CN102743327A - Nano-scale artificial oil body for targeted drug delivery system detection and treatment - Google Patents

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 oleZH2 protein, wherein the oleZH2 protein comprises oleosin and a ZHer2 peptide; a lipid, a lipid-coated liposoluble drug, and a lipid-coated known fluorescence molecule, wherein the materials are prepared into an oleZH2 oil body, an oleZH2 drug oil body and an oleZH2 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 utilizes the oil body memebrane protein to connect Z _Her2Peptide, and be assembled into oleZH ₂Protein, this oleZH ₂Albumen mass-energy coats lipid, fat-soluble medicine or known fluorescence signal molecule becomes oleZH ₂Oil body is again with oleZH ₂Oil body is applied to the application of targeted therapy or detection; The present invention utilizes the oil body memebrane protein to connect a specificity joint (linker) connection Z _Her2Peptide just can purification Z through separating _Her2Albumen, and obtain the high oleZH of purity ₂Oil body; The present invention utilizes excision enzyme peptide or restriction endonuclease peptide to connect oil body memebrane protein connection Z _Her2Peptide connects excision enzyme peptide or restriction endonuclease peptide again, and is assembled into oleZH ₂Oil body, this oleZH ₂Oil body can detect the canceration position, and gets into the cancerous cell of expressing and make cell division or self-apoptosis.

Background technology

According to statistics, cancer is one of the highest disease of whole world mortality rate, and cancer is also referred to as malignant tumor, is defined as: unknown cause is arranged or be to be caused by the multiple cause of disease, and malignant tumor on mankind takes place.Cancer can be grown in any tissue of health and the organ, and the ability of the cell of differentiation potential, uncontrollable growth and transfer is arranged, and makes that cell can't the normal regulating control growing, causes physiological function unusual, and can't normally cure.As far as the women, the coming out at the top of the fatality rate of breast carcinoma and cervical cancer.

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 subsequent use material of the different immunologic mechanism 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, poor appetite, hair loss, oral cavity or exanthemv, 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 lifting 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.

The present invention utilizes oil body to assemble as carrier and linking objective albumen to form oleZH ₂Oil body, the present invention attempts to utilize oleZH ₂Oil body coats 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 be used in effectively on targeted therapy or the cancer detection, and have the specificity of height.

The present invention be by difference can synthesize medicine analog peptide or disease had the peptide (above-mentioned peptide be called for short medicine peptide) of therapeutic effect; Form medicine or the synthetic or the synthetic polyprotein that have treatment to imitate to disease through genetic engineering, and connect and go up the oil body egg and connecting Z _Her2Peptide, this oil body can coat known fluorescence signal molecule and be assembled into self assembly targeted drug albumen, this albumen can specificity ground identification cancerization position and can assemble pharmaceutical protein voluntarily and carry out cancerization location detection, inhibition and Drug therapy.

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 is covalently bonded in oleZH ₂Proteinic N-end 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, and needs add excessive adjustment buffer (pH value) and dash the highly purified fusion rotein of proposition.OleZH ₂The separation of 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 oleZH ₂The oil body that the specificity joint is arranged utilizes specificity protease hydrolysis cutting specificity joint again, can be purified into the high Z of purity _Her2Protein.

Summary of the invention

The present invention provides a kind of fused protein, and (hereinafter referred is oleZH ₂Protein), this albumen comprises Z _Her2The combination of peptide and oil body memebrane protein.

The present invention also provides a kind of oil body memebrane protein carrier, and it comprises the oil body memebrane protein and combines Z _Her2Peptide and any oils and fats, and form narrow spectrum nanometer oil body, wherein this oleZH ₂The weight/volume ratio (microgram/microlitre) of protein and this lipid is at least about 1/25, and this oleZH ₂The mean diameter of oil body is a nano-scale.

The present invention utilizes oleZH again ₂Protein coats known fluorescence molecule, and the detection that can be used for the recognition specificity cancerous cell is used.

The present invention utilizes oleZH again ₂The protein coating medicine, the targeted therapy that can be used for the recognition specificity cancerous cell uses.

The present invention utilizes oleZH again ₂Protein coats known fluorescence molecule and coating medicine, and the detection and the targeted therapy that can be used for the recognition specificity cancerous cell use.

The present invention utilizes similar oleZH again ₂The medicine oil body, the targeted therapy that can be used for the recognition specificity cancerous cell uses.

The present invention utilizes oleZH again ₂The oil body that the specificity joint is arranged utilizes specificity protease hydrolysis cutting specificity joint again, can be purified into the high Z of purity _Her2Proteinic application.

Description of drawings

The structure of Fig. 1, oil body.

Fig. 2, oleZH ₂The preparation process of oil body.

Fig. 3, Z _Her2Protein purification prepares process.

Fig. 4, oleZH ₂The preparation process of medicine oil body.

Fig. 5, oleZH ₂Protein is formed and autonomous dress oleZH ₂The oil body laboratory observation.

Fig. 6, oleZH ₂Oil body coats GGK oil body yellow and Nile red and shows the observation of fluorescence micro mirror.

Fig. 7, oleZH ₂Oil body carries out the AFM test.

Fig. 8, oleZH ₂The different condition composition of oil body carries out fluorescence microscope (the diagram scale is 2 microns).

Fig. 9, oleZH ₂The different condition composition of oil body carries out the degree of stability test.

Figure 10, oleZH ₂The different condition composition of oil body carries out the particle diameter test.

Figure 11, oleZH ₂The testing in vitro fluorescence microscope of oil body pair cell-(fixing) cell.

Figure 12, oleZH ₂The testing in vitro fluorescence microscope of oil body pair cell-(work).

Figure 13, oleZH ₂Oil body is to SKOV3 cell tests fluorescence microscope-difference (concentration).

Figure 14, oleZH ₂Oil body is to SKOV3 cell tests fluorescence microscope-difference (time).

Figure 15, oleZH ₂The oil body function cells is carried out flow cytometer test-difference (concentration).

Figure 16, oleZH ₂The oil body function cells is carried out flow cytometer test-difference (time).

Figure 17, oleZH ₂Oil body effect SKOV3 cell utilizes conjugation fluorescence microscope XY axial plane to observe and the Z axial section is observed.

Figure 18, oleZH ₂The degree of stability experiment of oil body coating medicine.

Figure 19, oleZH ₂Oil body coats toxicity test cell counting (the 72nd hour)-lycopene, curcumin and the camptothecine of variable concentrations camptothecine.

Figure 20, oleZH ₂Oil body coats the camptothecine medicine in cell poisoning experiment (4 days).

Figure 21, oleZH ₂The oil body coating medicine is to blood cell blood dissolubility experiment-lycopene, curcumin and camptothecine.

Figure 22, oleZH ₂Oil body carries out oil body and melts blood experiment (blood cell).

Figure 23, oleZH ₂Oil body is in serum change of size test in time.

Figure 24, oleZH ₂Oil body is injected the back with different time IVSI observation test at mouse interior tumor.

Figure 25, oleZH ₂Oil body injected in mice after 24 hours internal organs with different time IVSI observation test.

