Enhanced drug loading in polymerized micellar cargo

Article abstract of DOI:10.1039/c004134c

A new drug carrier system based on self-assembly and polymerization of polydiacetylenic amphiphiles is described. Although classical amphiphiles can help in solubilizing hydrophobic molecules upon self-arrangement into a variety of nanometric structures, a greater effect on drug loading was observed for our polymerized micelles as compared to the non-polymerized analogues. This permitted higher aqueous solubilization of lipophilic drugs with low micelle concentration. ¹⁴C labeling of a model drug on one side and of the amphiphile on the other side permitted assessment, after intravenous injection, of biodistribution and excretion profiles of the drug cargo.

Full text of DOI:10.1039/c004134c

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Enhanced drug loading in polymerized micellar cargo†
a
b
c
b
b
b
Julien Ogier, Thomas Arnauld,* G e´ raldine Carrot, Antoine Lhumeau, Jean-Marie Delbos, Claire Boursier,
a
b
a
Olivier Loreau, Francois Lefoulon and Eric Doris*
Received 15th March 2010, Accepted 10th June 2010
First published as an Advance Article on the web 9th July 2010
DOI: 10.1039/c004134c
A new drug carrier system based on self-assembly and polymerization of polydiacetylenic amphiphiles
is described. Although classical amphiphiles can help in solubilizing hydrophobic molecules upon
self-arrangement into a variety of nanometric structures, a greater effect on drug loading was observed
for our polymerized micelles as compared to the non-polymerized analogues. This permitted higher
1
4
aqueous solubilization of lipophilic drugs with low micelle concentration. C labeling of a model drug
on one side and of the amphiphile on the other side permitted assessment, after intravenous injection,
of biodistribution and excretion profiles of the drug cargo.
Introduction
of specifically designed monomer surfactants and subsequent
polymerization.
The past two decades have witnessed significant progress in the de-
velopment of new drug delivery systems to carry active molecules
through different biological barriers and reach specific targets.
1
Results and discussion
Substantial efforts have been devoted to overcome drawbacks
associated with the intrinsic properties of therapeutic molecules,
such as solubility (toxicity and absorption issues), stability (in
vivo degradation), pharmacokinetics (rapid elimination) and/or
The micellar nano-structures were obtained by dispersion of
amphiphilic monomers at concentrations greater than the critical
micellar concentration (CMC). To stabilize the supramolecular
assembly, a photo-polymerizable amphiphile 1 was used. It
incorporates a photo-responsive diacetylenic group within the
C25-hydrocarbon chain and a hydrophilic nitrilotriacetic acid
14
2
biodistribution (non-specific distribution). Particularly, most of
the anticancer drugs exhibit low solubility in water due to their
inherent hydrophobic properties that, on the other hand, enhance
(
NTA) polar head (Scheme 1). The pH value of the solution has
3
their cell internalization and efficacy.
a key effect on the final structure of the self-assembly. At lower
pH (below 10), amphiphiles self-assemble as ribbons which, upon
Two main strategies have been developed to increase the
aqueous solubility of such compounds. The first approach consists
in modifying the chemical structure of the drug by attaching
hydrophilic functional groups or hydro-soluble molecules, i.e.
◦
15
heating to 70 C, coil-up into tubular nanostructures (see ESI†).
At higher pH values (e.g. pH = 12), well defined micelles were
obtained. To polymerize and uphold the micelle structure, the
4
5
targeting or cell-penetrating peptides, polyethylene glycols or
6
other hydrophilic ligands. However, the increase of the drug
solubility could disrupt therapeutic activity, and a balance be-
7,8
tween solubility and activity has to be found. The second
approach is based on the solubilization of the active molecules
9
10
using drug-cargo systems such as nanoparticles, micelles of
11
12
13
block copolymers, liposomes or dendrimers. This strategy
successfully increases the molecules’ stability and solubility, since
their activity is unaffected, their bio-availability improved by a
sustained release in time, and their toxicity lowered. Nevertheless,
the carrier has to be highly soluble in water with a significant drug
loading capacity.
In the present paper, we report a new cargo system based
on polymerized micelles which exhibit high solubility and drug
loading. Our approach is based on the self-assembly into micelles
a
CEA, iBiTecS, Service de Chimie Bioorganique et de Marquage, 91191
Gif-sur-Yvette, France. E-mail: eric.doris@cea.fr
b
Technologie Servier, 27 rue Eug e` ne Vignat, 45000 Orl e´ ans, France. E-mail:
thomas.arnauld@fr.netgrs.com
c
CEA, Laboratoire L e´ on Brillouin, 91191 Gif-sur-Yvette, France
†
Electronic supplementary information (ESI) available: Experimental
1
procedures, TEM pictures, H NMR drug loading data. See DOI:
0.1039/c004134c
1
Scheme 1 Polymerization of diacetylenic amphiphile.
3
