CN117618387A - Preparation method of film-coated calcium carbonate nanoparticles - Google Patents
Preparation method of film-coated calcium carbonate nanoparticles Download PDFInfo
- Publication number
- CN117618387A CN117618387A CN202311699599.8A CN202311699599A CN117618387A CN 117618387 A CN117618387 A CN 117618387A CN 202311699599 A CN202311699599 A CN 202311699599A CN 117618387 A CN117618387 A CN 117618387A
- Authority
- CN
- China
- Prior art keywords
- cell membrane
- calcium carbonate
- precipitate
- solution
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 title claims abstract description 103
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 78
- 229910000019 calcium carbonate Inorganic materials 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 91
- 239000002244 precipitate Substances 0.000 claims abstract description 60
- 239000012528 membrane Substances 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 29
- 238000010257 thawing Methods 0.000 claims abstract description 28
- 239000006228 supernatant Substances 0.000 claims abstract description 21
- 239000000819 hypertonic solution Substances 0.000 claims abstract description 11
- 229940021223 hypertonic solution Drugs 0.000 claims abstract description 11
- 239000000815 hypotonic solution Substances 0.000 claims abstract description 10
- 238000007710 freezing Methods 0.000 claims abstract description 7
- 230000008014 freezing Effects 0.000 claims abstract description 7
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 239000000644 isotonic solution Substances 0.000 claims abstract description 6
- 239000007864 aqueous solution Substances 0.000 claims description 38
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 29
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 21
- 239000008103 glucose Substances 0.000 claims description 21
- 102000018697 Membrane Proteins Human genes 0.000 claims description 19
- 108010052285 Membrane Proteins Proteins 0.000 claims description 19
- 239000011780 sodium chloride Substances 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 238000005119 centrifugation Methods 0.000 claims description 12
- 239000002504 physiological saline solution Substances 0.000 claims description 8
- 238000000464 low-speed centrifugation Methods 0.000 claims description 5
- 238000002844 melting Methods 0.000 claims description 4
- 230000008018 melting Effects 0.000 claims description 4
- 230000010355 oscillation Effects 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 2
- 230000003204 osmotic effect Effects 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 21
- 108090000623 proteins and genes Proteins 0.000 abstract description 21
- 239000002245 particle Substances 0.000 abstract description 15
- 230000008685 targeting Effects 0.000 abstract description 12
- 238000001125 extrusion Methods 0.000 abstract description 11
- 239000007888 film coating Substances 0.000 abstract description 6
- 238000009501 film coating Methods 0.000 abstract description 6
- 239000008188 pellet Substances 0.000 abstract description 5
- 239000000203 mixture Substances 0.000 description 30
- 230000000052 comparative effect Effects 0.000 description 29
- 210000004027 cell Anatomy 0.000 description 22
- 238000011534 incubation Methods 0.000 description 19
- 239000005457 ice water Substances 0.000 description 14
- 229920000515 polycarbonate Polymers 0.000 description 14
- 239000004417 polycarbonate Substances 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 238000001471 micro-filtration Methods 0.000 description 13
- 239000011248 coating agent Substances 0.000 description 9
- 238000000576 coating method Methods 0.000 description 9
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 230000008827 biological function Effects 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 239000004530 micro-emulsion Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 239000013078 crystal Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 210000002390 cell membrane structure Anatomy 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- 229960004679 doxorubicin Drugs 0.000 description 3
- 238000012377 drug delivery Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000003617 erythrocyte membrane Anatomy 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 239000002539 nanocarrier Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- PITRRWWILGYENJ-UHFFFAOYSA-N 2-[2-[2-[2-[2-(4-nonylphenoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCCC1=CC=C(OCCOCCOCCOCCOCCO)C=C1 PITRRWWILGYENJ-UHFFFAOYSA-N 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 201000007983 brain glioma Diseases 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- SPFMQWBKVUQXJV-BTVCFUMJSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;hydrate Chemical compound O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O SPFMQWBKVUQXJV-BTVCFUMJSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 102000013462 Interleukin-12 Human genes 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- -1 freezing temperature Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229940117681 interleukin-12 Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000000409 membrane extraction Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 229920006289 polycarbonate film Polymers 0.000 description 1
- 229920005668 polycarbonate resin Polymers 0.000 description 1
- 239000004431 polycarbonate resin Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000001132 ultrasonic dispersion Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The application discloses a preparation method of U87 cell membrane coated calcium carbonate nanoparticle, after extracting U87 cell membrane, re-suspending the cell membrane in hypotonic solution; measuring the protein concentration of the cell membrane by using a BCA method, mixing the cell membrane with calcium carbonate nanoparticles, completely freezing in an ultralow temperature environment, and then rapidly thawing; centrifuging, removing the supernatant, and collecting the precipitate; resuspending the precipitate with a hypertonic solution and oscillating at low temperature; centrifuging, removing supernatant, and collecting precipitate; the pellet was resuspended in isotonic solution, sonicated and extruded sequentially through 0.8 μm, 0.4 μm and 0.2 μm filter membranes. The U87 film-coated calcium carbonate nanoparticle prepared by the method can obtain a nanoparticle carrier with uniform particle size, high film coating success rate and good targeting even without repeated extrusion.
Description
Technical Field
The application relates to a preparation method of calcium carbonate nanoparticles, in particular to a preparation method of film-coated calcium carbonate nanoparticles.
Background
In recent years, the coating of nanoparticles with cell membranes has attracted attention. The membrane coated nano-particles are prepared by collecting cell membranes from natural cells and coating the cell membranes on the surfaces of the synthesized nano-particles, and the obtained membrane coated nano-particles not only have highly adjustable physicochemical properties of the synthesized nano-materials, but also have highly complex biological functions of host cell membranes, such as ligand identification, targeting, long blood circulation, immune escape and the like, and have great application prospects in nano-drug delivery.
