CN103849652B - A kind of nano-carrier complex for microRNA targeted delivery and preparation method thereof and application - Google Patents
A kind of nano-carrier complex for microRNA targeted delivery and preparation method thereof and application Download PDFInfo
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Abstract
The invention discloses a kind of nano-carrier complex for microRNA targeted delivery and preparation method thereof and application.Described carrier complexes is formed nano composite system by Protamine sulfates., hyaluronic acid and purpose microRNA, and wherein purpose microRNA suppresses function Microrna analog (microRNA mimics) for having cancer.Positively charged Protamine sulfates. can be self-assembly of nano-carrier by electrostatic interaction and electronegative hyaluronic acid, wraps up microRNA, give microRNA and be effectively protected, it is to avoid microRNA is degraded by RNase in forming process.The present invention is using hyaluronic acid and protamine as microRNA carrier, and material source is wide, price is low, and preparation process is simple simultaneously, gentle；Constituent hyaluronic acid in nano-carrier, has the ability of active targeting tumor simultaneously, treats at microRNA and has a good application prospect in cancer.
Description
Technical field:
The invention belongs to nano material biomedical sector.The present invention relates to a kind of targeted delivery microRNA nano-carrier complex, be specifically related to the preparation method and application of the hyaluronic acid/protamine poly ion complexes nano-carrier of the microRNA of a kind of parcel tumor suppression function for magnetic target therapy breast carcinoma.
Background technology:
MicroRNA is a class length is the strand non-coding RNA of 18-25 core former times acid, it is widely present in eukaryote, be combined mainly through 3' end untranslated region (3'UTR) complementary pairing wholly or in part with said target mrna, cause that said target mrna degraded or translation are suppressed, thus in the expression of post-transcriptional level negative regulation target gene.Substantial amounts of scientific investigations showed that, microRNA the wide participation multiple physiology of organism and pathological process, such as ontogeny, cell proliferation, adjust and die and break up, metabolism, the adjustment of immunity and stress, tumor formation etc..
MicroRNA is unstable, the nuclease degradation being very easily widely present.The major obstacle of exploitation microRNA molecule medicine is in that how to make microRNA pass through cell membrane, and is delivered to the effect playing degraded target gene mRNA in cell.At present, although virus type microRNA carrier transmission efficiency is high, but biological safety limits its application.Non-viral microRNA carrier, carries out the methods such as transport improve the effect of microRNA to a certain extent including synthesis Nanoparticle Aggregate and liposome vectors.MicroRNA can play good effect crucially to ensure before microRNA is transported to target tissue and target cell its activity, be not degraded.Therefore, how to select suitable carrier to improve target microRNA and be carried along into the efficiency of linked groups's cell, and protect microRNA not to be degraded, be the difficult problem realizing microRNA treatment.
Breast carcinoma is one of modal malignant tumor of women, and its sickness rate has leapt to female malignant first, for the number one killer of women.Breast carcinoma has easy aggressiveness, easily the feature of transfer and easily recurrence.Current chemotherapy is the treatment means that breast carcinoma is main, it is known that chemotherapy exists great side effect.CD44 molecule is a kind of transmembrane glycoprotein of cell surface, is primarily involved in the adhesion of tumor cell and host cell and host matrix.Research finds that CD44 CD44 in invasion and metastasis of tumor process has played important effect.The expression of CD44 is relevant to the growth of breast carcinoma, infiltration, transfer and prognosis.Research display CD44 process LAN in breast carcinoma, provides microenvironment for the propagation of tumor cell, differentiation, transfer and invasion and attack.
Polymer non-virus carrier becomes the study hotspot of current genophore.Wherein, poly ion complexes (Polyioncomplex) nano-carrier micelle is also a focus of non-virus carrier and nanoassemble area research in recent years.The Nano microsphere that it is the polymer by two kinds of oppositely chargeds or biomacromolecule is composited by electrostatic interaction.As nano-carrier, poly ion complexes micelle is except having the feature of traditional polymer micelle, also has the advantage of its uniqueness: (1) medicine carrying is in extensive range, both can pass through electrostatic interaction or hydrogen bond action load hydrophilic medicament, it is also possible to load gene class medicine；(2) nano-carrier forming process carries out substantially in aqueous, mild condition, and preparation method is simple, and avoids the use of organic solvent, it is possible to eliminate the toxic and side effects that dissolvent residual causes.
