Shell Cross-Linked Polymeric Micelles as Camptothecin Nanocarriers for Anti-HCV Therapy

Article abstract of DOI:10.1002/mabi.201500094

A suitable carrier for camptothecin to act as therapy against the hepatitis C virus is presented. The carrier relies on an amphiphilic hybrid dendritic-linear-dendritic block copolymer, derived from pluronic F127 and bis-MPA dendrons, that forms micelles

Full text of DOI:10.1002/mabi.201500094

Full Paper
Shell Cross-Linked Polymeric Micelles as
Camptothecin Nanocarriers for Anti-HCV
Therapya
ꢀ
ꢀ
ꢀ
Isabel Jimenez-Pardo, Rebeca Gonzalez-Pastor, Alexandre Lancelot,
ꢀ
Rafael Claveria-Gimeno, Adrian Velazquez-Campoy, Olga Abian,
M. Blanca Ros,* Teresa Sierra*
A suitable carrier for camptothecin to act as therapy against the hepatitis C virus is presented.
The carrier relies on an amphiphilic hybrid dendritic–linear–dendritic block copolymer,
derived from pluronic F127 and bis-MPA dendrons, that forms micelles in aqueous solution.
The dendrons admit the incorporation of multiple
photoreactive groups that allow the clean and
effective preparation of covalently cross-linked
polymeric micelles (CLPM), susceptible of loading
hydrophilic and lipophilic molecules. Cell-uptake
experiments using a newly designed fluorophore,
derived from rhodamine B, demonstrate that the
carrier favors the accumulation of its cargo within
the cell. Furthermore, loaded with camptothecin, it
is efficient in fighting against the hepatitis C virus
while shows lower cytotoxicity than the free drug.
1. Introduction
micelles (CLPM), formed by amphiphilic block copoly-
mers, have been successful for biomedical applications.[2]
First, they can fulfill those general requirements for drug
delivery systems: water solubility, low toxicity, to
Among the various nanocarriers described and assayed as
for drug delivery systems,[1] cross-linked polymeric
ꢀ
Dr. I. Jimenez-Pardo, Prof. M. B. Ros, Dr. T. Sierra
A. Lancelot
ꢀ
ꢀ
Departamento de Qu´ımica Organica, Instituto de Ciencia de
Departamento de Qu´ımica Organica, Instituto de Nanociencia de
ꢀ
ꢀ
Materiales de Aragon (ICMA) - Universidad de Zaragoza-CSIC,
Aragon, Universidad de Zaragoza, 50018, Zaragoza, Spain
50009, Zaragoza, Spain
E-mail: bros@unizar.es
E-mail: tsierra@unizar.es
R. Claveria-Gimeno, Dr. O. Abian
ꢀ
ꢀ
ꢀ
ꢀ
Centro de Investigacion Biomedica en Red en el Area Tematica de
ꢀ
Enfermedades Hepaticas y Digestivas (CIBERehd), Barcelona,
ꢀ
ꢀ
R. Gonzalez-Pastor, R. Claveria-Gimeno, Dr. A. Velazquez-Campoy,
Spain
ꢀ
Dr. O. Abian
R. Claveria-Gimeno, Dr. A. Velazquez-Campoy, Dr. O. Abian
Instituto de Investigaciones Sanitarias de Aragon (IIS-Aragon),
50009, Zaragoza, Spain
Institute of Biocomputation and Physics of Complex Systems
(BIFI), Joint Unit IQFR-CSIC-BIFI, Universidad de Zaragoza, 50018,
Zaragoza, Spain
ꢀ
R. Gonzalez-Pastor, R. Claveria-Gimeno, Dr. O. Abian
ꢀ
Instituto de Ciencias de la Salud (IACS), 50009, Zaragoza, Spain
Dr. A. Velazquez-Campoy
ꢀ
Fundacion ARAID, Government of Aragon, 50018, Zaragoza, Spain
aSupporting Information is available from the Wiley Online Library or
from the author.
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increase the stability of the drug inside
the living organisms, to facilitate cel-
lular uptake compared to free drug, and
to produce its controlled release at a
specific location. Second, the amphi-
philic nature of the constituent polymer
results in a hydrophobic core and a
hydrophilic shell that allow encapsula-
tion of both types of drug. Third, these
nanoparticles offer further stability
under high dilution conditions, below
the critical micellar concentration, com-
pared to other polymeric micelles.
Indeed, cross-linking avoids its disinte-
gration in the bloodstream and the
release of the drug before reaching the
target cell. Particularly, fixation of the
micelle structure by light-induced cova-
lent cross-linking, mostly employing
acrylate reactive groups,[3] represents
Figure 1. Chemical structure of the macromer F127-Ac-8. Schematic representation of
micelle formation, shell-photocross-linking, and CPT loading.
a
clean and effective procedure to
prepare stable polymer micelles that
can hold either water-soluble and non-water soluble
molecules and transport them through the bloodstream.
The features of nanocarriers, in general, and poly-
meric micelles, in particular, have been succesfully
leveraged for anticancer therapies,[4] but are also
attractive, and not extensively applied, for the develop-
ment of systems to treat viral diseases.[5] In this respect,
the lineal moiety, Pluronic F127 offers the ability to self-
arrange in aqueous solutions forming micelles, which have
proven to be excellent candidates for the physical
encapsulation of hydrophobic drugs,[13] with enhanced
drug transport across cellular barriers.[14] This lineal block
copolymer is functionalized at both ends with dendritic
blocks. The characteristic multivalency of dendritic blocks
affords the possibility of multifunctionalization with
photoreactive groups in the periphery to permit micelle
shell-photocross-linking. Dendritic blocks are based on 2,2-
bis(hydroxymethyl) propionic acid (bis-MPA) that provides
a biocompatible and biodegradable scaffold.[15] In vitro cell-
experiments have demonstrated that these nanoparticles
permit the accumulation of their cargo within the cell using
rhodamine B as a fluorescent probe. To further confirm cell
internalization of the polymeric micelles, a new rhodamine
Bderivativewithpoorwatersolubilityhasbeenpreparedto
prevent fast release during the cell-internalization experi-
ment. Finally, the potential of these nanoparticles as drug
carriers for anti-HCV therapy has been studied with the
poorly water-soluble drug CPT demonstrating that non-
toxic CPT-loaded systems display similar anti-HCV activity
than free, and more toxic, CPT.
camptothecin (CPT),
a
poorly water-soluble drug
extracted in 1966 from the plant Camptotheca acumi-
nate, which exhibits remarkable anticancer activity
against different types of cancer,[6] has been recently
described as a potent antiviral agent for hepatitis C
virus (HCV).[7] Indeed, a novel high throughput screen-
ing (HTS) strategy for identifying potential antivirals led
to the identification of this new potential pharmaco-
logical use of CPT.[8] However, CPT has some drawbacks
that limit its applications, i.e., very low water solubil-
ity[9] and chemical instability due to hydrolysis of its
lactone ring under physiological conditions. To over-
come this issue, different strategies have been adopted
by researchers and several carriers for CPT have been
described.[10] Nevertheless, none of them have been
explored for their potential for antiviral therapies.
