WO2019061790A1 - Nanoparticule, et son procédé de préparation - Google Patents

Nanoparticule, et son procédé de préparation Download PDF

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WO2019061790A1
WO2019061790A1 PCT/CN2017/113397 CN2017113397W WO2019061790A1 WO 2019061790 A1 WO2019061790 A1 WO 2019061790A1 CN 2017113397 W CN2017113397 W CN 2017113397W WO 2019061790 A1 WO2019061790 A1 WO 2019061790A1
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solution
liquid
reactant
nanoparticles
nanoparticle
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Chinese (zh)
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张龙
吴延恒
顾文艺
许志平
李疆
吴沛宏
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广州宏柯源生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles

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  • the invention relates to the field of life science technology, in particular to a nano particle and a preparation method thereof.
  • Nanoparticles are nanomaterials with a cavity in the middle. They can be encapsulated in small cavities, siRNAs, antibodies, etc. into the cavity and introduced into the target cells.
  • TIL cells T cells
  • T cells lymphocytes that are cultured together with the patient's cancer tissue and the patient's T cells, and then the lymphocytes are expanded and returned to the patient.
  • PD-L1 protein called PD-L1 on the surface of cancer cells. This PD-L1 protein binds to the PD-1 protein of T cells and inhibits the killing of cancer cells by T cells. Therefore, if you want T cells to kill cancer cells efficiently, you need to find ways to reduce the PD-1 of T cells and the PD-L1 of cancer cells.
  • One of the objects of the present invention is to provide a method for preparing nanoparticles, comprising the steps of:
  • a dispersion of the monolayer film nanoparticles is prepared, and a mixture of DOPC (1,2-dioleoylphosphatidylcholine), cholesterol, or DOTAP (2-dioleoylhydroxypropyl-) is added to the dispersion.
  • DOPC 1,2-dioleoylphosphatidylcholine
  • DOTAP 2,2-dioleoylhydroxypropyl-
  • a mixture of 3-N,N,N-trimethylammonium) and cholesterol, the particles in the resulting suspension particle solution are two-layer membrane nanoparticles.
  • the first reactant solution is a calcium salt solution
  • the second reactant solution is a phosphate solution or a pyrophosphate solution
  • the first reactant, the second reactant calcium The mass ratio of phosphorus is (25 to 400): 1.
  • a nanoparticle core can be obtained well, the uneven particle size is uniform, and the stability is good, and aggregation is not easy. If it is beyond the limits of the embodiments of the present application, either the nanoparticle core cannot be formed, or the formed particle core is agglomerated, and the subsequent coating operation is not easy.
  • the calcium salt solution has a concentration of 4.5 to 5.5 M; and the phosphate solution or pyrophosphate solution has a concentration of 45 to 55 mM.
  • the uniformity of the precipitate produced by the combination of the first reactant and the second reactant can be further improved, thereby improving the uniformity of the nanoparticles.
  • the calcium salt solution comprises a calcium chloride solution, a calcium nitrate solution, a calcium gluconate solution;
  • the phosphate solution comprises a dipotassium hydrogen phosphate solution, a diammonium hydrogen phosphate solution, and an ammonium dihydrogen phosphate solution.
  • the pyrophosphate solution comprises a calcium pyrophosphate solution, an acid sodium pyrophosphate solution, and a sodium pyrophosphate solution.
  • the concentration of DOPA in the DOPA solution is 15-25 mg/ml
  • the volume ratio of the added volume of the DOPA solution to the mixed solution of the A liquid and the C liquid is (70-80): 1.
  • the solvent of the DOPA solution includes chloroform, dichloromethane, ethyl acetate, tetrahydrofuran.
  • the mass ratio of DOPC to cholesterol in the mixture of DOPC and cholesterol is 1: (2.5 to 3.5); in the mixture of DOTAP and cholesterol, the quality of DOTAP and cholesterol The ratio is 2: (2.5 to 3.5).
  • the organic solvent is any one or more of cyclohexane, benzene, toluene, n-heptane, and carbon tetrachloride; and the surfactant includes polyoxygenation.
  • the invention adopts organic solvent screening and surfactant screening, and aims to introduce an oil-water system.
  • the oil-water system has the advantages of obtaining a precipitate which is combined with the second reactant by the high suspension and protection, and improves stability, so that the stability is improved. Coagulation does not easily occur between particles.
