Polymeric micelles using pseudo-amphiphilic block copolymers and their cellular uptake

Article abstract of DOI:10.1039/c0jm02519d

A PS-b-PMA block copolymer bearing a terminal carboxylic group has been synthesized using 3-(benzylsulfanyl thiocarbonylsulfanyl) propionic acid as RAFT agent. When dispersed in methanol, this block copolymer displays pseudo-amphiphilic behaviour providing micelles which can be suspended in water by osmosis. The carboxylic terminations are fundamental during the self-assembling process contributing to the formation of nanosized monodispersed particles. Furthermore, the anionic corona confers the to micelles stability in aqueous media. The core of the micelles was loaded with Nile Red to achieve the intracellular delivery of a lipophilic substance; this was demonstrated by Confocal Laser Scanning Microscopy. The particles, when loaded with doxorubicin, are able to overcome the intrinsic resistance of LoVo-MDR cancer cells. Further stabilization is given to the micelles by covalent shell crosslinking using triethylene glycol diacrylate. Because of the increased intracellular stability of the crosslinked micelles, they stain, when loaded with Nile Red, the aqueous cytosol instead of the lipophilic compartments. The unimers constituting the micelles were covalently labeled with fluorescein to follow their fate once incorporated into the cells.

Full text of DOI:10.1039/c0jm02519d

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PAPER
Polymeric micelles using pseudo-amphiphilic block copolymers and their
cellular uptake†
a
Massimo Benaglia, Angelo Alberti,a Enzo Spisni,b Alessio Papi,b Emanuele Treossia and Vincenzo Palermoa
*
Received 3rd August 2010, Accepted 9th November 2010
DOI: 10.1039/c0jm02519d
A PS-b-PMA block copolymer bearing a terminal carboxylic group has been synthesized using
3-(benzylsulfanyl thiocarbonylsulfanyl) propionic acid as RAFT agent. When dispersed in methanol,
this block copolymer displays pseudo-amphiphilic behaviour providing micelles which can be
suspended in water by osmosis. The carboxylic terminations are fundamental during the
self-assembling process contributing to the formation of nanosized monodispersed particles.
Furthermore, the anionic corona confers the to micelles stability in aqueous media. The core of the
micelles was loaded with Nile Red to achieve the intracellular delivery of a lipophilic substance; this was
demonstrated by Confocal Laser Scanning Microscopy. The particles, when loaded with doxorubicin,
are able to overcome the intrinsic resistance of LoVo-MDR cancer cells. Further stabilization is given
to the micelles by covalent shell crosslinking using triethylene glycol diacrylate. Because of the
increased intracellular stability of the crosslinked micelles, they stain, when loaded with Nile Red, the
aqueous cytosol instead of the lipophilic compartments. The unimers constituting the micelles were
covalently labeled with fluorescein to follow their fate once incorporated into the cells.
exploited in the synthesis of end-functional polymeric chains8
1
Introduction
and heterotelechelic polymers.9,10 An increasing number of
scientists have focused their attention on the RAFT technique
because it can be simply applied to a conventional radical
polymerization. This provides a very robust metal-free system
capable of producing well defined polymeric architectures in
a controlled way.
Nanotechnology sciences have recently benefited from
‘‘controlled/living radical polymerization’’,1 an extremely
powerful tool that has allowed new unexploited results to be
reached in polymer chemistry. Such techniques made it possible
to combine the versatility of radical polymerization, which is
suitable for a vast variety of differently functionalized mono-
mers, together with a precise control of the molecular weight and
its distribution.2–5 In particular, Atom Transfer Radical Poly-
merization (ATRP) and Radical Addition-Fragmentation chain
Transfer (RAFT) have emerged as the choice procedures for the
design and convenient synthesis of as-yet unexplored macro-
molecular architectures. These procedures allow for numerous
applications in the field of nanostructured functional materials
such as star polymers, bottle brush copolymers and amphiphilic
block copolymers which are capable of self-assembling mainly
into micelles or vesicles.6,7 ATRP and RAFT are also successfully
One of the most promising applications of the RAFT tech-
nique is its use in the preparation of functional polymeric
micelles11 that can be employed for the delivery of therapeutic
agents. This makes it possible to administer lipophilic drugs
while protecting unstable molecules from metabolic deactiva-
tion.12,13 Such characteristics, together with a reduced clearance,
increase the efficacy of drugs that are usually toxic, thus reducing
their effective dose.14 Another key feature of polymeric micelles is
