JP5713311B2 - Liposome complex, method for producing the same, and use thereof - Google Patents

Description

  The present invention relates to a liposome complex, a production method thereof, its introduction into cells, and use for the purpose of introduction into a tissue of an individual. In particular, the present invention relates to a liposome, a bio-nanocapsule, and a liposome complex containing a cell-introducing substance, a method for producing the same, and a use for the purpose of introducing it into cells.

  Bionanocapsule (BNC) is "biologically-derived hollow nanoparticles presenting a protein having membrane permeation activity (synonymous with membrane fusion activity) and its modified body", and its representative example is the infection mechanism of hepatitis B virus. There are hepatitis B virus surface antigen L protein particles developed as non-viral vectors having a cell and organ recognition mechanism (as other examples, influenza coat protein and Sendai virus coat protein are assumed). A DDS technique for delivering genes, drugs, and the like using this BNC (hereinafter referred to as hepatitis B virus surface antigen L protein particles) has been developed (Patent Document 1, Non-Patent Document 1). In order to enclose a substance such as a gene in the BNC, a method of introducing plasmid DNA into the BNC using an electroporation method has been developed. However, this method is inefficient in gene transfer, unsuitable for in vivo administration due to enlarging BNC particles by encapsulating genes, etc., and a huge electroporation material for producing pharmaceuticals There was a problem that it was difficult to manufacture.

  Therefore, as a method for encapsulating a substance such as a new gene in place of the electroporation method, a method of fusing the BNC with a cationic liposome and gene complex (lipoplex) has been established (Patent Document 2, Non-Patent Document 2). In addition, antibody-presenting BNC (Patent Document 3), lectin-presenting BNC (Patent Document 4), and growth factor-presenting BNC (Patent Document 5) have been developed as BNCs, and are specific to various cells or tissues. It is also possible to confer specific targeting ability.

  Cationic liposomes are liposomes composed of cationic lipids and auxiliary lipids that stabilize membranes, and application research is actively conducted as non-viral vectors with relatively high gene transfer efficiency. Cationic liposomes form a complex with a gene by electrostatic interaction, accumulate nonspecifically on the cell membrane, and are taken up into the cell by endocytosis. BNC fuses with lipoplex by its membrane fusion activity to form a BNC-lipoplex complex. The BNC-lipoplex complex acts specifically on liver cells by the action of a human liver-specific receptor present in the pre-S1 region of HBsAg L, a protein that constitutes BNC, and is present on the amino terminal side of HBsAg L. It is considered that escape from the late endosome is performed more rapidly by the action of the transmembrane peptide (Non-patent Document 3).

  Until now, in order to enhance the function of DNC as a DDS for a BNC-liposome complex for the purpose of introducing a substance into a cell, a technique (Patent Document 2) having a predetermined particle size, In view of the stability of this complex, a technique for making the surface charge close to zero (Patent Document 6) has been disclosed, but no technique for improving the substance introduction ability itself into cells has been developed. Further, as described above, since BNC can select a target to introduce a substance with high specificity, BNC-liposome complex having high specificity and improved ability to introduce a substance into cells. Development is desired.

JP 2001-316298 A JP 2007-106752 A WO2005 / 049824 JP 2009-120532 A JP 2003-286198 A JP 2010-077091 A Nature Biotechnology 21 (2003) 885-890.Nanoparticles for the Delivery of Genes and Drugs to Human Hepatocytes.Yamada, T., Iwasaki, Y., Tada, H., Iwabuki, H., Chuah, MKL, VandenDriessche, T. , Fukuda, H., Kondo, A., Ueda, M., Seno, M., Tanizawa, K., and Kuroda, S. Journal of Controlled Release 126 (2008) 255-264 Bio-nanocapsule conjugated with liposomes for in vivo pinpoint delivery of various materials Joohee Jung, Takashi Matsuzaki, Kenji Tatematsu, Toshihide Okajima, Katsuyuki Tanizawa, Shun'ichi Kuroda Methods in Enzymology 464 (2009) 147-166. Bio-nanocapsule-liposome conjugates for in vivo pinpoint drugs and gene delivery. Kasuya, T., Jung, J., Kinoshita, R., Goh, Y., Matsuzaki, T., Iijima, M., Yoshimoto, N., Tanizawa, K., and Kuroda, S.

  The main object of the present invention is to develop a technique for increasing the substance introduction efficiency while having high specificity to the cell of the BNC-liposome complex intended to introduce the substance into the cell.

