CN110179753A - Nano medication delivery system and its preparation and the application of a kind of targeting brain tumor and its tumor stem cell - Google Patents
Nano medication delivery system and its preparation and the application of a kind of targeting brain tumor and its tumor stem cell Download PDFInfo
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- CN110179753A CN110179753A CN201910499552.4A CN201910499552A CN110179753A CN 110179753 A CN110179753 A CN 110179753A CN 201910499552 A CN201910499552 A CN 201910499552A CN 110179753 A CN110179753 A CN 110179753A
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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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/186—Quaternary ammonium compounds, e.g. benzalkonium chloride or cetrimide
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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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
-
- 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
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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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
-
- 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 present invention discloses a kind of Nano medication delivery system for targeting brain tumor and its tumor stem cell, comprising: tumor therapeutic agent, carrier framework and the targeted molecular for being incorporated into carrier framework surface；Carrier framework is cationic-liposome, and targeted molecular includes at least the first targeted molecular and the second targeted molecular；First targeted molecular is surface modification polypeptide, and second targeted molecular is aptamer；The tumor therapeutic agent is selected from chemotherapeutics and/or gene target drug.The present invention discloses the preparation method and application of above-mentioned Nano medication delivery vector.Nano medication delivery vector of the invention can carry target gene or chemotherapeutics and treat for brain tumor, be increased to 67% or more from unmodified 5% through the dimolecular modified targeting efficiency for making system.
Description
Technical field
The present invention relates to the Nano medication delivery vectors of pharmaceutical field, more particularly to a kind of targeting brain tumor and its Tumor Stem
The Nano medication delivery vector of cell and its preparation and application.
Background technique
There are blood-brain barrier (blood brain barrier, BBB) and bloodtumorbarrier (blood tumor for brain tumor in situ
Barrier, BTB).The new vessels of low level brain tumor (I grade, II grade) are poorer, and there is no destructivenesses to change by BBB, stagnant
Stay effect (enhanced permeability and retention effect, EPR) unobvious, main barrier of drug delivery
Hinder for BBB；The new vessels of high-level brain tumor (III grade, IV grade) are abundant, and a series of changes, basement membrane office has occurred in Microvascular architecture
Portion has worm-eaten shape empty, fenestration and close connection expansion occurs, produces bloodtumorbarrier, there are EPR effects.BTB/BBB
Barrier is the vital barrier of drug therapy.
At present for BTB/BBB barrier design pharmaceutical carrier using relatively common.But even if overcome BBB/BTB
Barrier, drug usually can also generate toxicity to normal cerebrovascular endothelial cell, and brain tumor easily obtains after multiple chemicotherapy
It must resist, those easily cause tumor recurrence in the cancer cell of height infiltrative growth.Therefore, suitable drug delivery system was found both
It is current brain tumor drug therapy difficult scientific problems urgently to be resolved that brain tumor can be targeted by blood-brain barrier and can reduce drug resistance and recurrence again.
It is increasingly concerned recently as the targeted therapy of glioma stem cells, is ground for the targeting drug delivery system of tumor stem cell
Study carefully beginning gradually to increase.But since the growth conditions of the particularity of brain tumor anatomical location and its height infiltration are in addition few
Tumor stem cell distribution is that targeted delivery brings difficulty, while being related to part for the targeted therapy scheme of brain tumor stem cell
Signal path lacks specificity, is easy to cause in therapeutic process that (such as neural stem cell, Hematopoietic Stem are thin to whole body other stem cells
Born of the same parents, embryonic stem cell etc.) it causes to kill, bring serious side effects.
Summary of the invention
The first technical problem to be solved by the present invention is, provides a kind of nanometer for targeting brain tumor and its tumor stem cell
Drug delivery vehicle with cerebrovascular endothelial and the effect of brain tumor dual-target, targets high-efficient, good biocompatibility spy
Point.
