CN104436201A - Preparation of dual-hepatic-targeting long-circulation gypenoside liposome and preparation method of liposome - Google Patents
Preparation of dual-hepatic-targeting long-circulation gypenoside liposome and preparation method of liposome Download PDFInfo
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Abstract
The invention discloses a method for preparing a dual-hepatic-targeting long-circulation gypenoside liposome by modifying beta-sitosterol by virtue of galacturonic acid, glycyrrhetinic acid and polyethylene glycol. Compared with a normal gynostemma liposome, the targeting efficiency to parenchymal hepatic cells and long-circulation effect of the gypenoside liposome are greatly improved. The novel long-circulation liposome is good in hepatic targeting property, and can also obviously prolong the retention time of the gypenoside in a body, so that the gypenoside can actively target to hepatic tumor cells, and an effect of treating tumors is enhanced.
Description
Technical field
The present invention relates to field of pharmaceutical preparations, particularly relate to a kind of method of producing dual Liver targeting long circulating gypenoside liposome, more particularly, the present invention relates to dual Liver targeting long circulating gypenoside liposome and the pharmaceutical preparation containing dual Liver targeting long circulating gypenoside liposome.
Background technology
Liver is made up of parenchyma and nonparenchymal cell (endotheliocyte, Kupffer Cell), and hepatocarcinoma, hepatitis all occur in hepatic parenchymal cells.Asialoglycoprotein receptor (Asialoglycoprotein receptor, ASGPR) in a large amount of existence in mammiferous hepatic parenchymal cells film surface, this receptor can identify in specific manner with galactose to be the liposome of end group, thus with such liposome for carrier, can by drug targeting hepatic parenchymal cells.But, only lean on ASGPR receptors bind to be inadequate.There are some researches show that the binding activities of ASGPR can change with the change of many pathological conditions, nearly all hepatic disease, can cause there is binding inhibitors in serum, and the binding activities of ASGPR can significantly reduce, under pathological state, the binding activities of ASGPR reduces very remarkable.As can be seen here, the ability realizing liver cell targeting administration by means of only ASGPR mediation is limited, strengthens part and hepatocellular binding ability under pathological state, very necessary.Negishi confirmed in rat hepatocytes membrane component containing a large amount of enoxolone (glycyrrhetinic acid, GA) specific binding site in early 1990s.So the galactose be combined with ASGPR and GA receptor-specific, enoxolone equal energy targeting is to hepatic parenchymal cells.
Liposome is a kind of novel form of targeting drug delivery system, and it is the inside be made up of lipid bilayer is the vesicle of aqueous phase.Liposome, as a kind of intelligent drug delivery carrier, has targeting, biocompatibility, improves medicine stability and reduces the remarkable advantages such as toxic and side effects, thus obtaining people and more and more pay close attention to.Although conventional liposome can improve the curative effect of medicine, but to enter after in body reticuloendothelial system phagocytic in very fast body, main targeting is in the reticuloendothelial system such as kidney,liver,spleen, bone marrow organ, medicine in vivo the holdup time short, and be subject to the effect of albumen, enzyme etc. in vivo and seepage occurs, affect the treatment, toxicity is increased.
In recent years, long circulating liposomes becomes study hotspot both domestic and external.Long circulating liposomes is the one of targeting preparation, and it has long-acting, advantage such as reduction toxicity and stable entrapped drug etc., by the targeting that improve medicine after long circulating liposomes encapsulating, reduces toxicity.To be a kind of finishing natural or the novel lipide of synthesis hydrophilic polymer for long circulating liposomes, the solid flexible water-wetted surface that these polymer are formed, add the hydrophilic of liposome and sterically hindered, prevent biomolecule, cell and liposome interact, make liposome not easily by the opsonin identification in blood, thus reticuloendothelial system engulfing liposome in body can be reduced, increase its stability in vivo in environment, make it have longer circulation time in vivo, higher blood drug level, therefore better curative effect can be played, reduce toxicity.In long circulating liposomes, most study be exactly liposome (the Advanced Drug Delivery Reviews that Polyethylene Glycol (PEG) is modified, 1997, 24, 235-242), the liposome of finishing Polyethylene Glycol has the feature of " stealth " after intravenous injection, the opsonic conditioning in blood plasma can be escaped, thus reduce macrophage engulfing medicine, hinder the combination of blood protein composition and phospholipid, prolong drug circulation time in vivo (Clinical and Experimental Pharmacology and Physiology, 2006, 33, 557-562).
Herb Gynostemmae Pentaphylli has another name called Herba Gynostemmatis, Pentapanax, principle active component is polysaccharide, flavonoid, saponins and microelement kind, its effect mainly promotes body fat class substance metabolism, nutrition human body cell, there is good detoxicating, relieving inflammation effect simultaneously, be widely used in hyperlipidemia, fatty liver, obesity, constipation, insomnia, hepatitis B, the treatment of chronic airway inflammation (pharyngolaryngitis, gastroenteritis etc.) and health care.Saponin is wherein a kind of effective ingredient.Why " mystery " is because its saponin component (84 kinds) exceedes the several times contained by Radix Ginseng to gypenoside, it is to cardiovascular disease such as treatment and prophylaxis of hypertension, diabetes, constipation, hemorrhoid, asthma, migraine, acne, the symptoms such as mottle have remarkable efficacy, and have comparatively significantly calm, hypnosis, allaying tiredness, appetite stimulator, antidotal effect, to tumor, hepatitis, gastritis, gastric and duodenal ulcers, gastroptosis, stomatitis, coronary heart disease, arteriosclerosis, cholelithiasis, numb limbs and tense tendons, pachylosis, male sterility, sexual disorder, fat and poliosis is bald etc. has better curative effect.Gypenoside also may become the major physiological active substance that the 21 century mankind conquer cardiovascular disease, diabetes, catatonia, cancer and acquired immune deficiency syndrome (AIDS).
In order to make gypenoside can slow releasing in vivo, the growth of long-time suppression inflammatory cell or tumor, we utilize targeting and long circulating principle, first galactose and enoxolone is utilized to realize the function of the targeting hepatic parenchymal cells of liposome, utilize the hydrophilic polymer PEG connected at surface of liposome, make liposome prolonged stay and not caught by mononuclear phagocyte system (MPS) in vivo, extend drug release time, improve the sustained release performance of medicine.
