Preparation method and application of graphene-CpG
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A graphene and graphene technology, applied in the field of medicine, can solve the problems of low cell uptake efficiency, limited biotherapeutic application, poor stability, etc., and achieve the effects of improved stability and uptake efficiency, unique preparation method, and easy coupling
Inactive Publication Date: 2014-06-18
JILIN UNIV
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Problems solved by technology
However, artificially synthesized CpG oligonucleotides are usually difficult to enter cells. How to find a stable, highly active and biocompatible delivery system is the key to the application of CpG drugs
The single application of graphene or CpG has poor stability and low cell uptake efficiency
The biotherapeutic application of the two is obviously limited
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Embodiment 1
[0029] Example 1 Functionalized Graphene (Graphene)
[0030] 1) Weigh 1mg of graphene and 5mg of PL-PEG5000-Amine or PL-PEG2000-Amine into a 20ml straight thick-walled glass tube, add 5ml of distilled water, and shake on a shaker until DSPE-PEG is completely dissolved.
[0031] 2) Put it in a Vrtis VirSonic 300S water bath ultrasonic machine for ultrasonic treatment for 60 minutes, adjust the output power to level 7 every 5 to 10 minutes, and then remove the water and add ice to the water bath box each time to avoid overheating.
[0032] 3) Centrifuge the Graphene suspension at room temperature for 3 hours at a speed of 24000g, and collect the supernatant.
[0033] 4) Record the UV-VIS-NIR absorption spectrum of the obtained Graphene solution. The normal range of final concentration of Graphene is 40-70mg / L. Store at 4°C.
[0034] 5) Take 1ml of the stock solution prepared in step 3 and put it into a 4ml Aicon centrifugal filter device with a molecular weight cut-off (MWCO)...
Embodiment 2
[0043] (1) In vitro experiment
[0044] 1. Different doses of Graphene-CpG and Free CpG were incubated with RAW Blue cells to detect NFκB activity and determine the effective concentration and activity of Graphene-CpG. Such as figure 1 Shown; Graphene-CpG can induce the production of nuclear factor NF-κB in a time- and dose-dependent manner, and when the concentration is 1mg / ml, it has the greatest biological activity and the lowest cytotoxicity.
[0045] 2. Coupling Graphene with CY3 and CY5.5, incubating with HMPC cells for 4 hours, and then observing the cell uptake under a fluorescence microscope. Such as figure 2 Shown; Cell uptake increased with time, and cell viability was good.
[0046] (2) In vivo experiments
[0047] 1. Colorectal cancer
[0048] (1) Experimental materials: colorectal cancer cells SW480; athymic nude mice (female, 4W, body weight 18-20g).
[0049] (2) Experimental method: the human colorectal cancer cell line SW480 was used, and after cel...
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Abstract
The invention relates to a preparation method and an application of a graphene-CpG. The preparation method is characterized by specifically comprising the steps of functionalizing the graphene, weighing 1mg of graphene and 5mg of PL-PEG5000-Amine (Biotin-Poly Ethylene Glycol) or PL-PEG2000-Amine and adding the two materials to a 20ml straight thick-wall glass tube, adding 5ml of distilled water to the 20ml straight thick-wall glass tube and putting the 20ml straight thick-wall glass tube in a shaker for shaking until DSPE-PEG (Distearoyl Phosphatidyl Ethanolamine-Poly Ethylene Glycol) is completely dissolved; centrifuging a graphene suspension at the room temperature for 3 hours at a rotating speed of 24000g and collecting the supernate, and recording the UV-VIS-NIR (Ultraviolet-Visual-Near Infrared) absorption spectrum of the obtained graphene solution, wherein the final concentration of the graphene is in a normal rang of 40-70mg/L; and preserving at 4 DEG C, and then bonding the graphene with an oligonucleotide CpG. The preparation method is unique and capable of obviously inhibiting the growth of the tumor of breast and the tumor of colorectum; compared with a PBS (Phosphate Buffer Solution) group, the graphene-CpG prepared by the preparation method disclosed by the invention has the advantages that the difference is significant and the growth of the tumors is obviously suppressed; besides, the preparation method has statistical significance.
Description
Technical field [0001] The invention involves a graphene -CPG preparation method and application for the field of pharmaceuticals. Background technique [0002] The application of graphene in the field of biomedicine is facing the shortcomings that cause inflammatory reactions.CPG oligonucleotide fragments are identifiable molecules that are found in bacteria that can stimulate the immune system.At present, it is often used in immunotherapy or immunoaste assistance.TLR-9 is an intracellular signal molecule for CPG.However, artificially synthesized CPG oligonucleotide is usually difficult to enter cells. How to find a stable, high activity and good biocompatibility loading system is the key to CPG drug applications.The separate application of graphene or CPG has poor stability and low cell intake efficiency.The biological therapy applications of the two are obviously limited. Invention content [0003] The purpose of the present invention is to provide a graphene -CPG preparation...
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