One of to solve above-mentioned technical problem, technical solution provided by the invention are as follows:
A kind of Nano medication delivery system targeting brain tumor and its tumor stem cell, comprising: including tumor therapeutic agent, carry
Body skeleton and the targeted molecular for being incorporated into carrier framework surface；
The carrier framework is cationic-liposome, and the cationic-liposome includes:
1) polyethyleneglycol modified phosphatide；
2) dioleoylphosphatidylethanolamine (dioleoylphosphatidylethanolamine, DOPE)；
Or (the oily oxygroup propyl of 2,3- bis-) trimethyl ammonium chloride (2,3-Dioleoyloxy-propyl)-
Trimethylammonium, DOTAP)
3) carbamoyl cholesterol (DC-chol)；
The targeted molecular includes at least the first targeted molecular and the second targeted molecular；First targeted molecular be with
The surface modification polypeptide of cerebrovascular endothelial and the effect of brain tumor dual-target, the amino acid sequence of the surface modification polypeptide are as follows:
TFFYGGSRGKRNNFKTEEY(SEQ ID NO.1)；Second targeted molecular is suitable with tumor stem cell targeting
Gamete；The nucleotide sequence of the aptamer are as follows: 5 '-NH2-CCCUCCUACAUAGGG-3 ' (SEQ ID NO.2)；
The tumor therapeutic agent is selected from chemotherapeutics and/or gene target drug；The chemotherapeutics is wrapped in described
In cationic-liposome；The gene target drug carries negative electrical charge, is combined with the absorption of cationic-liposome positive surface charge.
The polyethyleneglycol modified phosphatide is selected from distearoylphosphatidylethanolamine-polyethylene glycol 2000- carboxyl commissure object
(DSPE-PEG2000-COOH) or phosphatidyl-ethanolamine-polyethylene glycol 2000-maleimide (MAL-PEG2000-DSPE).
The chemotherapeutic can be fat-soluble chemotherapeutic and/or water-soluble chemotherapeutic.Wherein, fat-soluble chemotherapeutic can be distributed
It can be distributed in the water phase of liposome in the lipid phase of liposome, or water-soluble chemotherapeutic.It is described in some specific embodiments
Fat-soluble chemotherapeutic is selected from paclitaxel kind anti-cancer drugs, Temozolomide, vinca alkaloids, 5 FU 5 fluorouracil, anthracycline anticancer
One of medicine, pemetrexed, platinum class anticarcinogen, camptothecin analogues, cyclophosphamide are a variety of.In some specific realities
It applies in mode, the water solubility chemotherapeutic is selected from the monoclonal antibodies anticancers such as doxorubicin hydrochloride, gemcitabine, benefit support former times monoclonal antibody
One of medicine, Hycamtin are a variety of.
Preferably, the gene target drug is the controlling gene drug of tumor stem cell gene.Some specific
In embodiment, the gene target drug is in for Notch, Hh and Wnt/ β-catenin signal path targeting medicine
It is one or more, such as siSurvivin.
Preferably, the modification efficiency of the surface modification polypeptide is 7-10mg/Kg, and the modification efficiency of the aptamer is 6-
12mg/kg.Modification efficiency refers to: the quality of modifier and the ratio of delivery system gross mass.
Preferably, the chemotherapeutic is taxol, and the drugloading rate of taxol is 2.5 ± 0.6%, encapsulation rate is 97.1 ±
1.9%.
Specifically, the surface modification polypeptide is connected to distearoylphosphatidylethanolamine-polyethylene glycol by bridging agent
On the carboxyl (COOH) of 2000- carboxyl commissure object (DSPE-PEG2000-COOH)；The aptamer is connected to two by bridging agent
On the carboxyl (COOH) of stearyl phosphatidyl ethanol amine-polyethylene glycol 2000-carboxyl commissure object (DSPE-PEG2000-COOH).
Specifically, the surface modification polypeptide is connected to the poly- second two of phosphatidyl-ethanolamine-by bridging agent after sulfhydrylation
Alcohol 2000- maleimide (MAL-PEG2000-DSPE)；The aptamer is connected to phosphatide by bridging agent after sulfhydrylation
Acyl ethanol amine-polyethylene glycol 2000-maleimide (MAL-PEG2000-DSPE).
Preferably, the bridging agent include at least 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide (EDC) or its
Salt, n-hydroxysuccinimide or its salt.
