X-ray computed tomography
(CT) is a robust, precise, fast, and
reliable imaging method that enables excellent spatial resolution
and quantification of contrast agents throughout the body. However,
CT is largely inadequate for molecular imaging applications due mainly
to its low contrast sensitivity that forces the use of large concentrations
of contrast agents for detection. To overcome this limitation, we
generated a new class of iodinated nanoscale activity-based probes
(IN-ABPs) that sufficiently accumulates at the target site by covalently
binding cysteine cathepsins that are exceptionally highly expressed
in cancer. The IN-ABPs are comprised of a short targeting peptide
selective to specific cathepsins, an electrophilic moiety that allows
activity-dependent covalent binding, and tags containing dendrimers
with up to 48 iodine atoms. IN-ABPs selectively bind and inhibit activity
of recombinant and intracellular cathepsin B, L, and S. We compared
the in vivo kinetics, biodistribution, and tumor accumulation of IN-ABPs
bearing 18 and 48 iodine atoms each, and their control counterparts
lacking the targeting moiety. Here we show that although both IN-ABPs
bind specifically to cathepsins within the tumor and produce detectable
CT contrast, the 48-iodine bearing IN-ABP was found to be optimal
with signals over 2.1-fold higher than its nontargeted counterpart.
In conclusion, this study shows the synthetic feasibility and potential
utility of IN-ABPs as potent contrast agents that enable molecular
imaging of tumors using CT.
Tumor trafficking of liposomes is routinely monitored via fluorescence microscopy and imaging. To investigate whether an accumulation of liposomes depends on the type of fluorescent label, we prepared PEGylated liposomes dual-labeled with indocarbocyanine lipids (ICLs: DiD or DiI) and fluorescent phospholipids (FPLs: Cy3-DSPE or Cy5-DSPE) with similar cyanine headgroups but different spectra. Using ex vivo confocal microscopy and imaging, we compared tumor extravasation and accumulation of ICLs and FPLs. After systemic injection in a syngeneic mouse model of 4T1 breast cancer, ICLs and FPLs initially colocalized in tumor blood vessels and perivascular space. At later time points, ICLs spread over a significantly larger tumor area and accumulated in tumor macrophages, whereas FPLs were mostly restricted to the vasculature with limited extravascular signal. This phenomenon was independent of liposomal composition and ICL/FPL type and was also observed in syngeneic intracranial GL261 glioma and LY2 head and neck cancer models. The dual-labeled liposomes were stable in plasma and delivered both dyes to tumors at early time points. Notably, while the level of ICLs increased over time, FPLs gradually disappeared from tumors and other organs in vivo, likely due to degradation of the phospholipid. These findings demonstrate that trafficking and stability of the label is of critical importance when assessing extravasation and accumulation of nanocarriers in tumors and other organs by fluorescence microscopy and imaging.
Many aspects of innate immune responses to SARS viruses remain unclear. Of particular
interest is the role of emerging neutralizing antibodies against the receptor-binding
domain (RBD) of SARS-CoV-2 in complement activation and opsonization. To overcome
challenges with purified virions, here we introduce “pseudovirus-like”
nanoparticles with ∼70 copies of functional recombinant RBD to map complement
responses. Nanoparticles fix complement in an RBD-dependent manner in sera of all
vaccinated, convalescent, and naïve donors, but vaccinated and convalescent
donors with the highest levels of anti-RBD antibodies show significantly higher IgG
binding and higher deposition of the third complement protein (C3). The opsonization
via
anti-RBD antibodies is not an efficient process: on average, each
bound antibody promotes binding of less than one C3 molecule. C3 deposition is
exclusively through the alternative pathway. C3 molecules bind to protein deposits, but
not IgG, on the nanoparticle surface. Lastly, “pseudovirus-like”
nanoparticles promote complement-dependent uptake by granulocytes and monocytes in the
blood of vaccinated donors with high anti-RBD titers. Using nanoparticles displaying
SARS-CoV-2 proteins, we demonstrate subject-dependent differences in complement
opsonization and immune recognition.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.