CN101129335B - Use of nano structured lipid carrier drug feeding system - Google Patents
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- CN101129335B CN101129335B CN2007100700437A CN200710070043A CN101129335B CN 101129335 B CN101129335 B CN 101129335B CN 2007100700437 A CN2007100700437 A CN 2007100700437A CN 200710070043 A CN200710070043 A CN 200710070043A CN 101129335 B CN101129335 B CN 101129335B
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Abstract
The invention discloses an application of nanometer structure lipid carrier administration system in the antineoplastic drug to reverse the multiple-drug tolerance of tumour cell, which comprises the following parts: solid lipid material, liquid lipid material and antineoplastic drug, wherein the rate of the liquid lipid (such as oleic acid) is 0-30wt%; the solid lipid is selected from monoglyceride; the liquid lipid is oleic acid; the antineoplastic drug is Paclitaxel or adriablastina. The invention has highly effective cell uptaking and cytolymph condensing function with packing molecular target in the antineoplastic drug of cell, which avoids P-glucoprotein in the drug tolerant cytolymph from identifying the antineoplastic drug to reduce exclusion.
Description
Technical field
The present invention relates to the purposes of nano structured lipid carrier drug feeding system, relate generally to the application of nano structured lipid carrier drug feeding system in antineoplaston and reverse multiple drug resistance of tumor.
Background technology
Tumor is the major disease that directly threatens human health always, and chemotherapy of tumors is because the molecular targeted property of medicine shortage itself, thus occur that cure rate is low, great treatment problem such as multidrug resistance, toxic and side effects are huge.One of major reason of clinical chemotherapy failure is that tumor cell produces drug resistance to chemotherapeutics.Most tumors patient's the cause of the death is directly or indirectly relevant to the drug resistance of antitumor drug with tumor cell.Therefore, seeking and drug delivery system that research can the reversing drug resistance tumor cell drug resistance, is one of the Critical policies of chemotherapy of tumors research and development and direction.
Discover have multiple reason to cause the generation of multi-drug resistance of the tumor, and with P-glycoprotein (P-gp), glutathione s-transferase (GST-Л), and the abnormal expression of topoisomerase (TOPO-II) is relevant.P-glycoprotein (P-gp) mainly is distributed in cell membrane, is the medicine rear pump of dependency ATP enzyme, also is a kind of Ca
2+With Cl
-Passage.P-gp can combine with entering cytoplasmic antitumor drug, and the energy that hydrolysis discharges by ATP directly or indirectly pumps cell with medicine; Or borrow the channeling of P-gp itself, and medicine is pumped the extracellular in cell, cause that drug level reduces in the tumor cell, cytotoxicity weakens or lose, and then make tumor generation drug resistance.
Studies show that polymer support is the multidrug resistance of tumor remission to a certain extent.Nonionic surfactant gathers oxireme-polyphenylene oxide-poly-oxireme trimer (PEO-PPO-PEO), behind the parcel antitumor drug, when near polymer concentration critical micelle concentration or when following, can bring into play best antitumous effect, embody and reverse the multidrug resistance sexual orientation.Polymer support can effectively reduce the ATP amount of mdr cell, but can not cause the variation of the ATP amount of sensitive cells.The minimizing of ATP amount has directly reduced the tumour medicine amount that is pumped by mdr cell in the mdr cell, shows as the reverse to drug resistance of tumor.And for example, poly-l-histidine (PolyHis), " proton sponge body " mechanism by imidazole group can realize endosome film lytic activity.If designed polymeric micellar, can in the endosome in early days release of triggering property and internalization take place, make sophisticated endosome film rupture subsequently, medicine is escaped, and in cytoplasm and nucleus (potential drug effect site), showing higher drug level, such drug-supplying system will become the drug delivery model that effectively overcomes multidrug resistance.Therefore, have the polymeric micellar of poly-l-histidine core, also be expected to become the inversion agent of effective tumor multidrug-resistance.