Gypenoside long circulating liposomes first with galacturonic acid, enoxolone and polyethyleneglycol modified cupreol for film material, prepare gypenoside long circulating liposomes, medicine is made to arrive site of action with minimum loss, the drug effect of gypenoside can be improved, reduce consumption, being a kind of novel dosage form, is the result of technological innovation.Prior art adopts this targeting group of enoxolone to modify Polyethylene Glycol, to utilizing the Liver targeting of enoxolone to reach the targeting object of cell grade, but, enoxolone is a kind of hydrophobic compound, in water, dissolubility is poor, outside after forming liposome, enoxolone often can not be exposed to, the recipient cell be just not easy on liver is combined the object reaching targeting.And in this patent, we also adopt hydrophilic galactose to modify enoxolone in the periphery of enoxolone, galactose is exposed to the periphery of liposome, can be combined with its receptor ASGPR, in addition, further improve the hydrophilic of enoxolone, enoxolone is exposed, be easy to and receptors bind, improve targeting.Because it has dual-target, can reach the diseased region of cell grade exactly, this medication is that additive method can not be compared.This experiment adopts reverse evaporation, film dispersion method, alcohol injection has prepared dual Liver targeting long circulating gypenoside liposome, and has investigated its envelop rate, filter out a kind of best practice preparing dual Liver targeting long circulating gypenoside liposome, make its utilization ratio reach the highest.This dual Liver targeting long circulating gypenoside lipid physical ability is with minimum consumption, and reach best therapeutic effect, targeting and long circulating liposomes may as the new development directions of following a kind of administration.
Have not yet to see the relevant report about dual Liver targeting long circulating gypenoside liposome.
Summary of the invention
The object of the present invention is to provide a kind of utilization to carry out Polyethylene Glycol, galacturonic acid and enoxolone to cupreol and modify the prescription and method of producing long circulating gypenoside liposome, the method technique is simple.Owing to adopting galacturonic acid, enoxolone and Polyethylene Glycol in the method, cupreol is modified, not containing cholesterol in preparation, moreover, compare with the common long circulating liposomes that enoxolone is modified with only using galacturonic acid, the targeting efficiency of this novel long circulating liposomes hepatic parenchymal cells is higher, can also the significant prolongation gypenoside holdup time in vivo, make gypenoside more effectively passive target in tumor cell, improve tumour inhibiting rate, reduce the dosage of gypenoside, improve therapeutic efficiency.
There is provided a kind of such as formula the preparation method with galacturonic acid, enoxolone, polyethyleneglycol modified cupreol shown in I in the present invention, described method not only can effectively be carried out, and can obtain productive rate higher, be easy to be separated, low-cost formula I.
Wherein, n=30-60; Preferred n=40-50.
In the present invention, provide a kind of preparation method such as formula the compound shown in I, described method comprises step:
(1) cupreol, succinic anhydride, DMAP are mixed in dichloromethane, are obtained by reacting such as formula the compound shown in IV at 30-60 DEG C;
(2) by formula IV compound, amino-end peg, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, is obtained by reacting the compound as shown in formula III at 30-60 DEG C;
(3) by formula III compound, enoxolone, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, is obtained by reacting such as formula the compound shown in II at 30-60 DEG C;
(4) by formula II compound, galacturonic acid, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, is obtained by reacting such as formula the compound shown in I at 30-60 DEG C;
In the reaction of step (1), the mol ratio of cupreol, succinic anhydride, DMAP is 1: 0.8-10: 0.8-5;
The mol ratio of step (2) compound of formula IV, amino-end peg, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1: 0.8-3: 0.8-3: 0.8-3;
The mol ratio of the reaction compound of formula III of step (3), succinic anhydride, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1: 0.8-10: 0.8-5: 0.8-5;
The mol ratio of step (4) compound of formula H, galacturonic acid, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1: 0.8-3: 0.8-3: 0.8-3;
Described dual Liver targeting long circulating liposomes, is characterized in that it is mainly made up of gypenoside, phospholipid material, formula I.
Wherein, described phospholipid material is: the mixture of one or more in the acid of DSPE, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidylinositols, phosphatidyl glycerol, phosphoglyceride, diphosphatidylglycerol, sphingomyelins.
Wherein, the composition of described liposome also comprises one or more in mannitol, glucose, tween 80, vitamin E or Polyethylene Glycol.
Wherein, this liposome contains following component by weight percentage: gypenoside 2% ~ 45%, phospholipid material 40 ~ 85%, formula I accounts for 10 ~ 40% of weight of formulation.
Wherein, the preparation of dual Liver targeting gypenoside long circulating liposomes can comprise the steps:
(1) gypenoside being dissolved in pH is that 6.8 ~ 8.0 phosphoric acid rush in liquid, and making mass concentration is 0.1 ~ 20mg/mL gypenoside buffer;
(2) when solid unit is milligram hour, liquid unit in milliliter, by that analogy.Phospholipid material: formula I: ether: the ratio of gypenoside buffer soln is chosen by 100 ~ 300 parts: 15 ~ 200 parts: 2 ~ 5 parts: 5 ~ 30 parts, for subsequent use;
(3) preparation of dual Liver targeting long circulating gypenoside liposome: phospholipid material, formula I and ether are mixed, then rotary evaporation make its uniformly lipoid plastic film covering at pear shape bottle inner bottom part, gypenoside buffer soln is added in bottle, add bead, at 30 ~ 60 DEG C after rotational oscillation 15 ~ 60min, place the abundant hydration of 0.5 ~ 2h and get final product.
Wherein, the preparation of dual Liver targeting gypenoside long circulating liposomes can comprise the steps:
(1) when solid unit is milligram hour, liquid unit in milliliter, by that analogy.The preparation of oil phase: gypenoside: phospholipid material: formula I: the ratio of ethanol is in the ratio chosen material of 10 ~ 30 parts: 40 ~ 300 parts: 10 ~ 100 parts: 5 ~ 50 parts, and mix homogeneously, prepares oil phase;
(2) preparation of aqueous phase: aqueous phase is phosphate buffer 5 ~ 50 parts, the pH of buffer is 6.8 ~ 8.0;
(3) preparation of dual Liver targeting long circulating liposomes: under constant temperature stirring, oil phase syringe needle being at the uniform velocity injected into temperature is in the aqueous phase of 35 ~ 60 DEG C, and constant temperature stirs 1 ~ 4h except ethanol, to obtain final product.
