CN101288646A - Lipid nanoparticles with anti-hepatitis B activity and application - Google Patents
Lipid nanoparticles with anti-hepatitis B activity and application Download PDFInfo
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- CN101288646A CN101288646A CNA200810061446XA CN200810061446A CN101288646A CN 101288646 A CN101288646 A CN 101288646A CN A200810061446X A CNA200810061446X A CN A200810061446XA CN 200810061446 A CN200810061446 A CN 200810061446A CN 101288646 A CN101288646 A CN 101288646A
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Abstract
The invention provides a lipid nanoparticle with anti-HBV activity, the lipid nanoparticle is composed of anti-viral drug and lipid material, and the drug selects adefovir dipivoxil for usage. The invention utilizes rapid cell uptake and cytoplasmic detaining functions of the solid lipid nanoparticle and a nano-structure lipid carrier to envelope the anti-viral drug with a molecular target positioned in the cell, thus leading the viral cells to greatly increase the uptake of the drug and increasing the drug concentration at the drug molecular target site. The lipid nanoparticle can cause the viral cells to increase the uptake of the drug, be conductive to reducing the distribution of the drug in the normal tissues or cells and reduce the toxicity and the side effects of the drugs; the increase the drug concentration at the drug molecular target site is conductive to improving the efficacy of the anti-viral drug, so the lipid nanoparticle can be applied in the preparation of the anti-HBV drugs.
Description
Technical field
The invention belongs to the application of nanoparticle, related generally to a kind of load antiviral drugs lipid nanoparticle and in the application of preparation in the anti-hepatic-B virus medicine.
Background technology
Viral disease has become one of main threat of human health as infectious disease.Chronic hepatitis B is the serious viral infectious of harm, is the main reason that causes hepatic fibrosis, liver cirrhosis and hepatocarcinoma.The whole world has 500,000 people of surpassing die from primary hepatocarcinoma every year, and wherein nearly 80% primary hepatocarcinoma cause by chronic viral hepatitis B.Because existing antiviral drugs can not suppress, remove virus fully, exist that dosage is big, curative effect is low, toxic and side effects and easily produce drug-fast problem, therefore, for the treatment of viral disease, especially viral chronically infected treatment remains a difficult problem medically.
Existing antiviral chemotherapeutics, the interior non-specific characteristic distributions of body with general chemotherapeutics, be subjected to biomembrane absorption and transhipment barrier in the body simultaneously, the influence of enzymes metabolism, medicine can't enter its molecular target in a large number, thereby causes that curative effect is low, dosage is big, toxic and side effects and chemical sproof generation.By the targeting preparation technology, distribution pattern in the medicine body that change is taken as the leading factor with the medicine self property, ectogenic drug conveying to pharmaceutically-active molecular target, can be improved pharmaceutically-active curative effect to greatest extent, reduce distribution and the toxic and side effects of medicine at normal internal organs.The molecular action site of anti-HBV chemotherapeutics except that HBV absorption inhibitor, all acts on the subcellular organelle in the host cell, and therefore ideal anti-HBV targeting preparation should have liver, the intracellular subcellular organelle target function of hepatocyte regulating liver-QI efficiently.Scientist both domestic and external, by to the regulation and control of pharmaceutical carrier size, surface physicochemical property and part on this basis or antibody modification technology, at present realized the targeted of medicine to liver cell, subcellular organelle targeting vector technology has become the core link of current realization drug molecule targeted therapy.
The solid lipid drug-supplying system is the new colloidal drug-supplying system that grows up early 1990s, and it is after Emulsion, liposome, polymer nanoparticle, has the target controlling and releasing drug-supplying system of development potentiality.The solid lipid drug-supplying system adopts natural or synthetic lipid materials, is carrier as stearic acid, lecithin, monoglyceride etc., pharmaceutical pack is wrapped in the lipoid nuclear makes solid micelle drug-supplying system.It had both possessed polymeric drug delivery system controlled release, had avoided advantages such as drug leakage, and the toxicity that has had Emulsion, liposome again concurrently is low, good biocompatibility, advantage that bioavailability is high.But also there are some potential limitation in the solid lipid drug-supplying system, squeezes problems such as phenomenon as limited medicine carrying ability, the medicine of storage process.
The different liquid fatty of mixed form is as mixing lipid substrate in solid lipid, with this prepare novel nano structured lipid carrier (nanostructured lipid carrier, NLC).The adding of liquid fatty can be upset the lattice structure of solid lipid rule, increases the ratio of irregular crystal formation in the nanoparticle structure, and the spatial content of carrying fat-soluble medicine is increased, thereby improves the medicine carrying ability of carrier.By controlling liquid lipid ratio, also can make NLC under body temperature, keep the solid skeletal structure, realize that the NLC controlled delivery of pharmaceutical agents discharges.Discover that solid lipid nanoparticle and nano structured lipid carrier all have cellular uptake and cytoplasm retention characteristics fast.