Due to the special anatomical location that brain tumor occurs, drug is difficult to contact through BBB with brain tumor, and brain tumor stem cell
It is distributed less and has therefore how powerful outer row function makes nanoscale medicine delivery system pass through blood-brain barrier and enter brain tumor
And active targeting tumor stem cell is the critical problem that this field needs to solve.The present invention devises a kind of double modify of targeting and receives
Rice delivery system (carrier), can enter brain tumor, and active targeting brain tumor stem cell by blood-brain barrier.
In the present invention, the sequence of the surface modification polypeptide is TFFYGGSRGKRNNFKTEEY, which has apparent brain
Characteristic is targeted, enters 70 times that brain effect is transferrins, there is cerebrovascular endothelial and brain tumor " dual " targeting.
In the present invention, the aptamer with tumor stem cell targeting is A15, sequence are as follows: 5 '-NH2-
CCCUCCUACAUAGGG-3'；The current histopathological techniques for detecting and targeting CD133 positive cancer stem cell are benefits
With the conventional system based on antibody, but due to the size of available anti-CD133 antibody and they it is opposite cannot penetrate tissue,
The shortcomings that lacking sensitivity there is non-specific binding.And aptamer A15 is the aptamer for CD133 positive cell,
It can specifically bind CD133, and can be by rapid internalization, and show excellent tumour penetration power.
The second technical problem to be solved by the present invention is, provides a kind of nanometer for targeting brain tumor and its tumor stem cell
The preparation method of drug delivery system, comprising the following steps:
(1): synthesis carries the phosphatide of targeted molecular: surface modification polypeptide, aptamer are connected to distearyl with bridging agent
On the COOH base of acyl phosphatidyl-ethanolamine-polyethylene glycol 2000-carboxyl commissure object (DSPE-PEG2000-COOH), molar ratio is
COOH-PEG2000-DSPE:EDC:NHS (n-hydroxysuccinimide)=(0.01-0.03): (1-2): (1-3), incubation at room temperature
It is slowly stirred 8-24h；The amino acid sequence of the surface modification polypeptide are as follows: TFFYGGSRGKRNNFKTEEY (SEQ ID
NO.1)；The nucleotide sequence of the aptamer are as follows: 5 '-NH2-CCCUCCUACAUAGGG-3'(SEQ ID NO.2)；
(2): will the obtained phosphatide and DC-chol for carrying targeted molecular of step (1), two kinds of phospholipid compositions of DOPE by mole
Than (0.5-2): (1-3): for (0.02-0.06) solution in chloroform, acetone and other organic solvent, concentration forms dry film, dry film in pH 7.4,
Aquation 30min in 10mM4- hydroxyethyl piperazineethanesulfonic acid (HEPES) buffer or 7.4 phosphate buffered saline solution of pH (PBS), makes
Final phospholipid concentration is 10~30mg/mL, probe sonicator ultrasound 10min, then by phospholipid suspension by 200nm, 100nm and
The polycarbonate membrane of 50nm, obtains carrier framework；Chemotherapeutics is enclosed in the carrier framework, the chemotherapeutics is liposoluble
Property chemotherapeutic or water-soluble chemotherapeutics；The fat-soluble chemotherapeutics is distributed in the lipid phase of liposome, the water solubility chemotherapy
Drug can be distributed in water phase convenient for carrying；
(4): genomic medicine can be combined because it carries negative electrical charge with the absorption of cationic-liposome positive surface charge, and (3) are made
The cationic-liposome skeleton and gene target drug mixed at room temperature obtained is incubated for 30min, prepares nano-complex.
(5): the cationic-liposome that step (3) is enclosed with chemotherapeutics enters step (4) in conjunction with gene target drug,
Ultimately form compound cationic-liposome.Because chemotherapeutic and the synergy of genomic medicine act on, it will bring treatment outstanding
Effect.
Preferably, the bridging agent is 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide (EDC) or its salt.
Preferably, final phospholipid concentration is 15~25mg/mL；More preferable 20mg/mL.
The third object of the present invention is to propose a kind of Nano medication delivering load for targeting brain tumor and its tumor stem cell
Application of the body in the product of preparation treatment brain tumor.
Preferably, the brain tumor include spongiocytoma, meningioma, pituitary adenoma, neurinoma, congenital tumor, its
His rare tumor such as lipoma, lymthoma, melanoma, intracranial metastatic tumor etc..
