WO2015135432A1 - 靶向脂质体的制备及其应用 - Google Patents

靶向脂质体的制备及其应用 Download PDF

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WO2015135432A1
WO2015135432A1 PCT/CN2015/073610 CN2015073610W WO2015135432A1 WO 2015135432 A1 WO2015135432 A1 WO 2015135432A1 CN 2015073610 W CN2015073610 W CN 2015073610W WO 2015135432 A1 WO2015135432 A1 WO 2015135432A1
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formula
polypeptide
ntcp
dspe
myr
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French (fr)
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刘宏利
韩超
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上海吉贝医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331—Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C08G65/3311—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing a hydroxy group
    • C08G65/3312—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing a hydroxy group acyclic
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331—Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C08G65/332—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof
    • C08G65/3322—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof acyclic
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33303—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group
    • C08G65/33306—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group acyclic
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33331—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing imide group
    • C08G65/33337—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing imide group cyclic

Definitions

  • the present invention relates to the preparation of targeted liposomes and their use, in particular to the preparation of liver-targeted liposomes and their use.
  • Liver-related diseases are one of the most important diseases affecting human health. The number of deaths due to end-stage liver disease in China exceeds 300,000 per year.
  • most of the drugs currently used for the treatment of liver diseases are systemic administration, which have the disadvantages of less liver distribution, unstable body, and more extrahepatic side effects, which limits the clinical application of the drug. Therefore, how to effectively deliver drugs to the liver, better use of therapeutic effects, and reduction of extrahepatic side effects are major problems in the field of liver disease treatment (Poelstra K, Prakash J, Beljaars L. Drug targeting to the diseased liver [J] .Journal of controlled release: official journal of the Controlled Release Society, 2012, 161(2): 188-197).
  • the liver-targeted drug delivery system can specifically target the drug to the liver, which is beneficial to liver enrichment, reducing the dosage of the drug and reducing the toxic side effects of the system (He Ling, Li Jianhe. Research progress of liver-targeted drug delivery system [J] Research Progress, 2010, 17(9): 15-17).
  • Liposomes are one of the most commonly used drug-loading models because of their low immunogenicity, wide drug-loading range, protection of drug activity, stability and sustained release.
  • ordinary liposomes can not achieve the effect of targeted delivery, so it is necessary to target the common liposome to achieve active targeting (Pranali PD, Swati B, Vladimir PT, et al. Current trends in The use of liposomes for tumor targeting [J]. Nanomedicine, 2013, 8(9): 1509-1528).
  • Hepatitis B virus is a hepadnavirus, and the PreS1 region of its outer membrane protein is an important site for binding to hepatocyte receptors (Liang TJ. Hepatitis B: The Virus and Disease [J]. Hepatology, 2009, 49 ( 5): S13-S21). Recent studies have revealed that sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for HBV (Yan H, Zhong G, Xu G, et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for Human hepatitis B and D virus [J].
  • NTCP sodium taurocholate cotransporting polypeptide
  • a multi-segment polypeptide derived from PreS1 including the D genotype preS/2-48 can specifically bind to it (Gripon P, Cannie I, Urban S. Efficient inhibition of hepatitis B virus infection by acifying peptides derived from the large viral surface protein [J]. J Virol, 2005, 79(3): 1613-1622; Petersen J, Dandri M, Mier W, et al. Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein [J]. Nat Biotechnol, 2008, 26(3): 335-341; Schulze A, Schieck A, Ni Y, et al.
  • HBVpreS/13-32 myr is used as a targeting sequence (SEQ ID NO. 3), which is chemically coupled to Mal-PEG-DSPE (maleimide-polyethylene glycol) - stearoylphosphatidylethanolamine) to produce HBVpreS/13-32 myr -PEG-DSPE; liposome was prepared by ethanol injection, and sodium fluorescein sodium (FS) was formed to form HBVpreS/13-32 myr- FS- LCL was used to analyze the targeting of nanoliposomes with HepG2 stably expressing NTCP as a cell model.
  • SEQ ID NO. 3 Mal-PEG-DSPE (maleimide-polyethylene glycol) - stearoylphosphatidylethanolamine)
  • liposome was prepared by ethanol injection, and sodium fluorescein sodium (FS) was formed to form HBVpreS/13-32 myr- FS- LCL was used to analyze the targeting of nanoliposomes with HepG
  • the synthesized targeted liposome is round and uniform in size, and the particle size is (94.09 ⁇ 9.2) nm, reaching the nanometer level; effectively encapsulating sodium fluorescein, the encapsulation efficiency is 89.32 ⁇ 1.02%; more sodium fluorescein can be delivered into the cell compared to the non-targeted modified liposome.
  • HBVpreS/13-32 myr modified targeting liposome with high encapsulation efficiency is a nano-liposome, can specifically target liver cells through NTCP, and is expected to become a new type of hepatocyte targeting Drug system.
  • the present invention provides a compound that targets hepatocytes, and the structure of the compound is as shown in Formula 1:
  • X is a polypeptide derived from the PreS1 region of the hepatitis B virus outer membrane protein that specifically binds to the sodium ion-taurocholic acid cotransport polypeptide (NTCP);
  • B is a maleimide-polyethylene glycol-stearoylphosphatidylethanolamine polymer represented by the following formula 2:
  • n is an integer of from 2 to 100, and DSPE is stearoylphosphatidylethanolamine;
  • X is a fragment derived from amino acid 2-219 of the PreS1 region of the hepatitis B virus outer membrane protein.
  • the X is preferably a fragment derived from amino acids 13-119 of the PreS 1 region of the hepatitis B virus outer membrane protein.
  • the X is further preferably a fragment derived from amino acids 13-59 of the PreS1 region of the hepatitis B virus outer membrane protein.
  • the X is further preferably a fragment derived from amino acids 13-49 of the PreS1 region of the hepatitis B virus outer membrane protein.
  • the X is further preferably a fragment derived from amino acids 13-39 of the PreS1 region of the hepatitis B virus outer membrane protein.
  • the X is further preferably a fragment derived from amino acids 13-32 of the PreS1 region of the hepatitis B virus outer membrane protein.
  • the N-terminus of X is modified with a fatty acid or cholesterol.