For the preparation of cell membrane coated nanoparticles, the most common method is extrusion, i.e. mixing the cell membrane with the nanoparticles, followed by repeated extrusion through an extruder, typically more than ten times. As disclosed in patent CN116212116a, a preparation method of a receptor erythrocyte membrane coated drug-loaded nanoparticle is disclosed, a drug-loaded nanoparticle dispersion liquid and a collected erythrocyte membrane are mixed, and after ultrasound, the nanoparticle is repeatedly pushed and extruded for 10-40 times by a liposome extruder, so that the erythrocyte membrane can be ensured to fully coat the nanoparticle. Patent CN115025061B discloses a preparation method of a brain targeting membrane coated nano drug delivery system based on a virus-free bacteria outer membrane capable of penetrating the blood brain barrier, after ultrasonic treatment of 30 s of the obtained virus-free bacteria outer membrane vesicle, the method and the final nanoparticle are mixed according to a mass ratio of 1:5, mixing, extruding the mixture for 11 times by using a 200 nm polycarbonate film to obtain the brain targeting film coating nano system based on the outer film coating of the detoxified bacteria.
The applicant has prepared a kind of human brain glioma cell U87 membrane coated calcium carbonate nanoparticle in earlier stage, mix the cell membrane with calcium carbonate nanoparticle, squeeze through 200 nm polycarbonate membrane, squeeze 11 times repeatedly, get U87 cell membrane coated calcium carbonate nanoparticle.
However, repeated extrusion has the following problems: 1) The membrane holes in the extruder are easy to be blocked by multiple times of extrusion, particularly solid inorganic nano-carriers such as calcium carbonate nano-particles are needed to be replaced for several times during extrusion, so that the time and the labor are consumed, and the mass production is not facilitated; 2) Each extrusion can consume part of calcium carbonate nano particles and cell membranes, and the extrusion is accumulated for multiple times, so that the material loss of the whole process is extremely high, and the process is uneconomical and environment-friendly.
Therefore, development of a new preparation method is needed to be developed, extrusion times can be remarkably reduced, and meanwhile, the prepared nano-carrier can be guaranteed to be uniform in particle size, high in film coating success rate and good in targeting.
Disclosure of Invention
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of U87 cell membrane coated calcium carbonate nanoparticles, which comprises the following steps:
1) Extracting U87 cell membranes, resuspending said cell membranes in a hypotonic solution;
2) Mixing the cell membrane in the step 1) with the calcium carbonate nanoparticles, and then completely freezing in an ultralow temperature environment, and then rapidly thawing; centrifuging, removing the supernatant, and collecting the precipitate;
3) Re-suspending the precipitate in step 2) with a hypertonic solution, oscillating at low temperature; centrifuging, removing supernatant, and collecting precipitate;
4) The precipitate in step 3) was resuspended in isotonic solution, sonicated and pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm filter membranes.
Further, the hypotonic solution is NaCl water solution with mass concentration of 0-0.5% or glucose water solution with mass concentration of 0-3%. In hypotonic solution, cell membrane is ruptured, and is more easily coated on the surface of calcium carbonate nanoparticles.
Further, the temperature of the ultralow temperature environment is-40 to-100 ℃. The temperature of the ultra-low temperature environment should not be too high or too low: too high a temperature, the cell membrane cannot be frozen to form ice crystals or is insufficient in ice crystal formation, and the cell membrane is insufficient in expansion, so that the cell membrane cannot be fully coated on the calcium carbonate nanoparticles; excessive ice crystals are formed when the temperature is too low, the cell membrane structure is severely destroyed, a large amount of cell fragments are easily generated, and the cell membrane function is destroyed.
Further, the hypertonic solution is an aqueous NaCl solution with the mass concentration of 2-6%; or glucose aqueous solution with mass concentration of 10-20%. The concentration of the hypertonic solution is not too high or too low, and when the concentration of the hypertonic solution is too low, the cell membrane is not coated on the calcium carbonate nanoparticles, so that the membrane coated nanoparticles with uniform particle size and stable quality are formed; the high-permeability solution has high concentration, the cell membrane is seriously shrunken, and the high-permeability solution can not be coated on the calcium carbonate nano-particles, and can also cause the loss of the biological functions of cells.
Further, the low-temperature oscillation temperature is 0-4 ℃ and the oscillation time is 1-4 h. The low temperature environment is beneficial to maintaining the activity of cell membranes. By shaking, the cell membrane can be fully contacted with the calcium carbonate nanoparticle, thereby being more beneficial to coating.
Further, the centrifugation in steps 2) and 3) is a low temperature low speed centrifugation.
Further, the temperature of the low temperature is 0-4 ℃, and the centrifugal force of the low-speed centrifugation is 1000-5000 g. The low temperature environment is favorable for maintaining the activity of cell membranes, and the low-speed centrifugation can avoid agglomeration of nanoparticles.
Further, the cell membrane and the calcium carbonate nanoparticle are based on cell membrane protein mass: the mass of the calcium carbonate is 1-3:1, so that enough cell membranes are fully contacted with the calcium carbonate nano particles, and the coating effect is improved.
Further, the isotonic solution is physiological saline, PBS buffer solution or glucose aqueous solution with mass concentration of 5%.
Further, in the step 2), the melting temperature is 30-40 ℃. The melting temperature should not be too high or too low, and too high a melting temperature would damage the cell membrane structure, resulting in a disruption of the biological functions of the cell membrane. The frozen membrane coating nano particles cannot be quickly melted at the too low temperature, and the ice crystals in the cell membrane cannot be melted for a long time, so that the cell membrane structure can be damaged, and the biological function of the cell membrane is affected.
In summary, the application discloses a method for coating calcium carbonate nanoparticles by a film, which can obviously reduce extrusion times, and can ensure that the prepared nano-carrier has uniform particle size, high success rate of film coating and good targeting.
