CN102100914A

CN102100914A - Artificial oil body carrier for targeted therapy of mastocarcinoma as well as preparation method and application thereof

Info

Publication number: CN102100914A
Application number: CN2010105986502A
Authority: CN
Inventors: 朱宝美; 陈顺基
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-12-21
Filing date: 2010-12-21
Publication date: 2011-06-22
Also published as: CN102743326A; CN102743324A; CN102743327A; WO2012083592A1; CN102743325A; CN102743761A

Abstract

The invention discloses an artificial oil body carrier for the targeted therapy of mastocarcinoma as well as a preparation method and an application thereof. With the preparation method, a pJO1-oleZH2 plasmid is converted to enter the host cell of escherichia coli BL21 (DE3), oleZH2 proteins are produced by induction, and oil and the oleZH2 proteins are mixed to prepare an artificial oil body carrier. Invitro and invivo experiments confirm that the artificial oil body carrier disclosed by the invention can be effectively applied to target therapy, and the oil body carried covered with fluorescent substances can reflect HER2/neu positive cancer cells so as to specifically detect the mastocarcinoma cell excessively expressing HER2/neu. The artificial oil body carrier has the advantages of simple preparation process, extra small environmental pollution, good stability in blood cells or serum, small volume and the like, is cheap and efficient, is convenient to operate and is easy to transport. The artificial oil body carrier can be used for covering anti-mastocarcinoma drugs of different types of oil solubility, can effectively kill mastocarcinoma cells after being coated on drugs and can carry out specific detection and target therapy for mastocarcinoma cells.

Description

People's oleoplast carrier of energy targeted therapy breast carcinoma and its production and application

Technical field

The invention belongs to genetic engineering and Medical Biology field, relate to the proteic method of escherichia coli expression oleZH2 of using, the application in specificity detecting and target therapy breast carcinoma of the preparation method of people's oleoplast carrier and this carrier.

Background technology

Cancer also is called malignant tumor, and according to present understanding, its cause of disease is because cancerous cell abnormal division and shifting everywhere in vivo, causes physiological function unusual and be difficult to cure, and becomes in recent years first of the global human cause of the death.From 2003, breast carcinoma became modal cancer among the women of Taiwan, and 50% breast carcinoma sufferer has been diagnosed out the HER2/neu positive in the world.These sufferers lack clinical symptoms, and are difficult to find in early days.Therefore, urgent needs breast carcinoma early diagnosis instrument.

Therapeutic Method mainly is to utilize the surgical operation mode with tumor resection traditionally, or comes kill cancer cell with chemicals or lonizing radiation means.But the surgical operation mode is difficult to eradicate cancerous cell and is difficult for implementing, general chemicals and radiation cure do not have specificity, in therapeutic process, tend to kill and keep the necessary normal cell of general physiological function, thereby produce many side effect, for example immunologic function degression (white blood cell count decline), feel sick, phenomenons such as vomiting, alopecia, the decline of the intestines and stomach absorption function and anemia.

Find that after deliberation cancerous cell can have some particular molecule biomarker usually, the method for target therapy (targeting therapy) in recent years at treatment of cancer development.So-called target therapy is a kind of medicine that can specificity identification cancerous cell surface molecular biomarker of design, can effectively block the cancerous cell activation or suppress its growth, thereby reach the purpose of treatment.The target therapy medicine can be directly hit cancerous cell accurately, less for Normocellular influence in the body.Therefore with respect to traditional therapy, target therapy has advantages such as hypotoxicity, low side effect, high efficiency and implementation convenience.

Target therapy has multiple mode, as utilize a carrier to coat, link or inlay a drug molecule with therapeutic activity, use various mechanism with this carrier and drug molecule specificity be delivered to targeted cancerous cells, this kind pattern is commonly referred to as drug delivery system (drug delivery system).This carrier has the target function, and this material can be protein (for example antibody), steroid, saccharide or other chemical compound etc.

Known carrier has high molecular polymerization granule, micella (micell), little ester grain (liposome) and viral carrier etc. at present, the different shortcomings of each tool of these carriers, for example particle diameter excessive and be difficult for being absorbed by the body, have toxicity, in blood or body fluid instability etc., limitation is arranged on practice more.In addition, but, need complicated and chemosynthesis step consuming time usually, not only improved manufacturing cost, and can cause environmental pollution when this type carrier during in conjunction with the material of above-mentioned identification cancerous cell.So urgent needs has nominal particle size, avirulence, stablizes and is easy to the carrier in conjunction with the identification cancerous cell.

At the demand, the present invention utilizes molecular biotechnology, and a kind of people's oleoplast carrier can be provided, and its preparation process is easy and environmental pollution is very low.Via in vitro (in vitro) and in vivo (in vivo) experimental results show that oil body carrier of the present invention can apply to target therapy or detecting effectively, and the bio-compatibility of tool excellence.

Summary of the invention

The purpose of this invention is to provide a kind of fusion rotein, it comprises an oil body protein, a part peptide or an antibody peptide, a cell-penetrating peptide or aforementioned combination.

Another object of the present invention provides a kind of people's oleoplast carrier, and it comprises an above-mentioned fusion rotein and a lipid, and fusion rotein and lipid weight/volume ratio (microgram/microlitre) are at least about 1/25, and this oil body mean diameter is 50 nanometer to 2000 nanometers.

Another object of the present invention provides the preparation method of above-mentioned fusion rotein and people's oleoplast carrier.

A further object of the invention provides a kind of drug delivery system that can the targeted therapy breast carcinoma, it comprises above-mentioned people's oleoplast carrier and a medicine, a signal molecule or aforementioned combination, in vitro (in vitro) and in vivo (in vivo) breast carcinoma is carried out specificity detecting and target therapy.

The preparation method of people's oleoplast carrier of a kind of energy targeted therapy breast carcinoma of the present invention may further comprise the steps:

(1) Expression of Fusion Protein.Reorganization strain BL21 (DE3)/pJO1-oleZH2 cultivates antibacterial initial density OD down at 37 ℃ ₅₅₀Value is 0.08.Be inoculated into the fresh LB culture fluid that contains ampicillin, as bacterial density OD ₅₅₀Value is 0.3 o'clock, induces 4 hours for 37 ℃, collects thalline, with 4000 rpm, 4 ℃ of frozen centrifugation 10 min, reuse 0.1 M pH7.5 sodium phosphate buffer cleans once, collects thalline, is condensed into bacterial density OD with 0.1M pH7.5 sodium phosphate buffer ₅₅₀Value is 10 bacterium liquid.

With bacterial density OD ₅₅₀To be 10 bacterium liquid carry out ultrasonication on ice (power: 3.5%, time: 2 minutes, run:0.5 second, rest:0.5 second) with ultrasonic cell disruptor to value.After the ultrasonic end,, collect centrifugal back supernatant and precipitate not aqueous soluble protein with 13000 rpm, 4 ℃ of frozen centrifugation 10 min.