Figure 26, Mus are got the fluorescence microscope of tumor tissues frozen section.

Figure 27, mouse tumor detect tissue slice and utilize oleZH ₂Oil body detects to be observed.

Figure 28, oleZH ₂Tumor load Mus-the tumor of oil body targeting type oil body treatment changes (size: cm ³).

Figure 29, oleZH ₂Tumor load Mus-the weight change of oil body targeting type oil body treatment (weight: kilogram).

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 present invention provides the nanoscale people oleoplast of the nanoscale people oleoplast of detection of targeted delivery of drugs system and treatment, and its composition comprises: an oleZH ₂Protein, this protein comprise an oil body memebrane protein and a Z _Her2Peptide; One lipid, this lipid adds said oleZH ₂Process oleZH behind the protein ₂Oil body; But and a lipid coating medicine, be called oleZH ₂The medicine oil body.

The present invention utilizes Z _Her2The preparation process easily of carrying out provides a kind of carrier of people's oleoplast (hereinafter referred is oleZH after peptide and oil body memebrane protein (oleosin) the binding molecule biotechnology ₂Oil body).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 with Z _Her2Peptide and oil body memebrane protein insert host cell, induce oleZH ₂Protein, 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 of Semen Sesami, Fructus Canarii albi, Semen sojae atricolor, Semen arachidis hypogaeae, numb seed, Brassica campestris L, sunflower, mustard, 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, oleZH ₂The weight/volume ratio (microgram/microlitre) of protein and this lipid is at least about 1/25, and this oleZH ₂The mean diameter of 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 oleZH ₂Z in the protein _Her2Peptide can be discerned the receptor on cancerous cell surface accurately.

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 utilizes oleZH ₂OleZH in the oil body ₂Protein; 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 oleZH ₂Protein, and oil body memebrane protein aminoacid sequence is shown in SEQ ID NO:1.For oil body memebrane protein mean size is 100 to 1,500 nanometers.

The nanoscale people oleoplast that the present invention detects and treats as the targeted delivery of drugs system, wherein, oleZH ₂Protein can comprise the lipid coating medicine again, is assembled into oleZH with Protocols in Molecular Biology ₂The medicine oil body, this medicine can be selected from the medicine of following group: lycopene, curcumin, camptothecine, antibiotic, cucurbitacin, Wen Nuoping (commodity are called Navelbin), paclitaxel (Paclitaxel), Western medicine, Chinese herbal medicine or its combination; Or oleZH ₂Protein can comprise that lipid coating medicine and lipid coat fluorescence signal molecule again, with Protocols in Molecular Biology and be assembled into oleZH ₂Fluorescence medicine oil body, 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), Nile red (Nile Re) or its combination; Or oleZH ₂Pharmaceutical protein can comprise that lipid coats known fluorescence signal molecule, is assembled into oleZH with Protocols in Molecular Biology again ₂Fluorescence medicine oil body, this known fluorescence signal molecule can be selected from following group: caesium cadmium quantum dot, Fluorescein isothiocyanate, alizarin yellow, Nile red or its combination; Above-mentioned one of which, as shown in Figure 2.

OleZH of the present invention ₂The oleZH of oil body ₂Protein comprises Z _Her2Peptide makes oleZH ₂Oil body becomes and has narrow spectrum delivery vector, claims targeting vector again, and oleZH ₂Oil body also can coating medicine be formed initiatively the targeted delivery of drugs system, and (hereinafter referred is oleZH ₂The medicine oil body).And Z _Her2Toplink is discerned the receptor on cancerous cell surface accurately, and hence one can see that, and the oil body memebrane protein combines Z _Her2People's oleoplast of peptide and coating medicine directly is delivered to the canceration position or acts on cancerous cell, can promote the concentration of canceration position or cancerous cell zone administration greatly, and can not have influence on normal cell.Also can the irritation cancer cell to oleZH ₂Oil body carries out phagocytosis and fusion, makes cancer therapy drug get into cancerous cell fast, can reach the Drug resistance of treating and avoiding producing medicine.

When treatment breast carcinoma or ovarian cancer, can use the ligand peptide of HER2/neu protein acceptor.And the HER2/neu protein acceptor is epithelical cell growth factor receptor (EGFR), is present in many cancerous cell surface, as the biomarker on cancerous cell surface.But people such as Nord have developed the peptide of a kind of specificity ground and HER2/neu protein acceptor key knot, are called Z _Her2Z _Her2Peptide comprises 58 aminoacid, and aminoacid sequence is shown in SEQ ID NO:2, and molecular weight is about 7 to 15 kilodaltons, and is also littler than the molecular weight (150 kilodalton) of monoclonal antibody, hence one can see that Z _Her2The easy permeates cell membranes of peptide.

OleZH of the present invention ₂Oil body 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 oleZH is provided ₂Protein, and utilize the multiple material of cell ligand peptide portability, and can directly pass cell membrane and get into cell, make oleZH ₂Oil body does not need to get into cell via receptor, reaches the passive target administration, and is as shown in Figure 2.

OleZH of the present invention ₂Oil body can prepare (but not as limit) by following preparation method: utilize gene recombination technology on expression vector, to combine the Z of oil body protein _Her2Peptide is again with oleZH ₂Oil body is sent in the host cell and is induced, and preparation contains oil body memebrane protein and Z _Her2The oleZH of peptide ₂Protein.Add buffer then and mix this oleZH ₂Protein and lipid utilize the ultrasonic processor oscillation mixture again, just can produce artificial oleZH ₂Oil body, as shown in Figure 2.

OleZH of the present invention ₂The lipid that oil body 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 adjustment oleZH ₂The ratio of protein and lipid, and can prepare the oleZH of the mean diameter that varies in size ₂Oil body.OleZH ₂The mean diameter of oil body and oleZH ₂The ratio of protein and lipid is inversely proportional to, oleZH ₂The oil body amount is fixed, and lipid components is fewer, oleZH ₂The mean diameter of oil body is littler.In oleZH of the present invention ₂In the oil body, oleZH ₂The weight/volume ratio (microgram/microlitre) of 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, oleZH ₂Protein can comprise the lipid coating medicine again, is assembled into oleZH with Protocols in Molecular Biology ₂The medicine oil body; Or oleZH ₂Protein can comprise that medicine and lipid that lipid coats coat known fluorescence signal molecule again, with Protocols in Molecular Biology and be assembled into oleZH ₂Fluorescence medicine oil body; Above-mentioned one of which, as shown in Figure 2.

Utilization of the present invention contains oil body memebrane protein and Z _Her2The oleZH of peptide ₂Protein adds different pH buffer then and mixes this oleZH ₂Protein and lipid utilize the ultrasonic processor oscillation mixture again, and the pH value of buffer can influence the oleZH of manufacturing ₂The mean diameter of oil body and stability.Preferable through the pH value that experiment showed, buffer more than 7.5, be more preferred from about 7.5 to about 9.0.

