CN106109417A - A kind of bionical lipidosome drug carrier of liver plasma membrane, manufacture method and application thereof - Google Patents
A kind of bionical lipidosome drug carrier of liver plasma membrane, manufacture method and application thereof Download PDFInfo
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
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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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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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
- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
- A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
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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
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
Abstract
The present invention relates to a kind of bionical lipidosome drug carrier of liver plasma membrane, further relate to the preparation method of a kind of bionical lipidosome drug carrier of above-mentioned liver plasma membrane, further relate to the application of a kind of bionical lipidosome drug carrier of above-mentioned liver plasma membrane.The bionical lipidosome drug carrier of described liver plasma membrane, it is characterised in that (1) this lipidosome drug carrier has epicyte protein component；(2) this lipidosome drug carrier can external loading medicine, for cell-targeting merge release；(3) the epicyte protein component that this liposome is had comes from the human liver cell of immortalization, for the targeting of virogene of hepatitis group is sheared the conveying of plasmid；(4) the epicyte protein component that this liposome is had comes from the hepatoma cell line of immortalization, and the targeted drug for liver neoplasm carries.The bionical lipidosome drug carrier of described liver plasma membrane can be used for the conveying of internal liver cell targeting and the conveying of medicine of CRISPR gene targeting plasmid.
Description
Technical field
The invention belongs to cell therapy and liposome medicament accurate conveying technology field, relate to a kind of bionical fat of liver plasma membrane
Liposome medicament carrier, further relates to the preparation method of a kind of bionical lipidosome drug carrier of above-mentioned liver plasma membrane, further relates in one
State the application of the bionical lipidosome drug carrier of liver plasma membrane, can be used for medicine conveying and the treatment of accurate targeting liver.
Background technology
The targeting induction system of medicine can reduce the usage amount of medicine, improves the utilization rate of medicine, reduces medicine non-
The toxic and side effects of targeting, increases the effectiveness of medicine.The targeting drug delivery system of current medical include liposome, Emulsion, microsphere,
Nanoparticles etc., wherein liposome administration system is to be loaded in by medicine by the spherical vesicle of ultra micro of lipid molecule parcel (liposome)
In, inside can load hydrophilic medicament, and outer layer lipid molecular layer can load lipophilic drugs, size typically at several nm to several
μm.Traditional each lipoid plastid is bigger to the fusion randomness of target cell, and specificity is relatively low, owing to liposome membrane lacks
The albumen device that full-time mediation film merges, the fusion probability of liposome is relatively low, causes this artificial liposome thin with target
The fusion probability of intercellular is relatively low, increases, it addition, traditional liposomal is easily by internal with the nonspecific fusion of non-target cell
Reticuloendothelial system is swallowed and cleaned, causes the half-life of medicine and effectiveness to be substantially reduced.Design is for specific cells
The liposome of type is an important development target in this field.
The progress of the targeting operating technology of gene at present, the dream removing virogene of hepatitis papova source is increasingly becoming
Reality, such as TALEN technology and CRISPR technology.The two is respectively arranged with pluses and minuses, and such as TALEN technology miss rate is the lowest, is limited to target
The DNA element of tropism is relatively big, beyond the capacity of majority carrier, thus cannot be used for multiple target practice；And CRISPR technology only with
One nucleic acid cleaving enzymatic CAS9, carries multiple gRNA, it is possible to achieve many target position cut, but CRISPR existence at present lacks
Point, effect of i.e. missing the target, the cutting to non-specific sites sequence can cause bigger trouble and side effect.
A no matter difficult problem for the orientation conveying in TALEN and CRISPR technology, all body, currently used retrovirus retrovirus,
Slow virus and the virus-mediated internal targeting of AAV, have higher infection rate, but the targeting of virus system be poor, and some is sick
Poison meeting integrator gene group causes the ANOMALOUS VARIATIONS of gene, thus there is also certain risk.And the risk of ordinary plasmids is big
The big risk less than virus, but plasmid exists cell and imports inefficient, shorter etc. in the intracellular persistent expression time
Shortcoming, and targeting can't resolve, and non-specific side effect is also a urgent problem, hampers the treatment of clinic
Application.
Hepatitis virus is divided at least 5 kinds, i.e. first, second, third, fourth, hepatitis E virus, cause respectively first, second, third, fourth,
Hepatitis E, the most domestic harm heavier for hepatitis B.Hepatitis B virus (HBV) is divided into the genes such as ABCDEF
Type, the area such as East Asia predominantly Type B hepatitis B virus genotypes.The genome (HBV-DNA) of hepatitis B virus is by two spiral shells
The loop configuration that the DNA of rotation surrounds.Wherein longer minus strand has been formed complete ring-type；Another length is shorter
Normal chain, in semicircular.After infected liver cell, this Semicircular DNA will be polymerized in hepatitis B with minus strand as template
Extend under the effect of enzyme (pol), ultimately form complete ring-type.This DNA is referred to as covalently closed circular DNA (i.e.