  • the ratio of the amount of the liquid A and the liquid B is (50 to 200): 1; the ratio of the amount of the liquid A and the liquid C is (50 to 200): 1; the liquid of the E and the liquid D
  • the dosage ratio is (1 to 3): (1 to 3).
  • the dispersing the monolayer film nanoparticles is specifically dispersing the monolayer film nanoparticles in chloroform, dichloromethane, ethyl acetate or tetrahydrofuran.
  • the functional substance in the functional substance solution includes a functional nucleic acid sequence (dsDNA, siRNA, etc.), a protein antibody, a drug molecule.
  • the functional substance in the functional substance solution comprises a functional nucleic acid sequence.
  • Another object of the present invention is to provide a nanoparticle obtained by the above production method.
  • the embodiment of the invention has the following beneficial effects:
  • the organic solvent, the surfactant, the first reactant solution, the functional substance solution, the second reactant solution, and then the double outer membrane are mixed stepwise, thereby improving the nanoparticle carrying the functional substance to the target. Transfection efficiency of cells.
  • the organic solvent, the surfactant, the first reactant solution, the functional substance solution, and the second reactant solution are mixed stepwise, so that the first reactant and the second reactant are formed after carrying the functional substance.
  • the nanoparticle core is small and uniform, and it is not easy to agglomerate.
  • the particle size of the obtained nanoparticle product can be controlled to a small scale (about 20 nm), and the film is also improved while coating the film.
  • the hydrophilicity on the outer side, the dispersibility and stability of the particles are increased, aggregation is less likely to occur, and the affinity with the cell surface is enhanced, and it is easily absorbed by the cells, thereby increasing the transfection efficiency.
  • FIG. 1A is a transmission electron microscope (TEM) photograph of a nanoparticle prepared according to an embodiment of the present invention
  • FIG. 1B is a dynamic light scattering diagram of a nanoparticle prepared according to an embodiment of the present invention
  • FIG. 2A is a cell uptake rate of nanoparticles prepared at different concentrations according to an embodiment of the present invention
  • FIG. 2B is a cell uptake rate of nanoparticles prepared according to an embodiment of the present invention during advancement;
  • FIG. 3A is a PD1 mRNA level of a cell after transfecting a TIL cell with a nanoparticle prepared according to an embodiment of the present invention
  • FIG. 3B is a PD1 protein level of the cell after transfecting the TIL cell with the nanoparticle prepared by the embodiment of the present invention
  • FIG. 4A is a PDL1 mRNA transcription level of a cell after transfecting a nanoparticle into a breast cancer cell according to an embodiment of the present invention
  • FIG. 4B is a PDL1 protein expression level of the cell after transfecting the nanoparticle prepared by the embodiment of the present invention
  • Figure 5 is a test for the lethality of TIL cells of different ratios and different degrees of silence on breast cancer cells
  • FIG. 6A and 6B are graphs showing the results of test results of transfection on TIL cell subsets
  • FIG. 6C and FIG. 6D are graphs showing test results of cytokine production by TIL cells before and after transfection
  • Figure 7 is a schematic view showing the structure of the obtained nanoparticles according to an embodiment of the present invention.
  • nanoparticles of the present invention and a method for preparing the same are described in further detail below in conjunction with specific examples.
  • Example 1 Nanoparticles containing silencing siRNA (PD1 silencing TIL cells) and preparation thereof method
  • This section provides a method for preparing nanoparticles, comprising the following steps:
  • liquid A mixing cyclohexane with polyoxyethylene (5) nonylphenyl ether, the ratio of the two is 70:30 volume ratio; cyclohexane can be replaced by benzene, toluene, n-glycan Alkane, carbon tetrachloride;
  • disodium hydrogen phosphate solution can be replaced with Dipotassium hydrogen phosphate solution, diammonium hydrogen phosphate solution, ammonium dihydrogen phosphate solution, calcium hydrogen phosphate solution, calcium phosphate solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium phosphate solution, calcium pyrophosphate solution, acid form Sodium pyrophosphate solution or sodium pyrophosphate solution;
  • E solution Take a portion of solution A (the volume used is 15 ml), add 150 ⁇ l of C solution, stir for 5 min, then add DOPA in chloroform solution (concentration of DOPA is 20 mg/ml, the volume of the solution is 200 ⁇ L), and continue stirring for 15 min.
