Multifunctional polymeric micelles for enhanced intracellular delivery of doxorubicin to metastatic cancer cells

Article abstract of DOI:10.1007/s11095-008-9673-5

Purposes. To develop multifunctional RGD-decorated poly(ethylene oxide)-b-poly(ester) based micelles and assess their pH-triggered core degradation and targeted drug release in tumor cells that overexpress RGD receptors. Methods. Novel poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL) based copolymers modified with RGD ligands on PEO and pendent functional groups on PCL, i.e., GRGDS-PEO-b-poly(α-benzylcarboxylate- ε-caprolactone) (GRGDS-PEO-b-PBCL) and GRGDS-PEO-b-poly(α-carboxyl- ε-caprolactone) (GRGDS-PEO-b-PCCL), were synthesized. Chemical conjugation of doxorubicin (DOX) to PCCL core produced GRGDS-PEO-b-P(CL-DOX) micellar conjugates, while GRGDS-PEO-b-PBCL were used to physically encapsulate DOX. For both systems, micellar core degradation, drug release, intracellular drug uptake/disposition, and cytotoxicity against B16F10 metastatic cells were investigated. Results. The PBCL and P(CL-DOX) cores were found resistant to degradation in pH 7.2, but showed 10% and 40% loss in core molecular weight in pH 5.0 within 144 h, respectively. Preferential release of DOX and DOX derivatives from PBCL and P(CL-DOX) cores was noted in pH 5.0, respectively. The GRGDS-modified micelles showed enhanced cellular internalization through endocytosis, increased intracellular DOX release, nuclear localization, and improved cytotoxicity against metastatic B16F10 cells compared to their unmodified counterparts. Conclusions. The results clearly suggest a promise for the development of multifunctional polymeric micelles with RGD ligand decorated shell and endosomal pH-triggered degradable core for selective DOX delivery to metastatic cancer cells.

Full text of DOI:10.1007/s11095-008-9673-5

#
Pharmaceutical Research, Vol. 25, No. 11, November 2008 ( 2008)
DOI: 10.1007/s11095-008-9673-5
Research Paper
Multifunctional Polymeric Micelles for Enhanced Intracellular Delivery
of Doxorubicin to Metastatic Cancer Cells
Xiao-Bing Xiong,1 Abdullah Mahmud,1 Hasan Uludağ,2 and Afsaneh Lavasanifar1,3
Received April 19, 2008; accepted June 19, 2008; published online July 18, 2008
Purposes. To develop multifunctional RGD-decorated poly(ethylene oxide)-b-poly(ester) based micelles
and assess their pH-triggered core degradation and targeted drug release in tumor cells that overexpress
RGD receptors.
Methods. Novel poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL) based copolymers modified
with RGD ligands on PEO and pendent functional groups on PCL, i.e., GRGDS-PEO-b-poly(α-
benzylcarboxylate-ε-caprolactone) (GRGDS-PEO-b-PBCL) and GRGDS-PEO-b-poly(α-carboxyl-ε-cap-
rolactone) (GRGDS-PEO-b-PCCL), were synthesized. Chemical conjugation of doxorubicin (DOX) to
PCCL core produced GRGDS-PEO-b-P(CL-DOX) micellar conjugates, while GRGDS-PEO-b-PBCL
were used to physically encapsulate DOX. For both systems, micellar core degradation, drug release,
intracellular drug uptake/disposition, and cytotoxicity against B16F10 metastatic cells were investigated.
Results. The PBCL and P(CL-DOX) cores were found resistant to degradation in pH 7.2, but showed
10% and 40% loss in core molecular weight in pH 5.0 within 144 h, respectively. Preferential release of
DOX and DOX derivatives from PBCL and P(CL-DOX) cores was noted in pH 5.0, respectively. The
GRGDS-modified micelles showed enhanced cellular internalization through endocytosis, increased
intracellular DOX release, nuclear localization, and improved cytotoxicity against metastatic B16F10
cells compared to their unmodified counterparts.
Conclusions. The results clearly suggest a promise for the development of multifunctional polymeric
micelles with RGD ligand decorated shell and endosomal pH-triggered degradable core for selective
DOX delivery to metastatic cancer cells.
KEY WORDS: block polymer; drug delivery; doxorubicin; micelles; poly(ethylene oxide)-b-poly(ε-
caprolactone); RGD peptides.
INTRODUCTION
flexibility of block copolymer chemistry that permits simulta-
neous modification of the micellar core and shell structure
The effectiveness of chemotherapy in cancer treatment is without one affecting the other. In the context of drug
significantly impaired due to non-selectivity of anticancer targeting, these modifications are aimed at designing poly-
drugs for malignant cells. A growing body of evidence meric micellar systems that can provide optimum disposition
supports a great promise for nano-technology approaches in of the incorporated drug in the biological system, i.e., within
improving the outcome of cancer chemotherapy through the reach of its molecular targets in cancerous tissue and away
tumor-selective drug delivery (1–4). Development of carriers from non-target healthy cells. Taking advantage of the
endowed with tumor targeting functions will enable specific chemical flexibility of core/shell structure has resulted in the
delivery of cytotoxic agent to malignant tissues, thereby development of multifunctional polymeric micelles that bear
increasing their local efficacy, while sparing peripheral tissue multiple targeting functionalities on an individual carrier and
from toxic side effects of the chemotherapeutic agent.
are expected to achieve superior selectivity for the diseased
In recent years polymeric micelles have attracted a lot of tissue (7).
attention as efficient delivery systems for the solubilization
The objective of this study was to develop multifunctional
and tumor-targeted delivery of chemotherapeutic agents after polymeric micelles based on RGD decorated poly(ethylene
systemic administration (5,6). The unique feature that has oxide)-b-poly(ester) block copolymers with potential for selec-
made polymeric micelles attractive in this regard is the tive delivery of doxorubicin (DOX) to metastatic tumor cells.
Our research group has reported on the development of novel
methoxypoly(ethylene oxide)-b-poly(ester) based micelles, i.e.,
methoxypoly(ethylene oxide)-b-poly(α-benzyl-carboxylate-ε-
caprolactone) (MePEO-b-PBCL) and MePEO-b-poly(α-car-
boxyl-ε-caprolactone) (MePEO-b-PCCL), which were used to
incorporate DOX through physical encapsulation or chemical
conjugation, respectively (4,8). To provide selective intracellu-
1 Faculty of Pharmacy and Pharmaceutical Sciences, University of
Alberta, Edmonton, Alberta T6G 2N8, Canada.
2 Department of Chemical & Materials Engineering, Faculty of
Engineering, University of Alberta, Edmonton, AB, Canada.
3 To whom correspondence should be addressed. (e-mail: alavasani
far@pharmacy.ualberta.ca)
#
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0724-8741/08/1100-2555/0
2008 Springer Science + Business Media, LLC