CccDNA), for the primary template of virus replication.After template is formed, viral gene can be with a cccDNA therein as template, profit
With the enzyme in hepatocytic genes and " catalysis " of archaeal dna polymerase, a fragment gene another fragment gene ground replicates, and forms minus strand and just
Chain.Finally refill to be fitted on and form new HBV-DNA granule together.HBV DNA minus strand has four open zones, is called S, C, P
And X (Figure 26-2), the most known HBV protein can be encoded.S district can be divided into two parts, S gene and pre-S gene.S gene (core
Thuja acid 155～833) major surface protein can be encoded.Before S gene it is one and can encode 163 aminoacid (2,848-154)
Pre-S gene, coding Pre S1 and Pre S2 albumen.C district gene includes anterior c gene and C gene, be separately encoded HBeAg and
HBcAg；(HBV polymerase pol) is the longest in P district, accounts for genome more than 75%, encodes virion DNA polymerase.The X district of HBV
(nucleotide 1,374～1,835) may have 154 amino acid whose basic polypeptides by coding, and the breach of long-chain is positioned at this district.
The HBsAg of purification contains lipoids, saccharide, lipid, protein and glycoprotein.It is by white group of 8 peptide species and albumen
Become: (1) major surface protein (S protein, little molecule HBsAg), be made up of 226 aminoacid of S gene code.(2) Middle molecule
Albumen (Middle molecule HBsAg), by front S2, S gene code, adds one containing 55 amino at the amino acid whose N end of S protein 226
The Pre S2 albumen composition of acid, totally 281 aminoacid.(3) high molecular weight protein (macromole HBsAg), by S, front S1 and front S2 base
Because of coding, add one at the amino acid whose N end of middle molecule protein 281 and form containing 119 amino acid whose Pre S1 albumen, altogether
400 aminoacid.The immune complex of HBsAg Anti-HBsAg antibody often can be measured in hepatitis B patients blood circulation.Immune complex
Ⅲ type allergy can be caused, wherein most commonly seen with arthritis and nephritis.The most also can be same from blood in acute severe hepatitis disease
Time survey HBsAg Anti-HBsAg antibody, this patient's prognosis mala, mortality rate is high.It is therefore contemplated that immune complex can cause disease outside liver
The series of symptoms of people.Owing to HBV will not crack infected hepatocyte, body is removed hepatitis B virus and is relied primarily on T cell (T
Kill cell) or kill target cell by antibody-mediated K cell, virus is released in body fluid, makees through antibody the most again
With and kill the HBV in blood.Therefore for said gene, the targeting of especially polymerase pol is sheared, and eliminates this gene
Permanent normal expression, will eliminate this disease health threat to people from source.
The carcinogenecity of HBv: the hepatocarcinoma of about 80% 90% has HBv background, and HBv is the key factor causing hepatocarcinoma.Have
People adds up discovery, and 20 years HBv infect history person, there are about the generation canceration of 5% 10%, and the reason of canceration is the x gene integration of HBv
On hepatocytic genes, there occurs sudden change, cause hepatocarcinoma.What people were concerned about the most is also the carcinogenic problem of HBV, the most asymptomatic
HBv carrier, hepatocarcinogenesis of especially takeing care.Therefore for X gene targeting shear sudden change, it will help eliminate hepatitis B patient or
The hepatocarcinoma potential threat of the asymptomatic HBVer of person.
The mortality rate of hepatocarcinoma arranges the 3rd in alimentary system malignant tumour, and China dies from hepatocarcinoma about 110,000 people every year, accounts for complete
The 45% of world's PLC mortality number, and had the trend increased year by year in recent years.The average ill age is 44 years old.Hepatocarcinoma is pernicious
Degree is high, quickly grows, if not in time or therapeutic scheme selection is improper in treatment, the mean survival time is half a year.Early hepatocarcinoma can
Asymptomatic, manifest symptom once occurs, there are about 1/3 and belonged to late period that our people is brought pole by the king during therefore hepatocarcinoma is known as cancer
Big life threat.The measures such as the most conventional operative treatment, interventional therapy, chemotherapy.One problem of Post operation maximum is thorough
Eliminate the cancerous cell of remaining, prevent recurrence and transfer.Target-oriented drug for hepatoma carcinoma cell carries, and is an important treatment
Means, it is to avoid nonspecific toxic and side effects, improve the utilization rate of medicine, and the bionical liposome medicament for hepatoma carcinoma cell carries
System is an important technological innovation.
Summary of the invention
The purpose of the present invention solves above-mentioned technical problem, setting of lipidosome drug carrier based on the fusion of homocellular film
Meter, it is provided that a kind of targeting, personalized bionical lipidosome drug carrier of liver plasma membrane and its preparation method and application, is used for
The internal liver cell targeting conveying of CRISPR gene targeting plasmid and the conveying of medicine.
In order to solve above-mentioned technical problem, the present invention by the following technical solutions: a kind of bionical liposomal body of liver plasma membrane
Thing carrier, it is characterised in that (1) this lipidosome drug carrier has epicyte protein component；(2) this lipidosome drug carrier energy
Enough external loading medicines, merge release for cell-targeting；(3) the epicyte protein component that this liposome is had comes from forever
Biochemical autologous hepatocyte, for shearing the conveying of plasmid to the targeting of virogene of hepatitis group；(4) this liposome is had
Epicyte protein component comes from the human liver tumor cell system of immortalization, and the targeted drug for liver neoplasm carries.