  • the chloroform of this step can also be replaced by dichloromethane, ethyl acetate or tetrahydrofuran;
  • E-liquid was dropped into D solution one by one, and stirred for 20 minutes, and the resulting precipitate was a single-layer membrane nanoparticle, that is, the first reactant, the second reactant, and the functional siRNA formed only outside the core.
  • the drop rate of E liquid is controlled at 1-3 ml/min; this step also includes cleaning and collecting the single-layer film nanoparticles.
  • the method includes the following steps: the step of washing comprises: adding 10 ml of ethanol to the mixture of the E solution and the D solution in a volume ratio of 1:1, stirring for 5 minutes, centrifuging at 10,000 g for 20 min, and discarding. Supernatant, leaving precipitate; add 10 ml of ethanol to the precipitate, centrifuge at 10,000 g for 20 min, then discard the supernatant and leave a precipitate; the collection step includes: adding 1 ml of chloroform to collect the monolayer membrane nanoparticles;
  • PBS phosphate buffer
  • the structure of the two-layer membrane particles prepared in this embodiment is shown in Figure 7.
  • the first reactant, the second reactant, the core of the nuclear nanoparticles formed by the functional siRNA, the DOPA formation includes the inner membrane outside the core, and DOPC and DOTAP are formed. Outer membrane.
  • Antisense strand 5'-UUUGAAAGUAUCAAGGUCUdTsdT-3';
  • the nanoparticles obtained by the preparation method of the present embodiment have good morphology, the size is in a controllable range, the particles are not adhered together, and the dispersion is uniform, as shown in FIG. 1A; the size of the nano material particles is about 20 nm, and the dispersion can be uniformly dispersed. Again, the proper size and good dispersibility of the particles were verified, see Figure 1B.
  • This section deals with the transfection of TIL cells with nanoparticles prepared in 1.1, including the following steps:
  • Step one inoculate a 6-well plate at a density of 1.5 ⁇ 10 5 TIL cells/well, place in an incubator (condition: 37° C., 5% CO 2 ) overnight, and remove the cell culture medium;
  • Step 2 Add fresh cell culture medium containing 1.1 nm particles to the 6-well plate treated in the first step, and let stand at 37 ° C; wherein the nanoparticles are coated with 50 nM of siRNA (the amount is: added siRNA) The total amount minus the amount of siRNA not encapsulated into the nanoparticles);
  • Step 3 Discard the medium in the 6-well plate treated in the second step, wash it three times with fresh phosphate buffer (PBS), and wash away the nanoparticles that were not absorbed by the TIL cells.
  • PBS phosphate buffer
  • the concentration of the fresh cell culture medium containing the nanoparticles of Example 1 in step 2 was set to three concentration levels, namely 20 nM, 40 nM, and 80 nM, and three nanoparticle concentrations were measured.
  • the rate of extraction of nanoparticles by TIL cells was set to three concentration levels, namely 20 nM, 40 nM, and 80 nM, and three nanoparticle concentrations were measured. The rate of extraction of nanoparticles by TIL cells.
  • Fig. 2A The results are shown in Fig. 2A. It can be seen from the figure that when the content of the nanoparticles in the medium is 40 nM, the uptake rate of the nanoparticles by the TIL cells can reach about 65%.
  • This example also tests the preferred uptake time for this preferred concentration (nanoparticle content 40 nM) at a preferred nanoparticle concentration.
  • Fig. 2B The results are shown in Fig. 2B. It can be seen from the figure that about 65% of TIL cells can be transfected in 2 hours.
  • TILs a, normal state of TIL cells that have not been transfected, labeled "TILs";
  • control siRNA is: 5'-UUCUCCGAACGUGUCACGUTT-3';
  • TIL cells transfected with the nanoparticles of the present example at a concentration of 40 nM labeled as "TILs + LCP + siRNA-40 nM";
  • TIL cells transfected with the nanoparticles of the present example at a concentration of 80 nM labeled as "TILs + LCP + siRNA-80 nM";
  • TIL cells transfected with the nanoparticles of the present application at a concentration of 160 nM labeled "TILs + LCP + siRNA - 160 nM”.