  The inventor of the present application has conducted extensive research to solve the above problems, and as a result, a liposome complex having a specific range of density, a bio-nanocapsule, and a liposome complex containing a cell introduction substance have high efficiency and high specificity. And found that a desired substance can be introduced into a specific cell. Furthermore, it has been found that the liposome complex produced under a specific environment exhibits particularly high substance introduction efficiency. The present invention has been completed based on such knowledge, and includes the following aspects.

Item 1. A liposome complex comprising a liposome and a bio-nanocapsule, or a liposome complex comprising a liposome, a bio-nanocapsule, and a cell introduction substance, wherein the liposome complex has a density of 1.07-1.19 mg / ml. Complex.
Item 2. The liposome complex according to Item 1, wherein the bio-nanocapsule is a hollow nanoparticle containing a hepatitis B virus protein or a variant thereof.
Item 3. The liposome complex according to Item 1 or 2, wherein the cell introduction substance is at least one selected from the group consisting of nucleic acids, proteins, drugs, carbohydrates, lipids, and labeling substances.
Item 4 The liposome complex according to Item 3, wherein the liposome is a cationic liposome, and the cell introduction substance is a nucleic acid.
Item 5. The liposome complex according to Item 3, wherein the liposome is an anionic liposome, and the cell introduction substance is a drug or a labeling substance.
Item 6. The liposome complex according to Item 1, wherein the liposome complex has a surface charge of -30 to +10 mV.
Item 7 The liposome complex according to Item 1, wherein the liposome complex has a particle size of 50 to 500 nm.
Item 8 A method of introducing the cell introduction substance into a cell by bringing the liposome complex according to Item 1 into contact with the cell.
Item 9 is a method for producing a liposome complex,
(1) Step 1 for mixing liposome and bio-nanocapsule in liquid and (2) Step 2 for separating the mixture obtained in Step 1 based on density
A method for producing a liposome complex, comprising:
Item 10 The method according to Item 9, wherein in the step 2, a liposome complex having a density of 1.07 to 1.19 mg / ml is separated.
Item 11 The method according to Item 9 or 10, wherein the mixing step in Step 1 is performed in a liquid having a pH of 5 or less, or 10 or more.
Item 12 The method according to any one of Items 9 to 11, wherein the bio-nanocapsule is a hollow nanoparticle containing a hepatitis B virus protein or a variant thereof.
Item 13 The method according to any one of Items 9 to 12, wherein a cell introduction substance is further mixed in the step 1.
Item 14 The method according to any one of Items 9 to 12, further comprising a step 3 of mixing a cell introduction substance after the step 2.
Item 15 The method according to Item 13 or 14, wherein the cell introduction substance is at least one selected from the group consisting of nucleic acids, proteins, drugs, carbohydrates, lipids, and labeling substances.
Item 16 The method according to Item 15, wherein the liposome is a cationic liposome, and the cell introduction substance is a nucleic acid.
Item 17 The method according to Item 15, wherein the liposome is an anionic liposome, and the cell introduction substance is a drug or a labeling substance.

  The liposome complex of the present invention can introduce a substance into cells with high efficiency and high specificity. In addition, the cationic liposome complex has a function of lowering the zeta potential from around 0 mV to minus, and suppressing the particle diameter to 300 nm or less, and can produce a complex suitable for in vivo administration.

The figure regarding manufacture of the liposome composite_body | complex of this invention. The liposome complex produced under the conditions of pH 2, 4, 6, 8, 10 and 12 is fractionated, and the fluorescence intensity exhibited by each fraction is shown. The figure regarding manufacture of the liposome composite_body | complex of this invention. The liposome complexes prepared under conditions of pH 3, 5, 7, 9 and 11 are fractionated, and the fluorescence intensity exhibited by each fraction is shown. The figure which shows the cell introduction | transduction efficiency of the liposome complex of this invention. The cell transfer efficiency of each liposome complex prepared under conditions of pH 2, 4, 6, 8, 10 and 12 is shown by luciferase assay. The amount of DNA introduced into each cell is all uniform. The figure which shows the cell introduction | transduction efficiency of the liposome complex of this invention. The cell transfer efficiency of each liposome complex prepared under conditions of pH 3, 5, 7, 9, and 11 is shown by luciferase assay. The amount of DNA introduced into each cell is all uniform. The figure regarding manufacture of the liposome composite_body | complex of this invention. The liposome complexes prepared under conditions of pH 3, 5, 7, and 9 are fractionated, and the fluorescence intensity exhibited by each fraction is shown. In the figure, □ (open square) indicates the fluorescence intensity of Cy3, and ● (black circle) indicates the fluorescence intensity of NBD. The figure which shows the cell introduction | transduction efficiency of the liposome complex of this invention. The cell introduction efficiency of the liposome complex produced under the condition of pH 3 is indicated by the fluorescence intensity exhibited by the cell into which the complex is incorporated.