The molecular action target of most antitumor drug is positioned at cell.Existing chemotherapy of tumors technology, substantially continued to use the non-targeting mode of administration of medicine, by the suitable carriers technology,, be to solve the cancer chemotherapy cure rate to hang down one of important means with toxic and side effects with the direct targeting pathological tissues of medicine (organ), cell and subcellular organelle.At present, both at home and abroad scientist is by the nano-carrier technology, obtained certain progress on the tissue (organ) of antitumor drug and cell-targeting, but the curative effect of making a breakthrough property not.One of its fundamental cause is that the molecular action target spot of most antitumor drug is positioned at cell.Therefore, at the targeted nanometer carrier material Study on Technology exploitation of medicine molecular action target spot (subcellular organelle) in the tumor cell, be the key that breaks through chemotherapy of tumors bottleneck technology.
The solid lipid nanoparticle drug-supplying system is the new colloidal drug-supplying system that grows up early 1990s, and it is after Emulsion, liposome, polymer nanoparticle, has the target controlling and releasing drug-supplying system of development potentiality.The solid lipid nanoparticle drug-supplying system adopts natural or synthetic lipid materials, is carrier as stearic acid, lecithin, monoglyceride etc., pharmaceutical pack is wrapped in the lipoid nuclear makes solid micelle drug-supplying system.It had both possessed polymeric drug delivery system controlled release, had avoided advantages such as drug leakage, and the toxicity that has had Emulsion, liposome again concurrently is low, good biocompatibility, advantage that bioavailability is high.But also there are some potential limitation in the solid lipid nanoparticle drug-supplying system, squeezes problems such as phenomenon as limited medicine carrying ability, the medicine of storage process.
In solid lipid nanoparticle, add the different liquid fatty of form as mixing lipid substrate, can prepare novel nano structured lipid carrier (nanostructured lipid carrier, NLC).The adding of liquid fatty can be upset the lattice structure of solid lipid rule, increases the ratio of irregular crystal formation in the nanoparticle structure, the spatial content of entrapped drug is increased, and then improve the medicine carrying ability of carrier.By controlling liquid lipid ratio, also can make NLC under body temperature, keep the solid skeletal structure, realize that the NLC controlled delivery of pharmaceutical agents discharges.
Summary of the invention
First purpose of the present invention provides the application of a kind of nano structured lipid carrier drug feeding system in the antineoplaston medicine.
Another object of the present invention provides the application of a kind of nano structured lipid carrier drug feeding system in the reversing drug resistance tumor multidrug-resistance.
Nano structured lipid carrier drug feeding system of the present invention is made up of solid-state matrix material, liquid matrix material and antitumor drug.Solid-state lipid is a monoglyceride, and liquid lipid is an oleic acid, and antitumor drug can be paclitaxel and amycin.Oleic ratio can be 0-30%, and the ratio of antitumor drug is 1-5%.Nano structured lipid carrier drug feeding system adopts the preparation of aqueous solvent diffusion method.The composition of the nano structured lipid carrier among the present invention and preparation method are contained by patent " a kind of nano structured lipid carrier with highly effective antineoplastic activity " (number of patent application 200610155605.3).
The present invention utilizes to have the concentrate nano structured lipid carrier of function of efficient cellular uptake and cytoplasm, envelope paclitaxel, amycin equimolecular target are positioned at intracellular antitumor drug, when improving such medicine antitumor curative effect, the drug resistance of reversing drug resistance tumor cell and multidrug resistance.
Usefulness of the present invention, be by having the concentrate nano structured lipid carrier of function of efficient cellular uptake and cytoplasm, seal molecular target and be positioned at intracellular antitumor drug, can significantly increase the picked-up of tumor cell to antitumor drug, improve the drug level at molecular drug target position, strengthen the curative effect of antitumor drug.By the nano structured lipid carrier entrapped drug, can avoid in the mdr cell cytoplasm P-glycoprotein to the identification of antitumor drug, reduce medicine and efflux from intracellular, when improving the antitumor drug curative effect, the drug resistance of reversing drug resistance tumor cell and multidrug resistance.