  • the N-terminus of X is modified with myristic acid.
  • amino acid sequence of the fragment is as set forth in any one of SEQ ID NOs: 1-3.
  • n is an integer from 2 to 80, preferably an integer from 5 to 50, preferably an integer from 10 to 40.
  • the polyethylene glycol has a molecular weight in the range of from 600 to 2,500, preferably from 1,000 to 2,500, more preferably from 1,500 to 2,000.
  • the polypeptide of X further comprises a cysteine residue at its C-terminus, This cysteine residue is linked to the maleimide moiety of B.
  • polypeptide of X further comprises a cysteine residue at its C-terminus, the amino acid sequence of which is set forth in SEQ ID NO:4.
  • the N-terminus of the polypeptide of X is modified by myristoylation.
  • the compound of the invention is as shown in Formula 3 below:
  • X, n and DSPE are as defined above.
  • the compound of the invention is as shown in Formula 4 below:
  • the polyethylene glycol in the formula has a molecular weight of 2,000.
  • the present invention relates to a composition for delivering a drug comprising the compound of the formula 1, 2, 3 or 4 of the present invention, and any one of phosphatidylcholine, cholesterol and PEG-DSPE, optionally Two or all three.
  • the invention is used to deliver a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the invention and a phosphatidylcholine, optionally further comprising cholesterol and/or PEG-DSPE.
  • the present invention is used in the delivery of a pharmaceutical composition
  • the weight ratio of the compound of the present invention, phosphatidylcholine, cholesterol and PEG-DSPE is from 1 to 100:1 to 1000:0 to 500:0 to 500. .
  • the composition for delivering a drug is a liposome.
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a liposome of the invention and a therapeutically effective amount of a medicament.
  • the invention further relates to the use of a compound of the formulae 1, 2, 3 and 4 of the invention for the manufacture of a medicament for the treatment of hepatitis B or for the inhibition of hepatitis B virus.
  • Figure 1 shows the HBVpreS/13-32 myr -PEG-DSPE synthesis equation.
  • Figure 2 shows that HepG2-NTCP cells successfully expressed NTCP.
  • a shows the use of RT-PCT
  • b-picture shows the results of NTCP expression on the cell surface by flow cytometry
  • c-picture shows the expression of NTCP on the cell surface by confocal laser scanning microscopy ( ⁇ 400). the result of.
  • Figure 3 shows the binding of HBVpreS/13-32 myr to cells by confocal microscopy.
  • HepG2-NTCP was co-incubated with HBVpreS/13-32 myr- FITC (a), Pep47-FITC (b), or untreated (c1-c3); HepG2-pCMV cells were also associated with HBVpreS/13-32, respectively.
  • Myr- FITC (d map), Pep47-FITC (e map) were co-incubated, or untreated (f map).
  • Figure 4 shows the HPLC and MALDI-TOF-MS detection of HBVpreS/13-32 myr -PEG 2000 -DSPE synthesis.
  • a figure shows the analysis of HBVpreS/13-32 myr before the reaction
  • b shows the analysis of HBVpreS/13-32 myr after the reaction
  • c shows the MALDI-TOF-MS analysis of the reactant Mal-PEG 2000 -DSPE
  • d shows the MALDI-TOF-MS analysis of the product HBVpreS/13-32myr-PEG 2000 -DSPE.
  • Figure 5 shows ultrastructural morphological observation and particle size analysis of liposomes.
  • a shows the ultrastructure morphology of HBVpreS/2-21 myr modified liposome measured by electron microscope ( ⁇ 210000)
  • b shows unmodified liposome measured by electron microscope ( ⁇ 210000).
  • Ultrastructural morphology c-graph shows the particle size of HBVpreS/13-32 myr modified liposomes obtained by particle size analysis
  • the d-graph shows the particles of unmodified liposomes obtained by particle size analysis. path.
  • FIG. 6 shows that HBVpreS/13-32 myr modified liposomes specifically target HepG2-NTCP cells by NTCP, using flow cytometry (a) and confocal laser scanning microscopy (b1-b8, x 400) The efficiency of delivery of FS into cells is obtained.
  • the present invention utilizes a polypeptide derived from the PreS 1 region of the outer membrane protein of hepatitis B virus and a maleimide-polyethylene glycol-stearyl lipid by specifically binding to a sodium ion-taurocholic acid cotransport polypeptide (NTCP).
  • NTCP sodium ion-taurocholic acid cotransport polypeptide
  • Polypeptides suitable for use in the present invention include, inter alia, fragments of the PreS1 region of the outer membrane protein of hepatitis B virus. It will be understood that a "fragment" of a polypeptide is referred to herein, for example, as a truncated sequence of the PreS1 region. The length of the fragment is usually shorter than the length of the full length sequence.
  • the fragment of the PreS1 region may contain amino acids 2-219 of the PreS1 region, preferably amino acids 13-119 of the PreS 1 region, and further preferably amino acids 13-59 of the PreS 1 region, further preferably Contains amino acids 13-49 of the PreS1 region, further It preferably contains amino acids 13 to 39 of the PreS1 region, and further preferably contains amino acids 13 to 32 of the PreS1 region.
  • the amino acid sequence of position 2-219 of the PreS1 region suitable for use in the present invention can be as shown in SEQ ID NO: 1.
  • examples of the polypeptide of the present invention may include amino acids 13 to 32 (SEQ ID NO: 3) and amino acids 13 to 59 (SEQ ID NO: 2) of PreS1 represented by SEQ ID NO: 1.
  • examples of the polypeptide of the present invention include amino acids 13 to 119, amino acids 13 to 49, and amino acids 13 to 39 represented by SEQ ID NO: 1.
  • the polypeptide fragment of the invention comprises at least SEQ ID NO: 3 and is capable of specifically binding to a sodium ion-taurocholic acid co-transport polypeptide (NTCP).
  • the fragment is usually 20-55 amino acid residues in length, preferably 20-50 amino acid residues, preferably 20-47 amino acid residues, preferably 20-40 amino acid residues.