(2) preparation of people's oleoplast carrier.Get 100 μ g oleZH2 albumen, add 950 μ l 0.01M pH7.5 sodium phosphate buffer, add 50 μ l Oleum sesami again, carry out ultrasonic mixing (power: 3.0% on ice with ultrasonic cell disruptor, time:20 second, run:0.5 second, rest:0.5 second), with 13000 rpm, 4 ℃ of frozen centrifugation 10 min, the oil cake of one deck white above taking out adds 0.01M pH7.5 sodium phosphate buffer 1 ml, breaks up with ultrasonic cell disruptor, same steps as is carried out three times, constructs people's oleoplast carrier.

People's oleoplast carrier of the present invention has passed through various performance tests, have volume little, in blood cell or serum good, the hypotoxicity of stability, low side effect, advantage such as efficient, easy to operate.

People's oleoplast carrier of the present invention is (in vitro) and in vivo all have targeting in (in vivo) experiment in vitro, can specificity the identification target cell, behind coating medicine, can suppress tumor growth.

In vivo in the anti-tumor activity test, get the nude mice in about 8-10 week, carry out the subcutaneous tissue injection with cell strain (SKOV3 and MDA-MB-231), make the hypodermic tumor size of nude mice about more than 0.5 centimetre, jede Woche double injection 100mg/ml coats the oil body carrier of camptothecine on tumor, and the result shows, increase in time, the oil body carrier that coats camptothecine can suppress tumor growth, and the nude mice body weight does not reduce.This illustrates that this research successfully makes up a kind of people's oleoplast carrier of energy targeted therapy breast carcinoma.

Unless otherwise indicated herein, in this description, use it " one " or " being somebody's turn to do " and similar term to be interpreted as comprising odd number and plural form.

The oil body of plant seed has sizable surface area, can be used for coating or inlaying a large amount of signal molecules, to follow the trail of as real-time (real time) signal amplifier that moves in the organism and to confirm focus, also can be used for coating fat-soluble medicine, treat disease as the specificity drug delivery system.The present invention utilizes the above-mentioned characteristic of oil body, and the binding molecule biotechnology provides a kind of people's oleoplast carrier (hereinafter referred is the oil body carrier) by simple method for preparing.

Oil body carrier of the present invention comprises a fusion rotein and a lipid, and wherein, this fusion rotein comprises an oil body protein, a part peptide or an antibody peptide, a cell-penetrating peptide or aforementioned combination.

The contained oil body protein of oil body carrier fusion rotein of the present invention is not limited to them.Oil body protein from plant seed is preferable, for example its combination of Oleum sesami, olive oil, soybean oil, Oleum Arachidis hypogaeae semen and mineral oil, and wherein the oil body protein of Semen Sesami seed is better.

Part peptide or antibody peptide that oil body carrier fusion rotein of the present invention is contained make the oil body carrier become specificity and transmit carrier (being the target carrier).Part peptide or antibody peptide be identification cancerous cell surface receptor (receptor) accurately, by part peptide or antibody peptide and receptors bind, the oil body carrier that is coated with cancer therapy drug directly can be delivered to the canceration position or act on cancerous cell, improve zonal drug level, do not influence normal cell.In addition, utilize above-mentioned mechanism, but also the irritation cancer cell absorbs the oil body carrier by gulping down formula effect (phagocytosis) with fusion (fusion), makes cancer therapy drug enter cancerous cell, thus the purpose that reaches treatment and avoid developing immunity to drugs.

Part that fusion rotein of the present invention comprises wins peptide or antibody victory peptide is not limited to them, as long as have the function of specificity recognizing cells.With regard to the part peptide, when treatment breast carcinoma or ovarian cancer, can adopt the part peptide of HER2/neu protein acceptor.Known HER2/neu protein acceptor is to belong to epithelical cell growth factor receptor (epidermal growth factor receptor, EGFR), it is present in multiple cancerous cell surface, in carcinogenic mechanism, play an important role, but people such as Nord have developed a kind of specificity ground and the bonded ZHer2 peptide of HER2/neu protein acceptor, with the part as this receptor.The ZHer2 peptide has 58 aminoacid, and its molecular weight (being about 7 to 15 kilodaltons) is easy to permeates cell membranes much smaller than monoclonal antibody molecular weight (150 kilodalton), makes the oil body carrier enter breast cancer cell, carries out targeting detection and treatment.

The present invention has the following advantages:

The present invention through inducing, carries out the proteic production of ole ZH2 after utilizing the conversion of pJO1-oleZH2 plasmid to enter e. coli bl21 (DE3) host cell, oil and ole ZH2 albumen is mixed again, and constructs people's oleoplast carrier.Via in vitro (in vitro) and in vivo (in vivo) experimental verification oil body carrier of the present invention can apply to target therapy or detecting effectively, and the bio-compatibility of tool excellence.Preparation process of the present invention is easy and pollution on the environment is very low.Oil body carrier stability in blood cell or serum is good, cheaply, hypotoxicity, low side effect, high efficiency, easy to operate, volume is little, transport easily, can coat various different oil-soluble cancer therapy drugs, effective kill cancer cell behind the coating medicine, and can carry out specificity detecting and treatment to cancerous cell.

Description of drawings

Fig. 1 is the electrophoretogram of fusion rotein after escherichia coli expression and oil body preparing carriers.In Fig. 1, marker protein standard: 116kDa, 66.2kDa, 45kDa, 35kDa, 25kDa, 18.4kDa, 14.4kDa represent different molecular weight size criteria albumen respectively; Oleosin: oil body protein; Oleosin-ZH2: fusion rotein; AOB: oil body carrier;-IPTG: before inducing; After+IPTG induces; Sup-1: escherichia coli expression fusion rotein, ultrasonication, the supernatant after centrifugal; Ppt-1: the escherichia coli expression fusion rotein, ultrasonication, the precipitation after centrifugal is aqueous soluble protein not; Sup-2: oil and fusion rotein, ultrasonic mixing, the supernatant after centrifugal; Ppt-2: oil and fusion rotein, ultrasonic mixing, the precipitation after centrifugal is aqueous soluble protein not.

Fig. 2 is the microscopic examination that the oil body carrier coats fluorescent material yellow GGK and Nile red.In Fig. 2, left column figure is that bright field is observed figure.Right row figure observes figure for the fluorescence visual field.

Fig. 3 is the AFM test of oil body carrier.

Fig. 4 is the microscopic examination of oil body carrier under different preparation conditions.

Fig. 5 is the degree of stability test of oil body carrier under different preparation conditions.

Fig. 6 is that the blood that melts of oil body carrier is tested (last figure); The change of size test (figure below) that the oil body carrier increases in time under the different temperatures.