In preferable result of the present invention, adopt following condition combination with preparation oleZH ₂Oil body: the oil body memebrane protein and the Z of Semen Sesami seed used in (1) _Her2Peptide makes up oleZH ₂Protein; (2) with olive oil as lipid; (3) adopt oleZH ₂The weight/volume ratio (microgram/microlitre) of protein and lipid is about 20/1; And the pH value of (4) buffer is about 7.5.

The present invention is stable more easily and adjustment oleZH than prior art ₂The size of oil body, and the oleZH that is applicable to various fat-soluble medicines and different lipids can be provided ₂Oil body.OleZH of the present invention ₂The oil body mean diameter is highly stable, and big I reaches tens nanometer to time size of micron, is easy to be absorption of human body.The oleZH of preparation tool injection type ₂During oil body, and can control the particle diameter 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, use oleZH of the present invention ₂Oil body detects the HER2/neu signaling molecule, in conjunction with the combination of different pharmaceutical and different lipids.And formation oleZH ₂The 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 oleZH ₂Oil body, as shown in Figure 2.

Assembling oleZH of the present invention ₂Oil body also can combine any known signaling molecule, and (hereinafter referred is oleZH ₂And can reach testing goal the signal oil body).Signaling molecule can be selected from following group: caesium cadmium quantum dot, Fluorescein isothiocyanate, alizarin yellow, Nile red and other known different wave length fluorescence signal that can send are known signaling molecule.For example: caesium cadmium quantum dot 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 oleZH that launches different fluorescence colors on using ₂Oil body.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 Z again _Her2Peptide is to host cell, and assembling is formed with the oleZH of specificity joint ₂Albumen adds lipid and is assembled into the oleZH of specificity joint ₂Oil body is through the centrifugal oleZH that the specificity joint is arranged ₂The oil body solution surface again that can suspend can reclaim very easily, utilizes the cutting of specificity protease hydrolysis to make the specificity joint breaking, makes Z _Her2Albumen separates from the oil body memebrane protein, through centrifugal and recovery, just can obtain Z _Her2Albumen (can discern HER2/neu and cross 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 Z _Her2Peptide is to host cell, and assembling forms oleZH ₂Albumen adds the lipid and the lipid that coats known fluorescence signal molecule of coating medicine, and is assembled into oleZH ₂Detect and the medicine oil body, with the oleZH that is coated with cancer therapy drug ₂Detection and medicine oil body directly are delivered to the canceration position or act on cancerous cell, can utilize different excitation wavelengths to detect oleZH ₂Detect and medicine oil body combination cancerization position, can know identification, can also improve zonal drug level the cancerization position, and the unlikely normal cell that influences.In addition, through above-mentioned mechanism, but also the irritation cancer cell to oleZH ₂The phagocytosis of medicine oil body and fusion make cancer therapy drug get into cancerous cell, 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; Through genetic engineering, this synthetic proteins is for for example by the medicine peptide: 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 peptide or its combination of the disease-resistant disease drug peptide of forming with molecular biology, disease-resistant disease medicine and connect and go up the oil body egg and connecting Z _Her2Peptide to host cell and assembling form similar oleZH ₂Pharmaceutical protein adds lipid and is assembled into and is self-assembled into similar oleZH ₂The 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 manufacture process is as shown in Figure 4.

The present invention assembles oleZH ₂Oil body can be demarcated cancerous cell accurately, can use the detection at cancerization position in vivo in real-time ground, also can be assembled into oleZH by coating medicine ₂Medicine oil body, 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.

The present invention assembles oleZH ₂Medicine oil body and oleZH ₂The medicine or the medicine peptide of fluorescence medicine oil body are not limited to above-mentioned 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, oleZH ₂The protein utilization technique for gene engineering connects the specificity joint, and is assembled into oleZH ₂Specificity joint oil body, oleZH ₂Specificity joint oil body can utilize the cutting of specificity protease hydrolysis, can be purified into the high Z of purity _HerProteinic application, this method are to make things convenient for simply again to influence proteic character, and be as shown in Figure 3.

The present invention detects as the targeted delivery of drugs system and the nanoscale people oleoplast of treatment, and 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:5; Z _HerPeptide is to comprise listed aminoacid sequence and Z by SEQ ID NO:2 _Her2The oil body memebrane protein of the DNA sequence that sequence SEQ ID NO:6 is listed.And assembling oleZH ₂Medicine oil body and oleZH ₂The medicine of 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 be worth explanation 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 oleZH ₂Oil 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, biological medicine material, animal vaccine, biotechnology.With following concrete description of test, narrate the present invention further.

[embodiment 1] preparation oleZH ₂Oil body

Make up oleZH of the present invention according to making preparation flow shown in Figure 2 ₂Oil body.

< step 1, construction of expression vector >

Utilize gene recombination technology with following three kinds gene constructed on expression vector:

Oil body memebrane protein (N end)-Z _Her2Peptide (C end) oleZH ₂Proteinic gene: with one contain the listed nucleotide sequence of SEQ IDNO:3 joint (comprising the listed aminoacid sequence of SEQ ID NO:4) gene to combine the oil body membrane protein gene (comprising the listed nucleotide sequence of SEQ ID NO:5) and the proteinic ligand peptide of HER2/neu of Semen Sesami seed (be Z _Her2Peptide) gene (comprising the listed nucleotide sequence of SEQ ID NO:6).Detailed operating procedure is following.At first; But people such as Nord develop the ZHer2 peptide of a kind of specificity ground and HER2/neu protein acceptor key knot, with the part as this receptor, referring to Nord et al.; Binding proteins selected from combinatorial libraries of an α-helical bacterial receptor domain.Nat Biotechnol.1997; 15:772-777, the document quotes in full in this for your guidance, is assembled into pET-Z with Protocols in Molecular Biology _Her2Carrier, purification pET-Z _Her2Carrier utilizes primer to obtain Z via the polymerase chain reaction to be used as template DNA again _Her2Genetic fragment, its size is 507bp.Then, with Nco I and Hind III restriction endonuclease enzyme action Z _Her2Gene, and be connected on a pBluescriptII (SK+) (available from the Novagen company) carrier, again the recombinant vector that makes is converted in E.coli.DH5 α (available from Foodstuff Industrial Development Inst. of Financial Group Legal Persons (the FIRDI)) host cell.Contain in one and to cultivate this host cell in LB (Luria-Bertani) 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 and is comprised pBluescript II-Z to obtain one _Her2The transformant of recombinant vector.At last, with the Z on Nco I and this recombinant vector of Hind III restriction endonuclease enzyme action _Her2Genetic fragment, and it is connected to one comprises on the recombinant vector of pJO1-oil body membrane protein gene, can make one and comprise pJo1-oil body membrane protein gene-Z _Her2Expression vector, be called pJO1-oleZH ₂(be called for short oleZH ₂Be 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.:4957), classification name ETEC (Escherichia coli)).

After accomplishing the structure of above-mentioned expression vector; 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 quotes in full here for your guidance.