2556
Xiong, Mahmud, Uludag and Lavasanifar
lar delivery of DOX in tumor over healthy cells, in this study, hexane were slowly added at −30°C under vigorous stirring
the shell of PEO-b-P(CL-DOX) and PEO-b-PBCL micelles with continuous argon supply. The solution was cooled to
(containing chemically conjugated and physically encapsulated −78°C and kept stirring for additional 20 min. Freshly distilled
DOX in their core, respectively) was decorated with an ε-caprolactone (30 mmol or 3.42 g) was dissolved in 8 mL of
internalizing antagonist peptide for αVβ3 integrin, i.e., dry THF and added to the above mentioned mixture slowly,
GRGDS. The overexpression of αVβ3 integrins on the surface followed by the addition of benzyl chloroformate (30 mmol,
of tumor endothelial cells and metastatic cancer cells has been 5.1 g). The temperature was allowed to rise to 0°C after 1.5 h
well documented (9–11). The GRGDS-PEO-b-PBCL micellar and the reaction was quenched with 5 ml of saturated
nanocontainers and GRGDS-PEO-b-P(CL-DOX) micellar ammonium chloride solution. The reaction mixture was
drug conjugates were hypothesized to act as multifunctional diluted with water and extracted with ethyl acetate (3×
polymeric micelles, where RGD peptide endows micelles 40 ml). The combined extracts were dried over Na2SO4 and
preferential entrance to the acidic endosomes of metastatic purified by column chromatography using an eluant of 25%
cancer cells, and the poly(ester) core ensures intra-endosomal EtOAc in hexane. Acetal-PEO-b-PBCL was synthesized by
pH triggered core degradation and preferential release of ring opening polymerization of α-benzylcarboxylate-ε-capro-
DOX or active DOX derivatives in those cells. A similar lactone using acetal-PEO as initiator and stannous octoate as
concept was the basis for the development of multifunctional catalyst (16). For comparison, acetal-PEO-b-PCL was also
polymeric micelles composed of folate-PEO-b-poly(aspartic synthesized using the same method.
acid) conjugated to DOX through acid cleavable hydrazone
bonds producing folate-PEO-b-P(Asp)-hyd-DOX by Bae et al. Synthesis of Acetal-PEO-b-P(CL-DOX) Block Copolymer
(12,13). The higher thermodynamic stability reflected by the
lower critical micelle concentration (CMC) of PCL based
Acetal-PEO-b-P(CL-DOX) was prepared from acetal-
micelles versus P(Asp) micelles (4) as well as acid catalyzed PEO-b-PBCL in two steps (Scheme 1A). In the first step,
degradability of the poly(ester) core in comparison to more acetal-PEO-b-poly(α-carboxyl-ε-caprolactone) (acetal-PEO-b-
stable poly(amide) core of P(Asp) are the distinguished PCCL) was obtained by removal of the benzyl groups of
characteristics of the multifunctional polymeric micelles devel- acetal-PEO-b-PBCL in the presence of H2. For this purpose,
oped here.
acetal-PEO-b-PBCL (1 g) was dissolved in 25 ml of THF and
placed into a 100 ml round bottom flask. Then, charcoal-
coated with palladium (300 mg) was dispersed in this solution
and H2 was introduced to the reaction flask. After stirring for
24 h in the presence of H2, the reaction mixture was
centrifuged at 3,000 rpm to remove the catalyst. The superna-
MATERIALS AND METHODS
Materials and Cell Lines
Diisopropyl amine (99%), benzyl chloroformate (tech. tant was collected, condensed under reduced pressure, precip-
95%), sodium (in Kerosin), butyl lithium (Bu-Li) in hexane itated in diethyl ether and washed repeatedly to remove
(2.5 M solution), 3, 3-diethoxy-1-propanol (DEP), naphtha- impurities. The final product was dried under vacuum at room
lene, ethylene oxide (EO) and potassium were purchased temperature for 48 h. In the second step, DOX was conjugated
from Sigma Chemicals (St. Louis, MO, USA). ε-caprolactone to the acetal-PEO-b-PCCL by amide bond formation between
was purchased from Lancaster Synthesis, UK and distilled by the amino group of DOX and the free carboxyl groups on the
calcium hydride upon use. Stannous octoate was purchased PCCL chain using N, N-dicylcohexyl carbodiimide (DCC) and
from MP biomedicals Inc., Germany. Linear GRGDS was N-hydroxysuccinimide (NHS) as the coupling agents. Briefly,
purchased from Bachem (Torrense, CA, USA). Potassium to a solution of acetal-PEO-b-PCCL (50 mg, ∼0.01 mmol) in
naphthalene solution was prepared by conventional method 10 ml of dry THF, DCC and NHS in THF was added, and the
and the concentration was determined by titration (14). Cell reaction mixture was left under constant stirring for 2 h till
culture media RPMI 1640, penicillin-streptomycin, fetal bovine precipitate was formed. The precipitate was removed by
serum, L-glutamine and HEPES buffer solution (1 M) were filtration. The solution of DOX.HCl (10 mg, 0.017 mmol)
purchased form GIBCO, Invitrogen Corp (USA). Doxorubicin and triethylamine (1.74 mg, 0.017 mmol) in methanol (5 ml)
(DOX.HCl) was purchased from Hisun Pharmaceutical Co. was added drop-wise to the polymer solution. The reaction
(Zhejiang, China). All other chemicals were reagent grade. proceeded for another 24 h under stirring at room tempera-
B16F10 Cells were grown as adherent cultures and maintained ture. The resulting solution was centrifuged to remove the
in RPMI 1640 supplemented with 10% fetal bovine serum at precipitate followed by evaporation under vacuum to remove
37°C and 5% CO2. Acetal-PEO was synthesized by anionic the solvents. Methanol (10 ml) was then introduced to dissolve
ring-opening polymerization of ethylene oxide at room tem- the product. The resulting solution was first purified by
perature under argon stream adopting a previously reported Sephadex LH-20 column using methanol as the eluent, and
method with some modifications (15).
then dialyzed (Mw cut off of 3,500 Da, Spectrum Laboratories,
USA) extensively against water to remove free DOX. The
resulted polymeric DOX solution was finally freeze-dried for
further use. The red powder was dissolved in methanol and
Synthesis of Acetal-PEO-b-PBCL Block Copolymer
The functionalized monomer, i.e., α-benzylcarboxylate-ε- evaluated by reversed phase-HPLC (RP-HPLC) to confirm
caprolactone, was synthesized according to the method the absence of unbound free DOX. Reversed phase chroma-
reported elsewhere (4). Briefly, to a solution of 60.0 mmol tography was carried out with a 10 μm C18–125 Ǻ column
(8.4 ml) of dry diisopropylamine in 60 ml of dry THF in a (3.9×300 mm, Waters) using a Waters 625 LC system. Twenty
three neck round bottom flask, 60.0 mmol (24 ml) of BuLi in microliter of the sample were injected in a gradient elution