Hyaluronic acid (Hyaluronicacid, HA) it is a kind of natural polyanionic polymer, have avirulence, non-immunogenicity, without causing the advantages such as inflammatory, good biocompatibility, biodegradable, it has become one of antitumor drug study hotspot as medicine and genophore.Hyaluronic acid can also combine with CD44 molecular specificity, has the ability of targeting breast carcinoma.Protamine sulfates. (Protaminesulfate), is a kind of cationic polypeptide, and its biological safety is good, is usually used in DNA concentration and transfection.Electronegative hyaluronic acid and positively charged protamine form nano-carrier by electrostatic interaction and wrap up microRNA.This nano-particle being self-assembly of by electrostatic interaction, avoid the bioactive reduction of microRNA that organic solvent causes with complicated preparation process, simultaneously, constituent hyaluronic acid in nano-carrier is a kind of natural targeting group, be conducive to targeting transport microRNA, improve the picked-up of target cell and target tissue, and carry out the research of antitumous effect, up to now, there is not yet report.
Summary of the invention:
It is an object of the invention to provide a kind of hyaluronic acid and Protamine sulfates. passes through electrostatic interaction, it is self-assembly of polyion nano-particle, wrap up microRNA simultaneously, forming the nano-carrier with microRNA delivery functions, it is quick and easy that this carrier has safe preparation process, and nanometer particle size is uniform, surface potential is suitable, microRNA bag load efficiency is high, and gene delivery is effective, gene silencing efficiency advantages of higher.
The present invention wraps up the hyaluronic acid/protamine polyion nano-carrier of microRNA, is according to certain mass ratio by hyaluronic acid and two components of Protamine sulfates., and by electrostatic interaction in water, autonomous dress forms many poly ion complexes.
Preferably, above-mentioned hyaluronan molecule amount is 230,000, and it has the function of targeting breast carcinoma MDA-MB-231 cell CD44 transmembrane protein, Protamine sulfates. derives from salmon, microRNA is low expression in breast carcinoma, has the miR-34a suppressing breast carcinoma to occur and develop, and its sequence is:
MiR-34a:Sense:UUCUCCGAACGUGUCACGUdTdT
Antisense:ACGUGACACGUUCGGAGAAdTdT
Wherein, hyaluronic acid concentration in aqueous is formulated as 2mg/mL, Protamine sulfates. concentration in aqueous is formulated as 1mg/mL, hyaluronic acid solution all mixes according to 1mL mutually slowly with protamine solution, wherein before mixing, microRNA join hyaluronic acid mutually in, microRNA makes consumption be 20 μ g, and forming hyaluronic acid and protamine mass ratio is 2:1.In conjunction with test result indicate that in the embodiment of the present invention, adopt above-mentioned quality proportioning, it is possible to make the present invention hyaluronic acid/protamine polyion nano-carrier particle diameter is little and is uniformly dispersed, nanometer productivity is the highest, microRNA parcel most effective.
The preparation method of a kind of hyaluronic acid/protamine nano combined carrier of many polyions wrapping load tumor suppression function microRNA, comprises the following steps:
(1) being joined by hyaluronic acid without in the water of RNase, making hyaluronic acid mass concentration is 2mg/ mL, crosses the biological filter membrane of 220nm.
(2) being joined by Protamine sulfates. without in the water of RNase, making hyaluronic acid mass concentration is 1mg/mL, crosses the biological filter membrane of 220nm.
(3) microRNA is dissolved in without, in the water of RNase, being configured to the concentration of 20 μ g/mL.
(4) being mixed with the hyaluronic acid solution in 1mL step (1) by the microRNA solution in 1mL step (3), vortex concussion uniformly, obtains mixed liquor.
(5) by the protamine solution in 1mL step (2), it is added drop-wise to slowly in step (4) in the mixed liquor obtained, and vortex concussion is uniformly, hatches 20-30 minute.
In preparation process of the present invention, in step (4), preferred hyaluronic acid and microRNA liquor capacity are 1mL, in step (5), the protamine solution of 1mL joins in above-mentioned mixed solution so that hyaluronic acid and protamine mass ratio are 2:1.
In preparation process of the present invention, microRNA used is the miR-34a of low expression in breast carcinoma, has the microRNA of the specific tumor suppression function for CD44 and notch-1.
Preferably, hyaluronic acid and protamine mass ratio control to be 2:1, and the mean diameter of the nano-particle of above-mentioned formation is 201.4nm, and surface zeta potential current potential is-23.6mV, and nano-particle productivity is 39%, and it is 87.2% that microRNA-34a wraps up efficiency.