  • FIG. 3A The results of the mRNA transcription level are shown in Figure 3A. As the concentration of nanoparticles increases from 20 nM to 160 nM, the transcription of PD1 decreases from 86% to about 15%. It can be seen that the nanoparticles can efficiently transfer PD1 siRNA into TIL cells, thereby silencing the expression of PD1 in TIL cells.
  • Fig. 3B The results of detecting the expression level of PD1 protein in TIL cells are shown in Fig. 3B.
  • concentration of the nanoparticles was 40 nM, the expression level of the PD1 protein was significantly reduced. It can be seen that when the concentration of nanoparticles is 40 nM, the expression of PD1 protein in TIL cells can be effectively reduced.
  • Example 2 Nanoparticles containing silencing siRNA (PDL1 silencing breast cancer cell MCF7) And preparation method thereof
  • This section provides a method for preparing nanoparticles, comprising the following steps:
  • Preparation liquid B 5M calcium chloride solution (volume used is 75 ⁇ L) mixed with 100 ⁇ M siRNA solution (volume used is 100 ⁇ L), and added 50 ⁇ l of ultrapure water;
  • liquid C 50 mM disodium hydrogen phosphate (volume used is 75 ⁇ L) mixed with 100 ⁇ M siRNA (volume used is 100 ⁇ L), and added 50 ⁇ l of ultrapure water;
  • E solution Take a portion of solution A (the volume used is 15 ml), add 150 ⁇ l of C solution, stir for 5 min, then add DOPA in chloroform solution (concentration of DOPA is 20 mg/ml, the volume of the solution is 200 ⁇ L), and continue stirring for 15 min. ;
  • E-liquid was dropped into D solution one by one, and stirred for 20 minutes, and the resulting precipitate was a single-layer membrane nanoparticle, that is, the first reactant, the second reactant, and the functional siRNA formed only outside the core.
  • the drop rate of E liquid is controlled at 1-3 ml/min; this step also includes cleaning and collecting the single-layer film nanoparticles.
  • the method includes the following steps: the step of washing comprises: adding 10 ml of ethanol to the mixture of the E liquid and the D liquid in a volume ratio of 1:1, stirring for 5 minutes, centrifuging at 10,000 g for 20 min, and discarding. Clear and leave a precipitate; add 10 ml of ethanol to the precipitate and centrifuge at 10,000 g for 20 min. Discarding the supernatant and leaving the precipitate; the collecting step includes: adding 1 ml of chloroform to collect the monolayer film nanoparticles;
  • PBS phosphate buffer
  • the structure of the two-layer membrane particles prepared in this embodiment is shown in Figure 7.
  • the first reactant, the second reactant, the core of the nuclear nanoparticles formed by the functional siRNA, the DOPA formation includes the inner membrane outside the core, and DOPC and DOTAP are formed. Outer membrane.
  • the sequence of the siRNA is PD-L1 siRNA:
  • Antisense strand 5'-UUCAACACUGCUUACGUCUdTsdT-3';
  • the nanoparticles obtained in the preparation method of this part are the same as in the first embodiment: the morphology is very good, the size is in a controllable range, the particles are not adhered together, and the dispersion is uniform, as shown in FIG. 1A; the size of the nano material particles is about 20 nm. And it can be evenly dispersed, and the proper size and good dispersibility of the particles are verified again, as shown in Fig. 1B.
  • Transfection of breast cancer cells with the nanoparticles prepared in 2.1 includes the following steps:
  • Step one inoculate a 6-well plate at a density of 1.5 ⁇ 10 5 breast cancer cells/well, place in an incubator (condition: 37° C., 5% CO 2 ) overnight, and remove the cell culture medium;
  • Step 2 Adding fresh cell culture medium containing the nanoparticles prepared in 2.1 above to the 6-well plate treated in the first step, and allowing to stand at 37 ° C for 4 h; wherein the nanoparticles are coated with 40 nM of siRNA (the amount is : the total amount of siRNA added minus the amount of siRNA not encapsulated into the nanoparticles);
  • Step 3 Discard the medium in the 6-well plate treated in the second step and wash it three times with fresh phosphate buffer (PBS) to wash away the nanoparticles that were not absorbed by the breast cancer cells.