Description of drawings
Fig. 1 is that fluorescent labeling monoglyceride nano structured lipid carrier is at MCF-7 cell and MCF-7-adr cellular uptake fluorescence photo.
Fig. 2 is that fluorescent labeling monoglyceride nano structured lipid carrier is at SKOV3 cell and SKOV3-adr cellular uptake fluorescence photo.
The specific embodiment
Embodiment 1: the preparation and the application of amycin monoglyceride nanostructured carrier drug feeding system
1) amycin monoglyceride nanostructured carrier drug feeding system preparation
Get monoglyceride 60mg and amycin 3mg respectively, the accurate title, decide, and adds the 6mL dehydrated alcohol, and 70 ℃ of dissolvings of water-bath form organic facies.With the distilled water is decentralized photo, puts in 70 ℃ of water-baths, at 400rmin
-1Under the mechanical agitation condition, organic facies is injected in the 60mL decentralized photo, stirs 5min, prepare amycin monoglyceride nano structured lipid carrier drug feeding system dispersion liquid, dispersion liquid 3molL
-1HCl solution is regulated pH to 1.2, with 20000rmin
-1Centrifugal 10min, precipitation adds 0.1% poloxamer (Poloxamer) (w/v) behind the redispersion, uses 1.0molL
-1NaOH solution is regulated pH to 7.0, obtains amycin monoglyceride nano structured lipid carrier drug feeding system.
Particle diameter, surface potential and the entrapment efficiency of amycin monoglyceride nano structured lipid carrier drug feeding system see Table 1.
Particle diameter, surface potential and the entrapment efficiency of table 1 amycin monoglyceride nano structured lipid carrier drug feeding system
2) drug resistance of the antitumor curative effect of monoglyceride nano structured lipid carrier drug feeding system and reversing drug resistance tumor cell
Be model cell with breast cancer cell MCF-7 cell and adriamycin-resistant breast cancer cell MCF-7-adr cell respectively, the antitumor curative effect of amycin monoglyceride nano structured lipid carrier drug feeding system, and to the chemical sproof reverse efficient of drug-resistant tumor cell, the IC after hatching altogether by drug-supplying system and cell
50Value (the half fatality rate of cell) is estimated.The cell survival rate test adopts tetrazolium salts colorimetry (MTT Assay) to measure.Cultivate 24h in advance in 24 orifice plates, after the cell attachment growth, add blank monoglyceride nano structured lipid carrier suspension, amycin solution and the amycin monoglyceride nano structured lipid carrier drug feeding system suspension of variable concentrations respectively.Control wells is established in experiment, and every group is repeated 3 times.After hatching 48 hours, every hole adds the MTT solution of 60 μ L, hatches abandoning supernatant after 4 hours, PBS solution flushing 2 times, and every hole adds the DMSO solution of 500 μ L, cessation reaction.With culture plate level vibration 10min, at the 570nm place, measure trap with enzyme connection detector, calculate cell survival rate by (1) formula:
Cell survival rate (%)=A
570(sample)/A
570(contrast) * 100% (1)
A wherein
570(sample) is the trap of the cell behind the adding suspension, A
570(contrast) is the trap of the cell of blank.
The IC of the blank monoglyceride nano structured lipid carrier of being measured, amycin solution and amycin monoglyceride nano structured lipid carrier drug feeding system
50The value result sees Table 2.
The IC of table 2 monoglyceride nano structured lipid carrier, amycin solution and amycin monoglyceride nano structured lipid carrier drug feeding system
50Value
Result of study shows that the monoglyceride nano structured lipid carrier is the hypotoxicity carrier material, and amycin on breast carcinoma sensitive cells and mdr cell, can improve 0.5 times of antitumor curative effect and 5.3 times respectively after the monoglyceride nano structured lipid carrier is sealed.The drug resistance multiple of breast carcinoma mdr cell is about 35.2 times, and amycin is after the monoglyceride nano structured lipid carrier is sealed, but the drug resistance of Partial Inverse projectile vomiting of milk adenocarcinoma mdr cell.
