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viability obtained using the MTS assay method in HeLa cells
were decreased by DOX-loaded polymeric micelles P3, and the
cytotoxicity of DOX-HCl and free DOX was examined by the
same method, as shown in Fig. S13.† All of the concentration of
the free DOX exhibited low cytotoxicity towards HeLa cells,
while the cell viability decreased sharply by 95% for DOX-HCl,
while for DOX-loaded polymeric micelles P3, the cell viability
decreased sharply by 70% aer 48 h of incubation; these results
provide further proof that DOX-loaded polymeric micelles P3
have sustained release effect on DOX.
In vitro cellular uptake of DOX
The intracellular uptake of DOX-HCl, free DOX and DOX-loaded
P3 was investigated using confocal lasing scanning microscopy
(CLSM) (Fig. 3). When the cells were incubated with DOX-HCl,
the cellular uptake of DOX-HCl was the same, regardless of
the different time duration of 2, 4, 8, 12, 24 and 48 h. DOX-HCl
was distributed in the nuclear region within 2 h of incubation,
and strong uorescence was observed in the nucleus, as re-
ported previously.21 From column DOX-HCl (P)-DOX-HCl (R),
aer 48 h incubation, it was clearly observed that the HeLa cell
morphology was completely destroyed. However, when the cells
were incubated with free DOX, the cellular uptake of free DOX
was also the same, regardless of different time durations of 2 h,
4 h, 8 h, 12 h, 24 h and 48 h, and free DOX was distributed in the
cytoplasm region during long-time incubation, with the aggre-
gation of free DOX with increasing time. Moreover, it was
difficult to for the free DOX to transfer into the nucleus even
aer 48 h. When the cells were incubated with DOX-loaded
polymeric micelle P3 in two hours, considerable uorescence
intensity was detected mainly in the cytoplasm, suggesting
novel cellular uptake ability of DOX-loaded polymeric micelle
P3, and with the prolongation of the incubation time, the
cellular uorescence was much higher. We found that aer 4
hours, the DOX molecules were localized in the cell nuclei, and
as expected from the drug release in vitro experiment, DOX
localized in the cell nuclei is likely to be intercalated into DNA
strands, thereby showing its toxicity against tumour cells. This
dual cellular uptake prole of DOX molecules is likely due to the
rapid demicellization of the DOX-loaded polymeric micelles
under cellular conditions, leading to the rapid release of DOX
from polymeric micelles and its subsequent internalization into
cell nuclei. As shown in Fig. S12,† the average uorescence
intensity in the nucleus is a measure of the amount of DOX that
enters the nucleus. When the cells were incubated with DOX-
HCl, the intensity mean value was approximately 80% aer
24 h incubation, and with the incubation time extended to 48 h,
the average uorescence intensity of HeLa nuclei was higher
than 200%. However, even aer 48 hours, the average uores-
cence intensity of the cells incubated with free DOX remained at
20%. The value obtained for DOX-loaded P3 was between these
values for free DOX and DOX-HCl and increased with incuba-
tion time, so that aer 48 h, the uorescence intensity increased
signicantly to 80%. These ndings suggested that DOX-loaded
polymeric micelles may be suitable for stimulus-responsive
nanocarriers.
Conclusions
In summary, we have successfully constructed a novel high-
drug-loading, dual stimulus-responsive drug-release system
based on a rearrangement of borate ester bond and a hydrolysis
of an ester bond. Compared to the common drug control release
systems, P3 exhibits high entrapment efficiency and drug
loading capacity, and the synergetic characteristics of two
stimuli. Such a dramatic loading capacity and synergetic char-
acteristics is anticipated to achieve unprecedented advances in
the development of stimuli-sensitive drug-delivery systems that
are deemed to be a promising vehicle for drug-delivery
nanocarriers.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
This work was nancially supported by the National Natural
Science Foundation of China (No. 21572147 and 21877082).
Notes and references
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