Wherein, the preparation of dual Liver targeting gypenoside long circulating liposomes can comprise the steps:
(1) when solid unit is milligram hour, liquid unit in milliliter, by that analogy.Phospholipid material: formula I: the ratio of dichloromethane is prepared oil phase by 40 ~ 300 parts: 10 ~ 200 parts: 50 ~ 500 parts, oil phase is placed in round-bottomed flask, rotates steaming vibrating dichloromethane;
(2) when solid unit is milligram hour, liquid unit in milliliter, by that analogy.Be dissolved in 10 ~ 500 parts of phosphate buffers by 10 ~ 200 parts of gypenosides, make aqueous phase;
(3) add in oil phase by aqueous phase, under ice bath, Probe Ultrasonic Searching 5 ~ 30min, forms uniform Emulsion;
(4) revolve steaming removing organic facies, add phosphate buffer 10 ~ 150 part after forming colloidal state, continue reduction vaporization 0.5 ~ 2h, Probe Ultrasonic Searching 3 ~ 20min, to obtain final product.
Wherein, dual Liver targeting gypenoside long circulating liposomes can make lyophilized injection, injection, preparation capable of permeating skin, aerosol, oral liquid, solution, ophthalmic preparation, tablet or capsule further.
In the present invention, additionally provide a kind of preparation method such as formula the compound shown in V, prepare single galacturonic acid Liver targeting gypenoside long circulating liposomes with formula V compound, described method comprises step:
(1) cupreol, succinic anhydride, DMAP are mixed in dichloromethane, are obtained by reacting such as formula the compound shown in IV at 30-60 DEG C;
(2) by formula IV compound, amino-end peg, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, is obtained by reacting the compound as shown in formula III at 30-60 DEG C;
(3) by formula III compound, galacturonic acid, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, is obtained by reacting such as formula the compound shown in V at 30-60 DEG C;
In the reaction of step (1), the mol ratio of cupreol, succinic anhydride, DMAP is 1: 0.8-10: 0.8-5;
The mol ratio of step (2) compound of formula H, Polyethylene Glycol, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1: 0.8-3: 0.8-3: 0.8-3;
Step (3) compound of formula III, galacturonic acid, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, is obtained by reacting such as formula the compound shown in V at 30-60 DEG C.
(1) gypenoside being dissolved in pH is that 6.8 ~ 8.0 phosphoric acid rush in liquid, and making mass concentration is 0.1 ~ 20mg/mL gypenoside buffer;
(2) when solid unit is milligram hour, liquid unit in milliliter, by that analogy.Phospholipid material: formula V compound: ether: the ratio of gypenoside buffer soln is chosen by 100 ~ 300 parts: 15 ~ 200 parts: 2 ~ 5 parts: 5 ~ 30 parts, for subsequent use;
(3) preparation of dual Liver targeting long circulating gypenoside liposome: phospholipid material, formula V compound and ether are mixed, then rotary evaporation make its uniformly lipoid plastic film covering at pear shape bottle inner bottom part, gypenoside buffer soln is added in bottle, add bead, at 30 ~ 60 DEG C after rotational oscillation 15 ~ 60min, place the abundant hydration of 0.5 ~ 2h and get final product.
The present invention is that one has reinforcement hepatocytes-targeting efficiency, and the dual Liver targeting long circulating liposomes of significant prolongation gypenoside circulation time in vivo, rat pharmacokinetic studies shows that the long circulating liposomes targeting efficiency of dual Liver targeting long circulating liposomes ratio galacturonic acid in the present invention and the single targeting of enoxolone is high a lot, moreover, the half-life also extends greatly.Pharmacodynamic study shows, dual Liver targeting long circulating liposomes of the present invention is significantly better than the long circulating liposomes of single targeting to the inhibitory action of hepatocarcinoma, stability experiment also shows that the percolation ratio of the dual Liver targeting long circulating liposomes Gynostemma pentaphyllum Makino saponin in this invention is wanted significantly lower than long circulating liposomes and the conventional liposome group of other single targeting.
In sum, dual Liver targeting gypenoside long circulating liposomes prepared by the present invention, the hydrophobic chain portion of formula I can enter the hydrophobic layer of liposome, strengthen the stability of liposome interior structure, hydrophilic chain segment part is positioned at liposome hydrophilic outer layer, can play long circulating action in targeting, stereoscopic stable effect and body.In preparation process, owing to selecting cupreol preparation I compound as film material, not containing cholesterol, effective reduction is harmful to the level (decreasing the impact of cholesterol on human body) of low density cholesterol, play the effect of angiocardiopathy preventing, expand the application of this one dosage type low temperature of liposome in cardiovascular disease field.With the dual Liver targeting gypenoside long circulating liposomes that the formula I introduced in the method is produced, compared with the long circulating liposomes of single targeting, the efficiency of targeting hepatic parenchymal cells significantly improves.
Detailed description of the invention
Subordinate's example is used for illustrating the detailed description of the invention of technical scheme of the present invention, but is not used in and limits the scope of the invention.
In the present invention, provide a kind of preparation method such as formula the compound shown in I, described method comprises step:
Embodiment 1
(1) by (4.15g, 0.01mol) cupreol, (0.80g, 0.008mol) succinic anhydride, (0.98g, 0.008mol) DMAP are mixed in 100ml dichloromethane, 45 DEG C of reactions, stir 48h.Dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Obtain such as formula the compound as white solid shown in IV;
(2) by (5.14g, 0.01mol) formula IV compound, (16g, 0.008mol) amino-end peg 2000, (0.98g, 0.008mol) DMAP, (1.53g, 0.008mol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, 45 DEG C of reactions, stir 48h.Dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Crude product through silica gel (300-400 order) flash column chromatography, methylene chloride-methanol (100: 1) eluting, concentrating under reduced pressure collect liquid, obtain the compound of yellow solid as shown in formula III;
(3) by (25.14g, 0.01mol) formula III compound, (3.77g, 0.008mol) enoxolone, (0.98g, 0.008mol) DMAP, (1.53g, 0.008mol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, being obtained by reacting at 30 DEG C is dissolved in 40ml dichloromethane such as formula the compound shown in II, 30 DEG C of backflows, stirs 5h.Dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Crude product through silica gel (300 ~ 400 order) flash column chromatography, methylene chloride-methanol (80: 1) eluting, concentrating under reduced pressure collect liquid, obtain yellow solid formula II compound;
(4) by (29.85g, 0.01mol) formula II compound, (1.70g, 0.008mol) galacturonic acid, (0.98g, 0.008mol) DMAP, (1.53g, 0.008mol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, at 45 DEG C of reaction 48h, dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Crude product is through silica gel (300-400 order) flash column chromatography, and methylene chloride-methanol (100: 1) eluting, concentrating under reduced pressure collects liquid, must such as formula the compound shown in I.