We herein present a new shell CLPM that shows high
loading capacity for camptothecin while maintaining its
antiviral activity and decreasing toxic effects.
2. Results and Discussion
Based on our previous results about effective nano-
carriers for antiviral drugs,[11] we have selected a hybrid
dendritic–linear–dendritic block copolymer (HDLDBC)
structure[12] for the design of a suitable amphiphilic
macromer to form polymeric micelles with a hydrophobic
core and a hydrophilic cross-linkable shell (Figure 1). As for
2.1. Synthesis and Characterization of the
Amphiphilic HDLDBC Macromer
The macromer F127-Ac-8 was synthesized by divergent
growing of second-generation bis-MPA dendrons at both
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Figure 2. (a) Synthesis of F127-Ac-8. Reagents and reaction conditions: (i) benzylidene-2,2-bis(oxymethyl) propionic anhydride, DMAP,
dichloromethane; (ii) Pd/C/H2, ethyl acetate; (iii) acryloyl chloride, TEA, dichloromethane. (b) 1H-NMR spectra of F127-Ac-8 in CDCl3, 300 MHz.
(c) CMC calculation: intensities ratio I335/I332 versus log concentration (log C) of F127-Ac-8.
ends of commercial Pluronic F127 (Figure 2a). The synthetic
pathway consists of five steps that provide each inter-
mediate with high yields after easy purification processes
when needed, i.e., F127-Bn-2, F127-Bn-4, and F127-Ac-8
were precipitated with cold diethyl ether from the reaction
mixture, and F127-OH-4 and F127-OH-8 were used as
obtained without further purification.
and correct integration of the signals corresponding to the
protons of the acrylate group, labeled as q, q0, and r in
Figure 2b.
Finally, theabilityoftheamphiphilicmacromerF127-Ac-
8 to self-aggregate in water so as to form micelles was
determined by the pyrene method. This method is based on
the displacement of the excitation maximum of pyrene
from lmax ¼ 332 to 335 nm when it is in a hydrophilic or a
hydrophobic environment, respectively. Once formed the
aggregates, pyrene locates in the hydrophobic core of the
micelles and hence the intensity of the excitation
maximum at 335 nm (I335) increases in detriment of the
band at 332 nm (I332). The representation of the I335/I332
intensity ratio versus logarithm of the concentration of
pyrene leads to a curve, where CMC is determined by
calculating the intersection of the two straight lines.
In order to obtain the corresponding curve, F127-Ac-8
solutions were prepared at 0.001, 0.01, 0.1, 0.5, 1, 3, 5, and
10 mg ꢀ mLꢁ1. Then, a similar amount of pyrene was added
in each sample, with a final pyrene concentration of
6 ꢂ 10ꢁ8 M. The excitation spectrum of the samples was
The terminal hydroxyl groups of Pluronic were esterified
using the benzyl-protected anhydride derived from 2,2-bis
(hydroxymethyl) propionic acid. This acylating agent was
used in each step to grow the generation of the dendron
following a debenzylation reaction to deprotect the
terminal hydroxyl groups of the bis-MPA dendron, as
described by Frechet and co-workers.[16] In the final stage,
ꢀ
the terminal hydroxyl groups of both dendritic blocks were
functionalized with acryloyl chloride in the presence of
triethylamine. The final compound was fully characterized
by 1H-NMR, 13C-NMR, IR, and MALDI-TOF. The complete
conversion of the octahydroxy intermediate, F127-OH-8,
into the octa-acrylate derivative, F127-Ac-8, was assessed
1
by H-NMR, Figure 2b (see also ESI), with the appearance
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2.3. Encapsulation of Fluorescent Probes and
Cell-Uptake Studies
For cell-uptake studies of the F127-Ac-8 CLPMs, water-
soluble rhodamine B [Rho-B] (Figure 4a) was first used as a
fluorescent probe. The encapsulation of Rho-B into the
CLPM was carried out by the solvent evaporation method,
using ethanolasthevolatile solvent. Rho-B wasdissolved in
ethanol at 0.15 mg ꢀ mLꢁ1 concentration. A 0.15 mg ꢀ mLꢁ1
solution of Rho-B was added over CLPMs F127-Ac-8 solution
at a feed ratio of 0.15 mg of Rho-B ꢀ mgꢁ1 of CLPM. The
Figure 3. (a) TEM image and (b) SEM image for F127-Ac-8 CLPMs.
measured in the range of 300–350 nm
with lemission ¼ 390 nm. A critical micel-
lar concentration of 0.11 mg ꢀ mLꢁ1 was
determined for F127-Ac-8 (Figure 2c).
2.2. Preparation and Morphological
Characterization of CLPMs
Upon formation of the micelles of F127-
Ac-8, these could be stabilized by intra-
micellar photocross-linking providing
stable polymeric nanoparticles.[3] The
photo-induced polymerization was car-
ried out using 7.7 mg ꢀ mLꢁ1 aqueous
solutions of the macromer containing
1 mg ꢀ mLꢁ1 of Irgacure 2959 as a bio-
compatible photoinitiator. The solution
was irradiated with UV light, l ¼ 365 nm,
at 25 8C for 10 min.[3b,17] Subsequent
dialysis at 4 8C for 72 h, and filtration
through 0.2 mm cellulose acetate filters
yielded a solution of nanoparticles at a
final concentration of1.9 ꢃ 0.1 mg ꢀ mLꢁ1
,
which was used for further studies.
The so-prepared CLPMs were observed
by TEM and SEM, and showed a spherical
morphology (Figure 3).
The size of the photo-polymerized
particles was studied by DLS. A mean
diameter of 127 ꢃ 9 nm (polydispersity:
0.56 ꢃ 0.07) was determined at 25 8C,
whereas a smaller mean diameter of
102 ꢃ 1 nm (polydispersity: 0.36 ꢃ 0.02)
was determined at 37 8C. The observed
size variation with temperature con-
firmed that the nanoparticles display
thermosensitive behavior, provided by
Pluronic-F127. This temperature-depend-
ent feature should also help to validate
these CLPMs as suitable candidates for
responsive drug carriers.[18]
Figure 4. (a) Chemical structure of rhodamine B; (b) and (c) confocal microscopy images
for HeLa cells incubated with Rho-B-loaded nanoparticles. (d) Chemical structure of the
derivative of rhodamine B incorporating a hydrophobic block, Rho-B-2C18; (e) and (f)
confocal microscopy images for HeLa cells incubated with Rho-B-2C18-loaded
nanoparticles. (b) and (e) images correspond to cellular nuclei stained with DAPI in
blue and Rho-B or Rho-B-2C18 appear in pink. (c) and (f) images exhibit also the green
fluorescence due to cytoskeleton stained with Alexa Fluor 488 Phalloidin.
