CN102743326A - Nano-scale artificial oil body for targeted drug delivery system detection and treatment - Google Patents

Nano-scale artificial oil body for targeted drug delivery system detection and treatment Download PDF

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CN102743326A
CN102743326A CN2011103271054A CN201110327105A CN102743326A CN 102743326 A CN102743326 A CN 102743326A CN 2011103271054 A CN2011103271054 A CN 2011103271054A CN 201110327105 A CN201110327105 A CN 201110327105A CN 102743326 A CN102743326 A CN 102743326A
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陈顺基
朱宝美
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陈致融
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    • A61K31/47—Quinolines; Isoquinolines
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Abstract

The present invention relates to a nano-scale artificial oil body for targeted drug delivery system detection and treatment. The nano-scale artificial oil body comprises: an oleEGFR protein, wherein the oleEGFR protein comprises oleosin and a EGFR peptide; a lipid, a lipid-coated liposoluble drug, and a lipid-coated known fluorescence molecule, wherein the materials are prepared into an oleEGFR oil body, an oleEGFR drug oil body and an oleEGFR fluorescence oil body so as to provide an active targeted drug delivery system and a detection system.

Description

The nanoscale people oleoplast that the targeted delivery of drugs system detects and treats
Technical field
The present invention relates to targeting property people oleoplast; It relates to a kind of receptor that can discern the cancerous cell surface through EGFR peptide in the oleEGFR protein accurately; And process oleEGFR medicine oil body or oleEGFR with lipid, fat-soluble medicine and/or known fluorescence signal molecule and detect oil body, and be able to identification cancerization position, specificity ground and can assemble pharmaceutical protein voluntarily and carry out the nanoscale people oleoplast that the targeted delivery of drugs system of cancerization location detection, inhibition and Drug therapy detects and treats.
The present invention utilizes the oil body memebrane protein to connect the EGFR peptide and is assembled into the oleEGFR oil body, and this oil body ability coating medicine or known fluorescence signal molecule can be used in the application of oleEGFR oil body in targeted therapy or detection.
Background technology
Cancer is to threaten global human life and healthy main killer at present, and according to American Cancer Society's statistics, the whole world had 7,600,000 people to die from all kinds of cancers approximately in 2007.According to Taiwan Department of Health statistics, the full court gulf had 38,913 people to die from malignant tumor approximately in 2008.In ten big underlying cause of deaths; It is the first that malignant tumor occupy continuously, and ten big main cancers and mortality rate cis-position are respectively: (1) pulmonary carcinoma, (2) hepatocarcinoma, (3) colorectal carcinoma, (4) female lung cancer, (5) gastric cancer, (6) oral cancer, (7) prostate parcinoma, (8) cervical cancer, (9) esophageal carcinoma, (10) pancreatic cancer.Because the rate occurred frequently and the relapse rate of cancer; The research and development of antitumor drug come into one's own; According to the data show of united states drug research, till 31 days March in 2008, have 750 kinds of cancer treatment drugs and be in the clinical experiment stage with AEM (PhRMA); Wait for the approval (PhRMA, 2008) of U.S. food Drug Administration (FDA).
Traditional cancer treatment method roughly is divided into following several kinds, i.e. surgical operation therapy, X-ray therapy, chemotherapy and immunity (gene) therapy.
The surgical operation therapy: mainly can remove malignant tumor directly, apace, be the most ancient, tradition and the most frequently used treatment of cancer mode, and when body part one place, directly excision mode has good therapeutic effect for tumor growth.If cancerous cell diffusion, the surgical operation sequela is often to need tumor resection adjacent tissue and lymphoid tissue, usually because the too much tissue of excision can cause damaged, the obstacle of patient's local function, also has the operation sudden death or leads to complications and cause death etc.
X-ray therapy: claim electrotherapy again, mainly be to use the radiation damage cancerous cell, have two kinds to the method for cancerous cell: external X-ray therapy and inherent X-ray therapy.External X-ray therapy is to utilize instrument that X-ray or gamma-rays are released on the tumor and kill cancer cell.Inherent X-ray therapy is that radioactive substance is inserted in the cancer human body with granule or capsule mode; Tissue treatment regular meeting for being not suitable for operating on maybe should not excising selects this kind X-ray therapy for use; Sometimes X-ray therapy also can merge with surgical treatment or chemotherapy and uses, the sequela of this kind method be feel sick, vomiting, cough or dyspnea, dysphagia, tired, lose hair and cause visceral rupture etc.
Chemotherapy: chemotherapy is to utilize chemical substance or hormone kill cancer cell, the method can administered through oral or injection system treat, chemotherapy can be widely used on the various tumor.To being difficult to control and shifting on a large scale or the cancerous cell of general kills, chemotherapy is unique, last selection, and sequela is that systemic cell is damaged, and particularly to immune lymph corpuscle, treatment time is long, can cause the Drug resistance of systemic cell.
Immunotherapy: immunotherapy be utilize the different immunologic mechanism working substance of patient body such as interleukin, tumor necrosis factor, monoclonal antibody and vaccine to wait to resist, tumoricidal quick growth.Immunotherapy also can merge with above-mentioned three kinds of cancer treatment methods commonly used to be used and can significantly reduce side effect such as tired, inappetence, alopecia, oral cavity or skin ulceration, feel sick and tooth drops etc. and significantly to reduce; And can assist normal cell slowly to repair interval in treatment; Slowly promote immunity; And promote the treatment for cancer effect, but treatment time is long, and effect is also very limited.
Find that through observing for a long time the cancerous cell surface can have some particular molecule biomarker usually and describe characteristic before being, in these years developed the targeted therapy method and be used for treatment for cancer.The targeted therapy method be exactly utilize experimental design a kind of can specificity ground the pharmaceutical methods of this type of biomarker of identification, the targeted therapy method can be blocked the activation of cancerous cell effectively and growth of cancer cells is suppressed, and reaches therapeutic purposes.Target therapeutic agent can be directly and hit cancerous cell accurately; Anticancer, less to Normocellular influence in the body, than above-mentioned Therapeutic Method; Targeted therapy has hypotoxicity, low side effect, high efficiency, reaches and implement advantages such as convenient, has caused everybody concern.
The method of targeted therapy is a lot of at present; The first utilizes carrier to assemble, coat, link or inlay the drug molecule of the therapeutic activity that forms; And use various mechanism will have narrow spectrum active drug molecule to be delivered to the targeted cancerous cells effect, this kind delivery system is called " drug delivery system ".Can but the material of carrier with specificity ground identification cancerous cell surface biological labelling be combined usually, and make this carrier have target function, said material can be protein, steroid, saccharide, chemical compound or antibody etc.
Carrier has high molecular polymerization composition granule, micella (micell), liposome at present, reaches viral vector etc.; Also there are different shortcomings in these carriers; As the carrier particle diameter excessive and be not easy to be absorbed by the body, carrier is assembled easily and be not easy to be absorbed by the body, have toxicity and can't excrete, can stable existence in blood or body fluid etc., also very limited in practice.And when the above-mentioned material of discerning cancerous cell combines with carrier, usually need the very chemosynthesis step of complicated and time consumption, and tending to increase manufacturing cost, more can pollute environment.So, in drug delivery system, also need very large improvement space and improve into that particle diameter is small, avirulence, stable in blood or body fluid, combination can be discerned the pharmaceutical carrier system of cancerous cell easily.
Tumor cell proliferation needs the new life of blood vessel; In recent years; Research relevant for the blocking-up angiogenesis has confirmed that lung carcinoma cell usually has the situation of overexpression vascular endothelial growth factor receptor (VEGFR) and epithelical cell growth factor receptor (EGFR); VEGFR and EGFR are quite relevant with the grade malignancy and the transfer ability of tumor, detect and find that lung cancer patient all has VEGFR and the EGFR that highly expresses.
The present invention utilizes the transgenic technology of E.coli to produce egf protein (EGFR); And in the oil body manufacture process, skin factor (EGFR) is marked on the nanometer oil body; Hope can make the nanometer oil body come the highly EGFR of expression of lung cancer-targeted cell institute, reaches the effect of pulmonary carcinoma cancer target.
The present invention utilizes oil body to produce epidermal growth factor (EGFR) and form oleEGFR as carrier and with transgenic technology; Add lipid again and assemble formation oleEGFR oil body, the present invention attempts to utilize the oleEGFR oil body to coat fat-soluble medicine (being designated hereinafter simply as coating medicine) and forms the artificial nanometer oil body of targeting drug delivery system.The present invention is through in vivo (in vivo) and in vitro the related experiment of (in vitro) confirms that this system toxicity is low, side effect is low, efficient is high, stable, preparation process convenient reaches environment is polluted very low or can ignore.The present invention utilizes the artificial nanometer oil body of targeting drug delivery system, can apply to targeted therapy or cancer detection effectively, and has the specificity of height.
The present invention be by difference can synthesize medicine analog peptide or disease had the peptide of therapeutic effect; Synthetic or synthetic polyprotein that therapeutic effect is arranged through genetic engineering composition medicine or to disease; And the last oil body memebrane protein of connection connects the EGFR peptide again; This oil body can coat known fluorescence signal molecule and be assembled into self assembly targeted drug oil body memebrane protein, and this albumen can also can be assembled pharmaceutical protein and carry out cancerization location detection, inhibition and Drug therapy in identification cancerization position, specificity ground voluntarily.
The purification mode is very many, as: the sedimentation method, molecular sieve chromatography, electrophoresis, affinity chromatography and covalent chromatography are known technologies and are used for the cell extract protein purification.And need the relevant protease inhibitor of expression to be used as a kind of fusion rotein or chimeric protein.Fusion protein technology provides a kind of " labelling " or " operation " in being commonly used in the protein purification process.
Generally speaking; The fusion rotein the inside of expressing with these systems comprises affinity labeling; For example: glutathione S-transferase, maltose-binding protein, cellulose binding domain, polyhistidyl, gather cysteine; Protein A and Streptavidin or the like, said labelling are covalently bonded in proteinic N-end of oleEGFR or C-end.For making things convenient for affinity purification, this labelling is had narrow spectrum part be fixed in the solid-state of a tubing string and go up mutually.Fusion rotein is bonded to affinity column, adds excessive adjustment buffer (pH value) and dash to propose highly purified fusion rotein.The separation of oleEGFR albumen on the labelled molecule cut in its specificity joint sequence with Proteolytic enzyme.In the preparation and operation of use affinity column, though easier, price is very expensive, does not meet economic benefit.
The affine technology of antibody immobilization immunity is to utilize a kind of combination of fusion rotein of expression, and antigenic oligopeptide labelling is contained in the inside, and this is marked with utilization antigenicity front end (antigenic head) part and connects tail end (linking tail) part.It is the hydrophilic amino acid that can cause the antigenicity reaction with some fast that this antigenic portions is formed.And join domain can be marked at fusion rotein, and fusion rotein is cut again after the cell separation, and obtains this albumen.Use the affine technology of antibody immobilization immunity also very limited for purification technique.
The present invention utilizes the AOB system to carry out purification, can obtain fusion rotein quicker and more economically.Utilize oleEGFR that the oil body of specificity joint is arranged, utilize proteolytic enzyme cutting specificity joint again, can be purified into the high EGFR protein of purity.
