Background. We endeavored to devise a stable method for demonstrating local delivery of fibrin-targeted, anti-inflammatory drug (pioglitazone)-loaded echogenic liposomes (PAFb-PGN-ELIP) to balloon-injured porcine arterial wall segments without interference by tissue autofluorescence. We report here a method for successful immunohistochemical (IHC) delivery demonstration with novel stable labeling strategies. Methods. N-(2,4-dinitrophenyl)-L-alanine (DNP-L-ala) was conjugated to L-cysteine via a peptide bond, followed by overnight Michael addition of the DNP-L-ala-L-cys to the pioglitazone pyridyl function. The DNP-labeled drug was loaded into biotinylated liposomes derivatized with a maleimido linker (MPB). The MPB-PGN-ELIP was conjugated to a fibrin-binding peptide, H-GPRPPGGGC-NH2 HCl, via the N-terminal cysteine to form a thioether linkage. The DNP-labeled, biotinylated PAFb-PGN-ELIP (5 mg lipid dose) was injected into Sinclair pigs proximal to balloon-injured segments of carotid and subclavian arteries. After euthanization, arterial sections were incubated with rabbit anti-DNP, followed by both fluorescent and IHC staining procedures to demonstrate drug localization. ELIP localization was demonstrated by incubation of sections with AlexaFluor 594-streptavidin. Results. The final content of DNP conjugated to PGN in PAFb-PGN-ELIP was 0.12-0.13 µg DNP/mg lipid. Subsequent demonstration of drug and liposome localization in the porcine arterial wall after in vivo administration of PAFb-PGN-ELIP is shown in Figure 1. Conclusions. A novel method for stable dual labeling of PGN and liposomes has been used to demonstrate successful in vivo delivery.

A novel molecular labeling strategy for demonstrating targeted, drug-loaded liposome localization to injured porcine arterial endothelium

ACS Spring Meeting 2021

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