I would like to check if the nanoparticles I am working with induce thrombogenicity, platelet aggregation or complement activation following its systemic administration in vivo (mouse model). I am thinking of collecting the blood plasma 4h and 24h following IV administration of the nanoparticles and checking for protein markers specific to thrombogenicity (CXCL4, total prothrombin/ thrombin), platelet activation (p-selectin, CD-41) or complement activation (C3). Does it make sense? Are the time-points for sample collection alright.
Concerning complement, please check: Passirani C., Barratt G., Devissaguet J-P., Labarre D. Long-circulating nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate), Pharm. Res., 15, 1046-1050, 1998
I do not think that collecting the blood at 24h would be useful. It's a very long time concerning reactions of blood to foreign materials. Start as early as possible (1 min if possible) and then 5, 10, 30, 60 min. At 4h, everyting is finished.
However, as this is an rather old story for me, I did not remember that we had not the required reagents to perform complement activation in mice blood. Complement activation was assessed in vitro with human serum (same authors, same year in Life Sciences)
Thank you for your suggestion. Maybe I should draw blood at earlier time-points. I noticed that there are many papers reporting in-vitro complement analysis using human serum (CH50 test). As I want to perform direct in-vivo analysis, I was wondering If I could draw blood from mice after IV administration of nanoparticles, isolate serum and look for respective markers by Western blots, like complement C3 with anti-C3 antibody. Not sure if that will be sensitive enough.
If you can find anti-C3 antibodies from mice, you can try. I have no idea concerning sensitivity
CH50 is really tedious. We preferred to use two-dimensional immunoelectrophoresis. A reference describing the technique is attached. Again, you need to use anti-C3 antibodies of the right species.
The best way to study thrombogenic material is to perform a recalcified coagulation activity assay (RECA) with freshest citrated plasma exposed to it.
I agree with Thomas W Stief. Of course this is an in vitro assesment with human plasma but a general approach for evaluation of thrombogenicity.
Glucose is an intrinsic trigger of coagulation. We performed oral glucose tolerance tests with ourselves and found interesting contact activating activities in blood at about 1h.
You can test nanoparticle-dependent complement activation in the mouse by following the protocol in Pham et al., attached.
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