Liposome complex of the present invention The liposome complex of the present invention is a liposome complex containing a liposome and a bio-nanocapsule, or a liposome complex containing a liposome, a bio-nanocapsule, and a cell introduction substance, and the density of the liposome complex is It is a liposome complex which is 1.07-1.19 mg / ml.

  The surface potential of the liposome complex of the present invention is not particularly limited, but is usually about −30 to +10 mV. A liposome complex having a surface potential in such a range can function effectively when administered to a living body as DDS.

  The liposome of the present invention may be any of an anionic liposome, a neutral liposome, and a cationic liposome, and is not particularly limited.

  Liposomes may be monolayer liposomes or multilamellar liposomes and are usually about 40 to 300 nm in size. Preferably it is about 50-200 nm, More preferably, it is about 60-150 nm. The size of the liposome is preferably about 0.5 to 2 times that of the hollow nanoparticle. If the liposome is too large or too small relative to the nanoparticle, formation of a smooth composite particle is prevented.

  Liposomes can be produced by ultrasonic treatment, reverse phase evaporation, freeze-thaw, lipid dissolution, spray drying, and the like.

  Examples of liposome components include phospholipids, cholesterols, fatty acids, and the like. Specifically, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, cardiolipin, sphingomyelin, egg yolk lecithin, soybean In addition to natural phospholipids such as lecithin and lysolecithin, or those hydrogenated by conventional methods, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, dipalmitoyl phosphatidylserine, eleostearoyl phosphatidylcholine, eleostearoyl phosphatidylethanol Amine, eleostearoyl phosphatidylserine, etc. Synthetic phospholipids, and the like. It is desirable to use phospholipids in combination with lipids having various degrees of saturation. In addition, cholesterols include cholesterol, phytosterol, and the like, and fatty acids include oleic acid, palmitooleic acid, linoleic acid, or a fatty acid mixture containing these unsaturated fatty acids. Liposomes containing unsaturated fatty acids with small side chains are effective in producing small liposomes because of curvature.

  An example of a method for producing liposomes will be described in detail. For example, the above-described phospholipids, cholesterol and the like are dissolved in an appropriate organic solvent, put in an appropriate container, the solvent is distilled off under reduced pressure, and the inner surface of the container is removed. A phospholipid membrane is formed, and an aqueous solution containing an introduction substance, preferably a buffer solution, is added thereto and stirred to obtain a liposome encapsulating the introduction substance. The liposome is directly or once freeze-dried, and then mixed with the freeze-dried nanoparticles of the present invention to obtain composite particles of liposomes and nanoparticles.

  The bio-nanocapsule of the present invention is a biological hollow nanoparticle that presents a protein having membrane permeation activity or a variant thereof, and a lipid membrane, and examples thereof include hepatitis B virus surface antigen particles, influenza coat protein, Sendai virus coat protein and the like. Among these, hollow nanocapsules containing hepatitis B virus L (HBsAg L) protein having particle size performance as a constituent element are preferable.

  HBsAgL protein acts specifically on liver cells by the action of a human liver-specific receptor present in the pre-S1 region, and escapes from late endosomes by the action of a transmembrane peptide present on the amino terminal side of HBsAgL protein. Will be done more quickly.

  Examples of the modified HbsAg protein include hepatitis B virus surface antigen protein with reduced antigenicity described in Patent Document 1, and hepatitis B virus surface antigen protein having an antibody binding region described in Patent Document 3. Etc.

  The cell introduction substance of the present invention is a substance for achieving, for example, the purpose of changing the function of a cell, the purpose of labeling a cell, and the purpose of imparting a new function to a cell. Examples of these substances include nucleic acids, proteins, lipids, sugar chains, drugs, and labeling substances.

  The nucleic acid described above is DNA (for example, DNA to which a plasmid, protein or peptide is bound, genomic DNA, synthetic DNA or a fragment thereof), RNA (for example, t-RNA, m-RNA, siRNA, miRNA, synthetic RNA or These fragments), nucleic acid analogs having adenine, guanine, cytosine, thymine or uracil. By introducing these nucleic acids into cells, the purpose of exerting a function of regulating gene expression is achieved.