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mixture was incubated at room temperature for 24 h.
Ethanol was evaporated with orbital agitation. After
dialysis for 24 h, the amount of encapsulated Rho-B was
calculated indirectly as the difference between the initial
quantityofRho-BaddedandtheamountofRho-Bpresentin
the dialysis solution, determined by fluorescence. Accord-
ingly, an encapsulation efficiency (EE) of 50.6% was
calculated.[2g]
(Spectra/Por MWCO 2000, Spectrum), causing non-encap-
sulated CPT precipitation. The elimination of non-encapsu-
lated CPT was carried out by filtration with 0.45 mm Teflon
filters. Encapsulated CPT quantification was made directly
by taking 5 mL of filtered solution, which was previously
lyophilized and re-dissolved in a known volume of DMSO.
CPT was quantified by fluorescence emission spectrum
(lmax ¼ 436 nm in DMSO with lexc ¼ 368 nm) by using a
calibration curve in the concentration range 11.2–39.2
mg ꢀ mLꢁ1 in DMSO. The final concentrations of the solution
were 0.42 mg ꢀ mLꢁ1 of CLPMs and 18.8 mg ꢀ mLꢁ1 of CPT.
ThisresultcorrespondstoanEE%of32.7%,[2g] adrugloading
content of 4.5% and a loading capacity of 44.6 mg of
CPT ꢀ mgꢁ1 of F127-Ac-8 CLPMs.
Cell-uptake experiments were performed on HeLa cells.
The cells were incubated with a solution of 0.6 mg ꢀ mLꢁ1
Rho-BloadedCLPMsfor4 hat37 8C.Fortheco-localizationof
Rho-B labeled CLPMs using confocal microscopy, staining of
actin was performed with Alexa Fluor 488 Phalloidin.
Confocal images (Figure 4b and c) suggest that F127-Ac-8
CLPMs allow the accumulation of Rho-B within the cell.
Nevertheless, sinceRho-Bishighlysolubleinwater, itisstill
possible that, in the high dilution conditions of culture
media, Rho-B releases from the nanoparticles and enters
into the cells by simple diffusion induced by a concen-
tration gradient.
The encapsulation process of CPT within the F127-Ac-8
CLPMs was studied by isothermal titration calorimetry, ITC.
Experiments were performed at 25 8C in aqueous media
with 3% of DMSO to increase solubility. 100 mM CPT
solution in the calorimetric cell was titrated with F127-Ac-8
polymer 30 mM solution. Control experiments were
performed under the same experimental conditions. The
heat evolved after each ligand injection was obtained from
the integral of the calorimetric signal (Figure 5).
In order to prevent fast release of the fluorescent probe
from the CLPMs during the experiment time, a newly
designed Rho-B fluorescent probe [Rho-B-2C18], which
contains a hydrophobic block that confers it negligible
solubilityinwater,wasdesigned(Figure4d).Rho-B-2C18 was
synthesized by copper-catalyzed 1,3-dipolar azide–alkyne
cycloaddition between an azide derivative of rhodamine-B
and a first generation bis-MPA dendron functionalized by
esterification of the hydroxyl groups with stearic acid.
The encapsulation of Rho-B-2C18 was carried out using
the same methodology described for Rho-B. The analysis of
The association constant (Ka) and the enthalpy change
(DH) were obtained through non-linear regression of
experimental data to a model considering one class of
ligand-binding sites. The results reflected that CPT encap-
sulation is energetically favored with an association
constant Ka ¼ 1.5 ꢀ 107 Mꢁ1, corresponding to a moderate-
to-high Gibbs energy of interaction (DG ¼ –9.8 kcal ꢀ molꢁ1
)
dominated by the entropic contribution (–TDS ¼ –16.6 kcal
ꢀ molꢁ1), with an unfavorable interaction enthalpy (DH
¼ 6.8 kcal ꢀ molꢁ1), as expected for a predominantly hydro-
phobic interaction between the drug and the hydrophobic
region of the CLPMs. Thus, the F127-Ac-8 CLPM-drug
calorimetric titrations show that CPT interacts specifically
with F127-Ac-8.
the dialysis solution revealed the absence of Rho-B-2C18
,
yielding an EE of 100%, likely due to the enhanced
interactions between its hydrophobic block and the
hydrophobic regions in the CLPM. Cell-uptake experiments
were performed under the same conditions as for Rho-B-
loaded CLPMs. Similarly, confocal images showed accumu-
lation of fluorescence within the cell after 4 h incubation at
37 8C (Figure 4e and f), thus confirming internalization of
nanoparticles into the cells.
2.5. In-vitro Anti-HCV Studies
Once confirmed cell internalization of the CLPMs to deliver
a cargo and the possibility of loading CPT into these
nanoparticles, the next step was to study the efficacy of the
CPT-carrier system as for the inhibition of HCV replicon.
Nevertheless, prior to this study and in order to check the
cytotoxicity of the system, we performed cell viability
studies on HeLa cells for a range of concentrations of free
CPT and F127-Ac-8/CPT CLPMs, close to those used in
subsequent antiviral experiments. Figure 6 represents the
viability of HeLa cells upon incubation for 3 d. The results
show that the cytotoxic effect of free CPT is reduced when
CPT is encapsulated into F127-Ac-8 CLPMs. The CC50 of
encapsulatedCPTis0.32 ꢃ 0.03mM,comparedto0.26 ꢃ 0.03
for free CPT.
2.4. Camptothecin Encapsulation
In order to explore the possibility of these CLPMs to act as
nanocarriers for anti-HCV drugs, we undertook cytotoxicity
and anti-HCV activity experiments based on encapsulated
CPT.
Due to the insolubility of CPT in volatile water-soluble
solvents, the evaporation method could not be employed
for the encapsulation. Instead, a solution (0.15 mg ꢀ mLꢁ1) of
CPT in DMSO was added into the F127-Ac-8 CLPMs solution
(1.9 ꢃ 0.1 mg ꢀ mLꢁ1) until a ratio of 0.15 mg of CPT per mg of
CLPM was reached. The mixture was incubated at room
temperature for 24 h. DMSO was eliminated by dialysis
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time (min)
10 20
100
80
60
40
20
0
100
80
60
40
20
0
0
30
0.08
0.06
0.04
0.02
0.00
-0.02
8.0
0
0,016 0,032 0,06 0,12 0,25 0,5
1
6.0
CPT (µM)
4.0
Figure 7. Inhibition of HCV replicon in Huh5-2 cells. Evaluation of
potency and cytotoxicity in Huh5-2 cells. Cell survival (bars) and
HCV replicon replication rate (lines and symbol) were assessed in
cell culture at increasing compound concentration to determine
CC50 and EC50: free CPT (grey bars and green line) and F127-Ac-8/
CPT CLPMs (red bars and violet line). *UTC: untreated controls.
