reaction to obtain disulfide bond modified mesoporous silica, adding the disulfide bond modified mesoporous silica into a doxorubicin water solution, then adding thiolated siRNA, and conducting separation, thus obtaining the composite material of the functional mesoporous silica loaded drug and siRNA. The composite material provided by the invention can be applied to preparation of anticancer drugs, can load drugs into cells, significantly improves the enrichment amount of drugs in cells, plays a good mesopore blocking role, and reaches the effect of silencing gene when released in cells.
Description
Technical field
The invention belongs to functional material carrying medicament and gene technology field, particularly a kind of functional mesoporous silicon dioxide carried medicine and siRNA composite and preparation method thereof and preparing the application in cancer therapy drug.
Background technology
The anticancer market of recent year is growing situation, and in hospital administration, cancer therapy drug has been leaped second, only lower than anti-infective.At present, the means of Therapeutic cancer mainly comprise surgical operation therapy, radiotherapy and chemotherapy, but these Therapeutic Method easy damaged normal cell and immunocyte again while killing cancerous cell, lack selectivity, and causing a series of toxic and side effects, some comparatively serious untoward reaction also can threaten the life of patient.
At present, the treatment of local and diffused cancer mainly relies on chemotherapy.But, Therapeutic cancer chemotherapeutics used ubiquity limits to as follows: (1) lacks targeting: normally used cancer therapy drug has general cytotoxicity, lack the selectivity to cancerous cell and targeting, normal cell is caused also to there is non-specific endocytosis, damage Normocellular propagation, thus produce serious side effect, therefore medicine shows the shortcomings such as therapeutic index is low, curative effect is not obvious; (2) multidrug resistance: multidrug resistance makes the cancer therapy drug entered in cancerous cell constantly pump cell, adds pump output, causes Intracellular drug to reduce, reduces the effect of cancer therapy drug to cancerous cell; (3) bioavailability is low: most of cancer therapy drug has the effect of good anticancer, but lower bioavailability have impact on the clinical effectiveness of medicine to a great extent.
Because drug loading is high, biological agent is strong, and can promote cell endocytic thus avoid the advantages such as multidrug resistance, in recent years Nano medication delivery system, especially the treatment developing into cancer of nano target medicine delivery system provides the very promising approach of one.Desirable nano target medicine delivery system should possess: the easy functionalization of (1) material surface; (2) high cell-specific and effective cell endocytic; (3) ability of low early seepage and lasting controllable release; (4) carrier self low toxicity or good biocompatibility; (5) medicine can be loaded in carrier and to protect by carrier; (6) dispersibility that had in Physiological Medium of carrier; (7) carrier is little to protein adsorption, can not affect the effect of targeting part to receptor targeted; (8) carrier can not gather at the organ such as kidney, liver, and can catabolism gradually.What be applied to nano target medicine delivery system at present comprises microemulsion, polymer micelle, liposome and vesicle etc., and under the outside stimulus such as temperature, pH, structure changes and realizes drug controllable release.Because they all belong to soft material category, structural instability in aqueous medium, cannot stop targeted prodrug thing seepage, causes realizing effective conveying and controllable release.Therefore people are used for having carried out much research in drug conveying and release at the constitutionally stable material of use in recent years.In these materials, silicon materials are stablized due to good biocompatibility, structure and surface nature and are used to conveying and the controlled release research of various medicine.In addition, silicon is also used to the biocompatibility increasing other delivery vehicles such as magnetic nano-particle, polymer.In silicon materials family, mesoporous silicon oxide has developed into the most important and the most promising class drug conveying carrier.
SiRNA is the ribonucleotide of one section of particular sequence, in the expression being transfected into reticent corresponding gene after in cell, can reach the effect (RNAi) of Gene interfere.But report in the past shows that the siRNA dissociated cannot enter in cell, need to carry out chemical modification or nano-particle load could effectively enter cells play effect.