Compared with prior art, the invention has the following advantages that
(1) present invention will have cerebrovascular endothelial and brain tumor dual-target effect using cationic-liposome as carrier framework
Surface modification polypeptide as one of targeted molecular, the aptamer with tumor stem cell targeting as the second targeted molecular
The Nano medication delivery vector of preparation targeting brain tumor and its tumor stem cell.It is swollen for brain to carry target gene or chemotherapeutics
Tumor treatment, is increased to 67% or more from unmodified 5% through the dimolecular modified targeting efficiency for making system.
(2) preparation condition of nano-carrier used in the present invention is mild, and step is simple, easy to operate.
(3) nano-medicament carrier prepared in the present invention has good biocompatibility.
(5) it is thin can to specifically bind target for the target polypeptide in nano-medicament carrier prepared in the present invention and aptamer
Born of the same parents, Targeting Effect are good.
(6) nano-medicament carrier prepared in the present invention can carry chemotherapeutics simultaneously and genomic medicine to enter tumour thin
Born of the same parents, combination therapy have synergistic function, and efficient than independent chemotherapeutic improves 30% or more, more independent gene
The therapeutic effect of siSurvivin improves 70% or more, and therapeutic effect significantly improves.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of Nano medication delivery vector of the invention.
Fig. 2A is the TEM and particle diameter distribution phenogram (one) of Nano medication delivery vector of the invention.
Fig. 2 B is the TEM and particle diameter distribution phenogram (two) of Nano medication delivery vector of the invention.
Fig. 2 C is the TEM and particle diameter distribution phenogram (three) of Nano medication delivery vector of the invention.
Fig. 2 D is the TEM and particle diameter distribution phenogram (four) of Nano medication delivery vector of the invention.
Fig. 3 is animal tissue's fluorescence imaging figure of Nano medication delivery vector of the invention.
Specific embodiment
Clear, complete description will be carried out to technical solution of the present invention below, it is clear that described embodiment is this hair
Bright a part of the embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art exist
Every other embodiment obtained under the premise of creative work is not made, shall fall within the protection scope of the present invention.
Embodiment 1
A kind of preparation method for the Nano medication delivery vector targeting brain tumor and its tumor stem cell, steps are as follows:
(1): synthesis carries the phosphatide of targeted molecular: by surface modification polypeptide (SEQ ID NO.1) and aptamer (SEQ ID
It NO.2 is) that bridging agent is connected on the COOH base of COOH-PEG2000-DSPE with EDC and NHS, molar ratio COOH-
PEG2000-DSPE:EDC:NHS (n-hydroxysuccinimide)=0.03:1.25:2.1, mmol/mmol, incubation at room temperature are slow
Stir 8h.The sum of mole of surface modification polypeptide and aptamer is equal with the mole of COOH-PEG2000-DSPE.
(2): two kinds of phospholipid compositions of phosphatide and DC-chol, DOPE 1:2:0.03 in molar ratio will be targeted made from step (1)
It is dissolved in chloroform, 40 DEG C of rotary evaporation in vacuo 2h, the dry film of formation aquation in HEPES (10mM, pH 7.4) buffer
30min makes final phospholipid concentration 20mg/mL, probe sonicator ultrasound 10min, then phospholipid suspension is passed sequentially through
The polycarbonate membrane of 200nm, 100nm and 50nm.
(3): in step (2) preparation process, fat-soluble chemotherapeutics and water-soluble chemotherapeutics can be distributed in liposome
Lipid phase or water phase convenient for carrying, the drugloading rate of taxol is 2.5 ± 0.6%, and encapsulation rate is 97.1 ± 1.9%.
(4): genomic medicine can be combined because it carries negative electrical charge with the absorption of cationic-liposome positive surface charge, and (3) are made
The cationic-liposome skeleton and gene target drug mixed at room temperature obtained is incubated for 30min, prepares nano-complex.
(5): the cationic-liposome that step (3) is enclosed with chemotherapeutics enters step (4) in conjunction with gene target drug,
Compound cationic-liposome is ultimately formed, because chemotherapeutic and the synergy of genomic medicine act on, it will bring treatment outstanding
Effect.
The Nano medication delivery vector is injected using tail vein injection mode, nude mice 16-23g, and relative medicine dosage is
2.5 μM of Survivin siRNA, 5 μ g taxols.