Fig. 7 is that the oil body carrier function is in the fixedly specificity test of tumor cell (Fixed cell).

Fig. 8 is the specificity test of oil body carrier function in tumor living cell (Living cell).

Fig. 9 is the specificity test of the oil body carrier function of variable concentrations in the SKOV3 cell strain.

Figure 10 is the specificity test of oil body carrier function in SKOV3 cell strain different time.

Figure 11 is that the oil body carrier function of variable concentrations is tested in the specificity FLOW of different tumor living cell strains.

Figure 12 is that the oil body carrier function is in the specificity FLOW of different tumor living cell strain different times test.

Figure 13 is that the oil body carrier function is observed and the observation of Z axial section in the conjugation fluorescence microscope XY of SKOV3 cell strain axial plane.

Figure 14 is that the FTIR that coats the oil body carrier of variable concentrations camptothecine observes.

Figure 15 is the stable turbidity test that coats the oil body carrier of variable concentrations camptothecine.

Figure 16 is the cell survival test that coats the oil body carrier of variable concentrations camptothecine.

Figure 17 is in time IVSI observation test behind the mice with tumor injection oil body carrier.

Figure 18 is that mice with tumor was injected the oil body carrier after 24 hours, the fluorescence microscope of test of internal organs cumulative bad and tumor tissues frozen section.

Figure 19 is that the tumor body of oil body vehicle treatment tumor load Mus changes and body weight change observation figure.

Figure 20 is the tumor growth observation figure of oil body vehicle treatment tumor load Mus.

Table 1 is the different proportion of oil and fusion rotein.

Table 2 is the size tests under different preparation conditions of oil body carrier.

Table 3 is the electronegativity tests under different preparation conditions of oil body carrier.

Table 4 is size and the electronegativity tests that coat the oil body carrier of variable concentrations camptothecine.

The specific embodiment

The preparation of embodiment 1 oil body carrier

Reorganization strain BL21 (DE3)/pJO1-oleZH2 cultivates antibacterial initial density OD down at 37 ℃ ₅₅₀Value is 0.08.Be inoculated into the fresh LB culture fluid that contains ampicillin, as bacterial density OD ₅₅₀Value is 0.3 o'clock, induces 4 hours for 37 ℃, collects thalline, with 4000 rpm, 4 ℃ of frozen centrifugation 10 min, reuse 0.1 M pH7.5 sodium phosphate buffer cleans once, collects thalline, is condensed into bacterial density OD with 0.1M pH7.5 sodium phosphate buffer ₅₅₀Value is 10 bacterium liquid.

With bacterial density OD ₅₅₀To be 10 bacterium liquid carry out ultrasonication on ice (power: 3.5%, time: 2 minutes, run:0.5 second, rest:0.5 second) with ultrasonic cell disruptor to value.After the ultrasonic end,, collect centrifugal back supernatant and precipitate not aqueous soluble protein with 13000 rpm, 4 ℃ of frozen centrifugation 10 min.Get the supernatant of 30 μ l and add the 2X Sample Buffer of 10 μ l, utilize Ox blood serum quantitatively to obtain sup1.Precipitate the 0.1M pH7.5 sodium phosphate buffer that aqueous soluble protein not adds 30 μ l, and add the 2X Sample buffer of 10 μ l, utilize Ox blood serum quantitatively to obtain ppt1, and analyze (Fig. 1) with SDS-PAGE, carry out the quantification of protein analysis with Quantity One (Bio-RAD) software, by fusion rotein amount conversion fused protein concentration.

Get 100 μ g oleZH2 albumen, add 950 μ l 0.01M pH7.5 sodium phosphate buffer, add 50 μ l Oleum sesami again, carry out ultrasonic mixing (power: 3.0% on ice with ultrasonic cell disruptor, time:20 second, run:0.5 second, rest:0.5 second), with 13000 rpm, 4 ℃ of frozen centrifugation 10 min, the oil cake of one deck white above taking out adds 0.01M pH7.5 sodium phosphate buffer 1 ml, breaks up with ultrasonic cell disruptor, same steps as is carried out three times, constructs the oil body carrier.Whether with the SDS-PAGE analysis and observation is the constructed target oil body carrier (Fig. 1) that comes out.

The result shows, with the pJO1-oleZH2 plasmid transform enter BL21 (DE3) bacterial strain after, can express ole ZH2 albumen, can clearly see through after inducing, be equipped with target protein in prediction bits and produce.Then, after the step that makes up the oil body carrier, target protein oleZH2 can pass through ole oil body structural protein, is fixed on the oil body, and the oleZH2 position is at 33KDa as can be seen.

Embodiment 2 oil body carriers coat fluorescent material and oil body AFM test

Get oleZH2 albumen, add 0.01M pH7.5 sodium phosphate buffer, add again and contain nile red(label: SIGMA, 0.5 μ g/ml) or Yellow GGK(label concentration:: Widetex Biotech Co., Ltd., concentration: Oleum sesami 1 μ g/ml), carry out ultrasonic mixing (power: 3.0% on ice with ultrasonic cell disruptor, time:20 second, run:0.5 second, rest:0.5 second), with 13000 rpm, 4 ℃ of frozen centrifugation 10 min, the oil cake of one deck white above taking out adds 0.01M pH7.5 sodium phosphate buffer, breaks up with ultrasonic cell disruptor, same steps as is carried out three times, constructs fluorescence oil body carrier.Dilute with 0.01M pH7.5 sodium phosphate buffer, and using fluorescence microscope, Yellow GGK excites with 488 nm, can see oil body carrier green-emitting fluorescence, nile red excites with 454-458 nm, can see the rubescent color fluorescence of oil body carrier (Fig. 2).

The mica sheet water is cleaned three times, each three minutes, the MgCl2 bubble of reuse 0.02M 2-3 minute, the reuse clear water cleans once.The oil body carrier is added on the mica sheet, left standstill 2 hours, the oil body carrier is adsorbed on the mica sheet, clean twice with ddH2O then, be positioned over drying in 37 ℃ of baking ovens, last AFM observes oil body kenel and size (Fig. 3).Utilize the AFM test to draw big or small about 200～500 nanometers of oil body carrier.

Embodiment 3 tests of oil body carrier under different preparation conditions

The oil of different proportion and albumen make up the oil body carrier.With oil and oleZH2 albumen is respectively 10:1 with the ratio, 2:1,1:1,1:5,1:10 mixes, get oleZH2 albumen, add 0.01M pH7.5 sodium phosphate buffer, add Oleum sesami (table 1) again, carry out ultrasonic mixing (power: 3.0% on ice with ultrasonic cell disruptor, time:20 second, run:0.5 second, rest:0.5 second), with 13000 rpm, 4 ℃ of frozen centrifugation 10 min, the oil cake of one deck white above taking out, add 0.01M pH7.5 sodium phosphate buffer 1 ml, break up with ultrasonic cell disruptor, same steps as is carried out three times, makes up the oil body carrier.

