A functional platelet bionic smart carrier and its application in anti-ischemic stroke
A platelet, functionalized technology, applied in the field of medicine, can solve the problems of decreased binding force and targeting, reduced tPA thrombolytic effect, short circulation time, etc., achieves good biocompatibility, improves therapeutic effect, and avoids phagocytosis. Effect
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Embodiment 1
[0032] Preparation of ZL006e-loaded polymer nanoparticles:
[0033] Take 2 mg of neuroprotective agent ZL006e and 20 mg of m-Dextran into a 15 ml centrifuge tube, add 2 ml of dichloromethane, vortex to dissolve completely, and add 4 ml of PVA solution prepared in 3% 1X PBS (pH 7.4). Ultrasonic cell crusher was sonicated in ice bath (35% power, ultrasonic for 2s / stop for 2s, 5min). The sonicated white emulsion was slowly introduced into 15 ml of a PVA solution prepared with 0.3% 1X PBS (pH 7.4) in high-speed stirring (800 rpm). After continuous stirring for about 1-2 hours, the dichloromethane was completely evaporated, and the solution was clear and transparent, showing blue nanoparticle opalescence. High-speed centrifugation (12000rpm, 40min), discard the supernatant, add water to reconstitute, repeat 2-3 times to wash off the emulsifier, and finally disperse in 1ml of deionized water to obtain a ZL006e-loaded polymer nanoparticle solution.
Embodiment 2
[0035] Preparation of ZL006e-loaded Polymer Nanoparticles Coated with Platelet Membrane
[0036] Take fresh blood and centrifuge at 500g, centrifuge the upper plasma at 2000g, discard the supernatant to obtain the lower layer of red blood cells, then add deionized water and mix with red blood cells, let stand for 1-2 hours at room temperature; 5 times), stored in 1X PBS (pH 7.4), and crushed by ultrasonic cell crusher to obtain platelet membrane stock solution.
[0037] The platelet membrane stock solution was added to the ZL006e-loaded polymer nanoparticle solution prepared in Example 1 (4 ml of platelet membrane stock solution was added to each 20 mg of ZL006e-loaded polymer nanoparticle solution), and slowly stirred for 8-10 hours to obtain coated platelets Membrane-coated polymer nanocarriers. Then add NHS-PEG 3500 -N 3 (Add 10 mg of NHS-PEG per 20 mg of ZL006e-loaded polymer nanoparticle solution 3500 -N 3 ), stirring rapidly for 2 to 3 hours. High-speed centrifugat...
Embodiment 3
[0039] Preparation of polypeptide-protein conjugates linked with thrombolytic drugs rtPA and Tat penetrating peptides and thrombin substrate peptides:
[0040] Dissolve 1 mg of tPA protein in deionized water, add Sulfo-SMCC, the molar ratio of tPA:SMCC is 1:15-20, stir at 800 rpm for 2-3 hours, and add excess Pro-Tat-LTPRGWRLGGC that can be sheared and cleaved by Thrombin Polypeptide (the molar ratio of tPA:peptide segment is 1:20~30), under the condition of 4℃, continue stirring at 800rpm for 2-3h, the reaction solution is passed through MidiTrap TM G-25 desalting column eluted to remove unreacted SMCC and polypeptides, dispersed in 20 mmol HEPES buffer (Ph7.2-7.4), and obtained the penetrating peptides connected with thrombolytic drugs tPA and Tat and peptides that can be cleaved by thrombin -Protein conjugate (Pro-Tat-LTPRGWRLGGC-tPA).
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