Further, the medicine of loading is general chemistry medicine, or DNA medicine, or protein drug, or polypeptide drug or
SiRNA medicine, or mRNA medicine, or the mixing of one or more in said medicine.
Further, the bionical lipidosome drug carrier of described liver plasma membrane is to encapsulate selectively targeted virogene of hepatitis group
The CRISPR target practice element of DNA, for shearing and activated virus genome, for patient or the carrier of C positives.
Further, the medicine of loading is plasmid vector, the most non-viral type plasmid vector, expresses effect to improve it
Rate, increases and passes on chance, and plasmid adds S/MAR gene order element, in order to improve hepatocellular expression specificity, reduces non-target
The expression of cell, uses hepatocyte specific promoter, such as AAT promoter, it is ensured that only could open expression in hepatocyte, increases
Add safety；In order to increase the efficiency of cutting virogene of hepatitis group, use CRISPR technique construction plasmid, use multiple
GRNA element, cuts multiple site and multiple gene；Entrapped drug can also is that nucleoside medicine, is directly targeted suppression hepatitis
Poison；Entrapped drug for liver tumor can be all kinds of chemotherapeutics for hepatocarcinoma, chemotherapeutics therein can be hydrophilic,
Hydrophobic drug or amphiphilic chemicals.
Further, the bionical lipidosome drug carrier of described liver plasma membrane contains single or multiple gRNA element, or
Containing multiple gRNA promoteres, or containing a CAS9 reading frame.Further, the expression of CAS9 can be specific by hepatocyte
Gene promoter starts, for increasing liposome in vivo specific expressed.
In order to improve the targeting of epicyte protein liposome further, during making the bionical liposome of film, add magnetic
Property nano-particle.The bionical lipidosome drug carrier of described liver plasma membrane, except targeting and gene-splicings such as encapsulating plasmids
DNA element, it is also possible to encapsulate magnetic nanoparticle simultaneously, in vitro by under the effect in magnetic field, increase the target of liver further
Tropism.Magnetic medicine carrier is reactive owing to having magnetic field, can enter target position, reduce and prolong under the guiding of externally-applied magnetic field
The probability removed by reticuloendothelial system late, increases the liposome holdup time of local target position, improves topical liposomal thin with target
The fusion probability of born of the same parents.Magnetic nano particle mainly ferroso-ferric oxide and iron sesquioxide material are main, the magnetic of ferroso-ferric oxide
More than iron sesquioxide, it is more that ferroso-ferric oxide uses, and conventional chemical precipitation method prepares ferric oxide nano particles, is according to gold
Belong to ion under alkaline environment, to form the principle of metal-oxide and design, but anaerobic must be kept in preparation process and add guarantor
Protecting agent, such as tetramethylphosphonihydroxide hydroxide level ammonium etc., prevent the gathering of oxide and the generation of precipitation, reaction temperature maintains 85-95 DEG C.Reaction
Molecular formula 2Fe3++Fe2++8OH-→Fe3O4↓+4H2O.Owing to magnetic nano-particle has absorption to electromagnetic wave thus produce heat
Amount, and the somatic thermostability of tumor cell compared with normal is low, it is possible to use this principle carries out magnetic mediation thermotherapy, utilizes alternating magnetic field
Effect kill phagocytosis magnetic nano-particle tumor cell.The bionical liposome medicament of magnetic of tumor-associated cell thus give real
The treatment of body tumor brings a brand-new therapeutic strategy and method.
Further, the bionical lipidosome drug carrier of described liver plasma membrane, it is also possible to load nucleoside medicine, be used for resisting
Virus, can be with CRISPR targeting DNA element coencapsuiation, for magnetic target therapy hepatitis.
Further, the bionical lipidosome drug carrier of described liver plasma membrane is in the application for the treatment of hepatocarcinoma, it is characterised in that
The bionical lipidosome drug carrier of described liver plasma membrane is encapsulated with medicines resistant to liver cancer.
Further, the bionical lipidosome drug carrier of described liver plasma membrane is in the application for the treatment of hepatocarcinoma, it is characterised in that
The bionical lipidosome drug carrier of described liver plasma membrane is except encapsulating the combination of one or more chemotherapeutics, it is also possible to encapsulating
Magnetic nanoparticle, increases the targeting of liver further.