Summary of the invention
The present invention provides a kind of fused protein (hereinafter referred is an oleEGFR protein), and this albumen comprises the combination of EGFR peptide and oil body memebrane protein.
The present invention also provides a kind of oil body memebrane protein carrier; It comprises the oil body memebrane protein and combines EGFR peptide and any oils and fats; And form narrow spectrum nanometer oil body; Wherein the weight/volume ratio (microgram/microlitre) of this oleEGFR protein and this lipid is at least about 1/25, and the mean diameter of this oleEGFR oil body is a nano-scale.
The present invention utilizes oleEGFR protein to coat known fluorescence molecule again, and the detection that can be used for the recognition specificity cancerous cell is used.
The present invention utilizes oleEGFR protein coating medicine again, and the targeted therapy that can be used for the recognition specificity cancerous cell uses.
The present invention utilizes oleEGFR protein to coat known fluorescence molecule and coating medicine again, and the detection and the targeted therapy that can be used for the recognition specificity cancerous cell use.
The present invention utilizes similar oleEGFR medicine oil body again, and the targeted therapy that can be used for the recognition specificity cancerous cell uses.
The present invention utilizes oleEGFR that the oil body of specificity joint is arranged again, utilizes specificity protease hydrolysis cutting specificity joint again, can be purified into the proteinic application of the high EGFR of purity.
Description of drawings
The structure of Fig. 1, oil body.
The preparation process of Fig. 2, oleEGFR oil body.
Fig. 3, EGFR protein purification prepare process.
The preparation process of Fig. 4, oleEGFR medicine oil body.
Fig. 5, oleEGFR protein are formed and autonomous dress oleEGFR oil body laboratory observation.
The oil body that Fig. 6, oleEGFR oil body coat GGK Huang and Nile red shows the observation of fluorescence micro mirror.
Fig. 7, oleEGFR oil body carry out the AFM test.
The different condition composition of Fig. 8, oleEGFR oil body carries out fluorescence microscope (2 microns on diagram scale).
The different condition composition of Fig. 9, oleEGFR oil body carries out the degree of stability test.
The testing in vitro fluorescence microscope of Figure 10, oleEGFR oil body pair cell-(fixing) cell.
The testing in vitro fluorescence microscope of Figure 11, oleEGFR oil body pair cell-(work) cell.
Figure 12, oleEGFR oil body are to A549 cell tests fluorescence microscope-difference (concentration).
Figure 13, oleEGFR oil body are to A549 cell tests fluorescence microscope-difference (time).
Figure 14, oleEGFR oil body function cells are carried out flow cytometer test-difference (concentration).
Figure 15, oleEGFR oil body function cells are carried out flow cytometer test-difference (time).
Figure 16, oleEGFR oil body effect A549 cell utilize conjugation fluorescence microscope XY axial plane to observe and the Z axial section is observed.
The degree of stability test of Figure 17, oleEGFR oil body coating medicine.
Figure 18, oleEGFR oil body coat toxicity test cell counting (the 72nd hour)-lycopene, curcumin and the camptothecine of variable concentrations camptothecine.
Figure 19, oleEGFR oil body coat the camptothecine medicine in cell poisoning experiment (4 days).
Figure 20, oleEGFR oil body coating medicine are to blood cell blood dissolubility experiment-lycopene, curcumin and camptothecine.
Figure 21, oleEGFR oil body carry out oil body and melt blood experiment (blood cell).
Figure 22, oleEGFR oil body are in serum change of size test in time.
Figure 23, oleEGFR oil body are injected the back with different time IVSI observation test at mouse interior tumor.
Figure 24, oleEGFR oil body injected in mice after 24 hours internal organs with different time IVSI observation test.
Figure 25, Mus are got the fluorescence microscope of tumor tissues frozen section.
Figure 26, mouse tumor detect tissue slice and utilize the oleEGFR oil body to detect observation.
Tumor load Mus-the tumor of Figure 27, the treatment of oleEGFR oil body targeting type oil body changes (size: cm 3).
Tumor load Mus-the weight change of Figure 28, the treatment of oleEGFR oil body targeting type oil body (weight: kilogram).
Figure 29, targeting type oleEGFR fluorescence oil body detect in the functional body of targeting in the mouse lung body.
The luminous signal value that Figure 30, targeting type oleEGFR fluorescence oil body detect in the functional body of targeting in different time mouse lung body.
The luminous signal value that detects in the body of Figure 31, targeting type oleEGFR fluorescence oil body mouse lung organ target function property after 48 hours.
The specific embodiment
Unless otherwise indicated herein, (especially in claims) employed " one ", " being somebody's turn to do " and similar term are interpreted as comprising odd number and plural form otherwise in this description.
The nanoscale people oleoplast that the present invention provides the targeted delivery of drugs system to detect and treat, its composition comprises: an oleEGFR protein, this protein comprise an oil body memebrane protein and an EGFR peptide; One lipid, this lipid are processed the oleEGFR oil body after adding said oleEGFR protein; But and a lipid coating medicine, be called oleEGFR medicine oil body.
The present invention utilizes the preparation process easily of carrying out after EGFR peptide and oil body memebrane protein (oleosin) the binding molecule biotechnology that a kind of carrier (hereinafter referred is the oleEGFR oil body) of people's oleoplast is provided.This person's oleoplast can coat or inlay a large amount of known fluorescence signal molecules and can follow the trail of and confirm focus as real-time (real-time) signal amplifier that moves in the organism; Perhaps; Be used for coating medicine, with as drug delivery system, moreover also can distinguish the function of oil-soluble medicine.
The present invention utilizes gene recombination technology that EGFR peptide and oil body memebrane protein are inserted host cell; Induce oleEGFR protein, this oil body calcium protein can be selected from the oil body protein of the plant seed of following group: a part or its combination in Semen Sesami, Fructus Canarii albi, Semen sojae atricolor, Semen arachidis hypogaeae, numb seed, Brassica campestris L, sunflower, mustard, the Flos Carthami; Host cell can be eukaryotic host cell and prokaryotic host cell; Eukaryotic host cell is selected from following group: unicellular organism body such as yeast cells, be derived from higher organism body such as plant; Insecticide or mammiferous not dead cell or its combination; Prokaryotic host cell is selected from following group: bacillus, coccus, spirillum, vibrio or its combination; The best is escherichia coli, and escherichia coli are worked as host cell and can be: E.coli.DH5 α, E.coli.BL21 (DE3), E.coli nissle (λ G2) or its combination.
This lipid can be selected from following group: triglyceride, olive oil, Oleum sesami, soybean oil, Oleum Arachidis hypogaeae semen, mineral oil, oleum lini, safflower oil or its combination; Wherein, the weight/volume ratio (microgram/microlitre) of oleEGFR protein and this lipid is at least about 1/25, and the mean diameter of this calEGFR oil body is a nano-scale.
Through above-mentioned composition, owing to the EGFR peptide in the oleEGFR protein can be discerned the surperficial receptor of cancerous cell accurately, make it to become to have narrow spectrum delivery vector, so that an active targeted delivery of drugs system to be provided.
Vegetable oils is a kind of neutral fat molecule, mainly contains three important compositions, triglyceride (TAG), phospholipid (PL) and oil body protein, and can the stabilize oil body structure.And oil body protein mainly contains the oil body calcium protein (caleosin) and oil body sterin albumen (steroleosin) of oil body memebrane protein (oleosin) and trace, and is as shown in Figure 1.
The present invention is the oleEGFR protein that utilizes in the oleEGFR oil body; The oil body protein that is comprised is the plant seed oil body protein; But be not limited thereto; Plant seed is if there is oil body protein to make up, for example: Semen Sesami, Fructus Canarii albi, Semen sojae atricolor, Semen arachidis hypogaeae, Brassica campestris L, sunflower, mustard, Caulis et Folium Lini, Flos Carthami (safflower), reach the proteic combination of other vegetable oils.And the present invention utilizes the oil body protein of Semen Sesami seed to make up oleEGFR protein, and oil body memebrane protein aminoacid sequence is shown in SEQ ID NO:1.Oil body memebrane protein mean size is 50 to 200 nanometers.
The nanoscale people oleoplast that the present invention detects and treats as the targeted delivery of drugs system; Wherein, OleEGFR protein can comprise the lipid coating medicine again; With Protocols in Molecular Biology and be assembled into oleEGFR medicine oil body, this medicine can be selected from the medicine of following group: lycopene, curcumin, camptothecine, antibiotic, cucurbitacin, Wen Nuoping (vinorelbine, commodity are called Navelbin), paclitaxel (Paclitaxel), Western medicine, Chinese herbal medicine or its combination; Or calEGFR protein can comprise that lipid coating medicine and lipid coat known fluorescence signal molecule again; Be assembled into oleEGFR fluorescence medicine oil body with Protocols in Molecular Biology, this medicine can be selected from the medicine of following group: lycopene, curcumin, camptothecine, antibiotic, cucurbitacin, Wen Nuoping, paclitaxel, Western medicine, Chinese herbal medicine or its combination; And; This known fluorescence signal molecule can be selected from following group: caesium cadmium quantum dot (quantum dot), Fluorescein isothiocyanate (FITC), alizarin yellow (Alizarine Yellow R; 5-[(p-nitrophenyl) azo] salicylic acid sodium salt), Nile red (Nile Red, 9-diethylamino-5H-benzo [α] phenoxazine-5-one) or its combination; Or the calEGFR pharmaceutical protein can comprise that lipid coats known fluorescence signal molecule again; Be assembled into oleEGFR fluorescence medicine oil body with Protocols in Molecular Biology, this known fluorescence signal molecule can be selected from following group: caesium cadmium quantum dot, Fluorescein isothiocyanate (FITC), alizarin yellow (Alizarine Yellow R), Nile red (Nile Red) or its combination; Above-mentioned one of which, as shown in Figure 2.
The oleEGFR protein of oleEGFR oil body of the present invention; Comprise the EGFR peptide; The oleEGFR oil body is become have narrow spectrum delivery vector, claim targeting vector again, and the oleEGFR oil body also can be formed initiatively targeted delivery of drugs system (hereinafter referred is an oleEGFR medicine oil body) by coating medicine.And the EGFR Toplink is discerned the receptor on cancerous cell surface accurately; Hence one can see that; The oil body memebrane protein combines people's oleoplast of EGFR peptide and coating medicine directly to be delivered to the canceration position or to act on cancerous cell; The administration concentration in canceration position or cancerous cell zone can be promoted greatly, and normal cell can be do not had influence on.Also can carry out phagocytosis and fusion to the oleEGFR oil body by the irritation cancer cell, make cancer therapy drug get into cancerous cell fast, can reach the Drug resistance of treating and avoiding producing medicine, as shown in Figure 2.
When controlling adenocarcinoma of lung, can use the ligand peptide of EGFR protein acceptor.And the EGFR protein acceptor is the epithelical cell growth factor receptor, is present in many cancerous cell surface, as the biomarker on cancerous cell surface.But forefathers develop the peptide of a kind of specificity ground and EGFR protein acceptor key knot, are called the EGFR peptide.The EGFR peptide comprises 98 aminoacid, and aminoacid sequence is shown in SEQ ID NO:2, and molecular weight is about 7 to 15 kilodaltons, and also littler than the molecular weight (150 kilodalton) of monoclonal antibody, hence one can see that, the easy permeates cell membranes of EGFR peptide.