Summary of the invention
An object of the present invention is to provide a kind of lipid nanoparticle with anti-hepatitis B activity, described lipid nanoparticle is made up of antiviral drugs and matrix material, this lipid nanometer particle loading molecular target be positioned at intracellular antiviral drugs, described antiviral drugs comprises medicines such as adefovir ester, and the content of adefovir ester is the 1%-4% of lipid nanoparticle.Matrix material wherein can be made up of one or both lipids such as glyceryl monostearate, tripalmitin, stearic acid or glyceryl monostearate and oleic acid etc., and its weight accounts for the 96-99% of nanoparticle; When wherein being made up of two kinds of matrix materials, the ratio that oleic acid accounts for matrix material is 5-20%;
Another object of the present invention provides the application of this lipid nanoparticle in the preparation anti-hepatic-B virus medicine.After this lipid nanoparticle and virus infected cell are hatched altogether, compare, can significantly suppress surface antigen (HBsAg), e antigen (HBeAg) and DNA (HBV DNA) content of virus, embody good antiviral curative effect with the solution contrast of medicine.
Usefulness of the present invention is: be detained the lipid nanoparticle of function by having efficient cellular uptake and cytoplasm, molecular target is positioned at sealing of intracellular antiviral drugs, can increase the picked-up of virocyte greatly to medicine, and the drug level at molecular drug target position.Increase the picked-up of virocyte, help reducing the distribution of medicine, reduce the toxic and side effects of medicine at normal structure or cell to medicine; And the increase of the drug level at molecular drug target position helps improving the curative effect of antiviral drugs.
The present invention utilizes solid lipid nanoparticle and nano structured lipid carrier all to have cellular uptake and cytoplasm delay function fast, antiviral drugs such as load adefovir ester, improving the drug level at molecular drug target position, thereby reach the purpose of the anti-hepatitis virus curative effect that improves such medicine.
Description of drawings
Fig. 1 is for after the adefovir ester lipid nanoparticle of different pharmaceutical concentration and adefovirdipivoxil ester solution hatch altogether with the HepG2.2.15 cell, to the suppression ratio variation of HBsAg.
Fig. 2 is for after the adefovir ester lipid nanoparticle of different pharmaceutical concentration and adefovirdipivoxil ester solution hatch altogether with the HepG2.2.15 cell, to the suppression ratio variation of HBeAg.
Fig. 3 is for after the adefovir ester lipid nanoparticle of different pharmaceutical concentration and adefovirdipivoxil ester solution hatch altogether with the HepG2.2.15 cell, the suppression ratio variation that HBV-DNA is expressed.
The specific embodiment
The present invention is described further in conjunction with the accompanying drawings and embodiments.
Embodiment 1: the preparation and the application of load adefovir ester glyceryl monostearate lipid nanoparticle
1) preparation of load adefovir ester monoglyceride lipid nanoparticle
Precision takes by weighing glyceryl monostearate 50mg and 10mg adefovir ester respectively, places the 2mL dehydrated alcohol, 50 ℃ of dissolvings of water-bath.Phosphate buffer with PH7.2 is a decentralized photo, puts in the ice bath.At 400rmin
-1Under the mechanical agitation condition, organic facies is injected the 10mL decentralized photo, stir 5min, obtain the lipid nanoparticle dispersion liquid of load adefovir ester.The lipid nanoparticle dispersion liquid 3molL of resultant adefovir ester
-1HCl solution is regulated pH to 1.2, with 20000rmin
-1Centrifugal 10min, precipitation adds 0.1% poloxamer (Poloxamer) (w/v) behind the redispersion, uses 1molL
-1NaOH solution is regulated pH to 7.0.Promptly obtain the lipid nanoparticle dispersion liquid of adefovir ester.
Table 1 is the physicochemical properties such as particle diameter, surface potential and entrapment efficiency of preparation-obtained load adefovir ester glyceryl monostearate lipid nanoparticle.
Table 1: particle diameter, surface potential and the entrapment efficiency of load adefovir ester glyceryl monostearate lipid nanoparticle.
