Quantitative Pharmacokinetics Reveal Impact of Lipid Composition on Microbubble and Nanoprogeny Shell Fate

Abstract

Microbubble‐enabled focused ultrasound (MB‐FUS) has revolutionized nano and molecular drug delivery capabilities. Yet, the absence of longitudinal, systematic, quantitative studies of microbubble shell pharmacokinetics hinders progress within the MB‐FUS field. Microbubble radiolabeling challenges contribute to this void. This barrier is overcome by developing a one‐pot, purification‐free copper chelation protocol able to stably radiolabel diverse porphyrin‐lipid‐containing Definity® analogues (pDefs) with >95% efficiency while maintaining microbubble physicochemical properties. Five tri‐modal (ultrasound‐, positron emission tomography (PET)‐, and fluorescent‐active) [64Cu]Cu‐pDefs are created with varying lipid acyl chain length and charge, representing the most prevalently studied microbubble compositions. In vitro, C16 chain length microbubbles yield 2–3x smaller nanoprogeny than C18 microbubbles post FUS. In vivo, [64Cu]Cu‐pDefs are tracked in healthy and 4T1 tumor‐bearing mice ± FUS over 48 h qualitatively through fluorescence imaging (to characterize particle disruption) and quantitatively through PET and γ‐counting. These studies reveal the impact of microbubble composition and FUS on microbubble dissolution rates, shell circulation, off‐target tissue retention (predominantly the liver and spleen), and FUS enhancement of tumor delivery. These findings yield pharmacokinetic microbubble structure‐activity relationships that disrupt conventional knowledge, the implications of which on MB‐FUS platform design, safety, and nanomedicine delivery are discussed.

Effect of Poly(ethylene glycol) Configuration on Microbubble Pharmacokinetics

“…The activation of the C3 pathway triggers the cleavage of C3 into C3b and C3a, initiating the phagocytic process and mediating inflammation or immune responses . Also, it has been reported that the presence of anti-PEG antibodies reduces the half-life of PEGylated drugs by 2–10-fold and increases hepatic and spleen uptake by 2–5 and 1–2-fold, respectively. , This observation corroborates our findings, which predict enhanced MB retention in C 2 , likely the lungs, spleen, and liver, ,,− which may be mediated by the complement activation. Thus, the secondary mechanism of MB clearance in C 2 can be attributed to phagocytosis by resident macrophages within the clearance organs.…”

“…Overall, increasing the MVD to 40 μL/kg extended circulation time from ∼20 to ∼30 min. MB clearance primarily involved dissolution and elimination by the lungs in C 1 , while MB retention is expected to occur in the liver, lungs, and spleen in C 2 . ,,,− Both the 10 and 5 mol % configurations displayed slightly accelerated biexponential decay when compared to the 2 mol % configuration.…”

“…The two-compartment model simply refers to the central compartment (i.e., the blood) and the peripheral compartment (all tissues such as the lung, liver, and spleen that may temporarily trap and/or permanently eliminate MBs). We do not mean to infer that the MBs are extravasating, nor do we refer to the biodistribution of the lipid shell debris as has been considered in other studies. , For the one-compartment model (eq ), C 1 = C 0 e − k e t …”

“…We do not mean to infer that the MBs are extravasating, nor do we refer to the biodistribution of the lipid shell debris as has been considered in other studies. 45,46 For the one-compartment model (eq 5), 56 C C e k t…”

“…In our previous work, we introduced MB volume dose (MVD) as a standard metric, unifying the MB size distribution and concentration into a single dose parameter (μL/kg) . This method allows direct assessment of the pharmacokinetics of intact MBs, comprising both the lipid shell and gaseous core, rather than separately evaluating the lipid , or gas components, , as conducted in prior studies. Moreover, this innovation allowed us to directly explore in vivo MB pharmacokinetics across various sizes and MVDs, identifying a transition from mono- to biexponential clearance from 20 to 40 μL/kg .…”