2.0
0.0
-2.0
0.0
0.1 0.2 0.3 0.4 0.5
Molar Ratio
For inhibition of HCV replicon assays, Huh 5-2 cells were
incubated with a control of free CPT and F127-Ac-8/CPT
CLPMs. Cell viability data for Huh 5-2 and % of viral load
reduction are represented in the same double axis graph
(Figure 7).
Figure 5. Camptothecin/F127-Ac-8 interaction by isothermal
titration calorimetry. Calorimetric titrations were performed by
programming sequential injections of camptothecin solution
(0.035 mg ꢀ mLꢁ1) into F127-Ac-8 solution (0.42mgꢀ mLꢁ1) in the
calorimetric cell. Upper plot shows the thermogram (thermal
power to maintain a zero temperature difference between the
reference and sample cells as a function of time) and lower plot
shows the binding isotherm (normalized heat per injection as a
function of molar ratio). From a simple analysis considering n
identical and independent binding sites for CPT in a given F127-Ac-
Forbothcelltypes(HeLaandHuh5-2), aslightdecreasein
toxicity of F127-Ac-8/CPT CLPMs in comparison to free CPT
was observed (Figure 6 and 7). Also, the anti-HCV activity
was not the result of a cytostatic or cytotoxic effect, since
cell viability remained near 100% at the EC50 values. Non-
loaded F127-Ac-8 CLPMs, at the same conditions, did not
promote any effect in the virus replication cycle or viability
(data not shown). The behavior of free CPT and F127-Ac-8/
CPT CLPMs is very similar, and their EC50 values, i.e.,
0.013 ꢃ 0.002 and 0.032 ꢃ 0.01 mM, respectively, are very
close in nanomolar order. Interestingly, cell viability is
always slightly higher for encapsulated CPT; in particular,
an increase from 40 ꢃ 10 to 50 ꢃ 7% of the initial cell
viability can be observed at 1 mM concentration, when the
viral load has been nearly totally reduced. This would be
specially interesting if CPT is intended as an antiviral agent,
instead of an antitumor drug (its approved pharmaceutical
application based on its high cytotoxicity in mammalian
cells).[19]
8
nanoparticle, the equilibrium association constant (Ka ¼
1.5 ꢀ 107 Mꢁ1) and the interaction enthalpy (DH¼ 6.8 kcal ꢀ molꢁ1
)
could be estimated.
100
F127-Ac-8
80
60
F127-Ac-8/CPT
40
CPT
20
3. Conclusion
0
1
2
3
4
Concentration (µM)
In conclusion, these results open new possibilities for the
molecular design of cross-linked polymeric micelles for
antiviral therapy applications. Particularly, the macromer
F127-Ac-8, with a hybrid dendritic–lineal–dendritic block
copolymer structure provides a molecular design that takes
Figure 6. Cell viability for F127-Ac-8 CLPMs on HeLa cells: free F127-
Ac-8 CLPMs (black), F127-Ac-8/CPT CLPMs (green), and free CPT
(red). Continuous lines are non-linear fittings to the dose–
response equation.
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–CH3), 1.13 (m, 201H, –CH3), 3.37–3.82 (m, ꢄ1 100H, –O–CH2–CH2–
O–), 4.35 (m, 4H, –CH2–CH2–OC(O)–), 4.66 (d, J ¼ 11.6 Hz, 4H, –CH2–
OC(O)–), 5.44 (s, 2H, –CH–Ph), 7.32 (m, 6H, Ar H), 7.42 (m, 4H, Ar H);
13C–NMR (100 MHz, CDCl3, d): 17.3, 17.4, 17.9, 42.4, 64.2, 68.5, 68.6,
69.1, 70.5, 72.9, 73.3, 75.1, 75.3, 75.5, 101.7, 126.2, 128.2, 128.9, 137.9,
173.9; IR (molten polymer over NaCl): n ¼ 2 883 (C–H st), 1 737 (C¼O
st), 1 114 (C_O_C st) ꢀ cmꢁ1; MALDIþ maximum m/z 5050.2 and
13297.2. Anal. calcd. for C621H1220O272: C 57.29, H 9.38; found: C
56.68, H 9.55.
advantage of the amphiphilic nature of Pluronic and the
multivalency provided by the dendritic blocks to achieve
micelle stabilization by photocross-linking and high load-
ing capacity. The cytotoxicity of the drug is reduced when
encapsulated, while the same efficacy of the free drug
against the hepatitis-C virus is maintained. Interestingly,
the multivalency of this dendritic block molecule can be
exploited toward the multiconjugation of the polymeric
micelles with active groups such as targeting moieties,
offering future prospects for this sort of amphiphilic
reactive compounds.
4.2.2. F127-OH-4
F127-Bn-2 (9.5 g, 0.73 mmol) was dissolved in ethyl acetate
(200 mL). Then, Pd/C (10% by weight) was added. After three
vacuum–argon cycles, the reaction mixture was stirred at room
temperature in hydrogen atmosphere overnight. The Pd/C was
filtered off with Celite and the filtrate was evaporated to give a
white solid. Yield: Quant. 1H NMR (300 MHz, CDCl3, d): 1.11 (s, 6H,
–CH3), 1.13 (m, 201H, –CH3), 3.37–3.82 (m, ꢄ1 100H, O–CH2–CH2–
O–), 4.34 (m, 4H, G, –CH2–CH2–OC(O)–); 13C NMR (75 MHz, CDCl3, d):
17.1, 17.3–17.4, 49.5, 63.2, 67.3, 68.7, 70.5, 72.9, 73.3, 75.1, 75.3, 75.5,
175.6; IR (molten polymer over NaCl): n ¼ 3 482 (OH), 2 884 (C–H st),
1 728 (C¼O), 1 112 (C_O_C) ꢀ cmꢁ1; MALDIþ: maximum m/z 5 213.9
and13780.1;Anal. calcd. forC607H1212O272:C56.42,H9.44;found:C
56.04, H 9.98.