their specific passive targeting to tumor tissues through the
enhanced permeability and retention (EPR) effect.15,16 Active
targeting is obtainable through linkages with monoclonal anti-
bodies,17 aptamers18 or small molecules like biotin8 and folic
acid.19
aISOF - Consiglio Nazionale delle Ricerche, Area della Ricerca, 40129
Bologna, Italy. E-mail: massimo.benaglia@cnr.it; Fax: +390516398349;
Tel: +390516398257
bDipartimento di Biologia Evoluzionistica Sperimentale, Universitaꢀ di
Bologna, 40126 Bologna, Italy
† Electronic supplementary information (ESI) available: 1H-NMR
spectra of 3-(benzylsulfanyl thiocarbonylsulfanyl) propionic acid, PS
macroRAFT and PS-b-PMA block copolymer. UV-Vis spectra of
3-(benzylsulfanyl thiocarbonylsulfanyl) propionic acid, PS-b-PMA
block copolymer and styrene. Fluorescence assays details of fluorescein
micelles labeling. See DOI: 10.1039/c0jm02519d
Once its role as RAFT mediator has ended, the thio-
carbonylthio function, carried by the polymer chains, is easily
convertible through reduction reactions or nucleophilic substi-
tution (by amines) to a thiol group.20,21 Since the thiol func-
tionality is easily oxidizable by atmospheric oxygen causing the
formation of the polymeric disulfane dimer, the nucleophilic
displacement must be carried out in an inert atmosphere. A
recent work reported the nucleophilic displacement of the RAFT
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function by hydrazine achieving a fast reaction and, at the same
time, avoiding the oxidation of the thiol.22 The obtained –SH is
reactive towards a large number of functionalities and can be
exploited in bioconjugation processes via disulfane bridge
formation or for linking with functional molecules bearing
moieties that can undergo mercaptan addition.23,24
mLꢀ1 streptomycin and grown at 37 ꢁC in a humidified atmo-
sphere with 5% CO2. The LoVo cell line was derived from
a metastatic human colon carcinoma and LoVo-MDR was the
derivative MDR clone. THF was distilled from LiAlH4, meth-
anol was distilled after reaction with sodium metal. Dialysis tube
(cutoff 12000–14000 Dalton) was purchased from Medicell
International Ltd.
Polymeric micelles are characterized by a low critical micelle
concentration (CMC), which makes them stable under diluted
conditions. Nevertheless, their high stability may not be suffi-
cient for the in vivo physiological extreme dilution. The polymeric
micelles can be further stabilized by crosslinking, a process that
can involve either the core or the shell. Core crosslinking (CCL)
is a hyperbranching process that, being confined to the core of
the micelles, does not bring complications; on the other hand, it
has the disadvantage of reducing the loading capacity of the
micelles. Shell crosslinked (SCL) micelles have a higher loading
capacity but the hyperbranching process is more delicate due to
the possibility of inter-micellar crosslinking. To prevent such an
inconvenience, triblock copolymers have been used;25 here the
third block is usually poly(ethylene glycol) that, being highly
hydrated, prevents micelle aggregation and more importantly
provides steric protection against inter-micellar crosslinking.
SCL micelles composed of a diblock copolymer have also been
made.26 The second block consists of a statistical block copol-
ymer where the reactive monomeric units, which interacts with
the crosslinker, resides in the inner shell of the assembled micelle,
preventing the inter-micellar crosslinking.
Instrumentation
1H-NMR spectra were recorded at 400 MHz on a Varian
Mercury 400. Polymer molecular weights and their distribution
were determined using a GPC instrument (MSI Concept PU III
and refractive index detector Shodex RI-71) equipped with 2 ꢂ
PL ResiPore 300 ꢂ 7.5mm (200–400 000 Daltons), the columns
were calibrated using polystyrene standards. DLS and Z poten-
tial measurements were taken with a Malvern Instruments -
Zetasizer 3000 HAS. Atomic Force Microscopy measurements
were taken with a Veeco - Multimode IIIA microscope. The
confocal imaging was performed on a Laica TCS SP2 confocal
microscope equipped with an argon/krypton ion laser. Scanning
Electron Microscopy micrographs were taken using a Philips
Model XL 30 scanning electron microscope.
Synthesis of 3-(benzylsulfanyl thiocarbonylsulfanyl) propionic
acid19,31,32
As described by Maysinger,27 the internalization of micelle-
incorporated fluorescent probes into cells has been successfully
followed using Confocal Laser Scanning Microscopy (CLSM),
a technique that is also suitable for studying the subcellular
localization of single molecules.28 Functionalized polymeric
micelles obtained by other techniques have been already used for
the delivery of doxorubicin into cells.29,30
3-Mercapto propionic acid (10 mmol, 0.87 mL) was added to
a solution of sodium hydroxide (20 mmol, 0.8 g) in methanol
(20 mL), then carbon disulfide (50 mmol, 3.08 mL) was added.