OleEGFR oil body of the present invention can be used as a kind of passive target drug delivery system (passive targeting drug delivery system), that is, and and the narrow spectrum drug delivery system of tool not.Wherein, Via combining oil body protein and above-mentioned cell ligand peptide that one oleEGFR protein is provided, and utilize the multiple material of cell ligand peptide portability, and can directly pass cell membrane and get into cell; Make the oleEGFR oil body not need to get into cell, reach the passive target administration via receptor.
OleEGFR oil body of the present invention can prepare by following preparation method; But not as limit: utilize gene recombination technology on expression vector, to combine the EGFR peptide of oil body protein; Again the oleEGFR oil body is sent in the host cell and expressed, and preparation contains the oleEGFR protein of oil body memebrane protein and EGFR peptide.Add buffer then and mix this oleEGFR protein and lipid, utilize the ultrasonic processor oscillation mixture again, just can produce artificial oleEGFR oil body, as shown in Figure 2.
The lipid that oleEGFR oil body of the present invention is comprised is also unrestricted, illustrates, and lipid can utilize triglyceride, olive oil, Oleum sesami, soybean oil, Oleum Arachidis hypogaeae semen, mineral oil, oleum lini, safflower oil, reach the combination of other plant seed lipid; Acting on more stable is the combination of triglyceride, Oleum sesami or soybean oil, and the best is the combination of Oleum sesami.
The present invention utilizes the ratio of adjustment oleEGFR protein and lipid, and can prepare the oleEGFR oil body of the mean diameter that varies in size.The ratio of the mean diameter of oleEGFR oil body and oleEGFR protein and lipid is inversely proportional to, and oleEGFR oil body amount is fixed, and lipid components is fewer, and the mean diameter of oleEGFR oil body is littler.In oleEGFR oil body of the present invention, the weight/volume ratio (microgram/microlitre) of oleEGFR protein and lipid is generally at least about 1/25, and is preferable at least about 1/1.
The nanoscale people oleoplast that the present invention detects and treats as the targeted delivery of drugs system, wherein, oleEGFR protein can comprise the lipid coating medicine again, is assembled into oleEGFR medicine oil body with Protocols in Molecular Biology; Or oleEGFR protein can comprise that medicine and lipid that lipid coats coat known fluorescence signal molecule, are assembled into oleEGFR fluorescence medicine oil body with Protocols in Molecular Biology again; Above-mentioned one of which, as shown in Figure 2.
Utilization of the present invention contains the oleEGFR protein of oil body memebrane protein and EGFR peptide.Add different pH buffer then and mix this oleEGFR protein and lipid, utilize the ultrasonic processor oscillation mixture again, the pH value of buffer can influence the mean diameter and stability of the oleEGFR oil body of manufacturing.Preferable through the pH value that experiment showed, buffer more than 7.5, be more preferred from about 7.5 to about 9.0.
The present invention with preferable result in, adopt the combination of following condition with preparation oleEGFR oil body: (1) uses the oil body memebrane protein and the EGFR peptide of Semen Sesami seed to make up oleEGFR protein; (2) with olive oil as lipid; (3) adopting the weight/volume ratio (microgram/microlitre) of oleEGFR protein and lipid is about 20/1; And the pH value of (4) buffer is about 7.5.
The present invention is more more convenient than prior art, the size of stable and adjustment oleEGFR oil body, and the oleEGFR oil body that is applicable to various fat-soluble medicines and different lipids can be provided.OleEGFR oil body mean diameter of the present invention is highly stable, and big I reaches tens nanometer to time size of micron, is easy to be absorption of human body.When preparing the oleEGFR oil body of tool injection type, and can control the particle diameters that its mean diameter is about 20 nanometers to about 300 nanometers.
The present invention provides the excellent delivery characteristics of a kind of tool, and is used for the targeted therapy of disease or the combination of detection, and the present invention uses the oleEGFR oil body to detect the EGFR signaling molecule, in conjunction with the combination of different pharmaceutical and different lipids, and forms oleEGFR medicine oil body.
The present invention's combination can comprise any medicine, is not limited to cancer therapy drug, and the best is to contain fat-soluble medicine.Composition of medicine is for example: the combination of lycopene, curcumin, camptothecine, antibiotic, cucurbitacin, Wen Nuoping, paclitaxel, Western medicine, Chinese herbal medicine and other different pharmaceutical also can add the combination of combined type medicine or other medicines.The present invention utilizes lycopene, curcumin and camptothecine to assemble the oleEGFR oil body, and is as shown in Figure 2.
Assembling oleEGFR oil body of the present invention also can combine any known signaling molecule (hereinafter referred is an oleEGFR signal oil body), and can reach testing goal.Signaling molecule can be selected from following group: caesium cadmium quantum dot, Fluorescein isothiocyanate (FITC), alizarin yellow (Alizarine Yellow R), Nile red (Nile Red) and other known signaling molecule that can send the different wave length fluorescence signal.For example: caesium cadmium quantum dot is on using; When utilizing the light of different wave length to excite the caesium cadmium quantum dot of different size; Can scatter the fluorescence of different wave length, so can utilize characteristic caesium cadmium quantum dot to prepare the oleEGFR oil body of launching different fluorescence colors.Therefore, assembling people oleoplast also combines any known signaling molecule, and can reach and reach in vitro testing goal in vivo, has the function that targeting detects in that the present invention is made up, and is to be used to demarcate the position of cancerous cell or focus, as shown in Figure 2.
The nanoscale people oleoplast that targeted delivery of drugs system of the present invention detects and treats; Wherein, the specificity joint (linker) that the oil body memebrane protein can connect a cutting unique sequence earlier connects the EGFR peptide again to host cell, and assembling is formed with the oleEGFR albumen of specificity joint; Add lipid and be assembled into the oleEGFR oil body of specificity joint; There is the oleEGFR oil body of specificity joint can be suspended in solution surface through centrifugal, can reclaims easily, utilize the cutting of specificity protease hydrolysis to make the specificity joint breaking; EGFR albumen is separated from the oil body memebrane protein; Through centrifugal and recovery, obtain EGFR albumen (can discern the EGFR expressed proteins), preparation method is as shown in Figure 3.
The nanoscale people oleoplast that targeted delivery of drugs system of the present invention detects and treats, wherein, the oil body memebrane protein connects the EGFR peptide to host cell; And assembling forms oleEGFR albumen; Add the lipid and the lipid that coats known fluorescence signal molecule of coating medicine, and be assembled into oleEGFR detection and medicine oil body, the oleEGFR detection and the medicine oil body that are coated with cancer therapy drug directly are delivered to the canceration position or act on cancerous cell; Can utilize different excitation wavelengths to detect oleEGFR detection and medicine oil body combination cancerization position; Identification can be known, zonal drug level can also be improved the cancerization position, and the unlikely normal cell that influences.In addition, through above-mentioned mechanism, but also the irritation cancer cell makes cancer therapy drug get into cancerous cell to the phagocytosis and the fusion of oleEGFR medicine oil body, with the purpose that reaches treatment and avoid developing immunity to drugs.
The nanoscale people oleoplast that targeted delivery of drugs system of the present invention detects and treats; The peptide (above-mentioned peptide is called the medicine peptide) that therapeutic effect is arranged by the peptide of the different analog that can synthesize medicine or to disease; Through genetic engineering; This synthetic proteins is for for example: the protein of curcumin, Ai Linuodegan, lycopene, bata-carotene, foot flavin, 3,4,3',4'-tetraketo-.beta.-carotene, maize quality, hirudin, insulin, inhibition disease, make disease cell oneself apoptosis peptide or/and the disease-resistant disease drug peptide of forming with molecular biology, peptide or its combination of disease-resistant disease medicine) and connect and go up that oil body protein connects EGFR peptide to host cell again and assembling forms similar oleEGFR pharmaceutical protein, add lipid and be assembled into to be self-assembled into similar oleEGFR medicine oil body.This oil body can also can be assembled pharmaceutical protein in identification cancerization position, specificity ground voluntarily, can in vivo reach and in vitro detect the cancerization position, carries out the cancerization position and suppresses and Drug therapy, and the preparation process is as shown in Figure 4.
The present invention assembles the oleEGFR oil body can demarcate cancerous cell accurately; Can use the detection at cancerization position in vivo in real-time ground; Also can be assembled into oleEGFR medicine oil body by coating medicine; And delivering drugs exactly, reduce the killed side effect of normal cell to reach accurate kill cancer cell, so the present invention has the effect that real-time ground detects monitoring and treatment.
Medicine or medicine peptide that the present invention assembles oleEGFR medicine oil body and oleEGFR fluorescence medicine oil body are not limited to said medicine; Can have suppress that disease cell, poisoning disease cell, the one or more genes of insertion/changes/removals make the sudden change of disease cytogene and can't be normally or fast division cause disease cellular atrophy or self-apoptosis, make the disease cell process step to interrupt and can't carry out the division of cell cycle, in the disease cell, produce and suppress or the material of destruction disease cell growth conditions and can't carry out the division of cell cycle and make the disease cell can't infect Normocellular material and can both assemble with this oil body or combine, have the effect that inhibition of cancerization position and Drug therapy are discerned in specificity ground.
The nanoscale people oleoplast that the present invention detects and treats as the targeted delivery of drugs system; Wherein, oleEGFR protein utilization technique for gene engineering connects the specificity joint, and is assembled into oleEGFR specificity joint oil body; OleEGFR specificity joint oil body can utilize the cutting of specificity protease hydrolysis; Can be purified into the proteinic application of the high EGFR of purity, this method is convenient simply again can't to influence proteic character, as shown in Figure 3.
This oil body memebrane protein of the present invention is the oil body memebrane protein that comprises by the listed DNA sequence of the listed aminoacid sequence of SEQ ID NO:1 and SEQ ID NO:3; The EGFR peptide is the oil body memebrane protein that comprises by the listed DNA sequence of the listed aminoacid sequence of SEQ ID NO:2 and SEQ ID NO:4.And assembling oleEGFR medicine oil body and the medicine of oleEGFR fluorescence medicine oil body or the medicine of medicine peptide; Can have suppress that disease cell, poisoning disease cell, the one or more genes of insertion/changes/removals make the sudden change of disease cytogene and can't be normally or fast division cause disease cellular atrophy or self-apoptosis, make the disease cell process step to interrupt and can't carry out the division of cell cycle, in the disease cell, produce and suppress or the material of destruction disease cell growth conditions and can't carry out the division of cell cycle and make the disease cell can't infect Normocellular material and can both assemble with this oil body or combine, have the effect that inhibition of cancerization position and Drug therapy are discerned in specificity ground.
What deserves to be mentioned is; The nanoscale people oleoplast that the present invention detects and treats as the targeted delivery of drugs system; Wherein, be a kind of escherichia coli of probiotic bacteria as the E.coli nissle (λ G2) of host cell, and diabetes, disease of stomach, heart disease or its disease are had therapeutic effect.
The present invention uses the oleEGFR oil body, has ease of Use property and excellent carrier characteristics, therefore can be widely used in industries such as protein purification, plant tissue culture, Chinese herbal medicine, Western medicine, medical test, biomedical material, animal vaccine, biotechnology.With following concrete description of test, narrate the present invention further.
Embodiment 1, preparation oleEGFR oil body
Make up oleEGFR oil body of the present invention according to preparation flow shown in Figure 2.