4. Experimental Section
4.1. Materials
Pluronic F127 (average Mw ¼ 12 600), acryloyl chloride, 4-
dimethyl-aminopyridine (DMAP), 2,2-bis(hydroxymethyl) pro-
pionic acid (bis-MPA), acryloyl chloride, triethylamine (TEA),
1,3-dicyclohexylcarbodiimide (DCC), benzaldehyde dimethyl ace-
tal, p-toluensulfonic acid monohydrate, 2-hydroxy-1-(4-(hydrox-
yethoxy) phenyl)-2-methyl-1-propanone (Irgacure 2959, I2959),
rhodamine B, CuSO4 ꢀ 5H2O, (l)-ascorbate, and camptothecin were
obtainedfromSigma–Aldrich.PluronicF127wasdriedduring3 hat
100 8Cundervacuumpriortouse. Allsolventswerepurchasedfrom
Sigma–Aldrich. 4-(dimethylamino) pyridinium 4-toluenesulfonate
(DPTS), 6-azido-hexan-1-ol, 2-(but-3-ynoyl)-2-methylpropane-1,3-
4.2.3. F127-Bn-4
F127-OH-4 (4 g, 0.31 mmol) was dissolved in dry dichloro-
methane (15 mL). Benzylidene-2,2-bis(oxymethyl) propionic
anhydride (2.13 g, 4.99 mmol) and DMAP (122 mg, 1 mmol) were
added after dissolution. The reaction was stirred at room
temperature for 72 h. The excess of anhydride was quenched
by adding methanol (6 mL). The mixture was stirred for 7 h and
the crude was precipitated in cold diethyl ether (1 L). The
product was isolated after filtration as white solid. Yield: 95%.
1H NMR (300 MHz, CDCl3, d): 0.93 (s, 12H, –CH3), 1.13 (m, 201H,
–CH3), 1.24 (s, 6H, –CH3), 3.37–3.82 (m, ꢄ1 100H, –O–CH2–CH2–O–
, and –CH2–OC(O)–), 4.11 (m, 4H, –CH2–CH2–OC(O)–), 4.37 (s, 8H,
–CH2–OC(O)–), 4.56 (d, J ¼ 11.5 Hz, 8H, –CH2–OC(O)–), 5.40 (s, 4H,
–CH–Ph), 7.28 (m, 12H, Ar H), 7.38 (m, 8H, Ar H); 13C–NMR
(75 MHz, CDCl3, d): 17.4, 17.6, 17.7, 42.5, 46.7, 64.1, 65.4, 68.5, 70.5,
72.9–73.3, 75.1–75.3–75.5, 101.6, 126.1, 128.0, 128.8, 137.7, 173.1;
IR (molten polymer over NaCl): n ¼ 2 883 (C–H st), 1 741 (C¼O), 1
113 cmꢁ1 (C_O_C). MALDIþ: maximum m/z 5 615.9 and 13 844.4.
Anal. calcd for C655H1260O284: C 57.59, H 9.23; found: C 56.89, H,
9.74.
diyl distearate (HCꢁC-2C18),[11] and tris(benzyltriazolylmethyl)
ꢁ
ꢁ
amine[20] (TBTA)weresynthesizedaccordingtopreviouslyreported
procedures.
ꢁ1
0
́
DMEM (Dulbeccos modified Eagle s medium, 4.5 g ꢀ L
glu-
cose), DPBS (Dulbecco’s phosphate-buffered saline), l-glutamine,
1ꢂ non-essential amino acids, Geneticin (G418), and Alamar Blue
reagent were purchased from Gibco. Penicillin/streptomycin (5
000 U ꢀ mLꢁ1), amphotericin
B
(250 mg ꢀ mLꢁ1), and trypsin
(trypsin–versene 10ꢂ) were obtained from Lonza. Alexa-
Fluor488-labeled-phalloidin and DAPI (40,6-diamidino-2-phenyl-
indole, dilactate) were purchased from Invitrogen. Paraformal-
dehyde (PFA) and bovine serum albumin (BSA) were supplied by
Sigma–Aldrich. Mowiol and saponin from Merck Millipore. Fetal
bovine serum (PAN-Biotech GmbH, Germany). Bright-GloTM
Luciferase Assay System (Promega Corporation), CellTiter 96
AQueous One Solution Cell Proliferation Assay (Promega
Corporation).
4.2. Synthesis and Characterization of F127-Ac-8
4.2.4. F127-OH-8
4.2.1. F127-Bn-2
F127-Bn-4(2.8g, 0.21mmol) was dissolvedinethyl acetate (100mL).
Then, Pd/C (10% by weight) was added. After three vacuum–argon
cycles, the reaction mixture was stirred at room temperature in
hydrogen atmosphere overnight. The Pd/C was filtered off with
Celite and the filtrate was evaporated to give the product as a white
solid. Yield:Quant%. 1H-NMR(400MHz, CDCl3, d):1.05(s,12H,–CH3),
1.13 (m, 201H, –CH3), 1.28 (s, 6H, –CH3), 3.37–3.79 (m, ꢄ1 100H, O–
CH2–CH2–O–), 4.26–4.38 (q, 8H, Dv0 ¼ 35Hz, J ¼ 11.2Hz, –CH2–
OC(O)–), 4.26 (m, 4H, –CH2–CH2–OC(O)–); 13C-RMN (100MHz, CDCl3,
Pluronic F127 (10.0 g, 0.79 mmol) was dissolved in dry dichloro-
methane (20 mL). DMAP (0.12 g, 0.95 mmol) and benzylidene-2,2-
bis(oxymethyl) propionic anhydride (2.0 g, 4.76 mmol) were added.
Themixturewas stirredat room temperature overnight. Theexcess
of anhydride was quenched by adding methanol (6 mL). The
mixture was stirred overnight and the crude was precipitated in
cold diethyl ether (1 L). The product was isolated as a white powder
after filtration. Yield: 95%. 1H-NMR (400 MHz, CDCl3, d): 1.04 (s, 6H,
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d): 17.1, 17.3–17.4, 17.9, 46.4, 49.7, 64.3, 64.9, 67.3–67.4, 68.5–68.8,
70.5, 72.9–73.3, 75.1–75.3–75.5, 172.9, 174.9; IR (molten polymer
over NaCl): n ¼ 3 457 (OH), 2 884 (C–H st), 1 733 (C ¼ O), 1 113 ꢀ cmꢁ1
(C_O_C);MALDIþ:maximumm/z5286.1and13693.8;Anal.calcdfor
observed in 1H NMR (2.26 (s, 2.4H), 7.07 (d, J ¼ 8 Hz, 1.6H), 7.9 (d,
J ¼ 8 Hz, 1.6H), and 13C NMR (21.3, 127.0, 129.7, 139.4, and 158.9)). IR (ATR
mode): n ¼ 2 928 and 2 852 (C–H st), 2 095 (N3), 1 715 (C¼ O), 1 645–1
585 ꢀ cmꢁ1 (C–Car). MS(ESIþ): m/z: [Mþ] calcdforC34H42N5O3 568,32;
found, 568,39. Anal. calcd for C34H42N5O3þ, Cl– / C7H7SO3–: C 66.8, H
6.7, N 9.9, S 3.5; found: C 66.3, H 7.0, N 10.0, and S 3.7.
C627H1244O284: C 56.59, H 9.36, found: C 56.17, H 9.98.
