Disulfide-Mediated Bioconjugation: Disulfide Formation
and Restructuring on the Surface of Nanomanufactured (Microfluidics)
Nanoparticles
Posted on 2019-07-18 - 19:04
This
study is about (1) nanomanufacturing (focusing on microfluidic-assisted
nanoprecipitation), (2) advanced colloid characterization (focusing
on field flow fractionation), and (3) the possible restructuring of
surface disulfides. Disulfides are dynamic and exchangeable groups,
and here we specifically focus, first, on their use to introduce biofunctional
groups and, second, on their re-organization, which may lead to variable
surface chemistries and uncontrolled cell interactions. The particles
were obtained via microfluidic-assisted (flow-focused) nanoprecipitation
of poly(ethylene glycol)-b-poly(ε-caprolactone)
bearing or not a 2-pyridyl disulfide (PDS) terminal group, which quantitatively
exchanges with thiols in solution. In this study, we have paid specific
attention to size characterization, thereby also demonstrating the
limitations of dynamic light scattering (DLS) as a stand-alone technique.
By using asymmetric flow field flow fractionation coupled with DLS,
static light scattering (SLS), and refractive index detectors, we
show that relatively small amounts of >100 nm aggregates (cryogenic
transmission electron microscopy and SLS/DLS comparison suggesting
them to be wormlike micelles) dominated the stand-alone DLS results,
whereas the “real” size distributions picked <50
nm. Our key result is that the kinetics of the conjugation based on
PDS–thiol exchange was controlled by the thiol pKa, and this also determined the rate of the exchange between
the resulting disulfides and glutathione (GSH). In particular, more
acidic thiols (e.g., peptides, where a cysteine is flanked by cationic
residues) react faster with PDS, but their disulfides hardly exchange
with GSH; the reverse applies to thiols with a higher pKa. Disulfides that resist against restructuring via thiol–disulfide
exchange allow for a stable bioconjugation, although they may be bad
news for payload release under reducing conditions. However, experiments
of both thiol release and nanoparticles uptake in cells (HCT116) show
that also the disulfides formed from less-acidic and, therefore, less-reactive,
and more exchangeable thiols were stable for at least a few hours
even in a GSH-rich (10 mM) environment; this suggests a sufficiently
long stability of surface groups to achieve, for example, a cell-targeting
effect.
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Geven, Mike; Luo, Hanying; Koo, Donghun; Panambur, Gangadhar; Donno, Roberto; Gennari, Arianna; et al. (2019). Disulfide-Mediated Bioconjugation: Disulfide Formation
and Restructuring on the Surface of Nanomanufactured (Microfluidics)
Nanoparticles. ACS Publications. Collection. https://doi.org/10.1021/acsami.9b07972
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AUTHORS (9)
MG
Mike Geven
HL
Hanying Luo
DK
Donghun Koo
GP
Gangadhar Panambur
RD
Roberto Donno
AG
Arianna Gennari
RM
Roberto Marotta
BG
Benedetto Grimaldi
NT
Nicola Tirelli
KEYWORDS
GSHcryogenic transmission electron microscopyDisulfide-Mediated Bioconjugationsurface chemistriessurface disulfidescationic residuesp Kcolloid characterizationacidic thiolsHCTthiol p KDLS resultscell-targeting effectcell interactionspayload releasesize characterizationterminal groupwormlike micellesindex detectorsflow field flow fractionationbiofunctional groupssurface groupsDisulfide FormationPDS2- pyridyl disulfidethiol releaseSLSnanoparticles uptake