Embodiment 2
(1) (4.15g, 0.01mol) cupreol, (1.0g, 0.0125mol) succinic anhydride, (2.44g, 2mol) DMAP are mixed in 100ml dichloromethane, 45 DEG C of reactions, stir 48h.Dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Obtain such as formula the compound as white solid shown in IV;
(2) by (5.14g, 0.01mol) formula IV compound, (60g, 0.03mol) amino-end peg 2000, (3.67g, 0.03mol) DMAP, (5.75g, 0.03mol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, 45 DEG C of reactions, stir 48h.Dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Crude product through silica gel (300-400 order) flash column chromatography, methylene chloride-methanol (100: 1) eluting, concentrating under reduced pressure collect liquid, obtain the compound of yellow solid as shown in formula III;
(3) by (25.14g, 0.01mol) formula III compound, (14.12g, 0.03mol) enoxolone, (3.67g, 0.03mol) DMAP, (5.75g, 0.03mol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, being obtained by reacting at 60 DEG C is dissolved in 40ml dichloromethane such as formula the compound shown in II, 30 DEG C of backflows, stirs 5h.Dichloromethane solution 1mol/L (3 × 100 mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Crude product through silica gel (300 ~ 400 order) flash column chromatography, methylene chloride-methanol (80: 1) eluting, concentrating under reduced pressure collect liquid, obtain yellow solid formula II compound;
(4) by (29.85g, 0.01mol) formula II compound, (6.36g, 0.03mol) galacturonic acid, (3.67g, 0.03mol) DMAP, (5.75g, 0.03mol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, at 45 DEG C of reaction 48h, dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2s0
4drying, filters, is spin-dried for.Crude product is through silica gel (300-400 order) flash column chromatography, and methylene chloride-methanol (100: 1) eluting, concentrating under reduced pressure collects liquid, must such as formula the compound shown in I.
Embodiment 3
(1) by (4.15g, 0.01mol) cupreol, (5.0g, 0.05mol) succinic anhydride, (3.05g, 0.025mol) DMAP are mixed in 100ml dichloromethane, 45 DEG C of reactions, stir 48h.Dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Obtain such as formula the compound as white solid shown in IV;
(2) by (5.14g, 0.01mol) formula IV compound, (30g, 0.015mol) amino-end peg 2000, (1.83g, 0.015mol) DMAP, (2.88g, 0.015mol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, 45 DEG C of reactions, stir 48h.Dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Crude product through silica gel (300-400 order) flash column chromatography, methylene chloride-methanol (100: 1) eluting, concentrating under reduced pressure collect liquid, obtain the compound of yellow solid as shown in formula III;
(3) by (25.14g, 0.01mol) formula III compound, (7.06g, 0.015mol) enoxolone, (1.83g, 0.015mol) DMAP, (2.88g, 0.015mol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, being obtained by reacting at 50 DEG C is dissolved in 40ml dichloromethane such as formula the compound shown in II, 30 DEG C of backflows, stirs 5h.Dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Crude product through silica gel (300 ~ 400 order) flash column chromatography, methylene chloride-methanol (80: 1) eluting, concentrating under reduced pressure collect liquid obtain yellow solid formula II compound;
(4) by (29.85g, 0.01mol) formula II compound, (3.18g, 0.015mol) galacturonic acid, (1.83g, 0.015mol) DMAP, (2.88g, 0.015mol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, at 45 DEG C of reaction 48h, dichloromethane solution 1mo1/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Crude product is through silica gel (300-400 order) flash column chromatography, and methylene chloride-methanol (100: 1) eluting, concentrating under reduced pressure collects liquid, must such as formula the compound shown in I.
Embodiment 4
| Prescription | Consumption |
| DSPE | 200mg |
| Formula I | 100mg |
| Ether | 3mL |
| Gypenoside | 20mg |
| Phosphate buffer (pH=7.0) | 15mL |
Technique
(1) the choosing of raw material: get phosphate buffer and gypenoside by above-mentioned prescription, gypenoside is dissolved in phosphate buffer, for subsequent use;
(2) preparation of gypenoside liposome: get DSPE, formula I and ether by above-mentioned prescription, mix homogeneously, then rotary evaporation make its uniformly lipoid plastic film covering at pear shape bottle inner bottom part, the phosphate buffer containing gypenoside is added in bottle, add bead, at 60 DEG C after rotational oscillation 30min, place the abundant hydration of 2h, to obtain final product.
Embodiment 5
| Prescription | Consumption |
| Gypenoside | 20mg |
| Lecithin | 200mg |
| Formula I | 50mg |
| Ethanol | 10mL |
| Phosphate buffer (pH=6.8) | 10mL |
Technique
(1) preparation of oil phase: get gypenoside, lecithin, formula I, ethanol by above-mentioned prescription, mix homogeneously, as oil phase;
(2) preparation of aqueous phase: get phosphate buffer as aqueous phase by above-mentioned prescription;
(3) preparation of liposome: under constant temperature stirring, oil phase syringe needle being at the uniform velocity injected into temperature is in the aqueous phase of 45 DEG C, and constant temperature stirs 2h except ethanol, to obtain final product.
Embodiment 6
| Prescription | Consumption |
| Gypenoside | 200mg |
| Sphingomyelins | 200mg |
| Formula I | 50mg |
| Dichloromethane | 50mL |
| Phosphate buffer (pH=8.0) | 40mL |
Technique
(1) get sphingomyelins by above-mentioned prescription: formula I: dichloromethane=200mg: 50mg: 50mL prepares oil phase, and oil phase is placed in round-bottomed flask, rotate steaming vibrating dichloromethane;
(2) 200mg gypenoside is dissolved in 20mL phosphate buffer (pH=8.0), as aqueous phase;
(3) add the phosphate buffer (pH=8.0) of 20mL containing gypenoside 200mg in oil phase, under ice bath, Probe Ultrasonic Searching 10min, forms uniform Emulsion;
(4) revolve steaming removing organic facies, continue to add phosphate buffer 20mL after forming colloidal state, continue reduction vaporization 0.5h, Probe Ultrasonic Searching 10min, to obtain final product.
Embodiment 7
| Prescription | Consumption |
| Phosphatidylcholine | 200mg |
| Formula I | 120mg |
| Ether | 4mL |
| Gypenoside | 30mg |
| Phosphate buffer (pH=7.0) | 20mL |
Technique
(1) the choosing of raw material: get phosphate buffer and gypenoside by above-mentioned prescription, gypenoside is dissolved in phosphate buffer, for subsequent use;
(2) preparation of gypenoside liposome: get phosphatidylcholine, formula I and ether by above-mentioned prescription, mix homogeneously,
Then rotary evaporation make its uniformly lipoid plastic film covering at pear shape bottle inner bottom part, in bottle, add the phosphate buffer containing gypenoside, add bead, at 60 DEG C after rotational oscillation 30min, place 2h abundant hydration, to obtain final product.
