CN102178954A - Recombinant high density lipoprotein (HDL) medicament delivery system with functions of targeted and reverse cholesterol transport (RCT) on vascular wall and application thereof - Google Patents
Recombinant high density lipoprotein (HDL) medicament delivery system with functions of targeted and reverse cholesterol transport (RCT) on vascular wall and application thereof Download PDFInfo
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Abstract
The invention belongs to the medical technical field, and relates to a new function of high density lipoproteins (HDLs) of different structures as a targeted carrier for a liposoluble cardiovascular medicament. The recombinant HDL can be taken as a cholesterol receptor for receiving excess extra-hepatic cellular cholesterol and transporting the cholesterol to liver for metabolism, thus achieving the purpose of treating cardiovascular or arteriosclerotic disease. In the invention, the HDLs or lipid components thereof are recombined into the carrier for the liposoluble medicament in vitro so as to achieve dual functions of medicament delivery to an atherosclerosis focal area on a vascular wall and reverse cholesterol transport (RCT), which provides a new effective way for treating cardiovascular disease.
Description
Technical field
The present invention relates to medical technical field, be specifically related to the preparation of different structure reorganization high density lipoprotein and, reach the function of transportation drug targeting blood vessel wall atherosis focus zone and antiport cholesterol as the new function of fat-soluble cardiovascular drugs carrier.
Background technology
Studies show that in a large number (high density lipoprotein, HDL) (atherosclerosis, As) generation and the order of severity with coronary heart disease is negative correlation to plasma high density lipoprotein level for level and atherosclerosis.The important mechanisms of the anti-As of HDL is exactly that HDL participates in the cholesterol antiport, the superfluous cholesterol of extrahepatic tissue is shifted out and is transported to liver transform, remove.In addition, HDL also has effects such as the expression of the oxidation of lipotropism matter, anticoagulant and adhesion molecule, the propagation that influences cell, protection endothelium and promotion vasodilation.HDL has new life's (plate-like) and ripe (spherical) two types, and the former is mainly by containing apolipoprotein A-1 (apolipoproteinA I, the phospholipid bilayer film that apoA-I) surrounds composition; The latter is made up of the monolayer immobilized artificial membrane and the apolipoprotein of nonpolar lipid core and periphery.At present, (reconstituted HDL rHDL) is used for the treatment of atherosclerosis, has obtained good curative effect at vitro recombination HDL for existing research and utilization apoA-I and phospholipid.In addition, the characteristic that the hydrophilic-hydrophobic structure of HDL uniqueness, bigger lipid core (can be used as the storage space of fat-soluble medicine), endogenous can be degraded fully and do not discerned and remove by reticuloendothelial system, make the carrier of rHDL, demonstrate special advantages at aspects such as improving drug targeting distributes, increase drug effect as medicine.Foreign patent US5128318 discloses the method that a kind of apoA-I of utilization and phospholipid prepare plate-like HDL, purpose is to remove unnecessary liposoluble substance in the body, and do not use its medicine carrying, foreign patent US6514523B1 discloses the preparation method of a kind of medicine carrying rHDL, be used to improve drug effect, reduce toxicity and reduce engulfing of reticuloendothelial system, and targeting is by the tissue or the cell of apoA-I mediation, but rHDL and other machine-processed targeting of not relating to different structure, CN1307906A discloses a kind of new drug carrier, utilize the rHDL medicine carrying to improve the targeting transhipment of liver or tumor tissues, but liver is as one of target organ maximum in the body, the targeting of itself is very remarkable, and the application of therefore developing the rHDL novel targets will have bigger meaning.
