CN102429868A - Liposome medicinal composition with tumor targeting, in-vivo tracing and treating functions and preparation method thereof - Google Patents
Liposome medicinal composition with tumor targeting, in-vivo tracing and treating functions and preparation method thereof Download PDFInfo
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- CN102429868A CN102429868A CN2011104113359A CN201110411335A CN102429868A CN 102429868 A CN102429868 A CN 102429868A CN 2011104113359 A CN2011104113359 A CN 2011104113359A CN 201110411335 A CN201110411335 A CN 201110411335A CN 102429868 A CN102429868 A CN 102429868A
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Abstract
The invention discloses a liposome medicinal composition with tumor targeting, in-vivo tracing and treating functions and a preparation method thereof. The medicinal composition is a liposome targeting medicament which is resistant to CD44 antibody coupling, has a molecular imaging function simultaneously, and can be used for monitoring the in-vivo distribution of a medicament in real time in a living body state. In particular, plasmids containing three fusion genes, including renilla luciferase, red fluorescent proteins and suicide gene thymidine kinase, are coupled to CD44 antibody mediation-resistant immunoliposome, the specificity of liposome nanoparticles in a liver cancer in-situ model of an in-vivo targeting NOD/SCID (Non-Obese Diabetic/Severe Combined Immune-Deficiency) mouse is monitored by detecting a renilla luciferase signal with a living body imaging system, and apoptosis of liver cancer cells is induced by applying target thymidine kinase of ganciclovir; and moreover, a targeted liposome can be coated with adriamycin for inducing apoptosis of the liver cancer cells. The liposome medicinal composition provided by the invention does not have any toxic or side effect, has small damage and a good effect, and is suitable to be applied for a long time.
Description
Technical field
The invention belongs to biological pharmacy technical field, be specifically related to a kind of new liposome drug combination with cancer target, the interior spike of body and therapeutical effect.Coupling contains the plasmid of renilla luciferase, red fluorescent protein and suicide gene thymidine kinase three fusion genes on the antibody-mediated immunoliposome of anti-CD44 specifically; Renilla luciferase and red fluorescent protein have the spike effect, judge target liposomes distribution in vivo; The suicide gene thymidine kinase has therapeutical effect, uses the target thymidine kinase of ganciclovir, induces hepatocarcinoma tumor stem cell apoptosis.Simultaneously, the antibody-mediated immunoliposome of anti-CD44 parcel amycin can the male hepatocarcinoma tumor stem cell of targeting CD44, cell death inducing.
Background technology
At present, tumor has become the important diseases that threatens human health, and present Therapeutic Method is main with operation, radiotherapy, chemotherapy mainly, and postoperative recurrence, drug resistance, transfer, complication are the main causes of treatment failure and patient death.Most chemotherapeutics does not have selectivity, in killing tumor cell, can produce lethal effect to normal cell yet, therefore can produce serious adverse.Utilize the liposome chemotherapeutics, like Evacet, daunorubicin liposome and Paclitaxel liposome, though can reduce toxicity to a certain extent, the non-specific absorption that can't overcome medicine with can't be in the accumulative problem of tumor locus.Targeted therapy has more the effect of " effecting a permanent cure " with respect to other traditional treatment means, has the high and selective killing characteristics of tumor cells of efficiency, and side effect is less, reduces normal tissue injury, treats tumor more targetedly.The key of the molecular targeted treatment of tumor will find the target spot (target) of tumor exactly, and what the molecular targeted treatment of just a kind of tumor was attacked is some target spots or a plurality of target spot of tumor cell, as long as tumor contains this target spot, just is fit to this targeted therapy.Molecular imaging then is the new technique that a kind of further microcosmic is estimated curative effect in the body simultaneously; It can detect the change of precancerous lesion molecule abnormality, growth kinetics of cells, angiogenesis factor, tumor cell label, gene; This imaging means can be used for the instrument that carries out the curative effect assessment and the developing new drug thing is provided if combine with novel targeted technology such as molecular targeted treatment.
Summary of the invention
It is big to the objective of the invention is to solve chemical therapy toxic side effect, can't monitor the problem of its distribution in vivo, provide have cancer target, the liposome drug combination and the method for preparing of spike and therapeutical effect in the body.This safety of medicine, effectively, can the target tumor position and can its distribution and gathering in vivo of spike.
