hydrolysis kinetics of the hydrazine bond. Based on the results,
it might be inferred that the ‘nano-flower’ had a half-open
state in the extracellular environment of tumor tissues, held
the cargos through the cell membrane, and bloomed fully
under endosomal acidic conditions to release the drugs finally.
In Fig. 3b, the MTT assay was used to estimate the cytotoxi-
city of the mixed-micelles in MCF-7 cells. As the Dox concen-
tration increased, the mixed micelles of CPCB/CPD incubated
under acidic conditions showed significantly higher cytotoxi-
city than that under neutral conditions. It was also found that
the cytotoxicity of CPC/CPD mixed micelles at pH 6.8 was
slightly higher than that of the CPCB/CPD sample at pH 7.4.
Although the hydrolysis of hydrazine bonds at pH 6.8 resulted
in a slight release of Dox, the biotin receptor-mediated endo-
cytosis was the main reason for the apoptosis of cancer cells.
In summary, the ‘nano-flower’ prepared by CPCB/CPD
mixed-micelles here was proven to be a smart drug carrier.
Due to the covalent conjugation of Dox on polymer chains
and the shielding of target ligands by folded PEG chains, the
‘nano-flower’ might effectively prevent the premature leak of
Dox and decrease the specific contact with receptors outside the
tumor tissues. With the decrease in pH value, the ‘nano-flower’
exhibited a half-open state to expose the target ligands on the
surface under tumor acidic conditions and fully bloomed to
release Dox under endosomal acidic conditions. The pH-triggered
blooming behavior made the ‘nano-flower’ accomplish a
process of tumor-triggered cell uptake and intercellular drug
release, which might be a good candidate for chemotherapy.
This work was financially supported by National Basic Research
Program of China (2011CB606202 and 2009CB930300).
Fig. 3 (a) The cumulative release of Dox from CPCB/CPD mixed-
micelles. (b) Cytotoxicity of the mixed-micelles in MCF-7 cells for 24 h
incubation.
There were some other factors, such as the strength of binding
affinity, the density of the PEG shell and the conformation of
PEG, which might also influence shielding effects on the
interaction of ligand–protein.19,20 Additionally, we measured
the CPB micelle (the reactant of CPCB before click chemistry),
in which all the biotin moieties were exposed on the micelle
surface, and found that 81.8 nmol per milligram of CPB was
available to avidin. By calculation, it was equivalent to 48.5%
of biotin available to avidin (Fig. 2b). It indicated that the
aggregated conformation of micelle might sterically prohibit
interaction of biotin with avidin. Although we did not estimate
the optimum percentage of CPCB in mixed micelle for tumor
targeted here, the strategy of single-component micelle (one
biotin per macromolecule) seemed to be a waste of target
ligands, which was the main reason for choosing a mixed micelle
rather than a single component micelle loaded with drugs.
The cell uptake capacity of ‘nano-flower’ on MCF-7 cells
was measured here to estimate acid-triggered ‘blooming’
again. To acquire desired fluorescence effects, 5 wt% of Brij
modified with FITC was used to form the mixed-micelle
(wCPCB/wCPD/wCPFITC = 1/1/0.1). We compared the cell
uptake behaviors of the mixed-micelles at pH 6.8 and 7.4. In
Fig. 2c, strong fluorescence was observed in the MCF-7 cells
incubated at pH 6.8, while very slight fluorescence was found
at pH 7.4. This result was consistent with that of the HABA/
avidin assay. The biotin receptor-mediated endocytosis was
significantly enhanced under tumor acidic conditions that
triggered the ‘nano-flower’ blooming. A controlled trial was
also carried out by treating the MCF-7 cells with other mixed
micelles consisting of CPC/CPD/CPFITC at the same weight
ratio as CPCB/CPD/CPFITC mixed micelles. In Fig. S12
(ESIw), there was no obvious fluorescence observed in cells
either at pH 6.8 or 7.4. It further proved that the internaliza-
tion process of the ‘nano-flower’ was mediated by the inter-
action between biotin and its receptor. Moreover, it was also
noted that Dox was not significantly internalized by the MCF-7
cell without the aid of biotin even at pH 6.8, which indicated
that the hydrolysis of the hydrazine bond needed lower
pH value or more time in terms of dynamics. A long term
cumulative drug release measurement was carried out and the
results are shown in Fig. 3a. After 4 days of release at 37 1C,
only 17.2% Dox was released from the CPCB/CPD mixed-
micelles at pH 7.4 and the value increased to 22.7% at pH 6.8.
When the pH was set to 5.4, which was similar to that under
the acidic conditions of endosomes, 71.5% Dox was released
finally. Since Dox was covalently linked onto the polymer
chains, the drug release behavior could be controlled by the
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