  • the amino acid sequence of the PreS 1 region of the outer membrane protein of hepatitis B virus is known, and a fragment of the PreS1 region which specifically binds to the sodium ion-taurocholic acid cotransport polypeptide (NTCP) is also known in the prior art and its determination. A method of whether a fragment specifically binds to NTCP. Using the prior art, it is not difficult for the skilled artisan to identify other fragments of the PreS1 region that specifically bind to the sodium ion-taurocholic acid co-transport polypeptide (NTCP). These fragments are all useful in the present invention.
  • the N-terminus of the polypeptide can be modified with a fatty acid, and an example of the invention is the modification of the N-terminus of the polypeptide with myristate (myr).
  • the maleimide-polyethylene glycol-stearoylphosphatidylethanolamine suitable for use in the compound of the present invention may have the structural formula shown in the following formula 2:
  • n is an integer of from 2 to 100
  • DSPE is stearoylphosphatidylethanolamine
  • n may be an integer of from 2 to 80, preferably an integer of from 5 to 50, preferably an integer of from 10 to 40.
  • the molecular weight of the polyethylene glycol is in the range of from 600 to 2,500, preferably from 1,000 to 2,500, more preferably from 1,500 to 2,000.
  • polyethylene glycol having a molecular weight of 2000 is used, i.e., the compound of formula 2 is Mal-PEG 2000 - DSPE.
  • a cysteine residue can be added to the C-terminus of the polypeptide of the present invention, and a thiol group of the cysteine residue can be coupled to the maleimide moiety of Formula 2.
  • An example of such coupling is shown in Figure 1.
  • Lipid compositions can be prepared using the compounds of Formulas 1, 2, 3, and 4 of the present invention to deliver a sense of delivery Drugs of interest.
  • the liposome composition of the present invention may contain a compound of the formula 1, 2, 3 or 4 of the present invention, phosphatidylcholine, cholesterol, PEG-DSPE.
  • PEG-DSPE phosphatidylcholine
  • the definition of PEG in the above PEG-DSPE is the same as above.
  • the molecular weight of the PEG is in the range of from 600 to 2,500, preferably from 1,000 to 2,500, more preferably from 1,500 to 2,000.
  • PEG 2000 - DSPE is used.
  • the present invention also encompasses a pharmaceutical composition comprising the liposome composition of the present invention and a drug of interest.
  • the pharmaceutical composition of the present invention can be prepared by a conventional method, for example, a pharmaceutical composition of the present invention in the form of a liposome by ethanol injection.
  • the pharmaceutical composition in the form of a liposome of the present invention has a diameter of at most 100 nm.
  • the particle size of the liposome in the resulting composition can be adjusted by adjusting various parameters in the preparation process, such as agitation mode, rate, time, and the like. Generally, the average particle size can be controlled to be below 120 nm.
  • the prepared liposomes were detected by laser particle size analyzer, and the ultrastructure morphology was observed by transmission electron microscopy. The relative fluorescence intensity of each group of fluorescein sodium was detected by multi-function microplate reader and HPLC, and sodium fluorescein was calculated. Content and encapsulation efficiency. These are all within the knowledge of those skilled in the art.
  • RNeasy Mini Kit was purchased from QIAGEN; Premix Ex Taq Version 2.0 was purchased from Takara; FastDigest Xho I and FastDigest EcoR I were purchased from ThermoFisher; Mal-PEG 2000- DSPE and mPEG 2000 -DSPE was purchased from NANOCS, USA; soybean phosphatidylcholine DSPC S-100 was purchased from Lipoid, Germany; cholesterol (Cholesterol) and sodium fluorescein were purchased from SIGMA; the peptides used in the experiment were entrusted to Hangzhou Zhongpept Biochemical Co., Ltd. synthesis.
  • the PCR primers used were 5'-CCCTCGAGAAAGAAGGCATCCAGCAA-3' and 5'-GGAATTCGGTTAGAACT TCTGAAGTTTAATTC-3'.
  • the plasmids pCMV-NTCP and pCMV were transfected into HepG2 cells, respectively, and the stably expressed cell lines HepG2-NTCP and control HepG2-pCMV cells were screened with a final concentration of 1 ⁇ g/ml puromycin. Then, the expression of NTCP in HepG2-NTCP cells was detected by RT-PCR method and FITC-labeled HBVpreS/13-59 myr , respectively.
  • the primers used in the RT-PCR assay were the primers used in the above experiments for constructing the NTCP expression vector.
  • the polypeptides Pep47 (SEQ ID NO: 5)-FITC and HBVpreS/13-59 myr- FITC were separately combined with HepG2- using the FITC-labeled irrelevant peptide Pep47-FITC. After incubation with NTCP and HepG2-pCMV cells for 30 minutes, flow cytometry and laser confocal microscopy were performed.
  • HBVpreS/13-32 myr Coupling a cysteine at the carbon terminus of HBVpreS/13-32 myr by dehydration condensation, covalently bonding to the terminal group maleimide of Mal-PEG 2000 -DSPE via its side chain thiol (eg Figure 1 shows) HBVpreS/13-32 myr -PEG 2000 -DSPE.
  • Liposomes were prepared by ethanol injection, and sodium fluorescein was encapsulated as a model drug.
  • the specific method is as follows: 80 ml of a 2 ⁇ g/ml sodium fluorescein aqueous solution is prepared as an aqueous phase; the components of the liposome are accurately weighed according to Table 1, and dissolved in 6 ml of absolute ethanol as an organic phase.
  • the aqueous phase and the organic phase water bath were heated to 46 ° C, then the aqueous phase was stirred and stirred, and the organic phase was quickly injected; after stirring for 30 minutes, the free sodium fluorescein was removed by ultrafiltration centrifugation, and finally the obtained HBVpreS/13- 32 myr modified targeting fluorescein sodium liposome HBVpreS/13-32 myr- FS-LCL (long circulating liposome, LCL) and normal liposome FS-LCL without targeting peptide.
  • the prepared liposomes were detected by laser particle size analyzer, and the ultrastructure morphology was observed by transmission electron microscopy. The relative fluorescence intensity of each group of fluorescein sodium was detected by multi-function microplate reader and HPLC, and sodium fluorescein was calculated. Content and encapsulation efficiency.