Embodiment 2: the preparation and the application of amycin monoglyceride/oleic acid nanostructured carrier drug feeding system
1) amycin monoglyceride/oleic acid nanostructured carrier drug feeding system preparation
Get monoglyceride 60mg, oleic acid 6mg and amycin 3mg respectively, the accurate title, decide.Add the 6mL dehydrated alcohol, 70 ℃ of dissolvings of water-bath.With the distilled water is decentralized photo, puts in 70 ℃ of water-baths, at 400rmin
-1Under the mechanical agitation condition, organic facies is injected in the 60mL decentralized photo, stirs 5min, obtain amycin monoglyceride/oleic acid nano structured lipid carrier drug feeding system dispersion liquid, dispersion liquid 3molL
-1HCl solution is regulated pH to 1.2, with 20000rmin
-1Centrifugal 10min, precipitation adds 0.1% poloxamer (Poloxamer) (w/v) behind the redispersion, uses 1molL
-1NaOH solution is regulated pH to 7.0, obtains amycin monoglyceride/oleic acid nano structured lipid carrier drug feeding system.
Particle diameter, surface potential and the entrapment efficiency of amycin monoglyceride/oleic acid nano structured lipid carrier drug feeding system see Table 3.
Particle diameter, surface potential and the entrapment efficiency of table 3 amycin monoglyceride nano structured lipid carrier drug feeding system
2) drug resistance of the antitumor curative effect of monoglyceride/oleic acid nano structured lipid carrier drug feeding system and reversing drug resistance tumor cell
Be model cell with breast cancer cell MCF-7 cell and adriamycin-resistant breast cancer cell MCF-7-adr cell respectively, the antitumor curative effect of amycin monoglyceride/oleic acid nano structured lipid carrier drug feeding system, and to the chemical sproof reverse efficient of drug-resistant tumor cell, the IC after hatching altogether by drug-supplying system and cell
50Value (the half fatality rate of cell) is estimated.The cell survival rate test adopts tetrazolium salts colorimetry (MTT Assay) to measure.Cultivate 24h in advance in 24 orifice plates, after the cell attachment growth, add suspension, amycin solution and the amycin monoglyceride/oleic acid nano structured lipid carrier drug feeding system suspension of the blank monoglyceride/oleic acid nano structured lipid carrier of variable concentrations respectively.Control wells is established in experiment, and every group is repeated 3 times.After hatching 48 hours, every hole adds the MTT solution of 60 μ L, hatch 4 hours after, abandoning supernatant, PBS solution flushing 2 times, every hole adds the DMSO solution of 500 μ L, cessation reaction.With culture plate level vibration 10min, at the 570nm place, measure trap with enzyme connection detector, calculate cell survival rate by (1) formula.
The IC of blank monoglyceride/oleic acid nano structured lipid carrier of being measured, amycin solution and amycin monoglyceride/oleic acid nano structured lipid carrier drug feeding system
50Value sees Table 4.
The IC of table 4 monoglyceride/oleic acid nano structured lipid carrier, amycin solution and amycin monoglyceride/oleic acid nano structured lipid carrier drug feeding system
50Value
Result of study shows that monoglyceride/oleic acid nano structured lipid carrier is the hypotoxicity carrier material, and amycin can improve antitumor curative effect 5% and 7.7 times respectively on breast carcinoma sensitive cells and mdr cell after monoglyceride/the oleic acid nano structured lipid carrier is sealed.The drug resistance multiple of breast carcinoma mdr cell is about 35.2 times, and amycin is after monoglyceride/the oleic acid nano structured lipid carrier is sealed, but the drug resistance of Partial Inverse projectile vomiting of milk adenocarcinoma mdr cell.