Embodiment 2
(1): synthesis carries the phosphatide of targeted molecular: by surface modification polypeptide (SEQ ID NO.1) and aptamer (SEQ ID
NO.2 it) is connected to after sulfhydrylation on phosphatidyl-ethanolamine-polyethylene glycol 2000-maleimide (MAL-PEG2000-DSPE),
Target molecule is dissolved in 0.15mol/L borate+0.1mmol/L edta buffer liquid (pH8.5), 2-IT is then added, room temperature is incubated
Reaction solution is concentrated by ultrafiltration after educating 60min, buffer exchange is examined at the PBS (pH8.0) of 0.1mol/L by Ellman reagent
It surveys, feed ratio is 1:40 (mol/mol).
(2): by step (1) the targeting phospholipid compositions such as phosphatide and DC-chol, DOPE obtained, 1:2:0.03 is molten in molar ratio
Solution is in chloroform, 40 DEG C of rotary evaporation in vacuo 2h, the dry film of formation aquation in HEPES (10mM, pH 7.4) buffer
30min makes final phospholipid concentration 20mg/mL, probe sonicator ultrasound 10min, then phospholipid suspension is passed sequentially through
The polycarbonate membrane of 200nm, 100nm and 50nm.
(3): in step (2) preparation process, fat-soluble chemotherapeutics and water-soluble chemotherapeutics can be distributed in liposome
Lipid phase or water phase convenient for carrying, the drugloading rate of taxol is 2.5 ± 0.6%, and encapsulation rate is 97.1 ± 1.9%.
(4): genomic medicine can be combined because it carries negative electrical charge with the absorption of cationic-liposome positive surface charge, and (3) are made
The cationic-liposome skeleton and gene target drug mixed at room temperature obtained is incubated for 30min, prepares nano-complex.
(5): the cationic-liposome that step (3) is enclosed with chemotherapeutics enters step (4) in conjunction with gene target drug,
Compound cationic-liposome is ultimately formed, because chemotherapeutic and the synergy of genomic medicine act on, it will bring treatment outstanding
Effect.
The Nano medication delivery vector is injected using tail vein injection mode, nude mice 16-23g, and relative medicine dosage is
2.5 μM of Survivin siRNA, 5 μ g taxols.
Embodiment 3
Characterize the partial size and mode of appearance of targeted nano carrier system.
(1) using dynamic light scattering (Zetasizer, Nano-ZS, Malvern, UK) measurement particle diameter distribution and surface electricity
Position.It has detected simultaneously and prepares the variation that partial size before and after 10%BSA is added in sample.
(2) it takes sample appropriate, is added dropwise on the copper mesh of covering carbon film, 2.0% phosphotungstic acid negative staining, drying at room temperature 0.5h is set
The formalness of liposome is observed under transmission electron microscope.
Experimental result is as shown in Fig. 2A~2D.By Fig. 2A~2D it is found that can be seen that system partial size point in conjunction with DLS and TEM
It dissipates uniformly, with the increase of plasmid amount, the partial size of system increases, and ZETA current potential reduces, but the partial size in 10mg/mL BSA slightly has
It reduces.Illustrate that stability of the cationic-liposome in BSA can be improved in the negative electrical charge of plasmid.The modification of polypeptide and aptamer simultaneously
Increase partial size, ZETA current potential decreases, the grain that unmodified drug delivery system causes aggregation to make it in BSA due to charge
Diameter obviously increases, and can then improve aggregation caused by charge after modification, the aggregation in BSA also reduces.,
Embodiment 4
Evaluate the fluorescence imaging that targeted nano carrier system is used for tumour.
(1): the foundation of glioma animal model in situ:
It is in logarithmic growth phase to U251 cell, is digested with pancreatin and cell is collected by centrifugation.With PBS cell dispersion to suitable
Density (5 × 105A/5 μ L).It after nude mouse anesthesia, is fixed with stereotaxic apparatus, cell inoculation is in corpus straitum (on the right side of bregma
1.8mm, deep 3mm), every nude mouse is inoculated with 5 μ L cell suspensions.