  • the HepG2-NTCP and HepG2-pCMV control cells were seeded in a 24-well plate at a concentration of 5 ⁇ 10 4 /well, and cultured to a cell concentration of 75% to 85%, respectively, and the liposome containing the targeting sequence and the non-containing liposome was added.
  • a simple DMEM medium was set as a blank control group. After incubation for 1 hour in the dark, flow cytometry and laser confocal microscopy were performed.
  • NTCP expression plasmid pCMV-NTCP was transfected into HepG2 cells, and the expression of NTCP was identified after screening with puromycin.
  • NTCP expression was analyzed at the mRNA level by RT-PCR. As a result, as shown in Fig. 2(a), there was a specific band at 1050 bp, which was consistent with the size of the NTCP coding sequence.
  • Fig. 2(a) there was a specific band at 1050 bp, which was consistent with the size of the NTCP coding sequence.
  • the expression of NTCP on the cell surface was detected by fluorescently labeled peptide HBVpreS/13-59 myr- FITC: the results of flow cytometry (Fig. 2, b) and confocal microscopy (Fig.
  • HBVpreS/13-59 myr- FITC specifically binds to HepG2-NTCP cells, suggesting the expression of NTCP on the cell membrane surface. Therefore, mRNA levels and protein levels showed the expression of NTCP in the transfected cells, suggesting that the HepG2-NTCP cell line was successfully constructed.
  • HBVpreS/13-32 myr-FITC was co-incubated with HepG2-NTCP cells and control cells HepG2-pCMV, and the binding of the polypeptide to the cells was observed by confocal microscopy.
  • the results indicate (Fig. 3) that HBVpreS/13-32 myr- FITC can specifically bind to HepG2-NTCP cells (Fig. 3, a), while the unrelated control polypeptide Pep47-FITC has no significant binding (Fig. 3, b).
  • the control cell HepG2-pCMV Fig. 3, d
  • HBVpreS/13-32 myr binds to the NTCP receptor on the surface of hepatocytes and acts as a mediator for hepatocyte targeting.
  • HBVpreS/13-32 myr -PEG 2000- DSPE The synthesis of hepatocyte-targeted phospholipid complex material HBVpreS/13-32 myr -PEG 2000- DSPE was carried out slowly at 4 °C. Excess HBVpreS/13-32 myr was added to the reaction, and the content was detected by HPLC. process.
  • the HPLC results before and after the reaction (Fig. 4, a, b) showed that the peak height of HBVpreS/13-32 myr detected in the pre-reaction system was 123.045, and the reaction was 9.387, which decreased by 92.5%, which proved the occurrence of covalent addition reaction. And proceeded more completely.
  • the MALDI-TOF-MS mass spectrometry results of the reactant Mal-PEG 2000 -DSPE and the reaction product HBVpreS/13-32 myr -PEG 2000 -DSPE are shown: the reactant HBVpreS/13-32 myr and The average molecular mass of Mal-PEG 2000 -DSPE was 2478.9 and 3028.4, and the average molecular mass of the reaction product was 5278.7, which was consistent with the molecular weight of HBVpreS/13-32 myr -PEG 2000 -DSPE, suggesting that HBVpreS/13-32 myr -PEG 2000 - DSPE was successfully synthesized.
  • Liposomes were prepared using HBVpreS/13-32 myr -PEG 2000 -DSPE and the components in Table 1, and their physical and chemical properties were analyzed. Under the transmission electron microscope, the liposomes were spherical, the surface was smooth, the size distribution was uniform, and the diameter was below 100 nm (Fig. 5, a, b). The particle size analysis of the laser particle size analyzer showed that there was no HBVpreS/13-32 myr targeting. The modified liposomes were of comparable size, with particle sizes of (94.09 ⁇ 10.5) nm and (117.08 ⁇ 11) nm, normal distribution (Fig.
  • the encapsulation efficiency of the HBVpreS/13-32 myr modified liposome group and the encapsulation efficiency of the common liposome group were (89.32 ⁇ 1.02)% and (85.78 ⁇ 2.23)%, respectively.
  • HBVpreS/13-32 myr modified liposome HBVpreS/13-32 myr -FS-LCL can target hepatocytes more efficiently and deliver its contents compared to unmodified liposome FS-LCL
  • HBVpreS/13-32 myr -FS-LCL and FS-LCL were incubated with HepG2-NTCP cells, respectively, for flow cytometry and laser confocal detection.
  • the results of flow cytometry showed that the HBVpreS/13-32 myr modified liposome group HBVpreS/13-32 myr- FS-LCL can be compared with the normal liposome group FS-LCL and fluorescein sodium aqueous solution group FS.