Hepatocyte, as the source of artificial cell film, needs to reach certain quantity, also to keep hepatocellular peculiar film
Composition, from the primary hepatocyte of healthy volunteer, must carry without hepatitis virus, can pass through external evoked immortalization, improves thin
The quantity of born of the same parents, owing to growth and the passage number of primary cell are very limited, needs it is carried out immortalization, some tumor cells
Energy for growth in vitro is the most very limited, it is also desirable to it is carried out immortalization process, the means of current immortalization relatively horn of plenty,
Technical difficulty is the most little, and the technology of immortality mainly uses the big T of process LAN telomerase catalytic subunit Tert, SV40 virus to resist at present
The methods such as former, c-myc, Epstein-Barr virus, proto-oncogene process LAN, roentgenization.Through the cell that immortalization processes, passage capacity is big
For strengthening, some cell can realize immortalization smoothly, and this is the confession that bionical liposome technology provides more epicyte protein
Give source.It addition, the source of tumor cell of liver film, it is possible to use the immortalization hepatoma cell line having built up, due to many livers
Cancerous cell line contains hepatitis virus, needs the gene targetings such as CRISPR, eliminates endogenic hepatitis virus and threatens, sets up nothing
Virocyte system；By stages, the cell line selecting grade malignancy different is used for the source of cell membrane, root to the state of an illness according to hepatocarcinoma patient
According to whether being whether hepatitis virus causes, select different hepatoma cell line as film donor.
Accompanying drawing explanation
Fig. 1 is the pattern collection of illustrative plates of immortalized liver cell inducing plasmid reading frame；
Fig. 2 is the reading frame forming types figure of HBV gene group targeting CRISPR target practice plasmid；
Fig. 3 is the collection of illustrative plates of plasmid P1；
Fig. 4 is the collection of illustrative plates of plasmid P5；
Fig. 5 is the grain size distribution of the bionical liposome of immortalised stem cell (unloaded)；
Fig. 6 is the entrapment efficiency determination figure of plasmid, chemical small molecule medicine, magnetic nano particle；
Fig. 7 is plasmid and the bionical liposome of HBV gene group plasmid (3:1) of the different HBV gene group targeting element of encapsulating
Impact on HepG2 emiocytosis HBeAG antigen；
Fig. 8 is the impact on HepG2 emiocytosis HBeAG antigen of the plasmid of the different HBV gene group targeting element of encapsulating；
Fig. 9 is that mtt assay measures the plasmid of the different HBV gene group targeting element of encapsulating and the imitative of HBV gene group plasmid (3:1)
The raw liposome proliferative effect to HepG2 cell；
Figure 10 is the bionical liposome the loading different plasmid impact on PLC/PRF/5 endogenous cellular secretion HBsAG；
Figure 11 is that PLC/PRF/5 endogenous cellular is secreted by the bionical liposome of tenofovir disoproxil fumarate encapsulating
The impact of HBsAG；
Figure 12 is the propagation of mtt assay detection cell.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.
1, the immortalization of primary hepatocyte
The present invention carries out the process of cell by traditional cellular immortalization method, and hepatocyte comes from autologous hepatocyte,
Or healthy volunteer hepatocyte, preferably healthy volunteer hepatocyte, carries without hepatitis virus；Mainly use hTERT, SV40 big
The methods such as T antigen, Epstein-Barr virus, c-MYC process, the individual processing of said method and/or combination application.Preferential use hTERT and
SV40 large T antigen Combined Treatment, the expression plasmid of hTERT and SV40 large T antigen, individually by plasmid-encoded or by one
Plasmid-encoded, the present invention preferentially uses hTERT (GenBank:NM_198253 or NM_001193376) and SV40 large T antigen
(GenBank:J02400.1) by same plasmid-encoded mode, therebetween use 2A autotomy peptide interval, for the two
Independent expression, in order to improve the survival of primary hepatocyte, HGF gene (GenBank:NM_000601) is also inserted into this carrier, has
2A autotomy peptide interval, see Fig. 1 Plasmid pattern figure；2A peptide of autotomying can use from porcine teschovirus P2A, or from four precursor virus
T2A, horse rhinitis A virus E2A, hand-foot-mouth disease virus F2A, the BmCPV2A of Cytoplasm polyhedrosis virus or silkworm infectiousness
Flacherie virus BmIFV2A, preferentially uses P2A；Expression vector is non-virus carrier or viral vector, and preferential employing virus carries
Body, such as slow virus carrier, the packaging for slow virus infects.The method improves the efficiency of infection to primary cell with the most steady
Fixed expression.Genome conformity type or genome circles viral vector that we build can be used, for virus packaging and
Infect.This carrier can be containing screening mark, such as antibiotic-screening mark, and this experiment preferentially uses band puromycin screening mark
The carrier of will carries out the resistance screening of follow-up stable express cell.
Primary cell can use the histiocyte of the cell of biopsy, excision, carries out Isolation and culture.Work as work
Inspection tissue is less, and the cell that is sorted on of cell cultivates row sorting again after some；Some be difficult to single culture primary carefully
Born of the same parents, easily use mixed culture, even stereoscopic culture.
The primary cell of sorting, or primary cell mixture, or the tissue of In vitro culture, or the group of In vitro culture
Knitting and cell mixture, the transfection or the virus infection that carry out immortalization plasmid vector process.The coding big T of hTERT-SV40 resists
The slow virus infected cell of former-HGF, after 24-48 hour, removes virus, continues to cultivate, and according to experiment needs, can suitably add anti-
Raw element screening, such as adds the puromycin screening cell of 0.3-5ug/ml, and the puromycin of the preferential 0.3-2ug/ml of employing processes,
The cell being uninfected by with removing, whether the screening of antibiotic determines according to the speed of growth of primary cell and density, to maintain
Cell reaches certain density and quantity, and when primary cell density is too low and quantity is very few when, antibiotic-screening can push away
Late or cancel.