Intracellular Delivery of Doxorubicin to Metastatic Cancer Cells
2557
A)
block copolymer. The content of conjugated DOX in synthe-
sized polymer, i.e., acetal-PEO-b-P(CL-DOX), was determined
by measuring its absorbance at 485 nm, on the assumption that
molar absorptivity of DOX residue bound to the polymer was
identical to that of free DOX at 485 nm.
O
O
C O
CH2
O
CH3CH2O
CH3CH2O
H
CH
CH2CH2O CH2CH2O
n
α
ε
-benzylcarboxylate-
-caprolactone (ε-BCL)
Acetal-PEO
Preparation of GRGDS-modified Polymeric Micelles
140˚ C, 4 h
stannous octoate
The RGD-peptide was conjugated to the micellar surface
by the functional acetal groups on the micellar shell
(Scheme 1B). Prepared block copolymers, i.e., acetal-PEO-
b-PBCL and acetal-PEO-b-P(CL-DOX), were assembled to
polymeric micelles by dissolving the polymers (20 mg) in
acetone (1 mL) and drop-wise addition (∼1 drop/15 s) of
polymer solutions to doubly distilled water (6 ml) under
moderate stirring at 25°C, followed by the evaporation of
acetone under vacuum. The aqueous solution of polymeric
micelles was then acidified to pH 2.0 with diluted HCl
(0.5 mol/l) and kept stirring for 2 h at room temperature to
produce aldehyde-polymeric micelles. The resulted solution
was then neutralized with NaOH (0.5 mol/l). The micellar
solution was extensively dialyzed (Mw cut-off 3,500) against
water to remove the salt, concentrated by ultrafiltration
with MILLIPORE Centrifugal Filter Device (Mw cut-off
100,000 Da), and freeze-dried. The dry powder was then
dissolved in CDCl3 (40 mg/ml) for 1H NMR analysis. For
GRGDS conjugation, the micellar aqueous solution was
concentrated followed by the addition of an appropriate
volume of concentrated sodium phosphate buffer solution to
obtain a 4 mg/ml polymer concentration (pH=7.0, ionic
strength 0.1 M). GRGDS was added and incubated with
aldehyde-PEO-b-PBCL and aldehyde-PEO-b-P(CL-DOX)
micelles at GRGDS:polymer molar ratio of 1:2 at room
temperature for 2 h under moderate stirring. Subsequently,
NaBH3CN (10 eq.) was added to the polymer to reduce the
Schiff base. After 90 h of reaction, unreacted peptide and
reducing reagent were removed by extensive dialysis against
water. The conjugation efficiency of GRGDS to polymeric
micelles was assessed by a gradient reverse HPLC method as
previously reported (17).
O
C
CH3CH2O
CH3CH2O
CH
CH2CH2O CH2CH2O
CHCH2CH2CH2CH2O
H
m
n
C O CH2
O
Acetal-PEO-b-PBCL
H2/Pd
O
C
CH3CH2O
CH
CHCH2CH2CH2CH2O
COOH
H
m
CH2CH2O CH2CH2O
DOX
n
CH3CH2O
Acetal-PEO-b-PCCL
DCC/NHS
O
C
O
CH3CH2O
CH
CHCH2CH2CH2CH2O
H
m
CH2CH2O CH2CH2O
n
CH3CH2O
C
Acetal-PEO-b-P(CL-DOX)
OH
HN
O
CH3
CH3
O
DOX
O
O
OH
H
O
HO
HO
OH
O
B)
DOX loading
a
Diffusion
pH triggered
GRGDS
conjugation
Micellarization
core degradation
DOX
b
Characterization of Prepared Block Copolymers
and Polymeric Micelles
DOX-HA
Synthesized copolymers were analyzed by gel perme-
ation chromatography (GPC) and 1H NMR. GPC was carried
out at 25°C with an HP instrument equipped with Waters
Styragel HT4 column (Waters Inc., Milford, MA, USA). The
elution pattern was detected at 35°C by refractive index
(PD2000, Percision Detectors, Inc.) and light scattering
detectors (Model 410, Waters Inc). THF was used as eluent
Scheme 1. A Synthetic scheme for the preparation of acetal-PEO-b-
PBCL and acetal-PEO-b-P(CL-DOX) block copolymers; B Models
for the preparation and modes of DOX release from a GRGDS-
PEO-b-PBCL/DOX micelles which contain physically encapsulated
DOX and b GRGDS-PEO-b-P(CL-DOX) micelles which contain
chemically conjugated DOX.
1
at a flow rate of 1.0 ml/min. H NMR spectra were measured
using 0.05% trifluroacetic acid aqueous solution and acetoni-
trile at a flow rate of 1.0 ml/min. The percentage of acetonitrile
in the mobile phase was increased from 15% to 85% within
15 min at a constant rate. The detection was performed at
485 nm using a Waters 486 UV/Vis absorbance detector. The
degree of DOX conjugation was expressed in mol % with
respect to the α-carboxylic-ε-caprolactone residue of acetal-
PEO-b-PCCL, which was estimated from the peak intensity
ratio of the aromatic protons (δ 7.35 ppm) of DOX to that of
methylene (–CH2O–, δ 4.05 ppm) in the PCCL segment of the
by a Bruker Unity-300 1H NMR spectrometer at room
temperature, using CDCl3 or DMSO-d6 as solvent and
tetramethylsilane as internal reference.
Average diameter and size distribution of prepared
micelles were estimated by dynamic light scattering (DLS)
using Malvern Zetasizer 3000 at a polymer concentration of
4 mg/ml in water at 25°C after filtration through 0.45 μm
filters (Fisher Scientific, USA). A change in the fluorescence
excitation spectra of pyrene in the presence of varied