Drawings
FIG. 1 is a graph of fluorescence results of isotype-targeted experiments.
Detailed Description
General experiment
1. U87 cell membrane extraction
1) Human brain glioma U87 cell culture: u87 cells were isolatedCultured in 75 cm 2 The cells were placed in a cell culture flask and placed in a cell incubator (5% CO) 2 Culturing at 37 ℃, adding trypsin to digest cells after the cells in the culture flask grow fully, adding a culture medium with serum to neutralize and blow after the cells become round, and collecting the cells; cells were collected by centrifugation (1000 rpm,5 min);
2) Adding 10 mL hypotonic lysate to resuspend cells, the hypotonic lysate consisting of 20 mM Tris-HCl, 10 mM KCl, 2 mM MgCl 2 And 1 tablet of protease inhibitor (Sieimer product No. A32955). And left to stand at 4℃for 5 hours. After completion of lysis, the lysate containing the cells was left to freeze completely at-80℃and then thawed completely in a 37℃water bath. After repeating the freeze thawing process for 3-5 times, the cell suspension is strongly shaken for 1 minute, and then centrifuged at 1000 rpm for 3 minutes to remove intracellular components. Finally, the supernatant was further centrifuged at 20000-30000 rpm for 1 hour at 4℃to obtain U87 cell membrane.
The cell membrane is prepared by repeated freeze thawing method, and has more complete cell membrane and less fragments.
The cell membrane can also be extracted by ultrasonic disruption, but the cell membrane obtained in advance by the method has more fragments.
2. Blank calcium carbonate nanoparticle preparation
CaCl 300 [ mu ] L 2 The (500 mM) aqueous solution was added to an oil phase of 15 mL, which was composed of cyclohexane/Igepal CO-520 (volume ratio 71:29), with stirring, to form a well dispersed "calcium phase microemulsion". Na of 300 mu L 2 CO 3 The aqueous solution (250 mM) was added to another 15 mL oil phase and stirred to form a well dispersed "carbonic acid phase microemulsion". Mixing and stirring the two-phase microemulsion for 45 minutes at room temperature, adding 30 mL ethanol for demulsification, centrifuging for 30 minutes at 12000 g to obtain blank calcium carbonate nanoparticles, and washing off an organic solvent and a surfactant by using absolute ethanol to obtain the calcium carbonate nanoparticles.
3. Preparation of calcium carbonate nanoparticles containing fluorescent markers
To verify the film-coated calcium carbonate nanoparticles (Cell membrane Coated Calcium carbonate NPs, hereinafter referred to as CM-CaCO 3 ) Can target U87 cell line, adopts homotypic targeting experiment, and uses CM-CaCO with fluorescence 3 Added to U87 cells and the average fluorescence intensity was measured using a flow cytometer (Becton Dickinson, USA).
Due to blank CM-CaCO 3 (i.e., nanoparticles that are not drug loaded and do not modify the cell membrane) do not contain fluorescence, and therefore, it is necessary to label CM-CaCO with a fluorescent material 3 . Common labeling methods are: 1) Cell membrane labelling, adding a lipophilic reagent such as ICG to the cell membrane; 2) The calcium carbonate nanoparticle carries medicine, and the medicine itself carries fluorescence or fluorescent marker, such as the calcium carbonate nanoparticle carries doxorubicin, and the doxorubicin itself contains fluorescence; or calcium carbonate nanoparticles load mRNA, and the mRNA is labeled with a fluorescent group Cy3, cy5 or FITC.
The inventors have previously used calcium carbonate nanoparticles to support Cy3-IL-12-mRNA (messenger RNA encoding interleukin 12 labeled with Cy 3), and the present application also used the nanoparticles, and the specific preparation method is as follows:
cy 3-IL-12-mRNA-calcium carbonate (hereinafter referred to as Cy 3-mRNA-CaCO) 3 ) Preparation of nanoparticles
CaCl 300 [ mu ] L 2 (500 mM) aqueous solution and 50 μg of IL-12-mRNA were mixed and added to an oil phase of 15 mL, which was composed of cyclohexane/Igepal CO-520 (volume ratio 71:29), with stirring, to form a well dispersed "calcium phase microemulsion". Na of 300 mu L 2 CO 3 The aqueous solution (250 mM) was added to another 15 mL oil phase and stirred to form a well dispersed "carbonic acid phase microemulsion". Mixing and stirring the two-phase microemulsion for 45 minutes at room temperature, adding 30 mL ethanol to demulsify, centrifuging 12000 g for 30 minutes to obtain Cy3-IL-12-mRNA loaded calcium carbonate nanoparticles, washing off the organic solvent and the surfactant with absolute ethanol to obtain purified Cy3-mRNA-CaCO 3 。
Specifically, by adopting the preparation method, the blank CM-CaCO coated by U87 cell membrane can be prepared 3 The method comprises the steps of carrying out a first treatment on the surface of the Can also be used for preparing U87 cell membrane coated CM-CaCO for drug delivery 3 The type of drug carrier is not particularly limited and may be a common chemotherapeutic drug such as doxorubicin; can also be a genetic drug, e.gVarious mRNAs. To simplify the patent text, the following examples and comparative examples each employ Cy3-mRNA-CaCO 3 An explanation is given.