Amycin (Dox) is a kind of antibiotic killing cancerous cell efficiently, is widely used at present in the treatment of cancer.But owing to not having selectivity, side effect larger in treatment of cancer inhibits it to apply.The present invention utilizes mesoporous silicon oxide plastic packaging Dox, and use other materials to carry out mesoporous shutoff can effectively by Dox load, utilize EPR effect medicine-carried system to be enriched to cancer region and cause cell endocytic, reach best anticancer effect and minimized side effects in Intracellular drug release.After in Dox plastic packaging to mesoporous silicon oxide, utilize disulfide bond to connect siRNA at meso-porous titanium dioxide silicon face, utilize steric effect shutoff mesoporous, form function mesoporous silicon oxide medicine-carried system.So far the report of functional mesoporous silica medicine-carried system of the present invention for treatment of cancer is found no.
Summary of the invention
In order to overcome the shortcoming of above-mentioned prior art with not enough, primary and foremost purpose of the present invention is the composite providing a kind of functional mesoporous silicon dioxide carried medicine and siRNA.
Another object of the present invention is the preparation method of the composite providing a kind of above-mentioned functions mesoporous silicon oxide carrying medicament and siRNA.
Still a further object of the present invention is to provide the composite of above-mentioned functions mesoporous silicon oxide carrying medicament and siRNA preparing the application in cancer therapy drug.
Object of the present invention is realized by following proposal:
A composite of functional mesoporous silicon dioxide carried medicine and siRNA, is prepared by the method comprised the following steps:
Mesoporous silicon oxide is added in 3-mercaptopropyl trimethoxysilane solution, reacting by heating, is separated, obtains sulfhydrylation mesoporous silicon oxide, be distributed to 2, in 2-bis-sulfur two pyridine solution, reacting by heating, obtains the mesoporous silicon oxide that disulfide bond is modified, added in amycin aqueous solution, add the siRNA of sulfhydrylation again, be separated, obtain the composite of functional mesoporous silicon dioxide carried medicine and siRNA.
In 3-mercaptopropyl trimethoxysilane used and mesoporous silicon oxide, the mol ratio of element silicon is preferably 2:1 ~ 4:1.
In 2,2-bis-sulfur two pyridines used and sulfhydrylation mesoporous silicon oxide, the mol ratio of element sulphur is preferably 2:1 ~ 1:1.
In the mesoporous silicon oxide that siRNA used and disulfide bond are modified, the mol ratio of element sulphur is preferably 1:2 ~ 1:1.
The concentration of described amycin aqueous solution is preferably 0.8 ~ 1g/L.
Described mesoporous silicon oxide is the mesoporous silicon oxide adopting standard technique to prepare.
The concentration of described 3-mercaptopropyl trimethoxysilane solution is preferably 1 ~ 1.2Mol/L.
Described 3-mercaptopropyl trimethoxysilane solution is preferably 3-mercaptopropyl trimethoxysilane toluene solution.
The concentration of described 2,2-bis-sulfur two pyridine solutions is preferably 0.1 ~ 0.12Mol/L.
2,2-bis-described sulfur two pyridine solutions are preferably 2,2-bis-sulfur two pyridine ethanol solution.
Described reacting by heating is all preferred reacts 12 ~ 24h at 80 ~ 83 DEG C.
The product that described separation obtains preferably uses deionized water wash and in vacuum 50 DEG C of dryings.
The functional mesoporous silicon dioxide carried medicine that the invention described above prepares and the composite of siRNA have the excellent biological activity killing cancerous cell, can be applicable to prepare in cancer therapy drug.
Mechanism of the present invention is:
The composite of functional mesoporous silicon dioxide carried medicine of the present invention and siRNA is with the mesoporous silicon oxide of carrying medicament effect for core, and surface connects siRNA by disulfide bond and carries out mesoporous shutoff, plays the effect of carrying medicament and siRNA simultaneously.The composite of functional mesoporous silicon dioxide carried medicine of the present invention and siRNA can be good at drug loading to enter in cell, significantly improves medicine at intracellular enriching quantity.Further, in composite, covalent bond connection siRNA can play the mesoporous effect of shutoff very well and can discharge the effect playing silent gene in cell.Preparation method of the present invention is simple, and the material obtained can directly be preserved and use.
The present invention, relative to prior art, has following advantage and beneficial effect:
(1) composite of functional mesoporous silicon dioxide carried medicine of the present invention and siRNA not only has the effect of killing cancerous cell, and has the effect causing cancer cell-apoptosis, is applicable to prepare anticancer medicine.