Embodiment 8
| Prescription | Consumption |
| Gypenoside | 100mg |
| Lecithin | 1000mg |
| Formula I | 300mg |
| Ethanol | 50mL |
| Phosphate buffer (pH=6.8) | 50mL |
Technique
(1) preparation of oil phase: get gypenoside, lecithin, formula I, ethanol by above-mentioned prescription, mix homogeneously, as oil phase;
(2) preparation of aqueous phase: get phosphate buffer as aqueous phase by above-mentioned prescription;
(3) preparation of liposome: under constant temperature stirring, oil phase syringe needle being at the uniform velocity injected into temperature is in the aqueous phase of 45 DEG C, and constant temperature stirs 2h except ethanol, to obtain final product.
Embodiment 9
| Prescription | Consumption |
| Gypenoside | 200mg |
| Phosphatidyl glycerol | 200mg |
| Formula I | 50mg |
| Dichloromethane | 50mL |
| Phosphate buffer (pH=8.0) | 40mL |
Technique
(1) get phosphatidyl glycerol by above-mentioned prescription: formula I: dichloromethane=200mg: 50mg: 50mL prepares oil phase, and oil phase is placed in round-bottomed flask, rotate steaming vibrating dichloromethane;
(2) 200mg gypenoside is dissolved in 20mL phosphate buffer (pH=8.0), as aqueous phase;
(3) add the phosphate buffer (pH=8.0) of 20mL containing gypenoside 200mg in oil phase, under ice bath, Probe Ultrasonic Searching 10min, forms uniform Emulsion;
(4) revolve steaming removing organic facies, continue to add phosphate buffer 20mL after forming colloidal state, continue reduction vaporization 0.5h, Probe Ultrasonic Searching 10min, to obtain final product.
Galacturonic acid list targeting long circulating liposomes: first adopt and synthesize formula V compound with the step in embodiment 1, step is as follows:
(1) by (4.15g, 0.01mol) cupreol, (0.80g, 0.008mol) succinic anhydride, (0.98g, 0.008mol) DMAP are mixed in 100ml dichloromethane, 45 DEG C of reactions, stir 48h.Dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Obtain such as formula the compound as white solid shown in IV;
(2) by (5.14g, 0.01mol) formula IV compound, (16g, 0.008mol) amino-end peg 2000, (0.98g, 0.008mol) DMAP, (1.53g, 0.008mol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, 45 DEG C of reactions, stir 48h.Dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Crude product through silica gel (300-400 order) flash column chromatography, methylene chloride-methanol (100: 1) eluting, concentrating under reduced pressure collect liquid, obtain the compound of yellow solid as shown in formula III;
(3) by (25.14g, 0.01mol) formula III compound, (1.70g, 0.008mol) galacturonic acid, (0.98g, 0.008mol) DMAP, (1.53g, 0.008mol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, at 45 DEG C of reaction 48h, dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Crude product is through silica gel (300-400 order) flash column chromatography, and methylene chloride-methanol (100: 1) eluting, concentrating under reduced pressure collects liquid, must such as formula the compound shown in V.
Record in the ratio in embodiment 4 and step and prepare galacturonic acid list targeting long circulating liposomes
| Prescription | Consumption |
| DSPE | 200mg |
| Formula V compound | 100mg |
| Ether | 3mL |
| Gypenoside | 20mg |
| Phosphate buffer (pH=7.0) | 15mL |
Technique
(1) the choosing of raw material: get phosphate buffer and gypenoside by above-mentioned prescription, gypenoside is dissolved in phosphate buffer, for subsequent use;
(2) preparation of gypenoside liposome: get DSPE, formula V compound and ether by above-mentioned prescription, mix homogeneously, then rotary evaporation make its uniformly lipoid plastic film covering at pear shape bottle inner bottom part, the phosphate buffer containing gypenoside is added in bottle, add bead, at 60 DEG C after rotational oscillation 30min, place the abundant hydration of 2h, to obtain final product.
Enoxolone list targeting long circulating liposomes: first adopt and synthesize formula II compound with the step in embodiment 1,
Step is as follows:
(1) by (4.15g, 0.01mol) cupreol, (0.80g, 0.008mol) succinic anhydride, (0.98g, 0.008mol) DMAP are mixed in 100ml dichloromethane, 45 DEG C of reactions, stir 48h.Dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Obtain such as formula the compound as white solid shown in IV;
(2) by (5.14g, 0.01mol) formula IV compound, (16g, 0.008mol) amino-end peg, (0.98g, 0.008mol) DMAP, (1.53g, 0.008mol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, 45 DEG C of reactions, stir 48h.Dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Crude product through silica gel (300-400 order) flash column chromatography, methylene chloride-methanol (100: 1) eluting, concentrating under reduced pressure collect liquid, obtain the compound of yellow solid as shown in formula III;
(3) by (25.14g, 0.01mol) formula III compound, (3.77g, 0.008mol) enoxolone, (0.98g, 0.008mol) DMAP, (1.53g, 0.008mol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, being obtained by reacting at 30 DEG C is dissolved in 40ml dichloromethane such as formula the compound shown in II, 30 DEG C of backflows, stirs 5h.Dichloromethane solution 1mol/L (3 × 100mL) hydrochloric acid solution washing, Na
2sO
4drying, filters, is spin-dried for.Crude product through silica gel (300 ~ 400 order) flash column chromatography, methylene chloride-methanol (80: 1) eluting, concentrating under reduced pressure collect liquid, obtain yellow solid formula II compound;
Record in the ratio in embodiment 4 and step and prepare enoxolone list targeting long circulating liposomes
| Prescription | Consumption |
| DSPE | 200mg |
| Formula II compound | 100mg |
| Ether | 3mL |
| Gypenoside | 20mg |
| Phosphate buffer (pH=7.0) | 15mL |
Technique
(1) the choosing of raw material: get phosphate buffer and gypenoside by above-mentioned prescription, gypenoside is dissolved in phosphate buffer, for subsequent use;
(2) preparation of gypenoside liposome: get DSPE, formula II compound and ether by above-mentioned prescription, mix homogeneously, then rotary evaporation make its uniformly lipoid plastic film covering at pear shape bottle inner bottom part, the phosphate buffer containing gypenoside is added in bottle, add bead, at 60 DEG C after rotational oscillation 30min, place the abundant hydration of 2h, to obtain final product.