(II) examples and comparative examples
Example 1
Adding 4 times of NaCl aqueous solution with the volume mass concentration of 0.2% into the U87 cell membrane, measuring the protein mass of the cell membrane by using a BCA method, and then measuring the protein mass of the cell membrane according to the protein mass of the cell membrane: cy3-mRNA-CaCO 3 Cy3-mRNA-CaCO was added at a mass ratio of 1:1 3 The mixture was placed in a-40 ℃ refrigerator and completely frozen, followed by rapid thawing in a 37 ℃ water bath. After thawing, centrifugation is carried out at low speed 2000 g for 5 minutes at 4 ℃, the supernatant is removed, and the precipitate is collected; adding 1 time volume of NaCl aqueous solution with the mass concentration of 4% into the precipitate, placing the precipitate in a refrigerator with the temperature of 4 ℃ for shaking and incubating for 2 h, and centrifuging at a low speed of 2000 g for 5 minutes at the temperature of 4 ℃ after incubation, and collecting the precipitate; the precipitate was resuspended in physiological saline, then sonicated in an ice-water mixture with a pulse of 100W "20 s on 20 s off" and finally pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm polycarbonate microfiltration membranes.
Example 2
3 volumes of ultrapure water (i.e., naCl aqueous solution with mass concentration of 0%) was added to the U87 cell membrane, and the cell membrane protein mass was measured by BCA method, followed by following the cell membrane protein mass: cy3-mRNA-CaCO 3 Cy3-mRNA-CaCO was added at a mass ratio of 1.5:1 3 The mixture was placed in a-80 ℃ refrigerator to be completely frozen, and then rapidly melted in a 45 ℃ water bath. After thawing, centrifuging at low speed 1000 g at 0deg.C for 10min, removing supernatant, and collecting precipitate; adding 2 times of NaCl aqueous solution with mass concentration of 2% into the precipitate, placing in a refrigerator with 0 ℃ for shaking incubation for 4 h, centrifuging at low speed 2000 g for 5 minutes at 0 ℃ after incubation, and collecting the precipitate; the pellet was resuspended in PBS and then sonicated in an ice-water mixture with a pulse of 100W "20 s on 20 s off" and finally pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm polycarbonate microfiltration membranes.
Example 3
Adding 2 times of NaCl aqueous solution with 0.6% volume and mass concentration into U87 cell membrane, and measuring by BCA methodDetermining the cell membrane protein mass, and then according to the cell membrane protein mass: cy3-mRNA-CaCO 3 Cy3-mRNA-CaCO was added at a mass ratio of 3:1 3 The mixture was placed in a-100 ℃ refrigerator to be completely frozen, and then rapidly melted in a water bath at 30 ℃. Centrifuging at low speed 5000 g at 0deg.C for 3 min after thawing, removing supernatant, and collecting precipitate; adding 1 time volume of NaCl aqueous solution with the mass concentration of 6% into the precipitate, placing the precipitate in a refrigerator with the temperature of 0 ℃ for shaking and incubating for 1h, centrifuging at a low speed of 2000 g for 5 minutes at the temperature of 0 ℃ after incubating, and collecting the precipitate; the pellet was resuspended in PBS and then sonicated in an ice-water mixture with a pulse of 100W "20 s on 20 s off" and finally pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm polycarbonate microfiltration membranes.
Example 4
Adding 1 time of glucose aqueous solution with the volume mass concentration of 2% into U87 cell membrane, measuring the protein mass of the cell membrane by using a BCA method, and then measuring the protein mass of the cell membrane according to the protein mass of the cell membrane: cy3-mRNA-CaCO 3 Cy3-mRNA-CaCO was added at a mass ratio of 2:1 3 The mixture was placed in a-80 ℃ refrigerator to be completely frozen, and then rapidly melted in a 37 ℃ water bath. After thawing, centrifugation at low speed 3000 g at 4 ℃ for 5 minutes, removal of supernatant and collection of precipitate; adding 2 times volume of glucose aqueous solution with mass concentration of 10%, shaking and incubating in a refrigerator at 0deg.C for 2 h, centrifuging at 0deg.C and low speed 2000 g for 5 min, and collecting precipitate; the precipitate was resuspended in 5% by mass glucose aqueous solution, then sonicated in an ice-water mixture with a pulse of "20 s on 20 s off" of 100W, and finally pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm polycarbonate microfiltration membranes.
Example 5
Adding 2 times of glucose aqueous solution with 3% volume mass concentration into U87 cell membrane, measuring cell membrane protein mass by BCA method, and then measuring cell membrane protein mass according to the cell membrane protein mass: cy3-mRNA-CaCO 3 Cy3-mRNA-CaCO was added at a mass ratio of 2:1 3 The mixture was placed in a-60 ℃ refrigerator and completely frozen, followed by rapid thawing in a 37 ℃ water bath. After thawing, centrifugation is carried out at low speed 2000 g for 5 minutes at 4 ℃, the supernatant is removed, and the precipitate is collected; adding 2 times of body into the precipitatePlacing the product of glucose aqueous solution with mass concentration of 20% in a refrigerator at 0 ℃ for shaking incubation for 1h, centrifuging at low speed of 2000 g at 0 ℃ for 5 minutes after incubation, and collecting precipitate; the precipitate was resuspended in 5% by mass glucose aqueous solution, then sonicated in an ice-water mixture with a pulse of "20 s on 20 s off" of 100W, and finally pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm polycarbonate microfiltration membranes.
Example 6
Adding 3 times of NaCl aqueous solution with the volume mass concentration of 0.3% into the U87 cell membrane, measuring the protein mass of the cell membrane by using a BCA method, and then measuring the protein mass of the cell membrane according to the protein mass of the cell membrane: cy3-mRNA-CaCO 3 Cy3-mRNA-CaCO was added at a mass ratio of 1.5:1 3 The mixture was placed in a-80 ℃ refrigerator to be completely frozen, and then rapidly melted in a 40 ℃ water bath. After thawing, centrifugation at low speed 3000 g at 4 ℃ for 5 minutes, removal of supernatant and collection of precipitate; adding 1 time volume of glucose aqueous solution with mass concentration of 15%, placing in a refrigerator at 0deg.C, shaking and incubating for 3 h, centrifuging at 0deg.C and low speed 2000 g for 5 min, and collecting precipitate; the precipitate was resuspended in physiological saline, then sonicated in an ice-water mixture with a pulse of 100W "20 s on 20 s off" and finally pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm polycarbonate microfiltration membranes.