Under different pH conditions, make up the oil body carrier.Fixed ratio with oil and fusion rotein, get oleZH2 albumen, the sodium phosphate buffer(pH6.5 that adds various pH, pH7.0, pH7.5, pH8.0, pH9.0), add Oleum sesami again, carry out ultrasonic mixing (power: 3.0% on ice with ultrasonic cell disruptor, time:20 second, run:0.5 second, rest:0.5 second), with 13000 rpm, 4 ℃ of frozen centrifugation 10 min, the oil cake of one deck white above taking out, 0.01M sodium phosphate buffer 1 ml that adds various pH, break up with ultrasonic cell disruptor, same steps as is carried out three times, makes up the oil body carrier.

Make up the oil body carrier with different types of oil.Fixed ratio with oil and fusion rotein, get oleZH2 protein, add 0.01M pH7.5 sodium phosphate buffer, add different oil (Oleum sesami again, mineral oil, Oleum Arachidis hypogaeae semen, olive oil and soybean oil), carry out ultrasonic mixing (power: 3.0% on ice with ultrasonic cell disruptor, time:20 second, run:0.5 second, rest:0.5 second), with 13000 rpm, 4 ℃ of frozen centrifugation 10 min, the oil cake of one deck white above taking out, add 0.01M sodium phosphate buffer 1 ml, break up with ultrasonic cell disruptor, same steps as is carried out three times, makes up the oil body carrier.

Make up the oil body carrier of above-mentioned different condition, with sodium phosphate buffer dilution, reuse fluorescence microscope (Fig. 4).

The size test of oil body carrier.The oil body carrier of different condition dilutes with sodium phosphate buffer, and the best dilution ratio that is fit to the particle size analyzer test is 1 * 10 ⁴To 1 * 10 ⁶Individual oil body is put into the oil body carrier of different condition in the 2ml cuvette, puts in the particle size analyzer again, tests (table 2).

The electronegativity test of oil body carrier.The oil body carrier of different condition dilutes with sodium phosphate buffer, and the best dilution ratio that is fit to the test of current potential measurement instrument is 1 * 10 ⁴To 1 * 10 ⁶Individual oil body is put into the oil body carrier of different condition in the 2ml cuvette, utilizes Zeta potential to measure instrument test oil body (table 3).

The result shows, oil and fusion rotein are mixed with following ratio (10:1,2:1,1:1,1:5,1:10), measure best ratio, use microscopic examination, fuel-displaced and optimal proportion fusion rotein is 1:1, this moment, the big low profile of oil body carrier was average, and the mutual fusion between the oil body carrier is less.When the buffer with different pH (6.5,7.0,7.5,8.0,9.0) constructs the oil body carrier, it is average to draw the big low profile of oil body carrier that the buffer with pH7.5 makes up, and the mutual fusion between the oil body carrier is less.Make up the oil body carrier with various oil (Oleum sesami, mineral oil, Oleum Arachidis hypogaeae semen, olive oil and soybean oil), the big low profile of the oil body carrier that Oleum sesami and olive oil are constructed is average, and the mutual fusion between the oil body carrier is less, opposite mineral oil oil body carrier granular maximum, and fusion is more mutually between the Oleum Arachidis hypogaeae semen oil body carrier, and it is spherical to present chain.

Oil and fusion rotein are when 1:1,1:5 and the 1:10 as can be known by granularmetric analysis, and the oil body carrier granular is less than normal.Less than normal with the oil body carrier granular that the buffer of pH7.5, pH8.0 and pH9.0 constructs.Less than normal with the oil body carrier granular that soybean oil, Oleum sesami and olive oil are constructed.The electronegativity of the oil body carrier under different preparation conditions is-45～-56 as can be seen in the zata analysis chart, and difference is little.

The degree of stability test of embodiment 4 oil body carriers

The oil body carrier of different condition carries out the degree of stability test with the turbidity (T) that sodium phosphate buffer adjusts its suspension at every fixed interval time remaining.In order to measure the oil body carrier, and use 2ml cuvette with spectrophotometer OD600, selected 20 minutes at interval, with the suspension degree of oil body carrier in sodium phosphate buffer of spectrophotometer record different condition.The turbidity (T) of oil body carrier suspension represents it is proportional with 10A, and turbidity relation expression T/T0=10A/10A0=10A/102.0 is diluted to Ao=2 with people's oleoplast, and its degree of stability (Fig. 5) is tested in beginning in time.The degree of stability result shows that the size of oil body carrier is big more unstable more, otherwise the size of oil body carrier is more little stable more.

Embodiment 5 oil body carriers are to test of blood cell hemolytic and serum stability test

The oil body carrier is tested the blood cell hemolytic.The oil body carrier is placed normal saline and 5% glucose solution, and place 37 ℃, centrifugal 10000rpm after 30 minutes, 10 minutes uses spectrophotometer measurement (Fig. 6) at last.

The oil body carrier is tested serum stability.The oil body carrier is placed the sodium phosphate buffer that contains 5% serum and break up, measure at different time and different temperatures, and observe and increase degree of stability and the size (Fig. 6) of oil body carrier in time at serum with particle size analyzer.

The result shows that the oil body carrier does not have injury to blood cell, can not make blood cell haemolysis.Granularmetric analysis shows that the oil body carrier is measured in different temperatures, and is still highly stable after 24 hours in 5% serum.

The targeting specificity functional test of embodiment 6 oil body carriers (in vitro detection)

The oil body carrier function is in the fixedly specificity test of tumor cell (Fixed cell).Get cell SKOV3 and MDA-MB-231, be seeded in 24 orifice plates in proxima luce (prox. luc), the next day with Phosphate Buffered Saline(PBS) pH7.5 cleans twice, with 2.5% Formaldehyde 30 ℃ fix 40 minutes after, reuse PBS pH7.5 cleans, and places 30 ℃ and allows PBS evaporate.The oil body carrier is added in the cell that fixes, 25 ℃ of reactions two hours, clean three times each 15 minutes with PBS pH7.5, be dissolved among the PBS with blocking solution(3% BSA) at room temperature reacted one hour, dilute an anti-anti-her2/neu(9G6 with 1:200), room temperature reaction at least one hour, PBS cleans three times again, each 15 minutes, dilute two anti-anti-mouse IgG-TRITC with 1:500, reacted one hour, reuse PBS cleans three times, each 15 minutes, DAPI transfect cell nuclear with the 1:15000 dilution dyeed 4 minutes, cleaned three times with PBS again, each 15 minutes, place drying, mounting utilizes fluorescence microscope (Fig. 7).