4.2.5. F127-Ac-8
4.3.2. Rho-B-2C18
F127-OH-8 (1.5 g, 0.11 mmol) was dissolved in of dry dichloro-
methane (20 mL). Then, triethylamine (0.55 mL, 3.98 mmol) and p-
methoxyphenol (200 mg, polymerization inhibitor) were added.
The reaction was cooled for 10 min in an ice bath and acryloyl
chloride (0.29 mL, 3.61 mmol) was added dropwise under argon
atmosphere. The mixture was stirred at room temperature for 14 h
in the dark. The solution was passed through a neutral alumina
column and the filtrate was dried with Na2CO3 during 2 h. The
productwaspurifiedbyprecipitationincolddiethylether(250 mL).
The product was obtained after filtration as a white solid. Yield:
80%. 1H NMR (300 MHz, CDCl3, d): 1.13 (m, 201H, –CH3), 1.23 (s, 6H,
–CH3), 1.27 (s, 12H, –CH3), 3.37–3.87 (m, ꢄ1 100H, –O–CH2–CH2–O–),
4.23–4.30 (m, 28H, –CH2–OC(O)–, and –CH2–CH2–OC(O)–), 5.85 (dd,
Jcis ¼ 10.4Hz, Jgem ¼ 1.3 Hz, 4H, H2C ¼ CH–), 6.10 (dd, Jtrans ¼ 17.3Hz,
Jcis ¼ 10.4Hz, 4H, H2C ¼ CH–), 6.39 (dd, Jtrans ¼ 17.3 Hz, Jgem ¼ 1.3 Hz,
4H, H2C ¼ CH–); 13C NMR (125MHz, CDCl3, d): 17.3–17.4, 17.7, 45.8,
46.5, 64.9, 65.3–65.6, 68.5–68.7, 70.5, 72.9–73.3, 75.1–75.3–75.5,
127.7,131.5,165.4,171.9–172.0;IR(moltenpolymeroverNaCl):n ¼ 2
867 (C–Hst), 1 733 (C¼ O), 1 109 ꢀ cmꢁ1 (C_O_C). MALDIþ: maximum
m/z 5 761.4 y 14 176.6; Anal. calcd for C651H1260O292: C 56.90, H 9.18;
found: C 56.48, H, 9.66.
Rho-B-N3 (200 mg, 0.33 mmol) and HCꢅC-2C18 (280 mg, 0.40 mmol)
were dissolved in dry dimethylformamide (7 mL) and the mixture
was stirred at 45 8C. Three cycles of vacuum–argon were made to
remove O2. CuSO4.5H2O (9.8 mg, 0.03 mmol), (l)-ascorbate (13.1 mg,
0.07 mmol), and TBTA (17.6 mg, 0.003 mmol) were dissolved in dry
dimethylformamide (3 mL) and the mixture was stirred at 45 8C.
Three cycles of vacuum–argon were made to remove O2. After
15 min, the Cu(I) solution was added to the first one through a
canula.Threecyclesofvacuum–argonweremadetoremoveO2.The
reaction mixture was stirred at 45 8C for 24 h protected from the
light. A mixture of brine and water (1:1) (100 mL) was added to
the reaction mixture. The product was extracted three times with
ethyl acetate (3 ꢂ 70 mL). The organic phases were dried over
anhydrous MgSO4 and the solvent was evaporated under reduced
pressure. The crude product was purified on silica gel (DCM:
MeOH ¼ ramp from 100:0 to 92:8) to give a purple solid. Yield: 46%.
1H NMR (400 MHz, CDCl3, d): 0.87 (t, J ¼ 6.4 Hz, 6H, –(CH2)17–CH3),
1.24(m,71H,–CH2–CH2–CH2–CH2,–(CH2)14–,–CH3),1.33(t,J ¼ 8 Hz,
12H, –N–CH2–CH3), 1.44 (m, 2H, –CH2–CH2–O–), 1.56 (m, 4H,
–(CH2)14–CH2–CH2–C(O)O–), 2.25 (t, J ¼ 7.2 Hz, 4H, –(CH2)14–CH2–
CH2–C(O)O–), 3.63 (q, J ¼ 6,8 Hz, 8H, –N–CH2–CH3), 4.01 (t, J ¼ 6 Hz,
2H, –CH2–CH2–O–), 4.21 (AB, 4H, –CH2–O–), 4.43 (t, J ¼ 5.6 Hz, 2H,
–C2H1N3–CH2–CH2–), 5.25(s, 2H, –O–CH2–C2H1N3–), 6.95(m, 4H, Ar
H), 7.10 (d, J ¼ 8 Hz, 2H, Ar H), 7.30 (d, J ¼ 8 Hz, 1H, Ar H), 7.35 (s, 1H,
–C2H1N3–), 7.77 (m, 2H, J, Ar H), 8.30 (d, J ¼ 8 Hz, 1H, Ar H), note:
the signal of the protons –C2H1N3–CH2–CH2– (near 1.90 ppm) is
overlapped with water signal and cannot be observed; 13C NMR
(75 MHz, CDCl3, d): 12.6, 14.1, 17.8, 22.6–24.9, 25.2, 26.2, 28.1, 28.0,
29.0–29.2–29.3–29.4–29.6–30.0–31.9–34.0, 46.3, 50.7, 64.9, 65.5,
95.6, 113.6, 114.3, 119.1, 124.3, 126.3, 128.5, 130.0–130.3–130.4,
131.3, 133.0, 133.4, 155.5, 157.7, 165.1, 172.6, 173.2; IR (ATR mode):
n ¼ 2 916ꢁ2 851 (C–H st), 1 740ꢁ1 715 (C ¼ O), 1 649ꢁ1 587 ꢀ cmꢁ1
4.3. Synthesis and Characterization of Rho-B-2C18
4.3.1. Rho-B-N3
Rhodamine B (1.00 g, 2.09 mmol) was dissolved in dry dichloro-
methane (70 mL). 6-azido-hexan-1-ol (602mg, 4.18 mmol) and DPTS
(492mg, 1.67 mmol) were added after the dissolution. The reaction
was stirred at 458C under argon atmosphere. DCC (861mg,
4.18mmol) was dissolved in dry dichloromethane (10 mL) and
was added dropwise to the reaction mixture. It was stirred at 458C
underargonatmosphereovernight,protectedfromthelight.Awhite
precipitate appeared, which was filtered off. Dichloromethane
(20 mL) was added to the filtrate. It was washed once with 100 mL of
HCl 1.0 M and twice with 100 mL of brine. The organic phase was
dried over anhydrous MgSO4 and the solvent was evaporated under
reduced pressure. The crude product was purified on silica gel (DCM:
MeOH¼ ramp from 10:0 to 9:1) to give a purple solid. Yield: 74%.