The color of the solution immediately turned deep yellow/
orange. After 30 min benzyl bromide (11 mmol, 1.31 mL) was
added and the mixture was stirred for two hours, the solvent was
then removed under vacuum in a rotating evaporator. The solid
residue was suspended in cyclohexane and filtered off. The
filtrate was dissolved in water and neutralized with 1 M HCl
(z10 mL) then extracted with dichloromethane, dehydrated
with Na2SO4 and, after solvent removal, purified by flash
chromatography using toluene/acetic acid (95/5) as eluent. A
This paper describes the formation of stable micelles starting
from a carboxyl terminated PS-b-PMA block copolymer that
shows pseudo-amphiphilic properties when dispersed in meth-
anol. Once assembled the micelles can be suspended in water by
osmosis. The particles were loaded with Nile Red or doxorubicin.
Cell internalization of the carried fluorescent compounds was
followed with CLSM. The micelles were stabilized by SCL using
triethylene glycol diacrylate as crosslinker. The unimers consti-
tuting the micelles were also labeled with fluorescein to follow
their fate once incorporated into the cells.
1
crystalline yellow solid (2.49 g, 91.4%) was obtained. H-NMR
(CDCl3, d): 7.3 (m, 5H; Ar H), 4.6 (s, 2 H; CH2), 3.63 (t, J ¼ 6.8
Hz, 2 H; CH2), 2.85 (t, J ¼ 6.8 Hz, 2 H; CH2).
Synthesis of polystyrene macro-RAFT
A
stock solution composed of 3-(benzylsulfanyl thio-
carbonylsulfanyl) propionic acid (100 mg, 3.7 ꢂ 10ꢀ4 mol), 2,20-
azobis(cyclohexanecarbonitrile) (24.4 mg, 1 ꢂ 10ꢀ4 mol) and
styrene to a volume of 10 mL (9.7 mL, 8.4 ꢂ 10ꢀ2 mol) was
prepared in a volumetric flask then divided in two ampoules
which were degassed with four freeze–thaw cyꢁcles. The ampoules
were placed in a thermostatic bath set at 90 C and left respec-
tively for 8 (a) and 16 (b) hours. Unreacted styrene was removed
under reduced pressure in a rotating evaporator; then the poly-
meric material was dissolved with toluene and evaporated, this
procedure was repeated three times or until complete styrene
depletion. Sample a yielded 2.5 g (55.5%) of polystyrene Mn ¼
6800, Mw/Mn ¼ 1.20. Sample b yielded 4 g (86.9%) of poly-
styrene Mn ¼ 11000, Mw/Mn ¼ 1.08.
Experimental
Materials
3-Mercapto propionic acid, benzyl bromide, 2,20-azobis(cyclo-
hexanecarbonitrile), styrene, 2,20-azobis(2-methylpropionitrile),
methyl acrylate, Nile Red, doxorubicin, hydroxyethyl acrylate,
2,20-azobis(2-methylpropionamidine) dihydrochloride, fluores-
cein diacetate 5-maleimide and morpholine were purchased from
Aldrich. Cell lines: colon cancer cell lines (LoVo) were purchased
from the American Type Culture Collection (Rockville, MD,
USA) and maintained in RPMI 1640 (Sigma, USA), supple-
mented with 10% fetal bovine serum (FBS) (Gibco, Grand
Island, N.Y., USA), 2 mM glutamine, 50 U/mL penicillin, 50 mg
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Synthesis of PS-b-PMA block copolymer
Atomic force microscopy measurements
A solution composed by the polystyrene macro-RAFT b (1.2 g,
1.1 ꢂ 10ꢀ4 mol), AIBN (0.82 mg, 5 ꢂ 10ꢀ6 mol), methyl acrylate
(7 mL, 7.8 ꢂ 10ꢀ2 mol) and benzene to a volume of 10 mL was
placed in an ampoule and degassed with four freeze–thaw cycles,
then placed in a thermostatic bath set at 70 ꢁC for 5.5 h.
Unreacted methyl acrylate was removed under reduced pressure
in a rotating evaporator; then the polymeric material was dis-
solved with chloroform and evaporated, this procedure was
repeated three times or until complete methyl acrylate depletion.
The polymerization yielded 2.8 g (41.2%) of PS-b-PMA block
copolymer GPCMn ¼ 35000, NMRMn ¼ 34000, Mw/Mn ¼ 1.09.