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Abstract

本发明涉及靶向脂质体的制备及其应用，具体涉及式1的靶向肝细胞的化合物、含有该化合物的用于递送药物的组合物以及药物组合物，式1中，X为与钠离子-牛磺胆酸共转运多肽(NTCP)特异性结合的源于乙型肝炎病毒外膜蛋白PreS1区的多肽；B为式2所示的马来酰亚胺-聚乙二醇-硬脂酰基磷脂酰乙醇胺聚合物；式2中，n为2－100的整数，DSPE为硬脂酰基磷脂酰乙醇胺；其中，X通过式2的马来酰亚胺部分与B相连。

Description

靶向脂质体的制备及其应用 技术领域
本发明涉及靶向脂质体的制备及其应用，具体涉及肝靶向脂质体的制备及其应用。
背景技术
肝脏相关疾病是影响人类健康的重要疾病之一，我国每年因终末期肝病死亡人数超过30万。然而目前用于治疗肝病的药物多为系统给药，具有肝脏分布少、体内不稳定、肝外副作用较多等缺点，限制了药物的临床应用。因此，如何将药物高效地传递至肝脏，更好地发挥治疗作用、降低肝外副作用是肝病治疗领域亟待解决的重大问题(Poelstra K,Prakash J,Beljaars L.Drug targeting to the diseased liver[J].Journal of controlled release:official journal of the Controlled Release Society,2012,161(2):188-197)。肝脏靶向给药系统可以将药物特异性靶向肝脏，有利于肝脏富集，减少药物用量及降低全身的毒副作用(贺玲,李健和.肝靶向给药系统的研究进展[J].研究进展,2010,17(9):15-17)。脂质体由于其免疫原性低、载药范围广，具有保护药物活性、提高稳定性、缓释等优点，是最常用的载药模型之一。然而，普通的脂质体并不能完成靶向传递的功效，因此需对普通脂质体进行靶向修饰，使其达到主动靶向(Pranali PD,Swati B,Vladimir PT,et al.Current trends in the use of liposomes for tumor targeting[J].Nanomedicine,2013,8(9):1509-1528)。
乙型肝炎病毒是一种嗜肝类病毒，其外膜蛋白的PreS1区是与肝细胞受体结合的重要部位(Liang TJ.Hepatitis B:The Virus and Disease[J].Hepatology,2009,49(5):S13-S21)。最近研究揭示，钠离子-牛磺胆酸共转运多肽NTCP(Sodium taurocholate cotransporting polypeptide)是HBV的功能性受体(Yan H,Zhong G,Xu G,et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus[J].Elife,2012,1(e00049)， 包括D基因型preS/2-48在内的源于PreS1的多段多肽均可以与其特异性结合(Gripon P,Cannie I,Urban S.Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein[J].J Virol,2005,79(3):1613-1622；Petersen J,Dandri M,Mier W,et al.Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein[J].Nat Biotechnol,2008,26(3):335-341；Schulze A,Schieck A,Ni Y,et al.Fine mapping of pre-S sequence requirements for hepatitis B virus large envelope protein-mediated receptor interaction[J].J Virol,2010,84(4):1989-2000)。在本发明申请的一个实施例中，以C基因型HBVpreS/13-32myr(myr-GTNLSVPNPLGFFPDHQLDP)作为靶向序列，偶联Mal-PEG-DSPE作为导向性材料，制备具有肝细胞靶向性的纳米脂质体，构建一种新型的肝细胞靶向给药系统，并在细胞模型上对其靶向性进行验证(任浩洋、常红霞、蔡雯雯，一种具有肝细胞选择性的脂质体的导向性评价[J].解放军药学学报,2007,23(1):37-40)。
发明内容
在本发明的一个实施例中，以HBVpreS/13-32myr作为靶向序列(SEQ ID NO.3)，将其化学偶联到Mal-PEG-DSPE(马来酰亚胺-聚乙二醇-硬脂酰基磷脂酰乙醇胺)，生成HBVpreS/13-32myr-PEG-DSPE；采用乙醇注入法制备脂质体，包裹荧光素钠(Fluorescein sodium，FS)形成HBVpreS/13-32myr-FS-LCL，以稳定表达NTCP的HepG2为细胞模型，分析纳米脂质体靶向性。
本发明的一个实施例结果显示，合成的靶向脂质体呈类圆形，大小均匀，粒径为(94.09±9.2)nm，达到纳米级别；有效包裹荧光素钠，包封率为89.32±1.02％；与无靶向修饰脂质体相比，可递送更多的荧光素钠进入细胞内。HBVpreS/13-32myr修饰的靶向脂质体，有较高的包封率，为纳米脂质体，能特异性地通过NTCP靶向肝细胞，有望成为一种新型的肝细胞靶向给药系统。
因此，本发明提供一种靶向肝细胞的化合物，该化合物的结构如下式1所示：
X-B  (式1)
式1中，
X为与钠离子-牛磺胆酸共转运多肽(NTCP)特异性结合的源于乙型肝炎病毒外膜蛋白PreS1区的多肽；和
B为下式2所示的马来酰亚胺-聚乙二醇-硬脂酰基磷脂酰乙醇胺聚合物：
Figure PCTCN2015073610-appb-000001
式2中，n为2－100的整数，DSPE为硬脂酰基磷脂酰乙醇胺；
其中，X通过式2的马来酰亚胺部分与B相连。
在一个具体实施例中，X为源于乙型肝炎病毒外膜蛋白PreS1区的第2-119位氨基酸的片段。
在一个具体实施例中，所述X优选地为源于乙型肝炎病毒外膜蛋白PreS 1区的第13-119位氨基酸的片段。
在一个具体实施例中，所述X进一步优选地为源于乙型肝炎病毒外膜蛋白PreS1区的第13-59位氨基酸的片段。
在一个具体实施例中，所述X进一步优选地为源于乙型肝炎病毒外膜蛋白PreS1区的第13-49位氨基酸的片段。