  The protein is not particularly limited, but may be a protein such as an antibody, a growth factor, a hormone, or the like that stimulates the cell from the outside to change the function of the cell, or a protein that functions inside the cell. That's fine. The protein in the present invention may be a peptide having a low amino acid polymerization degree.

  As with the above protein, lipids and sugar chains are not particularly limited as long as they stimulate the cell from outside the cell and change the function of the insertion rod, or function in the cell part. Not done.

  The drug is a low molecular compound and is not particularly limited. However, since the liposome complex of the present invention has a function as a DDS, a drug that needs to be specifically delivered to a tissue, or has a sustained release property. The required drug or the like is preferably used.

  The labeling substance is a substance that enables cells to be recognized from the outside, and is not particularly limited as long as it is generally called a labeling substance. The method for recognizing is not particularly limited, and may be any known means for recognizing a cell at a specific site from outside the body. Specific examples of such a labeling substance include fluorescent substances, radioactive substances, and magnetic substances.

  In addition, the particle size of the liposome complex of the present invention can be appropriately adjusted depending on the application. For example, if it is intended for administration into a living body, it is preferable to adjust the particle size to 200 nm or less. As a specific method for adjusting the particle diameter, the particle diameter can be adjusted by passing through a filter having a small pore diameter using an extruder.

  In the liposome complex of the present invention, the liposome and the bio-nanocapsule are bonded via a lipid membrane or the like that each has to form a complex. In the case of a liposome complex containing the liposome, the bio-nanocapsule, and the cell-introducing substance, the cell-introducing substance may be contained inside the liposome or the bio-nanocapsule, and is on the surface of the liposome and / or bio-nanocapsule. It may be in the form of a bond or a lipid membrane.

  The weight ratio of the liposome and the bio-nanocapsule constituting the liposome complex of the present invention is usually about liposome: bio-nanocapsule = 1: 0.2 to 1.5. From the viewpoint of obtaining high cell introduction efficiency, it is more preferably about 1: 0.7 to 1.5.

  Particularly preferred liposome complexes in the present invention include cationic liposomes as liposomes, hollow nanoparticles containing hepatitis B virus protein as bio-nanocapsules, and liposome complexes containing DNA as a cell introduction substance. The density of such a liposome complex is usually about 1.07 to 1.15 g / ml.

  In addition, an anionic liposome as a liposome and a liposome complex containing hollow nanoparticles containing a hepatitis B virus protein as a bio-nanocapsule are also particularly preferred liposome complexes in the present invention. The density of such a liposome complex is usually about 1.17 to 1.19 g / ml.

Method for Producing Liposome Complex of the Present Invention The method for producing the liposome complex of the present invention includes the following two steps.
(1) Step 1 of mixing liposomes and bio-nanocapsules in a liquid.
(2) Step 2 for separating the mixture obtained in Step 1 based on density.

<About Step 1>
In step 1, the liposome and the bio-nanocapsule are mixed in a liquid. What was mentioned above should just be used for a liposome and a bio-nanocapsule. Although the time to mix is not specifically limited, Usually, what is necessary is just about 10 to 30 minutes. Moreover, the temperature at the time of mixing is not specifically limited, Usually, what is necessary is just to set it as room temperature about 15-37 degreeC.

  For the purpose of increasing the cell introduction efficiency of the resulting liposome complex, it is preferable to mix in a liquid having a pH of 5 or less, or 10 or more. In order to further improve the cell introduction efficiency, the pH is preferably about 1 to 5 or about 10 to 14. At this time, the liposome or bio-nanocapsule may be prepared in a liquid having a pH in the above-mentioned range and then mixed, but immediately after mixing the liposome and the bio-nanocapsule in a liquid having an appropriate pH, the above-mentioned range. A liquid having a pH may be added.

  Such a liquid is not particularly limited, but a buffer solution is preferably used, and Britton Robinson buffer having a buffering ability at a relatively wide pH is particularly preferable.

  In step 1, the aforementioned cell introduction substance may be mixed simultaneously with the mixing of the liposome and the bio-nanocapsule. Alternatively, liposomes that have previously formed a complex with a cell introduction substance and bio-nanocapsules may be mixed, or bio-nanocapsules that have previously formed a complex with a cell introduction substance and liposomes may be mixed. Here, the cell introduction substance and the liposome, and the complex of the cell introduction substance and the bio-nanocapsule may be in a form in which the cell introduction substance is encapsulated in the liposome or the bio-nanocapsule. It may be in the form of binding to or intervening in a lipid membrane.