< step 1, construction of expression vector >
Utilize below the gene recombination technology general gene constructed on expression vector:
Oil body memebrane protein (N end)-EGFR peptide (C end) the proteinic gene of oleEGFR: the gene (comprising nucleotide sequence shown in the SEQ ID NO:4) that combines the proteinic ligand peptide of EGFR (being the EGFR peptide) with the oil body membrane protein gene (comprising nucleotide sequence shown in the SEQ ID NO:3) of a Semen Sesami seed.Detailed operating procedure is following.At first, utilize the sesame seed to carry out genetic recombination, purifying and extraction; And obtain oil body calcium protein peptides), to be assembled into pET-oil body calcium protein peptides, purifying pET-oil body membrane protein polypeptide; With as template DNA, utilize primer to obtain an oil body membrane protein gene fragment again via PCR.Then; With this genetic fragment of Nde I restriction endonuclease enzyme action; And it is connected on pET-29a (+) carrier (available from Novagen company), more prepared recombinant vector is converted in E.coli.DH5 α (available from Foodstuff Industrial Development Inst. of Financial Group Legal Persons (the FIRDI)) host cell.In the LB (Luria-Bertani) of kanamycin (available from Sigma company) solid medium, cultivate this host cell, the row filter of going forward side by side, with obtain one comprise the recombinant vector of pET-29a-oil body membrane protein gene transformant.
Come, purification pBlue-EGFR NS carrier utilizes primer to obtain the EGFR genetic fragment via polymerase chain reaction (PCR) to be used as template DNA more again.Then,, and be connected on pBluescript II (SK-) (available from the Novagen company) carrier, the recombinant vector that makes is converted in the E.coli.DH5 α host cell with EcoR V restriction endonuclease enzyme action EGFR gene.Contain in one and to cultivate this host cell in the LB solid medium of penicillin and X-gal (available from Sigma company), the row filter of going forward side by side wherein, is selected white bacterial strain to obtain the transformant of pBluescript II-EGFR recombinant vector.At last; With the EGFR genetic fragment on Nco I and this recombinant vector of HindIII restriction endonuclease enzyme action, and it be connected to one comprise on the recombinant vector of pJo1-oil body membrane protein gene, can make one comprise pJo1-oil body membrane protein gene-EGFR expression vector; Be called pJO1-oleEGFR and (be called for short oleEGFR; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica on June 15th, 2011; CGMCC No.:4959, classification name ETEC (Escherichia coli)).
After accomplishing the above-mentioned expression vector of structure; Respectively it is converted into e. coli host cell (E.coli BL21 (DE3) (available from Novagen company); With E.coli nissle (λ G2)) in (available from Foodstuff Industrial Development Inst. of Financial Group Legal Persons (FIRDI)), and extract plasmid to confirm the nucleotide sequence of expression vector.
Above-mentioned experimental technique can be referring to Sambrook et al., The CondenSEQ Protocols From Molecular Cloning:A Laboratory Manual 2006, and the document is incorporated this paper into for your guidance in full.
< step 2, expression oleEGFR albumen >
Induce prepared host cell in the step 1 with isopropyl-(IPTG, 0.05 millimolar concentration (available from USB company)), make its great expression oleEGFR albumen, and collect bacterium liquid.With 6; The centrifugal bacterium liquid of 500 rev/mins rotating speed 10 minutes; Suspend through the host cell of centrifugation with the long-pending TE buffer (TE (Tris-EDTA) buffer (available from Sigma company)) of about 1/10 times of bacteria liquid again, then, be added in the sample buffer (SDS-PAGE 4X sample buffer (available from Sigma company)) of sodium dodecyl sulfate-polyacrylamide gel electrophoresis it and mix homogeneously; Last 95 ℃ of heating are carried out the protein electrophorese analysis after about 10 minutes.The result is as shown in Figure 5.
< step 3, interpolation lipid and signaling molecule >
Prepared oleEGFR protein (50 milligrams) in the step 2 is added in the test tube; And add phospholipid and the 2.5 microgram fluorescein stains (alizarin yellow, (available from bosom moral biotechnology company) or Nile red (available from Sigma company)) of 50 milligrams of triglyceride, 150 micrograms, (model: Sonics VCX130) five times (condition was 10 seconds with several minutes interval vibration to utilize ultrasonic sample processor; 20amplitude; 0.5pulser), to carry out the oil body reorganization and to assemble out oleEGFR fluorescence oil body, make this oil body pass through 0.22 millimeter filter membrane; Utilize fluorescence microscope (model: Nikon 104) to observe the preparation result, the result is as shown in Figure 6.
General biochemical investigation analytical method capable of using is measured three kinds of bases in the oleEGFR oil body.In this, obtain content of triglyceride via detecting ester bond and calculating; Use the detectable (BCA protein assay) of BioRad company to calculate the acquisition protein content.
The AFM test of embodiment 2, oleEGFR oil body
Mica sheet (available from helping great enterprise stock company limited) water is cleaned three times, each three minutes, the MgCl infusion of reuse 0.02M 2-3 minute, and with the clear water cleaning once.Prepared oleEGFR protein (50 milligrams) in the step 2 is added in the test tube; And add the phospholipid of 50 milligrams of triglyceride, 150 micrograms and be assembled into the oleEGFR oil body, make this oil body pass through 0.22 millimeter filter membrane, the oil body after will filtering again is added to and leaves standstill 2 hours on the mica sheet; Targeting type people oleoplast is adsorbed on the mica sheet; Clean twice with distilled water afterwards, be positioned over drying in 37 ℃ of baking ovens, use AFM (Atomic Force Microscope at last; AFM) (model: NS4/D3100CL/MultiMode) observe oil body kenel and size, the result is as shown in Figure 7.
The influence of embodiment 3, lipid and the proteinic ratio of oleEGFR
100 micrograms oleEGFR add 950 microlitres in the protein (pJo1-oleEGFR) sodium phosphate buffer (0.01 molar concentration; PH 7.5) olive oil (available from Sigma company) of (available from Sigma company) and 50 microlitres, the weight/volume ratio (microgram/microlitre) that obtains containing oleEGFR protein/lipid (olive oil) is 2/1 mixture.Then, and the sonic oscillation mixture (mixture places on ice, power: 15%, and the time: 20 seconds, run:0.5 second, rest:0.5 second, vibrate three times), obtain desired oleEGFR oil body.Repeat aforementioned operation; But use 400,200,100 and 100 microgram oleEGFR protein respectively; And corresponding 20,20, the 100 and 500 microlitre olive oil that use are respectively 20/1,10/1,1/1, reach 1/5 oleEGFR oil body with the weight/volume ratio (microgram/microlitre) that obtains oleEGFR protein/lipid (olive oil).
Utilize the form (as shown in Figure 8) and the turbidity of Nikon 104 type observation by light microscope different condition oleEGFR oil bodies (as shown in Figure 9; Computational methods such as following examples 6 are said); And use particle size analyzer (Beckman Coulter, N4Plus) particle diameter of analysis oleEGFR oil body.Wherein, ionic strength is fixed as 0.1, and under 25 ℃ with dynamic light scattering (dynamic light scanning, the DLS) (argon laser beam: 633 nanometers of particle size analyzer; Angle of scattering: 90 °; Analytical method: Contin) analyze size and particle size distribution, the result is as shown in table 1.
Table 1, oleEGFR oil body are tested at the different condition particle diameter.
Figure BSA00000598149500141
The influence of embodiment 4, pH value
OleEGFR protein (100 microgram oleEGFR) is added in the sodium phosphate buffer (0.01 molar concentration) of different pH value (pH 6.5, pH 7.0, pH 7.5, pH 8.0 or pH 9.0) of 950 microlitres, adds the olive oil of 50 microlitres again, (mixture places on ice the sonic oscillation mixture; Power: 15%; Time: 20 seconds, run:0.5 second, rest:0.5 second; Vibrate three times), with preparation oleEGFR oil body.Utilize the form (as shown in Figure 8) and the turbidity (as shown in Figure 9) of Nikon 104 type observation by light microscope oleEGFR oil bodies; Analyze the particle diameter (as shown in table 1) of oleEGFR oil body with particle size analyzer.
The influence of embodiment 5, lipid species
OleEGFR protein (100 microgram pJo1-oleEGFR) is added to sodium phosphate buffer (0.01 molar concentration of 950 microlitres; PH 7.5) in, adding the different lipids (Oleum sesami (available from Sigma company), olive oil, soybean oil (available from Sigma company), Oleum Arachidis hypogaeae semen (available from Sigma company) or mineral oil (available from Sigma company)) of 50 microlitres again, (mixture places on ice the sonic oscillation mixture; Power: 15%; Time: 20 seconds, run:0.5 second, rest:0.5 second; Vibrate three times), with preparation oleEGFR oil body.Utilize the form (as shown in Figure 8) and the turbidity (as shown in Figure 9) of Nikon 104 type observation by light microscope oleEGFR oil bodies; Analyze the particle diameter (as shown in table 1) of oleEGFR oil body with particle size analyzer (model: Zetasizer Nano, Malvern #ZS90).
Shown in Fig. 8 and the table 1, the mean diameter of obtained oleEGFR oil body is between 10 to 2,000 nanometers, and along with the proteinic ratio of lipid and oleEGFR reduces and diminishes.Therefore, can adjust lipid and the proteinic ratio of oleEGFR to prepare the oleEGFR oil body of different mean diameters.In addition, the mean diameter of oleEGFR oil body is less relatively under alkaline environment.
The degree of stability of embodiment 6, mensuration oleEGFR oil body
Measure the degree of stability of oleEGFR oil body with following three kinds of modes
A, observation negative electricity repulsion
Can record its degree of stability through the three-dimensional barrier effect of observing surperficial negative electricity repulsion of oleEGFR oil body or protein covering, wherein, can observe the oil body gathering that negative electricity repulsion fades away and caused through the pH value that reduces solution.Therefore, the oleEGFR oil body is positioned in the phosphate buffer of different pH value, and under room temperature, leaves standstill 12 hours, again with its variation of observation by light microscope.Can find out by Fig. 8, in pH 7.5 held after 12 hours, being kept perfectly property all still.
B, measurement turbidity
If the oleEGFR oil body is complete, then the surface is a hydrophilic, and can dissolve each other with water effectively and be suspended state.If the oleEGFR oil body is incomplete, then its proteins on surfaces can cause polymerization each other between the oleEGFR oil body because of can't correctly folding, and makes the oleEGFR oil body float over solution surface.Therefore, can understand the integrity of oleEGFR oil body indirectly by the turbidity of measuring the solution bottom.Therefore, at first 1 milliliter oleEGFR oil body is placed in the disposable measuring tube, and with ferrule and avoid vibrations, be statically placed in room temperature after following 140 minutes, with absorbing wavelength 600 nano measurement turbidity.Turbidimeter is shown relatively: T/T0=10A/10A0=10A/102.0, wherein A0 is 2.0.Can be found out that by Fig. 9 after 140 minutes, the oleEGFR oil body is being kept perfectly property still, this explanation oleEGFR oil body has splendid stability.
C, measurement zeta current potential
The oleEGFR oil body is scattered under the varying environment (the proteinic weight/volume ratio of different lipids and oleEGFR, pH value or lipid species); And utilize surface potential analyser (Zetasizer Nano; Malvern #ZS90) variation of the surface zeta potential current potential of the artificial oleEGFR oil body of measurement; Measurement result is as shown in table 2, and the degree of stability of oleEGFR oil body reduces along with lipid and the proteinic ratio of oleEGFR and promotes, and comparatively stable under alkaline environment.
Table 2, oleEGFR oil body are tested at different condition surface potential analyser.
Figure BSA00000598149500161
The target function gonosome of embodiment 7, oleEGFR oil body is tested outward-fixedly tumor cell test
With 5 * 10 4Individual cell NCI-H520 (people's Lung Squamous Carcinoma Cells), A431 (glandular integumentaria cancerous cell) and A549 (lung adenocarcinoma cell) plant to one 24 hole culture plates, and place 37 ℃ cell culture incubator (containing 5 volume % carbon dioxide) to cultivate 24 hours.The next day, with phosphate buffer (PBS buffer) (available from the Sigma company) cleaning many times of pH 7.4, under room temperature, fix 20 minutes again with 2.5 weight/volume % formaldehyde (available from Sigma company), the phosphate buffer with pH 7.4 cleans again.To coat alizarin yellow oleEGFR oil body (comprising the every ml phosphate buffer of 2.5 microgram pJo1-oleEGFR/) is added to through fixed cell; In the phosphate buffer of pH 7.4; After reacting 1 hour under 25 ℃; Phosphate buffer (comprising 1/1000Tween-20 (available from USB company)) with pH 7.4 cleans three times, and the phosphate buffer with pH 7.4 cleans three times again.Afterwards, add lock solution (fetal bovine serum albumin of 3% weight/volume (BSA) (available from Sigma company) is dissolved in the phosphate buffer), and under room temperature, reacted 1 hour; With anti-EGFR one-level antibody (9G6; Santa Cruz Biotechnology company, Santa Cruz, California; The U.S.), under room temperature, reacted at least 1 hour again with after 1: 500 the dilution proportion.After cleaning three times with phosphate buffer again, utilize again Ab30 (with 1: 500 dilution proportion, Jackson ImmunoResearch Laboratories company; West Grove; Pennsylvania, the U.S.) reacted 1 hour, clean with phosphate buffer once more.Nucleus dyeing is then used 15,000 times DAPI (diamidino-2-phenylindole) to dye and is cleaned three times, mounting after dyeing is accomplished, and (model: Olympus IX71) observes to utilize fluorescence microscope.Experimental result is shown in figure 10, and the oleEGFR oil body can specificity ground be demarcated with formaldehyde fixed and passed through A431, A549 and the NCI-H520 cell of the EGFR receptor that scale reaches.
The target function gonosome of embodiment 8, oleEGFR oil body is tested outward-the tumor living cell test
With 5 * 10 4Individual cell NCI-H520 (people's Lung Squamous Carcinoma Cells), A431 (glandular integumentaria cancerous cell) and A549 (lung adenocarcinoma cell) plant to one 24 hole culture plates, and place 37 ℃ cell culture incubator (containing 5 volume % carbon dioxide) to cultivate 24 hours.The next day; After DMEM/F12 culture fluid (GIBCO Invitrogen Corporation, New York, the U.S.) cleaning; To coat alizarin yellow oleEGFR oil body (comprising the every ml phosphate buffer of 2.5 microgram pJo1-oleEGFR/) is added to through fixed cell; And in the DMEM/F12 culture fluid, in 37 ℃ incubators (containing 5 volume % carbon dioxide), reacted 2 hours, clean three times with pH 7.4 phosphate buffers again.Then,, clean after 30 minutes in fixed cell under the room temperature, add lock solution (3% weight/volume fetal bovine serum albumin is dissolved in the phosphate buffer) again, and under room temperature, reacted 1 hour with pH 7.4 phosphate buffers with 2.5 weight/volume % formaldehyde.With after 1: 500 the dilution proportion, reaction is at least 1 hour under room temperature with anti-EGFR one-level antibody (9G6, Santa Cruz Biotechnology company, Santa Cruz, California, the U.S.).Then, after phosphate buffer cleaning three times,, clean three times with phosphate buffer with Ab30 (with 1: 500 dilution proportion, Jackson ImmunoResearch Laboratories company, West Grove, Pennsylvania, the U.S.) reaction 1 hour.Last nucleus dyeing then is to use 15,000 times of DAPI to dye and cleans three times, give mounting after the completion dyeing, and (Olympus observes IX71) to utilize fluorescence microscope.Experimental result is shown in figure 11, and the oleEGFR oil body can specificity ground be demarcated with formaldehyde fixed and passed through A431, A549 and the NCI-H520 cell of the EGFR receptor that scale reaches.
The righttest activity of embodiment 9, oleEGFR oil body and tumor cell
With 5 * 10 4Individual cell NCI-H520 (people's Lung Squamous Carcinoma Cells), A431 (glandular integumentaria cancerous cell) and A549 (lung adenocarcinoma cell) plant to one 24 hole culture plates, and place 37 ℃ cell culture incubator (containing 5 volume % carbon dioxide) to cultivate 24 hours.The next day; With DMEM/F12 culture fluid (GIBCO Invitrogen Corporation; New York, U.S.) clean after, utilize infectious agent value (the MOI value that coats variable concentrations alizarin yellow oleEGFR oil body and cell; Multiplicity of infection) (MOI 100, MOI 200, MOI 400) measures the righttest activity between the two.Therefore; The cell strain that uses is the glandular integumentaria JEG-3 A431 of overexpression EGFR receptor, people's Lung Squamous Carcinoma Cells strain NCI-H520 that the no mistake in treatment scale reaches the EGFR receptor, and the lung adenocarcinoma cell line A549 of overexpression EGFR receptor; Be added to through fixed cell; And in the DMEM/F12 culture fluid, in 37 ℃ incubators (containing 5 volume % carbon dioxide), reacted 2 hours, clean three times with pH 7.4 phosphate buffers again.Then, after 30 minutes, clean with pH 7.4 phosphate buffers in fixed cell under the room temperature with 2.5 weight/volume % formaldehyde, following fluorescence microscope is all different with the experimental procedure of flow cytometer test, explanation respectively.
Fluorescence microscope: the cell strain of effect oleEGFR oil body, add lock solution (3% weight/volume fetal bovine serum albumin is dissolved in the phosphate buffer) again, and under room temperature, reacted 1 hour.With after 1: 500 the dilution proportion, reaction is at least 1 hour under room temperature with anti-EGFR one-level antibody (9G6, Santa Cruz Biotechnology company, Santa Cruz, California, the U.S.).Then, after phosphate buffer cleaning three times,, clean three times with phosphate buffer with Ab30 (with 1: 500 dilution proportion, Jackson ImmunoResearch Laboratories company, West Grove, Pennsylvania, the U.S.) reaction 1 hour.Last nucleus dyeing is then used 15,000 times of DAPI to dye and is cleaned three times, give mounting after the completion dyeing, and (model: Olympus observes IX71) to utilize fluorescence microscope.
The flow cytometer test: the cell strain of effect oleEGFR oil body, add pH 7.4 phosphate buffers again and utilize flow cytometer (BD FACSCanto (Argon-Ion Laser 488nm, He-Ne Laser 633nm)) test.
Wherein, the MOI value defined is the ratio of oleEGFR oil body number and cell number, and it can convert concentration unit into according to following formula:
MOI 100=1.25 * 10 -2Mcg/ml
MOI 200=2.5 * 10 -2Mcg/ml
MOI 400=5 * 10 -2Mcg/ml
Experimental result is shown in figure 12; Under fluorescence microscope, observe; The oleEGFR oil body that discovery comprises the EGFR peptide has narrow spectrum identification ability to the cell strain of overexpression EGFR receptor; And the oleEGFR oil body number that gets into cell has the trend of lifting along with the increase of MOI value, is then to present saturated situation at 200 o'clock to the MOI value.
Utilize fluorescence microscope oleEGFR oil body whether to get into the influence of cell and MOI value.The result shows that the oleEGFR oil body gets in the cell really, and its quantity that gets into cell increases along with the lifting of MOI value equally.The flow cytometer test result; (wherein, cell is defined as with the percentage rate that combines of oleEGFR oil body: through combining cell number/10,000 cell * 100 of oleEGFR oil body) shown in figure 14; Above-mentioned experimental result, fluorescence microscope is consistent with the analysis of flow cytometer.
The righttest action time of embodiment 10, oleEGFR oil body and tumor cell
With the righttest action time of observing oleEGFR oil body and tumor cell with embodiment 9 identical methods; Wherein the MOI value is fixed as 200; The cell strain that uses is for reaching A549 cell, the A431 cell of EGFR receptor through scale; And without the NCI-H520 cell of overexpression EGFR, and act in different time points (0,15,30,60,120 and 240 minute).The result is shown in figure 13; The oleEGFR oil body of under fluorescence microscope, all finding to comprise the EGFR peptide has narrow spectrum identification ability to the cell strain of overexpression EGFR receptor; And the number that the oleEGFR oil body gets into cell increases the trend that lifting is arranged with works with the time, when be 2 hours action time, then presents saturated situation.
The influence that utilizes fluorescence microscope oleEGFR oil body whether to get into cell and action time.The result shows that the quantity of oleEGFR oil body entering cell promotes with the increase of action time, and presents saturated situation in 2 hours.The flow cytometer test result; (wherein, cell is defined as with the percentage rate that combines of oleEGFR oil body: through combining cell number/10,000 cell * 100 of oleEGFR oil body) shown in figure 15; Above-mentioned experimental result, fluorescence microscope is consistent with the analysis of flow cytometer.
Embodiment 11, oleEGFR oil body are invaded tumor cell
Invade tumor cell to observe the oleEGFR oil body with embodiment 8 identical methods, wherein the MOI value is fixed as 200, and the time was fixed as 2 hours, and the cell strain of use is for reaching the SKOV3 cell effect of EGFR receptor through scale.The result is shown in figure 16, utilizes conjugation fluorescence microscope (model: Leica TCS SP2) get X, Y and Z axle, all find to comprise the narrow spectrum identification ability of oleEGFR oil body tool of EGFR peptide and can invade the cell the inside.
The composition of embodiment 12, oleEGFR medicine oil body, size and zeta current potential
Prepared oleEGFR protein (50 milligrams) in the step 2 is added in the test tube; Add the lipid that 50 microlitres coat different pharmaceutical again; (model: Sonics VCX130) five times (condition is 10 seconds with the vibration of several minutes interval to utilize ultrasonic sample processor again; 20amplitude, 0.5pulser), to carry out the oil body reorganization.Assemble out oleEGFR medicine oil body.Utilize particle size analyzer to analyze the particle diameter of oleEGFR medicine oil body, as shown in table 3.OleEGFR medicine oil body is utilized surface potential analyser (Zetasizer Nano) (model: the variation of the surface zeta potential current potential of the artificial oleEGFR medicine oil body of measurement Malvern #ZS90); Measurement result is as shown in table 3; The degree of stability of oleEGFR medicine oil body, shown in figure 17.
If oleEGFR medicine oil body is complete, then the surface is a hydrophilic, and can dissolve each other with water effectively and be suspended state.If oleEGFR medicine oil body is incomplete, then its proteins on surfaces can cause polymerization each other between oleEGFR medicine oil body because of can't correctly folding, and makes oleEGFR medicine oil body float over solution surface.Therefore, can understand the integrity of oleEGFR medicine oil body indirectly by the turbidity of measuring the solution bottom.Therefore, at first 1 milliliter oleEGFR oil body is placed in the disposable measuring tube, and with ferrule and avoid vibrations, be statically placed in room temperature after following 140 minutes, with absorbing wavelength 600 nano measurement turbidity.Turbidimeter is shown relatively: T/T0=10A/10A0=10A/102.0, wherein A0 is 2.0.Can be found out that by Figure 17 after 24 hours, the oleEGFR oil body is being kept perfectly property still, this explanation oleEGFR oil body has splendid stability.
Table 3, oleEGFR oil body coating medicine size and surface potential analyser test-lycopene, curcumin and camptothecine.
Figure BSA00000598149500201
Figure BSA00000598149500202
The experiment of embodiment 13, targeted therapy and detection-cell survival test
At first; Coat antitumor drug (lycopene (available from Sigma company), curcumin (available from Sigma company) or the camptothecine (CPT of variable concentrations according to the method preparation of embodiment 9; Available from Sigma company) the oleEGFR oil body, with as the combination that supplies targeted therapy and/or detection.With 1 * 10 4Individual cell (NCI-H520 and A549 cell) plants in the one 24 hole culture plates, places 37 ℃ cell culture incubator (comprising 5 volume % carbon dioxide) to cultivate 24 hours.The next day, the combination that makes more than the interpolation, and effect removed culture medium with suction pipe after 2 hours in incubator, and clean three times with phosphate buffer, with the oleEGFR oil body of flush away effect.Then, add fresh culture medium, after cultivating 72 hours; Staining cell utilizes microscope to calculate the number result of dead cell and living cells again, and is shown in figure 18; And utilize flow cytometer to detect different time oleEGFR fluorescence oil body in the A549 cell residue time, shown in figure 19.Show that by experiment oleEGFR oil body of the present invention does not have obvious cytostatic situation.
The hemolytic test of embodiment 14, oleEGFR oil body
To coat different types of oleEGFR medicine oil body (comprising the every ml phosphate buffer of 2.5 microgram pJo1-oleEGFR/) as for normal saline solution that mice whole blood (5%) (commercially available purchase) is arranged and 5% glucose solution (available from Sigma company); And place 37 ℃ centrifugal after 30 minutes; And with dividing luminometer to measure, shown in figure 20.With fluorescence microscope erythrocyte situation, shown in figure 21.The result shows that oil body is not have injury to blood cell, can not make blood cell haemolysis.
Embodiment 15, serum stability experiment
Utilize and coat different types of oleEGFR medicine oil body (comprising the every ml phosphate buffer of 2.5 microgram pJo1-oleEGFR/) as for containing 5% mice serum (commercially available purchase) adding sodium phosphate buffer and breaing up; Measure at different time and different temperatures with particle size analyzer; And observe and to increase degree of stability and the size of targeting people oleoplast at serum in time, shown in figure 22.The particle size analyzer result shows, it 24 hours was unusual stable status that the oleEGFR oil body is measured in different temperatures at 5% serum.
Experiment-the interior animal experiment of embodiment 16, targeted therapy and detection
The mice that utilization suffers from pulmonary carcinoma carries out zoopery.Raise about 8 weeks of mice (BALB/cAnN.Cg-Foxnlnu/CrlNarl, commercially available purchase) greatly, utilize the subcutaneous injection technology that the A549 lung carcinoma cell is injected to the subcutaneous of back, mice left side again, and carry out the pulmonary carcinoma induction culturing phase in about 2 weeks.After inducing the tumor of pulmonary carcinoma pattern to generate to reach for 4 weeks, tumor size is about 1,000 cubic millimeter, starts from mouse subcutaneous injection oleEGFR oil body of the present invention (comprising 1.0 microgram pJo1-oleEGFR and the every ml phosphate buffer of pJo1-ole/) again.Utilize IVIS 200 System 3D living body molecule image systems (hereinafter to be referred as: IVIS); Respectively at 1 hour, 4 hours, 8 hours and 24 hours run-down images, to observe the situation that the oleEGFR oil body is followed the trail of and distributed in each organ in the intravital blood circulation of mice, lung carcinoma cell.
The result is shown in figure 23, and the interior fluorescence intensity of body of the mice of matched group injection oil body memebrane protein fluorescence oil body increases along with the time and fades away, and experimental group injects oleEGFR fluorescence oil body mice then still can detect clearly signal.
Then, make mice carry out carbon dioxide narcosis and disconnected neck execution, take out tumor and organ again and carry out tissue slice, to observe the distribution scenario of oleEGFR oil body.Experimental result is shown in figure 24; In the body of control group mice; The oleEGFR oil body mainly is accumulated in liver (carrying out the organ of drug metabolism) position; Explain that the intravital oil body memebrane protein of control group mice oil body does not rest in the A549 tumor tissues, the intravital oleEGFR oil body of experimental mice then rests in the A549 tumor tissues, demarcates and got into the SKOV3 tumor tissues so show its specificity ground.
In addition; Shown in figure 25; The intravital oil body memebrane protein of matched group Mus oil body does not rest on A549 tumor tissue section and shows that the distribution of oil body memebrane protein oil body is also not obvious, and the intravital oleEGFR oil body of experimental mice then rests on the A549 tumor biopsy can find that then the oleEGFR oil body is retained in the tumor tissues.
The effect of the experiment of embodiment 17, targeted therapy and detection-oleEGFR oil body tumor tissues
After taking out the A549 tumor of the mice back leg among the embodiment 16, carry out OCT (the tissue refrigerant is available from LEICA company) embedding, (model: LEICA CM3050S) carries out frozen section to utilize freezing microtome.The tissue slice that cuts out is attached on the microscope slide; Clean section three times with phosphate buffer; With the OCT on the flush away histiocyte; Again with 2.5% formalin solution (0.5 kilogram of formalin powder, 2 milliliters through the phosphate buffer of dilution (10X) and 5 centinormal 1 sodium hydroxide of 50 microlitres) fixing organization cell, and act on 40 minutes.Then; With phosphate buffer cleansing tissue section three times; With the unnecessary formalin solution of flush away tissue, add oil body memebrane protein fluorescence oil body and oleEGFR fluorescence oil body (comprising 2.5 microgram oleEGFR and oil body memebrane protein/every ml phosphate buffer) and histiocyte effect 120 minutes again.Effect finishes, with phosphate buffer cleansing tissue three times, and the oleEGFR fluorescence oil body of flush away effect and oil body memebrane protein fluorescence oil body; Again with 1: 15, after the DAPI staining cell of 000 ratio multiple nuclear reaches 5 minutes, with phosphate buffer cleansing tissue section three times; With the unnecessary DAPI of flush away; Mounting more at last, and utilize the situation of fluorescence microscope oleEGFR fluorescence oil body and oil body memebrane protein fluorescence oil body and histiocyte effect, shown in figure 26.
Detect in the body of embodiment 18, targeting type oil body drug delivery vehicle target function property
The nude mice in about 8-10 age in week utilizes cell strain (NCI-H520 and A549) to carry out hypodermic injection; Make the hypodermic tumor size of nude mice about more than 0.5 centimetre; Twice injection 100mg/ml of jede Woche targeting type coats the oil body of camptothecine on tumor, and observes tumor size (result is shown in figure 27) and body weight (result is shown in figure 28).In zoopery, can be observed nanometer oil body targeting property learning; Show by the result, increase in time, the oil body that targeted nano coats camptothecine can suppress tumor growth; The nude mice body weight is not reduced, more can reach the effect that the effect of treatment is dwindled tumor.
Embodiment 19, targeting type oleEGFR fluorescence oil body detect in the functional body of targeting in the mouse lung body
The nude mice in about 8-10 age in week utilizes cell strain (NCI-H520 and A549) to carry out the injection of tail; Make nude mice lung cancer caused in pulmonary; Injection 100mg/ml oleEGFR fluorescence oil body is on tail afterwards; Through 12,24 and 48 hours, whether nude mouse in can detection of lung cancer cell, shown in figure 29 if observing oleEGFR fluorescence oil body with IVIS.Utilize IVIS to observe oleEGFR fluorescence oil body detection of lung cancer cell in nude mouse again, and utilize numerical value to make rectilinear most with the numerical value that IVIS detects signalling, shown in figure 30.Take out nude mice internal organs (lung) and detect the numerical value of signalling, utilize numerical value to make rectilinear at last with IVIS, shown in figure 31.
Experimental result and discussion
OleEGFR oil body of the present invention is to utilize oil body memebrane protein and EGFR peptide system host cell (escherichia coli) and form oleEGFR protein, and this protein adds lipid, and is assembled into the oleEGFR oil body, and as shown in Figure 5, oleEGFR protein position is at 29KDa.
Utilize the optimum condition of people's oleoplast (AOB) and add known fluorescence signal molecule; Also can make oil body for example send fluorescence: Nile red; Wavelength 384mm can send red fluorescence, and Yellow GGK wavelength 484mm can send green fluorescence, and uses fluorescence microscope; As shown in Figure 6, can make targeting people oleoplast send fluorescence signal so that observe.As shown in Figure 7, can utilize former seed microscope to measure oil body is to meet nano-scale.
Oil and fused protein are mixed with following ratio (10: 1,2: 1,1: 1,1: 5,1: 10); Measure best ratio; And with microscopic examination oleEGFR oil body; As shown in Figure 8, also be the best ratio of oil and fusion rotein 1: 1, the big low profile of oil body the most on average reaches oil body and oil body merges less each other.Utilize granularmetric analysis less than normal at 1: 1,1: 5 and 1: 10 oil body granule again as seeing fuel-displaced and fusion rotein.Buffer with different pH (6.5,7.0,7.5,8.0,9.0) constructs the oleEGFR oil body, and is as shown in Figure 8, measures best pH value, and the big low profile of buffer structure oil body of pH 7.5 the most on average reaches oil body and oil body merges less each other.Use various oil (olive oil, Oleum Arachidis hypogaeae semen, soybean oil, mineral oil, Oleum sesami) to make up oil body; As shown in Figure 8; Measure optimum oil, the big low profile of the constructed oil body of Oleum sesami and olive oil the most on average reaches oil body and oil body merges less, opposite each other; Mineral oil oil body granule is maximum, and to present chain spherical and the Oleum Arachidis hypogaeae semen oil body merges more meeting mutually.
In particle size analyzer experiment, as shown in table 1, institute is shown in sees on the ratio that fuel-displaced and fusion rotein are less than normal at 1: 1,1: 5 and 1: 10 targeting people oleoplast granule.Learn that at different pH pH 7.5, pH 8.0 and pH 9.0 targeting people oleoplast granules are less than normal.Learn that at inhomogeneous oil it is less than normal that soybean oil, Oleum sesami and olive oil manufacture targeting people oleoplast granule.Can find out that at surface potential analyser analysis chart on average-45～-56, difference is little, and is as shown in table 2 at the targeting people oleoplast electronegativity of different condition.Shown in the degree of stability result, targeting people oleoplast size is big more relatively more unstable, and targeting people oleoplast size is more little more stable, as shown in Figure 9.
Whether utilize the mode of cell fixation, testing the EGFR peptide that merges oleEGFR fluoroleum surface has function and specificity ground identification EGFR.Whether the EGFR peptide that merge test oleEGFR fluoroleum surface has the overexpression of function and specificity ground identification EGFR; So we discern the NCI-H520 cell strain that EGFR overexpression utilization has the A431 that highly expresses EGFR and A549 cell strain and no EGFR expression; OleEGFR fluorescence oil body is affacted A431, A549 and NCI-H520 cell strain; The result finds that the NCI-H520 cell strain do not observed fluorescence people oleoplast signal and produced; And can observe a lot of green fluorescence oil body signals at A431 and A549 cell strain; So the representative of green fluorescence oil body signal is because cell EGFR overexpression can recognize oil body; Can obtain the specificity that oleEGFR fluorescence oil body has identification EGFR overexpression cell by experiment, shown in figure 10, can find out that the cell that the EGFR overexpression is arranged can make oleEGFR fluorescence oil body specificity combine.
Learn that by The above results targeting people oleoplast can be discerned the specificity of overexpression cell at fixed cell, and further understand whether targeting people oleoplast discerns the overexpression cell at living cells specificity in depth.Utilize A431 and A549 cell strain result to learn that oleEGFR fluorescence oil body and oil body memebrane protein fluorescence oil body act on EGFR overexpression cell strain A549 and A431; Not observed fluorescence oil body signal by fluorescence microscope discovery oil body memebrane protein fluorescence oil body function cells produces; And can observe a lot of green fluorescence oil body signals at A431 and A549 born of the same parents' strain effect oleEGFR fluorescence oil body, by shown in Figure 11.
Learn by The above results; The oleEGFR oil body can be discerned the specificity of overexpression cell, and the oleEGFR fluorescence oil body that carries out different MOI acts on EGFR overexpression A549 and A431 cell strain, finds that by fluorescence microscope fixedly MOI is high more along with time that people's oleoplast adds at A549; It is many more that people's oleoplast has been combined in EGFR overexpression cell; The ability of recognition specificity EGFR overexpressing cell is good more, but oil body has when affacting MOI 200 and presents saturated situation, and is shown in figure 12; Different concentration is high more, and the ability of oleEGFR oil body recognition specificity EGFR overexpressing cell is many more.
Learn by The above results; The specificity of oleEGFR fluoroleum physical ability identification overexpression cell, and the oleEGFR fluorescence oil body that carries out different time acts on EGFR overexpression A549 cell strain, finds that by fluorescence microscope SKOV3 is along with the concentration fixed time that people's oleoplast adds is long more; It is many more that people's oleoplast has been combined in EGFR overexpression cell; The ability of recognition specificity EGFR overexpressing cell is good more, but oil body has when affacting two hours and presents saturated situation, and is shown in figure 13.It is long more in the time to add oleEGFR fluorescence oil body fixed amount, and the ability of oleEGFR recognition specificity EGFR overexpressing cell is many more.
Utilize flow cytometer to detect the oleEGFR oil body at the number of different time with different MOI entering; It is many more that the result obtains getting into cell concentration at the longer oleEGFR of causing oil body of concentration fixed time; The time fixedly MOI to cause the oleEGFR oil body to get into cell concentration many more more, being that longer or concentration of time is high more all can present the trend that quantity that the oleEGFR oil body gets into cell has increase, to MOI 200 with act on 2 hours and have and present saturated situation; The oleEGFR variable concentrations that do not coexist; Shown in figure 14, oleEGFR oil body concentration is high more, and the ability of oleEGFR recognition specificity EGFR overexpressing cell is many more.OleEGFR oil body different time, shown in figure 15, oleEGFR oil body fixed amount is long more in the time, and the ability of oleEGFR oil body recognition specificity EGFR overexpressing cell is many more.
Observe the oleEGFR oil body and whether can get into cell.Utilize the conjugation fluorescence microscope that single the cell that the oleEGFR oil body acts on recognition specificity overexpression cell carried out parallel cutting; Shown in figure 16, be to enter into the cell the inside really at the gather into folds oleEGFR oil body that can find out green-emitting fluorescence of different aspects.
The oleEGFR oil body can be discerned the best specificity of overexpression cell, utilizes oleEGFR albumen to coat the medicine (camptothecine, lycopene and curcumin) of variable concentrations.(the maximum molten oily dissolubility of camptothecine is 500mg/ml; The maximum molten oily dissolubility of lycopene is 9mg/ml; The maximum molten oily dissolubility of curcumin is 20mg/ml) learn that by particle diameter oleEGFR medicine oil body is can coat oil-soluble medicine can not make oleEGFR medicine oil body size bigger, as shown in table 3 because of coating medicine.But oleEGFR medicine oil body coats that camptothecine increases with covering amount that particle diameter increases and elecrtonegativity then is to reduce, and oleEGFR medicine oil body coats lycopene and curcumin can not increased and make negative charge increase or minimizing because of medicated bag covers medication amount, as shown in table 3.Through the degree of stability test, the nanometer oil body coats camptothecine because size is less than normal, so be very stable, shown in figure 17.
The oleEGFR oil body can be discerned the best specificity of overexpression cell; And the fat-soluble medicine (curcumin, lycopene and camptothecine) that utilizes the oleEGFR oil body to coat variable concentrations acts on EGFR overexpression cell (A431 and A549) and does not have EGFR overexpression cell strain NCI-H520; Add oleEGFR oil body coating medicine function cells; Shown in figure 18, and observe different time apoptosis degree, the result obtains coating the high more oil medicine (curcumin, lycopene and camptothecine) of concentration at the set time oil body; The apoptosis amount is high more, and the apoptosis amount is high more.
The oleEGFR oil body can be discerned the best specificity of overexpression cell; And utilize the oleEGFR oil body to coat known fluorescence signal molecular action at EGFR overexpression A549 cell strain; At the 0th day, the 1st day, the 2nd day, the 3rd day and the 4th day; Observe the distribution of oleEGFR medicine oil body in cell with flow cytometer, shown in figure 19, Zhao's elaioplast impels fluorescence signal to disappear at cell rupture to oleEGFR fluoroleum cognition along with the time increase.
Whether the oleEGFR oil body can haemolysis: add the oleEGFR oil body in the whole blood the inside and use spectrophotometer measurement, result to show that oil body being not have to injure to blood cell, can not make blood cell haemolysis, like Figure 20 and shown in Figure 21.Whether the oleEGFR oil body is stable in the serum the inside: shown that by the particle size analyzer result oleEGFR oil body 24 hours was very stable and the nano-scale of reaching is all arranged 37 ℃ of measurements at 5% serum, and is shown in figure 22.
The nude mice in about 8-10 age in week utilizes different cell strains that EGFR overexpression A549 cell strain is arranged; Carry out hypodermic injection, make the hypodermic tumor length and width of nude mice about more than 0.5 cubic centimetre, and injection has oleEGFR fluorescence oil body and oil body memebrane protein fluorescence oil body on tumor with high size; Utilize IVIS to observe; The result shows that the tumor brightness that oleEGFR fluorescence oil body and the effect of oil body memebrane protein fluorescence oil body are increased in the EGFR overexpression along with the time can not reduce, and does not have the tumor of EGFR overexpression along with the increase of time, and oleEGFR fluorescence oil body signal luminous intensity can slowly disappear; The result is presented at oleEGFR fluorescence oil body has specificity in the cancerous cell tumor; Observed 24 hours with IVIS, the result learns that EGFR overexpression A549 tumor combines the oleEGFR fluoroleum to know from experience residual signal always; Then overexpression effect A549 tumor can't combine oil body memebrane protein fluoroleum to know from experience because the time is long slowly disappearance, and is shown in figure 23.
The tumor, heart, liver, spleen, lungs and the kidney that take out mice again carry out each internal organs IVIS to be observed; Can find out with time observation and fluorescence oil body signal arranged at the tumor cell that has EGFR to cross expression; Shown in figure 24, carry out frozen tissue section again, utilize the distribution of fluorescence microscope oleEGFR fluorescence oil body; Shown in figure 25, can find out that the tumor cell that has EGFR to cross expression has oleEGFR fluorescence oil body residual.
Take out the A549 tumor of mice again, carry out frozen tissue section, act on oleEGFR fluorescence oil body and oil body memebrane protein fluorescence oil body, shown in figure 26, learn that by fluorescence microscope the tumor cell that has EGFR to cross expression has oleEGFR fluorescence oil body residual.
Learn by experiment in zoopery, can be observed nanometer oil body targeting property, whether the oleEGFR medicine oil body of further observing coating medicine can reach the treatment of cancer effect; Show by the result; Increase in time, the oleEGFR medicine oil body that coats camptothecine can suppress tumor growth, and the nude mice body weight is not reduced on the contrary to be increased; The effect that more can reach treatment is dwindled tumor, and is shown in figure 27 and shown in Figure 28.OleEGFR fluoroleum physical ability is transported via blood; And can detect lung carcinoma cell in the nude mouse in specificity ground; Utilizing IVIS to observe nude mice has oleEGFR fluorescence oil body signal to produce in pulmonary; Cut nude mice and take out internal organs oil IVIS and observe and to find tangible signal is arranged, and illustrative oleEGFR fluorescence oil body has targeting property, like Figure 29, Figure 30 and shown in Figure 31 in pulmonary.
Conclusion
Utilize the proteic production of pJo1-oleEGFR; Through obtaining the oleEGFR oil body after the step that makes up oil body; Utilize the oleEGFR oil body to be optimized test again; Carry out at different proportion, different pH and different types of oil, by fluorescence microscope, particle size analyzer and degree of stability can obtain people's oleoplast optimum condition has following points:
One, assembles the most stable mean size of oil body about 400 nanometers with Oleum sesami.
Two, oleEGFR oil body size is more unstable more greatly, and is more little stable more.
Three, the lipid of the known fluorescence signal molecule of oleEGFR oil body adding coating also can make oil body send the fluorescence of different wave length, observes more easily.
Four, utilized the film experiment can obtain less oleEGFR oil body.
Five, different condition is made people's oleoplast negative charge greatly between-45～-56, to reach the condition of optimizing people's oleoplast.
Condition that utilize to optimize the oleEGFR oil body produces oil body and carries out cell experiment, but can obtain tumor cell line A431 and A549 that oleEGFR oil body specificity ground combines overexpression EGFR, and not can with do not express EGFR tumor cell combination NCI-H520.The time that the oleEGFR oil body adds is longer or MOI is high more; It is many more that the oleEGFR oil body has been combined in EGFR overexpression cell; Recognition specificity EGFR overexpression cell ability is good more, but the oleEGFR oil body has when affacting MOI 200 and presents saturated situation.Utilize conjugation fluorescence microscope X, Y and Z axle can learn that the oleEGFR oil body of known fluorescence signal molecule is to be attached to the cell the inside really.Longer to cause people's oleoplast to get into cell concentration many more with flow cytometer also illustrative concentration fixed time, but the oleEGFR oil body has when affacting MOI 200 and presents saturated situation, consistent with microscopic examination.To cause people's oleoplast to get into cell concentration at the time fixed concentration many more more, are the trend that the longer or high more quantity that all can present oil body entering cell of concentration of time has increase, and hence one can see that, and people's oleoplast can reach nanometer property and specificity ground targeting.
Utilize assembling oleEGFR medicine oil body, the cell strain that this oil body acts on the EGFR overexpression can make the cell strain of EGFR overexpression can produce toxic effect to make cell have phenomenon, the oleEGFR medicine oil body of apoptosis to act on not have EGFR to cross that the cell strain of overexpression is then less sees having toxic effect to make apoptotic phenomenon.Utilize hemolytic experiment can make people's oleoplast coat camptothecine erythrocyte is broken, shown in the proof oil body coat camptothecine, lycopene and curcumin and do not have toxicity.
The oleEGFR oil body is expelled to mouse tumor cell and utilizes IVIS to learn, the oleEGFR oil body has signal to produce on the tumor tissues that the EGFR overexpression is arranged, in the tumor tissues that does not have EGFR overexpression no signal then.Learn via tissue slice that again the tumor that oil body meeting that the EGFR peptide merges and specificity ground combine overexpression EGFR combines, and can not combine with the tumor of normal EGFR.In zoopery, can be observed nanometer oil body targeting property; Coating medicine, can effectively dwindle tumor, and can make the nude mice weight increase after 40 days oncotherapy.The oleEGFR oil body also can be in vivo via the blood transportation specificity ground detection lung adenocarcinoma cell and reach targeting and therapeutic effect.
Comprehensive above result, the present invention has realized making up the nanometer oleEGFR oil body system that a target medicine is sent and detected.
Application case of the present invention such as above is too numerous to enumerate; The above only is to specify the present invention through preferred embodiment; Yet for any modification and the variation that this embodiment did, for example the variation of the material of the length of the kind of cultivating container, material, shape or water pipe, short tube, bore or filter screen, kind or the like does not all break away from spirit of the present invention and scope.
Can make the clear the present invention of those skilled in the art can reach aforesaid purpose really by above detailed description.
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Figure ISA00000598149700011

Claims (8)

1. one kind is detected as the targeted delivery of drugs system and the nanoscale people oleoplast of treatment, and its composition comprises:
OleEGFR protein; It is to utilize gene recombination technology that EGFR peptide and oil body memebrane protein insertion host cell are induced; This oil body memebrane protein can be selected from the seed oil body protein of the plant of following group: a part or its combination of Semen Sesami, Fructus Canarii albi, Semen sojae atricolor, Semen arachidis hypogaeae, numb seed, Brassica campestris L, sunflower, mustard, Flos Carthami; Said host cell can be eukaryotic host cell and prokaryotic host cell; Eukaryotic host cell is selected from following group: unicellular organism body such as yeast cells, be derived from higher organism body such as plant; Insecticide or mammiferous living cells or its combination; Prokaryotic host cell is selected from following group: bacillus, coccus, spirillum, vibrio or its combination, and the best is escherichia coli, escherichia coli are worked as host cell and can be: E.coli.DH5 α, E.coli.BL21 (DE3), E.coli nissle (λ G2) or its combination;
Lipid, it processes the oleEGFR oil body after adding said oleEGFR protein; This lipid can be selected from following group: triglyceride, olive oil, Oleum sesami, soybean oil, Oleum Arachidis hypogaeae semen, mineral oil, oleum lini, safflower oil or its combination; Wherein, the weight/volume ratio (microgram/microlitre) of oleEGFR protein and this lipid is at least about 1/25, and the mean diameter of this oleEGFR oil body is a nano-scale;
Through above-mentioned composition, become and have narrow spectrum delivery vector, so that a targeted delivery of drugs system initiatively to be provided, because the EGFR peptide in the oleEGFR protein can be discerned the receptor on cancerous cell surface accurately.
2. the nanoscale people oleoplast that detects and treat as the targeted delivery of drugs system as claimed in claim 1; Wherein, Can comprise further that lipid coats known fluorescence signal molecule; Be assembled into oleEGFR fluorescence oil body with Protocols in Molecular Biology, this known fluorescence signal molecule can be selected from following group: caesium cadmium quantum dot, Fluorescein isothiocyanate, alizarin yellow, Nile red or its combination;
Through above-mentioned composition; Become and have narrow spectrum delivery vector, so that a targeted delivery of drugs system initiatively to be provided, because the EGFR peptide in the oleEGFR protein can be discerned the receptor on cancerous cell surface accurately; Therefore see through oleEGFR and detect oil body; The oleEGFR that is coated with known fluorescence signal molecule is detected oil body directly be delivered to the canceration position or act on cancerous cell, and can detect and the identification diseased region on specificity ground, and the unlikely normal cell that influences; In addition, but also the irritation cancer cell makes fluorescence molecule get into cancerous cell to the phagocytosis and the fusion of oleEGFR fluorescence oil body, to reach the purpose that targeting specificity ground detects cancerous cell.
3. the nanoscale people oleoplast that detects and treat as the targeted delivery of drugs system as claimed in claim 1; Wherein, OleEGFR protein can further comprise the lipid coating medicine; Be assembled into oleEGFR medicine oil body with Protocols in Molecular Biology, this medicine can be selected from the medicine of following group: lycopene, curcumin, camptothecine, antibiotic, cucurbitacin, Wen Nuoping, paclitaxel, Western medicine, Chinese herbal medicine or its combination;
Through above-mentioned composition; Become and have narrow spectrum delivery vector, so that a targeted delivery of drugs system initiatively to be provided, because the EGFR peptide in the oleEGFR protein can be discerned the receptor on cancerous cell surface accurately; Therefore see through oleEGFR medicine oil body; The oleEGFR medicine oil body that is coated with disease-resistant disease drug directly is delivered to the canceration position or acts on cancerous cell, improve zonal drug level, and the unlikely normal cell that influences; In addition, also can stimulate phagocytosis and the fusion of disease cell, make cancer therapy drug get into cancerous cell, to reach the purpose that detects treatment and avoid developing immunity to drugs oleEGFR medicine oil body.
4. the nanoscale people oleoplast that detects and treat as the targeted delivery of drugs system as claimed in claim 1; Wherein, OleEGFR protein can further comprise the known fluorescence signal molecule that lipid coating medicine and lipid coat; Be assembled into oleEGFR fluorescence medicine oil body with Protocols in Molecular Biology, this medicine can be selected from the medicine of following group: lycopene, curcumin, camptothecine, antibiotic, cucurbitacin, Wen Nuoping, paclitaxel, Western medicine, Chinese herbal medicine or its combination; And this known fluorescence signal molecule can be selected from following group: caesium cadmium quantum dot, Fluorescein isothiocyanate, alizarin yellow, Nile red or its combination;
Through above-mentioned composition; Become and have narrow spectrum delivery vector, so that a targeted delivery of drugs system initiatively to be provided, because the EGFR peptide in the oleEGFR protein can be discerned the receptor on cancerous cell surface accurately; Therefore see through oleEGFR fluorescence medicine oil body; The oleEGFR fluorescence medicine oil body that is coated with disease-resistant disease drug directly is delivered to the canceration position or acts on cancerous cell, improve zonal drug level, and the unlikely normal cell that influences; In addition, also can stimulate phagocytosis and the fusion of disease cell, make cancer therapy drug get into cancerous cell, to reach the purpose that detects treatment and avoid developing immunity to drugs oleEGFR fluorescence medicine oil body.
5. the nanoscale people oleoplast that detects and treat as the targeted delivery of drugs system as claimed in claim 1; Wherein, OleEGFR protein utilization technique for gene engineering connects the medicine peptide; And be assembled into the oleEGFR pharmaceutical protein; Be assembled into oleEGFR medicine oil body with Protocols in Molecular Biology, this medicine peptide is not limited to them and can be selected from following group: the protein of curcumin, Ai Linuodegan, lycopene, bata-carotene, foot flavin, 3,4,3',4'-tetraketo-.beta.-carotene, maize quality, hirudin, insulin, inhibition disease, make disease cell oneself apoptosis peptide or/and the disease-resistant disease drug peptide of forming with molecular biology, peptide or its combination of disease-resistant disease medicine;
Through above-mentioned composition; Become and have narrow spectrum delivery vector, so that a targeted delivery of drugs system initiatively to be provided, because the EGFR peptide in the oleEGFR protein can be discerned the receptor on cancerous cell surface accurately; Therefore see through oleEGFR medicine oil body; The oleEGFR medicine oil body that is coated with disease-resistant disease drug directly is delivered to the canceration position or acts on cancerous cell, improve zonal drug level, and the unlikely normal cell that influences; In addition, also can stimulate phagocytosis and the fusion of disease cell, make cancer therapy drug get into cancerous cell, to reach the purpose that detects treatment and avoid developing immunity to drugs oleEGFR medicine oil body.
6. the nanoscale people oleoplast that detects and treat as the targeted delivery of drugs system as claimed in claim 5; Wherein, The oleEGFR pharmaceutical protein can comprise further that lipid coats known fluorescence signal molecule; Be assembled into oleEGFR fluorescence medicine oil body with Protocols in Molecular Biology, this known fluorescence signal molecule can be selected from following group: caesium cadmium quantum dot, Fluorescein isothiocyanate, alizarin yellow, Nile red or its combination;
Through above-mentioned composition; Become and have narrow spectrum delivery vector, so that a targeted delivery of drugs system initiatively to be provided, because the EGFR peptide in the oleEGFR protein can be discerned the receptor on cancerous cell surface accurately; Therefore see through oleEGFR fluorescence medicine oil body; The oleEGFR fluorescence medicine oil body that is coated with disease-resistant disease drug directly is delivered to the canceration position or acts on cancerous cell, improve zonal drug level, and the unlikely normal cell that influences; In addition, also can stimulate phagocytosis and the fusion of disease cell, make cancer therapy drug get into cancerous cell, to reach the purpose that detects treatment and avoid developing immunity to drugs oleEGFR fluorescence medicine oil body.
7. the nanoscale people oleoplast that detects and treat as the targeted delivery of drugs system as claimed in claim 1; Wherein, OleEGFR protein utilization technique for gene engineering connects the specificity joint, and is assembled into oleEGFR specificity joint oil body, and oleEGFR specificity joint oil body can utilize the cutting of specificity protease hydrolysis; Can be purified into the proteinic application of the high EGFR of purity, this method is to make things convenient for simply again to influence proteic character.
8. like claim 1,2,3,4,5, the 6 and 7 described nanoscale people oleoplast that detect and treat as the targeted delivery of drugs system; Wherein, this oil body memebrane protein is the oil body memebrane protein that comprises by the listed DNA sequence of the listed aminoacid sequence of SEQ ID NO:1 and SEQ ID NO:3; The EGFR peptide is the oil body memebrane protein that comprises by the listed DNA sequence of the listed aminoacid sequence of SEQ ID NO:2 and SEQ ID NO:4; And assembling oleEGFR medicine oil body and the medicine of oleEGFR fluorescence medicine oil body or the medicine of medicine peptide; Can have suppress that disease cell, poisoning disease cell, the one or more genes of insertion/changes/removals make the sudden change of disease cytogene and can't be normally or fast division cause disease cellular atrophy or self-apoptosis, make the disease cell process step to interrupt and can't carry out the division of cell cycle, in the disease cell, produce and suppress or the material of destruction disease cell growth conditions and can't carry out the division of cell cycle and make the disease cell can't infect Normocellular material and can both assemble with this oil body or combine, have the effect that inhibition of cancerization position and Drug therapy are discerned in specificity ground.
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