Comparative example 1
4 times of physiological saline is added into the U87 cell membrane, the protein mass of the cell membrane is measured by the BCA method, and then the protein mass of the cell membrane is measured according to the following steps: cy3-mRNA-CaCO 3 Cy3-mRNA-CaCO was added at a mass ratio of 1:1 3 The mixture was placed in a-40 ℃ refrigerator and completely frozen, followed by rapid thawing in a 37 ℃ water bath. After thawing, centrifuging at low speed 2000 Xg at 4deg.C for 5 min, removing supernatant, and collecting precipitate; adding 1 time volume of NaCl water solution with mass concentration of 5% into the precipitate, placing in a refrigerator with the temperature of 4 ℃ for shaking incubation for 2 h, centrifuging at the low speed of 2000 g for 5 minutes at the temperature of 4 ℃ after incubation, and collecting the precipitate; adding physiological saline into the precipitate for resuspension, performing ultrasonic dispersion in ice-water mixture with pulse 100W of 20 s on 20 s off, and sequentially passing through 0.8 μm, 0.4 μm and 0.2 μm microporous filter membrane of polycarbonate fiberAnd (5) extruding.
Comparative example 2
Adding 4 times of NaCl aqueous solution with the volume mass concentration of 0.2% into the U87 cell membrane, measuring the protein mass of the cell membrane by using a BCA method, and then measuring the protein mass of the cell membrane according to the protein mass of the cell membrane: cy3-mRNA-CaCO 3 Cy3-mRNA-CaCO was added at a mass ratio of 1:1 3 The mixture was placed in a-20 ℃ refrigerator and completely frozen, followed by rapid thawing in a 37 ℃ water bath. After thawing, centrifugation is carried out at low speed 2000 g for 5 minutes at 4 ℃, the supernatant is removed, and the precipitate is collected; adding 1 time volume of NaCl water solution with mass concentration of 5% into the precipitate, placing in a refrigerator with the temperature of 4 ℃ for shaking incubation for 2 h, centrifuging at the low speed of 2000 g for 5 minutes at the temperature of 4 ℃ after incubation, and collecting the precipitate; the precipitate was resuspended in physiological saline, then sonicated in an ice-water mixture with a pulse of 100W "20 s on 20 s off" and finally pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm polycarbonate microfiltration membranes.
Comparative example 3
Adding 4 times of NaCl aqueous solution with the volume mass concentration of 0.2% into the U87 cell membrane, measuring the protein mass of the cell membrane by using a BCA method, and then measuring the protein mass of the cell membrane according to the protein mass of the cell membrane: cy3-mRNA-CaCO 3 Cy3-mRNA-CaCO was added at a mass ratio of 1:1 3 The mixture was placed in a-120 ℃ refrigerator and completely frozen, followed by rapid thawing in a 37 ℃ water bath. After thawing, centrifugation is carried out at low speed 2000 g for 5 minutes at 4 ℃, the supernatant is removed, and the precipitate is collected; adding 1 time volume of NaCl water solution with mass concentration of 5% into the precipitate, placing in a refrigerator with the temperature of 4 ℃ for shaking incubation for 2 h, centrifuging at the low speed of 2000 g for 5 minutes at the temperature of 4 ℃ after incubation, and collecting the precipitate; the precipitate was resuspended in physiological saline, then sonicated in an ice-water mixture with a pulse of 100W "20 s on 20 s off" and finally pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm polycarbonate microfiltration membranes.
Comparative example 4
3 volumes of ultrapure water (i.e., naCl aqueous solution with mass concentration of 0%) was added to the U87 cell membrane, and the cell membrane protein mass was measured by BCA method, followed by following the cell membrane protein mass: cy3-mRNA-CaCO 3 Cy3-mRNA-CaCO was added at a mass ratio of 1.5:1 3 Placing the above mixture at-80deg.CThe ice was completely frozen in the refrigerator and then rapidly melted in a 45 ℃ water bath. Centrifuging at low speed 1000 g at 0deg.C for 10min after thawing, removing supernatant, and collecting precipitate; adding 2 times of NaCl aqueous solution with mass concentration of 1% into the precipitate, placing in a refrigerator with 0 ℃ for shaking incubation for 4 h, centrifuging at low speed 2000 g for 5 min at 0 ℃ after incubation, and collecting the precipitate; the pellet was resuspended in PBS and then sonicated in an ice-water mixture with a pulse of 100W "20 s on 20 s off" and finally pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm polycarbonate microfiltration membranes.
Comparative example 5
3 volumes of ultrapure water (i.e., naCl aqueous solution with mass concentration of 0%) was added to the U87 cell membrane, and the cell membrane protein mass was measured by BCA method, followed by following the cell membrane protein mass: cy3-mRNA-CaCO 3 Cy3-mRNA-CaCO was added at a mass ratio of 1.5:1 3 The mixture was placed in a-80 ℃ refrigerator to be completely frozen, and then rapidly melted in a 45 ℃ water bath. After thawing, centrifuging at low speed 1000 g at 0deg.C for 10min, removing supernatant, and collecting precipitate; adding 2 times of NaCl aqueous solution with mass concentration of 8% into the precipitate, placing in a refrigerator at 0 ℃ for shaking incubation for 4 h, centrifuging at low speed 2000 g at 0 ℃ for 5 minutes after incubation, and collecting the precipitate; the pellet was resuspended in PBS and then sonicated in an ice-water mixture with a pulse of 100W "20 s on 20 s off" and finally pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm polycarbonate microfiltration membranes.