Analyze from molecular structure, microRNA nano-carrier provided by the invention is the hyaluronic acid by band negative some lotus and positively charged protamine, by charge interaction, forms nano-particle, in the process forming nano-particle, wrap up into same electronegative microRNA.Wherein, hyaluronic acid and protamine molecule are wound around mutually, form spherical nanoparticle, can analyze from the surface zeta potential current potential-23.6mV of the nano-particle obtained, and nano grain surface is mainly made up of electronegative hyaluronic acid.Negative charge hyaluronic acid is in the periphery of nano-particle, toxicity can be reduced on the one hand, improve nano-carrier stability in vivo, on the other hand, hyaluronic acid can provide good targeting, the breast cancer cell MDA-MB-231 that targeting surface C D44 transmembrane protein is abundant.
The microRNA nano-particle of the present invention has good biocompatibility, and cytotoxicity is less.Nano-particle can be formed by self assembly mode, there is suitable physicochemical property, good stability; preparation method is simple, repeatable high, microRNA can be protected from degraded as nano-carrier; can, in conjunction with the distinctive dimensional effect of nano material, be a kind of good microRNA genophore again.
On the other hand, the invention provides a kind of novel targeted complex for therapy of tumor, to solve the difficult problem in the Biological target therapy of oncotherapy particularly three negative breast cancer.Wherein protamine plays function served as bridge, it has the function of bind nucleic acid, other two kinds of electronegative compositions can be combined formation complex, hyaluronic acid has the effect identifying target cell surface specific receptor, entrained microRNA then can play the effect of reticent genes of interest, thus this novel complexes can target recognition of tumor cell and reticent key gene thus playing the effect of Diseases Tumor treatment.
Advantages of the present invention
Nano-carrier raw material hyaluronic acid used in the present invention and protamine, be all that bio-safety performance is good, biodegradable natural polymer, and it is to be widely used in biological industry.Meanwhile, parcel microRNA nano-carrier preparation process is simple, convenient, it does not have to introduce synthesis and the organic solvent of any complexity, can be effectively protected the microRNA impact by these complicated factors.Meanwhile, it is capable in a straightforward manner, the function of targeting transport microRNA is introduced.By the parcel microRNA nano-carrier of tumor suppression function prepared by the present invention, its can wrap up can specificity for the microRNA of various diseases and siRNA (siRNA), by the mode of tail vein injection, the treatment of disease particularly cancer can be carried out.
Accompanying drawing explanation
The dynamic optical of the nano-carrier that Fig. 1 is formed in optimal conditions shines and transmission electron microscope picture.
Fig. 2 A is after parcel miR-34a hyaluronic acid/protamine nano-particle transfectional cell, it was demonstrated that miR-34a proceeds to intracellular result figure, takes pictures under fluorescence microscope, and DAPI is by nuclei dyeing au bleu, and the miR-34a of red fluorescence labelling occurs in Cytoplasm.
Fig. 2 B is before parcel miR-34a hyaluronic acid/protamine nano-particle transfectional cell, first the CD44 receptor of cell surface is made to neutralize with hyaluronic acid incubated cell, take pictures under fluorescence microscope, DAPI is by nuclei dyeing au bleu, and the miR-34a of red fluorescence labelling occurs in quantity in Cytoplasm and significantly reduces.Visible parcel miR-34a hyaluronic acid/protamine nano-particle tool contacts with each other with CD44, thus having target function.
Fig. 3 is the result figure of the suppression of parcel miR-34a hyaluronic acid/protamine nano-particle on cell proliferation.Compared with PBS control, nano-carrier substantially suppresses cell proliferation.
Fig. 4 is the result figure that parcel miR-34a hyaluronic acid/protamine nano-particle on cell migration suppresses.Compared with PBS control, nano-carrier substantially suppresses cell migration.
Fig. 5 is parcel miR-34a hyaluronic acid/protamine nano-particle inhibition to cell target protein.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
The preparation of the hyaluronic acid of embodiment 1 blank/protamine nano-particle.
The hyaluronic acid (east, Jiangsu biology company limited of unit) that 2mg molecular weight is 230,000 being dissolved in 1mL without, in the water of RNase, being configured to the liquid of 2mg/mL, by this solution by 200nm filter membrane, removing impurity, thus obtaining the working solution of 2mg/mL.1mg Protamine sulfates. (catalog number: PS4020, SigmaAdrich company) being dissolved in 1mL without in the water of RNase, by 200nm filter membrane, removing impurity, thus obtaining the working solution of 1mg/mL.In order to prepare blank hyaluronic acid/protamine nano-particle (hereinafter referred to as BlankHA/PS), according to different mass ratio (HA:PS=1:4,1:3,1:2,1:1,1:1.5,2:1,3:1,4:1) mixed transparent matter acid solution and protamine solution slowly, stirring mixed solution 20min, allows the sufficient of the two interact and the stable nano-particle formed.Afterwards the mixed solution obtained is centrifuged 15min with the centrifugal speed of 13000rpm, removes supernatant, the nano-particle stayed after being centrifuged.The centrifugal product lyophilizing that will finally give, weighs, and compares calculating with the raw material quality added, obtains nano-particle productivity.