Concrete composition with liposome drug combination of cancer target, the interior spike of body and therapeutical effect provided by the invention comprises:
Liposome drug combination is made up of the liposome core and the anti-CD44 antibody of target molecules of phospholipid double sublayer; Carry the reporter gene expression plasmid that contains renilla luciferase, red fluorescent protein and suicide gene thymidine kinase three fusion genes simultaneously, perhaps by the liposome core parcel amycin that contains anti-CD44 antibody.
Provided by the inventionly have cancer target, carry in target liposomes preparation of drug combination method and the body of spike effect and therapeutical effect reporter gene tracing method and realize by following steps:
1. carry target liposomes preparation of drug combination method, realize through following steps with spike effect and therapeutical effect reporter gene:
(1) 1,2-oleoyl phosphatidylcholine (DOPC), 1,2-oleoyl PHOSPHATIDYL ETHANOLAMINE (DOPE), cholesterol; 1; 2-oleoyl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000 (DOPE-PEG2000), 1,2-oleoyl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleimide (DOPE-PEG2000-Mal); According to original mol ratio is 40: 40: 40: 6: 3 mixed is dissolved in the methanol;
(2) utilize Rotary Evaporators to make the solution evaporation of step (1), form liposome membrane;
(3) liposome membrane that forms of step (2) is with (NH4) of every liter of phosphate buffer (PBS) or 300 mM
2HPO
4Aquation one hour makes that TL concentration is every liter of 3.32 mM;
(4) the ultrasonic solution that step (3) is obtained becomes the lipid ball of small homogeneous, crosses the poly carbonic ester film of 100 nanometers, makes it become the non-targeting homogeneous liposome of particle diameter about 100 nanometers;
(5) in the non-target liposomes solution that step (4) obtains, be to add anti-CD44 antibody at 1: 50 by the mol ratio of CD44 antibody and DOPE-PEG2000-Mal, utilize the sulfydryl of anti-CD44 antibody and the PEG chain combination on the liposome, obtain target liposomes;
(6) make up the reporter gene expression plasmid that contains renilla luciferase, red fluorescent protein and suicide gene thymidine kinase three fusion genes simultaneously;
(7) the reporter gene expression plasmid of resulting target liposomes of above-mentioned steps (5) and step (6) structure is by the mixed of the per 20 microgram plasmids of 100 microlitre liposomees; Utilize the positive charge of surface of liposome to combine, form target liposomes-DNA complex with the negative charge that plasmid carries.
2. the target liposomes preparation of drug combination of carrying amycin realizes through following steps:
(1) 1,2-oleoyl phosphatidylcholine (DOPC), 1,2-oleoyl PHOSPHATIDYL ETHANOLAMINE (DOPE), cholesterol; 1; 2-oleoyl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000 (DOPE-PEG2000), 1,2-oleoyl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleimide (DOPE-PEG2000-Mal); According to original mol ratio is 40: 40: 40: 6: 3 mixed is dissolved in the methanol;
(2) utilize Rotary Evaporators to make the solution evaporation of step (1), form liposome membrane;
(3) liposome membrane that forms in the step (2) is with (NH4) of every liter of 300 mM
2HPO
4Aquation one hour makes that TL concentration is every liter of 3.32 mM;
(4) the ultrasonic solution that step (3) is obtained becomes the lipid ball of small homogeneous, crosses the poly carbonic ester film of 100 nanometers, makes it become the non-target liposomes of particle diameter about 100 nanometers;
(5) ratio according to 1 milligram of amycin of every milliliter of liposome (10mg/ml) adds amycin in the liposome that step (4) obtains; Mix homogeneously, 55 ℃ were reacted 1 hour, and room temperature is placed and is spent the night; Utilize the inside and outside phosphate gradient of liposome, make amycin get into liposome;
(6) cross chromatographic column PD-10 purification, use water elution, remove the amycin that is not wrapped;
(7) be that 1: 50 ratio adds anti-CD44 antibody in the liposome that step (6) obtains in the mol ratio of antibody and DOPE-PEG2000-Mal, utilize the PEG chain combination on sulfydryl and the liposome, obtain the adriablastina target liposome.
3. vivo medicine-feeding
(1) with the HepG2 cell strain of well-grown labelling LUC Photinus pyralis LUC Photinus pyralis FL of trypsinization and green fluorescent protein, PBS washes one time, and is resuspended with the RMPI1640 minimal medium, makes that cell density is every milliliter of culture medium 1 * 10
7Individual cell;
(2) cut the left upper abdomen of the NOD/SCID combined immunodeficiency mouse of water and chloralization, expose the lobe of the liver part, the above-mentioned cell suspension of 10 microlitres is expelled in the mouse lobe of the liver, stitching, after the general week, growing in the liver original position can detected tumor;
Non-target liposomes and the target liposomes that (3) will carry reporter gene have in the NOD/SCID combined immunodeficiency mouse body of tumor to long through tail vein injection respectively.