Comparative example 6
Adding 1 time of glucose aqueous solution with the volume mass concentration of 5% into U87 cell membrane, measuring the protein mass of the cell membrane by using a BCA method, and then measuring the protein mass of the cell membrane according to the protein mass of the cell membrane: cy3-mRNA-CaCO 3 Cy3-mRNA-CaCO was added at a mass ratio of 2:1 3 The mixture was placed in a-80 ℃ refrigerator to be completely frozen, and then rapidly melted in a 37 ℃ water bath. After thawing, centrifugation at low speed 3000 g at 4 ℃ for 5 minutes, removal of supernatant and collection of precipitate; adding 2 times volume of glucose aqueous solution with mass concentration of 10%, shaking and incubating in a refrigerator at 0deg.C for 2 h, centrifuging at 0deg.C and low speed 2000 g for 5 min, and collecting precipitate; adding 5% of the mass concentration into the precipitateThe aqueous glucose solution was resuspended and then sonicated in an ice-water mixture with a 100W pulse of "20 s on 20 s off" and finally pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm polycarbonate resin microfiltration membranes.
Comparative example 7
Adding 2 times of glucose aqueous solution with 3% volume mass concentration into U87 cell membrane, measuring cell membrane protein mass by BCA method, and then measuring cell membrane protein mass according to the cell membrane protein mass: cy3-mRNA-CaCO 3 Cy3-mRNA-CaCO was added at a mass ratio of 2:1 3 The mixture was placed in a-60 ℃ refrigerator and completely frozen, followed by rapid thawing in a 37 ℃ water bath. After thawing, centrifugation is carried out at low speed 2000 g for 5 minutes at 4 ℃, the supernatant is removed, and the precipitate is collected; adding 2 times volume of glucose aqueous solution with mass concentration of 8% into the precipitate, placing in a refrigerator at 0 ℃ for shaking and incubating for 1h, centrifuging at low speed 2000 Xg for 5 minutes at 0 ℃ after incubating, and collecting the precipitate; the precipitate was resuspended in 5% by mass glucose aqueous solution, then sonicated in an ice-water mixture with a pulse of "20 s on 20 s off" of 100W, and finally pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm polycarbonate microfiltration membranes.
Comparative example 8
Adding 2 times of glucose aqueous solution with 3% volume mass concentration into U87 cell membrane, measuring cell membrane protein mass by BCA method, and then measuring cell membrane protein mass according to the cell membrane protein mass: cy3-mRNA-CaCO 3 Cy3-mRNA-CaCO was added at a mass ratio of 2:1 3 The mixture was placed in a-60 ℃ refrigerator and completely frozen, followed by rapid thawing in a 37 ℃ water bath. After thawing, centrifugation is carried out at low speed 2000 g for 5 minutes at 4 ℃, the supernatant is removed, and the precipitate is collected; adding 2 times of glucose aqueous solution with mass concentration of 25% into the precipitate, placing in a refrigerator at 0 ℃ for shaking incubation for 1h, centrifuging at low speed 2000 g at 0 ℃ for 5 minutes after incubation, and collecting the precipitate; the precipitate was resuspended in 5% by mass glucose aqueous solution, then sonicated in an ice-water mixture with a pulse of "20 s on 20 s off" of 100W, and finally pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm polycarbonate microfiltration membranes.
(III) film coated nanoparticle functional testing
1. Characterization of film coated calcium carbonate nanoparticles
The average particle size and potential of the film-coated calcium carbonate nanoparticles prepared in the above examples and comparative examples were measured using a photodynamic scattering particle sizer (Malvern Zetasizer Nano ZS, uk).
2. Homotypic targeting experiment of film coated calcium carbonate nanoparticle
After the calcium carbonate nanoparticles are verified to be coated by the U87 cell membrane, the U87 cell line can be targeted by measuring the uptake of Cy3-mRNA in the U87 cell line, and the prepared membrane coated nanoparticle cell membrane has better biological function. U87 cells were seeded on a 6-well plate, and nanoparticles prepared in the above examples and comparative examples were added after the cells were attached. After incubation of 4 h, the resuspension was digested with trypsin and the average fluorescence intensity of each well cell was measured using a flow cytometer (Becton Dickinson, usa).
Experimental results and discussion
1. Particle size results
Table 1: particle size results
| Name of the name | Particle size (nm) | PDI |
| Example 1 | 150.1 | 0.181 |
| Example 2 | 165.0 | 0.062 |
| Example 3 | 159.5 | 0.197 |
| Example 4 | 163.8 | 0.035 |
| Example 5 | 155.7 | 0.160 |
| Example 6 | 160.2 | 0.121 |
| Comparative example 1 | 167.2 | 0.374 |
| Comparative example 2 | 159.8 | 0.402 |
| Comparative example 3 | 188.6 | 0.396 |
| Comparative example 4 | 176.0 | 0.539 |
| Comparative example 5 | 239.5 | 0.725 |
| Comparative example 6 | 173.8 | 0.348 |
| Comparative example 7 | 170.2 | 0.391 |
| Comparative example 8 | 206.3 | 0.601 |
In the above table, PDI represents a dispersion index, and the larger the PDI is, the wider the molecular weight distribution is; the smaller the PDI, the more uniform the molecular weight distribution. According to the preparation process of the application, the particle size of the nanoparticle is within 200 nm, and the dispersion index PDI is less than 0.3
In comparison with example 1, the nanoparticles of comparative example 1 had a particle diameter of 200 nm or less, but had a PDI > 0.3. It was demonstrated that the coating of the cell membrane with calcium carbonate nanoparticles in an isotonic solution was less effective than the resuspension of the cell membrane with a hypotonic solution in example 1. Presumably, the reason is that in hypotonic solutions, cells are prone to swelling and are more prone to coating the surface of calcium carbonate nanoparticles after freeze thawing. The experimental results of example 4 and comparative example 6 also demonstrate that hypotonic solutions are beneficial for cell membrane coating of calcium carbonate nanoparticles.