在一个具体实施例中，所述X进一步优选地为源于乙型肝炎病毒外膜蛋白PreS1区的第13-39位氨基酸的片段。
在一个具体实施例中，所述X进一步优选地为源于乙型肝炎病毒外膜蛋白PreS1区的第13-32位氨基酸的片段。
在一个具体实施例中，所述X的N端被脂肪酸或胆固醇修饰。
在一个具体实施例中，所述X的N端被豆蔻酸修饰。
在一个具体实施例中，所述片段的氨基酸序列如SEQ ID NO:1－3中任一项所示。
在一个具体实施例中，n为2－80的整数，优选5－50的整数，优选10－40的整数。
在一个具体实施例中，聚乙二醇的分子量在600－2500的范围之内，优选为1000－2500，更优选为1500－2000。
在一个具体实施例中，X所示多肽在其C末端还含有半胱氨酸残基，通过 该半胱氨酸残基与B的马来酰亚胺部分相连。
在一个具体实施例中，X所示多肽在其C末端还含有半胱氨酸残基，其氨基酸序列如SEQ ID NO:4所示。
在一个具体实施例中，X所示多肽的N末端被豆蔻酰化修饰。
在一个具体实施例中，本发明的化合物如下式3所示：
Figure PCTCN2015073610-appb-000002
式3中，X、n和DSPE如上文所定义。
在一个具体实施例中，本发明的化合物如下式4所示：
Figure PCTCN2015073610-appb-000003
式中的聚乙二醇的分子量为2000。
本发明涉及一种用于递送药物的组合物，该组合物含有本发明式1、2、3或4所示的化合物，以及磷脂酰胆碱、胆固醇和PEG-DSPE中的任一种、任意两种或全部三种。
在一个具体实施例中，本发明用于递送药物组合物含有本发明化合物和磷脂酰胆碱，还任选地含有胆固醇和/或PEG-DSPE。
在一个具体实施例中，本发明用于递送药物组合物中，本发明化合物、磷脂酰胆碱、胆固醇和PEG-DSPE的重量比为1～100:1～1000:0～500:0～500。
在一个具体实施例中，所述用于递送药物的组合物是脂质体。
本发明还涉及一种药物组合物，该药物组合物含有本发明的脂质体及治疗有效量的药物。
本发明还涉及本发明式1、2、3和4所示化合物在制备治疗乙型肝炎或抑制乙型肝炎病毒的药物中的用途。
附图说明
图1显示HBVpreS/13-32myr-PEG-DSPE合成方程式。
图2显示HepG2-NTCP细胞成功表达NTCP。其中，a图显示采用RT-PCT 和琼脂糖凝胶电泳对NTCP DNA进行分析的结果，b图显示采用流式细胞术检测细胞表面上NTCP表达的结果，c图显示采用共聚焦激光扫描显微镜(×400)检测细胞表面上NTCP表达的结果。
图3显示共聚焦显微镜观察HBVpreS/13-32myr与细胞结合情况。HepG2-NTCP分别与HBVpreS/13-32myr-FITC(a图)、Pep47-FITC(b图)共孵育，或未处理(c1－c3图)；HepG2-pCMV细胞也分别与HBVpreS/13-32myr-FITC(d图)、Pep47-FITC(e图)共孵育，或未处理(f图)。
图4显示HPLC和MALDI-TOF-MS检测HBVpreS/13-32myr-PEG2000-DSPE合成过程。其中，a图显示反应前HBVpreS/13-32myr的分析，b图显示反应后HBVpreS/13-32myr的分析，c图显示反应物Mal-PEG2000-DSPE的MALDI-TOF-MS分析，d图显示产物HBVpreS/13-32myr-PEG2000-DSPE的MALDI-TOF-MS分析。
图5显示脂质体的超微结构形态学观察和粒径大小分析。其中，a图显示采用电子显微镜(×210000)测定的HBVpreS/2-21myr修饰的脂质体的超微结构形态学，b图显示采用电子显微镜(×210000)测定的未修饰的脂质体的超微结构形态学，c图显示采用粒径大小分析获得的HBVpreS/13-32myr修饰的脂质体粒径，d图显示显示采用粒径大小分析获得的未修饰的脂质体的粒径。
图6显示HBVpreS/13-32myr修饰脂质体通过NTCP特异性靶向HepG2-NTCP细胞，其中，采用流式细胞术(a图)和共聚焦激光扫描显微镜(b1-b8，×400)检测得到FS递送到细胞中的效率。
具体实施方式
本发明通过采用与钠离子-牛磺胆酸共转运多肽(NTCP)特异性结合的源于乙型肝炎病毒外膜蛋白PreS 1区的多肽与马来酰亚胺-聚乙二醇-硬脂酰基磷脂酰乙醇胺聚合物而完成本发明。
适合用于本发明的多肽尤其包括乙肝病毒外膜蛋白PreS1区的片段。应理解，多肽的“片段”在本文中指，例如PreS1区的截短序列。片段的长度通常短于全长序列的长度。例如，本申请中，PreS1区的片段可含有PreS1区第2-119位氨基酸，优选地含有PreS 1区第13-119位氨基酸，进一步优选地含有PreS 1区第13-59位氨基酸，进一步优选地含有PreS1区第13-49位氨基酸，进一步 优选地含有PreS1区第13-39位氨基酸，进一步优选地含有PreS1区第13-32位氨基酸。
适用于本发明的PreS1区第2－119位氨基酸序列可如SEQ ID NO:1所示。因此，本发明多肽的例子可包括SEQ ID NO:1所示的PreS1第13－32位氨基酸(SEQ ID NO:3)、和第13－59位氨基酸(SEQ ID NO:2)。在其它实施例中，本发明多肽例子包括SEQ ID NO:1所示的第13-119位氨基酸、13-49位氨基酸，和第13-39位氨基酸等。在优选的实施例中，本发明的多肽片段至少含有SEQ ID NO:3，并能特异性与钠离子-牛磺胆酸共转运多肽(NTCP)结合。片段长度通常为20－55个氨基酸残基，优选20-50个氨基酸残基，优选20-47个氨基酸残基，优选20-40个氨基酸残基。
应理解，人们已知乙肝病毒外膜蛋白PreS 1区的氨基酸序列，且现有技术也已知能特异性与钠离子-牛磺胆酸共转运多肽(NTCP)结合的PreS1区的片段及其确定某片段是否与NTCP特异性结合的方法。采用现有技术，技术人员不难确定能特异性与钠离子-牛磺胆酸共转运多肽(NTCP)结合的PreS1区的其它片段。这些片段都可用于本发明。多肽的N端可进行脂肪酸修饰，本发明的一个例子就是对多肽的N端进行豆蔻酸(myr)修饰。
适用于本发明化合物的马来酰亚胺-聚乙二醇-硬脂酰基磷脂酰乙醇胺可具有如下式2所示的结构式：
Figure PCTCN2015073610-appb-000004
式2中，n为2－100的整数，DSPE为硬脂酰基磷脂酰乙醇胺。