The target liposomes that (4) will carry amycin has in the NOD/SCID combined immunodeficiency mouse body of tumor to long through tail vein injection respectively.
4. chemical luminescence imaging in the body
(1) injected the plain h (500 micrograms/every kilogram of mouse) of the mouse intravenous injection renilla luciferase substrate of non-target liposomes and target liposomes-enteric cavity, utilized Xenogen200 living imaging system to detect chemiluminescence signal, time of exposure is 5 minutes.
(2) injected the mouse of carrying amycin or reporter gene liposome and target liposomes, given lumbar injection D-fluorescein sodium salt (150 mg/kg body weight), utilized Xenogen200 living imaging system to detect chemiluminescence signal, time of exposure is 1 minute.
5. external immunofluorescence imaging
The mouse of (1) putting to death is got tumor tissues, and paraformaldehyde is fixed, and 5 microns frozen section is cut in the OCT embedding.
(2) PBS gives a baby a bath on the third day after its birth time, each 5 minutes;
(3) with 5% BSA sealing 30 minutes;
(4) discard redundant solution, drip anti-RFP antibody (1: 100), room temperature left standstill 1 hour;
(5) the TBST buffer is given a baby a bath on the third day after its birth time, each 5 minutes;
(6) drip fluorescence two anti-(1: 1000), the room temperature lucifuge leaves standstill half an hour;
(7) the TBST buffer is given a baby a bath on the third day after its birth time, each 5 minutes;
(8) DAPI redyes nucleus, and the room temperature lucifuge left standstill 5 minutes;
(9) the TBST buffer is washed once;
(10) mounting, the fluorescence microscope microscopy is observed.
Advantage of the present invention and good effect:
It is big that this liposome drug combination has solved existing liposome medicament toxic and side effects; Non-specific absorption and be difficult to directed accumulative problem; It can be directionally in the effect that accumulates in tumor locus performance killing tumor cells, and can utilize tracer technique to monitor liposome medicament distribution in vivo in real time.This medicine has effective targeted therapy effect, has no side effect, damages little, suitable prolonged application.
Description of drawings
Fig. 1 carries the plasmid map of reporter gene;
The grain size analysis scattergram of Fig. 2 target liposomes;
The transmission electron micrograph of Fig. 3 target liposomes;
Fig. 4 renilla luciferase chemiluminescence spike target liposomes is in the intravital distribution of mouse;
The variation of the mouse tumor of Fig. 5 LUC Photinus pyralis LUC Photinus pyralis FL chemiluminescence tracer targets behind liposome therapeutic;
Fig. 6 red fluorescent protein immunofluorescence spike target liposomes is in the intravital distribution of mouse;
The non-target liposomes of Fig. 7 renilla luciferase chemiluminescence spike is in the intravital distribution of mouse;
The variation of the mouse tumor after the non-target liposomes treatment of Fig. 8 LUC Photinus pyralis LUC Photinus pyralis FL chemiluminescence spike;
The present invention combines the accompanying drawing and the specific embodiment to explain further details.
The specific embodiment
Embodiment 1
Carry the target liposomes preparation of drug combination of reporter gene
(1) DOPC, DOPE, cholesterol, DOPE-PEG2000, DOPE-PEG2000-Mal is 40: 40: 40 according to original mol ratio: 6: 3 mixed is dissolved in the methanol;
(2) utilize Rotary Evaporators to make the solution evaporation of step (1), form liposome membrane;
(3) liposome membrane that forms of step (2) makes that with phosphate buffer (PBS) aquation one hour TL concentration is every liter of 3.32 mM;
(4) the ultrasonic solution that step (3) is obtained becomes the lipid bubble of small homogeneous, crosses the poly carbonic ester film of 100 nanometers, makes it become the homogeneous liposome of particle diameter about 100 nanometers;
(5) mol ratio by anti-CD44 antibody and DOPE-PEG2000-Mal is 1: 50 adding CD44 antibody, utilizes the PEG chain combination on sulfydryl and the liposome, obtains target liposomes;
(6) contain the structure of reporter gene plasmid, on commercialization plasmid vector pCDNA3.1, insert and contain renilla luciferase, red fluorescent protein and suicide gene thymidine kinase three fusion genes, reporter gene expression plasmid pCDNA3.1-RL-RFP-tTK; (as shown in Figure 1)
(7) coupling of reporter gene and liposome; With liposome and plasmid pCDNA3.1-RL-RFP-tTK mixed by per 100 microlitre liposomees, 20 microgram plasmids; Utilize the set of the positive charge of surface of liposome and negative charge that plasmid carries, form target liposomes-DNA complex.
The liposome suspension is used transmission electron microscope observation, and the result is as shown in Figure 2, uniform particle diameter, and liposome is the phospholipid bilayer of interior sky.
Embodiment 2
Carry the non-target liposomes preparation of drug combination of reporter gene
(1) DOPC, DOPE, cholesterol, DOPE-PEG2000, DOPE-PEG2000-Mal is 40: 40: 40 according to original mol ratio: 6: 3 mixed is dissolved in the methanol;
(2) utilize Rotary Evaporators to make its solution evaporation, form liposome membrane;
(3) liposome membrane that forms makes that with phosphate buffer (PBS) aquation one hour TL concentration is every liter of 3.32 mM;
(4) ultrasonic its lipid that becomes small homogeneous that makes steeps, and crosses the poly carbonic ester film of 100 nanometers, makes it become the non-target liposomes of the homogeneous of particle diameter about 100 nanometers;
(5) coupling of reporter gene and liposome; The plasmid pCDNA3.1-RL-RFP-tTK that liposome and embodiment 1 made up is by the mixed of per 100 microlitre liposomees, 20 microgram plasmids; Utilize the set of the positive charge of surface of liposome and negative charge that plasmid carries, form non-target liposomes-DNA complex.
Embodiment 3
The target liposomes curative effect is identified and the interior spike of body
Set up the former bit model of hepatocarcinoma of NOD/SCID combined immunodeficiency mouse.With the HepG2 cell strain of well-grown labelling LUC Photinus pyralis LUC Photinus pyralis FL of trypsinization and green fluorescent protein, PBS washes one time, and is resuspended with the RMPI1640 minimal medium, makes that cell density is every milliliter of culture medium 1 * 10
7Individual cell.Water and chloralization, the left upper abdomen of incision NOD/SCID combined immunodeficiency mouse exposes the lobe of the liver part, and the above-mentioned cell suspension of 10 microlitres is expelled in the mouse lobe of the liver, sews up, and after the general week, growing in the liver original position can detected tumor.With 100 microlitres target liposomes-DNA complex of preparing among the embodiment 1, through tail vein injection in lotus tumor mouse body, subsequently every day the lumbar injection ganciclovir, ID is 50 mg/kg body weight, once a day.
Chemical luminescence imaging in the body
(1) renilla luciferase imaging monitoring liposome location and gathering
The plain h (500 micrograms/every kg body weight) of the rat tail intravenous injection renilla luciferase substrate of injecting lipid body-enteric cavity utilizes Xenogen200 living imaging system to detect chemiluminescence signal, and time of exposure is 5 minutes.As shown in Figure 3, luminous signal concentrates on the epigastrium of mouse, is the liver position, explains that the antibody-mediated target liposomes enrichment of anti-CD44 is to liver TIS place.Come the spike target liposomes in the intravital distribution of mouse with this.
(2) LUC Photinus pyralis LUC Photinus pyralis FL imaging monitoring tumor growth situation
Form the NOD/SCID combined immunodeficiency mouse of tumor, give lumbar injection D-fluorescein sodium salt (150 mg/kg body weight), utilize Xenogen200 living imaging system to detect chemiluminescence signal, time of exposure is 1 minute.Second day from the injection tumor begins to monitor the fluorescein signal, and monitoring once monitored for 5 weeks weekly.As shown in Figure 4, after giving the ganciclovir processing, to compare with the matched group of not administration, the tumor growth of experimental group mouse obviously is suppressed.
External immunofluorescence imaging
Method with immunofluorescence detects red fluorescent protein in the intravital location of mouse, and concrete steps are following:
The mouse of (1) putting to death is got tumor tissues, and paraformaldehyde is fixed, and 5 microns frozen section is cut in the OCT embedding.
(2) PBS gives a baby a bath on the third day after its birth time, each 5 minutes;
(3) with 5% BSA sealing 30 minutes;
(4) discard redundant solution, drip anti-RFP antibody (1: 100), room temperature left standstill 1 hour;
(5) the TBST buffer is given a baby a bath on the third day after its birth time, each 5 minutes;
(6) drip fluorescence two anti-(1: 1000), the room temperature lucifuge leaves standstill half an hour;
(7) the TBST buffer is given a baby a bath on the third day after its birth time, each 5 minutes;
(8) DAPI redyes nucleus, and the room temperature lucifuge left standstill 5 minutes;
(9) the TBST buffer is washed once;
(10) mounting, the fluorescence microscope microscopy is observed.