2558
Xiong, Mahmud, Uludag and Lavasanifar
concentrations of block copolymers was used to measure their polymer concentration of 1 mg/ml (above CMC). At different
CMC (18). The viscosity of micellar cores was estimated by time points, 5 ml of incubation medium was withdrawn and
measuring excimer to monomer related peaks (at 373 and extensively dialyzed against water to remove all possible
480 nm, respectively) intensity ratios (Ie/Im) from the degradation products. The remaining polymer in the dialysis
emission spectra of 1,3-(1,1′-dipyrenyl)propane (18).
bag was lyophilized, and redissolved in CDCl3 for calculation
of block copolymer Mn by H NMR analysis. The percentage
1
Physical Encapsulation of DOX in Acetal- and GRGDS-
PEO-b-PBCL Micelles
of poly(ester) block remained in the copolymers of acetal-
PEO-b-PBCL or acetal-PEO-b-P(CL-DOX) at different
degradation time points was estimated from the following
Acetal- or GRGDS-PEO-b-PBCL (10 mg) and DOX equation:
(2 mg) were dissolved in acetone (2 ml) in a glass vial
Remained polyðesterÞð%Þ
followed by the addition of 3.0 equivalent of triethylamine
(TEA). This solution was added drop-wise to distilled water
(12 ml) under moderate stirring at 25°C. The glycosidic amino
group in DOX was deprotonated in the presence of TEA.
The micellar solution was then left stirring overnight, allow-
ing slow evaporation of acetone. Vacuum was then applied to
ensure the removal of residual acetone. DOX-loaded poly-
meric micellar solutions were then dialyzed against a large
quantity of water for 8 h using a pre-swollen semi-permeable
membrane (Spectra/Pro 6, Mw cut-off 3,000, Spectrum,
Houston, USA) to remove unloaded DOX. Average diameter
and size distribution of prepared micelles were estimated by
DLS. The extent of DOX encapsulation in micelles (wt%)
was quantified photometrically at 485 nm after redissolving
the freeze-dried sample in a DMSO-CHCl3 mixture (1:1, v/v).
The extent of DOX incorporation in polymeric micelles in
terms of weight and molar percent of DOX to polymer was
calculated based on the following equations:
Mn of block copolymer after hydrolysis ꢁ Mn of PEO
Mn of block copolymer before hydrolysis ꢁ Mn of PEO
ꢀ 100 %
¼
At the end point of the study (144 h), micellar solution
was lyophilized to a dry powder, redissolved in 1 ml THF,
passed through a 0.4 μm syringe filter and analyzed by GPC
as described under polymer characterization. The fractions
generated from GPC column after injection of block copol-
ymer samples of acetal-PEO-b-P(CL-DOX) incubated in
pH 5.0 for 144 hs were collected and analyzed by LC/MS
(Waters Micromass ZQ 4000, MA, USA). The latter exper-
iment was carried out on acetal-PEO-b-P(CL-DOX) and
acetal-PEO-b-PBCL incubated in PBS (pH 7.4) for 144 h, as
well.
Cellular Uptake of Polymeric Micelles by B16F10 Cells
amount of loaded DOX in mg
weight %ðdrug=polymerÞ ¼
molar %ðdrug=polymerÞ ¼
ꢀ100 %
Flow cytometry studies were conducted to determine the
association of DOX incorporated polymeric micelles by
B16F10 cells. Cells grown in the six-well plate as a monolayer
were incubated with free DOX, GRGDS- and acetal- micelles
of PEO-b-P(CL-DOX) or PEO-b-PBCL containing chemi-
cally or physically incorporated DOX (5 μg equivalent DOX/
mL), respectively, for 4 h at 37°C. Single cell suspension were
prepared by brief treatment with trypsin, washed with PBS,
filtered through 35 μm nylon mesh, and finally examined on a
FACsort™ flow cytometer (Becton-Dickinson Instruments,
Franklin Lakes, NJ, USA). Ten thousand cells were counted
with logarithmic settings. The cell-associated DOX was
excited with an argon laser (488 nm) and the fluorescence
was detected at 560 nm.
Confocal fluorescent microscopy was used to compare
the intracellular uptake of DOX from GRGDS- or acetal-
PEO-b-P(CL-DOX) and GRGDS- or acetal-PEO-b-PBCL
micelles containing physically encapsulated DOX. The intra-
cellular disposition of polymeric micelles in B16F10 cells was
also evaluated. B16F10 cells were grown on coverslips to 50%
confluence and incubated with free DOX and micelles
containing chemically or physically loaded DOX (5 μg
equivalent DOX/ml) diluted in culture medium at 37°C for
4 h, separately. Cells were incubated with LysoTracker blue
DND-22 (50 nM, Molecular Probe, OR, USA) for 2 h for
endosome/lysosome labelling. The cells were then washed
three times with PBS, fixed in paraformaldehyde in PBS for
10 min. For nucleus labelling, fixed cells were washed with
PBS and then incubated with DAPI (Molecular Probes) for
15 min. In the competition test, B16F10 cells were first
amount of copolymer in mg
Molecular weight of polymer
Molecular weight of DOX
ꢀweight %ðdrug=polymerÞ
Drug Release from DOX Loaded Micelles
The in vitro release from micelles was investigated by
dialysis method. The dialysis was conducted in Dulbecco’s
phosphate buffered saline (PBS, pH 7.2), 0.1 M acetate buffer
(pH 5.0) or 10% fetal bovine serum in PBS (pH 7.2). Micellar
solution containing physically encapsulated DOX or chemi-
cally conjugated DOX diluted in the media at initial
concentration of 300–400 μg/ml was placed in a dialysis bag
(Mw cut-off: 3,000 g mol−1, Spectrum Laboratories, USA).
The dialysis bag was immersed in the media at 37NC at ten
times volume of micellar solution inside the dialysis bag. At
certain time intervals, micellar solution in the dialysis bag was
sampled, dissolved in DMSO, centrifuged and subjected to
photometrical assay at 485 nm to determine the residual
DOX content. To calculate the amount of released drug,
DOX remained in the dialysis bag was subtracted from the
initial DOX amount used in the release experiment.
Assessing the Degradation Rate of Poly(ester) Core in Acidic
and Physiological pH
Acetal-PEO-b-P(CL-DOX) and acetal-PEO-b-PBCL
copolymers were incubated in acetate buffer (pH 5.0) at a incubated with excess of free GRGDS (2 mM) for 30 min and