Natural HDL finds to maturation circulation transforming process from new life in research, and most cholesteryl esters can be transferred to very low density lipoprotein (VLDL) rapidly (very low density lipoprotein VLDL) waits on other apolipoprotein in the sophisticated HDL granule.In this process, VLDL can bring cholesteryl ester into the extrahepatic tissue that comprises arterial wall, and HDL is reinvented discoid newborn HDL simultaneously, continues the effect of performance antiport cholesterol, and this process helps antiatherogenic treatment.The cardiovascular drugs bag is written into reorganization HDL; in its metabolic process, be transferred to VLDL; be carried into arterial wall by the VLDL receptor acting; this targeting almost is negligible to normal body; but for atherosclerotic patient; VLDL not only participates in the formation of foam cell in the Atheromatosis reason process, and blood vessel endothelium increases the permeability of VLDL, and VLDL is subjected to physical ability medicine to be brought into the lesions position of arterial wall by VLDL.Therefore, the objective of the invention is to utilize reorganization HDL bag to carry cardiovascular drugs, under the condition of not introducing exogenous VLDL, both brought into play the targeting of VLDL blood vessel focus, make rHDL after reconstruct, produce the antiport cholesterol function of similar newborn HDL again, from increasing the therapeutic effect that target site drug level and antiport cholesterol two aspects improve medicine.
As everyone knows, cardiovascular drugs especially some Chinese medicine ingredients mostly is fat-soluble, and bioavailability is low, this brings many inconvenience to clinical practice, consider the chronicity of cardiovascular patient medication, at present, existing document and patent report the sustained-release microparticle preparation of cardiovascular drugs, as liposome, solid lipid nanoparticle etc., but the common matrix material that these preparations adopted, multi-source is used oils and fats in intestinal and non-enteral nutrition Emulsion, can there be potential harm in life-time service or intravenous injection.As glyceryl monostearate, be used for food additive; Triglyceride and cholesterol content and the hyperlipemia, atherosclerosis in blood is closely related, and long-term a large amount of these ectogenic fat of taking in will jeopardize human health, and are for the patient of cardiovascular and cerebrovascular disease, more harm than good especially.Difference of the present invention is its bio-imitability, is to adopt the composition of natural high density lipoprotein and the drug-loading system that approaching ratio is recombinated, and has both avoided the unfavorable factor of common lipid materials, has brought into play the targeting and the physiological function of carrier again.
In addition,, the invention also discloses a kind of rHDL that does not contain apolipoprotein, promptly only adopt the lipid components of HDL, make it, produce similar physiological function and effect in vivo in conjunction with the apolipoprotein of natural HDL in order to simplify the preparation process of reorganization HDL.
Summary of the invention
The carrier that the present invention is a fat-soluble medicine with high density lipoprotein or its lipid components vitro recombination, bring into play its dual function to the atherosis focus of blood vessel wall zone transmission medicine and antiport cholesterol, for the foregoing invention purpose, the inventor adopts following technical scheme:
A kind of discous reorganization high density lipoprotein drug-loading system, it is characterized in that comprising following several component: fat-soluble medicine, phospholipid, cholesterol, apolipoprotein in surfactant and the natural high density lipoprotein, have the plate-like form, similar new life's HDL has the function of plate-like form and antiport cholesterol.
According to the proportioning of natural each composition of HDL, above-mentioned plate-like rHDL is in the prescription gross weight, the shared percentage ratio of each component is: phosphatidase 13 0%～60%, cholesterol 1%～10%, surfactant 1%～5%, apolipoprotein 20%～60%, fat-soluble medicine 1%～5%.
The preparation method of above-mentioned plate-like rHDL is with tanshinone (also can select other fat-soluble medicines for use), and phospholipid and cholesterol are dissolved in organic facies (ethanol for example, acetone, chloroform, methanol) in, in water (as the buffer of pH=8.0), add surfactant (as sodium lauryl sulphate, sodium deoxycholate, sodium cholate, benzalkonium bromide), adopt thin film to disperse or the preparation of emulsifying evaporation, form transparent suspension, suspension and apolipoprotein are hatched, dialysis is removed surfactant promptly.It is characterized in that one or more surfactants are added aqueous phase, improved the combination rate of albumen and lipid, and albumen incubation conditions gentleness, guaranteed the biological activity of apolipoprotein.