<step 2, expression oleZH ₂Dan Bai>

Induce prepared host cell in the step 1 with isopropyl-(IPTG, 0.05 millimolar concentration (available from USB company)), make its great expression oleZH ₂Albumen, 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 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 >

With prepared oleZH in the step 2 ₂Protein (50 milligrams) 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 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 and to assemble out oleZH ₂The fluorescence oil body makes this oil body pass through 0.22 millimeter filter membrane, utilizes fluorescence microscope (model: Nikon 104) to observe the preparation result, and the result is as shown in Figure 6.

General biochemical investigation analytical method capable of using is measured oleZH ₂Three kinds of bases in the 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.

[embodiment 2] oleZH ₂The AFM test of 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.With prepared oleZH in the step 2 ₂Protein (50 milligrams) is added in the test tube, and adds the phospholipid of 50 milligrams of triglyceride, 150 micrograms and be assembled into oleZH ₂Oil body makes this oil body cross 0.22 millimeter filter membrane, will pass through filtering oil body again and be added to and leave 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.

[embodiment 3] lipid and oleZH ₂The influence of proteinic ratio

At 100 microgram oleZH ₂Protein (pJO1-oleZH ₂) the middle sodium phosphate buffer (0.01 molar concentration, pH 7.5) (available from Sigma company) of 950 microlitres and the olive oil (available from Sigma company) of 50 microlitres of adding, obtain containing oleZH ₂The weight/volume ratio (microgram/microlitre) of 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 oleZH ₂Oil body.Repeat aforementioned operation, but use 400,200,100 and 100 microgram oleZH respectively ₂Protein, and corresponding 20,20, the 100 and 500 microlitre olive oil that use are to obtain oleZH ₂The weight/volume ratio (microgram/microlitre) of protein/lipid (olive oil) is respectively 20/1,10/1,1/1, reaches 1/5 oleZH ₂Oil body.

Utilize Nikon 104 type observation by light microscope different condition oleZH ₂Form of oil body (shown in Figure 8) and turbidity (as shown in Figure 9, computational methods such as following examples 6 are said) as the, and use particle size analyzer (Beckman Coulter, N4 Plus) to analyze oleZH ₂The particle diameter of 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 shown in figure 10.

The influence of [embodiment 4] pH value

With oleZH ₂Protein (100 microgram oleZH ₂) be 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, adding 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 oleZH ₂Oil body.Utilize Nikon 104 type observation by light microscope oleZH ₂Form of oil body (as shown in Figure 8) and turbidity (as shown in Figure 9); Analyze oleZH with particle size analyzer ₂The particle diameter of oil body (shown in figure 10).

The influence of [embodiment 5] lipid species

With oleZH ₂Protein (100 microgram pJO1-oleZH ₂) be added in the sodium phosphate buffer (0.01 molar concentration, pH 7.5) of 950 microlitres, add 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; 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), with preparation oleZH ₂Oil body.Utilize Nikon 104 type observation by light microscope oleZH ₂Form of oil body (as shown in Figure 8) and turbidity (as shown in Figure 9); Analyze oleZH with particle size analyzer (model: Zetasizer Nano, Malvern #ZS90) ₂The particle diameter of oil body (shown in figure 10).

Fig. 8 and shown in Figure 10, obtained oleZH ₂The mean diameter of oil body is between 10 to 2,000 nanometers, and along with lipid and oleZH ₂Proteinic ratio reduces and diminishes.Therefore, can adjust lipid and oleZH ₂Proteinic ratio is to prepare the oleZH of different mean diameters ₂Oil body.In addition, oleZH ₂The mean diameter of oil body is less relatively under alkaline environment.

OleZH is measured in [embodiment 6] ₂The degree of stability of oil body

Measure oleZH with following three kinds of modes ₂The degree of stability of oil body

A, observation negative electricity repulsion

Can be through observing oleZH ₂The three-dimensional barrier effect that the negative electricity repulsion on oil body surface or protein cover and record its degree of stability wherein, can be observed the negative electricity repulsion oil body that is caused that fades away through the pH value that reduces solution and assemble.Therefore, with oleZH ₂Oil 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 oleZH ₂Oil body is complete, and then the surface is a hydrophilic, and can dissolve each other with water effectively and be suspended state.If oleZH ₂Oil body is incomplete, and then its proteins on surfaces can cause oleZH because of can't correctly folding ₂Polymerization each other makes oleZH between oil body ₂Oil body floats over solution surface.Therefore, can understand oleZH indirectly by the turbidity of measuring the solution bottom ₂The integrity of oil body.Therefore, at first with 1 milliliter oleZH ₂Oil body places 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 find out by Fig. 9, after 140 minutes, oleZH ₂Oil body is being kept perfectly property still, and this explains oleZH ₂Oil body has splendid stability.

C, measurement zeta current potential

With oleZH ₂Oil body is scattered in varying environment (different lipids and oleZH ₂Proteinic weight/volume ratio, pH value or lipid species) following, and utilize surface potential analyser (Zetasizer Nano, Malvern #ZS90) to measure artificial oleZH ₂The variation of the surface zeta potential current potential of oil body, measurement result is as shown in table 1, oleZH ₂The degree of stability of oil body is along with lipid and oleZH ₂Proteinic ratio reduces and promotes, and comparatively stable under alkaline environment.

Table 1, oleZH ₂Oil body is tested at different condition surface potential analyser

[embodiment 7] oleZH ₂The target function gonosome of oil body is tested outward-fixedly tumor cell test

With 5 * 10 ⁴Individual cell (MCF7, MCF7/Her18, SKOV3 (reaching the ovarian cancer cell of HER2/neu receptor through scale), SKBR3 (reaching the breast cancer cell of HER2/neu receptor through scale) or MDA-MV-231 (the no mistake in treatment scale reaches the breast cancer cell of HER2/neu receptor) cell) plants to one 24 hole culture plates, and places 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 oleZH ₂Oil body (comprises 2.5 microgram pJO1-oleZH ₂/ every ml phosphate buffer) 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-HER2/neu 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 anti-Mus IgG-TRITC (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 11, oleZH ₂Oil body can specificity ground be demarcated with formaldehyde fixed and is passed through the MCF7/Her18 and the SKOV3 cell of the HER2/neu receptor that scale reaches.

[embodiment 8] oleZH ₂The target function gonosome of oil body is tested outward-the tumor living cell test

With 5 * 10 ⁴Individual cell (MCF7, MCF7/Her18, SKOV3 (reaching the ovarian cancer cell of HER2/neu receptor through scale), SKBR3 (reaching the breast cancer cell of HER2/neu receptor through scale) or MDA-MV-231 (the no mistake in treatment scale reaches the breast cancer cell of HER2/neu receptor) cell) plants to one 24 hole culture plates, and places 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, will coat alizarin yellow oleZH ₂Oil body (comprises 2.5 microgram pJO1-oleZH ₂/ every ml phosphate buffer) 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-HER2/neu one-level antibody (9G6, Santa Cruz Biotechnology company, Santa Cruz, California, the U.S.).Then, clean three times with phosphate buffer after, with anti-Mus IgG-TRITC (with 1: 500 dilution proportion; Jackson ImmunoResearch Laboratories company, West Grove, Pennsylvania; The U.S.) reaction is 1 hour, cleans three times with phosphate buffer.Then use 15,000 times of DAPI to dye with nucleus dyeing at last and clean three times, give mounting after accomplishing dyeing, and (Olympus observes IX71) to utilize fluorescence microscope.Experimental result is shown in figure 12, oleZH ₂Oil body can specificity ground be demarcated with formaldehyde fixed and is passed through the MCF7/Her18 and the SKOV3 cell of the HER2/neu receptor that scale reaches.