n的范围可为2－80的整数，优选5－50的整数，优选10－40的整数。
或者，式2中，聚乙二醇的分子量在600－2500的范围之内，优选为1000－2500，更优选为1500－2000。
在本发明的一个具体实施例中，使用分子量为2000的聚乙二醇，即式2化合物为Mal-PEG2000-DSPE。
可通过在本发明多肽的C末端加入一个半胱氨酸残基，并通过该半胱氨酸残基的巯基与式2的马来酰亚胺部分偶联。图1示出了这种偶联的一个例子。
可利用本发明的式1、2、3和4化合物来制备脂质体组合物，用以递送感 兴趣的药物。本发明的脂质体组合物可含有本发明的式1、2、3或4化合物，磷脂酰胆碱、胆固醇、PEG-DSPE。上述PEG-DSPE中的PEG的定义和上文相同。优选的是，PEG的分子量在600－2500的范围之内，优选为1000－2500，更优选为1500－2000。
在一个具体实施例中，使用PEG2000-DSPE。
本发明还包括一种药物组合物，该药物组合物含有本发明的脂质体组合物及感兴趣的药物。
可采用常规的方法制备本发明的药物组合物，例如采用乙醇注入法成脂质体形式的本发明药物组合物。本发明脂质体形式的药物组合物的直径多在100nm以下。可通过调整制备过程中的各项参数，例如搅拌方式、速率、时间等，来调整所得组合物中脂质体的粒径大小。通常，可将其平均粒径大小控制在120nm以下。制备的脂质体通过激光粒度分析仪检测脂质体颗粒粒径大小，透射电镜观察超微结构形态，多功能酶标仪和HPLC检测各组荧光素钠的相对荧光强度，并计算荧光素钠的含量和包封率。这些都在本领域技术人员所掌握的知识范围之内。
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅仅是阐述性的，而非限制性的。实施例中所用到的试剂及其用量、方法条件等，除非另有说明，否则都按制造商的建议或按照本领域常规的做法实施。
实施例
1.材料与方法
1.1细胞与主要试剂
人肝癌细胞株HepG2细胞由本实验室传代保存；RNeasy Mini Kit购自QIAGEN公司；Premix Ex Taq Version2.0购自Takara公司；FastDigest Xho I和FastDigest EcoR I购自ThermoFisher公司；Mal-PEG2000-DSPE和mPEG2000-DSPE购自美国NANOCS公司；大豆磷脂酰胆碱DSPC S-100购自德国Lipoid公司；胆固醇(Cholesterol)和荧光素钠购自SIGMA公司；实验中所用多肽均委托杭州中肽生化有限公司合成。
1.2HepG2-NTCP细胞构建及鉴定
从人肝组织提取总RNA，进行逆转录PCR，扩增NTCP编码序列；通过Xho I和EcoR I构建到pCMV载体，得到NTCP表达质粒pCMV-NTCP。在上述过程中，所用的PCR引物为5’-CCCTCGAGAAAGAAGGCATCCAGCAA-3’和5’-GGAATTCGGTTAGAACT TCTGAAGTTTAATTC-3’。
将质粒pCMV-NTCP和pCMV分别转染HepG2细胞，用终浓度1μg/ml嘌呤霉素(puromycin)筛选得到稳定表达的细胞株HepG2-NTCP及对照HepG2-pCMV细胞。然后，分别通过RT-PCR方法和FITC标记HBVpreS/13-59myr检测NTCP在HepG2-NTCP细胞中的表达。RT-PCR检测中所用引物为上述构建NTCP表达载体实验中所用引物。在FITC标记HBVpreS/13-59myr检测实验中，以FITC标记的无关肽Pep47-FITC为对照，将多肽Pep47(SEQ ID NO:5)-FITC和HBVpreS/13-59myr-FITC分别与HepG2-NTCP和HepG2-pCMV细胞孵育30分钟后，进行流式细胞仪和激光共聚焦显微镜检测。
1.3HBVpreS/13-32myr-PEG2000-DSPE的合成
利用脱水缩合的方法在HBVpreS/13-32myr的碳端偶联一个半胱氨酸，通过其侧链的巯基与Mal-PEG2000-DSPE的末端基团马来酰亚胺共价结合(如图1示)，得到HBVpreS/13-32myr-PEG2000-DSPE。具体方法为：将多肽HBVpreS/13-32myr溶于PBS溶液中(PH＝7.0)，Mal-PEG2000-DSPE溶于DMF(二甲基甲酰胺)，按Mal-PEG2000-DSPE:HBVpreS/13-32myr＝1:1.2物质的量比混合搅拌，4℃缓慢反应8小时。HPLC全程监测整个反应过程，MAIDI-TOF-MS鉴定反应产物。
1.4荧光靶向纳米脂质体的制备
通过乙醇注入法制备脂质体，包裹荧光素钠作为模式药物。具体方法如下：配制2μg/ml的荧光素钠水溶液80ml作为水相；将脂质体各组分按照表1精确称量，溶于6ml无水乙醇中作为有机相。水相和有机相水浴加热至46℃，然后将水相旋转搅拌，将有机相快速注射；持续搅拌30分钟后，通过超滤离心的方法去除游离的荧光素钠，最终得到的HBVpreS/13-32myr修饰的靶向荧光素钠脂质体HBVpreS/13-32myr-FS-LCL(长效循环脂质体，Long circulating  liposome，LCL)和无靶向肽的普通脂质体FS-LCL。制备的脂质体通过激光粒度分析仪检测脂质体颗粒粒径大小，透射电镜观察超微结构形态，多功能酶标仪和HPLC检测各组荧光素钠的相对荧光强度，并计算荧光素钠的含量和包封率。
表1脂质体配方
Figure PCTCN2015073610-appb-000005
1.5靶向性检测
将HepG2-NTCP及HepG2-pCMV对照细胞接种于24孔板，接种浓度5×104/孔，培养至细胞浓度75％～85％时，分别加入含靶向序列和不含的脂质体，设单纯的DMEM培养基为空白对照组。避光孵育培养1小时后,进行流式细胞分析和激光共聚焦显微镜检测。
2.结果
2.1 HepG2-NTCP细胞成功构建
将NTCP表达质粒pCMV-NTCP转染HepG2细胞，经嘌呤霉素筛选后，对其NTCP的表达进行鉴定。首先，通过RT-PCR方法在mRNA水平上分析NTCP表达，结果如图2(a)所示，在1050bp处有一特异性条带，与NTCP编码序列大小相符。其次，以荧光标记多肽HBVpreS/13-59myr-FITC检测NTCP在细胞表面的表达情况：流式细胞技术检测(图2，b)和共聚焦显微镜观察(图2，c)结果一致，均表明HBVpreS/13-59myr-FITC特异性地与HepG2-NTCP细胞相结合，提示了NTCP在细胞膜表面的表达。因此，mRNA水平和蛋白水平均显示NTCP在转染后细胞中的表达，提示HepG2-NTCP细胞株构建成功。