The oil body carrier function is in the specificity test of tumor living cell (Living cell).Get cell (MCF-7, MCF-7/Her18, SKOV3, SKBR3 and MDA-MB-231), in proxima luce (prox. luc) with every hole 1 * 10 ⁵The density of cell is seeded in 24 orifice plates, with DMEM/F12 culture fluid (GIBCO Invitrogen Corporation, New York, USA) cultivate, the next day clean twice with DMEM/F12, with DMEM/F12 culture fluid dilution oil body carrier, be added in the cleaned cell, at 37 ℃, 5% CO ₂Incubator cultivated 2 hours, clean three times with PBS pH7.4 again, fix 40 minutes with 2.5% Formaldehyde in 30 ℃, clean three times with PBS pH7.4, be dissolved among the PBS with blocking solution(3% BSA then) at room temperature reacted one hour, dilute an anti-anti-her2/neu(9G6 with 1:200), room temperature reaction one hour, PBS pH7.4 cleans three times again, each 15 minutes, dilute two anti-anti-mouse IgG-TRITC with 1:500, reacted one hour, reuse PBS pH7.4 cleans three times, each 15 minutes, DAPI transfect cell nuclear with the 1:15000 dilution dyeed 4 minutes, cleaned three times with PBS again, each 15 minutes, place drying, mounting utilizes fluorescence microscope (Fig. 8).

The oil body carrier function of variable concentrations (MOI) is tested in tumor living cell.Get cell (MCF-7, MCF-7/Her18, SKOV3, SKBR3 and MDA-MB-231), in proxima luce (prox. luc) with every hole 1 * 10 ⁵The density of cell is seeded in 24 orifice plates, cultivate with the DMEM/F12 culture fluid, the next day clean twice with DMEM/F12, with DMEM/F12 culture fluid dilution oil body carrier, add variable concentrations oil body carrier in cleaned cell, at 37 ℃, 5% CO ₂Incubator cultivated 2 hours, clean three times with PBS pH7.4 again, fix 40 minutes with 2.5% Formaldehyde in 30 ℃, clean three times with PBS pH7.4, be dissolved among the PBS with blocking solution(3% BSA then) at room temperature reacted one hour, dilute an anti-anti-her2/neu(9G6 with 1:200), room temperature reaction one hour, PBS pH7.4 cleans three times again, each 15 minutes, dilute two anti-anti-mouse IgG – TRITC with 1:500, reacted one hour, reuse PBS pH7.4 cleans three times, each 15 minutes, DAPI transfect cell nuclear with the 1:15000 dilution dyeed 4 minutes, cleaned three times with PBS again, each 15 minutes, place drying, mounting utilizes fluorescence microscope (Fig. 9).

The test of oil body carrier function tumor living cell different time.Cell in proxima luce (prox. luc) with every hole 1 * 10 ⁵The density of cell is seeded in 24 orifice plates, cultivate with the DMEM/F12 culture fluid, the next day clean twice with DMEM/F12, with DMEM/F12 culture fluid dilution oil body carrier, be added in the cleaned cell, at 37 ℃, 5% CO ₂Incubator cultivated different time (15 minutes, 30 minutes, 60 minutes, 120 minutes and 240 minutes), clean three times with PBS pH7.4 again, fix 40 minutes with 2.5% Formaldehyde in 30 ℃, clean three times with PBS pH7.4, be dissolved among the PBS with blocking solution(3% BSA then) at room temperature reacted one hour, dilute an anti-anti-her2/neu(9G6 with 1:200), room temperature reaction one hour, PBS pH7.4 cleans three times again, each 15 minutes, dilute two anti-anti-mouse IgG – TRITC with 1:500, reacted one hour, reuse PBS pH7.4 cleans three times, each 15 minutes, DAPI transfect cell nuclear with the 1:15000 dilution dyeed 4 minutes, cleaned three times with PBS again, each 15 minutes, place drying, mounting utilizes fluorescence microscope (Figure 10).

The oil body carrier function of variable concentrations is in the FLOW of tumor living cell test.Get cell (MCF-7, MCF-7/Her18, SKOV3, SKBR3 and MDA-MB-231), in proxima luce (prox. luc) with every hole 5 * 10 ⁵The density of cell is seeded in 12 orifice plates, cultivate with the DMEM/F12 culture fluid, the next day clean twice with DMEM/F12, with DMEM/F12 culture fluid dilution oil body carrier, add variable concentrations oil body carrier in cleaned cell, at 37 ℃, 5% CO ₂Incubator cultivated 2 hours, clean three times with PBS pH7.4 again, add trypsin effect 5 minutes, get cell to tube(label: BD, model: Falcon), 1000 rpm, centrifugal 10 minutes, remove supernatant, if at once with flow cytometer (label: BD, model: FACSCanto) detect, directly add PBS buffer.If test every other day, with 2.5% Formaldehyde in 4 ℃ fixing, cell is broken up in placement overnight, with flow cytometer detection (Figure 11).

The FLOW test of oil body carrier function tumor living cell different time.Get cell (MCF-7, MCF-7/Her18, SKOV3, SKBR3 and MDA-MB-231), in proxima luce (prox. luc) with every hole 5 * 10 ⁵The density of cell is seeded in 12 orifice plates, cultivate with the DMEM/F12 culture fluid, the next day clean twice with DMEM/F12, with DMEM/F12 culture fluid dilution oil body carrier, be added in the cleaned cell, at 37 ℃, 5% CO ₂Incubator cultivate different time (15 minutes, 30 minutes, 60 minutes, 120 minutes and 240 minutes), clean three times with PBS pH7.4 again, add trypsin effect 5 minutes, get cell to tube, 1000 rpm, centrifugal 10 minutes, remove supernatant,, directly add PBS buffer if detect with flow cytometer at once.If test every other day, with 2.5% Formaldehyde in 4 ℃ fixing, cell is broken up in placement overnight, with flow cytometer detection (Figure 12).

The oil body carrier is observed by the conjugation fluorescence microscope XY axial plane of SKOV3 cell strain and the Z axial section is observed.Get cell SKOV3, in proxima luce (prox. luc) with every hole 1 * 10 ⁵The density of cell is seeded in 24 orifice plates, cultivate with the DMEM/F12 culture fluid, the next day clean twice with DMEM/F12, with DMEM/F12 culture fluid dilution oil body carrier, be added in the cleaned cell, at 37 ℃, 5% CO ₂Incubator cultivated 2 hours, clean three times with PBS pH7.4 again, reuse 2.5% Formaldehyde fixes 40 minutes in 30 ℃, clean three times with PBS pH7.4, be dissolved among the PBS with blocking solution(3% BSA then) at room temperature reacted one hour, dilute an anti-anti-her2/neu(9G6 with 1:200), room temperature reaction one hour, PBS pH7.4 cleans three times again, each 15 minutes, dilute two anti-anti-mouse IgG – TRITC with 1:500, reacted one hour, reuse PBS pH7.4 cleans three times, each 15 minutes, the DAPI transfect cell nuclear with the 1:15000 dilution dyeed 4 minutes, clean three times with PBS again, each 15 minutes, place drying, mounting, utilize the photo of laser scanning confocal spectrophotometric spectra microscope photographing same position differing heights, reuse Leica system analyzes (Figure 13).