<About step 2>
In step 2, the mixture obtained in step 1 is separated based on density. That is, the composite obtained in Step 1 is a mixture formed with various compositions, and is a step of dividing the mixture according to the density of each composite.

  For the separation step, a separation method depending on the density may be used, and examples thereof include a density gradient centrifugation method. Examples of the density gradient solution used for the density gradient include a cesium chloride solution, a sucrose solution, a cesium sulfate solution, OptiPrep, Percol, and the like, and may be appropriately selected and used according to each application. The density gradient liquid may be a stepped density gradient or a linear density gradient, and may be appropriately selected and used.

  In the liposome production method of the present invention, when the cell introduction substance is not mixed with the liposome and the bio-nanocapsule in Step 1, the liposome complex obtained in Step 2 and the cell introduction substance are mixed, and the liposome and the bio-nanocapsule are mixed. And a liposome complex containing a cell-introducing substance. The conditions at the time of mixing are not specifically limited, For example, what is necessary is just to mix about 10 to 30 minutes at 15-37 degreeC. After mixing, the liposome complex or cell introduction substance obtained in Step 2 in which no complex was formed contains a liposome, a bio-nanocapsule, and a cell introduction substance as appropriate separated using a gel filtration column or the like. The liposome complex may be purified.

  The liposome complex obtained after step 2 may be subjected to dialysis treatment or the like according to the purpose of use.

Use of Liposome Complex of the Present Invention The liposome complex of the present invention has a cell introduction ability. Moreover, when the liposome complex of the present invention contains the above-mentioned cell introduction substance, the cell introduction substance can be introduced into the cells. The term “cell introduction” means that the entire liposome complex enters the cell, the cell membrane and the liposome complex are bound by lipid membrane fusion, etc., and a part of the liposome complex enters the cell, or It means that the liposome complex comes into contact with the vicinity of the cell membrane, and a part of the components forming the cell introduction substance complex enters the cell.

The liposome complex of the present invention can be used for, for example, DDS. That is, when the liposome complex of the present invention is administered in vivo, the liposome complex is introduced into cells constituting tissue in the body. The liposome complex of the present invention can also be used as a gene introduction reagent for cultured cells and the like.
By using the liposome complex of the present invention as DDS, it can be used for treatment or diagnosis of diseases.

  For example, a liposome complex using an anticancer drug such as doxorubicin as a cell introduction substance can introduce an anticancer drug into cells.

<Preparation of Bionanocapsule (BNC)>
BNC used in this example, Saccharomyces cerevisiae AH22R harboring BNC expression plasmid pGLDLIIP39-RcT ^- from strain, described in JP 2007-209307 was prepared according to an efficient method for purifying bio-nanocapsules. BNC was freeze-dried and stored.

  1 mL of the prepared BNC (1 mg / 1 mL as the amount of protein) was added to an Amersham FluoroLink (registered trademark) Cy3 Reactive Dye (GE Healthcare UK Ltd. Buckinghamshire UK) 1 tube at room temperature, and kept at room temperature for 60 minutes. Thereafter, 50 μl of 1 mM Tris-HCl (pH 7.4-7.5) was added, and the reaction was stopped by standing for 5 minutes. By this step, BNC labeled with Cy3 (Cy3-BNC) was produced.

  Subsequently, the reaction product was subjected to gel filtration chromatography using Sephadex G-25 Fine (GE Healthcare) and PBS to purify Cy3-BNC.

The obtained Cy3-BNC solution was measured for protein absorbance (A ₂₈₀ ) and Cy3 absorbance (A ₅₅₂ ) with a spectrophotometer to quantify the BNC concentration as the protein concentration. Specifically, Cy3-BNC 8% of _{A 552} from _{A 280} solution minus the (background due Cy3) as the absorbance of the protein, unlabeled measured in advance BNC (1 mg as protein amount / The BNC concentration was calculated using the A ₂₈₀ value of ml) as standard.

  Based on the quantification result, the Cy3-BNC solution was dispensed into a freeze-drying tube so that the BNC amount was 100 μg / tube (as the protein amount), 250 μg / ml sucrose solution was added at 20 μl / tube, and liquid nitrogen was added. And then freeze-dried for 48 hours in a square dry chamber (Tokyo Rika Kikai Co., Ltd.) connected to a freeze dryer (Tokyo Rika Kikai Co., Ltd.).