Common phospholipid modified long circulating liposomes (PEG-DSPE 2000 long circulating liposomes)
The common phospholipid modified long circulating liposomes of preparation is recorded in the ratio in embodiment 4 and step
| Prescription | Consumption |
| PEG-DSPE 2000 | 200mg |
| Ether | 3mL |
| Gypenoside | 20mg |
| Phosphate buffer (pH=7.0) | 15mL |
Technique
(1) the choosing of raw material: get phosphate buffer and gypenoside by above-mentioned prescription, gypenoside is dissolved in phosphate buffer, for subsequent use;
(2) preparation of gypenoside liposome: get PEG-DSPE 2000 and ether by above-mentioned prescription, mix homogeneously, then rotary evaporation make its uniformly lipoid plastic film covering at pear shape bottle inner bottom part, the phosphate buffer containing gypenoside is added in bottle, add bead, at 60 DEG C after rotational oscillation 30min, place the abundant hydration of 2h, to obtain final product.
The mensuration of envelop rate
The each sample solution 4mL that accurate absorption prepares, 16000Rmin
-1high speed centrifugation 15min.Aspirate supernatant is that the PBS buffer standardize solution of 7.0 is in 10mL volumetric flask with pH.Get 0, the above-mentioned solution of 1000 μ L is placed in the test tube of tool plug respectively, each precision adds 5% vanillin-glacial acetic acid and perchloric acid mixed liquor 2mL (2: 8), shake up, close plug, put in 60 DEG C of water-baths and heat 15min, room temperature is cooled to immediately with flowing water, each precision adds glacial acetic acid 10mL, shakes up, and at 555nm place, ultraviolet method records absorbance.Separately get centrifugal after precipitation, breakdown of emulsion in the Trition X-100 alcoholic solution (0.5mL) being dissolved in 10%, with ethanol standardize solution to 10mL, shakes up.Get 0,1000 μ L breakdown of emulsion solution respectively and be placed in tool plug test tube, in like manner add developer, survey absorbance.According to standard curve, computational envelope rate.
Significant difference is there is in single target liposomes with two target liposomes envelop rate.
Particle diameter, Zeta potential measure
Gypenoside long circulating liposomes in Example and common phospholipid modified gypenoside long circulating liposomes solution, laser particle analyzer measures its particle diameter and Zeta potential thereof.
Stability test:
(1) leak rate test: the dual Liver targeting gypenoside long circulating liposomes in the embodiment 1 in the present invention is placed on (pH 3,4,5 in the phosphate buffer of different pH value from enoxolone list targeting long circulating liposome preparation, 6,7,8,9,10), residual in liposome saponin amount (%) carries out measuring (0 after different number of days, 7,14,21,28,35,42,49,56,63 days).
A: represent the dual Liver targeting gypenoside long circulating liposomes contained in the embodiment 4 of formula I in the present invention.
B: represent gypenoside enoxolone list targeting long circulating liposomes.
Result shows that dual Liver targeting gypenoside long circulating liposomes is stablized at various ph values than enoxolone list targeting long circulating liposomes.
(2) oxidation stability test: the dual Liver targeting gypenoside long circulating liposomes in the present invention and enoxolone list targeting long circulating liposome preparation are placed in 25 DEG C, (0,7,14 are measured with thiobarbituric acid-reactive substances method (TBARS) after placing alloted days, 21,28,35,42,49,56,63 days) measure the degree of oxidation of liposome, value is higher shows that degree of oxidation is more serious.
| Sample | A | B |
| 0 day | 0.29 | 0.43 |
| 7 days | 0.32 | 0.7 |
| 14 days | 0.43 | 0.65 |
| 21 days | 0.42 | 0.79 |
| 28 days | 0.46 | 0.82 |
| 35 days | 0.51 | 0.94 |
| 42 days | 0.59 | 1 |
| 49 days | 0.76 | 1.04 |
| 56 days | 0.85 | 1.26 |
| 63 days | 0.89 | 1.39 |
A: represent the dual Liver targeting gypenoside long circulating liposomes contained in the embodiment 4 of formula I in the present invention.
B: represent gypenoside enoxolone list targeting long circulating liposomes.
Result shows that dual Liver targeting gypenoside long circulating liposomes is better than enoxolone list targeting long circulating liposomes oxidation stability.
Antitumaous effect is studied
(1) MTT experiment
Mtt assay detects and cultured cells is inoculated in the culture plate in 96 holes, is placed in the CO of saturated humidity
2continue in incubator to cultivate 24h (37 DEG C, 5%CO
2), discard culture fluid, then to add DMEM culture fluid appropriate in every hole, blank group adds the blank serum of not pastille, positive drug group adds containing 5-fluorouracil serum, two targeting long circulating liposomes, single targeting long circulating liposomes, conventional liposome, gypenoside group, add calf serum respectively.Continue to cultivate 24h, obtained subject cell liquid.Every hole adds MTT solution, hatches 4h for 37 DEG C, sucks the culture fluid in every hole, and every hole adds dimethyl sulfoxide (DMSO), adopts enzyme linked immunoassay detector to measure each hole OD value, measure under certain wavelength after vibration, and zeroing hole is only containing DMSO.Gained each hole mean OD value is by following formulae discovery inhibition rate of tumor cell:
Suppression ratio (%)=(matched group mean OD value-administration group mean OD value)/matched group mean OD value × 100%.
Result shows that dual Liver targeting gypenoside long circulating liposomes is higher than the inhibition rate of tumor cell of enoxolone list targeting long circulating liposomes, and the two exists pole significant difference statistically.
(2) cell cycle kinetics research
Get the subject cell liquid in above-mentioned experiment, add each group of sample and free gypenoside respectively in complete culture solution, separately establish cell blank matched group.Cultivate 48h.Collecting cell, detects with flow cytometer (COULTER company of the U.S.), and Cellfit software collects 1 × 10
5cell, MULTICYCLE software analysis data, record the ratio of each phase of each cell cycle, according to occurring in DNA content prescription figure that sub-peak, AP district cell percentage calculates apoptotic body percentage rate.
Result shows that dual Liver targeting gypenoside long circulating liposomes is higher than the apoptotic body percentage rate of enoxolone list targeting long circulating liposomes, and the two exists pole significant difference statistically.