Intracellular Delivery of Doxorubicin to Metastatic Cancer Cells
2559
a b
c
d
e
f
f
g
h
i
j k
then incubated with GRGDS-modified DOX incorporated
micelles. Cells were examined by a confocal microscope (Zeiss
510 LSMNLO, Jena, Germany). The excitation and emission
wavelengths were set at 480 and 540 nm, respectively.
O
CH3CH2O
CH CH2CH2O CH2CH2O
C CHCH2CH2CH2CH2O
H
n
m
CH3CH2O
R
Acetal-PEO-b-PCL
Acetal-PEO-b-PBCL
H
In vitro Cytotoxicity Study
O
n
m
O CH2
C
Cytotoxicity of chemically conjugated or physically
encapsulated DOX in acetal and GRGDS-micelles against
B16F10 cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay. Growth me-
dium RPMI-1640 (100 μl) containing 4,000 cells was placed in
each well in 96-well plates and incubated overnight to allow
cell attachment. Cells were then exposed to serial dilutions of
different formulations at 37°C for 24 or 48 h, followed by the
addition of 20 μl of MTT solution. Three hours later, medium
was aspirated and the precipitated formazan was dissolved in
200 μl of DMSO. Cell viability was determined by measuring
the optical absorbance differences between 570 and 650 nm
using a PowerwaveX340 microplate reader (BIO-TEK Instru-
ments, Inc., Nepean, Ontario, Canada). The mean and
standard deviation of cell viability for each treatment was
determined, converted to the percentage of viable cells
relative to the control. The concentration of drugs required
for 50% growth inhibition (IC50 values) was estimated from a
plot of the percentage of viable cells versus log DOX
concentration for each treatment.
Acetal-PEO-b-PCCL
COOH
R =
O
NH
O
C
r
OH
Acetal-PEO-b-P(CL-DOX)
s
CH3
t
CH3
O
OH O
OH O
O
p
o
HO
n
b,e,f
m
HO
O
k
CDCl3
h,j
g
i
i
a
a
c
Acetal-PEO-b-PCL
Acetal-PEO-b-PBCL
d,j
h
k
n
m
g
c
c
d,j
a
h
Statistical Analysis
i
k
g
Acetal-PEO-b-PCCL
Values are presented as mean standard deviation (SD)
of triple measurements. Statistical significance of differences
was tested using either unpaired Students’ t test or one-way
ANOVA test (SSPS for Windows v.13, Cary, NC, USA).
Differences between means of IC50 were assessed using One
way ANOVA followed by post hoc analysis using Dunnett T3
test. The level of significance was set at α=0.05.
n
a,s
DMSO-d6
h
i
d,j
g
Acetal-PEO-b-P(CL-DOX)
k,t
c
m
o
p,r
ppm
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
Fig. 1. 1H NMR spectra of acetal-PEO-b-PCL, acetal-PEO-b-PBCL,
acetal-PEO-b-PCCL in CDCl3, and acetal-PEO-b-P(CL-DOX) in
DMSO-d6.
RESULTS
molecular weight (2,670 g mol−1), suggesting incomplete
substitution of DOX or possible chain cleavage of poly
(ester) block during conjugation process. The degree of
DOX conjugation on the block copolymer was 11.1%
(molar ratio of DOX to CL unit) as estimated by comparing
the peak intensity of DOX (δ 7.35) to that of PCL-DOX (δ
Preparation and Characterization of Block Copolymers
The 1H NMR spectra of starting and intermediate
polymers in the synthetic process are shown in Fig. 1. In the
1H NMR spectrum of acetal-PEO-b-PBCL in CDCl3, peaks
appeared at δ 1.20 (t), 1.25–2.00 (m), 3.65 (s), 4.05 (t), 4.65 (t),
5.15 (s), 7.35 (s). The yield for the production of acetal-PEO-b-
PBCL from polymerization of α-benzylcarboxylate-ε-
1
4.05) in its H NMR spectrum in DMSO-d6.
Prepared block copolymers self assembled to micelles
and treated with acid so that the acetal groups on the micellar
surface were converted to functional aldehyde groups for
RGD peptide conjugation (Scheme 1B). In the 1H NMR
spectra of the acid treated polymers, the peaks corresponding
to the acetal group (methyl protons of CH3CH2O, δ 1.2 ppm
and methylene protons of OCHCH2, δ 4.6 ppm) disappeared
and the related peak for the aldehyde proton appeared at δ
9.8 ppm. Measurements by GPC revealed Mn values of 5,100,
4,020 and 5,200 g mol−1 for aldehyde-PEO-b-PBCL,
aldehyde-PEO-b-PCCL and aldehyde-PEO-b-P(CL-DOX)
block copolymers, respectively. The polydispersities of block
copolymers were 1.74, 1.40 and 1.37, respectively. The
similarity between the molecular weight and polydispersity
1
caprolactone with acetal-PEO was 92.4%. H NMR spectrum
of reduced block copolymer, acetal-PEO-b-PCCL, in CDCl3
revealed existence of peaks at δ 1.20 (t), 1.25–2.00 (m), 3.65 (s),
4.05 (t), 4.65 (t) and absence of the characteristic benzyl peak
at δ 7.35 ppm. The yield for the preparation of acetal-PEO-b-
PCCL block copolymer from acetal-PEO-b-PBCL was 80.0%.
The 1H NMR of acetal-PEO-b-P(CL-DOX) in DMSO-d6,
on the other hand, produced peaks at δ 1.20 (t), 1.25–2.0 (m),
3.65 (s), 4.05 (t), 4.55 (t), 5.60 (d, 7.35 (s) and the yield for the
production of acetal-PEO-b-P(CL-DOX) from acetal-PEO-b-
PCCL was 73%.
The measured molecular weight of P(CL-DOX) (Mn
∼1,800 g mol−1) is lower than the expected P(CL-DOX)

2560
Xiong, Mahmud, Uludag and Lavasanifar
of aldehyde- and acetal-polymers suggests limited chain
cleavage in the poly(ester) block during 2 h incubation at
pH 2.0. Measurement of the concentration of free GRGDS
remaining after 96 h reaction by HPLC revealed a conjugated
GRGDS/polymer molar ratio of 1:2 at a molar peptide/polymer
feed ratio of 1:2, corresponding to 100% conjugation efficiency.
Preparation and Characterization of Polymeric Micelles
The results of characterization studies on acetal- and
GRGDS-modified PEO-b-PBCL and PEO-b-P(CL-DOX)
micelles with respect to their CMC, particle size, and core
viscosity are summarized in Table I. In comparison to acetal-
PEO-b-PCL, introduction of hydrophobic benzyl groups
resulted in copolymer acetal-PEO-b-PBCL with a lower
CMC, revealing a better thermodynamic stability for the
latter structure. DOX-conjugated copolymers showed signif-
icantly higher CMC than PEO-b-PCL and PEO-b-PBCL due
to the introduction of more hydrophilic DOX and existence
of free carboxyl groups in the core-forming block. Polymeric
micelles under the study showed rigid cores as evidenced by a
low intensity of the excimer peak in the fluorescence emission
spectra of 1,3-(1,1′-dipyrenyl) propane in the presence of
block copolymers at concentrations above their CMC s
(∼1 mg/ml). Micellar cores consisting of PBCL and PCL
were found to be more rigid than P(CL-DOX) cores.
GRGDS-attached polymers displayed similar CMC and Ie/
Im to their acetal-counterparts, indicating the independence
of core properties from shell modification in the polymeric
micellar structure. The average diameter of micelles under
current study was between 48–92 nm and they illustrated no
sign of aggregation within one month of storage at 4°C.
The characteristics of DOX incorporated acetal- and
GRGDS-micelles are shown in Table II. No significant
difference in the level of DOX incorporation between
GRGDS- and acetal-PEO-b-PBCL micelles was observed
pointing to the incorporation of DOX in the PBCL core.
Average diameter of acetal- or GRGDS-PEO-b-PBCL
micelles containing physically encapsulated DOX was lower
than acetal- and GRGDS-PEO-b-P(CL-DOX) micelles. All
the prepared micellar solutions with physically loaded or
chemically conjugated DOX demonstrated good stability and
no sign of aggregation or precipitation within one month of
storage at 4°C.
In vitro DOX Release and Hydrolytic Degradation
of the Micellar Core in Acidic and Physiological pH
Physically encapsulated DOX was released from poly-
meric micelles in a typical two phase profile showing burst
release at initial time points and more sustained release after
6 h incubation in pH 7.2 (Fig. 2A). DOX release from PBCL
containing polymeric micelles was found to be significantly
higher at pH 5.0 compared to pH 7.2 for all time points within
72 h experiment. DOX release from PBCL core was slightly
but not significantly increased in the presence of 10% serum
(P>0.05). No significant difference was observed between
DOX releases from acetal- and GRGDS-PEO-b-PBCL
micelles at pH 7.2. DOX-conjugated micelles didn’t show
detectable DOX release in the used media based on
spectrophotometrical assay.