A kind of globular reorganization high density lipoprotein drug-loading system, it is characterized in that comprising following several component: fat-soluble medicine, phospholipid, cholesterol, glyceride, cholesteryl ester, the apolipoprotein in surfactant and the natural high density lipoprotein has spherical-like morphology, similar sophisticated HDL, after entering in the body, can form plate-like HDL, should have the function of antiport cholesterol through metabolism.
Ratio according to each component among the natural HDL, above-mentioned spherical rHDL, in the prescription gross weight, the shared percentage ratio of each component is: phosphatidase 11 0%～30%, cholesterol 1%～10%, glyceride 2%～10%, cholesteryl ester 1%～15%, surfactant 1%～3%, apolipoprotein 20%～60%, fat-soluble medicine 1%～3%.
The preparation method of above-mentioned spherical rHDL, be with tanshinone (also can select other fat-soluble medicines for use), phospholipid, cholesterol, glyceride and cholesteryl ester are dissolved in organic facies (ethanol for example, acetone, chloroform, methanol) in, one or more surfactants of adding in water (as the buffer of pH=8.0) (as sodium lauryl sulphate, sodium deoxycholate, sodium cholate, benzalkonium bromide), adopt the preparation of emulsifying evaporation, form transparent suspension, suspension and apolipoprotein are hatched, dialysis is removed surfactant promptly, its feature is characterized in that one or more surfactants are added aqueous phase with the preparation feature of plate-like rHDL, has improved the combination rate of albumen and lipid, and albumen incubation conditions gentleness has been guaranteed the biological activity of apolipoprotein.
A kind of non-proteic reorganization high density lipoprotein drug-loading system is characterized in that comprising following several component: fat-soluble medicine, phospholipid, cholesterol, glyceride and cholesteryl ester, and can compete specifically in conjunction with the apolipoprotein on the natural HDL, particle diameter with similar natural HDL, density and function.
Ratio according to each component among the natural HDL, above-mentioned non-albumen rHDL, in the prescription gross weight, the shared percentage ratio of each component is: phosphatidase 13 5%～55%, cholesterol 1%～20%, glyceride 5%～25%, cholesteryl ester 1%～30%, surfactant 1%～5%, lovastatin (also can select other fat-soluble medicines for use) 1%～5%.
The apolipoprotein of above plate-like or spherical rHDL is to comprise apolipoprotein apoA I, and is selected from other main apolipoprotein such as apoAII on the high density lipoprotein, apoAIV, apoD, apoE, apoC I, apoC II, any one or more among the apoCIII.
More than three kinds of rHDL drug-loading systems, wherein fat-soluble medicine can be cardiovascular drugs, cardiovascular drugs can be a tanshinone, lovastatin.
More than three kinds of rHDL, wherein phospholipid is selected from the natural phospholipid in Semen sojae atricolor, egg yolk, brain or the spinal cord.
The rHDL that more than contains cholesteryl ester, wherein the fatty acid in cholesteryl ester and the triglyceride is the middle long-chain fatty acid of 6-24 carbon, contain one or more unsaturated bonds, cholesteryl ester can be a cholesterol acid ester, the cholesterol palm acid ester, cholesterol arachidonate, cholesterol linoleate, cholesterol Semen Myristicae oil acid esters, one or more in the cholesterol linolenate; Glyceride can be Lipoid MCT, Labrafac CC, and glycerol trioleate, Dynasan 114, Miglyol812N, Softisan138, Softisan142, one or more among the Miglyol 840.
More than three kinds of reorganization high density lipoprotein drug-loading systems can be used for preparing the carrier of fat-soluble cardiovascular drugs, reach function to the atherosis focus of blood vessel wall zone transmission medicine and performance carrier antiport cholesterol.