2) the external anti-hepatitis virus curative effect of load adefovir ester monoglyceride lipid nanoparticle
Get the HepG2.2.15 cell, in the RPMI RPMI-1640 that contains 10% calf serum of having an appointment, cultivate (5%CO
2, 37 ℃ of incubators).When cell reaches exponential phase, can inoculate.The trophophase cell of taking the logarithm after the PBS rinse, adds trypsinization and with the culture fluid dilution, by every hole 1 * 10
4The density of individual cell is inoculated in 24 well culture plates, cultivates 24 hours in the incubator.After the 24 well culture plate inner cell adherent growth, discard old culture fluid, add adefovir ester lipid nanoparticle and the adefovirdipivoxil ester solution that contains different pharmaceutical concentration respectively after using buffer solution (PBS) rinse 2 times of PH7.4, continue to cultivate after 2-5 days, collect culture fluid, surface antigen (HBsAg) in the enzyme immunoassay (EIA) kit measurement cell culture fluid and e antigen (HBeAg) content, instant PCR quantitative method is measured hepatitis B virus DNA (HBV DNA) content.After the adefovir ester lipid nanoparticle of different pharmaceutical concentration and adefovirdipivoxil ester solution and HepG2.2.15 cell were hatched altogether, to HBsAg, the suppression ratio result of variations that HBeAg and HBV-DNA express was seen Fig. 1-3.
The result shows adefovir ester after lipid nanoparticle is sealed, and compares with the adefovirdipivoxil ester solution, and to HBsAg, the suppression ratio that HBeAg and HBV-DNA express significantly increases, and discloses the remarkable antiviral effect of adefovir ester through lipid nanoparticle.
Embodiment 2:
The preparation of load adefovir ester monoglyceride lipid nanoparticle
Precision takes by weighing glyceryl monostearate 50mg and 5mg adefovir ester respectively, places the 2mL dehydrated alcohol, 50 ℃ of dissolvings of water-bath.Phosphate buffer with PH7.2 is a decentralized photo, puts in the ice bath.At 400rmin
-1Under the mechanical agitation condition, organic facies is injected the 10mL decentralized photo, stir 5min, obtain the lipid nanoparticle dispersion liquid of load adefovir ester.The lipid nanoparticle dispersion liquid 3molL of resultant adefovir ester
-1HCl solution is regulated pH to 1.2, with 20000rmin
-1Centrifugal 10min, precipitation adds 0.1% poloxamer (Poloxamer) (w/v) behind the redispersion, uses 1molL
-1NaOH solution is regulated pH to 7.0.Promptly obtain the lipid nanoparticle dispersion liquid of adefovir ester.
Table 2 is the physicochemical properties such as particle diameter, surface potential and entrapment efficiency of preparation-obtained load adefovir ester glyceryl monostearate lipid nanoparticle.
Table 2: particle diameter, surface potential and the entrapment efficiency of load adefovir ester glyceryl monostearate lipid nanoparticle.
Embodiment 3:
The preparation of load adefovir ester stearic acid lipid nanoparticle
Precision takes by weighing monostearate 50mg and 10mg adefovir ester respectively, places the 2mL dehydrated alcohol, 50 ℃ of dissolvings of water-bath.Phosphate buffer with PH7.2 is a decentralized photo, puts in the ice bath.At 400rmin
-1Under the mechanical agitation condition, organic facies is injected the 10mL decentralized photo, stir 5min, obtain the lipid nanoparticle dispersion liquid of load adefovir ester.The lipid nanoparticle dispersion liquid 3molL of resultant adefovir ester
-1HCl solution is regulated pH to 1.2, with 20000rmin
-1Centrifugal 10min, precipitation adds 0.1% poloxamer (Poloxamer) (w/v) behind the redispersion, uses 1molL
-1NaOH solution is regulated pH to 7.0.Promptly obtain the lipid nanoparticle dispersion liquid of adefovir ester.
Table 3 is the physicochemical properties such as particle diameter, surface potential and entrapment efficiency of preparation-obtained load adefovir ester stearic acid lipid nanoparticle.
Table 3: particle diameter, surface potential and the entrapment efficiency of load adefovir ester stearic acid lipid nanoparticle.
Embodiment 4:
The preparation of load adefovir ester glyceryl monostearate oleic acid lipid nanoparticle
Precision takes by weighing glyceryl monostearate 47.5mg respectively, and oleic acid 2.5mg and 10mg adefovir ester place the 2mL dehydrated alcohol, 50 ℃ of dissolvings of water-bath.Phosphate buffer with PH7.2 is a decentralized photo, puts in the ice bath.At 400rmin
-1Under the mechanical agitation condition, organic facies is injected the 10mL decentralized photo, stir 5min, obtain the lipid nanoparticle dispersion liquid of load adefovir ester.The lipid nanoparticle dispersion liquid 3molL of resultant adefovir ester
-1HCl solution is regulated pH to 1.2, with 20000rmin
-1Centrifugal 10min, precipitation adds 0.1% poloxamer (Poloxamer) (w/v) behind the redispersion, uses 1molL
-1NaOH solution is regulated pH to 7.0.Promptly obtain the lipid nanoparticle dispersion liquid of adefovir ester.
