Targeted modified and drug-loaded exosome as well as preparation method and application thereof
An exosome and loaded technology, which is applied in the field of target-modified and drug-loaded exosomes and its preparation, can solve the problem that neurotrophic factors cannot efficiently cross the blood-brain barrier, and there is no neurotrophic factor delivery method , short half-life of neurotrophic factors, etc., to reduce toxicity and other unintended effects, reduce nerve tissue damage, and promote concentration
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Embodiment 1
[0051] Example 1. NGF@Exo RVG construction and identification of
[0052] In this application, we used the plasmid expressed by fusion of rabies virus glycoprotein (RVG) and exosomal protein lysosome-associated membrane glycoprotein 2b (Lamp2b) to carry out targeted modification on exosomes to prepare RVG-modified exosomes, and Neurotrophic factors and their mRNAs are loaded into the RVG-modified exosomes. In this example, the preparation process is described by taking the loading of the RVG-modified exosomes with human recombinant NGF protein and its mRNA as an example.
[0053] NGF@Exo RVG Construction of RVG and Lamp2b fusion expression plasmid pcDNA3.1(-)-RVG-Lamp2b (recorded in "Exosome Mediated Delivery of miR-124 Promotes Neurogenesis after Ischemia.", PMID: 28624203) and cloned neurotrophic factor The recombinant vector pCI-neo-NGF plasmid (CN 102898514 A) (in this application, RVG-Lamp2b+NGF will be used to represent these two plasmids) co-transfected human embryon...
Embodiment 2
[0057] Example 2. NGF@Exo RVG Contains NGF detection
[0058] qRT-PCR was used to detect the expression level of NGF mRNA in HEK293 cells without transfection, HEK293 cells co-transfected with RVG-Lamp2b+NGF, and HEK293 cells co-transfected with pcDNA3.1+pCI-neo. The results showed that co-transfection The level of NGF mRNA in HEK293 cells transfected with RVG-Lamp2b+NGF was significantly increased (see Figure 2A ).
[0059] qRT-PCR was used to detect exosomes harvested from the culture supernatant of untransfected HEK293 cells, HEK293 cells co-transfected with RVG-Lamp2b+NGF, and co-transfected with pcDNA3.1+pCI-neo HEK293 cells The expression level of NGFmRNA, the results show that the NGF@Exo harvested from the culture supernatant of HEK293 cells co-transfected with RVG-Lamp2b+NGF RVG The level of NGF mRNA was significantly increased (see Figure 2B ).
[0060]NGF protein in exosomes harvested from untransfected HEK293 cells, HEK293 cells co-transfected with RVG-Lamp2...
Embodiment 3
[0062] Example 3.NGF@Exo RVG Can deliver NGF to recipient cells
[0063] The aforementioned results prove that NGF@Exo RVG NGF mRNA and protein can be loaded, we also explored the exosome NGF@Exo RVG Whether NGF can be delivered to recipient cells when taken up by recipient cells. To this end, we added no or 20 μg, 50 μg, 100 μg, 200 μg or 300 μg of NGF@Exo to HEK293 cell culture medium RVG Incubate for 4h (eg Figure 3A shown). It was found by qRT-PCR detection without adding NGF@Exo RVG , HEK293 cells had no obvious expression of nerve growth factor; adding 100μg NGF@Exo RVG Can significantly increase the level of NGF mRNA in HEK293 recipient cells. With 20μg and 50μg NGF@Exo RVG After incubation, although nerve growth factor tended to increase, it did not significantly increase the expression of nerve growth factor mRNA; 100-300μg NGF@Exo RVG Can significantly increase NGF mRNA and protein levels in recipient cells HEK293, when 200μg and 300μg NGF@Exo RVG The incr...
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