Single apolipoprotein-the apoA-I of many uses during the HDL of preparation reorganization at present, but the apolipoprotein among the HDL has polytype, and have difference in functionality separately, can activate lecithin cholesterol acyltransferase as apoA-I, and can be used as the part of HDL receptor; ApoA-II have keep the HDL Stability Analysis of Structures and with the ability of phospholipids incorporate; ApoE can determine the binding site of cholesterol, mediates effects such as cholesterol outflow in addition.Therefore the present invention adopts the mixture of the multiple apolipoprotein that comprises apoA-I to prepare reorganization high density lipoprotein drug-loading system, will have higher physiologically active.
The present invention has adopted the method that adds one or more surfactants when preparing fat nuclear in aqueous phase in advance, this compares with the method that adds or do not add single surfactant when hatching albumen of bibliographical information in the past, has higher apolipoprotein combination rate, and the rHDL fat that forms nuclear drug loading height, thereby can more effectively realize protein mediated receptor acting, the physiological function of performance carrier and raising curative effect of medication.
The present invention has broken through and had simulated the conventional theory of natural HDL particle diameter in the past when preparation contained albumen rHDL, adopted comparatively gentle condition to hatch albumen, this is because proteic three dimensional structure is to guarantee its bioactive key factor, though conditions such as acutely ultrasonic can obtain smaller particle size, but can produce cavitation, make the albumen local temperature too high or generate free radical and degeneration.The prepared rHDL of the present invention, particle diameter have but still kept the physiologically active of natural HDL antiport cholesterol and have had the characteristic of targeting transportation medicine much larger than natural HDL.
With the preparation of existing rHDL carrier with use different, the invention also discloses a kind of non-proteic rHDL drug-loading system, it can be competed in vivo in conjunction with the apolipoprotein on the natural HDL, present the form similar, particle diameter, density and function to natural HDL, thereby avoided containing the antigenicity of albumen rHDL, also reduced the complexity of cost and technology.
The plate-like or the spherical medicine carrying rHDL of the present invention's preparation are respectively liposome and nanoparticle form before hatching with apolipoprotein, after hatching with albumen, liposome changes the plate-like of accumulation into, and nanoparticle still brush border occurred for sphere and surface, and Electronic Speculum figure sees Fig. 1.Mean diameter～160nm, envelop rate＞90%, drug loading＞4%, Zeta potential＜-16mV.
Because competence exertion mediated the effusive effect of cholesterol after the rHDL of spherical medicine carrying will change into plate-like through internal metabolism, therefore in external this function that can only investigate plate-like medicine carrying rHDL.Cholesterol flows out the experiment (see figure 2) and shows, the negative control bovine serum albumin is at each time point, the cell inner cholesterol does not have significant difference, and plate-like medicine carrying rHDL can flow out by the mediated cell inner cholesterol, it is 50.1% of cell inner cholesterol primary quantity that content lowers, similar to the ability of natural HDL cholesterol reducing (56.0%), the prepared plate-like medicine carrying rHDL of proof the present invention has the effusive ability of mediation cholesterol, lays a good foundation for further bringing into play antiatherogenic effect in the body.
The non-albumen medicine carrying rHDL (Fig. 3) that the present invention is prepared, mean diameter～8nm, envelop rate＞90%, drug loading＞4%, Zeta potential＜-29mV, the transmission electron microscope demonstration forms tangible core-shell structure after hatching with natural HDL, and form is similar to natural HDL.
With atherosis focus district in the foam cell modeling body; vitro data shows plate-like; the foam cell amount of engulfing of spherical HDL and non-albumen medicine carrying rHDL has increased by 10.3 respectively than macrophage; 5.0 and 4.8 times; proved that three kinds of medicine carrying rHDL all have good targeting; and phagocytosis depends on very low density lipoprotein (VLDL) (VLDL) and cholesterol ester transfer protein (CETP); in addition; plate-like medicine carrying rHDL targeting can produce higher targeting efficient greater than spherical medicine carrying rHDL after adding Lecithin-cholesterol acyltransferase. (LCAT).
Description of drawings
Fig. 1 is the transmission electron microscope picture of albumen rHDL.After adding cholesteryl ester and glyceride in the demonstration prescription among the figure, formed fat nuclear is nanoparticle structure (A), and when not using these two kinds of lipids, formed fat nuclear is liposome structure (B), and still be nanoparticle structure (C) after the fat of nanoparticle structure nuclear is hatched with apolipoprotein, liposome structure then become plate-like (D).
Fig. 2 is the effusive result of plate-like rHDL mediated cell inner cholesterol, shows among the figure and compare with the negative control bovine serum albumin that plate-like rHDL can reduce the content of cell inner cholesterol.
The transmission electron microscope picture of rHDL (B) after Fig. 3 is hatched for non-albumen rHDL (A) and with natural HDL shows the core-shell structure that presents similar natural HDL after rHDL and natural HDL are hatched among the figure.
The specific embodiment
In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative fully, and they only are used for the present invention is specifically described, and not should be understood to limitation of the present invention.
Embodiment 1:
Preparation technology: with the medicine of recipe quantity, egg phosphatide, cholesterol places cillin bottle, adds an amount of chloroform, ultrasonicly makes its dissolving, be transferred in the eggplant-shape bottle, place Rotary Evaporators, organic facies, vacuum drying 2h are removed in decompression, add the hydration medium that is dissolved with sodium cholate in right amount, ultra-sonic dispersion evenly becomes translucent suspension, re-uses the probe Ultrasonic Pulverization, makes it become the transparent opalescent liquid that has, cross 0.22 μ m filter membrane, add apolipoprotein, dissolving is placed on 4 ℃ of refrigerators, 300rpm magnetic agitation 3-6h, use 2L to contain the Tris-HCl buffer dialysed overnight of NaCl and EDTA sodium, promptly get rHDL.
Form before and after rHDL and albumen are hatched among the employing transmission electron microscope observing embodiment 1 is seen B, D in the accompanying drawing 1, is liposome structure before hatching, and hatching the back is the plate-like packed structures, and mean diameter is 158.5nm, and envelop rate is 60.12%, and protein binding rate is 60.8%.
Embodiment 2:
Preparation technology is with embodiment 1, and the mean diameter after prepared rHDL and albumen are hatched is 211.5nm, and envelop rate is 89.12%, and protein binding rate is 44.1%.
Cell in vitro experiment: adopt Mus source cell strain RAW264.7 macrophage, the foam cell model is duplicated in the oxidized low-density lipoprotein stimulation, in two kinds of cells, add plate-like rHDL respectively, investigation is at very low density lipoprotein (VLDL) (VLDL), Lecithin-cholesterol acyltransferase. (LCAT), under the effect of cholesterol ester transfer protein (CETP), the targeting of rHDL, the result shows that the engulf amount of plate-like rHDL in foam cell is 0.19 a μ g/1000000 cell, the intake of macrophage is 0.084 a μ g/1000000 cell, there is significant difference in the two, and phagocytosis depends on VLDL and CETP, the amount of engulfing of foam cell increases to 0.505 a μ g/1000000 cell behind the adding LCAT, shows that plate-like rHDL can issue the structure that changes in the effect of LCAT, brings into play more significant targeting; When apolipoprotein content increases to 0.55g by 0.16g, foam cell increases to 0.58 a μ g/1000000 cell to the intake of rHDL by 0.13, illustrates that the amount of apolipoprotein among the rHDL and cellular uptake amount are proportionate.
Cellular cholesterol flows out experiment: adopt Mus source cell strain RAW264.7 macrophage, be inoculated in 24 porocyte plates after the hatching, go down to posterity after 2～3 times, do the cell cholesterol and flow out experiment, add rHDL and bovine serum albumin in the cell culture medium respectively, after in incubator, hatching 9 hours, taking-up discards supernatant liquid, and wash cell plates repeatedly with cold PBS, add the dissolve with methanol attached cell, HPLC detects the content of cell inner cholesterol, compares with the bovine serum albumin negative control group, and the reduction of calculating the cell inner cholesterol is 63.5% of a primary quantity.Illustrate that rHDL has the effusive ability of mediation cholesterol.