NMR molecular weight was determined by comparing the inte-
grals of the areas between 3.2–4.0 ppm (PMA) and 6.2–7.6 ppm
The methanolic suspension resulting from the crosslinking
process was diluted four times with methanol, then spin-coated
(3000 RPM) on Si (100) substrates covered by native oxide. Prior
to deposition, the substrates were cleaned by SC1 treatment
ꢁ
(NH4OH 25% : H2O2 30% : H2O 1 : 1 : 5 at 70 C) and rinsed
with water. AFM topographical images were recorded in tapping
mode.
Scanning electron microscopy measurements
A methanolic suspension of micelles (SCL-M3) was deposited on
a silica plate which, after solvent evaporation, was plasma gold-
plated.
1
(PS) in a H-NMR experiment.
Apoptosis assay
Colon cancer cell (10.000/cmq) lines were grown on coverslips
and after 24 h of treatment cells were fixed with 4% para-
formaldehyde and, thereafter, permeabilized with Triton X-100
(0.1% in PBS). Cells were stained with 2.5 mg mLꢀ1 DNA dye
Hoechst 33258 in PBS for 30 min at 20 ꢁC and analyzed by
fluorescence microscopy. At 20-fold magnification cells with
condensed or fragmented nuclei were counted on adjacent four
fields of each coverslip for a total of 160–180 cells. The
percentage of cell death was determined through two coverslips
for each condition and compared with controls.
Micelle formation
PS-b-PMA block copolymer (30 mg) was dissolved in 0.5 mL of
THF/NEt3 solution (dry THF 5 mL, NEt3 1.2 mL), then dry
methanol (25 mL) was quickly added with a syringe. A clear
suspension formed. Methanol was replaced by water using
a dialysis tube (cutoff 12000–14000 Dalton). Nile Red (0.2 mg) or
neutralized doxorubicin (0.2 mg) were dissolved together with
the block copolymer when preparing the loaded micelles.
Shell crosslinked micelles (SCL-M)
Cell viability assay
PS-b-PMA block copolymer (30 mg) was dissolved in 0.5 mL of
THF/NEt3 (dry THF 5 mL, NEt31.2 mL) solution, then dry
methanol (25 mL) was quickly added with a syringe (0.2 mg of
Nile Red was dissolved together with the block copolymer when
preparing loaded SCL micelles). A clear suspension formed.
Triethylene glycol diacrylate33 (SCL-M1 1.04 mL, 4.5 ꢂ 10ꢀ6 mol;
SCL-M2 10.4 mL, 4.5 ꢂ 10ꢀ5 mol; SCL-M3 104 mL, 4.5 ꢂ 10ꢀ4
mol), 2-hydroxyethyl acrylate (SCL-M1 0.5 mL, 4.5 ꢂ 10ꢀ6 mol;
SCL-M2 5 mL, 4.5 ꢂ 10ꢀ5 mol; SCL-M3 50 mL, 4.5 ꢂ 10ꢀ4 mol)
and 2,20-azobis(2-methylpropionamidine) dihydrochloride (1 ꢂ
10ꢀ7 mol) were added to the methanolic suspension. The mixture
was degassed by bubbling argon for 20 min, then the sealed vials
were kept at 50 ꢁC for 24 h in an oil bath. An aliquot of 4.5 mL
was taken, methanol was removed and 1 mL of THF was added
to the residue; this solution was used for GPC measurements.
The methanolic suspension was put in a dialysis tube (cutoff
12000–14000 Dalton) and left in deionized water until complete
replacement of methanol with water.
The effect of doxorubicin on cell viability was evaluated by MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay, based on the reduction of the number of metabolically
active cells, and the results were expressed as percentage of the
controls. 3 ꢂ 10ꢀ3 cells/well were seeded into a 96-well plate and
treated with several concentrations of doxorubicin, for 24 h and
72 h, after which 0.5 mg mlꢀ1 of MTT (Sigma, USA) was added
to each well and incubated for 4 h at 37 ꢁC. Following the
incubation, a solution containing 10% Sodium Dodecyl Sulfate
(SDS) and 0.01M HCl was added. After at least 18 h at 37 ꢁC, the
absorbance of each well was measured in a microplate reader at
570 nm. The results were expressed as percentage of the controls.
Confocal microscopy and image processing analysis
Cells (10.000/cmq) were treated with labeled micelles, added to
the culture medium at dilution higher than 1 : 100, and grown on
coverslips for 0.5–12 h. Then, cells were washed five times with
PBS and fixed with 4% paraformaldehyde. After washing, cells
were mounted in glycerol–PBS medium containing 50 mg mlꢀ1
DABCO. The confocal imaging was performed on a confocal
microscope equipped with an argon/krypton ion laser. A green
laser line was used to visualize Nile Red and doxorubicin fluo-
rescence at respectively 535 nm and 545 nm. A blue laser line at
488 nm was used to visualize fluorescein fluorescence. Optical
sections were obtained at increments of 0.25 mm in the Z-axis and
were digitized with a scanning mode format of 1024 ꢂ 1024
pixels. The image processing and the volume rendering were
performed using the Leica TCS software. Negative controls
consisted of samples not incubated with labeled micelles or
Labeling of the micelles with fluorescein
To the methanolic suspension of micelles, obtained as described
above, fluorescein diacetate 5-maleimide (0.5 mg, 9.8 ꢂ 10ꢀ7 mol)
and morpholine (0.24 mL, 2.7 ꢂ 10ꢀ6 mol) were added. The
resulting suspension was degassed by bubbling argon for 20 min.
The sealed ampoule was heated at 50 ꢁC for 16 h. Then, methanol
was replaced by water using a dialysis tube (cutoff 12000–14000
Dalton). The deionized water of the dialysis bath was renewed
until the fluorescence release ceased (about one week). Fluores-
cence assays details are provided with the electronic supple-
mentary information.†
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incubated with unlabeled micelles. To visualise fluorescein-
labeled unimers, cells were treated with labeled micelles for 1 h,
then were washed three times with fresh medium, and grown on
coverslips for an additional 2–11 h. Four coverslips were ana-
lysed by CLSM for each condition tested.
terminated block copolymer is diluted with methanol confirmed
what was previously observed. This phenomenon, which is
characteristic of amphiphilic block copolymers, is observed
despite the fact that both polystyrene and poly(methyl acrylate)
are insoluble in methanol. We suppose that the driving force for
the self-assembling process of PS-b-PMA is due to a different
grade of (poor) solubility in methanol of the polystyrene block
with respect to the poly(methyl acrylate) one, and it is helped also
by the solvated charged heads. Hence, the particles formed are
composed of a PS core and a partially solvated PMA shell. Such
behaviour is consistent with the self-assembly of the polymeric
chains (unimers) and results in the formation of core–shell
particles (micelles). Beside the charged (or not charged) head, the
presence of water in the solvents also influences the process. In
fact, the particles formed in not anhydrous solvents (commercial
grade) by non-deprotonated carboxyl ended unimers are ther-
modynamically unstable and tend to increase their dimensions
from 350 nm to 1.5 mm in 1.5 h. The particles formed by unimers
with deprotonated carboxyl functions in not anhydrous solvents
are thermodynamically more stable, their hydrodynamic diam-
eter is 120 nm and their Z potential is ꢀ15 mV. Micelles formed
by diluting with dry methanol a solution of copolymer 2 in dry
THF containing one equivalent (with respect to the carboxylic
function) of triethylamine are characterized by a much smaller
hydrodynamic diameter, i.e. 20–30 nm. In brief, when the
solvents employed contain traces of water, larger particles are
formed; this is due to a less thermodynamically stable sol–gel
interface. Thus, well defined monodispersed spherical micelles
are obtained only when the acidic groups are deprotonated and
by using dry solvents.
Results and discussion
Synthesis of the block copolymer
The RAFT agent 3-(benzylsulfanyl thiocarbonylsulfanyl) pro-
pionic acid (1, Scheme 1) was synthesized modifying an already
reported procedure.30 This compound was used to control the
radical polymerization of styrene. The process, conducted at
90 ꢁC, is initiated by 2-20-azobis(cyclohexane-carbonitrile).
Carboxyl terminated polystyrene was obtained after a reaction
time of 8 and 16 h. The molecular weight and polydispersity were
respectively Mn ¼ 6800 g molꢀ1, Mw/Mn ¼ 1.20 (a) and Mn ¼
11000 g molꢀ1, Mw/Mn ¼ 1.08 (b). The macro-RAFT b was used
to synthesize the polystyrene-b-poly(methyl acrylate) block
copolymer 2 (Scheme 1), after accurate removal of the residual
styrene. Traces of styrene inhibited the subsequent polymeriza-
tion of methyl acrylate that requires milder conditions than
styrene (lower temperature and higher dilution). Similar behav-
iour has been observed in the synthesis of PS-b-PMA.34 The
polymerization of methyl acrylate, initiated by AIBN, proceeded
at 70 ꢁC for 5.5 h. The molecular weight and polydispersity of the
resulting block copolymer were Mn ¼ 35000 g molꢀ1, Mw/Mn ¼
1.09 from GPC analysis and Mn ¼ 34000 g molꢀ1 from NMR
analysis.
Micelle formation
Stability of the micelles in water. Once small micelles are
obtained by self-assembling of anionic unimers in dry methanol,
they are easily dispersible in water. The negatively charged
corona on the micelles, enhancing the thermodynamic stabili-
zation, causes the formed particles to remain stable even when
the dispersant medium (methanol) is replaced with water by
osmosis. After suspension in deionized water, the micelles were
dispersed in a 10 mM solution of NaHCO3 in the same dialysis
tube. The hydrodynamic diameter of the micelles was found to be
25 nm and the Z potential was ꢀ54 mV. To verify the stability of
the neutralized micelles in water, solutions of acetic acid at
different concentration were added. The addition of 2 mL of
acetic acid 20 mM to 2 mL of the micellar suspension in 10 mM
NaHCO3 causes rapid particle coalescence that makes it
impossible to determine the hydrodynamic diameter by DLS
analysis. The acidic medium shifts to neutrality the acid–base
equilibrium of the carboxylated corona which loses the negative
charge. Therefore, the lost thermodynamic stabilization causes
the rapid particle coagulation. The addition of 2 mL of 10 mM
acetic acid to 2 mL of the micellar suspension in 10 mM NaHCO3
causes slow particle coalescence. Indeed, three hours after
neutralization, their dimension was found to be 160 nm. In this
case, a neutral environment makes the micelle surface partially
ionized due to the weak acidity of the carboxylic groups. In
neutral medium the particles are characterized by a partial
thermodynamic stabilization that slows down the coagulation
process.
Self-assembly in methanol. We casually observed, working on
a different project, the formation of rather stable particles when
diluting with methanol a THF solution of PS100-b-PMA134 block
copolymer. This material was obtained while optimizing the
synthesis of PS-b-PMA-b-PVA triblock copolymer. The switch-
able RAFT agent S-(methyl propion-2-yl) N-methyl N0-(pyridin-
4-yl) carbamodithioate p-toluene sulfonic acid salt34,35 used for
this synthesis provides polymeric chains bearing a positively
charged head. The DLS characterization of the particles, which
were obtained in not dry solvents, gave 280 nm as hydrodynamic
diameter with low polydispersity and +10 mV as Z potential. The
observation of this phenomenon prompted us to use the easily
synthesizable
RAFT
agent
3-(benzylsulfanyl
thio-
carbonylsulfanyl) propionic acid19,31,32 in order to obtain ioniz-
able carboxyl ended polymeric species. The RAFT
polymerization of styrene gave a carboxyl ended PS-macro-
RAFT that, upon reaction with methyl acrylate, provided the
species PS106-b-PMA267-SC(S)S(CH2)2COOH (2, Scheme 1).
The particles obtained when a THF solution of carboxyl
Scheme 1 Synthesis of PS-b-PMA block copolymer.
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Shell-crosslinking
Another key feature of the carboxylic acid terminated unimers
that affords micelles with an anionic corona is the ability to direct
elongation processes that involve the RAFT function only at
the particle interface. This is particularly useful for starting on
the surface of the micelles (and only there) a crosslinking poly-
merization process that involves the bifunctional monomer
triethylene glycol diacrylate, the intercalating monomer
2-hydroxyethyl acrylate and the radical initiator 2,20-azobis(2-
methylpropionamidine) dihydrochloride. Because of electro-
static interactions this initiator settles, in its cationic form, only
in proximity to the micelle surface (Scheme 2) initiating there the
crosslinking polymerization process. To prove the importance of
the electrostatic interactions between the micelles and the initi-
ator, the azo compound 4,40-azobis(4-cyanopentanoic acid) was
instead used to initiate the crosslinking process, and a dramati-
cally different behaviour was observed. In this case, due to the
opposing interactions between the azo compound and the
particles, the initiation stage starts in the sol phase forming
amorphous micro-gel aggregates which, eventually, incorporate
and desegregate the micelles as evidenced by AFM analysis
(Fig. 1a). The charged corona of the micelles also has the func-
tion of hampering inter-micellar crosslinking because of the
electrostatic repulsion of the particles. Fig. 1 shows AFM
topography images of an amorphous microgel incorporating
a few micelles (a) and the well separated monodispersed SCL
micelles (b). The double function of the anionic corona that
constitutes the outer shell of the micelles must be emphasised:
one function is to direct the hyperbranching process only at the
surface of the particles, the other one is to prevent micelle
aggregation by electrostatic repulsion.
Fig. 1 AFM topography images of amorphous microgel (a, z-range 53
nm) and SCL-M1 micelles (b, z-range 31 nm); in c and d are shown the
corresponding height profiles, taken along the white lines.
crosslinking degree is observed with a reaction time of 24 h at 50
ꢁC. Thus, as evidenced by GPC analysis (Fig. 2), for SCL-M1
around 15% of unimers are dimerized, for SCL-M2 around 25%
of chains are dimerized (also a small amount of higher hyper-
branching grade is observed), and for SCL-M3 around 50% of
chains are hyperbranched (of which 50% is still in the dimeric
form). The diameters of the particles in methanol were
determined as 25 nm (SCL-M1), 30 nm (SCL-M2) and 50 nm
(SCL-M3) through DLS measurements. Fig. 3 shows a SEM
micrograph of SCL-M3.
SCL micelles can be suspended in an aqueous phase, as easily
as the uncrosslinked ones, by osmosis. The hydrodynamic
diameter of SCL-M3 micelles in 10 mM NaHCO3 was 50 nm and
the Z potential was ꢀ52 mV. The increased stability of the shell
crosslinked micelles SCL-M3 was tested by adding solutions of
acetic acid at different concentrations to the micelles suspended
in 10 mM NaHCO3. The acidic environment, resulting from the
addition of 2 mL of 20 mM acetic acid to 2 mL of the micellar
suspension in 10 mM NaHCO3, causes particle coalescence but
the dispersion is not destabilized. Indeed, after 16 h the dimen-
sion of the particles was found to be 130 nm. The neutral
The crosslinking degree is governed by the crosslinker amount
and three different crosslinker/polymer molar ratios have been
tested (5/1 SCL-M1; 50/1 SCL-M2; 500/1 SCL-M3). Because of
the dilution of the reagents (macro-RAFT 3.6 ꢂ 10ꢀ5 M) the rate
of the polymerization of the crosslinker is low and an appreciable
Fig. 2 GPC traces of the starting block copolymer (PS-b-PMA) and of
Scheme 2 Micelle shell crosslinking.
the crosslinked block copolymer (SCL micelles).
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Fig. 3 SEM micrograph of SCL-M3.
environment, resulting from the addition of 2 mL of 10 mM
acetic acid to 2 mL of the micellar suspension in 10 mM
NaHCO3, causes a little coalescence of the particles that were
found to be 70 nm after three hours. This behaviour shows the
increased stability of the SCL-M3 micelles compared to the
uncrosslinked ones. This increased stability in neutral or acidic
media is not attributable to thermodynamic stabilization but to
the covalent hyperbranching between the unimers.
Fig. 4 Confocal laser scanning microscopy analysis of human umbilical
venin endothelial (HUVE) cells (a, c) and GN11 mouse neurons (b, d)
treated with Nile Red loaded free micelles (a, b) or with Nile Red loaded
SCL-M1 micelles (c, d).
SCL micelles, even at the lower degree of shell crosslinking (SCL-
M1), showed intracellular resistance and did not desegregate
within 12 h keeping the dye confined in their core. This
confinement also prevents dye depletion by the laser energy as
shown by brighter images provided by cells treated with SCL
micelles (Fig. 4c and d).
Micelle loadability
The micelles obtained with this method can be easily loaded with
lipophilic compounds like fluorescent markers or therapeutic
agents. Nile Red or neutralized doxorubicin were dissolved
together with the block copolymer in the THF solution before
adding methanol. During the self-assembling process, the lipo-
philic environment of the forming micellar core segregates the
lipophilic substances that are not compatible with the dispersant
media. The Nile Red loaded micelles dispersed in methanol were
also crosslinked. The loaded particles, suspended in water by
osmosis, confined their lipophilic contents in the core.
Cellular uptake
Upon methanol replacement with water the micelles were ready
for dilution in cell medium and administration to cultured cells.
The micelles (uncrosslinked and SCL-M1) loaded with Nile Red
were incubated with Human Umbilical Venin Endothelial
(HUVE) cells, with GN11 neurons or with colorectal carcinoma
LoVo cells for times between 30 min and 12 h. After fixation with
paraformaldehyde, the cells were observed with CLSM at 535 nm
excitation. The obtained images of cells (Fig. 4) that have
internalized the micelles indicate the staining of different cellular
compartments according to the kind of micelles used. Cells
treated with uncrosslinked micelles are stained in their lipophilic
domains (e.g. membrane and intracellular organelle membranes),
Nile Red being highly lipophilic. Conversely, the treatment with
SCL micelles leaves Nile Red dispersed in the aqueous cytoplasm
(Fig. 4c and d). Although the time course of micelle internali-
zation shows that the particles undergo a rapid and massive
internalization in all cell types tested, in HUVE and LoVo cells
the maximum fluorescence intensity is reached after 30 min of
incubation, while in GN11 neurons it is reached after 60 min,
indicating different internalization rates in different cell types.
Fig. 5 Confocal laser scanning microscopy analysis of LoVo-MDR cells
treated with free doxorubicin (a) or doxorubicin loaded micelles (b).
Doxorubicin loaded into micelles is capable of inducing similar apoptosis
levels in LoVo and LoVo-MDR cells (c). Apoptosis was evaluated after
24 h exposure to different concentrations of doxorubicin loaded micelles.
Bars represents the mean (ꢃSEM) of three independent experiments,
performed in duplicate. * ¼ p < 0.05 with respect to controls.
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Doxorubicin delivery
fluorescein diacetate 5-maleimide resulting in unimers covalently
bound with the fluorescent dye (Scheme 3). It should be noticed
that the not fluorescent moiety (fluorescein diacetate) is cleaved
by a transesterification process that proceed in methanol and it is
catalyzed by morpholine; this turned the fluorescein moiety
fluorescent. This kind of micelle was administered to LoVo,
HUVE and GN11 cells that, after fixation, were observed with
CLSM at 488 nm excitation. The fluorescein labeled micelles
were promptly incorporated into the cells within one hour
(Fig. 6a) and localized everywhere in the cytosol and in the
nucleus. After 12 h most of the marked unimers were eliminated
by the cells as shown in Fig. 6b. The excretion of fluorescein
labeled unimers will be traced in future in vivo experiments.
Uncrosslinked micelles were loaded with doxorubicin and
administered to doxorubicin resistant cells (LoVo-MDR). The
comparison of the images of cells treated with free doxorubicin
(Fig. 5a) and with doxorubicin loaded micelles (Fig. 5b),
obtained by CLSM at 545 nm excitation, shows that in the
former case the fluorescent drug remains confined in the area
around the cell membrane whereas in the latter the drug is
allocated within the nucleus because of the DNA intercalating
properties of doxorubicin. This behaviour reveals that uncros-
slinked micelles are incorporated into cells, overcoming their
intrinsic resistance to doxorubicin, probably by endocytosis. The
apoptosis induction of doxorubicin loaded micelles in LoVo and
LoVo-MDR cells (Fig. 5c) is almost identical, confirming that
micelles containing doxorubicin bypass the drug efflux due to
MDR phenotype. Moreover, in LoVo cells the LD50 of doxo-
rubicin is 10 times lower when using doxorubicin loaded in
uncrosslinked micelles than free doxorubicin (respectively 3 mM
and 300 nM) directly added to the culture medium, as evaluated
by cell viability assay.
Conclusions
With this work, we exploited many of the RAFT technique
peculiarities ranging from controlled polymerization for
synthesizing functional polymers to post-polymerization conju-
gation reactions that involve the RAFT function. The synthe-
sized functional polymers conjugate self-assembling properties
with the needed post-micellization specific functions. So we
showed that micelles composed of PS-b-PMA block copolymer
can be easily suspended in water after their self-assembly in
methanol. The process is favoured and the nanoparticles are
stabilized by the anionic carboxylate function terminating each
unimer. The anionic corona also has the fundamental function to
direct the process of shell crosslinking only at the interface
between the micelles and the free solution. This prevents the
formation of an amorphous micro-gel and, at the same time,
inter-micellar crosslinking. Free and SCL micelles rapidly cross
the cellular plasma membrane of many cell types, if not all, and
thus can be very efficient vehicles for intracellular delivery of
lipophilic fluorescent probes and drugs. Indeed, free micelles
loaded with doxorubicin act as very efficient drug shuttles that
overcome the intrinsic resistance of LoVo-MDR cells. The SCL
process is capable of stabilizing the micelles in the intracellular
environment, as evidenced by the different cellular compart-
ments stained, and of also increasing the sensitivity of cellular
labeling. The stability conferred to the SCL micelles by covalent
crosslinking has been confirmed by changing the pH of the
dispersant medium. The micelles can also be labeled in their
constituent unimers which were covalently bound with fluores-
cein in order to trace the unimer fate in vitro, and possibly in vivo.
Unimer labeling
To trace the unimer fate after the internalization of micelles into
the cells we labeled the block copolymer chains exploiting the
electrophilic reactivity of the RAFT function. So the trithiocar-
bonate group located in the corona of the crosslinked micelles
(SCL-M3) suspended in degassed methanol was displaced by
morpholine. This leaves a free thiol group which reacts with
Scheme 3 Trithiocarbonate displacement by morpholine followed by
Acknowledgements
mercaptan addition to fluorescein diacetate 5-maleimide.
This work was supported by a grant from MIUR (PRIN
2008MT34AP).
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