(2): distribution is investigated in vivo:
Take lotus knurl model mouse, the double targeting drug deliveries prepared by embodiment 1 that tail vein injection DiR is marked after inoculation 16 days
System, injection dosage is every 50 μ g DNA and 5 μ g PTX, after mouse peritoneal injects 10% chloral hydrate anesthesia of 300mg/kg,
Be placed on 37 DEG C of hot plates, 2 after administration, 6,12, for 24 hours in taking pictures in CRi imager (620-900nm), set the time for exposure as
600ms collects the fluorescence signal at 780nm.After for 24 hours, take off neck and put to death mouse, take out lotus knurl brain tissue and major organs (including
The heart, liver, spleen, lung, kidney), normal saline flushing is placed on observation in living imaging instrument and takes pictures, as shown in Figure 3.
From the figure 3, it may be seen that tail vein injection drug delivery system for 24 hours after, observe apparent fluorescence in transplantable tumor nude mice brain
Signal.Illustrate that drug delivery system after double targeting modifications there is apparent brain to target characteristic.
In conclusion the various embodiments described above are only presently preferred embodiments of the present invention, it is not of the invention to limit
Protection scope, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should be all included in
In protection scope of the present invention.
Sequence table
<110>Shanghai City Beijing Tongren Hospital
<120>the Nano medication delivery system and its preparation of a kind of targeting brain tumor and its tumor stem cell and application
<130> CPC-NP-19-101508
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> PRT
<213>surface modification polypeptide (surface modification polypeptide)
<400> 1
Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Lys Thr
1 5 10 15
Glu Glu Tyr
<210> 3
<211> 15
<212> RNA
<213>aptamer (aptamer)
<400> 3
cccuccuaca uaggg 15
Claims (19)
1. the Nano medication delivery system of a kind of targeting brain tumor and its tumor stem cell characterized by comprising cancer therapeutics
Object, carrier framework and the targeted molecular for being incorporated into carrier framework surface；
The carrier framework is cationic-liposome, and the cationic-liposome includes: 1) polyethyleneglycol modified phosphatide；2) two oil
Acyl phosphatidyl-ethanolamine or (the oily oxygroup propyl of 2,3- bis-) trimethyl ammonium chloride；With 3) carbamoyl cholesterol；
The targeted molecular includes at least the first targeted molecular and the second targeted molecular；First targeted molecular is with brain blood
The surface modification polypeptide of endothelial tube and the effect of brain tumor dual-target, the amino acid sequence of the surface modification polypeptide such as SEQ ID
Shown in NO.1；Second targeted molecular is the aptamer with tumor stem cell targeting；The nucleotide of the aptamer
Sequence is as shown in SEQ ID NO.2；
The tumor therapeutic agent is selected from chemotherapeutics and/or gene target drug；The chemotherapeutics be wrapped in it is described sun from
In sub- liposome；The gene target drug carries negative electrical charge, is combined with the absorption of cationic-liposome positive surface charge.
2. Nano medication delivery system as described in claim 1, which is characterized in that the chemotherapeutic is fat-soluble chemotherapeutic.
3. Nano medication delivery system as claimed in claim 2, which is characterized in that the fat-soluble chemotherapeutic is distributed in lipid
The lipid phase of body.
4. Nano medication delivery system as claimed in claim 3, which is characterized in that the fat-soluble chemotherapeutic is selected from taxol
Kind anti-cancer drugs, Temozolomide, vinca alkaloids, 5 FU 5 fluorouracil, anthracyclines, pemetrexed, platinum class anticarcinogen,
One of camptothecin analogues, cyclophosphamide are a variety of.
5. Nano medication delivery system as described in claim 1, which is characterized in that the chemotherapeutic is water-soluble chemotherapeutic.
6. Nano medication delivery system as claimed in claim 5, which is characterized in that the water solubility chemotherapeutic is distributed in lipid
The water phase of body.
7. Nano medication delivery system as claimed in claim 6, which is characterized in that it is described water solubility chemotherapeutic be selected from hydrochloric acid Ah
One of mycin, gemcitabine, benefit support former times monoclonal antibody, Hycamtin are a variety of.
8. Nano medication delivery system as described in claim 1, which is characterized in that the gene target drug is that Tumor Stem is thin
The controlling gene drug of born of the same parents' gene.
9. Nano medication delivery system as claimed in claim 8, which is characterized in that the gene target drug is selected from and is directed to
Notch, Hh and Wnt/ β-catenin signal path target one of medicine or a variety of.