  • PBS phosphate buffer
  • control siRNA is: 5'-UUCUCCGAACGUGUCACGUTT-3';
  • the detection results of mRNA transcription levels are shown in Figure 4A. As the concentration of nanoparticles increases from 10 nM to 40 nM, the transcription of PD1 decreases from 80% to about 20%. It can be seen that the nanoparticles can efficiently transfer PDL1 siRNA into breast cancer cells, thereby silencing the expression of PDL1 in breast cancer cells.
  • Fig. 4B The results of the detection of protein expression levels are shown in Fig. 4B.
  • the inventors found that when the concentration of the nanoparticles was 40 nM, the expression level of PDL1 protein was significantly reduced. It can be seen that when the concentration of the nanoparticles is 40 nM, the expression of PDL1 protein in breast cancer cells can be effectively reduced.
  • the TIL cells involved in Example 1 were mixed with the breast cancer cell MCF7 of Example 2 to test the lethality of TIL cells against breast cancer cell MCF7.
  • Selected TIL cells include: untransfected TIL cells that highly express PD1, labeled "PD1+”; TIL cells that are lowly expressed PD1 transfected with 40 nM nanoparticles, labeled "PD1-”.
  • the selected breast cancer cells MCF7 include: untransfected breast cancer cells with high expression of PDL1, labeled as "PDL1+”; breast cancer cells with low expression of PDL1 transfected with 40 nM nanoparticles, labeled "PDL1-";
  • the selected TIL cells and the selected breast cancer cells MCF7 were mixed at different cell weight ratios, and a total of 12 different mixing treatments were included, including the following:
  • the cell killing efficiency test is detected by a lactate dehydrogenase detection kit. Ie using CytoTox Standard 4-hour lactate dehydrogenase release assay was performed by Non-Radioactive Cytotoxicity Assay (Promega, WI). Methods as below:
  • the breast cancer cells to be examined (silencing breast cancer cells MCF7 K and unsilent breast cancer cells MCF7) were inoculated into 96-well plates at a density of 1 ⁇ 10 4 per well, and after 18 hours of culture, the medium was changed to phenol-free. Red, RPMI 1640 medium (Gibco-BRL) containing 5% fetal bovine serum, continued to culture for 6 hours.
  • TIL cells (silent TIL cells TIL K and unsiltained TIL cells TIL) were cultured in CNE-2 medium for 24 hours, and then the cells were collected, in phenol-free red, RPMI 1640 containing 5% fetal bovine serum. The cells were resuspended in medium (Gibco-BRL).
  • % killing efficiency (experimental result - breast cancer cell basal release value - TIL cell basal release value) / (peak killing cell release peak - breast cancer cell basal release value) x 100.
  • the coated siRNA nanoparticles were prepared by referring to the preparation method of Example 1, and then the TIL cells were transfected with the nanoparticles at a concentration of 40 nM.
  • the pre-transfection TIL cells pre-silent TIL cells, labeled as TILs
  • the transfected TIL cells ie, silenced TIL cells, labeled TILs K
  • the results of the assay are shown in Figures 6A and 6B; Figure 6A shows TIL cells after silencing, and Figure 6B shows TIL cells before silencing. According to Figures 6A and 6B, there is no change in TIL cell subsets after and after PD1 silencing. This indicates that the entire silencing process has no effect on the cell subset of TIL cells.
  • TILs K transfected TIL cells
  • MCF7 MCF7 K and CTL6 cells
  • the results of the assay are shown in Figure 6C and Figure 6D.
  • the expression levels of IL17, IL10, INF ⁇ , and INF ⁇ were significantly increased in transfected TIL cells, especially INF ⁇ , indicating that the killing ability of TIL cells after transfection was significantly enhanced.
  • the first reactant solution is a calcium salt solution
  • the second reactant solution is a phosphate solution or a coke.
