Chlorotoxin (CTX), a disulfide-rich peptide from the scorpion Leiurus 
quinquestriatus, has several promising biopharmaceutical properties, including 
preferential affinity for certain cancer cells, high serum stability, and cell 
penetration. These properties underpin its potential for use as a drug design 
scaffold, especially for the treatment of cancer; indeed, several analogs of CTX 
have reached clinical trials. Here, we focus on its ability to internalize into 
cells-a trait associated with a privileged subclass of peptides called 
cell-penetrating peptides-and whether it can be improved through conservative 
substitutions. Mutants of CTX were made using solid-phase peptide synthesis and 
internalization into human cervical carcinoma (HeLa) cells was monitored by 
fluorescence and confocal microscopy. CTX_M1 (ie, [K15R/K23R]CTX) and CTX_M2 
(ie, [K15R/K23R/Y29W]CTX) mutants showed at least a twofold improvement in 
uptake compared to CTX. We further showed that these mutants internalize into 
HeLa cells largely via an energy-dependent mechanism. Importantly, the mutants 
have high stability, remaining intact in serum for over 24 h; thus, retaining 
the characteristic stability of their parent peptide. Overall, we have shown 
that simple conservative substitutions can enhance the cellular uptake of CTX, 
suggesting that such type of mutations might be useful for improving uptake of 
other peptide toxins.