The microscopy result is as shown in Figure 5; Can detect the expression of red fluorescent protein (RFP) at the tumor locus of expressing anti-CD44; Explain that the target liposomes that carries RFP is to concentrate on tumor locus, with the antibody-mediated target liposomes of the anti-CD44 of this spike in the intravital distribution of mouse.
Embodiment 4
Spike in the non-target liposomes body
Set up the former bit model of hepatocarcinoma of NOD/SCID combined immunodeficiency mouse.With the HepG2 cell strain of well-grown labelling LUC Photinus pyralis LUC Photinus pyralis FL of trypsinization and green fluorescent protein, PBS washes one time, and is resuspended with the RMPI1640 minimal medium, makes that cell density is every milliliter of culture medium 1 * 10
7Individual cell.Water and chloralization, the left upper abdomen of incision NOD/SCID combined immunodeficiency mouse exposes the lobe of the liver part, and the above-mentioned cell suspension of 10 microlitres is expelled in the mouse lobe of the liver, sews up, and after the general week, growing in the liver original position can detected tumor.With the non-target liposomes of 100 microlitres-DNA complex of preparing among the embodiment 2, through tail vein injection in lotus tumor mouse body, subsequently every day the lumbar injection ganciclovir, ID is 50 mg/kg body weight, once a day.
Chemical luminescence imaging in the body
(1) renilla luciferase imaging monitoring liposome location and gathering
The plain h (500 micrograms/every kg body weight) of rat tail intravenous injection renilla luciferase substrate behind the liposome therapeutic-enteric cavity utilizes Xenogen200 living imaging system to detect chemiluminescence signal, and time of exposure is 5 minutes.As shown in Figure 6, luminous signal is dispersed in the hypogastric region of mouse, and the non-target liposomes that carries reporter gene does not have orienting enriching at tumor locus, explains that the antibody-mediated non-target liposomes of not anti-CD44 can not enrichment arrive liver TIS place.Come the spike target liposomes in the intravital distribution of mouse with this.
(2) LUC Photinus pyralis LUC Photinus pyralis FL imaging monitoring tumor growth situation
Form the NOD/SCID combined immunodeficiency mouse of tumor, give lumbar injection D-fluorescein sodium salt (150 mg/kg body weight), utilize Xenogen200 living imaging system to detect chemiluminescence signal, time of exposure is 1 minute.Second day from the injection tumor begins to monitor the fluorescein signal, and monitoring once monitored for 5 weeks weekly.As shown in Figure 7, after giving the ganciclovir processing, the tumor growth of comparing mouse with matched group is not suppressed.
Embodiment 5
Carry the preparation and the treatment of the target liposomes of amycin
(1) 1,2-oleoyl phosphatidylcholine (DOPC), 1,2-oleoyl PHOSPHATIDYL ETHANOLAMINE (DOPE), cholesterol; 1; 2-oleoyl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000 (DOPE-PEG2000), 1,2-oleoyl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleimide (DOPE-PEG2000-Mal); According to original mol ratio is 40: 40: 40: 6: 3 mixed is dissolved in the methanol;
(2) utilize Rotary Evaporators to make its solution evaporation, form liposome membrane;
(3) liposome membrane that forms is with (NH4) of every liter of 300 mM
2HPO
4Aquation one hour makes that TL concentration is every liter of 3.32 mM;
(4) ultrasonic its lipid ball that becomes small homogeneous that makes, the poly carbonic ester film of 100 nanometers makes it become the non-target liposomes of particle diameter about 100 nanometers excessively;
(5) ratio according to 1 milligram of amycin of every milliliter of liposome (10mg/ml) adds amycin, mix homogeneously, and 55 ℃ were reacted 1 hour, and room temperature is placed and is spent the night, and utilizes the inside and outside phosphate gradient of liposome, makes amycin get into liposome;
(6) cross chromatographic column PD-10 purification, use water elution, remove the amycin that is not wrapped;
(7) mol ratio by antibody and DOPE-PEG2000-Mal is 1: 50 adding CD44 antibody, utilizes the PEG chain combination on sulfydryl and the liposome, obtains the adriablastina target liposome;
(8) LUC Photinus pyralis LUC Photinus pyralis FL imaging monitoring tumor growth situation
Form the NOD/SCID combined immunodeficiency mouse of tumor, give lumbar injection D-fluorescein sodium salt (150 mg/kg body weight), utilize Xenogen200 living imaging system to detect chemiluminescence signal, time of exposure is 1 minute.Second day from the injection tumor begins to monitor the fluorescein signal, and monitoring once monitored for 5 weeks weekly.As shown in Figure 8, carry after the target liposomes treatment of amycin, the tumor growth of comparing the processed group mouse with the matched group of not administration obviously is suppressed.