The result shows, utilizes the mode of cell fixation, and whether test the ZH2 pepteide that is merged by the constructed oil body carrier surface of oleZH2 can specificity identification HER-2/neu protein receptor.Utilization has the MCF7/Her18 cell of HER-2/neu and the MDA-MB-231 cell of no HER-2/neu, with these two kinds of cells of oil body carrier function, found that the MDA-MB-231 cell do not observed the fluorescence signal and produced, and can observe a lot of green fluorescence signals at the MCF7/Her18 cell.It is because of the HER-2/neu overexpression on the cell, so can allow the oil body carrier recognize that green fluorescence oil body carrier signal is arranged.As seen, the oleZH2 of oil body carrier can the excessive cell that shows of specificity identification HER-2/neu.

The oil body carrier can be discerned the excessively fixed cell of performance of HER-2/neu by specificity, and next step will understand the specificity identification of oil body carrier to living cells in depth.Utilize MCF-7, MCF-7/Her18, SKOV3, SKBR3 and MDA-MB-231 cell strain, the oil body carrier function can be observed a lot of green fluorescence signals in the cell (MCF7/Her18, SKBR3 and SKOV3) that HER-2/neu excessively shows by fluorescence microscope as can be known from the results.The oil body carrier can not act on does not have the excessively cell (MCF7 and MDA-MB-231) of performance of HER-2/neu, has not observed the fluorescence signal by fluorescence microscope and has produced.

Above result shows that proof oil body carrier can specificity identification target cell.When the SKOV3 cell of the oil body carrier function of using variable concentrations in the excessive performance of HER-2/neu, by fluorescence microscope, along with the adding concentration of oil body carrier is high more, it is many more that the oil body carrier is combined in cell surface, the ability of specificity recognizing cells is good more, but the oil body carrier function has during to MOI 200 and presents saturated trend.

Utilize the oil body carrier at different time effect SKOV3 cell, find by fluorescence microscope, when the oil body carrier adds concentration fixed, along with the time is long more, it is many more that the oil body carrier is combined in cell surface, the ability of specificity recognizing cells is good more, presents saturated trend but have during oil body carrier function to two hour.

Utilize flow cytometer to detect the oil body carrier and enter what of cell at different time and variable concentrations, the result shows that when concentration fixed the time is long more, and the amount that the oil body carrier enters cell is many more.Fix when the time, concentration is high more, and the amount that the oil body carrier enters cell is many more.No matter be that the time is long more or concentration is high more, the amount that the oil body carrier enters cell all has the trend of increase, all saturated situation can be arranged afterwards.

Utilize the conjugation fluorescence microscope that the single cell that the oil body carrier function is arranged is carried out parallel cutting, stack up, can see that the oil body carrier of green-emitting fluorescence enters cell really in different aspects.

The targeted drug of embodiment 7 oil body carriers transmits test (in vitro detection)

Get oleZH2 albumen, add 0.01M pH7.5 sodium phosphate buffer, add again and contain camptothecine (label: SIGMA, concentration: Oleum sesami 500 μ g/ml), carry out ultrasonic mixing (power: 3.0% on ice with ultrasonic cell disruptor, time:20 second, run:0.5 second, rest:0.5 second), with 13000 rpm, 4 ℃ of frozen centrifugation 10 min, the oil cake of one deck white above taking out, add 0.01M pH7.5 sodium phosphate buffer 1 ml, break up with ultrasonic machine, same steps as is carried out three times, constructs the oil body carrier that coats oil-soluble medicine.

The FTIR that coats the oil body carrier of variable concentrations camptothecine observes.The oil body carrier takes out the upper strata oil cake in 4 ℃ of frozen centrifugation 10 min, and the reuse freezer dryer is placed the lyophilization of oil body carrier in the salt sheet with the oil body carrier at last, and observes (Figure 14) with FTIR.

Coat the stable turbidity test of the oil body carrier of variable concentrations camptothecine.The oil body carrier carries out the degree of stability test with the turbidity (T) that sodium phosphate buffer adjusts its suspension at every fixed interval time remaining.In order to measure the oil body carrier, and use 2ml cuvette with spectrophotometer OD600, selected 20 minutes at interval, and with the suspension degree of oil body carrier in sodium phosphate buffer of spectrophotometer record different condition.The turbidity (T) of oil body carrier suspension represents it is proportional with 10A, and the turbidity relation table is shown T/T0=10A/10A0=10A/102.0, and the oil body carrier is diluted to Ao=2, and its degree of stability (Figure 15) is tested in beginning in time.

Coat the size test of the oil body carrier of variable concentrations camptothecine.The oil body carrier dilutes with sodium phosphate buffer, and the best dilution ratio that is fit to the particle size analyzer test is 1 * 10 ⁴To 1 * 10 ⁶Individual oil body is put into the oil body carrier of different condition in the 2ml cuvette, puts in the particle size analyzer, tests (table 4).

Coat the electronegativity test of the oil body carrier of variable concentrations camptothecine.The oil body carrier dilutes with sodium phosphate buffer, and the best dilution ratio that is fit to the test of current potential measurement instrument is 1 * 10 ⁴To 1 * 10 ⁶Individual oil body is put into 2ml cuvette with the oil body carrier of different condition, utilizes Zeta potential to measure instrument test (table 4).

Coat the cell survival property testing of the oil body carrier of variable concentrations camptothecine.Get cell (MCF-7, MCF-7/Her18, SKOV3, SKBR3 and MDA-MB-231), in proxima luce (prox. luc) with every hole 1 * 10 ⁴The density of cell is seeded in 24 orifice plates, cultivate with the DMEM/F12 culture fluid, the next day clean twice with DMEM/F12, and, be added in the cleaned cell with DMEM/F12 culture fluid dilution oil body carrier, at 37 ℃, 5% CO ₂Incubator was cultivated 2 hours, cleaned three times with PBS pH7.4 again, added the DMEM/F12 culture fluid that contains 10% FBS, at 37 ℃, and 5% CO ₂Incubator carry out successive transfer culture with standardization program, in 72 hours living cell countings and apoptosis cell, analyze (Figure 16).