(2) functional mesoporous silicon dioxide carried medicine of the present invention and the composite of siRNA utilize disulfide bond to be connected siRNA and serve the mesoporous effect of shutoff at mesoporous silicon oxide.Disulfide bonds can be caused to discharge amycin and siRNA to high expressed glutathione response in cancerous cell after cellular uptake simultaneously.
(3) composite of functional mesoporous silicon dioxide carried medicine of the present invention and siRNA can significantly improve the accumulation of amycin at cell, especially in the enrichment of nuclear area.Covalent bond siRNA can significantly improve gene silencing efficiency, reaches the effect of reticent target gene.
Accompanying drawing explanation
Fig. 1 is perspective Electronic Speculum (TEM) figure of the mesoporous silicon oxide of embodiment 1.
Fig. 2 is perspective Electronic Speculum (TEM) figure of the functional mesoporous silicon dioxide carried medicine of embodiment 1 and the composite of siRNA.
Fig. 3 is the functional mesoporous silicon dioxide carried medicine of embodiment 3 and the composite cytotoxicity figure of siRNA.Wherein, MSNs-SS-Py is the mesoporous silicon oxide of functionalization, and DOX is amycin, and MSNsDOX is the functional mesoporous silicon dioxide of load amycin, and MSNs-SS-siRNADOX is the composite of load amycin and siRNA.
Fig. 4 is that the composite of functional mesoporous silicon dioxide carried medicine and siRNA is at Intracellular drug enrichment figure.Wherein, FreeDOX is free amycin, and MSNs-SS-siRNADOX is the composite of load amycin and siRNA.
Fig. 5 is the design sketch that the composite silent gene of functional mesoporous silicon dioxide carried siRNA is expressed.Wherein, MSNs is mesoporous silicon oxide, MSNs-SS-siRNA is mesoporous silicon oxide load siRNA, MSNs-SS-NCsiRNA is the out of order siRNA of mesoporous silicon oxide load, FreesiRNA is free siRNA, Lipo2000+siRNA is business liposome 2000 load siRNA, as positive control.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
As long as the raw materials used purity of the present invention reaches more than chemical pure, source all can be buied from market.
Embodiment 1: the preparation of the composite of functional mesoporous silicon dioxide carried medicine and siRNA
The preparation of mesoporous silicon oxide (MSNs): by cetyl trimethyl ammonium bromide (CTAB, 500mg) be dissolved in distilled water (250mL), then NaOH aqueous solution (1.75mL is added, 2Mol/L), mixed solution is heated to 80 DEG C with vigorous stirring.When after temperature stabilization, ethyl orthosilicate (TEOS, 2mL) is slowly added mixed solution.After 2h, nanoparticle leaves the heart 3 minutes by 10000 and collects, with ethanol and distilled water wash three times.For removing template, collect nanoparticle Eddy diffusion at ethanol (80mL) containing concentrating hydrochloric acid (4mL, 37%) in, reflux 24h at 80 DEG C, leave the heart by 10000 to collect for 3 minutes, with ethanol and distilled water wash three times, dried in vacuo overnight at nanoparticle 50 DEG C.Its pattern is observed by TEANAI-10 type transmission electron microscope (TEM), as shown in Figure 1.Its particle diameter is at about 80nm.
The above-mentioned MSNs (50mg) prepared to be dissolved in 3-mercaptopropyi trimethoxy silane (MPTMS) mixed solution of ethanol (20mL) and 1.8mL in a nitrogen atmosphere 50 DEG C stir 24h.Then, granule, by collected by centrifugation (10000 turns, 5min), obtains sulfhydrylation mesoporous silicon oxide (MSNs-SH).Mesoporous silicon oxide (MSNs-SH) (60mg) of sulfhydrylation is dispersed in 5mL methanol again (containing 0.1g2,2-dithiodipyridine) in, mixture at room temperature stirs 24h, nanoparticle passes through collected by centrifugation, with ethanol and water washing three times, dried in vacuo overnight at 50 DEG C, obtains mesoporous silicon dioxide nano particle that disulfide bond is modified.
