Short designed peptide amphiphiles are attractive at killing bacteria and inhibiting cancer cell growth, and the flexibility in their structural design offers a great potential for improving their potency and biocompatibility to mammalian host cells. Amino acid sequences such as G(IIKK)nI-NH2 (n≥3) have been shown to be membrane lytic, but terminal amino acid modifications could impose a huge influence on their performance. We report in this work how terminal amino acid modifications to G(IIKK)3I-NH2 influence its α-helical structure, membrane penetrating ability, and selective actions against different cell types. Deletion of an N-terminal Gly or a C-terminal Ile did not affect their antibacterial activity much, an observation consistent with their binding behavior to negatively charged membrane lipid monolayers. However, the cytotoxicity against mammalian cells was much worsened by the N-terminal Gly deletion, consistent with an increase in its helical content. Despite little impact on the antibacterial activity of G(IIKK)3I-NH2, deletion of both terminal amino acids greatly reduced its antitumor activity. Cholesterol present in tumor cell membrane-mimic was thought to constrain (IIKK)3-NH2 from penetrating into the cancerous membranes, evident from its lowest surface physical activity at penetrating model lipid membranes. On the other hand, its low toxicity to normal mammalian cells and high antibacterial activity in vitro and in vivo made it an attractive antibacterial agent. Thus, terminal modifications can help rebalance the different interactions involved and are highly effective at manipulating their selective membrane responses.
G(IIKK) 3 I-NH 2 (G3) has been recently shown to be highly effective at killing bacteria and inhibiting tumor cell growth while remaining benign to normal host mammalian cells.The aim of this work is to evaluate how residue substitutions of Ala (A), Val (V), Glu (E), and Lys (K) for the N-terminal Gly (G) or C-terminal Ile (I) of G3 affect the physiochemical properties and bioactivity of the variants. All substitutions caused the reduction of peptide hydrophobicity whilst N-terminal substitutions had less noticeable effect on the surface activity and helix-forming ability than C-terminal substitutions.N-terminal variants held potent antitumor activity but exhibited much lower hemolytic activity; these actions were related to the maintenance of their moderate surface pressures (12 to 16 mN/m) whilst their hydrophobicity was reduced. Thus, N-terminal substitutions enhanced the cell selectivity of the mutants relative to the control peptide G3. In contrast, C-terminal variants exhibited lower antitumor activity and further reduced hemolytic activity except for G(IIKK) 3 V-NH 2 . These features were also correlated well with their lower surface pressures (≤10 mN/m) and further decreased hydrophobicity. In spite of its very low helical content, the C-terminal variant G(IIKK) 3 V-NH 2 still displayed potent antitumor activity whilst retaining high hemolytic activity as well, again correlating well with its relatively high surface pressure and hydrophobicity. These results together indicated that surface activity governs the antitumor activity of the peptides but relative hydrophobicity influences their hemolytic activity. In contrast, helicity appears to be poorly correlated to their bioactivity. This work has demonstrated that N-terminal modifications provide a useful strategy to optimize the antitumor activity of helical anticancer peptides (ACPs) against its potential toxicity to mammalian host cells.
Amino acid side chains of a short amphiphilic G(AABB)3A-NH2peptide affect its bioactivity.
A dual-sensitive polymeric drug conjugate (HA-SS-MP) was synthesized by conjugating hydrophobic 6-mercaptopurine (MP) to thiolated hyaluronic acid (HA) as the carrier and ligand to deliver doxorubicin (Dox) to parental colon cancer and colon cancer stem cells. Because of the amphiphilic nature of HA-SS-MP, it was self-assembled in the aqueous media, and Dox was physically encapsulated in the core of the micelles. The particle size and the zeta potential of the micelle were analyzed by dynamic light scattering (DLS), and the morphology of the micelle was investigated using transmission electron microscopy (TEM). Drug release study results revealed more drug release at pH 5.0 in the presence of GSH than that at the physiological pH value. The cytotoxicity of free Dox was slightly greater than that of Dox-loaded HA-SS-MP micelles. In vitro cytotoxicity of HA-SS-MP and Dox-loaded HA-SS-MP micelles was greater for cancer stem cells (HCT116-CSCs) than for parental HCT116 colon cancer cells and L929 normal fibroblast cells. The MTT and flow cytometry results confirmed that free HA competitively inhibited Dox-loaded HA-SS-MP uptake. Similarly, flow cytometry results revealed anti-CD44 antibody competitively inhibited cellular uptake of Rhodamine B isothiocyanate conjugated micelles, which confirms that the synthesized micelle is uptaken via CD44 receptor. Cell cycle analysis revealed that free drugs and Dox-loaded HA-SS-MP arrested parental HCT116 colon cancer cells at the S phase, while cell arrest was observed at the G0G1 phase in HCT116-CSCs. In addition, ex vivo biodistribution study showed that Dox-loaded HA-SS-MP micelles were accumulated more in the tumor region than in any other organ. Furthermore, the in vivo results revealed that Dox-loaded HA-SS-MP micelles exhibited more therapeutic efficacy than the free drugs in inhibiting tumor growth in BALB/C nude mice. Overall, the results suggested that the synthesized micelles could be promising as a stimuli carrier and ligand for delivering Dox to colon cancer cells and also to eradicate colon cancer stem cells.
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