Embodiment 3: the preparation and the application of paclitaxel monoglyceride nanostructured carrier drug feeding system
1) paclitaxel monoglyceride nanostructured carrier drug feeding system preparation
Get monoglyceride 60mg and paclitaxel 3 mg respectively, the accurate title, decide, and adds the 6mL dehydrated alcohol, 70 ℃ of dissolvings of water-bath.With the distilled water is decentralized photo, puts in 70 ℃ of water-baths, at 400 rmin
-1Under the mechanical agitation condition, organic facies is injected the 60mL decentralized photo, stir 5min, obtain paclitaxel monoglyceride nano structured lipid carrier drug feeding system dispersion liquid, dispersion liquid 3molL
-1HCl solution is regulated pH to 1.2, with 20000rmin
-1Centrifugal 10min, precipitation adds 0.1% poloxamer (Poloxamer) (w/v) behind the redispersion, uses 1molL
-1NaOH solution is regulated pH to 7.0, obtains paclitaxel monoglyceride nano structured lipid carrier drug feeding system.
Particle diameter, surface potential and the entrapment efficiency of paclitaxel monoglyceride nano structured lipid carrier drug feeding system see Table 5.
Particle diameter, surface potential and the entrapment efficiency of table 5 paclitaxel monoglyceride nano structured lipid carrier drug feeding system
2) drug resistance of the antitumor curative effect of monoglyceride nano structured lipid carrier drug feeding system and reversing drug resistance tumor cell
Respectively with breast cancer cell MCF-7 cell and adriamycin-resistant breast cancer cell MCF-7-adr cell, the ovarian cancer cell SKOV3 cell ovarian cancer cell of anti-paclitaxel SKOV3-adr cell is a model cell, the antitumor curative effect of paclitaxel monoglyceride nano structured lipid carrier drug feeding system, and to the chemical sproof reverse efficient of drug-resistant tumor cell, the IC after hatching altogether by drug-supplying system and cell
50Value (the half fatality rate of cell) is estimated.The cell survival rate test adopts tetrazolium salts colorimetry (MTT Assay) to measure.Cultivate 24h in advance in 24 orifice plates, after the cell attachment growth, (solvent is Cremophor: dehydrated alcohol mixed solvent (1: 1, v/v)) and paclitaxel monoglyceride nano structured lipid carrier (oleic acid content is 0) drug-supplying system suspension to add suspension, the paclitaxel solution of the blank monoglyceride nano structured lipid carrier of variable concentrations respectively.Control wells is established in experiment, and every group is repeated 3 times.After hatching 48 hours, every hole adds the MTT solution of 60 μ L, hatch 4 hours after, abandoning supernatant, PBS solution flushing 2 times, every hole adds the DMSO solution of 500 μ L, cessation reaction.With culture plate level vibration 10min, at the 570nm place, measure trap with enzyme connection detector, calculate cell survival rate by (1) formula.
The IC of the blank monoglyceride nano structured lipid carrier of being measured, paclitaxel solution and paclitaxel monoglyceride nano structured lipid carrier drug feeding system
50Value sees Table 6.
The IC of table 6 monoglyceride nano structured lipid carrier, paclitaxel solution and paclitaxel monoglyceride nano structured lipid carrier drug feeding system
50Value
Result of study shows, the monoglyceride nano structured lipid carrier is the hypotoxicity carrier material, paclitaxel on breast carcinoma sensitive cells and mdr cell, can improve 2.2 times of antitumor curative effects and 125.5 times respectively after monoglyceride nano structured lipid carrier (not containing oleic acid) is sealed.The breast carcinoma mdr cell is about 29.7 times to the multidrug resistance multiple of paclitaxel, and paclitaxel can reverse the multidrug resistance of breast carcinoma mdr cell fully after the monoglyceride nano structured lipid carrier is sealed.Paclitaxel on ovarian cancer sensitive cells and mdr cell, can improve 0.8 times of antitumor curative effect and 5.9 times respectively after monoglyceride nano structured lipid carrier (not containing oleic acid) is sealed, can partly reverse the drug resistance of ovarian cancer drug-resistant cell.