<Preparation of cationic liposome>
3.3mM DC-6-14 (Mutaku Pharmaceutical Co., Ltd.) 801.8 μL, 3.3 mM DOPE (NOF) 601.4 μL, 3.3 mM Cholesterol (Sigma) 581.3 μL, 2 mg / 16.4 μL of 22- (N- (7-nitrobenzo-2-oxa-1,3-diazol-4-yl) amino) -23,24-bisnor-5-cholen-3β-ol at a concentration of mL was prepared. All of these were put into a 50 mL eggplant flask, dried under reduced pressure, and then hydrated with 2 mL of distilled water.

  Subsequently, freezing and thawing with liquid nitrogen were repeated 5 times, and further, a sonication process was performed using an anthrason ultrasonic cell crusher (Misonix Inc.) to obtain an NBD-labeled cationic liposome having a final concentration of 2 mg / ml. .

<Preparation of cationic liposome complex>
Cationic liposomes labeled with NBD are dispensed in an amount of 200 μg of lipids into Eppendorf tubes, and 44.2 μg of pCAG-Luc3 vector (expression plasmid in which a luciferase gene is placed downstream of the CAG promoter) is added for 15 minutes at room temperature. The mixture was allowed to stand to prepare a DNA-cationic liposome complex (hereinafter referred to as lipoplex). At this time, the weight ratio of the two was pCAG-Luc3 vector: liposome = 1: 4.53.

  Next, the prepared lipoplex and 100 μg of Cy3-BNC already prepared were mixed, and Britton Robinson buffer (0.1 M boric acid, 0.1 M acetic acid, 0.1 M phosphoric acid, pH 3, 5, 7, and 9) The pH was adjusted with 0.5 M aqueous sodium hydroxide solution) and incubated at 37 ° C. for 30 minutes to prepare a BNC-lipoplex complex.

  Subsequently, the obtained BNC-lipoplex complex was subjected to a cesium chloride density gradient centrifugation to perform a fractionation operation. Fractionation by cesium chloride density gradient centrifugation is 10%, 15%, 20%, 30%, and 40% (w / v) cesium chloride solution (in this case, PBS is 2 ml each) for P40ST. Were centrifuged for 16 hours at room temperature at a rotational speed of 24,000 rpm using a separation ultracentrifuge CP100MX (Hitachi Koki).

  After centrifugation, 500 μl was collected from the upper layer of the centrifuge tube to obtain each fraction. Each fraction was measured by using Variosscan (Thermo Fisher Scientific) to measure the fluorescence derived from NBD that labels liposomes (measurement wavelength 530 nm) and the fluorescence derived from Cy3 that labels BNC (measurement wavelength 552 nm), and BNC-lipoplex complex The peak fraction containing the most body was collected. The fluorescence intensity of NBD and Cy3 in each fraction is shown in FIGS.

  In this example, the BNC-lipoplex complex prepared under pH 2 conditions is the 7th and 8th fractions, the pH 4 condition is the 8th and 9th fractions, and the pH 6 condition is the 9th position. The fraction of pH 8 is the 10th fraction, the pH 10 condition is the 8, 9 and 10th fractions, and the pH 12 condition is the 5th, 6th and 7th fractions. Was recovered. In addition, the BNC-lipoplex complex prepared under the condition of pH 3 shows the sixth, seventh and eighth fractions, the condition of pH 5 shows the eighth and ninth fractions, and the condition of pH 7 shows the ninth. Fractions were collected at pH 9 for the 10th fraction and at pH 11 for the 7th fraction. Each collected peak fraction was dialyzed several times with 500 ml of PBS and then stored at 4 ° C.

  Various properties of the BNC-lipoplex complex contained in these fractions were measured. BNC quantification (as protein amount) was evaluated by BCA assay using Micro BCA assay kit (Promega). In addition, DNA contained in the complex was quantified by an Applied Biosystems 7300 real-time PCR system (Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen)) using an Applied Biosystems 7300 real-time PCR system. Specifically, 12.5 μl of Platinum SYBR Green qPCR SuperMix-UDG with ROX, 10 μM each of Forward Primer (SEQ ID NO: 1) and Reverse Primer (SEQ ID NO: 2) were added to each microtube, and 0.5 μl each. 5 μl of each template was added thereto, and the volume up to 25 μl with distilled water was used for RT-QPCR (Real time-Quantitative PCR).

