312 Stefano Salmaso et al.
indicate that the internalization process undergoes cell
saturation. ese results were in apparent contradiction
with the flow cytofluorimetry and confocal microscopy
data, which showed that the micelles could be taken up
by all cells regardless the incubation pH. Nevertheless,
both cytofluorimetry and confocal microscopy analysis
clearly indicated that the higher cell uptake of SD-PEG-
DSPE micelles at pH 6.2 as compared to SD-PEG-DSPE
micelles at pH 7.4 or to mPEG-DSPE micelles could be
ascribed to a cell subpopulation, about 6% of the incu-
bated cells, which was significantly active in micelle
payload internalization under acidic conditions. is
unexpected behavior may be due to specific cell cycle
phase associated uptake. Additionally, the confocal
images showed that no fluorescence was associated to
the nucleus while it was located in the membrane and
cytosolic compartment.
e feasibility to use SD-PEG-DSPE micelles for drug
delivery was verified with paclitaxel, a poorly soluble
and unstable anticancer drug. Paclitaxel was efficiently
incorporated into the lipid core of micelles resulting in
dramatic solubility increase, from 0.3 μg/mL (Choa et al.,
2004) to 125 μg/mL. e slow drug release observed in
vitro highlights the high stability of paclitaxel-loaded
system, which is in agreement with the high micelle sta-
bility shown by the CMC value. Actually, the high stabil-
ity of the micelles at pH 7.4 is pre-requisite to avoid the
systemic drug release that may be responsible for unspe-
cific toxicity. On the other hand, the slow drug release
observed in buffer at pH 6.2 suggests that micelles are
stable under acidic conditions too, but the drug may be
released upon tumor cell interaction that is promoted by
the lower pH in this tissue. However, it should be noted
that, although the drug release has been evaluated in
buffer which simulates physiological conditions, a dif-
ferent behavior is expected in blood or the tumor tissue.
erefore additional investigations will be undertaken to
examine the micelle behavior either in ex vivo matrices
or in vivo.
a cell subpopulation as observed by flow cytometry and
confocal analysis.
Conclusions
e data described in this paper show that pH-
sensitive micelles can be obtained by conjugating SD
to amphiphilic molecules which spontaneously self
assemble into colloidal structures. e resulting materi-
als possess the requisites for pharmaceutical applica-
tion, namely biocompatibility, high drug payload, and
stability in the body compartment. Furthermore, the SD
decoration endows micelles that can interact with cells to
provide for selective drug delivery into tumor site.
However, although the results show the potential
applicability of these formulations for tumor targeting
opening interesting perspectives in the development of
these systems for chemotherapy, it was shown that the
attachment of one SD moiety is not per se sufficient to
guarantee extensive drug release into the tumor cells.
Declaration of interest
e authors report no conflicts of interest.
References
Choa YW, Leeb J, Leeb SC, Huhb KM, Parkb K. (2004). Hydrotropic
agents for study of in vitro paclitaxel release from polymeric
micelles. J Control Release, 97, 249–257.
De S, Robinson DH. (2004). Particle size and temperature effect on
the physical stability of PLGA nanospheres and microspheres
containing Bodipy. AAPS PharmSciTech, 5, e53.
Gao Z, Lukyanov AN, Chakilam AR, Torchilin VP. (2003). PEG-PE/
phosphatidylcholine mixed immunomicelles specifically deliver
encapsulated taxol to tumor cells of different origin and promote
their efficient killing. J Drug Target, 11, 87–92.
Gao Z, Lukyanov AN, Singhal A, Torchilin VP. (2002). Diacyllipid-
polymer micelles as nanocarriers for poorly soluble anticancer
drugs. Nano Lett, 2, 979–982.
Gerweck LE, Seetharaman K. (1996). Cellular pH gradient in tumor
versus normal tissue: potential exploitation for the treatment of
cancer. Cancer Res, 56, 1194–1198.
e drug delivery performance of the pH-sensitive
formulation was investigated by paclitaxel-loaded
SD-PEG-DSPE micelles with cells at pH 7.4 and 6.2. e
cytotoxicity profiles showed a slight but significant differ-
ence in paclitaxel delivery obtained with SD-PEG-DSPE
micelles at pH 6.2 as compared to the same formulation
at pH 7.4, indicating higher intracellular paclitaxel deliv-
ery at pH 6.2. Such a selectivity is also supported by the
data obtained with mPEG-DSPE micelles that did not
show any differences between the cytotoxicity profiles
obtained at the two pHs. ese results are once again in
good agreement with the physicochemical and biophar-
maceutical properties of the pH-sensitive micelles previ-
ously discussed. e higher paclitaxel delivery observed
at pH 6.2 as compared to that at pH 7.4 highlights the
pH-sensitive properties of the colloidal carriers showed
with the fluorescence studies. On the other hand, the
only limited increase in cell toxicity observed at pH 6.2
may be ascribed to the restricted micelles interaction to
Hansen MB, Nielsen SE, Berg K. (1989). Re-examination and further
development of a precise and rapid dye method for measuring cell
growth/cell kill. J Immunol Methods, 119, 203–210.
Kang SI, Bae YN. (2001). pH-induced volume-phase transition by
reversible crystal formation. Macromolecules, 34, 8173–8178.
Kang SI, Bae YH. (2002). pH-induced solubility transition of
sulfonamide-based polymers. J Control Release, 80, 145–155.
Kumari A, Yadav SK, Yadav SC. (2010). Biodegradable polymeric
nanoparticles based drug delivery systems. Colloids Surf
Biointerfaces, 75, 1–18.
B
Kwon G, Naito M, Yokoyama M, Okano T, Sakurai Y, Kataoka K. (1993).
Micelles based on AB block copolymers of poly(ethylene oxide)
and poly(beta-benzyl L-aspartate). Langmuir, 9, 945–949.
Lukyanov AN, Gao Z, Mazzola L, Torchilin VP. (2002). Polyethylene
glycol-diacyllipid micelles demonstrate increased acculumation
in subcutaneous tumors in mice. Pharm Res, 19, 1424–1429.
Lukyanov AN, Torchilin VP. (2004). Micelles from lipid derivatives
of water-soluble polymers as delivery systems for poorly soluble
drugs. Adv Drug Deliv Rev, 56, 1273–1289.
Makino K, Kado H, Ohshima H. (2001). Aggregation behavior of
poly(N-isopropylacrylamide) microspheres. Colloids Surf
Biointerfaces, 20, 347–353.
B
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