CN110327308A - A kind of recombination apoferritin nanocages and preparation method thereof being loaded with siRNA - Google Patents
A kind of recombination apoferritin nanocages and preparation method thereof being loaded with siRNA Download PDFInfo
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- CN110327308A CN110327308A CN201910592070.3A CN201910592070A CN110327308A CN 110327308 A CN110327308 A CN 110327308A CN 201910592070 A CN201910592070 A CN 201910592070A CN 110327308 A CN110327308 A CN 110327308A
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- 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
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5169—Proteins, e.g. albumin, gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses a kind of recombination apoferritin nanocages and preparation method thereof for being loaded with siRNA, the nanocages include recombinating apoferritin nanocages and containing in the siRNA molecule in recombination apoferritin nanocages；The recombination apoferritin nanocages surface modification has oligomerization lysine.The present invention is using the recombination apoferritin nanocages with lysosome escape function as carrier; siRNA is contained using depolymerization/recombination method that pH changes; obtained nanoparticle size uniformity; with active targeting; it can protect siRNA that enzyme is avoided to degrade, while there is lysosome escape capability and excellent biocompatibility.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of recombination apoferritin nanocages for being loaded with siRNA
And preparation method thereof.
Background technique
In recent years, gene therapy gradually causes the extensive concern of people as a kind of new tool for the treatment of.RNA interference
(RNA interference, RNAi) is the sequence specific gene silencing by double chain RNA mediate, in gene therapy and drug
It has been widely used in exploitation.There are three types of strategies by RNAi: short hairpin RNA (shRNA), endogenous microRNA(miRNA) He little Gan
Disturb RNA(siRNA).Wherein, siRNA is not because needing genome conformity and being readily synthesized, and is widely used as gene therapy medicament.
SiRNA is the double-stranded RNA of one section of about 21-25 base pairs length, enters after cell that be combined into RNA induction with enzyme-specific heavy
Silent compound (RNA-induced silencing complex, RISC), then in the effect of ATP- dependence helicase
Lower opening double-strand, antisense strand activate RISC, then in conjunction with specific purpose mRNA, so that mRNA decomposes to reach downward purpose
Protein quantity, cytostatic gene silencing effect.But because siRNA relative molecular mass is larger, has height negative electrical charge,
It is difficult to it across cell membrane, and it enters after born of the same parents easily by cellular enzymatic cleavage, to be difficult to realize specific gene silencing.Therefore structure
It is particularly critical to build safe and efficient siRNA delivery vector.The carrier material of delivering siRNA can be divided into viral vectors and non-at present
Viral vectors two major classes.Although viral vectors has extremely strong transfection efficiency, there are still certain limitations, such as immunogene for it
The problems such as property is strong, cytotoxicity is big；And non-virus carrier is better than viral vectors because its is at low cost, prepares simple and safety, and
It is increasingly being used for gene therapy, but there are still deficiencies at present for non-virus carrier --- transfection efficiency is low and exists certain
Safety issue.
As a kind of endogenous protein, the biocompatibility of apoferritin nanocages, internal stability are superior to most of
The nano-carrier of inorganic or organic material building；In addition, can mass production preparation and reorganization using genetic engineering recombination and expression techniques
Protein cage, and modification easily can be carried out to surface and make it have special function；It can be expressed with tumor cell surface height
TfR 1(transferrin receptors 1, TfR1) occur specific recognition, realize active targeting；Benefit
With its sensibility to pH, it can make protein cage that depolymerization and recombination occur by changing pH value of solution, to carry out containing for drug.This
A little superior performances make apoferritin nanocages become a kind of ideal Nano medication delivery vector.But unmodified de-iron
After protein nano cage swallows born of the same parents in, it will usually rest in lysosome, existing a large amount of enzymes will cause protein cage in lysosome
Structural damage, and then degrade or destroy and contain in drug wherein.So realizing that lysosome escape is by apoferritin nanometer
Cage is used for the steps necessary of siRNA delivering.
Summary of the invention
The purpose of the present invention is being directed to the deficiencies such as existing siRNA delivery vector safety is poor, transfection efficiency is low, one is provided
The new siRNA carrier system of kind.Using the recombination apoferritin nanocages with lysosome escape function as carrier, changed using pH
Depolymerization/recombination method contain siRNA.Obtained nanoparticle size uniformity has active targeting, siRNA can be protected to avoid
Enzyme degradation, while there is lysosome escape capability and excellent biocompatibility.
In order to solve the above technical problems, The technical solution adopted by the invention is as follows:
A kind of recombination apoferritin nanocages being loaded with siRNA, including recombinate apoferritin nanocages and contain in recombination de-iron
SiRNA molecule in protein nano cage；
The recombination apoferritin nanocages surface modification has oligomerization lysine.
Further, the number of the oligomerization lysine is 4 lysines.
Further, the recombination apoferritin nanocages partial size for being loaded with siRNA is 10-40 nm.
The above-mentioned recombination apoferritin nanocages for being loaded with siRNA, comprising the following steps:
Step 1, DNA recombinant expression technology preparation and reorganization apoferritin nanocages are utilized；
Step 2, the recombination apoferritin nanocages that step 1 obtains are configured to solution, adjust pH to 1.0-2.5, makes to recombinate
Apoferritin nanocages are sufficiently depolymerized to subunit；
Step 3, siRNA solution is added into the solution that step 2 obtains, is uniformly mixed；
Step 4, the pH to 7.0-10.0 of 3 mixed solution of regulating step, shaking, makes subunit be reassembled into recombination apoferritin
Nanocages to get.
Further, in step 1 preparation and reorganization apoferritin nanocages detailed process are as follows: first in FTH gene code sequence
5 ' terminal modified lysine encoding genes of column are subcloned into plasmid vector pET-30a (+), and are converted extremelyE.coli
ArcticExpressTMCompetent cell, by Kanamycin resistance screening positive monoclonal transformant, to obtain purpose recombination
Protein cage expresses engineering bacteria；Then expression engineering bacteria is inoculated in LB-Kan with certain proportion+In culture medium, IPTG induction is added
Expression, thalline were collected by centrifugation；Supernatant is collected by centrifugation after ultrasonic disruption, supernatant is placed in 50-70 DEG C of heating water baths, again from
The heart is obtained containing there is the supernatant of purpose fusion protein, using affinity chromatography method in supernatant foreign protein and purpose merge
Albumen carries out isolation and purification, obtains recombination apoferritin nanocages.
Further, the molar ratio that apoferritin nanocages and siRNA are recombinated in step 3 is 30:1-1:1.
Further, it is 10-37 DEG C, 2-24 h that condition is shaken in step 4.
SiRNA delivery system of the invention has excellent biocompatibility and stability；It can be with tumor cell surface height
Specific recognition occurs for the TfR1 of expression, and realizes under the action of receptor-mediated and effectively enter born of the same parents；Into after lysosome, surface is repaired
By " proton sponge effect " lysosome escape occurs for the recombination apoferritin nanocages for being decorated with lysine, protects in cage
SiRNA increases siRNA in intracytoplasmic concentration, improves therapeutic effect from degradation.
Preparation method is simple for delivery system of the invention, provides a kind of reason for the efficient targeting delivering of genomic medicine
The multifunctional nano-carrier thought, with good application prospect.
Detailed description of the invention
Fig. 1 is the SDS-PAGE figure of 4L-HFn each efflux in affinity chromatography method purification process in embodiment 1.
Fig. 2 is 4L-HFn@siRNA nanoparticle grain size distribution prepared in embodiment 1.
Fig. 3 is 4L-HFn@siRNA nanoparticle transmission electron microscope picture in embodiment 1.
Fig. 4 be embodiment 2 in 4L-HFn@siRNA nanoparticle digest stability experiment (A), serum stability test (B) and
Storage stability tests (C).
Fig. 5 is that 4L-HFn@siRNA nanoparticle lysosome escape capability investigates figure in embodiment 3.
Specific embodiment
Technical solution of the present invention is described further combined with specific embodiments below.
In the following embodiments, recombination apoferritin nanocages general formula is 4L-HFn, wherein L indicates lysine, and 4 be bad
For propylhomoserin in the modification number of single protein protomer N-terminal, HFn refers to the protein nano cage being self-assembly of by 24 FTH.
Embodiment 1
A kind of preparation method for the recombination apoferritin nanocages (4L-HFn@siRNA) being loaded with siRNA, comprising the following steps:
Step 1, DNA recombinant expression technology preparation and reorganization apoferritin nanocages are utilized
Detailed process is as follows:
(1) building of recombination apoferritin expression engineering bacteria: based on FTH encoding gene in NCBI, in the gene code sequence
5 ' ends of column are plus 4 lysine encoding genes (AAGAAAAAGAAA) shown in SEQ ID NO.1, then after the modification
5 ' the ends and 3 ' ends of gene coded sequence are respectively plus CCATGGCT and GCGGCCGC to obtain Nco I and Not I digestion identification
Site finally optimizes gene order, and the sequence is made to can be realized correct expression in E. coli system.Sequence length
The 4L-HFn protein protomer is encoded as shown in SEQ ID NO.2 for 580 bp.Using Nco I and Not I to synthesis
Sequence carries out double digestion, and carries out same double digestion to plasmid vector pET-30a (+), overnight by 4 DEG C of T4 DNA ligase
Two kinds of digestion products are connected, which is synthesized by Nanjing Jin Sirui company.Connection product plasmid is transferred to greatly by heat shock
Enterobacteria ArcticExpressTMIn, and positive monoclonal is gone out by kanamycin resistance screening and is saved, to obtain purpose
4L-HFn expresses engineering bacteria.
(2) expression, separation and the purifying of apoferritin are recombinated: micro positive engineering bacteria being taken to be inoculated in fresh LB-Kan+
In culture medium, 12-20 h of oscillation activation, take 4 mL bacterium solutions to be transferred to 200 mL LB-Kan under the conditions of 37 DEG C+Culture medium
In, 37 DEG C of 5 h of shaken cultivation.IPTG to final concentration of 0.1 mM, 37 DEG C of 8 h of shaken cultivation are added, de-iron egg is recombinated with induction
White expression.After induction, 30 mL Binding Buffer(20 mM Tris, 5 mM miaows are added to collect thallus in centrifugation
Azoles, 0.5 M NaCl, pH 7.9) it is resuspended, it is centrifuged again after resuspension, abandons supernatant and collect thallus, be resuspended in 30 mL again
Lysozyme is added in Binding Buffer, and final -20 DEG C freeze.It takes and freezes the thawing of thallus room temperature, in 70 DEG C of water after carrying out ultrasonic bacteria breaking
Bath heating removes heat labile foreign protein, after supernatant is collected by centrifugation, obtain the supernatant containing destination protein.It is affine using nickel column
Chromatography purifies destination protein.Resin is added into chromatographic column, using NiSO is added after sterile water wash4Solution carries out
In conjunction with after resin greening, using Binding Buffer cleaning balance chromatographic column.With certain flow rate loading, make albumen and tree
Rouge sufficiently combines.After end of the sample with Binding Buffer balance chromatographic column, then with gradient imidazole solution (30 mM, 50 mM,
70 mM, 90 mM, 300 mM) successively elute foreign protein and destination protein.300 mM eluents are taken to carry out ultrafiltration (100 kDa),
Imidazoles and other salt ions are removed, recombination apoferritin after purification is finally stored in storage Buffer(0.15 M NaCl,
20 mM Tris, pH 8.0) in.As shown in Figure 1, SDS-PAGE is at 29 kDa the result shows that there is apparent band, this with again
Quite, molecular weight is consistent group apoferritin molecular weight subunit (26.80 kDa), illustrates to recombinate apoferritin that successful expression has been simultaneously
High-purity purifying.Gained 4L-HFn amino acid sequence is as shown in SEQ ID NO.3.
The concentration of IPTG is 0.01-3 mmol/L in step (2), and centrifuging temperature is 0-37 DEG C, and supernatant water-bath adds
Hot temperature is 50-70 DEG C.
Step 2, the HFn(8.0 mg/mL of 120 μ L pH 8.0 is taken) solution, addition HCl solution adjusting pH value of solution to 2.0,
Albumen is set to be depolymerized to 24 subunits.
Step 3,40 μ L siRNA(10 nmol/mL are added into step 2) solution, 200 rpm, shakes under the conditions of 25 DEG C
Swing 2 h.
Step 4, it using the pH to 8.0 of solution in the slow regulating step 3 of NaOH solution, is uniformly mixed, in 200 rpm, 25
8 h of shaken cultivation under the conditions of DEG C.Oscillation is concentrated by ultrafiltration after terminating using the super filter tube of 100 kDa, finally will be obtained
Nanoparticle is stored in 4 DEG C of refrigerators.
Using the partial size of the prepared 4L-HFn@siRNA nanoparticle of Malvern particle size determination instrument measurement, as shown in Fig. 2, institute
The nanoparticle partial size of preparation is about 20 nm, and size distribution is uniform.4L-HFn@siRNA nanoparticle is carried out by transmission electron microscope
Morphological characterization, as shown in figure 3, about 12-16 nm of partial size of 4L-HFn@siRNA nanoparticle, uniform particle diameter are uniformly dispersed
And there is spherical cage structure, the albumen after illustrating depolymerization can succeed self assembly.
Embodiment 2
Enzyme stability to degradation, serum stability and the storage stability of 4L-HFn@siRNA is investigated
Enzyme degradation experiment: the principal element for hindering siRNA effectively to deliver in vivo is the degradation of nucleic acid in vivo enzyme.In order to assess this
Protective effect of the nanoparticle to siRNA, by naked siRNA and the nanoparticle (siRNA, 50 ng) of embodiment 1 respectively with RNase A
(5 μ L, 3 mg/mL) is incubated at 37 DEG C, in different time (0,2,4,8,16 and 24 h) sampling.Using 1% agarose
Gel electrophoresis is investigated.Gel runs 15 min under 100 V voltages, is imaged afterwards by Chemidoc XRS+ imager.Such as Fig. 4
Shown: the siRNA encapsulated by recombination apoferritin speed of service ratio siRNA in gel rubber system is slow, the band 2 for the siRNA that dissociates
H degrades complete；And compared with 0 h, significant change does not occur for the band brightness of nanoparticle obtained by 2,4,8,16,24 h, says
Bright 4L-HFn@siRNA stablizes under Nuclease R Nase A existence condition, can protect siRNA that it is made not degraded by nuclease.
Serum stability experiment: by the nanoparticle and the PBS solution (10% fetal calf serum, pH 7.4) containing serum in 37 DEG C
It is incubated with, in different time (0,2,4,8,16 and 24 h) sampling.Using in the agarose gel electrophoresis test sample of 1 %
SiRNA intensity is to assess the stability of nanoparticle.Gel runs 15 min under 100 V voltages, then passes through Chemidoc
XRS+ imaging.As shown in Figure 4: in the solution of BS containing blood-serum P, the band brightness of free siRNA extends at any time and gradually weakens,
Indicate that it is gradually degraded；And significant change does not occur at any time for the band brightness of 4L-HFn@siRNA, illustrates in physiological condition
In lower 24 h, 4L-HFn@siRNA nanoparticle is stablized.
Storage stability experiment: the nanoparticle is placed in 4 DEG C of refrigerators and is placed, is taken in different time (0,1,2,4 week)
Sample.SiRNA intensity in the agarose gel electrophoresis test sample of 1 % is used to assess the stability of nanoparticle.Gel is in 100 V
15 min are run under voltage, are then imaged by Chemidoc XRS+.As shown in Figure 4: the band brightness of 4L-HFn@siRNA is not
Significant change occurs at any time, the siRNA being encapsulated in 4L-HFn is stored 4 weeks at 4 DEG C, do not show detectable leakage or
Degradation illustrates that 4L-HFn@siRNA nanoparticle is stablized under condition of storage.
Embodiment 3
The lysosome escape Performance of 4L-HFn siRNA:
The lysine for recombinating the modification of apoferritin nanocages subunit N-terminal generates " proton sponge effect under acid lysosome environment
Answer " lysosome escape occurs.This example contains Cy5-siRNA in recombination apoferritin nanocages (4L-HFn), investigates 4L-
The lysosome escape capability of HFn@Cy5-siRNA nanoparticle, the nanoparticle include recombination apoferritin nanocages shell and
Cy5-siRNA kernel.
By Hela cell with 2 × 105The density of/ware is inoculated into culture dish and is incubated for 24 h.Then 4L-HFn@Cy5- is used
SiRNA(siRNA concentration is 200 nM) processing cell.Respectively after 2,4 and 6 h of incubation time, cell is mildly washed with PBS, and
Diluted with Lyso-Tracker Red(1: 5000) in 37 DEG C of 2 h of dyeing.Next with 4% paraformaldehyde, the cells are fixed 10
Min then uses Hoechst 33342(10 μ g/ mL at room temperature) 15 min are handled by nuclear targeting.Finally, being washed with PBS
Cell is washed, and shows lysosome flight behavior using Zeiss confocal laser scanning microscope, CLSM.The oil immersion eyeglass amplified with 63 times
Obtain image.As shown in figure 5, green is lysosome dye colour (Lyso-Tracker Red), blue is nucleus dyestuff face
Color (Hoechst 33342), red are Cy5-siRNA, and Merge is blue, green, red stacking chart.When 2 h, it is rendered as
Red fluorescence and part fluorescent orange, declaratives 4L-HFn@Cy5-siRNA successfully enter born of the same parents, and start to melt with lysosome
It closes, illustrates that the existing part of 4L-HFn@Cy5-siRNA nanoparticle enters lysosome at this time.With the extension of time, when 4 h, it is red
Fluorescence increases, and illustrates that 4L-HFn@Cy5-siRNA enters the amount of cell and increases, while more fluorescent orange occur, illustrates big
Part 4L-HFn@Cy5-siRNA nanoparticle enters in lysosome.Fluorescent orange is reduced when 6 h, while red in cell cytosol
Phosphor dot increases, and illustrates that 4L-HFn@Cy5-siRNA success is escaped from lysosome.
Sequence table
<110>China Medicine University
<120>a kind of recombination apoferritin nanocages and preparation method thereof for being loaded with siRNA
<130> 2019
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gggagagcgg tctgaacgcg atggagtgcg cgctgcacct ggaaaagaac gttaaccaga 360
gcctgctgga actgcacaag ctggcgaccg acaaaaacga tccgcacctg tgcgacttca 420
tcgagaccca ctacctgaac gaacaagtga aggcgattaa agagctgggt gatcacgtta 480
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Lys Lys Met Thr Thr Ala Ser Thr Ser Gln Val Arg Gln Asn Tyr His
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Gln Asp Ser Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu Glu Leu Tyr
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Claims (7)
1. a kind of recombination apoferritin nanocages for being loaded with siRNA, it is characterised in that: including recombination apoferritin nanocages and packet
The siRNA molecule being loaded in recombination apoferritin nanocages；
The recombination apoferritin nanocages surface modification has oligomerization lysine.
2. the recombination apoferritin nanocages according to claim 1 for being loaded with siRNA, it is characterised in that: the oligomerization relies
The number of propylhomoserin is 4 lysines.
3. the recombination apoferritin nanocages according to claim 1 for being loaded with siRNA, it is characterised in that: described to be loaded with
The recombination apoferritin nanocages partial size of siRNA is 10-40 nm.
4. the preparation method of the recombination apoferritin nanocages described in claim 1 for being loaded with siRNA, it is characterised in that: including
Following steps:
Step 1, DNA recombinant expression technology preparation and reorganization apoferritin nanocages are utilized；
Step 2, the recombination apoferritin nanocages that step 1 obtains are configured to solution, adjust pH to 1.0-2.5, makes to recombinate
Apoferritin nanocages are sufficiently depolymerized to subunit；
Step 3, siRNA solution is added into the solution that step 2 obtains, is uniformly mixed；
Step 4, the pH to 7.0-10.0 of 3 mixed solution of regulating step, shaking, makes subunit be reassembled into recombination apoferritin
Nanocages to get.
5. the preparation method according to claim 4, it is characterised in that: preparation and reorganization apoferritin nanocages in step 1
Detailed process are as follows: first in 5 ' terminal modified lysine encoding genes of FTH gene coded sequence, subclone to plasmid vector pET-
In 30a (+), and convert extremelyE.coliArcticExpressTMCompetent cell passes through Kanamycin resistance screening positive Dan Ke
Grand transformant, to obtain purpose recombinant protein cage expression engineering bacteria；Then expression engineering bacteria is inoculated in LB- with certain proportion
Kan+In culture medium, IPTG inducing expression is added, thalline were collected by centrifugation；Supernatant is collected by centrifugation after ultrasonic disruption, supernatant is set
In 50-70 DEG C of heating water baths, centrifugation is obtained containing the supernatant for having purpose fusion protein, using affinity chromatography method pair again
Foreign protein and purpose fusion protein in supernatant carry out isolation and purification, obtain recombination apoferritin nanocages.
6. the preparation method according to claim 4, it is characterised in that: in step 3 recombinate apoferritin nanocages with
The molar ratio of siRNA is 30:1-1:1.
7. the preparation method according to claim 4, it is characterised in that: the condition that shakes in step 4 is 10-37 DEG C, 2-
24 h。
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| CN112342207A (en) * | 2020-11-27 | 2021-02-09 | 华侨大学 | Method for purifying recombinant xylanase by low-speed centrifugation |
| CN112826943A (en) * | 2021-01-14 | 2021-05-25 | 齐鲁工业大学 | Protein nano-carrier, carrier loaded with targeting substance, preparation method and application |
| WO2023165467A1 (en) * | 2022-03-04 | 2023-09-07 | 南京纳么美科技有限公司 | Ferritin nanocage vector loaded with small nucleic acid drug in inner cavity and use |
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