For the template, 2 μl of BNC-lipoplex complex and 2% (v / v) TX-100 in the same amount were mixed, and placed at room temperature for 30 minutes, diluted 200 times and used as a standard pCAG-Luc3 Used concentrations of 0.025, 0.25, 2.5, and 25 pg / μl. The concentration of Triton X-100 mixed in the reaction solution was 5 × 10 ⁻³ % (v / v) or less. It has been separately confirmed that this concentration does not inhibit RT-QPCR. The PCR reaction thermal program was performed according to the protocol of the above system.

  The particle size and ζ potential of the BNC-lipoplex complex were measured using a Zetasizer Nano series ZS (Malvern Instruments Ltd.). Specifically, particle size measurement by DLS method (dynamic light scattering method) in which laser light is applied to a particle group in Brownian motion and the scattered light is detected by a photomultiplier tube, and mixed mode measurement (M3) measurement Ζ potential measurement was performed by M3-PALS, which is a combination of the technology and the PALS (Phase Analysis Light Scattering) method.

  Table 1 below shows various properties of the BNC-lipoplex complex contained in each of the above fractions, which were produced after each pH environment and then fractionated by density gradient centrifugation.

  As a comparative experimental example, the above-mentioned lipoplex and Cy3-BNC were mixed at a weight ratio of 4.53: 7.48, and left standing at room temperature for 10 minutes, and subjected to cesium chloride density gradient centrifugation. No sample was used as a comparative experimental example in a subsequent cell transfer efficiency experiment.

<Cell transfer efficiency experiment using liposome complex>
The efficiency of gene transfer into cells was measured based on intracellular luciferase activity.
As the introduced cells, Huh7 (human liver cancer-derived cells) and HEK293 cells (human kidney-derived cells) were used. These cells were seeded at 5 × 10 ⁴ / well in a 24-well well-plate (AGC technoglass), and 10% (v / v) FBS-containing DMEM medium was present at 37 ° C. and 5% (v / v) CO _2. The cells were cultured for 24 hours. Next, the BNC-lipoplex complex after being subjected to the above-described density gradient centrifugation was added to the cells so that the amount of DNA was 3 ng / well, and cultured for 72 hours.

In addition, as a comparative experimental example, lipoplex and a lipoplex-BNC complex not subjected to the above-described density gradient centrifugation were added to the cells so as to be 3 ng per well. Luciferase activity was measured using Luciferase Assay System (Promega). Specifically, luciferase-expressing cells were washed once with PBS, Passive Lysis Buffer was added at 100 μl / well, and shaken at room temperature for 5 minutes using BioShaker (TAITEC), and then the cell lysate was collected. Next, 100 μl of luciferase substrate was added to 5 μl of cell lysate, and the amount of luminescence for 10 seconds was measured using a luminometer (Lumat LB9507), and the luciferase activity was expressed in relative luminescence units (RLU).
In addition, the protein concentration of each sample was quantified by Micro BCA Protein Assay Kit (SIGMA), converted to reflect the number of cells, and the RLU value was corrected. The results are shown in FIGS. The measurement was performed 6 times, and the average value was calculated and used.

  The gene transfer efficiency of the BNC-lipoplex complex subjected to density gradient centrifugation was higher than that of the conventional method under all pH conditions at the time of preparing all the complexes. Among these, the BNC-lipoplex complex produced under low pH conditions or high pH conditions dramatically improved the efficiency. In addition, the conventional BNC-lipoplex complex tended to exhibit lower gene transfer efficiency than lipopolex, whereas the BNC-lipoplex complex subjected to density gradient centrifugation had a gene transfer efficiency higher than that of lipoplex. It became high. Moreover, the liver cell specificity of the BNC-lipoplex complex subjected to the density gradient centrifugation method was dramatically improved as compared with the conventional BNC-lipoplex complex.

  Specifically, in the experiment on Huh7 cells, the RLU value of only lipoplex was 4243, and the RLU value of the BNC-lipoplex complex that was not subjected to density gradient centrifugation was 2058, which was just mixed. Among the BNC-lipoplex complexes subjected to density gradient centrifugation as in this example, the RLU values of those adjusted under pH 2 conditions were 845559, pH 4 at 405935, pH 6 at 277470, pH 8 at 147597, pH 10 at 279063. It was 342588 at pH 12. From the above results, it was revealed that the BNC-lipoplex complex prepared under a pH environment other than neutral has particularly high gene transfer ability.