Intracellular Delivery of Doxorubicin to Metastatic Cancer Cells
2561
Table II. Characteristics of the Polymeric Micelles Containing Physically Loaded and/or Chemically Conjugated DOX (n=3)
DOX incorporation
DOX-loaded micelles
Average diameter (nm)a
PDIa, b
drug/polymer (wt%)
drug/polymer (molar%)
Acetal-PEO-b-PBCL/DOX
GRGDS-PEO-b-PBCL/DOX
Acetal-PEO-b-P(CL-DOX)
GRGDS-PEO-b-P(CL-DOX)
68.4 2.4*
69.6 3.2**
89.4 2.9
91.6 3.5
0.28
0.19
0.18
0.21
3.78 0.05
3.62 0.14
3.64 0.04
3.58 0.06
46.36 0.61
44.47 1.70
36.53 0.40
35.34 0.60
a Estimated by DLS technique
b Polydispersity index (PDI)
c estimated by UV/Vis method
*p<0.01, when compared to acetal-PEO-b-P(CL-DOX)
**p<0.01, when compared to GRGDS-PEO-b-P(CL-DOX)
Instead, under acidic or enzymatic environment such as containing physically incorporated DOX (Fig. 3A-a) or
in endosome, the poly(ester) block of the copolymer can be conjugated DOX (Fig. 3A-b) by B16F10 cells. Micelles of
degraded by hydrolysis and DOX derivative would be GRGDS-PEO-b-PBCL containing physically encapsulated
released as suggested in Scheme 1B. GPC and 1H NMR DOX provided slightly higher DOX intracellular concentra-
were used to confirm the core degradation. The molecular tions than GRGDS-PEO-b-P(CL-DOX) micelles containing
weight loss of PBCL and P(CL-DOX) based on 1H NMR chemically conjugated DOX.
analysis during acidic hydrolysis are shown in Fig. 2B. 1H
The cellular uptake was further studied by confocal
NMR analysis revealed a decrease in the Mn of polyester microscopy (Fig. 3B). Significant increase in the intracellular
block as a result of incubation of P(CL-DOX) and PBCL DOX fluorescent intensity was observed with GRGDS-
core-containing micelles in acetate buffer (pH 5.0), pointing modified micelles containing chemically or physically incor-
to the degradation of the poly(ester) core. The degradation of porated DOX (Fig. 3B-a and c, respectively) compared to
P(CL-DOX) core was much faster than that of PBCL core in their non-RGD counterparts (Fig. 3B-b and d). In the
corresponding polymeric micelles. A 40% decrease in the Mn presence of blocking ligand for competition experiment, the
of acetal-PEO-b-P(CL-DOX) (from 1,850 to 1,200) was cellular uptake of DOX from GRGDS-modified micelles
observed compared to the <10% decrease in the Mn of containing chemically or physically loaded DOX was dramat-
acetal-PEO-b-PBCL (from 3,600 to 3,200) for after 144 h ically reduced and became essentially equivalent to that of
incubation. The hydrolytic products from acetal-PEO-b- the unmodified micelles (data not shown), pointing to the
PBCL and acetal-PEO-b-P(CL-DOX) after 144 h of involvement of receptor mediated endocytosis in the uptake
incubation were further analyzed by GPC (Fig. 2C, D) and of GRGDS modified micelles. In contrast to the predominant
LC/MS. Incubation of acetal-PEO-b-P(CL-DOX) micelles in accumulation of free DOX in the nuclei (Fig. 3B-e), DOX
pH 5.0 after 144 h produced a new predominant peak with an fluorescence for DOX-chemically conjugated micelles was
increased retention time (11.03 min) compared to the original mainly located in the cytoplasm (Fig. 3B-a and b). For
polymer (Fig. 2C). Further analysis of the new peak by LC/ physically encapsulated DOX in PBCL based micelles,
MS revealed the presence of compounds with molecular DOX fluorescence was observed in cytoplasm as well as in
weights of 701.28 and 176.17 g mol−1, which matches the the nuclei (Fig. 3B-c and d). The endocytosis of GRGDS-
molecular weights of the expected degradation products modified micelles by B16F10 cells was demonstrated by
from P(CL-DOX), i.e., 2-DOX-6-hydroxyhexanoic acid LysoTracker staining (Fig. 4). The superimposed images
(DOX-HA) and 2-(4-hydroxybutyl)malonic acid (HBMA), following the co-localization of LysoTracker and DOX
respectively (data not shown). The GPC chromatogram of incorporated micelles (Fig. 4a and b) but not free DOX
acetal-PEO-b-PBCL after incubation also revealed an (Fig. 4c) showed pink spots in cytoplasm, indicating that
increase in the retention time for the polymer and a GRGDS-attached DOX-loaded micelles were taken up from
relatively weaker new peak at ∼11.08 min (Fig. 2D), which the extracellular fluid into the cells by endocytosis and
can be attributed to the degradation products such as 2- entrapped in endosomes/lysosomes compartments of the cells
(benzyloxy)carbonyl-6-hydroxyhexanoic acid or HBMA. In (17,19,20).
contrast, no noticeable degradation was observed after
incubation of acetal-PEO-b-P(CL-DOX) and acetal-PEO-b- In Vitro Cytotoxicity of DOX against B16F10 Cells
PBCL micelles at pH 7.2 for 144 h (inserted panel in Fig. 2C
and D, respectively).
Overall, free DOX was shown to be more effective as
indicated by the lower IC50 values (0.32 and 0.023 μM for 24
and 48 h incubation, respectively) than the polymeric micellar
formulations (IC50 values ranged from 1.22–6.45 and 0.315–
1.36 μM after 24 and 48 h incubation, respectively) (Fig. 5). In
Cellular Uptake and Localization of Acetal- and GRGDS-
polymeric Micellar DOX in B16F10 Cells
The cellular uptake of DOX from different formulations general, chemically conjugated DOX was found less cytotoxic
was studied by flow cytometry (Fig. 3A). As expected, the than physically encapsulated DOX. Modification of the
highest amount of cell-associated fluorescence was observed micelles with GRGDS resulted in improved efficacy for both
with free DOX. Decoration of polymeric micellar surface chemically conjugated and physically loaded DOX in poly-
with GRGDS increased the cellular uptake of micelles meric micelles. GRGDS modification of PEO-b-PBCL

2562
Xiong, Mahmud, Uludag and Lavasanifar
A)
B)
100
90
80
70
60
50
Acetal-PEO-b-PBCL/DOX, pH 5.0
40
30
20
10
0
Acetal-PEO-b-PBCL/DOX in PBS, pH 7.2
GRGDS-PEO-b-PBCL/DOX in PBS, pH 7.2
Acetal-PEO-b-PBCL/DOX in 10% FBS in PBS, pH 7.2
acetal-PEO-b-P(CL-DOX)
acetal-PEO-b-PBCL
0
20
40
60
80
20
40
60
80
100
120
140
160
Time (h)
Time (h)
D)
C)
Fig. 2. Acid-catalyzed hydrolysis of poly(ester) core resulted in drug release from micelles with PBCL core and P(CL-DOX) core. A Drug
release from DOX physically loaded micelles at pH 5.0 and pH 7.2 with or without 10% serum. Acetal-PEO-b-P(CL-DOX) micelles didn’t
show detectable drug release. Each point represents the average of three experiments SD. B The rate of poly(ester) core degradation in
acetate buffer (pH 5.0) based on 1H NMR analysis. C and D GPC analysis of acetal-PEO-b-P(CL-DOX) and acetal-PEO-b-PBCL block
copolymers after 144 h of degradation in acetate buffer (pH 5.0), respectively. The inserted panel shows the GPC plot of corresponded
copolymers after 144 h of incubation in PBS (pH 7.2).
micelles led to 2.6 and 2.0 fold decrease in the average IC50 of and 48 h incubation, respectively. In addition, free DOX plus
physically encapsulated DOX after 24 and 48 h incubation, empty acetal-PEO-b-PBCL or GRGDS-PEO-b-PBCL
respectively. For chemically conjugated DOX, 1.9 and 2.7 fold micelles demonstrated comparable IC50 to that of free DOX
decreases in the average IC50 of DOX was observed as a alone, suggesting these carriers themselves didn’t cause extra
result of micellar surface modification with GRGDS after 24 cytotoxicity to B16F10 cells.