2, CRISPR target practice plasmid, the structure of hepatitis B gene group plasmid
The gRNA design of targeting HBV gene group: for hepatitis B virus polymerase, X protein, hepatitis B virus core protein and second
Hepatovirus envelope protein design gRNA, according to the online website of CCTop (http://crispr.cos.uni-
Heidelberg.de/) design, this program is provided with the powerful genetic search that misses the target, and the similarity of the gene that misses the target is minimum 4
The difference of gene, selects position of missing the target herein at intron and intergenic sequence, misses the target position on gene extron
GRNA sequence is got rid of without exception.
PUC19c plasmid (2686bp, GenBANK:L09137.2) multiple clone site district transforms, KpnI and XbaI it
Between replace to 3 single restriction enzyme sites of AgeI-NheI-SpeI.Then by BGH-PA tailing signal high-fidelity PCR amplification, mould
Plate is the plasmid with BGH-PA；Primer, with SpeI and XbaI enzyme cutting site, inserts improved pUC19 plasmid, due to 2
The same tail of restriction enzyme site, identifies forward and reverse, the person that takes forward.
With forward primer with NheI and downstream primer with primer amplification S/MAR sequence (the Seq ID of SpeI
No.11), template is the genomic DNA of 293T cell, and after NheI and SpeI enzyme action, product inserts the plasmid being connected with BGH-PA, by
It is isocaudarner in NheI and SpeI, needs to identify forward and reverse, the person that takes forward.Without the sequence plasmid of S/MAR, this step is omitted.
Then by above-mentioned with S/MAR-BGH-PA or the plasmid of band BGH-PA further with AgeI and NheI enzyme action, use
With the primer amplification dCAS9 of AgeI and NheI restriction enzyme site, after enzyme action, insert the plasmid with S/MAR-BGH-PA.
By stand-by to dCAS9-S/MAR-BGH-PA KpnI and AgeI enzyme action, synthetic is for HBV cccDNA genome
Single gRNA and promoter sequence, two ends are with KpnI and AgeI restriction enzyme site；Synthetic hH1, hU6, mU63 is opened
The 3XgRNA reading frame sequence that mover starts, is separately encoded gRNA1, gRNA2, gRNA3 or gRNA2, gRNA3 and gRNA4, in
Between by 2A autotomy peptide segmentation (Seq ID No.5), this many reading frame gRNA two ends are with KpnI and AgeI restriction enzyme site, hU6
(Seq ID No.6)、mU6(Seq ID No.7)、hH1(Seq ID No.8).Insert with dCAS9-S/MAR-after enzyme action
The plasmid of BGH-PA；Synthetic 4XgRNA sequence, is respectively provided with hH1, hU6, mU6, hH1 promoter, and these promoteres are respectively
Starting gRNA1, gRNA2, gRNA3, gRNA4, two ends are with KpnI and AgeI restriction enzyme site.
By the plasmid with 4x/3xgRNA-dCAS9-S/MAR-BGH-PA, stand-by, by two ends with AgeI enzyme action further
With the primer amplification AAT promoter (Seq ID No.9) of AgeI restriction enzyme site, template is 293T cell genomic dna.Enzyme action
Rear insertion, with the plasmid of gRNA-dCAS9-S/MAR-BGH-PA, is identified forward and reverse, selects forward to insert clone.So
P4xgRNA (HBV)-dCAS9-S/MAR plasmid and p4xgRNA (HBV)-dCAS9 plasmid just can build, and critical sequences is all
Carried out sequence verification without sudden change.The encoder block of CAS9 can use consensus promoter hUBC (Seq ID No.10) to start.
Wherein 4 weights, the forming types figure of 3 weight gRNA plasmids are shown in Fig. 2, and wherein the collection of illustrative plates of 4 weight gRNA plasmids is shown in Fig. 3, sequence
For Seq ID No.12, hH1:414-793gRNA1:794-898；HU6:899-1146, gRNA2:1147-1252；MU6:
1253-1567, gRNA3:1568-1673；HH1:1674-2047, gRNA4:2048-2153；AAT promoter:887bp
2166-3052；DCAS9:4176bp 3059-7234；S/MAR:7241-9434；BGH-PA:9435-9659；Without S/MAR sequence
4 weight gRNA plasmid maps of row are shown in Fig. 4, hH1:420-793gRNA1:794-898；HU6:899-1146, gRNA2:1147-
1252；MU6:1253-1567, gRNA3:1568-1673；HH1:1674-2047, gRNA4:2048-2153；AAT
Promoter:887bp 2166-3052；DCAS9:4176bp 3059-7234；BGH-PA:7244-7468.
According to genotype B hepatitis B virus full-length genome (GenBank:AB775201) sequence, synthetic 1.3X genome
Sequence, it is ensured that the encoder block of all hepatitis B genes can be expressed, inserts pcDNA3.1 carrier, builds Hepatitis B virus-DNA and carries
Body.