  In addition, as a comparative experiment, the RLU value of Fugene, which is generally used as a gene introduction agent, was 84579.

<Preparation of anionic liposome>
Anionic liposomes were prepared as follows. Dipalmitoylphosphatidylcholine (DPPC; NOF), dipalmitoylphosphatidylethanolamine (DPPE; NOF), dipalmitoylphosphatidylglycerol sodium (DPPG-Na; NOF) and cholesterol (Chol; Nacalai Tesque) Is dissolved in a chloroform / methanol (4: 1 (v / v)) mixed solution, and DPPC: DPPE: DPPG-Na: Chol is added to the eggplant flask so that the molar ratio is 15: 15: 30: 40. In addition, a hemispherical lipid film was produced by drying under reduced pressure using a rotary evaporator. Distilled water was added to the film, followed by sonication for 6 minutes at room temperature using an anthrason ultrasonic crusher. In addition, an anionic liposome labeled with NBD is replaced with the above-mentioned DPPE by N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) -1,2-dihexadecanyl-sn-glycero- 3-phosphoethanolamine (NBD-DHPE; Invitrogen) was used.

<Preparation of BNC-anionic liposome complex>
The above-mentioned NBD-labeled anionic liposome and the above-mentioned Cy3-labeled BNC are added to Britton-Robinson buffer having a pH ratio of 3, 5, 7, and 9 in an amount of 2: 1, respectively. And incubated at 37 ° C. for 30 minutes.

  Later, as in <Preparation of Cationic Liposome Complex> described above, it was subjected to a cesium chloride density gradient centrifugation method. The fluorescence intensity exhibited by each fraction obtained by the cesium chloride density gradient centrifugation is shown in FIG. 5 for each pH condition at the time of preparing the complex.

  The BNC-anionic liposome complex prepared under pH 3 conditions is the 8th fraction, the pH 5 condition is the 8th fraction, the pH 7 condition is the 8th fraction, and the pH 9 condition. The bottom collected the 8th fraction. Each collected peak fraction was dialyzed several times with 500 ml of PBS and then stored at 4 ° C.

  Of the obtained BNC-anionic liposomes, those prepared under the condition of pH 3 were found to have a density of 1.18 g / mL.

<Introduction ability of BNC-anionic liposome complex into cells>
Among the above-mentioned BNC-anionic liposome complexes, those prepared under the condition of pH 3 and fractionated by cesium chloride density gradient centrifugation (protein concentration 10 μg / ml) were about 1 × 10 ⁴ cells. Added to Huh7 or HeLa cells and incubated on ice for 1 hour. As a comparative experiment, anionic liposomes labeled with Cy3-BNC with the same protein concentration and NBD with the same lipid concentration were added to the cells and incubated in the same manner. Thereafter, the cells were washed several times with cold PBS, fixed with 4% (w / v) paraformaldehyde, and used as each measurement sample.

  Each measurement sample was observed with a confocal laser microscope FV-1000D (Olympus), and the fluorescence intensity of Cy3 and NBD was measured. Each fluorescence intensity was analyzed using MetaMorph softwarewareversion 6.1r2. In addition, the obtained fluorescence intensity value was standardized by the number of cells. The results are shown in FIG.

  When the BNC-anionic liposome complex is allowed to act on cells, the amount of Huh7 cells is about 44 times higher than that of Hela cells, and about 59 times higher based on the fluorescence intensity of NBD. It was clarified that the BNC-anionic liposome complex was adsorbed or taken up into the cell in a certain amount. In contrast to Huh7 cells, the BNC-anionic liposome complex is adsorbed or taken up by cells in an amount of about 6 times that of BNC alone and about 200 times that of anionic liposome alone. There was found.

Claims (3)

A method for producing a liposome complex, comprising:
(1) Cationic liposomes, B-type hollow nanoparticles comprising hepatitis viral proteins, and step 1 is mixed nucleic acids at the liquid, and (2) separated on the basis of the mixture obtained in the step 1 to the density Including step 2
The mixing step in step 1 is performed in a liquid having a pH of 5 or less or 10 or more,
A method for producing a liposome complex. The method according to claim 1, wherein in the step 2, a liposome complex having a density of 1.07 to 1.19 g / ml is separated. Hollow nanoparticles, as a component of the lipid membrane, including hepatitis B virus protein, the production method according to claim 1 or 2.

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