Intracellular Delivery of Doxorubicin to Metastatic Cancer Cells
2563
Fig. 3. Cellular uptake of DOX formulated in micelles. A Flow cytometry showed enhanced association of GRGDS-modified micelles
containing a physically loaded DOX and b chemically conjugated DOX, with B16F10 cells in comparison to acetal micelles after 4 h incubation
at 37°C. Untreated cells served as negative control while free DOX solution was used as positive control. B Confocal fluorescent microscopy
showed enhanced intracellular uptake and nuclear localization of a GRGDS-PEO-b-P(CL-DOX) compared to b acetal-PEO-b-P(CL-DOX);
and c GRGDS-PEO-b-PBCL/DOX micelles compared to d acetal-PEO-b-PBCL/DOX micelles. Free DOX (e) was used as control. Cells were
incubated with DOX formulations (5 μg eq. DOX/ml) for 4 h, washed, fixed in 95% ethanol. Red Doxorubicin fluorescence; blue DAPI
fluorescence. A comparison between chemically conjugated DOX and physically encapsulated DOX (a versus c and b versus d) points to
enhanced nuclear localization of physically encapsulated DOX in comparison to chemically conjugated DOX.
DISCUSSION
ential micellar core degradation and enhanced release of drug or
active drug derivatives, exclusively, in the endosomal pH of cells
The objective of this study was to assess the feasibility of overexpressing αvβ3 integrins. Having this general goal in
multifunctional polymeric micelles containing acid cleavable poly mind, PEO-b-PBCL and PEO-b-P(CL-DOX) micelles con-
(ester) based core structures and, at the same time, decorated taining physically encapsulated and chemically conjugated
with RGD ligands on their surface for enhanced anticancer drug DOX in their poly(ester) core and modified on the surface
delivery to metastatic cancer cells. This specific design was with a model internalizing RGD peptide, i.e., GRGDS were
hypothesized to enhance the selective delivery of incorporated prepared (Scheme 1). The potential of this architecture in
anticancer drug (DOX) to metastatic tumor cells through providing preferential drug release inside metastatic melanoma
integration of two targeting strategies: (1) First, active targeting B16 cells through combination of aforementioned targeting
of RGD modified carrier to the endosme of metastatic tumor cells strategies was investigated. Due to their excellent biodegrad-
that overexpress RGD receptor, i.e., αvβ3 integrins; (2) prefer- ability and biocompatibility, PEO-b-polyesters represent one of

2564
Xiong, Mahmud, Uludag and Lavasanifar
and DOX release in endosomal pH from both PBCL and P
(CL-DOX) related systems. The results of studies on the rate
of PBCL and P(CL-DOX) core degradation in acidic
environments mimicking the pH of endosomal/lysosomal
organelles provided evidence for acid catalyzed hydrolysis of
the poly(ester) core as suggested in Scheme 1B. Overall, in
comparison to PBCL, the P(CL-DOX) core was more stable
in neutral pH to avoid premature drug release, and more
susceptible to degradation specially in pHs that mimic endo-
somal pH (Fig. 2A–D). This might be related to the higher
hydrophobicity and rigidity of PBCL in comparison to P(CL-
DOX) cores, as evidenced by lower CMC and Ie/Im ratios,
respectively (Table I), which restricts the movement of water
required for hydrolysis of the poly(ester) core (22).
Release of physically encapsulated DOX from its carrier
is dependent on both the rate of drug diffusion from micellar
core and core degradation; whereas for conjugated DOX,
core degradation plays a major role in drug release. We
observed a significant release of physically encapsulated
DOX in both physiological and endosomal pHs but not for
DOX conjugated micelles (Fig. 2A). Release of physically
encapsulated DOX was higher in pH 5.0, possibly due to the
higher water solubility of protonated DOX molecule in this
pH, which drives DOX diffusion and release out of its carrier.
A preferential degradation of PBCL core in acidic pH might
have accelerated DOX release from the PBCL core after 24 h
incubation time point leading to a three phasic release profile
for this structure (Fig. 2A). In contrast to physically
encapsulated DOX, no loss of chemically conjugated DOX
Fig. 4. Confocal fluorescent microscopy showed lysosomal/endo-
somal localization of a GRGDS-PEO-b-P(CL-DOX) micelles and b
GRGDS-PEO-b-PBCL/DOX micelles, compared to c free DOX.
Cells were incubated with DOX formulations (5 μg eq DOX/ml) for
4 h, washed, fixed in 95% ethanol. Red Doxorubicin fluorescence;
blue LysoTracker blue fluorescence.
the most promising amphiphilic copolymers for micellar self- from its carrier was detected during the dialysis study in
assembly in drug delivery. Research on cancer-targeted pH 5.0. The observation may reflect production of DOX
polymeric micelles has been greatly advanced in the past derivatives with either molecular weights larger than the
decade (7). However, the potential for simultaneous modifica- molecular weight cut-off of the dialysis membrane or very low
tion of the PEO block and polyester block and the application water solubility (e.g., DOX-HA) disrupting sink conditions in
of multifunctional PEO-b-poly(ester) based micelles for cancer the release experiment. Noticeably, both the drug release and
targeting has not been explored sufficiently. To the best of our degradation data showed that DOX conjugated micelles are
knowledge, this is the first report on the successful preparation more stable at pH 7.2 compared to DOX physically loaded
of PEO-b-polyester micelles simultaneously bearing functional micelles (Fig. 2A, C), therefore a greater change in the
groups on the PEO shell and several reactive side groups on pharmacokinetics and tumor-targeted delivery of DOX after
the polyester core and their application as multifunctional
polymeric micelles with controlled core degradation and drug
release properties for anticancer drug targeting.
Successful synthesis of GRGDS decorated PEO-b-PBCL
and PEO-b-P(CL-DOX) copolymers provided evidence for the
feasibility of acetal installation on the PEO end and Schiff base
chemistry in the preparation of multifunctionalized PEO-b-poly
(ester) based copolymers. This synthetic approach has previous-
ly been utilized to modify the surface of conventional PEO-b-
poly(ester) micelles such as PEO-b-poly(D, L-lactide) and PEO-
b-PCL micelles (17,21). Scission of the polyester core could
happen but would be minimized since the hydrophobic micellar
core is segregated from the aqueous environment during
conversion of acetal-ended micelles to aldehyde-ended micelles
under treatment of pH 2.0 for 2 h. However, due to the
relatively more hydrophiphilic core of P(CL-DOX) compared to
PBCL core, a drop in the molecular weight was observed for P
(CL-DOX) core (from 1,850 to 1,700 g.mol−1) but not for PBCL
core (from 3,060 to 3,040 g mol−1), which is consistent with the
results of core degradation under pH 5.0 (Fig. 2B).
Fig. 5. Cytotoxicity of various DOX formulations against B16-F10
cells by MTT assay. Each bar represents the average IC50 value of
The first part of our assessments on this system provided
DOX, estimated from a plot of the percentage of viable cells versus
evidence for preferential degradation of the poly(ester) core log DOX concentration, from three independent experiments SD.

Intracellular Delivery of Doxorubicin to Metastatic Cancer Cells
2565
systemic administration of GRGDS-PEO-b-P(CL-DOX) con-
The results of cytotoxicity studies agreed well with those
jugates compared to physically encapsulated DOX in of cell uptake, intracellular localization and biodegradation
GRGDS-PEO-b-PBCL micelles is envisioned (23). studies. For instance, consistent with its lower cellular uptake
In the second part, the experiments were designed to and nuclear localization, chemically conjugated DOX dem-
investigate the potential benefit of RGD modification of onstrated less cytotoxicity against B16F10 cells in comparison
PEO-b-poly(ester) micelles for DOX delivery to B16 cells. to physically encapsulated DOX (Fig. 5). Previous studies on
RGD-containing peptides are known to serve as recognition the in vitro efficacy of polymer-DOX conjugates have shown
motifs for multiple integrin ligands (24,25) and have been similar trend (32–34). By GRGDS modification, the cellular
extensively utilized for drug targeting to tumor endothelial uptake, nuclear localization and cytotoxicity of chemically
cells or metastatic tumor cells (26–29). The B16F10 murine conjugated DOX were improved. In addition, decoration of
cells are highly metastatic melanoma cells and overexpress polymeric micelles with GRGDS appeared more efficient in
multiple integrin-related receptors on their surface (30,31). improving the cytotoxicity of conjugated DOX at longer
Therefore, our observation on enhanced uptake of DOX as incubation times (48 h), while this strategy seem to provide
part of GRGDS decorated micelles compared to their acetal better cytotoxic effects for the physically encapsulated DOX
counterparts by B16F10 cells is not surprising (Fig. 3). The at earlier time points (24 h). This is due to the involvement of
cellular uptake for the GRGDS micelles led to their pH triggered diffusion of physically encapsulated DOX,
localization in endosomes where more rapid release of which enhances DOX release from micelles containing PBCL
DOX from its multifunctional polymeric micellar carriers core at early time points after endosomal localization. For
was expected (Fig. 4). Incubation of B16F10 cells with chemically conjugated DOX, release of DOX derivatives will
GRGDS decorated micelles also resulted in higher levels of depend on degradation of poly(ester) core at endosomal pH,
DOX in cell nucleus for both PEO-b-PBCL and PEO-b-P which is a slower process than diffusion. Besides, endosomal pH
(CL-DOX) micelles (Fig. 3B-a and c) compared to their triggered degradation of P(CL-DOX) core will leads to the
acetal-counterparts (Fig. 3B-b and d). The observation may release of DOX-HA derivatives that may undergo further
be attributed to the rapid uptake and localization of GRGDS- degradation of the amide linkage between DOX and poly(ester)
decorated micelles in endosome/lysosomes of B16F10 cells, backbone (which is an even slower process) to provide free
followed by facilitated core degradation and preferential DOX. Better results in terms of cytotoxicity for RGD decorated
release of DOX or DOX derivatives in endosomal pH, PEO-b-poly(ester) based DOX conjugates may be expected if
subsequent drug transfer to cytoplasm and partitioning DOX is attached to the PCCL backbone through an acid
through the nuclear membrane. Although the extracellular cleavable hydrazone bond instead of more stable amide
DOX leakage from the micelles may partly be responsible for linkage. On balance, a better therapeutic index for RGD-
the nuclear distribution of physically encapsulated DOX modified PEO-b-P(CL-DOX) micellar conjugates compared to
(Fig. 2A), the difference between the distribution pattern of RGD-PEO-b-PBCL micelles containing physically encapsulat-
physically encapsulated DOX and free DOX (Fig. 3B-b and d ed DOX in biological system is envisioned since the former is
versus e) clearly shows this mechanism not to be exclusively expected to prevent premature drug leakage from the carrier
responsible for the interaction of encapsulated DOX and in a biological system and is more susceptible to changes of pH
cells. Perhaps, part of encapsulated DOX will remain inside for triggered drug release. To test this hypothesis, in vivo
the micelles, get released inside the cells after micellar uptake studies to evaluate the RGD-modified micelles containing
and gradually penetrate to the cell nucleus.
chemically and physically incorporated DOX are undergoing
We also weighed on the potential significance of RGD in our lab. The results of in vivo studies should also shed light
modification in systems containing physically encapsulated on the efficacy of prepared multifunctional polymeric micelles
DOX over those with chemically conjugated DOX. Overall, for selective drug delivery to integrin overexpressing cells.
RGD modification appeared to affect the cellular internali-
zation and intracellular distribution of chemically conjugated
DOX to higher extent (Figs. 3 and 4). Without RGD
modification, cellular uptake, endosomal or nuclear localization
was either low or undetectable within 4 h of incubation.
Modification of micelles with RGD ligand drastically changed
this pattern. Conjugated DOX in RGD modified micelles was
mainly localized in the endosomes and was also detected in the
nucleus within 4 h. Cellular uptake of DOX physically loaded in
either acetal- or GRGDS- decorated PEO-b-PBCL micelles
was closer to that of free DOX when compared to chemically
conjugated DOX reflecting release of physically encapsulated
DOX from its carrier through diffusion into the extracellular
space at physiological pH as expected and subsequent parti-
CONCLUSION
The results of this study shows a great potential for RGD
modified PEO-b-poly(ester) based micellar DOX conjugates
and containers for pH triggered enhanced intracellular drug
delivery to metastatic tumor cells. Final conclusion on the
success of the present multifunctional polymeric micellar drug
conjugates and containers and possible superiority of one
over the other in enhancing the selective delivery of
encapsulated drug can only be made only by in vivo studies.
tioning of released DOX to intracellular environment through ACKNOWLEDGEMENTS
cell membrane diffusion. This was also evident from the results
of cell localization experiments, where lower endosomal
This study was supported by the Natural Sciences and
localization (Fig. 3) and higher nuclear accumulation (Fig. 4) Engineering Council of Canada (NSERC) grant Nos.
was observed for physically encapsulated DOX compared to G121210926 and G121220086. AM was supported by Rx
chemically conjugated DOX in polymeric micelles.
and D HRF/CIHR graduate student research scholarship.

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