[embodiment 9] oleZH ₂The righttest activity of oil body and tumor cell

With 5 * 10 ⁴Individual cell (MCF7, MCF7/Her18, SKOV3 (reaching the ovarian cancer cell of HER2/neu receptor through scale), SKBR3 (reaching the breast cancer cell of HER2/neu receptor through scale) or MDA-MV-231 (the no mistake in treatment scale reaches the breast cancer cell of HER2/neu receptor) cell) plants to one 24 hole culture plates, and places 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, utilize to coat variable concentrations alizarin yellow calZH ₂The infectious agent value of oil body and cell (MOI value, multiplicity of infection) (MOI 100, MOI 200, MOI 400) is measured the righttest activity between the two.Therefore; The cell strain that uses is the cell strain MCF7/Her18 of overexpression HER2/neu receptor, cell strain MCF7 that the no mistake in treatment scale reaches the HER2/neu receptor, and the ovarian cancer cell SKOV3 of overexpression HER2/neu 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: effect oleZH ₂The cell strain of oil body adds lock solution (3% weight/volume fetal bovine serum albumin is dissolved in the phosphate buffer) again, and under room temperature, reacts 1 hour.With after 1: 500 the dilution proportion, reaction is at least 1 hour under room temperature with anti-HER2/neu one-level antibody (9G6, Santa Cruz Biotechnology company, Santa Cruz, California, the U.S.).Then, clean three times with phosphate buffer after, with anti-Mus IgG-TRITC (with 1: 500 dilution proportion; Jackson ImmunoResearch Laboratories company, West Grove, Pennsylvania; The U.S.) reaction is 1 hour, cleans three times with phosphate buffer.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.

Flow cytometer test: effect oleZH ₂The cell strain of oil body adds pH 7.4 phosphate buffers again and utilizes flow cytometer (BD FACSCanto (Argon-Ion Laser 488nm, He-Ne Laser 633nm)) test.

Wherein, the MOI value defined is oleZH ₂The ratio of 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 13, under fluorescence microscope, observes, and finds to comprise Z _Her2The oleZH of peptide ₂Oil body has narrow spectrum identification ability to the cell strain of overexpression HER2/neu receptor, and oleZH ₂The number that oil body gets into cell has the trend of lifting along with the increase of MOI value, be then to present saturated situation at 200 o'clock to the MOI value.

Utilize fluorescence microscope oleZH ₂Whether oil body gets into the influence of cell and MOI value.The result shows, oleZH ₂Oil 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 and oleZH shown in figure 15 ₂The combination percentage rate of oil body is defined as: through combining oleZH ₂The cell number of oil body/10,000 cell * 100), above-mentioned experimental result, fluorescence microscope is consistent with the analysis of flow cytometer.

[embodiment 10] oleZH ₂The righttest action time of oil body and tumor cell

To observe oleZH with embodiment 9 identical methods ₂The righttest action time of oil body and tumor cell, wherein the MOI value is fixed as 200, and the cell strain of use is for reaching the SKOV3 cell of HER2/neu receptor through scale, and acts in different time points (0,15,30,60,120 and 240 minute).The result is shown in figure 14, under fluorescence microscope, all finds to comprise Z _Her2The oleZH of peptide ₂Oil body has narrow spectrum identification ability to the cell strain of overexpression HER2/neu receptor, and oleZH ₂The number that 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.

Utilize fluorescence microscope oleZH ₂The influence whether oil body gets into cell and action time.The result shows, oleZH ₂The quantity that oil body gets into cell promotes with the increase of works with the time, and presents saturated situation in 2 hours.The flow cytometer test result, (wherein, cell and oleZH shown in figure 16 ₂The combination percentage rate of oil body is defined as: through combining oleZH ₂The cell number of oil body/10,000 cell * 100), above-mentioned experimental result, fluorescence microscope is consistent with the analysis of flow cytometer.

[embodiment 11] oleZH ₂Oil body is invaded tumor cell

To observe oleZH with embodiment 8 identical methods ₂Oil body is invaded tumor cell, and 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 HER2/neu receptor through scale.The result is shown in figure 17, and (model: Leica TCS SP2) get X, Y and Z axle all find to comprise Z down to utilize the conjugation fluorescence microscope _Her2The oleZH of peptide ₂The narrow spectrum identification ability of oil body tool and can invade cell the inside.

[embodiment 12] oleZH ₂The composition of medicine oil body, size and zeta current potential

With prepared oleZH in the step 2 ₂Protein (50 milligrams) is added in the test tube; Add the lipid that 50 microlitres coat different pharmaceutical (lycopene, curcumin and camptothecine (CPT)) 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 and can assemble out oleZH ₂The medicine oil body.Utilize particle size analyzer to analyze oleZH ₂The particle diameter of medicine oil body, as shown in table 2.With oleZH ₂The medicine oil body utilizes surface potential analyser (Zeta size rNano) (model: Malvern #ZS90) measure artificial oleZH ₂The variation of the surperficial boundary current potential of medicine oil body, measurement result is as shown in table 2, oleZH ₂The degree of stability of medicine oil body, shown in figure 18.

If oleZH ₂The medicine oil body is complete, and then the surface is a hydrophilic, and can dissolve each other with water effectively and be suspended state.If oleZH ₂The medicine oil body is incomplete, and then its proteins on surfaces can cause oleZH because of can't correctly folding ₂Polymerization each other makes oleZH between the medicine oil body ₂The medicine oil body floats over solution surface.Therefore, can understand oleZH indirectly by the turbidity of measuring the solution bottom ₂The integrity of medicine oil body.Therefore, at first with 1 milliliter oleZH ₂Oil body places 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 find out by Figure 18, after 24 hours, oleZH ₂Oil body is being kept perfectly property still, and this explains oleZH ₂Oil body has splendid stability.

Table 2, oleZH ₂Oil body coating medicine size and surface potential analyser test-lycopene, curcumin and camptothecine

The experiment of [embodiment 13] targeted therapy and detection-cell survival test

At first, coat the oleZH of the antitumor drug (lycopene (available from Sigma company), curcumin (available from Sigma company) or camptothecine (CPT) (available from Sigma company)) of variable concentrations according to the method preparation of embodiment 9 ₂Oil body is with the combination as confession targeted therapy and/or detection.With 1 * 10 ⁴Individual cell (MCF7, MCF7/Her18, SKOV3, SKBR3 and MDA-MB-231 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 oleZH of flush away effect ₂Oil body.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 19; And utilize fluorescence microscope whether to make up the bio-toxicity of cell growth inhibiting and more various combinations, shown in figure 20.Show oleZH of the present invention by experiment ₂Oil body does not have obvious cytostatic situation.

[embodiment 14] oleZH ₂The hemolytic test of oil body

To coat different types of oleZH ₂The medicine oil body (comprises 2.5 microgram pJo1-oleZH ₂/ every ml phosphate buffer) 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 a luminometer measurement, shown in figure 21.With fluorescence microscope erythrocyte situation, shown in figure 22.The result shows that oil body being not have injury to blood cell, can not make blood cell haemolysis.

The experiment of [embodiment 15] serum stability

Utilize and coat different types of oleZH ₂The medicine oil body (comprises 2.5 microgram pJO1-oleZH ₂/ every ml phosphate buffer) as for containing 5% mice (commercially available purchase) serum 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 23.The particle size analyzer result shows, oleZH ₂It 24 hours was unusual stable status that 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 breast 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 MDA-MB-231 or SKOV3 breast cancer cell are injected to the subcutaneous of back, mice left side again, and carry out the breast carcinoma induction culturing phase in about 2 weeks.After inducing the tumor of breast carcinoma pattern to generate to reach for 4 weeks, tumor size is about 1,000 cubic millimeter, starts from mouse subcutaneous injection oleZH of the present invention again ₂Oil body (comprises 1.0 microgram pJO1-oleZH ₂/ every ml phosphate buffer).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 oleZH ₂The situation that oil body is followed the trail of and distributed in each organ in the intravital blood circulation of mice, breast cancer cell.

The result is shown in figure 24, and detecting in the body of control group mice (body contains the MDA-MB-231 cell) fluorescence intensity increases along with the time and fade away, and experimental mice (body contains the SKOV3 cell) 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 oleZH ₂The distribution scenario of oil body.Experimental result is shown in figure 25, in the body of control group mice, and oleZH ₂Oil body mainly is accumulated in liver (carrying out the organ of drug metabolism) position, explains that it does not get in the MAD-MB-231 tumor tissues on specificity ground, and the intravital oleZH of experimental mice ₂Oil body then rests in the SKOV3 tumor tissues, demarcates and got into the SKOV3 tumor tissues so show its specificity ground.

In addition, shown in figure 26, the MAD-MB-231 tumor biopsy of control group mice shows oleZH ₂The distribution of oil body is also not obvious, and the SKOV3 tumor biopsy of experimental mice then can be found oleZH ₂Oil body is retained in the tumor tissues.

Experiment-the oleZH of [embodiment 17] targeted therapy and detection ₂The effect of oil body tumor tissues

After taking out the MAD-MB-231 and SKOV3 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,, add oleZH again with the unnecessary formalin solution of flush away tissue ₂The fluorescence oil body (comprises 2.5 microgram pJO1-oleZH ₂/ every ml phosphate buffer) with histiocyte effect 120 minutes.Effect finishes, with phosphate buffer cleansing tissue three times, and the oleZH of flush away effect ₂Oil 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 fluorescence microscope oleZH ₂The situation of oil body and histiocyte effect, shown in figure 27.

Detect in the body of [embodiment 18] targeting type oil body drug delivery vehicle target function property

About 8-10 week, big nude mice utilized cell strain (SKOV3 and MDA-MB-231) 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 28) and body weight (result is shown in figure 29).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.

[experimental result and discussion]

OleZH of the present invention ₂Oil body is to utilize oil body memebrane protein and Z _Her2Peptide system host cell (escherichia coli) and form oleZH ₂Protein, this protein adds lipid, and is assembled into oleZH ₂Oil body, as scheme shown in Figure 5, oleZH ₂The protein position is at 36KDa.

Utilize the optimum condition of people's oleoplast (AOB) and add with 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 use fluorescence microscope, and 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 use microscopic examination oleZH ₂Oil 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 as seeing that fuel-displaced and fusion rotein are less than normal at 1: 1,1: 5 and 1: 10 oil body granule again.Buffer with different pH (6.5,7.0,7.5,8.0,9.0) constructs the oleZH2 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 and merges lessly each other, and opposite 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, shown in figure 10, 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 targeting people oleoplast electronegativity in different condition on average-45～-56 at surface potential analyser analysis chart, difference is little, and is as shown in table 1.Big more relatively more unstable in the oleoplast of targeting people shown in degree of stability result size, targeting people oleoplast size is more little more stable, as shown in Figure 9.

Utilize the mode of cell fixation, test oleZH ₂The Z that merge the fluoroleum surface _Her2Whether peptide has function and specificity ground identification HER2/neu.Test oleZH ₂The Z that merge the fluoroleum surface _Her2Whether peptide has the overexpression of function and specificity ground identification HER2/neu; So we discern HER2/neu overexpression utilization and have the cell (MDA-MB-231 and MCF7) that the cell of overexpression HER2/neu (MCF7/Her18 and SKOV3) and no mistake in treatment kilsyth basalt reach HER2/neu, with oleZH ₂The fluorescence oil body affacts the cell strain of SKOV3, MCF7, MCF7/Her18 and MDA-MB-231; The result finds that MDA-MB-231 and MCF7 cell strain do not observed fluorescence people oleoplast signal and produced; And can observe a lot of green fluorescence oil body signals at MCF7/Her18 and SKOV3 cell strain; So the representative of green fluorescence oil body signal is because cell HER2/neu overexpression can recognize oil body, we can obtain oleZH ₂The fluorescence oil body has the specificity of identification HER2/neu overexpression cell, and is shown in figure 11, can find out that the cell that the HER2/neu overexpression is arranged can make oleZH ₂Fluorescence oil body specificity ground combines.

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 (MCF7, MCF7/Her18, SKOV3, SKBR3 and MDA-MB-231) cell strain result to learn oleZH ₂The fluorescence oil body acts on HER2/neu overexpression cell strain (MCF7/Her18, SKBR3 and SKOV3) and does not have HER2/neu overexpression cell strain (MDA-MB-231 and MCF7) cell strain, has not observed oleZH by fluorescence microscope discovery MCF7 and MDA-MB-231 cell strain ₂Fluorescence oil body signal produces, and can observe a lot of green fluorescence oil body signals in MCF7/Her18, SKBR3 and SKOV3 born of the same parents' strain, by shown in Figure 12.

Learn oleZH by The above results ₂Oil body can be discerned the specificity of overexpression cell, and carries out the oleZH of different MOI ₂The fluorescence oil body acts on HER2/neu overexpression SKOV3; Sent out by fluorescence microscope that fixedly MOI is high more along with time that people's oleoplast adds at SKOV3, it is many more that people's oleoplast has been combined in HER2/neu overexpression cell, and the ability of recognition specificity HER2/neu overexpressing cell is good more; Present saturated situation but have when oil body affacts MOI 200; Shown in figure 13, different concentration is high more, oleZH ₂The ability of oil body recognition specificity HER2/neu overexpressing cell is many more.

Learn oleZH by The above results ₂The fluoroleum physical ability is discerned the specificity of overexpression cell, and carries out the oleZH of different time ₂The fluorescence oil body acts on HER2/neu overexpression SKOV3 cell strain; Find 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 HER2/neu overexpression cell; The ability of recognition specificity HER2/neu overexpressing cell is good more, but oil body has when affacting two hours and presents saturated situation, and is shown in figure 14.Add oleZH ₂Fluorescence oil body fixed amount is long more in the time, oleZH ₂The ability of recognition specificity HER2/neu overexpressing cell is many more.

Utilize flow cytometer to detect oleZH ₂Oil body is at the number of different time with different MOI entering, and the result obtains at the longer oleZH of causing of concentration fixed time ₂It is many more that oil body gets into cell concentration, the time fixedly MOI cause oleZH more ₂It is many more that oil body gets into cell concentration, is that the time is longer or concentration is high more all can to present oleZH ₂The quantity of oil body entering cell has the trend of increase, has in 2 hours with effect to MOI 200 to present saturated situation, oleZH ₂The variable concentrations that do not coexist, shown in figure 15, oleZH ₂Oil body concentration is high more, oleZH ₂The ability of recognition specificity HER2/neu overexpressing cell is many more.OleZH ₂The oil body different time, shown in figure 16, oleZH ₂The oil body fixed amount is long more in the time, oleZH ₂The ability of oil body recognition specificity HER2/neu overexpressing cell is many more.

Observe oleZH ₂Whether oil body can get into cell.Utilize the conjugation fluorescence microscope to oleZH ₂Single the cell that oil body acts on recognition specificity overexpression cell carries out parallel cutting, and be shown in figure 17, gathers into folds in different aspects and can find out the oleZH of green-emitting fluorescence ₂Oil body is to enter into the cell the inside really.

OleZH ₂Oil body can be discerned the best specificity of overexpression cell, utilizes oleZH ₂Albumen coats the medicine (camptothecine, lycopene and curcumin) of variable concentrations.(the maximum molten oily dissolubility of camptothecine is 500mg/ml, and the maximum molten oily dissolubility of lycopene is 9mM, and it is 20mg/ml that the curcumin maximum is dissolved oily dissolubility) learnt oleZH by particle diameter ₂The medicine oil body is can coat oil-soluble medicine can not make oleZH because of coating medicine ₂Medicine oil body size is bigger, and is as shown in table 2.But, oleZH ₂The medicine oil body coats camptothecine, and elecrtonegativity then is to reduce, oleZH with covering amount increase particle diameter increase ₂The medicine oil body coats lycopene and curcumin can not make negative charge increase or minimizing because of medicated bag covers the medication amount increase, and is as shown in table 2.Through the degree of stability test, nanometer oil is planted and is coated camptothecine because size is less than normal, so be very stable, shown in figure 18.

OleZH ₂Oil body can be discerned the best specificity of overexpression cell, and utilizes oleZH ₂The fat-soluble medicine (curcumin, lycopene and camptothecine) that oil body coats variable concentrations acts on HER2/neu overexpression cell strain (MCF7/Her18 and SKOV3) and does not have HER2/neu overexpression cell strain (MDA-MB-231 and MCF7), adds oleZH ₂Oil body coating medicine function cells, shown in figure 19, and observe different time apoptosis degree; Shown in; The result obtains coating the high more oil medicine (curcumin, lycopene and camptothecine) of concentration at the set time oil body, and the apoptosis amount is high more, and the apoptosis amount is high more.

OleZH ₂Oil body can be discerned the best specificity of overexpression cell, and utilizes oleZH ₂Oil body coats known fluorescence signal molecular action at HER2/neu overexpression SKOV3 cell strain, at the 0th day, the 1st day, the 2nd day, the 3rd day and the 4th day, observes oleZH with particle size analyzer ₂The distribution of medicine oil body in cell, shown in figure 20, oleZH ₂Fluoroleum is known from experience along with the time increase and fluorescence signal is disappeared.

OleZH ₂Whether oil body can haemolysis: add oleZH in the whole blood the inside ₂Oil body and with dividing luminometer to measure, the result show oil body to blood cell be do not have an injury can not make blood cell haemolysis, like Figure 21 and shown in Figure 22.OleZH ₂Whether oil body is stable in the serum the inside: shown oleZH by the particle size analyzer result ₂Oil 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 23.

The nude mice in about 8-10 age in week utilizes different cell strains that HER2/neu overexpression cell strain (SKOV3) is arranged and does not have HER2/neu overexpression cell strain (MDA-MB-231); Carry out hypodermic injection; Make the hypodermic tumor length and width of nude mice about more than 0.5 cubic centimetre, and injection have oleZH with high size ₂The fluorescence oil body utilizes IVIS to observe on tumor, and the result shows oleZH ₂The tumor brightness that the effect of fluorescence oil body is increased in the HER2/neu overexpression along with the time can not reduce, and does not have the tumor of HER2/neu overexpression along with the increase of time, oleZH ₂Fluorescence oil body signal luminous intensity can slowly disappear, and the result is presented at oleZH ₂The fluorescence oil body has specificity in the cancerous cell tumor; Observed 24 hours with IVIS, the result learns that HER2/neu overexpression SKOV3 tumor is residual signal always; Then there is not HER2/neu overexpression MDA-MB-231 tumor signal slowly to disappear because the time is long, shown in figure 24.

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 HER-2/neu to cross expression; Shown in figure 25, carry out frozen tissue section again, utilize the distribution of fluorescence microscope people oleoplast; Shown in figure 26, can find out that the tumor cell that has HER-2/neu to cross expression has ole ZH2 fluorescence oil body residual.

Take out MAD-MB-231 and the SKOV3 tumor of mice again, carry out frozen tissue section, act on oleZH ₂The fluorescence oil body, shown in figure 27, learn oleZH by fluorescence microscope ₂Fluoroleum combines or gets on the HER2/neu overexpression SKOV3 tumor, then can HER2/neu overexpression MDA-MB-231 tumor have the combination and the entering of oil body.

Learn by experiment; In zoopery, can be observed nanometer oil body targeting property, whether the oil body of further observing the targeted nano coating medicine can reach the treatment of cancer effect, is shown by the result; Increase in time; The oil body that targeted nano coats camptothecine can suppress tumor growth, and the nude mice body weight is not reduced, and the effect that more can reach treatment is dwindled tumor.Like Figure 28 and shown in Figure 29.

[conclusion]

Utilize BL21 (DE3)/pJo1-oleZH ₂Proteic production, and through obtaining oleZH after the step that makes up oil body ₂Oil body utilizes oleZH again ₂Oil body is optimized test, carries out at different proportion, different pH and oil not of the same race, and 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, oleZH ₂The oil body size is more unstable more greatly, and is more little stable more.

Three, oleZH ₂Oil body adds the lipid that coats known fluorescence signal molecule also can make oil body send the fluorescence of different wave length, observes more easily.

Four, utilized the film experiment can obtain less oleZH ₂Oil body.

Five, different condition is made people's oleoplast negative charge greatly between-45～-56, to reach the condition of optimizing people's oleoplast.

Utilize and optimize oleZH ₂The condition of oil body produces oil body and carries out cell experiment, can obtain oleZH ₂But oil body specificity ground combines tumor cell line SKOV3 and the MCF7/Her18 of overexpression HER2/neu, and not can with do not express the HER-2/neu tumor cell and combine MCF7 and MDA-MB-231.OleZH ₂The time that oil body adds is longer or MOI is high more, oleZH ₂Oil body has been combined in that HER2/neu overexpression cell is many more, and recognition specificity HER2/neu overexpression cell ability is good more, but oleZH ₂Have when oil body affacts MOI 200 and present saturated situation.Utilize conjugation fluorescence microscope X, Y and Z axle can learn the oleZH of known fluorescence signal molecule ₂Oil body is to be attached to the cell the inside really.With flow cytometer also illustrative: the concentration fixed time is longer, and to cause people's oleoplast to get into cell concentration many more, but oleZH ₂When affacting MOI 200, oil body has to present saturated situation, and 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 oleZH ₂The cell strain that medicine oil body, this oil body act on the HER2/neu overexpression can make the cell strain of HER2/neu overexpression can produce toxic effect to make cell that phenomenon, the oleZH of apoptosis arranged ₂The medicine oil body acts on that the cell strain that does not have the HER2/neu overexpression is then fewer 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.

OleZH ₂Oil body is expelled to mouse tumor cell and utilizes IVIS to learn, oleZH ₂Oil body has signal to produce having on the HER2/neu overexpression tumor tissues, do not having then no signal of HER2/neu overexpression tumor tissues.Learn Z again via tissue slice _Her2The tumor that oil body meeting that peptide merges and specificity ground combine overexpression HER2/neu combines, and can not combine with the tumor of normal HER2/neu.In zoopery, can be observed nanometer oil body targeting property; Coating medicine after 40 days, can effectively dwindle tumor to oncotherapy.

Comprehensive above result, the present invention has realized making up the nanometer oil body system that a target medicine is sent.

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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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:

OleZH ₂Protein, it is to utilize gene recombination technology with Z _Her2Peptide and oil body memebrane protein insert host cell and induce; This oil body memebrane protein can be selected from the oil body protein of seed 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 not dead cell 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 adds said oleZH ₂Process oleZH behind the protein ₂Oil body; 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, oleZH ₂The weight/volume ratio (microgram/microlitre) of protein and this lipid is at least about 1/25, and this oleZH ₂The mean diameter of 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 oleZH ₂Z in the protein _Her2Peptide 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, is assembled into oleZH with Protocols in Molecular Biology ₂The fluorescence oil body, 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 oleZH ₂Z in the protein _Her2Peptide can be discerned the receptor on cancerous cell surface accurately, therefore sees through oleZH ₂Detect oil body, with the oleZH that is coated with known fluorescence signal molecule ₂Detect oil body and 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 to oleZH ₂The phagocytosis of fluorescence oil body and fusion make fluorescence molecule get into cancerous cell, 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, oleZH ₂Protein can further comprise the lipid coating medicine, is assembled into oleZH with Protocols in Molecular Biology ₂The medicine oil body, 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 oleZH ₂Z in the protein _Her2Peptide can be discerned the receptor on cancerous cell surface accurately, therefore sees through oleZH ₂The medicine oil body is with the oleZH that is coated with disease-resistant disease drug ₂The medicine oil body directly is delivered to the canceration position or acts on cancerous cell, improves zonal drug level, and the unlikely normal cell that influences; In addition, also can stimulate the disease cell to oleZH ₂The phagocytosis of medicine oil body and fusion make cancer therapy drug get into cancerous cell, to reach the purpose that detects treatment and avoid developing immunity to drugs.

4. the nanoscale people oleoplast that detects and treat as the targeted delivery of drugs system as claimed in claim 1, wherein, oleZH ₂Protein can comprise further that lipid coating medicine and lipid coat fluorescence signal molecule, are assembled into oleZH with Protocols in Molecular Biology ₂Fluorescence medicine oil body; This medicine can be selected from the medicine of following group: 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; 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 oleZH ₂Z in the protein _Her2Peptide can be discerned the receptor on cancerous cell surface accurately, therefore sees through oleZH ₂Fluorescence medicine oil body is with the oleZH that is coated with disease-resistant disease drug ₂Fluorescence medicine oil body directly is delivered to the canceration position or acts on cancerous cell, improves zonal drug level, and the unlikely normal cell that influences; In addition, also can stimulate the disease cell to oleZH ₂The phagocytosis and the fusion of fluorescence medicine oil body make cancer therapy drug get into cancerous cell, to reach the purpose that detects treatment and avoid developing immunity to drugs.

5. the nanoscale people oleoplast that detects and treat as the targeted delivery of drugs system as claimed in claim 1, wherein, oleZH ₂The protein utilization technique for gene engineering connects the medicine peptide, and is assembled into oleZH ₂Pharmaceutical protein is with Protocols in Molecular Biology and be assembled into oleZH ₂The medicine oil body, 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 oleZH ₂Z in the protein _Her2Peptide can be discerned the receptor on cancerous cell surface accurately, therefore sees through oleZH ₂The medicine oil body is with the oleZH that is coated with disease-resistant disease drug ₂The medicine oil body directly is delivered to the canceration position or acts on cancerous cell, improves zonal drug level, and the unlikely normal cell that influences; In addition, also can stimulate the disease cell to oleZH ₂The phagocytosis of medicine oil body and fusion make cancer therapy drug get into cancerous cell, to reach the purpose that detects treatment and avoid developing immunity to drugs.

6. the nanoscale people oleoplast that detects and treat as the targeted delivery of drugs system as claimed in claim 5, wherein, oleZH ₂Pharmaceutical protein can comprise that lipid coats known fluorescence signal molecule again, with Protocols in Molecular Biology and be assembled into oleZH ₂Fluorescence medicine oil body, 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 oleZH ₂Z in the pharmaceutical protein _Her2Peptide can be discerned the receptor on cancerous cell surface accurately, therefore sees through oleZH ₂Fluorescence medicine oil body is with the oleZH that is coated with disease-resistant disease drug ₂Fluorescence medicine oil body directly is delivered to the canceration position or acts on cancerous cell, improves zonal drug level, and the unlikely normal cell that influences; In addition, also can stimulate the disease cell to oleZH ₂The phagocytosis and the fusion of fluorescence medicine oil body make cancer therapy drug get into cancerous cell, to reach the purpose that detects treatment and avoid developing immunity to drugs.

7. the nanoscale people oleoplast that detects and treat as the targeted delivery of drugs system as claimed in claim 1, wherein, oleZH ₂The protein utilization technique for gene engineering connects narrow spectrum joint, and is assembled into oleZH ₂Specificity joint oil body, oleZH ₂Specificity joint oil body can utilize the cutting of specificity protease hydrolysis, can be purified into the high Z of purity _Her2Proteinic application, this method are 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:5; Z _HerPeptide is to comprise listed aminoacid sequence and Z by SEQ ID NO:2 _Her2The oil body memebrane protein of the DNA sequence that sequence SEQ ID NO:6 is listed and assemble oleZH ₂Medicine oil body and oleZH ₂The medicine of 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, discern the effect of inhibition of cancerization position and Drug therapy with having.

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