Embodiment 4: the preparation and the application of paclitaxel monoglyceride/oleic acid nanostructured carrier drug feeding system
1) paclitaxel monoglyceride/oleic acid nanostructured carrier drug feeding system preparation
Get monoglyceride 60mg, oleic acid 6 or 18mg and paclitaxel 3mg respectively, the accurate title, decide, and adds the 6mL dehydrated alcohol, 70 ℃ of dissolvings of water-bath.With the distilled water is decentralized photo, puts in 70 ℃ of water-baths, at 400rmin
-1Under the mechanical agitation condition, organic facies is injected in the 60mL decentralized photo, stirs 5min, obtain paclitaxel monoglyceride/oleic acid nano structured lipid carrier drug feeding system dispersion liquid, dispersion liquid 3molL
-1HCl solution is regulated pH to 1.2, with 20000rmin
-1Centrifugal 10min, precipitation adds 0.1% poloxamer (Poloxamer) (w/v) behind the redispersion, uses 1molL
-1NaOH solution is regulated pH to 7.0, obtains paclitaxel monoglyceride/oleic acid nano structured lipid carrier drug feeding system.
Particle diameter, surface potential and the entrapment efficiency of the monoglyceride of paclitaxel/oleic acid nano structured lipid carrier drug feeding system see Table 7.
Particle diameter, surface potential and the entrapment efficiency of table 7 paclitaxel monoglyceride nano structured lipid carrier drug feeding system
2) lung cancer A549 cell of monoglyceride nano structured lipid carrier transhipment
The present invention adopts and contains fluorescein isothiocyanate (fitc) (FITC) and stearylamine chemistry grafting, the fluorescent marker that carries out lung cancer A549 cell transhipment research as the monoglyceride nano structured lipid carrier.The monoglyceride nano structured lipid carrier that contains fluorescein isothiocyanate (fitc) and stearylamine chemistry grafting, preparation by the following method: get monoglyceride 27mg and fluorescein isothiocyanate (fitc) and stearylamine chemistry grafting 4.5mg respectively, the accurate title, decide, and adds the 3mL dehydrated alcohol, 70 ℃ of dissolvings of water-bath.With the distilled water is decentralized photo, puts in 70 ℃ of water-baths.At 400rmin
-1Under the mechanical agitation condition, organic facies is injected the 30mL decentralized photo, stir 5min, obtain the dispersion liquid of fluorescent labeling monoglyceride nano structured lipid carrier, dispersion liquid 3molL
-1HCl solution is regulated pH to 1.2, with 20000rmin
-1Centrifugal 10min, precipitation adds 0.1% poloxamer (Poloxamer) (w/v) behind the redispersion, uses 1.0molL
-1NaOH solution is regulated pH to 7.0, and dispersion liquid is used for lung cancer A549 cell transhipment research.
Get breast cancer cell MCF-7 cell and adriamycin-resistant breast cancer cell MCF-7-adr cell respectively, in containing the RPMI RPMI-1640 of 10% calf serum, cultivate (5%CO
2, 37 ℃ of incubators).The trophophase cell of taking the logarithm after the PBS rinse, adds trypsinization and with the culture fluid dilution, by every hole 1 * 10
5The density of individual cell is inoculated in 24 well culture plates, and after the cell attachment growth in 24 well culture plates, (final concentration is 100 μ gmL to add fluorescently-labeled monoglyceride nano structured lipid carrier
-1), hatch 1,2,4,12, behind the 24h, wash cell 3 times with PBS, the fluorescence inverted microscope is observed also and is taken pictures, and the result is referring to Fig. 1.Wherein nano-carrier and cell were hatched 12 hours altogether, A be fluorescent labeling monoglyceride nano structured lipid carrier (containing 30% oleic acid) at MCF-7 cellular uptake fluorescence photo, B is that fluorescent labeling monoglyceride nano structured lipid carrier (containing 30% oleic acid) is at MCF-7-adr cellular uptake fluorescence photo.
Get breast cancer cell MCF-7 cell and adriamycin-resistant breast cancer cell MCF-7-adr cell respectively, the ovarian cancer cell SKOV3 cell ovarian cancer cell of anti-paclitaxel SKOV3-adr cell is cultivated (5%CO in containing the RPMI RPMI-1640 of 10% calf serum
2, 37 ℃ of incubators).The trophophase cell of taking the logarithm after the PBS rinse, adds trypsinization and with the culture fluid dilution, by every hole 1 * 10
5The density of individual cell is inoculated in 24 well culture plates, and after the cell attachment growth in 24 well culture plates, (final concentration is 100 μ gmL to add fluorescently-labeled monoglyceride/oleic acid nano structured lipid carrier
-1), hatch 1,2,4,12, behind the 24h, wash cell 3 times with PBS, the fluorescence inverted microscope is observed also and is taken pictures, the result is referring to Fig. 2, wherein nano-carrier and cell were hatched 12 hours altogether, for fluorescent labeling monoglyceride nano structured lipid carrier (containing 30% oleic acid) at SKOV3 cellular uptake fluorescence photo, B fluorescent labeling monoglyceride nano structured lipid carrier (containing 30% oleic acid) is at SKOV3-adr cellular uptake fluorescence photo.
3) drug resistance of the antitumor curative effect of monoglyceride/oleic acid nano structured lipid carrier drug feeding system and reversing drug resistance tumor cell
Respectively with breast cancer cell MCF-7 cell and adriamycin-resistant breast cancer cell MCF-7-adr cell, the ovarian cancer cell SKOV3 cell ovarian cancer cell of anti-paclitaxel SKOV3-adr cell is a model cell, the antitumor curative effect of paclitaxel monoglyceride/oleic acid nano structured lipid carrier drug feeding system, and to the chemical sproof reverse efficient of drug-resistant tumor cell, the IC after hatching altogether by drug-supplying system and cell
50Value (the half fatality rate of cell) is estimated.The cell survival rate test adopts tetrazolium salts colorimetry (MTT Assay) to measure.Cultivate 24h in advance in 24 orifice plates, after the cell attachment growth, (solvent is Cremophor: dehydrated alcohol mixed solvent (1: 1, v/v)) and paclitaxel monoglyceride/oleic acid nano structured lipid carrier (containing 10 and 30% oleic acid) drug-supplying system suspension to add blank monoglyceride/oleic acid nano structured lipid carrier suspension, the paclitaxel solution of variable concentrations respectively.Control wells is established in experiment, and every group is repeated 3 times.After hatching 48 hours, every hole adds the MTT solution of 60 μ L, hatch 4 hours after, abandoning supernatant, PBS solution flushing 2 times, every hole adds the DMSO solution of 500 μ L, cessation reaction.With culture plate level vibration 10min, at the 570nm place, measure trap with enzyme connection detector, calculate cell survival rate by (1) formula.
The IC of blank monoglyceride/oleic acid nano structured lipid carrier of being measured, paclitaxel solution and paclitaxel monoglyceride/oleic acid nano structured lipid carrier drug feeding system
50Value sees Table 8.
The IC of table 8 monoglyceride/oleic acid nano structured lipid carrier, paclitaxel solution and paclitaxel monoglyceride/oleic acid nano structured lipid carrier drug feeding system
50Value
Result of study shows, monoglyceride/oleic acid nano structured lipid carrier is the hypotoxicity carrier material, paclitaxel is after monoglyceride/oleic acid nano structured lipid carrier (containing 30% oleic acid) is sealed, on the mdr cell of breast carcinoma sensitive cells and adriamycin-resistant, can improve 3.5 times of antitumor curative effects and 123.8 times respectively.The breast carcinoma mdr cell is 29.7 times to the multidrug resistance multiple of paclitaxel, and paclitaxel can reverse the multidrug resistance of breast carcinoma mdr cell fully after the monoglyceride nano structured lipid carrier is sealed.Paclitaxel on ovarian cancer sensitive cells and mdr cell, can improve 2.0 times of antitumor curative effects and 175.4 times respectively after monoglyceride nano structured lipid carrier (not containing oleic acid) is sealed, can reverse the drug resistance of ovarian cancer drug-resistant cell fully.
Claims (2)
1. the application of nano structured lipid carrier drug feeding system in the preparation antitumor drug, it is characterized in that this drug-supplying system is to be prepared by following steps: (1) gets monoglyceride 60mg, oleic acid 6mg and amycin 3mg respectively; (2) add the 6mL dehydrated alcohol, 70 ℃ of dissolvings of water-bath are decentralized photo with the distilled water, put in 70 ℃ of water-baths, at 400rmin
-1Under the mechanical agitation condition, organic facies is injected in the 60mL decentralized photo, stirs 5min, obtain amycin monoglyceride/oleic acid nano structured lipid carrier drug feeding system dispersion liquid; (3) dispersion liquid 3molL
-1HCl solution is regulated pH to 1.2, with 20000rmin
-1Centrifugal 10min after precipitation adds 0.1% poloxamer (w/v) redispersion, uses 1molL
-1NaOH solution is regulated pH to 7.0, obtains amycin monoglyceride/oleic acid nano structured lipid carrier drug feeding system.
2. the application of nano structured lipid carrier drug feeding system in the preparation antitumor drug, it is characterized in that this drug-supplying system is to be prepared by following steps: (1) gets monoglyceride 60mg, oleic acid 6 or 18mg and paclitaxel 3mg respectively; (2) add the 6mL dehydrated alcohol, 70 ℃ of dissolvings of water-bath are decentralized photo with the distilled water, put in 70 ℃ of water-baths, at 400rmin
-1Under the mechanical agitation condition, organic facies is injected in the 60mL decentralized photo, stirs 5min, obtain paclitaxel monoglyceride/oleic acid nano structured lipid carrier drug feeding system dispersion liquid, (3) dispersion liquid 3molL
-1HCl solution is regulated pH to 1.2, with 20000rmin
-1Centrifugal 10min after precipitation adds 0.1% poloxamer (w/v) redispersion, uses 1molL
-1NaOH solution is regulated pH to 7.0, obtains paclitaxel monoglyceride/oleic acid nano structured lipid carrier drug feeding system.
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| ES2875824T3 (en) * | 2013-01-14 | 2021-11-11 | Infirst Healthcare Ltd | Solid solution compositions and their use in chronic inflammation |
| CN104013600A (en) * | 2014-06-05 | 2014-09-03 | 暨南大学 | Psoralen-enveloped nanostructure lipid carrier preparation and preparation method thereof |
| CN104367549B (en) * | 2014-08-22 | 2017-09-12 | 辽宁大学 | Contain psoralen adriamycin composite nanostructure lipid vector preparation and preparation method thereof |
| CN105030680A (en) * | 2015-08-19 | 2015-11-11 | 合肥华方医药科技有限公司 | Total bufogenin nano lipid carrier drug delivery system for injection and preparation method thereof |
| CN107115531A (en) * | 2017-05-05 | 2017-09-01 | 浙江大学 | A kind of glycolipid polymers modification nano structured lipid carrier and preparation method and application |
| CN108853056B (en) * | 2018-07-02 | 2021-09-14 | 天津中医药大学 | Folic acid targeted modification carried doxorubicin hydrochloride and gambogic acid nano-structure lipid carrier preparation and preparation method thereof |
| CN116173188B (en) * | 2023-02-07 | 2023-12-01 | 浙江大学 | Oral GLP-1 analogue solid lipid nanoparticle, and preparation method and application thereof |
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2007
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| Hong Yuan et al..Preparation and characteristics of nanostructured lipid carriers for control-releasing progesterone by melt-emulsification.《Colloids and Surfaces B: Biointerfaces》.Science Direct,2007,第60卷174-179. * |
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