Embodiment 3:
Preparation technology is with embodiment 1, and the mean diameter after prepared rHDL and albumen are hatched is 173.4nm, and envelop rate is 88.12%, and protein binding rate is 61.3%.
The cell in vitro test: with embodiment 2, the intake of plate-like rHDL foam cell is 0.21 a μ g/1000000 cell, and the intake of macrophage is 0.087 a μ g/1000000 cell.
Cellular cholesterol flows out experiment: with embodiment 2, rHDL mediated cell inner cholesterol flows out, content is reduced to 41.2% of cell inner cholesterol primary quantity, illustrates to use the rHDL that makes up with natural HDL apolipoprotein same composition to have the higher effusive ability of mediation cholesterol than the rHDL that contains an apoA-I.
Embodiment 4:
Preparation technology is same: with the medicine of recipe quantity, egg phosphatide, cholesterol, cholesterol Semen Myristicae oil acid esters, Miglyol 812N places cillin bottle, add adequate amount of ethanol, ultrasonicly make its dissolving, be heated to 60 ℃, splash into the aqueous phase that contains sodium cholate, magnetic agitation, the probe ultra-sonic dispersion places Rotary Evaporators then, organic facies is removed in decompression, and being concentrated into certain volume, 0.22 μ m filter membrane is crossed in cooling, add apolipoprotein, the dissolving back uses 2L to contain the Tris-HCl buffer dialysed overnight of NaCl and EDTA sodium in 37 ℃ of magnetic agitation 8-12h, promptly gets rHDL.
Form before and after rHDL and albumen are hatched among the employing transmission electron microscope observing embodiment 4 is seen A, C in the accompanying drawing 1, is the nanoparticle structure before and after hatching, and its particle surface of hatching has brush border, mean diameter is 162.3nm, and envelop rate is 72.37%, and protein binding rate is 48.7%.
Embodiment 5:
Preparation technology is with embodiment 4, and the mean diameter after prepared rHDL and albumen are hatched is 158.5nm, and envelop rate is 90.3%, and protein binding rate is 54.1%.
Cell in vitro experiment: adopt Mus source cell strain RAW264.7 macrophage, the foam cell model is duplicated in the OxLDL ELISA stimulation, in two kinds of cells, add spherical rHDL respectively, investigation is at VLDL, under the effect of CETP, the targeting of rHDL, the result shows that the engulf amount of spherical rHDL in foam cell is 0.057 a μ g/1000000 cell, significantly greater than the intake of macrophage (0.013 μ g/1000000), and phagocytosis depends on VLDL and CETP, but less than the targeting of plate-like rHDL.
Embodiment 6:
Preparation technology is with embodiment 4, and it is 155.1nm that prepared rHDL and albumen are hatched the back mean diameter, and envelop rate is 92.7%.
Cell in vitro test: with embodiment 5, the intake of spherical rHDL is 0.05 a μ g/1000000 cell, significantly greater than the intake of macrophage (0.011 μ g/1000000), when apolipoprotein content increases to 0.26g by 0.06g, the intake of foam cell increases to 0.13 a μ g/1000000 cell by 0.03, illustrates that the amount of apolipoprotein among the rHDL and cellular uptake amount are proportionate.
Embodiment 7:
Preparation technology is with embodiment 4, but need not to add surfactant and hatch apolipoprotein at aqueous phase, prepared rHDL is respectively 8.0nm and 8.5nm with the mean diameter that natural HDL competition combines the apolipoprotein front and back, and envelop rate is 94.2%, and density is in the 1.063-1.21g/ml scope.
Cell in vitro test: with embodiment 5, after non-albumen rHDL and natural HDL are hatched, the amount of being engulfed by foam cell is 0.06 a μ g/1000000 cell, significantly greater than the amount of being absorbed by macrophage (0.017 μ g/1000000), and phagocytosis depends on VLDL and CETP, has proved its targeting effect.