1HNMR(400MHz,CDCl3):d(ppm):1.32(t,J ¼ 8 Hz,12H,N–CH2–CH3),
1.49 (m, 4H, –CH2–CH2–CH2–CH2–), 1.65 (tt, J ¼ 12Hz, 2H, N3–CH2–
CH2–), 1.87 (tt, J ¼ 12 Hz, 2H, –CH2–CH2–O–), 3.23 (t, J ¼ 4 Hz, 2H, N3–
CH2–), 3.63 (q, J ¼ 8 Hz, 8H, –N–CH2–CH3), 4.02 (t, J ¼ 8 Hz, 2H, –CH2–
O), 6.82(d,J ¼ 4 Hz, 2H,ArH),6.89(dd, J ¼ 8 Hz, J< 4 Hz, 2H, ArH),7.07
(d, J ¼ 8 Hz, 2H, Ar H), 7.30 (dd, J ¼ 8 Hz, J < 4 Hz, 1H, Ar H), 7.75 (td,
J ¼ 8 Hz, J ¼ 4 Hz, 1H, Ar H), 7.82 (td, J ¼ 8 Hz, J ¼ 4 Hz, 1H, Ar H), 8.29
(dd, J ¼ 8 Hz, J < 4 Hz, 1H, Ar H); 13C NMR (75 MHz, CDCl3): d (ppm)
12.6, 25.4, 26.3, 28.2, 28.6, 46.2, 51.3, 65.5, 96.5, 113.6, 114.3, 126.3,
128.5, 130.0–130.3–130.4, 131.3, 133.1, 133.5, 155.5, 157.7, and 165.1.
Note: the counter-ions area mixture ofC7H7SO3– and Cl–; C7H7SO3– was
(C–Car), note: a peak corresponding to water is observed at 3 406 cmꢁ1
MALDIþ: m/z (%) 1 273.1 (100) [C78H122N5O8, H2O]þ.
.
4.4. Chemical Characterization Techniques
The 1H NMR and 13C NMR spectra were recorded from their
corresponding solutions in CDCl3, operating at 300/75 MHz (Bruker
AMX300) or 400/100 MHz (Bruker AV-400). IR spectra (Thermo
Nicolet Avatar 360 FT-IR spectrometer) of F127-Bn-2, F127-OH-4,
F127-Bn-4, F127-OH-8, and F127-Ac-8 were registered from molten
polymer over a NaCl tablet, whereas IR spectra (JACSO FT/IR-4100)
of Rh-B-N3 and Rh-B-2C18 were registered in ATR mode. Mass
spectrometry was performed using an ESI Bruker Esquire 3000 plus
spectrometer for Rh-B-N3 and with a MALDI/TOF-MS Bruker
Microflex system for the rest of the products. Elemental analysis
was obtained in a microanalyzer Perkin Elmer CHN 2400. GPC was
performed in a Waters 2695 apparatus equipped to a light
scattering detector Waters 2424.
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Shell Cross-Linked Polymeric Micelles as Nanocarriers . . .
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difference between the initial feed of Rho-B added and the amount
of Rho-B present in the dialyzed solution.
4.5. Preparation of CLPMs
A 10% (w/v) solution of F127-Ac-8 containing 0.1% of photoinitiator
I2959 was prepared in distilled water and kept overnight at 4 8C for
complete dissolution of the polymer. The final solution was filtered
through a 0.2 mm acetate cellulose filter to remove possible particles
in suspension. By dilution with filtered distilled water containing
0.1% of photoinitiator, 0.77% (w/v) solutions were prepared, which
were used for photopolymerization. 0.77% precursor solutions were
equilibrated for 1 h at room temperature. 5 mL of 0.77% precursor
solutions was light cured in a 6 cm diameter cylindrical glass
container, using UV light (l ¼ 365 nm, 258C, 10min) with a distance
of 8 cm between the lamp and the sample. The photopolymerized
solution was dialyzed at 4 8C for 72h, using a cellulose acetate
membrane (Spectra/Por Biotech 300,000 MWCO (Spectrum)), with
daily water changes. In order to remove larger aggregates, dialyzed
samples were filtered with 0.2 mm cellulose acetate filters. To
calculate the final concentration, known volumes of this dialyzed
and filtered solution of CLPMs were dried by lyophilization in order
to weigh the residue. A mean concentration of 1.9ꢃ 0.1 mg ꢀ mLꢁ1
was determined from three freeze-drying experiments.
For Rho-B-2C18, no further purification was necessary after
ethanol evaporation, due to no precipitation of Rho-B-2C18
,
indicating a complete encapsulation for Rho-B-2C18
.
4.8. Camptothecin Encapsulation and Loading
Content Determination
CPT was dissolved in DMSO at 0.15 mg ꢀ mLꢁ1. The CPT solution was
added over a F127-Ac-8 CLPMs solution (1.9 mg ꢀ mLꢁ1) at a feed
ratio of 0.15 mg of CPT ꢀ mgꢁ1 of CLPM. The mixture was incubated
at room temperature for 24 h. DMSO was eliminated by dialysis
(Spectra/Por MWCO 2000, Spectrum), causing non-encapsulated
camptothecin precipitation. Elimination of non-encapsulated CPT
was carried out by filtration with 0.45 mm Teflon filters.
Encapsulated CPT quantification was made directly by taking 5
mL of filtered solution, which was previously lyophilized and
redissolved in a known volume of DMSO. CPT was quantified by
fluorescence emission spectrum (lmax ¼ 436 nm in DMSO with
lexc ¼ 368 nm) by using a calibration curve in the concentration
range 11.2–39.2 mg ꢀ mLꢁ1 in DMSO.
4.6. CLPM Characterization
4.6.1. Dynamic Light Scattering (DLS)
4.9. In vitro Cellular Uptake
HeLa cells were seeded at a density of 40 ꢀ 103 cells per well in 24
multiwell culture plates over sterile glass covers. Cells were grown
for 24 h, and then themedium was replaced with 500 mL of Rho-B or
Rho-B-2C18 loaded CLPMs solution (0.6 mg ꢀ mLꢁ1) (prepared as
described before), whose concentration was adjusted by adding
DMEM containing 4.5 g ꢀ Lꢁ1 D-Glucose, during 4 h at 37 8C. Then,
CLPMs solution was removed and cells were washed three times
with PBS. Fixation of cells was performed by the addition of 300 mL
of paraformaldehyde 4% solution and incubation for 20 min at
room temperature. Cells were washed two times with PBS. For the
co-localization of Rho-B labeled CLPMs, staining of actin was
performed with Alexa Fluor 488 Phalloidin. Cells were first
permeabilized in PBS þ 1% BSA þ 0.1% saponin and then incubated
with40mLofAlexaFluor488Phalloidindilutedinpermeabilization
solution (1:200) for 1 h at room temperature in the dark. After
washing with PBS-BS (PBS þ 1% BSA þ 0.1% saponin), PBS-B (PBS
þ 1%BSA)anddistilledwater, coverslipsweremountedandthecell
nucleistainedatthesametimewith5mLsolutionofMOWIOL-DAPI
(1:1 000). After drying, coverslips were sealed with nail polish
around the edges and stored at 4 8C in the darkness until confocal
microscopy observation. Cellular uptake and localization were
explored by confocal microscopy using a 60ꢂ objective (Olympus
FV10-i Oil Type, Olympus, Spain). Green fluorescence was observed
under 499/520 (Ex/Em), red fluorescence was observed under 558/
575 (Ex/Em) and DAPI under 359/461 (Ex/Em). Image treatment
was done with FV10i-SW software (Olympus).
