Maleimide availability in thiol-maleimide peptide conjugation on PEGylated liposomes: a reactive-site accounting framework illustrated using hepatic drug-delivery systems

Peptide-targeted PEGylated liposomes are a widely investigated active-targeting strategy for hepatocellular carcinoma (HCC) and other hepatic indications. A critical but underreported formulation variable is effective maleimide availability: the number of outward-facing, unhydrolysed, sterically accessible maleimide groups present when peptide thiols are coupled. In the seven hepatic systems examined, authors often report nominal lipid or peptide input without measuring reactive sites, conjugation efficiency, or final surface density. Hydrolysis, steric access, bilayer orientation, and peptide-thiol oxidation are individually well-established features of thiol-maleimide chemistry; the contribution of this Perspective is not to identify these phenomena but to integrate them into a quantitative accounting framework that links nominal input to biological outcome. We therefore propose a reactive-site accounting framework that separates six distinct quantities often conflated in practice: nominal maleimide input (the formulation's stated lipid composition), reactive maleimide (the outward-facing, unhydrolyzed fraction actually present at coupling), available peptide thiol (the free-thiol fraction remaining after synthesis, reduction or thiolation, and handling), conjugation efficiency (the proportion of input peptide that is actually conjugated), final conjugated ligand density (molecules per particle), and biologically accessible ligand density (the fraction capable of engaging its receptor in the biological milieu). Route selection, such as distinguishing maleimide-lipid pre-incorporation, post-insertion of a lipid conjugate, and direct coupling on preformed liposomes, should be evaluated experimentally rather than assumed. The chemical limitations underlying this framework are not specific to hepatic targeting; the seven hepatic systems examined here serve as an illustrative case set for a broader reporting problem in thiol-maleimide functionalization of nanocarriers. Reporting these measures would make density-response relationships easier to interpret and peptide-liposome studies, hepatic and otherwise, easier to reproduce.