The amycin aqueous solution of preparation 1g/L, mesoporous silicon dioxide nano particle (50mg) modified by above-mentioned disulfide bond is distributed in solution, ultrasonic 12h, and granule is by collected by centrifugation (10000 turns, 5min).Meanwhile, the siRNA (the Shanghai biological company limited of lucky horse) of sulfydryl modification is hatched 2h with dithiothreitol, DTT (DTT), and break the disulfide bond formed between siRNA, and then excessive DTT adopts glucosan post to slough.By the nanoparticle Eddy diffusion of load amycin at PBS buffer (pH7.4), hatch 2 times of equivalent siRNA-SH48h at 4 DEG C, leave the heart 10000 and collect for 5 minutes, obtain the composite of functional mesoporous silicon dioxide carried medicine and siRNA.Its pattern is observed by TEANAI-10 type transmission electron microscope (TEM), as shown in Figure 2.Its particle diameter, at about 100nm, still keeps some features of mesoporous silicon oxide, such as cavernous structure and spherical morphology.
Embodiment 2: the composite of functional mesoporous silicon dioxide carried medicine and siRNA is as the experiment in vitro of cancer therapy drug
This experimental selection human breast cancer cell (MCF-7, neat (Shanghai) the biological engineering company limited of match) is as the anticancer experimental subject of functional mesoporous silica medicine-carried system.
MTT method of testing: with 0.25% trypsin digestion and cell 5 minutes, blow and beat cell gently and become cell suspension in suspension device centrifuge tube, get 10 μ L cell suspending liquid rolling counters forward viable counts with suction pipe, adjustment viable cell concentrations is 5 × 10
3/ mL is added on 96 well culture plates, every hole 100 μ L, and cultivate after 24 hours, add the composite that variable concentrations embodiment 1 prepares respectively, be placed in 37 DEG C, volume fraction is 5%CO
2cultivate 24 hours.Within 4 hours, add MTT20 μ L/ hole before the end, abandoning supernatant after 4 hours, add DMSO100 μ L/ hole, vibrate about 15 minutes, microplate reader measures OD value, and wavelength is 450nm.By following formulae discovery survival rate, evaluate medicine and kill cancerous cell effect.Experimental result as shown in Figure 3.
Survival rate %=medicine feeding hole mean OD value/control wells mean OD value × 100%
Embodiment 3: the composite of functional mesoporous silicon dioxide carried medicine and siRNA is in Intracellular drug enrichment
By being in the MCF-7 cell of logarithmic (log) phase with after the trypsin digestion cell 3 ~ 5min of 0.25%, after centrifugalize, mixed by culture medium, inoculation 1 × 10
4individual cell, in the little groove of laser co-focusing ware, adds the culture fluid of 2mL, 5%CO again after 1h
2, in the incubator of 37 DEG C, cultivate 24h.Add amycin process cell 1h and 3h of composite 10 μ g/mL that embodiment 1 prepares and a great deal of respectively.Suck the culture medium in ware subsequently, after then rinsing three times with PBS, then add the PBS infiltrating cells of 1mL, adopt confocal laser scanning microscope to take pictures.As shown in Figure 4, compared to free amycin, the amycin in composite of the present invention can significantly improve amycin at intracellular enriching quantity, also significantly improves at the enriching quantity of nuclear area simultaneously, this just amycin play the region of drug effect.
Embodiment 4: the efficiency of the composite silent gene expression of functional mesoporous silicon dioxide carried siRNA
By being in the MCF-7 cell of logarithmic (log) phase with after the trypsin digestion cell 3 ~ 5min of 0.25%, after centrifugalize, mixed by culture medium, adjustment viable cell concentrations is 5 × 10
3/ mL is added in 6 orifice plates, 5%CO
2, in the incubator of 37 DEG C, cultivate 24h.After adding various drug incubation 24h, directly use RIPA lysate (containing PMSF) by lysis on ice, after placing 30min on ice, 12000rpm, 4 DEG C, the centrifugal rear absorption supernatant of 10min.Total protein BCA test kit carries out quantitatively, gets 1.5mL total protein and boil 5min together with sample-loading buffer, then frozen in-80 DEG C, for westernblot.Westernblot is briefly as follows: albumen loading is carried out electrophoretic separation albumen to SDS polyacrylamide gels.Wherein resolving gel concentration 10%, concentrated gum concentration 5%.Then albumen is carried out transferring film (wet turn), transferring film condition is constant current 250mA, 2h.And then film is cleaned 3 times in TBST, each 5min.Then add 5% defatted milk powder and carry out closed 1h.Film and primary antibodie 4 DEG C of solutions are educated and spends the night, clean 3 times through TBST, each 10min.Then by film and the anti-incubated at room 1h of HRP labelling two, wash paint 3 times through TBST, after each 10min, carry out ECL colour developing.As shown in Figure 5, functional mesoporous silicon dioxide carried siRNA can make siRNA enter cancerous cell, overcomes the shortcoming of siRNA treatment.Compared to other matched groups, the siRNA of nano load has the effect of better reticent genes of interest Bcl-2 expression; Meanwhile, the expression that Bcl-2 expresses the associated protein pro-caspase-9 of path is also affected, and shows that functional mesoporous silica medicine-carried system has the effect of good reticent target gene further.