The size of the CLPMs was measured by DLS at two temperatures,
25 8C and 37 8C, using a Malvern Instrument Nano ZS that uses a
He–Ne laser, 633 nm, and a detection angle of 1738.
4.6.2. Transmission Electronic Microscopy (TEM) and
Scanning Electronic Microscopy (SEM)
The morphology of the CLMPs was observed by TEM (TECNAI G20,
200 kV) and SEM (Inspect F50). TEM samples were prepared by
adding a drop of CLMPs solution in a TEM grid (HOLLEY carbon film
300 meshCu, Agar, Scientific). Waterfromthesamplewasremoved
by capillarity with a filter paper. The sample was dried overnight in
the darkness. SEM samples were prepared by adding a drop of
CLMPs solution in a 12 mm glass coverslip. Sample was allowed to
dry overnight in the darkness and gold sputtered before SEM
observation.
4.7. Rho-B and Rho-B-2C18 Encapsulation and
Loading Content Determination
Rho-B or Rho-B-2C18 was dissolved in ethanol at 0.15 mg ꢀ mLꢁ1
concentration. Rho-B or Rho-B-2C18 solution was added over CLPMs
F127-Ac-8 solution at a feed ratio of 0.15 mg of Rho-B or Rho-B-2C18
mgꢁ1 of CLPM. The mixture was incubated at room temperaturefor
24 h. Ethanol was evaporated with orbital agitation.
Aqueous solution containing Rho-B-loaded CLPMs was dialyzed
(Slide-A-Lyzes dialysis Cassette G2 2,000 MWCO, Thermo Scientific)
withdistilledwaterfor24 htoremovethenon-encapsulatedRho-B.
Non-encapsulated Rho-B quantification was carried out by
fluorescence (lmax ¼ 576 nm in water) by using a calibration curve
in the concentration range of 2.5–38 mg ꢀ mLꢁ1 in water. The
amount of encapsulated Rho-B was calculated indirectly as the
4.10. Isothermal Titration Calorimetry (ITC) Assay
Binding of CPT to F127-Ac-8 polymer was determined with a high-
sensitivity isothermal titration VP-ITC microcalorimeter (MicroCal,
USA). Experiments were performed at 25 8C in aqueous media with
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I. Jimenez-Pardo et al.
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3% of DMSO to increase solubility. 100 mM compound solution in
the calorimetric cell was titrated with F127-Ac-8 polymer 30 mM
solution. Control experiments were performed under the same
experimental conditions. The heat due to the binding reaction was
obtained as the difference between the reaction heat and the
corresponding heat of dilution, the latter estimated as a constant
heat throughout the experiment, and included as an adjustable
parameter in the analysis. Data were analyzed using software
developed in our laboratory implemented in Origin 7 (OriginLab,
USA).
dilutions of the testcompounds in complete DMEM(without G418)
wereadded24 hafterseeding.Cellswereallowedtoproliferatefor3
d at 37 8C, after which the cell number was determined by CellTiter
96 AQueous One Solution Cell Proliferation Assay (Promega
Corporation). The 50% cytostatic concentration (CC50) was
determined employing the dose–response equation (i.e., Hill
equation). All the experiments with HeLa and Huh5-2 cells were
carried out in triplicate and each experiment was repeated three
different days.
Acknowledgements: This work was financially supported by the
MINECO-FEDER funds (projects MAT2012-38538-CO3-01, CTQ2012-
35692, and BFU2013-47064-P), Miguel Servet Program from
Instituto de Salud Carlos III (CP07/00289, CPII13/00017), Fondo
de Investigaciones Sanitarias (PI10/00186), and the Gobierno de
4.11. Cells and Replicon System
The highly permissive cell clone Huh 7-Lunet, as well as Huh 7 cells
containing subgenomic (HCV) replicons I389luc-ubi-neo/NS3-3’/
5.1 (Huh 5-2), I377NS3-3’/wt (Huh 9-13), or I389/hygro-ubi-NS3-3/
5.1 (a kind gift from Dr. V. Lohmann and Dr. R. Bartenschlager) has
been described recently.[21–24] Briefly, this system allowed the
efficient propagation of genetically modified HCV RNAs (replicons)
in a human hepatoma cell line (Huh). The amount of the RNA that
has been transcribed and translated is determined through the
quantification of a reporter contained in the replicon system
(luciferase). Theamountofluminescencedetected(afteraddingthe
substrate specific for this enzyme) is proportional to the virus
replication rate.[21–24] Cells were grown in Dulbecco’s modified
Eagle’s medium (DMEM; Gibco, Belgium) supplemented with 10%
heat-inactivated fetal bovine serum (PAN-Biotech GmbH, Ger-
many), 1ꢂ non-essential amino acids (Gibco), 100 IU mLꢁ1 pen-
icillin (Gibco), 100 mg ꢀ mLꢁ1 streptomycin (Gibco), and 250 mg ꢀ
mLꢁ1 geneticin (G418; Gibco).
ꢀ
Aragon-FSE (E04, B89 and B01 research groups). A. L. and R. C-G.
thank the MEC for their FPU grants, FPU12/05210 and FPU13/
3870, and R.GP and R. C.-G. thank the DGA for the EPIF grants,
B054/12 and B136/13. Authors would like to acknowledge the use
ꢀ
of ‘‘Servicios Cient´ıfico-Tecnicos’’ of CEQMA (UZ–CSIC) and of CIBA
(IACS), and the LMA from INA (UZ).
Received: March 17, 2015; Revised: May 14, 2015; Published
online: June 5, 2015; DOI: 10.1002/mabi.201500094
Keywords: anti-HCV; camptothecin; cross-linked micelles; den-
drimers; pluronic
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4.12. Antiviral Assay with Huh 5-2 Cells
Antiviral assays for assessing the efficacy of the F127-Ac-8/CPT
CLPMs system were performed as described in literature.[21–25]
Briefly, Huh5-2 cellswereseededat a densityof5 ꢀ 103 cellsperwell
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removed and luciferase activity was determined using the Bright-
Glo Luciferase Assay System (Promega Corporation, the Nether-
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