Comparative examples 2 and 3 changed the freezing temperature compared to example 1. The PDI of the prepared nano-particles is more than 0.3 and does not meet the preparation process requirement in comparative example 2 when the freezing temperature is too high (-20 ℃). The temperature in comparative example 3 was too low (-120 ℃) and the particle size of the prepared nanoparticle was large and PDI was > 0.3. It follows that either too high or too low a temperature is detrimental to the preparation of film coated calcium carbonate nanoparticles.
Comparative examples 4 and 5 changed the concentration of the hypertonic solution compared to example 2. Comparative example 4 the concentration of hypertonic solution is too low, the particle size of the prepared nano-particles is bigger, and PDI is more than 0.3. The concentration of the hypertonic solution in comparative example 5 is too high, the particle size of the prepared nano-particles is more than 200 nm, and PDI is more than 0.3. Therefore, the concentration of the hypertonic solution is too low or too high, and the prepared film-coated nano particles do not meet the production process requirements. The experimental results of example 5 and comparative examples 7 and 8 also confirm the above conclusion.
In summary, the preparation method of the film-coated calcium carbonate nanoparticle is summarized by setting parameters such as hypotonic solution, freezing temperature, hypertonic solution concentration and the like. The U87 film-coated calcium carbonate nanoparticle prepared by the method can obtain a nanoparticle carrier with uniform particle size and high film coating success rate even without repeated extrusion.
2. Homotypic targeting experimental results
The fluorescence results of the homotype targeting experiments prove that (see figure 1), by adopting the preparation method, the U87 cell membrane can successfully coat the calcium carbonate nanoparticle, and the obtained membrane-coated calcium carbonate nanoparticle has targeting property, which shows that by adopting the preparation method, although the membrane-coated calcium carbonate nanoparticle is subjected to freezing and thawing of low-permeability solution and incubation of high-permeability solution, the biological function of the cell membrane can not be damaged in the whole process flow, and the prepared nanoparticle still has better targeting property.
Claims (10)
1. The preparation method of the U87 cell membrane coated calcium carbonate nanoparticle is characterized by comprising the following steps of:
1) Extracting U87 cell membranes, resuspending said cell membranes in a hypotonic solution;
2) Mixing the cell membrane in the step 1) with the calcium carbonate nanoparticles, and then completely freezing in an ultralow temperature environment, and then rapidly thawing; centrifuging, removing the supernatant, and collecting the precipitate;
3) Re-suspending the precipitate in step 2) with a hypertonic solution, oscillating at low temperature; centrifuging, removing supernatant, and collecting precipitate;
4) The precipitate in step 3) was resuspended in isotonic solution, sonicated and pressed sequentially through 0.8 μm, 0.4 μm and 0.2 μm filter membranes.
2. The preparation method according to claim 1, wherein the hypotonic solution is an aqueous solution of NaCl with a mass concentration of 0-0.5% or an aqueous solution of glucose with a mass concentration of 0-3%.
3. The preparation method according to any one of claims 1 or 2, wherein the temperature of the ultralow temperature environment is-40 to-100 ℃.
4. The preparation method according to any one of claims 1 or 2, wherein the high osmotic pressure solution is an aqueous NaCl solution having a mass concentration of 2 to 6%; or glucose aqueous solution with mass concentration of 10-20%.
5. The production method according to any one of claims 1 or 2, wherein the low-temperature oscillation temperature is 0 to 4 ℃ and the oscillation time is 1 to 4 h.
6. The method of claim 1, wherein the centrifugation in steps 2) and 3) is a low temperature low speed centrifugation.
7. The method according to claim 6, wherein the low temperature is 0-4 ℃, and the centrifugal force of the low-speed centrifugation is 1000-5000 g.
8. The method of any one of claims 1 or 2, wherein the cell membrane and the calcium carbonate nanoparticle are based on cell membrane protein mass: the mass of the calcium carbonate is 1-3:1.
9. The preparation method according to any one of claims 1 or 2, wherein the isotonic solution is physiological saline, PBS buffer solution or glucose aqueous solution with a mass concentration of 5%.