Claims (13)
1. discous reorganization high density lipoprotein drug-loading system, it is characterized in that comprising following several component: fat-soluble medicine, phospholipid, cholesterol, apolipoprotein in surfactant and the natural high density lipoprotein has the function of plate-like form and antiport cholesterol.
2. the drug-loading system described in the claim 1, it is characterized in that ratio according to each component among the natural HDL, in the prescription gross weight, the shared percentage ratio of each component is: phosphatidase 13 0%～60%, cholesterol 1%～10%, surfactant 1%～5%, apolipoprotein 20%～60%, fat-soluble medicine 1%～5%.
3. according to the described drug-loading system of claim 1～2, its preparation method is a) with fat-soluble medicine and phospholipid, the cholesterol mixed together is dissolved in the organic facies, b) remove organic facies, prepare a dried mixture, c) use the buffer medium hydration that contains surfactant, form liquid mixture, cross 0.22 μ m filter membrane, d) add apolipoprotein, stirring or homogenizing or ultrasonic it is fully hatched, e) surfactant is removed in dialysis, it is characterized in that one or more surfactants are added aqueous phase, has improved the combination rate of albumen and lipid, and albumen incubation conditions gentleness has been guaranteed the biological activity of apolipoprotein.
4. globular reorganization high density lipoprotein drug-loading system, it is characterized in that comprising following several component: fat-soluble medicine, phospholipid, cholesterol, glyceride, cholesteryl ester, apolipoprotein in surfactant and the natural high density lipoprotein has spherical-like morphology, enter in the body after, form plate-like HDL through metabolism, should have the function of antiport cholesterol.
5. the drug-loading system described in the claim 4, it is characterized in that ratio according to each component among the natural HDL, in the prescription gross weight, the shared percentage ratio of each component is: phosphatidase 11 0%～30%, cholesterol 1%～10%, glyceride 2%～10%, cholesteryl ester 1%～15%, surfactant 1%～3%, apolipoprotein 20%～60%, fat-soluble medicine 1%～3%.
6. according to the described drug-loading system of claim 4～5, its preparation method is a) with fat-soluble medicine and phospholipid, cholesterol, glyceride, the cholesteryl ester mixed together is dissolved in the organic facies, b) at a certain temperature, organic facies is splashed into the water that contains surfactant, fully stir and form translucent milky liquid, c) Ultrasonic Pulverization, d) organic facies is removed in decompression, cross 0.22 μ m filter membrane, e) add apolipoprotein, stirring or homogenizing or ultrasonic it is fully hatched, f) surfactant is removed in dialysis, it is characterized in that one or more surfactants are added aqueous phase, improve the combination rate of albumen and lipid, and albumen incubation conditions gentleness, guaranteed the biological activity of apolipoprotein.
7. a non-proteic reorganization high density lipoprotein drug-loading system is characterized in that comprising following several component: fat-soluble medicine, phospholipid, cholesterol, glyceride and cholesteryl ester, and can compete specifically in conjunction with the apolipoprotein on the natural HDL, particle diameter with similar natural HDL, density and function.
8. the drug-loading system described in the claim 7, it is characterized in that ratio according to each component among the natural HDL, in the prescription gross weight, the shared percentage ratio of each component is: phosphatidase 13 5%～55%, cholesterol 1%～20%, glyceride 5%～25%, cholesteryl ester 1%～30%, surfactant 1%～5%, fat-soluble medicine 1%～5%.
9. according to the described drug-loading system of claim 1～6, wherein apolipoprotein is to comprise apolipoprotein apoA I, and is selected from other main apolipoprotein such as apoAII on the high density lipoprotein, apoAIV, apoD, apoE, apoC I, apoCII, any one or more among the apoCIII.
10. according to the described drug-loading system of claim 1～8, wherein fat-soluble medicine can be cardiovascular drugs, and cardiovascular drugs can be a tanshinone, lovastatin.
11. according to the described drug-loading system of claim 1～8, wherein phospholipid is selected from the natural phospholipid in Semen sojae atricolor, egg yolk, brain or the spinal cord.