(3) tumor inhibition
By male mouse of kunming by body weight random packet, often organize 15.Intraperitoneal injection sterile liquid paraffin, within the 3rd day, intraperitoneal injection people HepG2 hepatoma carcinoma cell suspension is appropriate.10th day, matched group intraperitoneal injection normal saline, the intraperitoneal injection of gypenoside group dissociates gypenoside, dual Liver targeting group intraperitoneal injection dual Liver targeting Herb Gynostemmae Pentaphylli liposome, single Liver targeting group, to group intraperitoneal injection list Liver targeting Herb Gynostemmae Pentaphylli liposome, is 2 times weekly.Within after tumor planting 8 weeks, put to death animal, observe lymph node, liver, spleen, peritoneum, kidney, mesenteric mesaraic transfer case, peel off tumor and claim tumor weight, calculate tumour inhibiting rate to judge its tumor-inhibiting action; Stripping spleen, thymus are weighed, Computation immunity systematic influence index.Computing formula is as follows:
The average tumor of tumour inhibiting rate (%)=(matched group average tumor weight-treatment group average tumor weight)/matched group heavy × 100%.
Intrusion Index (%)=(matched group average weight-treatment group average weight)/matched group average weight × 100%.
Result shows that dual Liver targeting gypenoside long circulating liposomes is higher than enoxolone list targeting long circulating liposomes tumour inhibiting rate, and the two exists pole significant difference statistically; Comparatively enoxolone list targeting long circulating liposomes is low for Intrusion Index, and there is significant difference statistically.Pharmacokinetic studies
Adopt LC-MS/MS, with ginsenoside Rb
1for contrast, ginsenoside Rb in Simultaneously test rat different time sections blood
1, content, calculate pharmacokinetic parameters.
(1) collection of administration and sample
Healthy Wistar rat, before experiment, water is can't help in 12h fasting, and experimental session is freely drunk water, and takes food after experiment 12h.If high, medium and low 3 dosage groups, each blood sampling point has 6 rats, and every rat is taken a blood sample 1 time.Get blood different time sections retroorbital venous clump respectively after administration, put in heparinised tubes, centrifugal, separated plasma, to be measured.
(2) plasma sample process
Get rat plasma sample, add the Hcl100 μ l protein precipitation of 1Mol/L, 10000 leave heart 10min, get supernatant, 0.45 μm of filtering with microporous membrane, sample introduction 5 μ l, analyze.
(3) in vivoassay method
Chromatogram is obtained, by ginsenoside Rb with rat blank plasma samples sample introduction
1reference substance series joins blank blood, according to carrying out sample treatment with method; The plasma sample same treatment of collecting after getting rat administration, obtains plasma sample chromatogram after rat administration, analyzes by external standard method.
DAS 2.0 program fitting result shows, after the gypenoside long circulating liposomes that the dual Liver targeting gypenoside long circulating liposomes containing formula I in the present invention and common phospholipid PEG-DSPE 2000 (DSPE-PEG2000) are modified, two kinds of preparations all meet two compartment models in rat body, and its average pharmacokinetic parameters is as shown in the table.
A: represent the dual Liver targeting gypenoside long circulating liposomes contained in the embodiment 1 of formula I in the present invention.
B: represent the gypenoside long circulating liposomes that common phospholipid PEG-DSPE 2000 (DSPE-PEG2000) is modified.
C: represent galacturonic acid list targeting long circulating liposomes.
D: represent enoxolone list targeting long circulating liposomes.
*: check significant difference (p < 0.05) with the pharmacokinetic parameters of A group through t.
*: check pole significant difference (p < 0.01) through t with the pharmacokinetic parameters of A group.
Result shows that the dual Liver targeting gypenoside long circulating liposomes clearance rate Cl in the present invention is lower compared with other group, statistically there is pole significant difference.AUC is higher compared with other group, statistically there is pole significant difference.Show that the dual Liver targeting gypenoside long circulating liposomes in the present invention has better long circulating effect.
Hepatic cell selective
(1) hepatic parenchymal cells: mice 30, be divided into 3 groups at random, long circulating GPS liposome modified by tail vein injection GPS solution, GPS conventional liposome, galactose-enoxolone respectively, after administration, different time sections is by mice etherization, portal catheterization injecting normal saline, blood in liver is removed clean, take off liver, shred, add collagenase solution and digest in 37 DEG C of shaking tables, then by Digestive system filtered through gauze, filtrate 1000r/min, centrifugal 1.5min, sucking-off supernatant is stand-by, precipitation washes 3 times, obtains hepatic parenchymal cells.
(2) liver non-parenchymal cell: the centrifugal 5min of supernatant 2000r/min, the precipitation obtained washes 3 times under similarity condition again, to obtain final product.
Respectively treating excess syndrome matter and nonparenchymal cell sample, with the D-Hanks solution washing 3 times of 4 DEG C, then adds D-Hanks solution, and culture plate is put into the refrigerator multigelation 3 times of-70 DEG C, piping and druming cell is secondary repeatedly makes cell breakage.Gypenoside content in hepatic parenchymal cells, nonparenchymal cell is measured, tries to achieve the uptake ratio of hepatic parenchymal cells ingestion of drugs.
Result shows that dual Liver targeting gypenoside long circulating liposomes is higher than enoxolone list targeting long circulating liposomes hepatic parenchymal cells uptake ratio, and the two exists pole significant difference statistically.
(3) tissue distribution experiment
Core, liver, spleen, lung, kidney, brain tissue homogenate's liquid, survey percent drug.
Result shows that dual Liver targeting gypenoside long circulating liposomes is more in the distribution of liver than enoxolone list targeting long circulating liposomes, and the two exists pole significant difference statistically, show that the dual Liver targeting gypenoside long circulating liposomes hepatic targeting in the present invention is better.
In sum: the present invention is a kind of novel dual Liver targeting long circulating liposomes, envelop rate is higher, slip, oxidation stability experimental result show, liposome stability of the present invention is better, MTT experiment, cell cycle kinetics research, tumor inhibition show that the novel dual Liver targeting long circulating liposomes tumor-inhibiting action in the present invention is significantly better than other experimental group, have better antitumaous effect.Immune system Intrusion Index shows that novel dual Liver targeting long circulating liposomes in the present invention is while tumor suppression, is also significantly better than other experimental group to immune impact.Pharmacokinetic studies, hepatic cell selective research, tissue distribution experimental result also show that the hepatic targeting of the novel dual Liver targeting long circulating liposomes in the present invention is stronger, the prolong drug holdup time in vivo, make medicine better reach hepatic parenchymal cells, reach better therapeutic effect.
