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first incubated with CPD only, RNase A only and RNase A would like to thank Dr. Renshuai Zhang for his assistance in the
DOI: 10.1039/D0CC04728G
protein-CPD conjugate, respectively. Cell viability was synthesis of the compounds.
determined using an MTT assay. After 24 h treatment, 500 nM
of RNase A protein-CPD conjugate exhibited notable cytotoxic Conflicts of interest
effect, while CPD and RNase A controls alone did not There are no conflicts to declare.
significantly lower cell viability (Fig. 4C).
To determine whether the dual-labeled RNase A prodrug
Notes and references
could be activated by endogenous ROS upon intracellular
delivery, CPD was conjugated to both the NTC-5-modified
RNase A prodrug (i.e. labeled with LBL) and the corresponding
control (i.e. NBL-labeled RNase A), followed by incubation with
HeLa cells. After 24 h, HeLa cells treated with 500 nM of the cell-
permeable RNase A prodrug had a much lower viability (~20%)
than that of the non ROS-responsive control (Fig. 4D). We
further determined if cancer cell-specific cytotoxicity can be
achieved using the cell-permeable RNase A prodrug. A panel of
cancer and non-cancerous cell lines, including NIH3T3
(embryonic fibroblast cells) and RAW264.7 (murine
macrophages) were screened for their intracellular ROS levels
by incubation with 2',7'-dichlorodihydrofluorescein diacetate
(DCFDA), a ROS-responsive fluorescent probe. FACS analysis
indicated elevated levels of ROS in all 4 cancer cells compared
to the other 2 non-cancerous cell lines (Fig. 4E). Following the
intracellular delivery of the RNase A prodrug, the viability of
cancer cell lines (HeLa, MDA-MB-231, A431, HEPG2) decreased
to ≤ 30%. On the other hand, non-cancerous cell lines, NIH3T3
and RAW264.7, displayed over 90% viability (Fig. 4E). To
eliminate any discrepancy due to uptake efficiency, the cellular
uptake of RNase A protein-CPD conjugates for each cell line was
determined and found to be similar (Fig. S9). These results
suggested that dual modification of RNase A successfully
endowed it with both cell-penetrating ability and ROS-
responsive prodrug function for targeted cancer therapy.
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In summary, we have reported a new approach for CPD-
facilitated cytosolic delivery of proteins using N-terminal site-
specific modification. As shown, proteins such as RNase A and
anti-NPC antibody retained their functions after intracellular
delivery. Hence, this strategy holds great potential in the
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believe that this dual-labeling approach involving N-terminal
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precision therapy, such as protein kinases, which require
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have key amino acid residues such as Cys or Ser in their active
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Trust Fund (R-143-000-88-592), the Synthetic Biology Research
& Development Programme (SBP) of National Research
Foundation (SBP-P4 and SBP-P8) of Singapore and the National
Natural Science Foundation of China (81672508). The authors
4 | J. Name., 2012, 00, 1-3
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