The transformation of PUC19 multiple clone site, replacement sequence is inserted in KpnI and XbaI site and restriction enzyme site is as follows:
KpnI AgeI NheI SpeI XbaI
GGTACC ATAACCGGT TAA GCTAGC ACAACTAGTACT TCTAGA
3, the extraction of epicyte protein
A large amount of extractions of cell membrane, general use Low Osmotic Method to process cell, hypotonic medium adds a little DNAse I and/or
RNAaseA, the cell membrane that Mechanical Crushing expands, then will remove relatively maxicell device by low-speed centrifugal (400-1000g), such as line
Plastochondria and nucleus, use ultracentrifugation method separation cell membrane further after being collected by supernatant, ultracentrifugation is frequently with sugarcane
Sugar gradient or Percoll gradient centrifugation, or the two mixing gradient centrifugation, centrifugal force is at 25000g-120000g, such as, sugarcane
Sugar discontinuous gradient, spreads 53%, 43% and 38% saccharose gradient respectively, the supernatant after cell homogenates is placed in low-density layer
Upper strata, 4 DEG C of ultracentrifugations 1-12 hour, finally it is layered collection, cell membrane is typically distributed across top regions, low density area (38% layer
Upper and 43-53% density interlayer), collect low density area, upper strata cell membrane, dilution, further ultracentrifugation, collect and precipitate,
Re-suspended cell film, applicable centrifugal head can be SW-27, SW-28, SW-40, SW-41 etc. of Beckman.The cell membrane of this method
Protein recovery, at 40-70%, is mixed with a small amount of organelle film.The membrane pellet collected can place one ,-80 DEG C of refrigerators
Month, liquid nitrogen container steam can be placed 3 months to 3 years, can be used for the preparation of follow-up membrane lipids, the egg that this method is extracted
It is mixed with the pollution of the memebrane protein from endoplasmic reticulum, a small amount of Golgi body and mitochondrial memebrane protein in vain to pollute.
The extraction of a large amount of cell membrane may be used without phytohemagglutinin magnetic bead and extracts, and utilizes epicyte protein to have higher
Glycosylation principle, it is possible to use some special glycosyl associated proteins carry out purifying cells film, uses plant lectin usually to adsorb carefully
After birth, such as WGA, concanavalin A, Con A (ConA) etc..After such as phytohemagglutinin ConA being coupled to magnetic bead, it is possible to use magnetic
Pearl sedimentation cell film, is finally combined with the ConA of magnetic bead by the mannose of excess, glucose competitiveness, can be the cell membrane combined
Albumen is replaced, thus eluting epicyte protein.The cracking of cell can use Low Osmotic Method and isotonic method, uses electronic or the evenest
Slurry device homogenate, the cell membrane supernatant liquid after low-speed centrifugal directly carries out the extraction of cell membrane, and the response rate of cell membrane is at 25-
75%, average out to about 30-40%, but the purity of cell membrane is Graded Density partition method can not compare, and substantially reduces
The chance of organelle fouling membrane cell membrane, the present invention preferentially uses phytohemagglutinin paramagnetic particle method to extract hepatocyte or hepatoma carcinoma cell
The memebrane protein of system.
The a small amount of of cell membrane is extracted, and phytohemagglutinin magnetic bead kit can be used to extract, and Related product has Qproteome matter
Memebrane protein separating kit (Qiagen) or the test kit Minute of similar separation mechanismTMPlasmalemma protein separating kit
(Invent Biotechnologies).This method is for preparing bionical liposome in a small amount.
Hepatoma cell line: PLC/PRF/5, MHCC97, Hep3B2.1-7, SNU-42, SNU-475, extrahepatic growing hepatocellular carcinoma cell
It is EGHC-9901, QGY-7703, SMMC-7721, KMCH-1, RBHF-1, Mz-Hep-1, HB611, Huh6-c15, HepG2 etc..
Select the principle of cell line, be first hepatocarcinoma by stages, select the same or like hepatoma cell line of grade malignancy or table
The cell line that face mark is similar, the present invention generally selects the PLC/PRF/5 (ATCC) of secretion hepatitis B surface antigen to be used for detecting, makes
With HepG2 (ATCC) as cell membrane donorcells strain, this strain cell is hepatitis B negative cells strain.
4, the preparation of the magnetic Nano material of ferroso-ferric oxide
Ferric ion and the ferrous ion of 2M of 1M is prepared respectively, by 20ml 1M ferric ion with the hydrochloric acid of 2M
Mix with the 2M divalent iron ion of 5ml, be slowly added into 42ml 25% (W/W) tetramethyl ammonium hydroxide, until PH reaches 13.Hold
Continue and be stirred vigorously 15-40 minute, with Magnet by the Nanoscale Iron precipitate and separate of black, remove supernatant, clean with deionized water and receive
Rice ferrum precipitates 2-4 time, and ferriferrous oxide nano precipitation is finally suspended in deionized water and saves backup.Above-mentioned deionized water is as far as possible
Preparing with deoxygenated water, reaction vessel is logical N2Under the conditions of operate.
5, the preparation of bionical liposome
Membrane process: dipalmitoyl phosphatidyl choline (DPPC), distearoyl phosphatidylcholine (DSPC), 1,2-dioleoyl
Phosphatidylcholine (DOPC), cholesterol (Avanti Polar Lipids), (6:1:2:3) is dissolved in chloroform by a certain percentage: methanol
(3:1 or 2:1 or 1:1, V/V), fat-soluble medicine can add in this step, such as Sorafenib Tosylate, then
Evaporate solvent in a rotary evaporator, form thin layer.Then lamellar lipid rehydration, the memebrane protein extracted from cell (is dissolved in
PBS) join in thin layer with 1:100-1:800 ratio (albumen: film fat)；Reconstitution process is initially added into hydrophilic little molecule
Medicine, plasmid, nanometric ferromagnetic particles etc..40-65 DEG C of heating vortex processes 2-6 minute, repeats 2-5 time.Albumen is at 40-65 DEG C
Squeeze through cellulose acetate sheets or the polycarbonate membrane in 200nm aperture, be repeated 10-20 time, to reduce the hole of liposome
Footpath and uniform particle sizes's degree of raising liposome, the unilamellar liposome film bubble obtained is through semi-permeable membrane dialysed overnight purification, or warp
Cross SephadexG-50 post or similar gels column purification, to remove free non-integral protein and impurity.The unloaded lipid of comparison
The making of body, in addition to not entrapped drug, other steps, technique are the most identical with content, the bionical liposome of non-entrapped drug, flat
All particle diameters are about 142.3nM (Fig. 5).
For the medicine of the treatment of hepatitis, the predominantly plasmid of target gene operation, for the multiple gRNA of hepatitis B virus
With the encoding plasmids of dCas9, and/or nucleoside analogue drugs, such as lamivudine, adefovir ester, Sebivo, entecavir
Wei, tenofovir disoproxil and Clevudine.
For the medicine of cancerous cell, 9-nitrocamptothecin, all-trans-retinoic acid, paclitaxel, Docetaxel, 5-fluorine urea are phonetic
Pyridine, capecitabine, ametycin, ifosfamide, cyclophosphamide, methotrexate, cisplatin, carboplatin, vinorelbine, Changchun are new
Alkali, amycin, epirubicin, Sorafenib Tosylate, bleomycin, exemestane, irinotecan hydrochloride, TGF-β suppress
Agent etc., said medicine can be the combination encapsulating of monomer medicine encapsulating or several drugs.
6, liposome Characteristics Detection
Laser particle analyzer the detection particle diameter of liposome, current potential.The data of several representative drugs encapsulating liposome, are shown in Table 1:
7, envelop rate detection
Measure the envelop rate of medicine, use 16000-18000g to be centrifuged 10-30 minute, phase of fetching water, measure the residual of aqueous phase
Drug level and content, total medicine deducts the amount of non-encapsulated part and is envelop rate divided by the percentage rate of total amount, the mensuration of plasmid
Then use cold ethanol precipitation and supercentrifugal process to carry out, measure the total amount of precipitation DNA, then use 360nm ultraviolet photometer to measure
Concentration；Direct 360nm ultraviolet photometer can also be used to measure the DNA remaining quantity of liquid phase, computational envelope rate.
For chemical small molecule medicine, measure and mainly use HPLC method to measure the left drug in non-liposomal liquid phase
Concentration, total amount deducts the percentage ratio not encapsulating residual quantity divided by total amount.
The mensuration of magnetic-particle, then the content of ferromagnetic material in employing phenanthroline method measures bionical liposome.
Drug encapsulation concentration range: plasmid encapsulating concentration 0.3-4mg/ml, tenofovir disoproxil fumarate 3-300 μ g/ml,
Amycin 0.5-10mg/ml, cisplatin 20-2000 μ g/ml, capecitabine 40.0 μ g/ml-40mg/ml, 5-fluorouracil 100mg/
Ml-40g/ml, vincristine 20 μ g/ml-2mg/ml, Sorafenib Tosylate 1-100 μM.
Result is shown in Fig. 6.
8, the virus genomic inactivation in the hepatoma cell line that hepatitis virus is positive processes
The cell line of the bionical liposome transfection hepatitis b surface antigen positive of P1 plasmid encapsulating, such as PLC/PRF/5, transfects 2
Secondary or 3 times, every minor tick 12 hours, after 48 hours, make cloning inoculation, after 1-2 week, ELISA measures what each clone cultivated
HBsAG in the supernatant of cell, selects expression low, the cell clone similar with the background expression of negative cells, for not
Carry out amplification culture, as the source of bionical liposome.In like manner, in addition to HBV virus, other viruses are such as HPV, EB, CMV, HIV etc.
Virus can also use this method to process, it is ensured that engineering cell is without harmful microorganism.After P1 plasmid encapsulating liposome-treated, PLC/
Clearly, result is shown in Figure 10 to the shearing Inactivation Effect of PRF/5 cell HBV gene group, and this result shows bionical liposome delivery
Plasmid is effective to hepatoma carcinoma cell, also show P1 multiple gRNA and dCAS9 system simultaneously and for activated virus genome is
Effective and feasible method.