Table 4 is the physicochemical properties such as particle diameter, surface potential and entrapment efficiency of preparation-obtained load adefovir ester lipid nanoparticle.
Table 4: particle diameter, surface potential and the entrapment efficiency of load adefovir ester lipid nanoparticle.
Embodiment 5:
The preparation of load adefovir ester glyceryl monostearate oleic acid lipid nanoparticle
Precision takes by weighing glyceryl monostearate 40mg respectively, and oleic acid 10mg and 10mg adefovir ester place the 2mL dehydrated alcohol, 50 ℃ of dissolvings of water-bath.Phosphate buffer with PH7.2 is a decentralized photo, puts in the ice bath.At 400rmin
-1Under the mechanical agitation condition, organic facies is injected the 10mL decentralized photo, stir 5min, obtain the lipid nanoparticle dispersion liquid of load adefovir ester.The lipid nanoparticle dispersion liquid 3molL of resultant adefovir ester
-1HCl solution is regulated pH to 1.2, with 20000rmin
-1Centrifugal 10min, precipitation adds 0.1% poloxamer (Poloxamer) (w/v) behind the redispersion, uses 1molL
-1NaOH solution is regulated pH to 7.0.Promptly obtain the lipid nanoparticle dispersion liquid of adefovir ester.
Table 5 is the physicochemical properties such as particle diameter, surface potential and entrapment efficiency of preparation-obtained load adefovir ester lipid nanoparticle.
Table 5: particle diameter, surface potential and the entrapment efficiency of load adefovir ester lipid nanoparticle.
Embodiment 6: the preparation of load adefovir ester tripalmitin lipid nanoparticle
The preparation of load adefovir ester monoglyceride lipid nanoparticle
Precision takes by weighing tripalmitin 50mg and 10mg adefovir ester respectively, places the 2mL dehydrated alcohol, 50 ℃ of dissolvings of water-bath.Phosphate buffer with PH7.2 is a decentralized photo, puts in the ice bath.At 400rmin
-1Under the mechanical agitation condition, organic facies is injected the 10mL decentralized photo, stir 5min, obtain the lipid nanoparticle dispersion liquid of load adefovir ester.The lipid nanoparticle dispersion liquid 3molL of resultant adefovir ester
-1HCl solution is regulated pH to 1.2, with 20000rmin
-1Centrifugal 10min, precipitation adds 0.1% poloxamer (Poloxamer) (w/v) behind the redispersion, uses 1molL
-1NaOH solution is regulated pH to 7.0.Promptly obtain the lipid nanoparticle dispersion liquid of adefovir ester.
Table 6 is the physicochemical properties such as particle diameter, surface potential and entrapment efficiency of preparation-obtained load adefovir ester tripalmitin lipid nanoparticle.
Table 6: particle diameter, surface potential and the entrapment efficiency of load adefovir ester tripalmitin lipid nanoparticle.
Claims (4)
1. lipid nanoparticle with anti-hepatitis B activity, described lipid nanoparticle is made up of medicine and matrix material, it is characterized in that: this lipid nanometer particle loading antiviral drugs, this antiviral drugs is that molecular target is positioned at intracellular medicine.
2. a kind of lipid nanoparticle with anti-hepatitis B activity according to claim 1 is characterized in that: described choice of drug adefovir ester, the content of adefovir ester are the 1%-4% of lipid nanoparticle.
3. a kind of lipid nanoparticle according to claim 1 with anti-hepatitis B activity, it is characterized in that: matrix material wherein can be made up of one or both lipids such as glyceryl monostearate, tripalmitin, stearic acid or glyceryl monostearate and oleic acid etc., its weight accounts for the 96-99% of nanoparticle, when wherein being made up of two kinds of matrix materials, the ratio that oleic acid accounts for matrix material is 5-20%.
4. a kind of application of lipid nanoparticle in the preparation anti-hepatic-B virus medicine according to claim 1 with anti-hepatitis B activity.
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Cited By (1)
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| CN115227672B (en) * | 2022-07-14 | 2023-10-13 | 浙江大学 | Solid lipid nanoparticle for promoting tumor vascular normalization and preparation and application thereof |
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| CN115227672B (en) * | 2022-07-14 | 2023-10-13 | 浙江大学 | Solid lipid nanoparticle for promoting tumor vascular normalization and preparation and application thereof |
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Open date: 20081022 |