The hyaluronic acid of embodiment 2 blank/protamine nano particle diameter size, the sign of surface zeta potential current potential and polydispersity coefficient.
Nanometer product that will obtain in 1, is redissolved in water, is configured to the concentration of 0.1mg/mL, by the particle diameter of ZetasizerNanoZS (Malvern, Worcestershire, U.K.) test compound thing and surface zeta potential current potential.The parameter of test particle diameter is 25 DEG C, 173 ° of angle of scatterings.The mensuration of surface potential is based on nanoparticle electrophoretic mobility in the solution, measures in automatic mode.
Embodiment 3 preferably goes out most suitable ratio, is used for wrapping up microRNA.
By particle diameter in the yield and 2 of the nano-particle of each ratio in 1, the comparative analysis of surface zeta potential current potential and polydispersity coefficient, result is as shown in table 1.Table 1 is the test of hyaluronic acid and protamine solution mixed proportion, and the particle diameter under various ratio, surface zeta potential current potential, the sign of polydispersity coefficient and nanometer productivity, and the most suitable nano-carrier of optimization forms ratio.
Drawing when HA:PS=2:1, obtaining nanometer particle size suitable (about 201.4nm), be uniformly dispersed (about PDI=0.2), and of a relatively high nano-particle productivity.When HA:PS<when 2:1, nano particle diameter is big and dispersion uneven with, at HA:PS>2:1, nano particle diameter is suitable, but nanometer yield is relatively low.Therefore, in the experiment and biotic experiment of follow-up parcel microRNA, carry out when choosing HA:PS=2:1.
Embodiment 4 wraps up the preparation of miR-34a hyaluronic acid/protamine nano-particle (hereinafter referred to as miR-HA/PS)
By 20 μ gmiR-34a(Guangzhou Rui Bo biotech companies) it is dissolved in 1mL without, in the water of RNase, making the working solution that concentration is 20 μ g/mL.MicroRNA working solution is mixed with the hyaluronic acid working solution of 1mL2mg/mL, then the protamine working solution of 1mL1mg/mL is added drop-wise to slowly in microRNA and hyaluronic acid working solution, be slowly stirred 20-30min.Same, the nano-particle obtained is centrifuged, retains supernatant and give over to follow-up test.Centrifugal product is dispersed in water again, namely obtains the nano-particle of parcel microRNA.
The test of embodiment 5MicroRNA bag load efficiency
By the supernatant analysis in embodiment, with ultraviolet spectrophotometer (PerkinElmer, Fremont, CA, USA) under 260nm wavelength, test the residual volume of microRNA in supernatant, compare with input amount, and then calculate the parcel efficiency of microRNA.
The hyaluronic acid of embodiment 5 cellular uptake parcel miR-HA/PS/protamine nano-particle ability detection
1, cell is cultivated and inoculation
The present invention uses breast cancer cell line MDA-MB-231 as cell experiment object, Chinese Academy of Sciences's cell centre provide, and uses containing 10% hyclone, DMEM culture medium (Invitrogen company), in 37 DEG C, and 5%CO2(v/v), the incubator (Thermo Fischer Scient Inc.) of 70% humidity is cultivated.By 2 × 105Individual MDA-MB-231 cell is inoculated in the culture dish (NEST Biotechnology Co., Ltd.) of diameter 20mm, glass thickness 0.13-0.17mm, incubated overnight.
2, miR-HA/PS nano-particle adds cell culture medium and observation
MiR-HA/PS nano-particle and 1ml plasma-free DMEM medium are mixed in 1.5ml is without RNase doffer's pipe, adding in the culture dish of MDA-MB-231 cell attachment growth, the final concentration of 100nM of miR-34a, to add PBS for negative control, it is placed in 37 DEG C, 5%CO2(v/v), the incubator of 70% humidity is cultivated, after 6h, culture medium is changed into containing 10% hyclone, DMEM complete medium, in 37 DEG C, 5%CO2(v/v), after the CMC model 24h of 70% humidity, old culture medium is removed, PBS washed cell 3 times, each culture dish adds 1ml4% paraformaldehyde fixative, fixing 20min, removes fixative, PBS washs 3 times, each culture dish adds 1mlDAPI dyeing liquor (green skies biotechnology research institute), and dye 5min, removes DAPI dyeing liquor, PBS washs 3 times, each 2min.Fluorescence inverted microscope (Lycra company of Germany) observes miR-34a in the intracellular distribution situation of MDA-MB-231.Result is as shown in Figure 2 A, it was shown that use miR-HA/PS material to be delivered in cell by the miR-34a that marked Cy3.Fig. 2 B is before miR-HA/PS nano-particle as above method transfectional cell, first within 1 hour, makes the CD44 receptor of cell surface neutralize with hyaluronic acid incubated cell, and the miR-34a of red fluorescence labelling occurs in quantity in Cytoplasm and significantly reduces.Illustrate that protamine-hyaluronic acid-miR-34a nano-particle has good targeting.