Claims (9)
1. galacturonic acid, enoxolone, polyethyleneglycol modified cupreol are a dual Liver targeting long circulating liposomes for film material, it is characterized in that, containing such as formula the compound shown in I in material, the preparation method of this compound comprises step:
(1) cupreol, succinic anhydride, DMAP are mixed in dichloromethane, are obtained by reacting such as formula the compound shown in IV at 30-60 DEG C;
(2) by formula IV compound, amino-end peg, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, is obtained by reacting the compound as shown in formula III at 30-60 DEG C;
(3) by formula III compound, enoxolone, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, is obtained by reacting such as formula the compound shown in II at 30-60 DEG C;
(4) by formula II compound, galacturonic acid, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed in dichloromethane, is obtained by reacting such as formula the compound shown in I at 30-60 DEG C;
In the reaction of step (1), the mol ratio of cupreol, succinic anhydride, DMAP is 1: 0.8-10: 0.8-5;
The mol ratio of step (2) compound of formula IV, amino-end peg, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1: 0.8-3: 0.8-3: 0.8-3;
The mol ratio of the reaction compound of formula III of step (3), enoxolone, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1: 0.8-10: 0.8-5: 0.8-5;
The mol ratio of step (4) compound of formula H, galacturonic acid, DMAP, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1: 0.8-3: 0.8-3: 0.8-3;
Wherein, n=30-60.
2. dual Liver targeting long circulating liposomes as claimed in claim 1, is characterized in that it is mainly made up of gypenoside, phospholipid material, formula I.
3. the dual Liver targeting long circulating liposomes as described in claim 1,2, it is characterized in that, described phospholipid material is: the mixture of one or more in the acid of sphingomyelins, DSPE, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidylinositols, phosphatidyl glycerol, phosphoglyceride, diphosphatidylglycerol.
4. the dual Liver targeting long circulating liposomes as described in claim 1,2, is characterized in that, the composition of described liposome also comprises one or more in mannitol, glucose, tween 80, vitamin E or Polyethylene Glycol.
5. the dual Liver targeting long circulating liposomes as described in claim 1,2, is characterized in that this liposome is containing following component by weight percentage: gypenoside 2% ~ 45%, phospholipid material 40 ~ 85%, formula I accounts for 10 ~ 40% of weight of formulation.
6. the preparation method of dual Liver targeting long circulating liposomes described in any one of claim 1-5, it is characterized in that it comprises the steps: (1) gypenoside being dissolved in pH is that 6.8 ~ 8.0 phosphoric acid rush in liquid, and making mass concentration is 0.1 ~ 20mg/mL gypenoside buffer;
(2) when solid unit is milligram hour, liquid unit in milliliter, by that analogy.Phospholipid material: formula I: ether: the ratio of gypenoside buffer soln is chosen by 100 ~ 300 parts: 15 ~ 200 parts: 2 ~ 5 parts: 5 ~ 30 parts, for subsequent use;
(3) preparation of dual Liver targeting long circulating gypenoside liposome: phospholipid material, formula I and ether are mixed, then rotary evaporation make its uniformly lipoid plastic film covering at pear shape bottle inner bottom part, gypenoside buffer soln is added in bottle, add bead, at 30 ~ 60 DEG C after rotational oscillation 15 ~ 60min, place the abundant hydration of 0.5 ~ 2h and get final product.
7. the preparation method of dual Liver targeting long circulating liposomes described in any one of claim 1-5, is characterized in that it comprises the steps:
(1) when solid unit is milligram hour, liquid unit in milliliter, by that analogy.The preparation of oil phase: gypenoside: phospholipid material: formula I: the ratio of ethanol is in the ratio chosen material of 10 ~ 30 parts: 40 ~ 300 parts: 10 ~ 100 parts: 5 ~ 50 parts, and mix homogeneously, prepares oil phase;
(2) preparation of aqueous phase: aqueous phase is phosphate buffer 5 ~ 50 parts, the pH of buffer is 6.8 ~ 8.0;
(3) preparation of dual Liver targeting long circulating liposomes: under constant temperature stirring, oil phase syringe needle being at the uniform velocity injected into temperature is in the aqueous phase of 35 ~ 60 DEG C, and constant temperature stirs 1 ~ 4h except ethanol, to obtain final product.
8. the preparation method of dual Liver targeting long circulating liposomes described in any one of claim 1-5, is characterized in that it comprises the steps:
(1) when solid unit is milligram hour, liquid unit in milliliter, by that analogy.Phospholipid material: formula I: the ratio of dichloromethane is prepared oil phase by 40 ~ 300 parts: 10 ~ 200 parts: 50 ~ 500 parts, oil phase is placed in round-bottomed flask, rotates steaming vibrating dichloromethane;
(2) when solid unit is milligram hour, liquid unit in milliliter, by that analogy.Be dissolved in 10 ~ 500 parts of phosphate buffers by 10 ~ 200 parts of gypenosides, make aqueous phase;
(3) add in oil phase by aqueous phase, under ice bath, Probe Ultrasonic Searching 5 ~ 30min, forms uniform Emulsion;
(4) revolve steaming removing organic facies, add phosphate buffer 10 ~ 150 part after forming colloidal state, continue reduction vaporization 0.5 ~ 2h, Probe Ultrasonic Searching 3 ~ 20min, to obtain final product.
9. dual Liver targeting long circulating liposomes as claimed in claim 1, is characterized in that it can make lyophilized injection, injection, preparation capable of permeating skin, aerosol, oral liquid, solution, ophthalmic preparation, tablet or capsule.
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| CN112778389A (en) * | 2019-12-31 | 2021-05-11 | 嘉应学院医学院 | Targeting ligand molecule, preparation method thereof and drug loading system |
| CN113318234A (en) * | 2021-06-11 | 2021-08-31 | 盐城师范学院 | Arginine and ursolic acid modified chitosan nano drug delivery carrier and preparation method and application thereof |
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2014
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112778389A (en) * | 2019-12-31 | 2021-05-11 | 嘉应学院医学院 | Targeting ligand molecule, preparation method thereof and drug loading system |
| CN113318234A (en) * | 2021-06-11 | 2021-08-31 | 盐城师范学院 | Arginine and ursolic acid modified chitosan nano drug delivery carrier and preparation method and application thereof |
| CN113318234B (en) * | 2021-06-11 | 2022-03-08 | 盐城师范学院 | Arginine and ursolic acid modified chitosan nano drug delivery carrier and preparation method and application thereof |
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