3 research outputs found
Enhanced siRNA Delivery Using a Combination of an Arginine-Grafted Bioreducible Polymer, Ultrasound, and Microbubbles in Cancer Cells
RNAi-based
gene therapy for cancer treatment has not shown significant
clinical impact due to poor siRNA delivery to the target site. Here,
we design a nonviral siRNA gene carrier using a combination of an
arginine-grafted bioreducible polymer (ABP), microbubbles (MB), and
ultrasound (US), for targeting vascular endothelial growth factor
(VEGF) in a human ovarian cancer cell line. Newly designed MBs with
a perfluorocrownether gas core show higher stability compared to controls.
Further, MBs in combination with polyplexes show a significant higher
loading capacity compared to naked siRNA. Lastly, only siRNA-ABP-MB
(SAM) complexes in combination with US show significant VEGF knock
down in A2780 human ovarian cancer cell line compared to naked siRNA
when incubated for a short time after sonication treatment
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Tuning the Diels–Alder Reaction for Bioconjugation to Maleimide Drug-Linkers
The thiol–maleimide
linkage is widely used for antibody–drug
conjugate (ADC) production; however, conjugation of maleimide–drugs
could be improved by simplified procedures and reliable conjugate
stability. Here, we report the evaluation of electron-rich and cyclic
dienes that can be appended to antibodies and reacted with maleimide-containing
drugs through the Diels–Alder (DA) reaction. Drug conjugation
is fast and quantitative due to reaction acceleration in water, and
the linkage is more stable in serum than in the corresponding thiol–maleimide
adduct with the same drug. ADCs produced using the DA reaction (DAADCs)
are effective in vitro and in vivo, demonstrating the utility of this
reaction in producing effective biotherapeutics. Given the large number
of commercially available maleimide compounds, this conjugation approach
could be readily applied to the production of a wide range of antibody
(or protein) conjugates
A Nanoparticle Platform To Evaluate Bioconjugation and Receptor-Mediated Cell Uptake Using Cross-Linked Polyion Complex Micelles Bearing Antibody Fragments
Targeted nanomedicines are a promising
technology for treatment
of disease; however, preparation and characterization of well-defined
protein-nanoparticle systems remain challenging. Here, we describe
a platform technology to prepare antibody binding fragment (Fab)-bearing
nanoparticles and an accompanying real-time cell-based assay to determine
their cellular uptake compared to monoclonal antibodies (mAbs) and
Fabs. The nanoparticle platform was composed of core-cross-linked
polyion complex (PIC) micelles prepared from azide-functionalized
PEG<i>-<i>b</i>-</i>polyÂ(amino acids), that is,
azido-PEG<i>-<i>b</i>-</i>polyÂ(l-lysine)
[N<sub>3</sub>–PEG<i>-<i>b</i>-</i>PLL]
and azido-PEG<i>-<i>b</i>-</i>polyÂ(aspartic acid)
[N<sub>3</sub>–PEG<i>-<i>b</i>-</i>PAsp].
These PIC micelles were 30 nm in size and contained approximately
10 polymers per construct. Fabs were derived from an antibody binding
the EphA2 receptor expressed on cancer cells and further engineered
to contain a reactive cysteine for site-specific attachment and a
cleavable His tag for purification from cell culture expression systems.
Azide-functionalized micelles and thiol-containing Fab were linked
using a heterobifunctional cross-linker (FPM-PEG<sub>4</sub>-DBCO)
that contained a fluorophenyl-maleimide for stable conjugation to
Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle
azide groups. Analysis of Fab–PIC micelle conjugates by fluorescence
correlation spectroscopy, size exclusion chromatography, and UV–vis
absorbance determined that each nanoparticle contained 2–3
Fabs. Evaluation of cellular uptake in receptor positive cancer cells
by real-time fluorescence microscopy revealed that targeted Fab–PIC
micelles achieved higher cell uptake than mAbs and Fabs, demonstrating
the utility of this approach to identify targeted nanoparticle constructs
with unique cellular internalization properties