The result shows, after coating the oil-soluble camptothecine of variable concentrations with the oil body carrier, utilizes the functional group result of FTIR test camptothecine to learn, the oil body carrier can coat oil-soluble medicine (the maximum molten oily dissolubility of camptothecine is 500mg/ml).Learn that by the particle diameter test oil body carrier can coat oil-soluble medicine, can not make the size of oil body carrier bigger because of coating medicine.But, after the oil body carrier coats camptothecine, increasing with covering amount, its particle diameter increases, and elecrtonegativity reduces.Through the degree of stability test, the size that the oil body carrier coats behind the camptothecine is less than normal, and is therefore highly stable.

Experimental results show that more than the warp, as can be known the oil body carrier can specificity recognizing cells, excessively show cell (MCF7/Her18 and SKOV3) and do not have HER-2/neu excessively to show cell (MDA-MB-231 and MCF7) when the oil-soluble medicine (camptothecine) that coats variable concentrations with the oil body carrier acts on HER-2/neu, observe different time cell apoptosis degree, the result obtained in the set time, the oil body carrier coats the high more oil-soluble medicine of concentration, and cell apoptosis degree is high more.

The targeting specificity functional test of embodiment 8 oil body carriers (in vivo detection)

Get the nude mice in about 8-10 week, utilize cell strain (SKOV3 and MDA-MB-231) to carry out the subcutaneous tissue injection, make the hypodermic tumor size of nude mice about more than 0.5 centimetre, inject 100 μ g/ml and contain the oil body carrier of fluorescein stain (Yellow GGK) on tumor, 0h after injection, 1h, 4h, 8h and 24h time, utilize IVIS to observe the residual signal (Figure 17) of oil body carrier.Take out tumor, heart, liver, spleen, lungs and kidney again, utilize non-intrusion type live body image system to observe the oil body carrier and remain in signal (Figure 18) in each internal organs.Again tumor and have the tissue of signal to carry out frozen tissue section.

Take off tumor earlier, utilize OCT embedding (bubble is not arranged during embedding), placing more than one hour in-20 ℃ the refrigerator, utilize freezing microtome (label: Leica, model: CM3050S) downcut the tissue of embedding and being attached on the slide, clean twice with PBS pH7.4, reuse 2.5% Formaldehyde fixes 40 minutes in room temperature, clean three times with PBS pH7.4, DAPI with 15000 times of dilutions carries out nucleus dyeing, clean three times with PBS pH7.4, mounting utilizes fluorescence microscope to observe (Figure 18).

The result shows, the big nude mice utilization of about 8-10 week has HER-2/neu excessively to show cell (SKOV3) and does not have HER-2/neu excessively to show cell (MDA-MB-231), carry out the subcutaneous tissue injection, make the hypodermic tumor of nude mice long, each is about more than 0.5 centimetre for wide and high size, and the oil body carrier of injection band fluorescence is on tumor, utilize IVIS to observe, along with the time increases, on the tumor that has HER-2/neu excessively to show, brightness can not reduce, and residual signal is arranged always, and the tumor that does not have HER-2/neu excessively to show, along with the increase of time, oil body carrier fluorescence signal luminous intensity can slowly disappear, and visible oil body carrier can specificity identification tumor.

Take out tumor, heart, liver, spleen, lungs and the kidney of nude mice, each internal organs is carried out IVIS observe, can see that in the tumor cell that has HER-2/neu excessively to show the fluorescence signal is arranged.Carry out frozen tissue section again, utilize the distribution of fluorescence microscope oil body carrier, the tumor cell that has HER-2/neu excessively to show as can be seen has fluorescence oil body carrier residual.

The targeted delivery of drugs test (in vivo detection) of embodiment 9 oil body carriers

Get the nude mice in about 8-10 week, utilize cell strain (SKOV3 and MDA-MB-231) to carry out the subcutaneous tissue injection, make the hypodermic tumor size of nude mice about more than 0.5 centimetre, jede Woche double injection 100mg/ml coats the oil body carrier of camptothecine on tumor, observation tumor size and body weight change (Figure 19, Figure 20).

Can be observed the targeting of oil body carrier, the treatment of cancer effect of further observing the oil body carrier of coating medicine by above zoopery.Show by the result, increase in time, the oil body carrier that coats camptothecine can suppress tumor growth, and tumor is dwindled, and the body weight of nude mice does not reduce, this more can reach the effect of treatment.

Table 1

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Table 2

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Table 3

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Table 4

Claims

1. people's oleoplast carrier of a targeted therapy breast carcinoma, it is characterized in that, described people's oleoplast carrier comprises a fusion rotein and a lipid, and fusion rotein and lipid weight/volume ratio (microgram/microlitre) are at least about 1/25, and this oil body mean diameter is 50 nanometer to 2000 nanometers.

2. people's oleoplast carrier according to claim 1 is characterized in that, the contained fusion rotein of this people's oleoplast carrier, and it comprises an oil body protein, a part peptide or an antibody peptide, a cell-penetrating peptide or aforementioned combination.

3. people's oleoplast carrier according to claim 1 and 2 is characterized in that, the contained fusion rotein of this people's oleoplast carrier, and its contained oil body protein is not limited to them; Oil body protein from plant seed is preferable, and for example its combination of Oleum sesami, olive oil, soybean oil, Oleum Arachidis hypogaeae semen and mineral oil is better from the oil body protein of Semen Sesami seed.

4. people's oleoplast carrier according to claim 1 and 2 is characterized in that, the contained fusion rotein of this people's oleoplast carrier, its contained part win peptide or antibody victory peptide is not limited to them, as long as have the function of specificity recognizing cells; With regard to the part peptide, when treatment breast carcinoma or ovarian cancer, can adopt the part peptide of HER2/neu protein acceptor.

5. the preparation method of people's oleoplast carrier of a targeted therapy breast carcinoma as claimed in claim 1, its feature may further comprise the steps:

(1) Expression of Fusion Protein; Reorganization strain BL21 (DE3)/pJO1-oleZH2 cultivates antibacterial initial density OD down at 37 ℃ ₅₅₀Value is 0.08; Be inoculated into the fresh LB culture fluid that contains ampicillin, as bacterial density OD ₅₅₀Value is 0.3 o'clock, induces 4 hours for 37 ℃, collects thalline, with 4000 rpm, 4 ℃ of frozen centrifugation 10 min, reuse 0.1 M pH7.5 sodium phosphate buffer cleans once, collects thalline, is condensed into bacterial density OD with 0.1M pH7.5 sodium phosphate buffer ₅₅₀Value is 10 bacterium liquid;

With bacterial density OD ₅₅₀To be 10 bacterium liquid carry out ultrasonication on ice (power: 3.5%, time: 2 minutes, run:0.5 second, rest:0.5 second) with ultrasonic cell disruptor to value; After the ultrasonic end,, collect centrifugal back supernatant and precipitate not aqueous soluble protein with 13000 rpm, 4 ℃ of frozen centrifugation 10 min;

(2) preparation of people's oleoplast carrier; Get 100 μ g oleZH2 albumen, add 950 μ l 0.01M pH7.5 sodium phosphate buffer, add 50 μ l Oleum sesami again, carry out ultrasonic mixing (power: 3.0% on ice with ultrasonic cell disruptor, time:20 second, run:0.5 second, rest:0.5 second), with 13000 rpm, 4 ℃ of frozen centrifugation 10 min, the oil cake of one deck white above taking out adds 0.01M pH7.5 sodium phosphate buffer 1 ml, breaks up with ultrasonic cell disruptor, same steps as is carried out three times, constructs people's oleoplast carrier.