10. The method according to any one of claims 1 or 2, wherein the melting temperature in step 2) is 30 to 40 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311699599.8A CN117618387A (en) | 2023-12-12 | 2023-12-12 | Preparation method of film-coated calcium carbonate nanoparticles |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311699599.8A CN117618387A (en) | 2023-12-12 | 2023-12-12 | Preparation method of film-coated calcium carbonate nanoparticles |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117618387A true CN117618387A (en) | 2024-03-01 |
Family
ID=90018163
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311699599.8A Pending CN117618387A (en) | 2023-12-12 | 2023-12-12 | Preparation method of film-coated calcium carbonate nanoparticles |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117618387A (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160136106A1 (en) * | 2013-08-08 | 2016-05-19 | The Regents Of The University Of California | Nanoparticles Leverage Biological Membranes to Target Pathogens for Disease Treatment and Diagnosis |
| CN107244687A (en) * | 2017-07-04 | 2017-10-13 | 河南师范大学 | A kind of method that nano-calcium carbonate is prepared by bioreactor of red blood cell |
| CN110859826A (en) * | 2019-12-09 | 2020-03-06 | 深圳先进技术研究院 | Brain tumor targeted bionic drug-loaded nanoparticle and preparation method and application thereof |
| CN113599368A (en) * | 2021-07-27 | 2021-11-05 | 东南大学 | Bionic drug-loading nano system combining cell membrane antagonism with nano enzyme, preparation method and application |
| CN114099470A (en) * | 2021-12-09 | 2022-03-01 | 济南优科医疗技术有限公司 | Method for uniformly and slowly releasing liquid magnetic induction medium for treating tumors |
| US20220362162A1 (en) * | 2018-11-26 | 2022-11-17 | Arytha Biosciences, Llc | Nanoparticles containing cellular membrane and uses thereof |
| CN115505570A (en) * | 2022-08-15 | 2022-12-23 | 东南大学 | Method for extracting tumor cell membrane by combining chemical induction apoptosis and physical freeze thawing |
| US20230226218A1 (en) * | 2020-05-11 | 2023-07-20 | Erytech Pharma | Red Cell Extracellular Vesicles (RCEVs) Containing Cargoes and Methods of Use and Production Thereof |
| CN116650673A (en) * | 2023-05-25 | 2023-08-29 | 赣南医学院 | Cell membrane coated medicine fat granule and its preparation method and application |
-
2023
- 2023-12-12 CN CN202311699599.8A patent/CN117618387A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160136106A1 (en) * | 2013-08-08 | 2016-05-19 | The Regents Of The University Of California | Nanoparticles Leverage Biological Membranes to Target Pathogens for Disease Treatment and Diagnosis |
| CN107244687A (en) * | 2017-07-04 | 2017-10-13 | 河南师范大学 | A kind of method that nano-calcium carbonate is prepared by bioreactor of red blood cell |
| US20220362162A1 (en) * | 2018-11-26 | 2022-11-17 | Arytha Biosciences, Llc | Nanoparticles containing cellular membrane and uses thereof |
| CN110859826A (en) * | 2019-12-09 | 2020-03-06 | 深圳先进技术研究院 | Brain tumor targeted bionic drug-loaded nanoparticle and preparation method and application thereof |
| US20230226218A1 (en) * | 2020-05-11 | 2023-07-20 | Erytech Pharma | Red Cell Extracellular Vesicles (RCEVs) Containing Cargoes and Methods of Use and Production Thereof |
| CN113599368A (en) * | 2021-07-27 | 2021-11-05 | 东南大学 | Bionic drug-loading nano system combining cell membrane antagonism with nano enzyme, preparation method and application |
| CN114099470A (en) * | 2021-12-09 | 2022-03-01 | 济南优科医疗技术有限公司 | Method for uniformly and slowly releasing liquid magnetic induction medium for treating tumors |
| CN115505570A (en) * | 2022-08-15 | 2022-12-23 | 东南大学 | Method for extracting tumor cell membrane by combining chemical induction apoptosis and physical freeze thawing |
| CN116650673A (en) * | 2023-05-25 | 2023-08-29 | 赣南医学院 | Cell membrane coated medicine fat granule and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| PENGXUAN ZHAO ET AL.: "Biomimetic calcium carbonate nanoparticles delivered IL-12 mRNA for targeted glioblastoma sono-immunotherapy by ultrasound-induced necroptosis", 《JOURNAL OF NANOBIOTECHNOLOGY》, vol. 20, 10 December 2022 (2022-12-10), pages 1 - 10 * |
| 周超培;刘芊芊;杨春荣;杨阳;高春生;: "红细胞载药递送系统的研究进展", 《中国药房》, no. 02, 30 January 2020 (2020-01-30), pages 116 - 123 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhu et al. | 3D bioprinting mesenchymal stem cell-laden construct with core–shell nanospheres for cartilage tissue engineering | |
| Scott et al. | Modular scaffolds assembled around living cells using poly (ethylene glycol) microspheres with macroporation via a non-cytotoxic porogen | |
| AU2017305948B2 (en) | Internally fixed lipid vesicle | |
| US6126936A (en) | Microcapsules and composite microreactors for immunoisolation of cells | |
| US5656469A (en) | Method of encapsulating biological substances in microspheres | |
| WO2008121447A1 (en) | Self-assembling membranes and related methods thereof | |
| WO2014025312A1 (en) | Methods of manufacturing hydrogel microparticles having living cells, and compositions for manufacturing a scaffold for tissue engineering | |
| SE454780B (en) | SET TO MANUFACTURE INTERFERONES AND MONOCLONAL ANTIBODIES BY SEMIPERMEABLE CELL-COVERED CELLS | |
| US10471016B2 (en) | Microparticles, methods for their preparation and use | |
| Cellesi et al. | Towards a fully synthetic substitute of alginate: Optimization of a thermal gelation/chemical cross‐linking scheme (“tandem” gelation) for the production of beads and liquid‐core capsules | |
| Shi et al. | Parametric analysis of shape changes of alginate beads | |
| Xue et al. | Controllable fabrication of alginate/poly-L-ornithine polyelectrolyte complex hydrogel networks as therapeutic drug and cell carriers | |
| Morelli et al. | Microparticles for cell encapsulation and colonic delivery produced by membrane emulsification | |
| US11667906B2 (en) | Magnetic microcarriers | |
| CN114796617A (en) | Composite 3D printing ink and application thereof | |
| CN117618387A (en) | Preparation method of film-coated calcium carbonate nanoparticles | |
| Zhang et al. | Water-in-water Pickering emulsion: A fascinating microculture apparatus for embedding and cultivation of Lactobacillus helveticus | |
| Patel et al. | An overview of resealed erythrocyte drug delivery | |
| US20120294903A1 (en) | Methods and systems of making nanostructures | |
| CN113201525B (en) | Stem cell microsphere group, stem cell in-vitro amplification method and application | |
| CN115990134B (en) | Injectable hydrogel/nanogel drug-loaded slow-release system and preparation method and application thereof | |
| CN117511851B (en) | Application of hydrogel scaffold in preparation of cell culture meat | |
| CN117018229B (en) | Application of over-expressed Clec1b protein cell membrane in preparation of knee osteoarthritis drugs | |
| CN114369565A (en) | Method for efficiently producing extracellular vesicles | |
| Park et al. | Injectable and Cryopreservable MSC-Loaded PLGA Microspheres for Recovery from Chemically Induced Liver Damage |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |