CN102614529A - Drug delivery system for treating nasopharyngeal carcinoma (NPC) and construction method and application method thereof - Google Patents
Drug delivery system for treating nasopharyngeal carcinoma (NPC) and construction method and application method thereof Download PDFInfo
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- CN102614529A CN102614529A CN2012101264652A CN201210126465A CN102614529A CN 102614529 A CN102614529 A CN 102614529A CN 2012101264652 A CN2012101264652 A CN 2012101264652A CN 201210126465 A CN201210126465 A CN 201210126465A CN 102614529 A CN102614529 A CN 102614529A
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Abstract
The invention discloses a drug delivery system for treating nasopharyngeal carcinoma (NPC) and a construction method and an application method thereof. Hydroxyapatite (HA) with high biocompatibility is used as a carrier, deoxyribozyme capable of targeting NPC latent membrane protein 1 (LMP1) is packaged on HA particles, a deoxyribozyme/HA complex is combined with cell-penetrating peptides (CPPs), and the drug delivery system capable of targeting NPC LMP1 is constructed. The drug delivery system can mediate the deoxyribozyme and plays a key role in the gene therapy of NPC.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of structure and application of gene therapy medicine for nasopharyngeal delivery system.
Background technology
(na sopharyngeal carcinoma is a kind of epithelial cell property malignant tumor with race and geographical distribution difference NPC) to nasopharyngeal carcinoma, and sickness rate is higher more than 100 times than white race crowd in the southern china crowd, is 25-30/10 ten thousand.This tumor is one and infects relevant multigenic disease with genetic predisposition, EBV.Epidemiological study shows that EBV infects very extensively, and adult infection rate can reach 98%; In most of the cases, exist with the latent infection state behind the EBV infection host, in nasopharyngeal carcinoma main express virus protein such as EBNA1, LMP1, LMP2 and non-coding RNA (EBV-encoded RNA, EBER).Research confirms that LMP1 (Latent membrane protein 1) mainly mediates the cross-talk between NF-κ B, AP-1 and STAT three bars pathways and the signal transduction pathway; Cause cell cycle disorder, apoptosis retardance, uncontrolled cellular proliferation; Be the tumorigenesis albumen with tumor gene function of having been proved conclusively at present of EBV coding, in the morbidity of EBV related neoplasms, play an important role.
DNAzyme (deoxyribozyme/DNAzyme) is a kind of short segments single stranded DNA with catalysis that utilizes external molecular evolution technique to obtain, and has catalytic activity and structure identification ability efficiently.DNAzyme catalytic degradation ability efficiently combines with the targeting identification ability of antisense, can combine and cutting target gene mRNA by specificity, thus the goal of regulation and control protein expression.Have great treatment, target checking, diagnosis potentiality.Our research team is a study model with EBV positive cells system and transplanted tumor in nude mice thereof, is the research target with LMP1, has set up and has used DNAzyme is closed Disease-causing gene from the mRNA level technology platform.Select activated DNAzyme at cell and animal horizontal screen; And, confirmed that the DNAzyme of targeting LMP1 can strengthen the sensitivity of nasopharyngeal carcinoma cell to radiotherapy first with DNAzyme and radiotherapy Combined application.The carrier great majority that adopt in the gene therapy clinical trial at present are viral vector, but due to illness there are problems such as safety, ethics and immunogenicity in poisonous carrier, and the transfection effect of non-virus carrier (like liposome, high molecular polymer etc.) is undesirable.It is carrier that the present invention adopts the good hydroxyapatite of biocompatibility; The DNAzyme of ability targeting nasopharyngeal carcinoma tumorigenesis albumen LMP1 is packaged on the HA granule; Again DNAzyme/HA complex is combined with cell-penetrating peptides, construct the drug delivery system of ability targeting nasopharyngeal carcinoma tumorigenesis albumen LMP1.Experiment shows: this drug delivery system ability transfection DNAzyme, the efficient of mediated gene transfection all is superior to the other drug delivery system.
Summary of the invention
The objective of the invention is to make up a kind of drug delivery system that can targeting LMP1, and this drug delivery system is applied to prepare the preparation of gene therapy nasopharyngeal carcinoma.
A kind of treatment medicine for nasopharyngeal delivery system is characterized in that, described drug delivery system is made up of DNAzyme (DZ1), hydroxyapatite (HA) and the cell-penetrating peptides (CPPs) of targeting LMP1; Wherein the DNAzyme of targeting LMP1 and hydroxyapatite form complex, combine to form the drug delivery system of ability targeting LMP1 then with cell-penetrating peptides.
DZ1 adsorbs, is wrapped in HA surface formation complex in the said medicine delivery system, and perhaps DZ1 is filled in nanometer HA hollow ball or the particulate hole of mesoporous HA and forms complex, then at HA/DZ1 complex surfaces branch knot or parcel cell-penetrating peptides.
The DNAzyme sequence of described targeting LMP1 is:
Sequence(5’to3’)gCA AAg?gAA?ggC?TAg?CTA?CAA?CgA AgA?ggA?CAA。
Described hydroxyapatite is shaft-like, spherical (comprising medicine ball, hollow ball or meso-porous nano ball), lamellar or cellular.
Described hydroxyapatite particle diameter is 5nm-1000nm.
Described hydroxyapatite can adopt PEI (PEI), Polyethylene Glycol or arginine to modify before use.
Described cell-penetrating peptide content is a 0-5 μ g/1mg hydroxyapatite.
The sequence of described cell-penetrating peptides be following any:
PEP-1：KETWWETWWTEWSQPKKKRKV
HIV-1protein?Tat(48-60)：GRKKRRQRRRPPQQ
Penetrat?in：RQIKIWFQNRRMKWKK
plsl：RVIRVWFQNKRCKDKK
Transportan：GWTLNSAGYLLGKINLKALAALAKKIL
P-b?e?t?a：GALFLGFLGAAGSTMGAWSQPKKKRKV
P-al?pha：GALFLAFLAAALSLMGLWSQPKKKRRV。
The construction method of described drug delivery system: the HA solution with behind the DNAzyme adding autoclaving of targeting LMP1, shake up, hatch, obtain DNAzyme and the hydroxylapatite compound of targeting LMP1; With adding cell-penetrating peptides behind the DNAzyme of targeting LMP1 and the hydroxylapatite compound eluting, the room temperature held gets final product behind the 20min at least.
Described drug delivery system is used to prepare the preparation of gene therapy nasopharyngeal carcinoma.
The constructed drug delivery system of the present invention is to be drug delivery system carrier, can targeting LMP1 with the good HA of biocompatibility; The problems such as safety, ethics and immunogenicity that it has avoided viral carrier system to exist; The metabolic problems of also having avoided other carrier systems to exist, it can not only be at in-vitro transfection, and the DNAzyme of transfection targeting LMP1 in can body; Transfection efficiency is high, can be used for the gene therapy of nasopharyngeal carcinoma.
Description of drawings
Fig. 1: nanometer HA transmission electron microscope picture of the present invention;
Fig. 2: the DNAzyme DZ1 mass spectrum of targeting LMP1;
Fig. 3: the transfection figure of drug delivery system of the present invention.
The specific embodiment
Be intended to further specify the present invention below in conjunction with embodiment, and unrestricted the present invention.
Embodiment 1
Mainly comprise: the HA/DZI/CPPs drug delivery system makes up (preparation of HA suspension, nano-hydroapatite particles combine with cell-penetrating peptides with combine experiment, the DNAzyme/HA complex of DNAzyme) and the gene transfection experiment is formed.
The HA/DZI/CPPs drug delivery system makes up
1, HA nanoparticle suspension preparation
The HA nanoparticle of preparation is divided into 3 groups, and one group is blank HA granule, and one group is that PEI modifies, and one group is that arginine is modified.The HA nanoparticle is made into 25mg/ml solution with deionized water.(Ultrasonic Hemogenizer-4710 USA), leaves standstill 2h with ultrasonic dispersing 60min.HA nanoparticle suspension does not have layering, is emulsus.Suspension is inserted the 50ml vial, preserve behind the autoclaving.
2, nano-hydroapatite particles and DNAzyme, cell-penetrating peptides combines experiment
The HA/DZI complex:
(RPMI is the abbreviation of Roswell Park Memorial Institute to A liquid 80 μ l HA (25mg/ml)+170 μ l RPMI1640, acute pyogenic infection of finger tip Loews dimension park memorial institute.RPMI is one type of cell culture medium of these institute research and development, the 1640th, and the culture medium code name.
B liquid 8 μ l DZ1 (concentration is 5 μ g/ml-60 μ g/ml)+242 μ l RPMI 1640
B liquid is added in the A liquid,, hatch on rocking evenly machine that (500rpm, room temperature 1h) obtain the HA/DZ1 complex solution with the liquid-transfering gun piping and druming 2min of sterilization.
With HA/DZ1 complex eluting (adopt D-hanks liquid to the HA/DZ1 complex flow wash make in the sample other components separate and stream come out) after add 0,1 μ l, 5 μ l cell-penetrating peptides (concentration is 5 μ g/ml-60 μ g/ml) respectively, room temperature held 20min obtains the HA/DZ1/CPPs drug delivery system.The gene transfection experiment
1) cell culture
With 2 * 10
5/ ml, 2.5 * 10
5/ ml and 3 * 10
5The CM cell of/ml [the CM cell is that CNE1 cell (KB cell) surely changes the cell line that LMP1 builds] density is inoculated on two 6 well culture plates, with there not being three anti-calf culture medium culturings, is used as the cell transfecting experiment after 24 hours.
2) gene transfection
With the cell in 6 orifice plates with twice of RPMI 1640 culture medium cells washed; The HA/DZI/CPPs drug delivery system of the present invention's preparation is dropwise added (the addition 1-5 μ g of the HA/DZI/CPPs drug delivery system of the present invention's preparation) in the hand-hole; Every hole adds 1.5ml 1640 culture medium again; Wave and culture plate, mixing gently.Put into the lunch box lucifuge, at 37 ℃, 5%CO
2Insulation is 4 hours in the incubator.After 4 hours, change the complete medium that contains serum, observation of cell transfection results (see figure 3) under fluorescence microscope.(attention lucifuge) is at 37 ℃, 5%CO
212h-24h in the incubator does the FCM flow cytometry then, detects the transfection level.
3) flow cytometry
(1). collecting cell: wash 2 times with D-hanks liquid, add 1 trypsinization, adding the 2ml/ hole does not again have three anti-calf culture medium, and piping and druming is evenly collected in vitro;
(2). centrifugal (800rpm, room temperature, 5min)
(3). with 100 μ l, 1 * PBS buffer re-suspended cell, use the micropipettor sucking-off, add in the EP pipe of 1.5ml, do Flow cytometry transfection level.Experimental result shows, adopts HA/DZI/CPPs drug delivery system transfection CM cell, and transfection efficiency reaches as high as 40%.
Claims (9)
1. a treatment medicine for nasopharyngeal delivery system is characterized in that described drug delivery system is made up of DNAzyme, hydroxyapatite and the cell-penetrating peptides of targeting LMP1; Wherein the DNAzyme of targeting LMP1 and hydroxyapatite form complex, combine to form the drug delivery system of ability targeting LMP1 then with cell-penetrating peptides.
2. drug delivery system as claimed in claim 1 is characterized in that, the DNAzyme sequence of described targeting LMP1 does
Sequence(5’to3’)gCA?AAg?gAA?ggC?TAg?CTA?CAA?CgA?AgA?ggA?CAA。
3. drug delivery system as claimed in claim 1 is characterized in that, described hydroxyapatite is shaft-like, spherical, lamellar or cellular.
4. drug delivery system as claimed in claim 3 is characterized in that, described hydroxyapatite particle diameter is 5nm-1000nm.
5. drug delivery system as claimed in claim 1 is characterized in that, described hydroxyapatite adopts PEI, Polyethylene Glycol or arginine to modify.
6. drug delivery system as claimed in claim 1 is characterized in that, described cell-penetrating peptide content is a 0-5 μ g/1mg hydroxyapatite.
7. like claim 1 or 6 described drug delivery systems, it is characterized in that, the sequence of described cell-penetrating peptides be following any:
PEP-1：KETWWETWWTEWSQPKKKRKV
HIV-1protein?Tat(48-60)：GRKKRRQRRRPPQQ
Penetratin：RQIKIWFQNRRMKWKK
plsl：RVIRVWFQNKRCKDKK
Transportan：GWTLNSAGYLLGKINLKALAALAKKIL
P-beta：GALFLGFLGAAGSTMGAWSQPKKKRKV
P-alpha：GALFLAFLAAALSLMGLWSQPKKKRRV。
8. the construction method of any described drug delivery system of claim 1-7 is characterized in that, the HA solution with behind the DNAzyme adding autoclaving of targeting LMP1 shakes up, hatches, and obtains DNAzyme and the hydroxylapatite compound of targeting LMP1; With adding cell-penetrating peptides behind the DNAzyme of targeting LMP1 and the hydroxylapatite compound eluting, the room temperature held gets final product behind the 20min at least.
9. the application process of any described drug delivery system of claim 1-7 is characterized in that, is used to prepare the preparation of gene therapy nasopharyngeal carcinoma.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111671921A (en) * | 2020-07-01 | 2020-09-18 | 中国科学院武汉病毒研究所 | Cell marking method and application thereof in rare cell MRI imaging |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999053961A1 (en) * | 1998-04-23 | 1999-10-28 | The Regents Of The University Of Michigan | Peptides for efficient gene transfer |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999053961A1 (en) * | 1998-04-23 | 1999-10-28 | The Regents Of The University Of Michigan | Peptides for efficient gene transfer |
Non-Patent Citations (3)
| Title |
|---|
| ZHONG-XIN LU ET AL: "DNAzymes targeted to EBV-encoded latent membrane protein-1 induce apoptosis and enhance radiosensitivity in nasopharyngeal carcinoma", 《CANCER LETTERS》, vol. 265, 31 December 2008 (2008-12-31) * |
| 孙虹 等: "用羟基磷灰石纳米粒作内耳基因治疗新载体的体内外研究", 《中华耳鼻咽喉头颈外科杂志》, vol. 43, no. 1, 31 January 2008 (2008-01-31), pages 51 - 57 * |
| 赵颜忠 等: "纳米羟基磷灰石的精氨酸表面修饰及其与基因的结合性能", 《中国有色金属学报》, vol. 20, no. 6, 30 June 2010 (2010-06-30), pages 1203 - 1208 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111671921A (en) * | 2020-07-01 | 2020-09-18 | 中国科学院武汉病毒研究所 | Cell marking method and application thereof in rare cell MRI imaging |
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