Wherein, MSNs-SS-NCsiRNA is the out of order siRNA of mesoporous silicon oxide load, its preparation method is: out of order siRNA (the Shanghai biological company limited of lucky horse) is hatched 2h with dithiothreitol, DTT (DTT), break the disulfide bond formed between siRNA, and then excessive DTT adopts glucosan post to slough.Mesoporous silicon dioxide nano particle (50mg) that disulfide bond embodiment 1 prepared is modified is suspended in PBS buffer (pH7.4), siRNA48h after hatching 2 times of above-mentioned process of equivalent at 4 DEG C, leave the heart 10000 to collect for 5 minutes, obtain the out of order siRNA of mesoporous silicon oxide load.Because this siRNA is out of order, it to targeted silent corresponding gene, therefore can not possess activity.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a composite of functional mesoporous silicon dioxide carried medicine and siRNA, is characterized in that the method by comprising the following steps prepares:
Mesoporous silicon oxide is added in 3-mercaptopropyl trimethoxysilane solution, reacting by heating, is separated, obtains sulfhydrylation mesoporous silicon oxide, be distributed to 2, in 2-bis-sulfur two pyridine solution, reacting by heating, obtains the mesoporous silicon oxide that disulfide bond is modified, added in amycin aqueous solution, add the siRNA of sulfhydrylation again, be separated, obtain the composite of functional mesoporous silicon dioxide carried medicine and siRNA.
2. the composite of functional mesoporous silicon dioxide carried medicine according to claim 1 and siRNA, is characterized in that: in 3-mercaptopropyl trimethoxysilane used and mesoporous silicon oxide, the mol ratio of element silicon is 2:1 ~ 4:1.
3. the composite of functional mesoporous silicon dioxide carried medicine according to claim 1 and siRNA, is characterized in that: in 2,2-bis-sulfur two pyridines used and sulfhydrylation mesoporous silicon oxide, the mol ratio of element sulphur is 2:1 ~ 1:1.
4. the composite of functional mesoporous silicon dioxide carried medicine according to claim 1 and siRNA, is characterized in that: in the mesoporous silicon oxide that siRNA used and disulfide bond are modified, the mol ratio of element sulphur is 1:2 ~ 1:1.
5. the composite of functional mesoporous silicon dioxide carried medicine according to claim 1 and siRNA, is characterized in that: the concentration of described amycin aqueous solution is 0.8 ~ 1g/L.
6. the composite of functional mesoporous silicon dioxide carried medicine according to claim 1 and siRNA, is characterized in that: the concentration of described 3-mercaptopropyl trimethoxysilane solution is 1 ~ 1.2Mol/L.
7. the composite of functional mesoporous silicon dioxide carried medicine according to claim 1 and siRNA, is characterized in that: the concentration of described 2,2-bis-sulfur two pyridine solutions is 0.1 ~ 0.12Mol/L.
8. the composite of functional mesoporous silicon dioxide carried medicine according to claim 1 and siRNA, is characterized in that: described 3-mercaptopropyl trimethoxysilane solution is 3-mercaptopropyl trimethoxysilane toluene solution; 2,2-bis-described sulfur two pyridine solutions are 2,2-bis-sulfur two pyridine ethanol solution.
9. the composite of functional mesoporous silicon dioxide carried medicine according to claim 1 and siRNA, is characterized in that: described reacting by heating all reacts 12 ~ 24h at 80 ~ 83 DEG C.
10. the application in cancer therapy drug prepared by the functional mesoporous silicon dioxide carried medicine according to any one of claim 1 ~ 9 and the composite of siRNA.
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