6. method according to claim 5 is characterized in that the described reorganization strain of step (1) is BL21 (DE3)/pJO1-oleZH2, cultivates down at 37 ℃, and initial concentration is OD ₅₅₀=0.08.

7. method according to claim 5 is characterized in that the described reorganization bacterial classification inoculation of step (1) is to the fresh LB culture fluid that contains ampicillin, when cell concentration reaches OD ₅₅₀=0.3 o'clock, induced 4 hours for 37 ℃, collect thalline, with 4000 rpm, 4 ℃ of frozen centrifugation 10 min, reuse 0.1 M pH7.5 sodium phosphate buffer cleans once, collects thalline, is condensed into cell concentration OD with 0.1M pH7.5 sodium phosphate buffer ₅₅₀=10 bacterium liquid.

8. method according to claim 5 is characterized in that the described cell concentration OD of step (1) ₅₅₀=10 bacterium liquid carries out ultrasonication on ice with ultrasonic cell disruptor; The ultrasonic power 3.5% that is set to, 2 minutes time, run 0.5 second, rest 0.5 second.

9. method according to claim 5, it is characterized in that the described 100 μ g oleZH2 albumen of getting of step (2), add 950 μ l 0.01M pH7.5 sodium phosphate buffer, add 50 μ l Oleum sesami again, carry out ultrasonic mixing on ice with ultrasonic cell disruptor.

10. method according to claim 5 is characterized in that the described ultrasonic power 3.0% that is set to of step (2), time 20 seconds, run 0.5 second, rest 0.5 second.

11. method according to claim 5, after it is characterized in that the described ultrasonic mixing of step (2) on ice, with 13000 rpm, 4 ℃ of frozen centrifugation 10 min, the oil cake of one deck white above taking out, add 0.01M pH7.5 sodium phosphate buffer 1 ml, break up with ultrasonic cell disruptor, same steps as is carried out three times, and construction goes out people's oleoplast carrier.

12. people's oleoplast carrier according to claim 5 acquisition, it is characterized in that, the optimization of (different proportion, different pH and oil not of the same race) under different preparation conditions of described people's oleoplast carrier is tested, can obtain people's oleoplast carrier optimum condition by fluorescence microscope, size analysis, electronegativity test and degree of stability test following points is arranged: the optimal proportion of (1) oil and fusion rotein is 1:1, this moment, the big low profile of people's oleoplast carrier was average, and the mutual fusion between people's oleoplast carrier is less;

(2) the big low profile of people's oleoplast carrier of the buffer structure of usefulness pH7.5 is average, and the mutual fusion between people's oleoplast carrier is less;

(3) the most stable with people's oleoplast carrier of Oleum sesami preparation;

(4) size of people's oleoplast carrier is big more unstable more, and is more little stable more;

(5) utilized the film experiment can obtain less people's oleoplast carrier;

(6) negative charge of people's oleoplast carrier of different condition structure is-45～-56, and difference is little.

13. people's oleoplast carrier according to claim 5 acquisition, after it is characterized in that described people's oleoplast carrier coats fluorescent material (as yellow GGK and Nile red), also can make people's oleoplast carrier send fluorescence, and be more conducive to observe, can be used for reflecting the HER2/neu positive cancer cell, thereby but specificity detects the breast cancer cell of overexpression HER2/neu.

14. people's oleoplast carrier according to claim 5 acquisition, it is characterized in that described people's oleoplast carrier in vitro (in vitro) and in vivo (in vivo) experiment in all have targeting, can discern target cell by specificity, can effectively kill breast cancer cell behind the coating medicine, can be used for the targeted therapy of breast cancer cell.

15. people's oleoplast carrier according to claim 5 acquisition, it is characterized in that described people's oleoplast carrier carries out cell experiment (fixed cell and living cells), but the identification of people's oleoplast carrier specificity excessively shows the tumor cell line (SKBR3 and SKOV3) of HER-2/neu as can be seen, can not discern the tumor cell (MCF7 and MDA-MB-231) that does not show HER-2/neu; The time that people's oleoplast carrier adds is long more or concentration is high more, the cell that people's oleoplast carrier is attached to the excessive performance of HER-2/neu is many more, the ability that specificity is discerned this cell is good more, but people's oleoplast carrier function time reaches two hours or concentration reaches at 200 o'clock and has and presents saturated situation; People's oleoplast carrier of green-emitting fluorescence enters cell to utilize the conjugation fluorescence microscope to stack up as can be seen in different aspects; Further specify as if concentration fixed with flow cytometer, the time is long more, and the amount that people's oleoplast carrier enters cell is many more, if the time is fixed, concentration is many more, and the amount that people's oleoplast carrier enters cell is many more.

16. people's oleoplast carrier according to claim 5 acquisition, it is characterized in that described people's oleoplast carrier coats oil-soluble medicine (camptothecine), when it acts on the cell of the excessive performance of HER-2/neu, can produce toxic effect, make cell that the apoptosis phenomenon be arranged; When acting on the cell that does not have the excessive performance of HER-2/neu, see less that then toxic effect is arranged.

17. people's oleoplast carrier according to claim 5 acquisition, it is characterized in that described people's oleoplast vector injection is to the nude mice tumor cell, utilizing IVIS to observe people's oleoplast carrier has signal to produce on the tumor tissues that has HER-2/neu excessively to show, and does not then have signal at the tumor tissues that is not having HER-2/neu excessively to show; Learn the excessively tumor of performance HER-2/neu of people's oleoplast carrier meeting specificity identification via tissue slice again, can not discern the tumor that does not show HER-2/neu.

18. people's oleoplast carrier according to claim 5 acquisition, it is characterized in that described people's oleoplast carrier coats oil-soluble medicine (camptothecine), in vivo in the anti-tumor activity test, get the nude mice in about 8-10 week, carry out the subcutaneous tissue injection with cell (SKOV3 and MDA-MB-231), make the hypodermic tumor size of nude mice about more than 0.5 centimetre, jede Woche double injection 100mg/ml coats people's oleoplast carrier of camptothecine on tumor, the result shows, increase in time, the people's oleoplast carrier that coats camptothecine can suppress tumor growth, and the nude mice body weight does not reduce; This illustrates that this research successfully makes up a kind of people's oleoplast carrier of energy targeted therapy breast carcinoma.