2.2 HBVpreS/13-32myr特异性结合NTCP
为了验证多肽HBVpreS/13-32myr与NTCP的结合，将HBVpreS/13-32myr-FITC与HepG2-NTCP细胞以及对照细胞HepG2-pCMV共孵育，共聚焦显微镜观察多肽与细胞结合情况。结果表明(图3)：HBVpreS/13-32myr-FITC可以与HepG2-NTCP细胞特异性结合(图3，a)，而无关对照多肽Pep47-FITC没有明显的结合作用(图3，b)，与对照细胞HepG2-pCMV也没有明显的结合(图3，d)，证实了HBVpreS/13-32myr与肝细胞表面NTCP受体结合，可作为肝细胞靶向的介质。
2.3 HBVpreS/13-32myr-PEG2000-DSPE合成成功
肝细胞靶向磷脂复合物材料HBVpreS/13-32myr-PEG2000-DSPE的合成在4℃条件下缓慢经行，反应中加入过量HBVpreS/13-32myr，利用HPLC检测其含量，全程监控反应进程。反应前后HPLC检测结果(图4，a、b)显示，反应前体系中HBVpreS/13-32myr检测的峰高为123.045，反应后为9.387，降低了92.5％，证明共价加成反应的发生且进行得较完全。反应物Mal-PEG2000-DSPE和反应产物HBVpreS/13-32myr-PEG2000-DSPE的MALDI-TOF-MS质谱结果(图4，c、d)所示：反应物HBVpreS/13-32myr与Mal-PEG2000-DSPE的平均分子质量为2478.9和3028.4，反应产物平均分子质量为5278.7，与HBVpreS/13-32myr-PEG2000-DSPE的分子量相符，提示HBVpreS/13-32myr-PEG2000-DSPE合成成功。
2.4脂质体形态及粒径分析
以HBVpreS/13-32myr-PEG2000-DSPE和表1中的各成分制备脂质体，然后对其理化性状进行分析。透射电镜下可见脂质体呈球状，表面光整，大小分布均匀，直径多在100nm以下(图5，a、b)；激光粒度仪进行粒径分析，可见有无HBVpreS/13-32myr靶向修饰的脂质体大小相当，粒径大小分别为：(94.09±10.5)nm和(117.08±11)nm，正态分布(图5，c、d)；经多功能酶标仪和HPLC检测，HBVpreS/13-32myr修饰的脂质体组的包封率和普通脂质体组的包封率分别为：(89.32±1.02)％和(85.78±2.23)％。
2.5HBVpreS/13-32myr-FS-LCL具有肝细胞靶向性
为了分析HBVpreS/13-32myr修饰脂质体HBVpreS/13-32myr-FS-LCL与无修 饰脂质体FS-LCL相比，能否更有效地靶向肝细胞、并将其包容物递送至细胞内部，将HBVpreS/13-32myr-FS-LCL和FS-LCL分别与HepG2-NTCP细胞孵育，进行流式细胞术和激光共聚焦检测。流式细胞仪检测的结果显示：相对于普通脂质体组FS-LCL和荧光素钠水溶液组FS，HBVpreS/13-32myr修饰的脂质体组HBVpreS/13-32myr-FS-LCL可以递送更多的荧光素钠进入细胞(图6，a)；而对照组细胞HepG2-pCMV细胞，脂质体HBVpreS/13-32myr-FS-LCL递送荧光素钠效率显著降低，提示在该过程是NTCP依赖的。激光共聚焦显微镜观察结果与流式结果相符，相对于荧光素钠的水溶液FS和单纯的普通脂质体，HBVpreS/13-32myr修饰的HBVpreS/13-32myr-FS-LCL靶向脂质体组有更强的荧光强度(图6，b1-b4)；而在无NTCP表达的HepG2-pCMV细胞中，荧光强度则显著降低(图6，b5-b8)。总之，流式和激光共聚焦显微镜结果均提示脂质体可以借助于HBVpreS/13-32myr的靶向作用，特异性地与肝细胞表面NTCP结合，更加高效地将其包容物递送至细胞内部。

Claims (10)

  1. 一种靶向肝细胞的化合物，该化合物的结构如下式1所示：
    X-B     (式1)
    式1中，
    X为与钠离子-牛磺胆酸共转运多肽(NTCP)特异性结合的源于乙型肝炎病毒外膜蛋白PreS1区的多肽；和
    B为下式2所示的马来酰亚胺-聚乙二醇-硬脂酰基磷脂酰乙醇胺聚合物(Mal-PEG-DSPE)：
    Figure PCTCN2015073610-appb-100001
    式2中，n为2－100的整数，DSPE为硬脂酰基磷脂酰乙醇胺；
    其中，X通过式2的马来酰亚胺部分与B相连。
  2. 如权利要求1所述的化合物，其特征在于，X为乙型肝炎病毒外膜蛋白PreS1区第2-119位氨基酸的多肽片段。
  3. 如权利要求2所述的化合物，其特征在于，X为乙型肝炎病毒外膜蛋白PreS1区第13-59位氨基酸或第13－32位氨基酸的多肽片段。。
  4. 如权利要求1－3中任一项所述的化合物，其特征在于，所述多肽的氨基酸序列如SEQ ID NO:1、2和3中任一项所示。
  5. 如权利要求1－4中任一项所述的化合物，其特征在于，式2中，n为2－80的整数，优选5－50的整数，优选10－40的整数；或式2中，聚乙二醇的分子量在600－2500的范围之内，优选为1000－2500，更优选为1500－2000。
  6. 如权利要求1－5中任一项所述的化合物，其特征在于，X所示多肽在其C末端还含有半胱氨酸残基，通过该半胱氨酸残基与B的马来酰亚胺部分相连。
  7. 如权利要求1－6中任一项所述的化合物，其特征在于，在一个具体实施例中，X所示多肽在其N末端被脂肪酸修饰，例如被豆蔻酸修饰。
  8. 如权利要求1所述的化合物，其特征在于，所述化合物如下式4所示：
    Figure PCTCN2015073610-appb-100002
    式中的聚乙二醇的分子量为2000，X的N端被豆蔻酸修饰。
  9. 一种用于递送药物的组合物，其特征在于，该组合物含有权利要求1－8中任一项所述的化合物，以及磷脂酰胆碱、胆固醇和PEG-DSPE中的一种、任意两种、或全部三种。
  10. 一种药物组合物，其特征在于，该药物组合物含有权利要求9所述的用于递送药物的组合物和药物。
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