Claims (3)
1. liposome drug combination with spike and therapeutical effect in the cancer target, body; It is characterized in that this liposome drug combination is made up of the liposome core and the anti-CD44 antibody of target molecules of phospholipid double sublayer; And can carry the reporter gene of spike effect and therapeutical effect, also can wrap up amycin.
2. target liposomes preparation of drug combination method that carries spike effect and therapeutical effect reporter gene is characterized in that the concrete steps of this method are:
(1) 1; 2-oleoyl phosphatidylcholine (DOPC), 1; 2-oleoyl PHOSPHATIDYL ETHANOLAMINE (DOPE), cholesterol, 1,2-oleoyl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000 (DOPE-PEG2000) and 1,2-oleoyl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleimide (DOPE-PEG2000-Mal); By original mol ratio is 40: 40: 40: 6: 3 mixed is dissolved in the methanol;
(2) utilize Rotary Evaporators to make the solution evaporation of step (1), form liposome membrane;
(3) liposome membrane that forms of step (2) makes that with phosphate buffer (PBS) aquation one hour TL concentration is every liter of 3.32 mM;
(4) the ultrasonic solution that step (3) is obtained becomes the lipid ball of small homogeneous, crosses the poly carbonic ester film of 100 nanometers, becomes the homogeneous liposome of particle diameter in 100 nanometers;
(5) be will resist CD44 antibody to add in the liposome that step (4) obtains at 1: 50 by the mol ratio of antibody and DOPE-PEG2000-Mal, utilize the PEG chain combination on sulfydryl and the liposome, obtain target liposomes;
(6) make up the reporter gene expression plasmid that contains renilla luciferase, red fluorescent protein and suicide gene thymidine kinase three fusion genes simultaneously;
(7) coupling of reporter gene and liposome; The reporter gene expression plasmid that liposome and step (6) are made up is by the mixed of per 100 microlitre liposomees, 20 microgram plasmids; Utilize the set of the positive charge of surface of liposome and negative charge that plasmid carries; Form target liposomes-DNA complex, be liposome drug combination.
3. target liposomes preparation of drug combination method that carries amycin is characterized in that the concrete steps of this method are:
(1) 1; 2-oleoyl phosphatidylcholine (DOPC), 1; 2-oleoyl PHOSPHATIDYL ETHANOLAMINE (DOPE), cholesterol, 1,2-oleoyl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000 (DOPE-PEG2000) and 1,2-oleoyl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleimide (DOPE-PEG2000-Mal); By original mol ratio is 40: 40: 40: 6: 3 mixed is dissolved in the methanol;
(2) utilize Rotary Evaporators to make the solution evaporation of step (1), form liposome membrane;
(3) liposome membrane that forms in the step (2) is with (NH4) of every liter of 300 mM
2HPO
4Aquation one hour makes that TL concentration is every liter of 3.32 mM;
(4) the ultrasonic solution that step (3) is obtained becomes the lipid ball of small homogeneous, crosses the poly carbonic ester film of 100 nanometers, makes it become the non-target liposomes of particle diameter in 100 nanometers;
(5) ratio according to 1 milligram of concentration of every milliliter of liposome amycin that is 10mg/ml adds amycin in the liposome that step (4) obtains; Mix homogeneously, 55 ℃ were reacted 1 hour, and room temperature is placed and is spent the night; Utilize the inside and outside phosphate gradient of liposome, make amycin get into liposome;
(6) cross chromatographic column PD-10 purification, use water elution, remove the amycin that is not wrapped;
(7) be to add anti-CD44 antibody in 1: 50 the liposome of ratio behind step (6) purification in the mol ratio of antibody and DOPE-PEG2000-Mal, utilize the PEG chain combination on sulfydryl and the liposome, obtain the adriablastina target liposome.
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| CN107441505A (en) * | 2017-09-11 | 2017-12-08 | 东南大学 | A kind of target magnetic nano liposome and preparation method thereof |
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