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Evaluation of Polymeric Micelles from Brush
Polymer with Poly(ε-caprolactone)-bPoly(ethylene glycol) Side Chains as Drug Carrier
Biomacromolecules 2009, 10, 2169–2174
Introduction
Polymeric micelles self-assembled from amphiphilic copolymers have attracted
significant attention in the fields of biomedical applications. They have been
developed as delivery systems of drug as well as contrast agents in diagnostic
imaging applications.
In an aqueous environment, the hydrophobic component of the copolymer is
expected to segregate into the core of the micelles, while the hydrophilic
component forms the corona or outer shell. The hydrophobic micelle core
serves as a microenvironment for incorporation of various therapeutic
compounds, while the corona can act as a stabilizing interface between the
hydrophobic core and the external medium.
Although various micelles have been developed for in vitro and in vivo studies
and applications, the majority of works have been focused on micelles formed
by intermolecular aggregation of amphiphilic linear block copolymers.
Micelles from polymers with other architectures as drug delivery vectors are
less studied, especially brush-like macromolecules.
However, it has been reported that the architecture of polymer may determine the
static and dynamic stability, morphology, size and size distribution of the
micelles, and further affect the performance of micelles including drug loading
and release rate, even in vivo circulation and distribution.
Cylindrical brush polymers have recently attracted considerable
attention from polymeric chemists.
In this work, we synthesized cylindrical brush polymers PHEMAg-(PCL-b-PEG) with poly(2-hydroxyethyl methacrylate)
(PHEMA) as the backbone and poly(ε-caprolactone)-bpoly(
ethylene glycol) (PCL-b-PEG) amphiphilic block copolymers
as the side chains.
The micelle formation of these polymers was studied, and
doxorubicin (DOX) was used to evaluate the drug loading and
release behavior from the micelles.
The internalization and cytotoxicity of drug-loaded micelles
against A549 human lung carcinoma cells were also
investigated.
Experiments and Results
Experimental
Materials
Different polymers and reagents.
Methods
Preparation of Micelles
Micelles were prepared by a dialysis method.
Briefly, PHEMA-g-(PCL-b-PEG) or PCL-b-PEG (10 mg) was dissolved
in 2 mL of dimethyl sulfoxide (DMSO) and stirred for 2 h at room
temperature.
Then, the polymer solution was added dropwise into 5 mL of ultrapurified
water under vigorous stirring. Two hours later, the solution was
transferred into dialysis membrane tubing and dialyzed for 24 h against
water to remove the organic solvent.
Transmission Electron Microscopy (TEM) TEM
was performed on a transmission electron microscope for the
measurement of micelle morphology and dimensions.
Result
The formation of micellar nanoparticles was
confirmed by TEM observations. Formed micelles
are well-dispersed and display spherical
morphology. They are relatively uniform in size
and the average diameters are approximately 45
nm.
Fluorescence Measurements.
The fluorescent probe method was employed to determine the
critical micellization concentration (CMC) of the micelles
as followed.
A predetermined amount of pyrene solution in acetone was
added into a series of volumetric flasks, and the acetone
was then evaporated completely.
A series of copolymer solutions at different concentrations
ranging from 1.0 × 10-5 to 1.0 mg mL-1 were added to the
flasks, while the concentration of pyrene in each flask was
fixed at a constant value (6.0 × 10-7 mol L-1).
The excitation spectra were recorded at 25 °C at λem of 390
nm.
Result
With increasing the polymer
concentration, several changes in the
fluorescence spectra of pyrene were
observed. The intensity ratio of bands
at 338 and 335 nm (I338/ I335) was
calculated and plotted against the
polymer concentrations, giving a
sigmoid curve as shown in Figure 3.
I338/I335 remained nearly unchanged
at low polymer concentrations and
increased dramatically when the
polymer concentration reached a
certain value, exhibiting the
characteristic of pyrene entirely in a
hydrophobic environment, which is
actually a reflection of micelle
formation.
Preparation of DOX-Loaded Micelles
DOX-loaded micelles were prepared similarly to the
blank micelles. Briefly, 10 mg of each polymer
was dissolved in 2 mL of DMSO, followed by
adding a predetermined amount of DOX· HCl and
two molar equivalents of triethylamine (TEA) and
stirred at room temperature for 2 h. Then, the
mixed solution was added dropwise to 5 mL of
water. After being stirred for an additional 2 h, the
solution was dialyzed against water for 24 h. The
solution was further centrifuged at 3000 g for 3
min to remove free DOX.
Determination of DOX Loading Content (DLC) and Loading
Efficiency (DLE)
To measure the amount of DOX trapped in the micelles, an aliquot of the
DOX-loaded micelle solution was lyophilized and dissolved in DMSO.
The concentration of DOX was measured by HPLC analyses as described
below. The percentages of DLC and DLE were calculated according to
the following equations:
Results
Size and size distributions of DOX-loaded
micelles by DLS measurements revealed that encapsulation of
DOX into micelles increased the diameters of micelles, as
summarized in Table 3.
In vitro Drug Release
DOX-loaded micelle solution was diluted to 1 mg
mL-1 in phosphate buffered saline, and transferred
into a dialysis membrane tubing.
The tubing was immersed in 20 mL of PBS and
shaken at 37 °C. At a predetermined time interval,
the external buffer of the tubing was collected, and
it was replaced by fresh PBS.
The concentration of DOX was determined by
HPLC analyses with a fluorescence detector.
Results
DOX release kinetics was indeed affected by
polymer architectures.
Cell Culture
A549 human lung carcinoma cells (ATCCs) were cultured in suitable
conditions.
Confocal Laser Scanning Microscopy (CLSM)
A549 cells were incubated at 37 °C. Twenty-four hours later, free
DOX (6 µM) and DOX-loaded micelles (final DOX concentration at 6
µM) were added to the cells, and the cells were incubated for 2 h. The
cells were then observed with a Zeiss LSM510 Laser Confocal Scanning
Microscope imaging system.
Results
In the Figure, cells incubated with free DOX showed strong fluorescence
in cell nuclei, while very weak fluorescence was observed in
cytoplasm, indicating DOX molecules entered the cells and rapidly
accumulated in the nuclei.
On the contrary, after 2 h incubation of DOX-loaded brush copolymer
micelles, intense DOX fluorescence was observed in the cytoplasm
rather than in cell nuclei. It implies that DOX loaded brush copolymer
micelles can be effectively internalized by A549 cells.
Cytotoxicity Study
Cytotoxicity of DOX-loaded micelles and free
DOX were measured against A549 cells where
the relative cell viability was calculated.
Results
As shown in Figure 6, DOX-loaded brush polymer micelles
exhibited relatively lower cytotoxicity to A549 cells when
compared with free DOX at the same dose.
Conclusions
The brush polymers self-assembled into spherical micelles with the
diameter less than 100 nm in aqueous solution.
The brush polymer micelles showed enhanced aqueous stability than linear
PCL-b-PEG diblock copolymer with similar structure to the side chains as
determined by fluorescent probe method. When used as drug delivery
carriers, the brush polymer micelles exhibited higher DOX loading
capacity than the micelles from linear PCLb- PEG, and the burst drug
release in the initial period was significantly suppressed.
The micelles can be effectively internalized by A549 cells and slowly
released the encapsulated drug molecules.
They showed less potent but effective cell proliferation inhibition compared
to free DOX.
With these advantages, the brush polymer micelles are potential carriers for
efficient drug delivery.