10. Nano medication delivery system as described in claim 1, which is characterized in that the modification hundred of the surface modification polypeptide
Dividing rate is 7-10mg/Kg, and the modification efficiency of the aptamer is 6-12mg/kg.
11. Nano medication delivery system as claimed in claim 10, which is characterized in that the chemotherapeutic is taxol, Japanese yew
The drugloading rate of alcohol is 2.5 ± 0.6%, and encapsulation rate is 97.1 ± 1.9%.
12. Nano medication delivery system as described in claim 1, which is characterized in that the polyethyleneglycol modified phosphatide is selected from
Distearoylphosphatidylethanolamine-polyethylene glycol 2000- carboxyl commissure object or phosphatidyl-ethanolamine-polyethylene glycol 2000-Malaysia
Acid imide.
13. Nano medication delivery system as claimed in claim 12, which is characterized in that the surface modification polypeptide passes through connection
Agent is connected on the carboxyl of distearoylphosphatidylethanolamine-polyethylene glycol 2000- carboxyl commissure object；The aptamer passes through company
Agent is connect to be connected on the carboxyl of distearoylphosphatidylethanolamine-polyethylene glycol 2000- carboxyl commissure object.
14. Nano medication delivery system as claimed in claim 12, which is characterized in that the surface modification polypeptide is through sulfhydrylation
It is connect afterwards by bridging agent with phosphatidyl-ethanolamine-polyethylene glycol 2000-maleimide；The aptamer leads to after sulfhydrylation
Bridging agent is crossed to connect with phosphatidyl-ethanolamine-polyethylene glycol 2000-maleimide.
15. Nano medication delivery system according to claim 13 or 14, which is characterized in that the bridging agent includes at least 1-
(3- dimethylamino-propyl) -3- ethyl carbodiimide or its salt, n-hydroxysuccinimide or its salt.
16. a kind of preparation method of the Nano medication delivery system of targeting brain tumor and its tumor stem cell, which is characterized in that including
Following steps:
(1): synthesis carries the phosphatide of targeted molecular: surface modification polypeptide, aptamer are connected to distearyl phosphorus with bridging agent
On the carboxyl of acyl ethanol amine-polyethylene glycol 2000-carboxyl commissure object, molar ratio COOH-PEG2000-DSPE:EDC:NHS
=(0.01-0.03): (1-2): (1-3), incubation at room temperature are slowly stirred 8-24h；The amino acid sequence of the surface modification polypeptide
As shown in SEQ ID NO.1；The nucleotide sequence of the aptamer is as shown in SEQ ID NO.2；
(2): will the obtained phosphatide and DC-chol for carrying targeted molecular of step (1), two kinds of phospholipid compositions of DOPE in molar ratio 1:
2:0.03 is dissolved in chloroform, and concentration forms dry film, and dry film aquation in 10mM, the HEPES buffer solution of pH7.4 makes final
Phospholipid concentration is 10~30mg/mL, again by phospholipid suspension by the aperture at least polycarbonate membrane of 50nm after ultrasound, is carried
Body skeleton；Chemotherapeutics is enclosed in the carrier framework, the chemotherapeutics is fat-soluble chemotherapeutic or water-soluble chemotherapeutic
Object；The fat-soluble chemotherapeutics is distributed in the lipid phase of liposome；The water solubility chemotherapeutics is distributed in the water phase of liposome；
(3): being enclosed with the cationic-liposome skeleton of chemotherapeutics made from step (2) and gene target drug mixed at room temperature is incubated
30min combination is educated, compound cationic-liposome is formed.
17. preparation method as claimed in claim 16, which is characterized in that the bridging agent includes at least 1- (3- dimethylamino
Propyl) -3- ethyl carbodiimide or its salt, n-hydroxysuccinimide or its salt.
18. a kind of Nano medication delivery system of targeting brain tumor and its tumor stem cell answering in the product of preparation treatment brain tumor
With.
19. application as claimed in claim 18, which is characterized in that the brain tumor includes spongiocytoma, meningioma, pituitary gland
Tumor, neurinoma, congenital tumor, lipoma, lymthoma, melanoma, intracranial metastatic tumor.
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