The suppression of embodiment 6miR-HA/PS nano-particle on cell proliferation
The present invention adopts CCK-8 cell viability test kit (east Renhua Science and Technology Ltd.) to detect the suppression of nano-particle on cell proliferation.It is embodied as step as follows: with every hole 1 × 104MDA-MB-231 is inoculated in 96 orifice plates (GreinerBio-one company) by the inoculum concentration of individual/100 μ l, uses containing 10% hyclone, DMEM culture medium (Invitrogen company), in 37 DEG C, and 5%CO2(v/v), overnight incubation in the incubator (Thermo Fischer Scient Inc.) of 70% humidity.Remove the old culture medium in 96 orifice plates, add 100 μ l serum-frees, DMEM culture medium.Adding in miR-HA/PS nano-particle extremely each hole by the amount of final concentration of 200nM, each concentration arranges 6 multiple holes, with PBS for comparison.37 DEG C, 5%CO2Change into after cultivating 6h containing 10% hyclone, DMEM culture medium, continue to cultivate 24h, 48h, 72h, respectively CCK-8 is added in each hole with the amount of the 10% of every hole culture volume, namely 100 μ l culture medium add 10 μ lCCK-8, hatch 1h for 37 DEG C.Multi-functional microwell plate detector is used to measure every hole absorbance at 450nm place.The result hyaluronic acid of miR-34a as shown in Figure 3/obvious cell growth inhibiting of protamine nano-particle.
The suppression of embodiment 7miR-HA/PS nano-particle on cell migration
After MDA-MB-231 cell transfects 24 hours in aforementioned manners, digest from 6 orifice plates, and resuspended by serum-free DMEM in high glucose culture medium, by 1x105Individual/hole is seeded in Transwell(coring company) the upper room of cell, add the 500ul DMEM culture medium containing 10% hyclone in lower room and induce it to migrate across the poly-carbonic acid vinegar film in 8 μm of apertures.37 DEG C, 5%CO2Cultivate 12 hours.Take out cell, wipe face, upper room cell with cotton swab gently.Fixing lower face, room cell 10min under the awake room temperature of 4% poly first.PBS washes 3 times, and face, room dried by cotton swab.0.1% violet staining 15min, PBS washes 3 times, air-dry under room temperature.It is placed in basis of microscopic observation to take pictures.Result as shown in Figure 4, compare PBS control and substantially suppress cell migration by miR-HA/PS nano-particle.
Last institute is it should be noted that, above example is only in order to illustrate technical scheme but not limiting the scope of the invention.All employings are equal to replacement or the technical scheme of equivalent transformation formation, all fall within the protection domain of application claims.Table 1
Claims (2)
1. the preparation method for the nano-carrier complex of microRNA targeted delivery, it is characterised in that comprise the following steps:
(1) being joined by hyaluronic acid without in the water of RNase, making hyaluronic acid mass concentration is 2mg/mL；Hyaluronan molecule amount is 230,000；
(2) being joined by Protamine sulfates. without in the water of RNase, making Protamine sulfates. concentration of polymer solution is 1mg/mL；
(3) microRNA is dissolved in without, in the water of RNase, being configured to the microRNA solution of 20 μ g/mL concentration；
(4) being mixed with the hyaluronic acid solution in 1mL step (1) by the microRNA solution in 1mL step (3), vortex concussion uniformly, obtains mixed liquor；
(5) by the Protamine sulfates. solution in 1mL step (2), it is added drop-wise in step (4) in the mixed liquor obtained, and vortex concussion is uniformly, hatches 20-30 minute；
MicroRNA used is miR-34a, the MiR-34a:Sense:UUCUCCGAACGUGUCACGUdTdT of low expression in breast carcinoma
Antisense:ACGUGACACGUUCGGAGAAdTdT。
2. the nano-carrier complex described in claim 1 is being used for preparing the application of mastocarcinoma gene treatment targeted drug.
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