  • a phosphate solution the first reactant, the second reactant has a calcium to phosphorus mass ratio of (25 to 400):1;
  • the calcium salt solution has a concentration of 4.5 to 5.5 M; and
  • the phosphate solution Or the concentration of the pyrophosphate solution is 45-55 mM;
  • the mass ratio of DOPC and cholesterol in the mixture of DOPC and cholesterol is 1: (2.5 to 3.5);
  • the mass ratio of DOTAP to cholesterol is 2: ( 2.5 to 3.5);
  • the organic solvent is any one or more
  • Nanoparticles containing the silencing siRNA (PD1 silencing TIL cells) used in this comparative example were obtained by the following preparation methods:
  • Step 1 10.5 ml of cyclohexane, 4.5 ml of polyoxyethylene (5) nonylphenyl ether, 5 M calcium chloride solution (volume 50 ⁇ L), 100 ⁇ M siRNA solution (volume used is 66.7 ⁇ L), ultrapure Mixing 33.3 ⁇ L of water to obtain a mixture I;
  • Step 2 10.5 ml of cyclohexane, 4.5 ml of polyoxyethylene (5) nonylphenyl ether, 50 mM disodium hydrogen phosphate (volume 50 ⁇ L), 100 ⁇ M siRNA solution (volume used is 66.7 ⁇ L), ultrapure 33.3 ⁇ L of water was mixed to obtain a mixture II;
  • the mixture I was dropped into the mixture II at a rate of 1 to 3 ml/min, and stirred for 20 minutes, and the resulting precipitate was a nanoparticle.
  • step one in step two, the volume ratio of cyclohexane to polyoxyethylene (5) nonylphenyl ether is 70:30, and the sequence and concentration (50 nM) of the encapsulated siRNA are the same as in the first embodiment.
  • the nanoparticles prepared in this comparative example have a single-layer membrane structure, the particle size is uneven, the particle diameter is larger than 20 nm, and the dispersibility is poor.
  • the concentration of the obtained nanoparticles was adjusted to 40 nM, and TIL cells were transfected with reference to section 1.2 in Example 1, and samples were taken at different time periods to measure transfection efficiency.
  • the results are shown in Table 1. According to Table 1, it can be seen that the extraction rate of the nanoparticle of Comparative Example 1 does not change significantly with time, and the corresponding extraction rate is not high for 8 hours of transfection.
  • the concentration of the obtained nanoparticles was adjusted to 40 nM, and the breast cancer cells MCF7 were transfected with reference to section 2.2 in Example 1, and samples were taken at different time periods to measure the transfection efficiency.
  • the results are shown in Table 2. According to Table 2, the extraction rate of the nanoparticle of Comparative Example 1 did not change significantly with time, and the corresponding extraction rate was not high for 8 hours of transfection.
  • Example 4 The method of Example 4 with reference to embodiments, TIL cells were transfected with the above-mentioned (1) obtained in the MCF7, MCF7 K, CTL6 three cell co-cultures and tested before and after transfection in culture before and after The amount of cytokine expression. The test results are shown in Table 4.

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Abstract

La présente invention concerne une nanoparticule, et son procédé de préparation. Le procédé comprend : le mélange d'un solvant organique et d'un tensioactif pour obtenir une solution A ; le mélange d'une première solution de réactif avec une solution de substance fonctionnelle pour obtenir une solution B ; le mélange d'une seconde solution de réactif avec une solution de substance fonctionnelle pour obtenir une solution C ; le fait de permettre au second réactif d'entrer en contact avec le premier réactif pour former une précipitation ; le mélange de la solution A et de la solution B uniformément pour obtenir une solution D ; le mélange de la solution A et de la solution C uniformément, l'addition d'une solution de DOPA à la solution mixte résultante, et le mélange uniforme pour obtenir une solution E ; l'addition de la solution E à la solution D, et la réalisation de la centrifugation sur le mélange pour recueillir une substance précipitée, afin d'obtenir des nanoparticules ayant un film en couche unique ; et la préparation d'une dispersion des nanoparticules ayant un film en couche unique, et l'addition, à la dispersion, d'un mélange de DOPC et de cholestérol ou d'un mélange de DOTAP et de cholestérol, les particules dans la suspension de particules résultante étant des nanoparticules ayant un film double couche.
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CN105873569A (zh) * 2013-11-06 2016-08-17 芝加哥大学 用于化疗剂、核酸和光敏剂的递送或共递送的纳米级载体
CN105878047A (zh) * 2014-12-23 2016-08-24 广州暨南大学医药生物技术研究开发中心 一种包裹细胞生长因子的脂质磷酸钙纳米粒的制备及应用
CN105168151A (zh) * 2015-08-07 2015-12-23 哈尔滨工业大学 一种氧化还原敏感型纳米靶向载体的制备方法

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