9, ELISA detects single gRNA, 3 weights or the HBV gene group shear effect mensuration of 4 weight gRNA
The targeting of genome is sheared the HBV gene group plasmid by measuring cotransfection and is produced hepatitis B surface antigen and hepatitis B
The amount of E antigen judges, empty carrier pUC19, gRNA1, gRNA2, gRNA3, gRNA4, and multiple gRNA combination plasmid divides
Not with HBV gene group plasmid co-transfection (mol ratio is 3:1), after transfecting 72 hours, the hepatitis B antigen in ELISA mensuration supernatant,
Measure hepatitis B surface antigen (HBsAg) and hepatitis B E antigen (HBeAg) ELISA kit (the beautiful pearl in Zhuhai) respectively.Result is shown in
Fig. 7 and Fig. 8.
10, the expression of endogenous hepatitis B virus gene is affected by tenofovir disoproxil fumarate encapsulating liposome
The liposome of encapsulating concentration 10 μ g/ml tenofovir disoproxil fumarate and unloaded liposome transfection PLC/PRF/5, often
Every transfection in 24 hours once, the content of the HBsAg in mensuration supernatant after 72 hours.Result shows that the activity of (Figure 11) virus obtains
Effective suppression.Future can optimize further for zoopery and clinical trial, can join with CRISPR target practice plasmid further
Close encapsulating liposome application.
11, the propagation of mtt assay detection cell
Empty carrier pUC19, gRNA1, gRNA2, gRNA3, gRNA4, and multiple gRNA combination plasmid respectively with HBV gene
Group plasmid co-transfection (mol ratio is 3:1), transfection HepG 2 cell, after 72 hours, adds 5mg/ml MTT tetrazolium bromide and hatches 4 hours
After, sopping up supernatant, add DMSO and dissolve, decolorization swinging table vibrates 10 minutes, then survey light absorption value at 490nm, result is shown in Fig. 9.
This experiment shows that constructed plasmid does not show cytotoxicity, thus gets rid of the Cytotoxic existence caused of missing the target.
The bionical liposome of the little molecular encapsulation of the cancer therapy drug proliferative effect to HepG2, is also adopted by mtt assay and measures cell
Upgrowth situation, result is shown in Figure 12, and this result shows that bionical wrapping kmedicine by liposome delivers, and is effective to hepatitis cell, future
Can optimize further for zoopery and clinical trial.
Claims (10)
1. the bionical lipidosome drug carrier of liver plasma membrane, it is characterised in that (1) this lipidosome drug carrier has cell membrane
Protein component；(2) this lipidosome drug carrier can external loading medicine, for cell-targeting merge release；(3) this liposome
The epicyte protein component being had comes from the autologous hepatocyte of immortalization, for shearing the targeting of virogene of hepatitis group
The conveying of plasmid；(4) the epicyte protein component that this liposome is had comes from the human liver tumor cell system of immortalization, is used for
The targeted drug conveying of liver neoplasm.
The bionical lipidosome drug carrier of liver plasma membrane the most according to claim 1, it is characterised in that the medicine of loading is general
Logical chemicals, or DNA medicine, or protein drug, or polypeptide drug, or siRNA medicine, or mRNA medicine, or above-mentioned medicine
The mixing of one or more in thing.
The bionical lipidosome drug carrier of liver plasma membrane the most according to claim 1, it is characterised in that described liver plasma membrane is imitated
Raw lipidosome drug carrier is the CRISPR target practice element encapsulating selectively targeted virogene of hepatitis group DNA, suffers from for hepatitis
Person or the hepatitis virus carrier without disease symptoms.
The bionical lipidosome drug carrier of liver plasma membrane the most according to claim 1, it is characterised in that the medicine of loading is matter
Grain carrier.
The bionical lipidosome drug carrier of liver plasma membrane the most according to claim 4, it is characterised in that described plasmid vector is
Non-viral type plasmid vector.
The bionical lipidosome drug carrier of liver plasma membrane the most according to claim 1, it is characterised in that described liver plasma membrane
Bionical lipidosome drug carrier contains single or multiple gRNA element, or containing multiple gRNA promoteres, reads containing a CAS9
Frame, CAS9 can start via the gene promoter that hepatocyte is special.
The bionical lipidosome drug carrier of liver plasma membrane the most according to claim 1, it is characterised in that described liver plasma membrane
Bionical lipidosome drug carrier, except encapsulating plasmid and/or gene-splicing DNA element, encapsulates magnetic nanoparticle the most simultaneously.
The bionical lipidosome drug carrier of liver plasma membrane the most according to claim 1, it is characterised in that described liver plasma membrane
Bionical lipidosome drug carrier, also loads nucleoside medicine.
9. the answering in treatment hepatocarcinoma of the bionical lipidosome drug carrier of liver plasma membrane according to any one of claim 1-8
With.
10. the bionical lipidosome drug carrier of liver plasma membrane described in claim 9 is in an application for treatment hepatocarcinoma, and its feature exists
In, the bionical lipidosome drug carrier of described liver plasma membrane is encapsulated with medicines resistant to liver cancer and/or encapsulating magnetic nanoparticle.
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