12. according to the described drug-loading system of claim 4～8, wherein the fatty acid of cholesteryl ester is the middle long-chain fatty acid of 6～24 carbon, contain one or more unsaturated bonds, can form by one or more cholesteryl esters, wherein glyceride is selected from the list of fatty acid or in two or the triglyceride one or more, wherein fatty acid is 6～24 carbon, contains the middle long-chain fatty acid of one or more unsaturated bonds.
13. according to the described reorganization high density lipoprotein of arbitrary claim drug-loading system in the claim 1～12 in the application aspect the preparation fat-soluble medicine blood vessel wall targeting vector.
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| CN104324007A (en) * | 2014-09-15 | 2015-02-04 | 中国药科大学 | Preparation technology and application of natural recombinant nanostructured lipid carrier |
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2011
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Patent Citations (1)
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| WO2001058492A2 (en) * | 2000-02-14 | 2001-08-16 | Ottawa Heart Institute Research Corporation | Carrier particles for drug delivery and process for preparation |
Non-Patent Citations (1)
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| 《International Journal of Pharmaceutics》 20100531 Wen-Li Zhang et al. Nanostructured lipid carriers constituted from high-density lipoprotein components for delivery of a lipophilic cardiovascular drug 313-321 7-8,10-13 第391卷, 第1-2期 * |
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| CN102846551A (en) * | 2011-06-28 | 2013-01-02 | 复旦大学 | Liver-targeting high-density lipoprotein analogue nano-particles, preparation method thereof, and application thereof |
| CN102614515A (en) * | 2012-04-16 | 2012-08-01 | 中国药科大学 | Novel chemical restructuring high-density lipoprotein drug carrying system with targeted and double anti-tumor effects and application |
| CN103505720A (en) * | 2012-06-30 | 2014-01-15 | 复旦大学 | Application of apolipoprotein A-I in preparation of medicine used for preventing and treating liver disease metabolic syndrome |
| CN104324007A (en) * | 2014-09-15 | 2015-02-04 | 中国药科大学 | Preparation technology and application of natural recombinant nanostructured lipid carrier |
| CN104324007B (en) * | 2014-09-15 | 2017-04-12 | 中国药科大学 | Preparation technology and application of natural recombinant nanostructured lipid carrier |
| CN104784188A (en) * | 2015-03-26 | 2015-07-22 | 辽宁中医药大学 | Application of tanshinone IIA in preparing medicine for regulating blood serum HDL subclass distribution |
| CN112218619A (en) * | 2017-11-20 | 2021-01-12 | 西奈山伊坎医学院 | Suppression of acclimatized immunity by therapeutic nanobiological formulation compositions |
| CN108144070A (en) * | 2017-12-06 | 2018-06-12 | 常州朋悦纺织品有限公司 | A kind of preparation method of lipoprotein cladding hydridization gadolinium oxide micelle contrast agent |
| CN108404139A (en) * | 2018-01-29 | 2018-08-17 | 南昌大学 | The preparation method and applications of the recombination lipoprotein of low concentration Monostalotetrahexosylgangliside modification |
| CN108508102A (en) * | 2018-02-05 | 2018-09-07 | 贵州医科大学 | The assay method of nine kinds of effect components plasma protein binding rates in Sheepear Inula Herb extract |
| CN111973759A (en) * | 2020-09-10 | 2020-11-24 | 吉林大学 | Preparation method of diagnosis and treatment integrated recombinant high-density lipoprotein |
| CN112999351A (en) * | 2021-03-11 | 2021-06-22 | 华中农业大学 | Preparation method and application of artificial lipid drops and freeze-dried preparation thereof |
| CN112999351B (en) * | 2021-03-11 | 2022-06-10 | 华中农业大学 | Preparation method and application of artificial lipid drops and freeze-dried preparation thereof |
| US11859021B2 (en) | 2021-03-19 | 2024-01-02 | Icahn School Of Medicine At Mount Sinai | Compounds for regulating trained immunity, and their methods of use |
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