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solution was irradiated at 254 nm for 5 h. The polymerization
takes place by a topochemical 1,4-addition mechanism.
samples (Fig. 2a) and DLS data (Fig. 2b) showed spherical
structures with an average diameter of ca. 6 nm (2–9 nm size
distributions as determined by DLS), compared to that of non-
polymerized micelles of 7 nm.
16–20
In the course of the polymerization step, the micelle solution
changed from colorless to yellowish. This indicates an increase in
length of the conjugated polymer backbone (Fig. 1a,b). Kinetic
studies of the micelle polymerization showed fast polymerization
of the monomer in the initial five hours with up to 75% conversion.
Beyond this point, the polymerization rate decreased and reached
9
0% conversion after 15 h (Fig. 1d). The polymerization step
makes the micelles robust, stable for months and insensitive to
-
1
dilution below CMC (0.11–0.12 mg mL ). To meet physiological
conditions, the pH was finally adjusted to 7.5 and osmolality to
ca. 290 mOsm by addition of NaCl.
Fig. 2 (a) TEM picture of polymerized micelles. Size distribution in
number (%) of non-polymerized (b) and polymerized (c) micelles solutions.
The polymerized micelles were then used as a solubility
enhancer of hydrophobic therapeutic molecules (TM) toward
potential drug delivery applications. Molecules involved in this
study are: an E-ring keto analogue of camptothecin (CPTD),
a flavone derivative (FLD), and paclitaxel (Fig. 3). They all
-
1
intrinsically exhibit very low solubility in water (≤2 mg mL ,
Table 1).
Experiments showed a general increase of the solubility of the
different therapeutic molecules (TM) by a factor ranging from
Fig. 1 Pictures of (a) unpolymerized and (b) polymerized micelle
solutions. Evolution of absorbance as a function of polymerization time
of amphiphile 1 (c), (d).
The structure of the polymerized micelles was characterized
by transmission electron microscopy (TEM) and dynamic light
scattering (DLS). Both TEM observation of negatively stained
Fig. 3 Chemical structures of E-ring keto analog of camptothecin
(CPTD), flavone derivative (FLD), and paclitaxel.
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Table 1 Solubilization of therapeutic molecules by polymerized micelles
non-irradiated micelle. The non-irradiated micelle formed mainly
a microsuspension of the TM that is filtered off after the inclusion
process. This demonstrates that polymerization of the micelle is
crucial for the loading and withholding of the drugs.
TM solubility in TM solubility in
Drug loading
(mass balance, %)
-3
-3
TM
water/mg cm
micelle soln/mg cm
3
CPTD
FLD
Paclitaxel 0.40
0.09
0.20
9.00 ¥ 10
47
24
31
Preliminary studies were then undertaken to evaluate the
pharmacokinetics of the polymeric micelles, more particularly
their toxicity, biodistribution, and elimination. Acute toxicity was
assessed in a single dose administration study to Wistar rats after
bolus injection of the polymeric micelles in the caudal vein. No
adverse event was observed over a three-day period at doses
3
3.15 ¥ 10
3
4.55 ¥ 10
1
1 000 to 100 000 and drug loading from 24% to 47%, which
correspond to a drug intake of 3.15 mg (FLD) to 9 mg (CPTD) per
0 mg of micelles. It is to be noted that, in the case of paclitaxel,
-
1
below 100 mg kg . For distribution/elimination studies of the
1
1
4
polymerized micelles, a C-labeled amphiphile 1 (compound 10,
values of 11 000 and 31% were obtained for solubility enhancement
and loading capacity, respectively. These values are comparable
to those of other analogous nanometric carriers described in
25,26
see ESI†) was synthesized
and assembled into polydiacetylenic
micelles. The latter (incorporating no drug) were administered to
two male Wistar rats by bolus injection in the caudal vein at a
21–24
the literature
and designate our polydiacetylenic micelles as
-
1
-1
dose of 100 mg kg corresponding to 4 MBq kg of radioactive
material. The whole body tissue distribution of radioactivity
was performed by radioluminography from saggital sections
efficient cargo for the encapsulation of hydrophobic drugs.
1
In the mean time, H NMR spectroscopy was used to study
the drug/micelle complex. To this end, the overall sequence
(
20 mm thick) at 10 min and 24 h after dosing (Fig. 5a). The
(
preparation of the micelles and loading of the TM) was repeated
1
radioluminograms showed wide and rapid distribution of the
micelles in major tissues, but not in the brain (Fig. 5b). Slow
renal elimination of the micelle was observed as only ca. 5% of the
administered dose was detected in the urine, after 24 h. In addition,
biliary excretion and faecal elimination seems to be operative since
radioactivity was also detected in the faeces at 24 h.
in D O/NaOD and the samples were analyzed by H NMR. For
2
1
example, the H NMR spectrum of the FLD/micelle complex
exhibited intense broad aliphatic peaks corresponding to the
micelle and weak broad aromatic signals that were attributed
to FLD (Fig. 4b). The drug signals are masked by the micelle
since the encapsulated molecules relax at the same rate as the
surrounding polymer. To visualize both the load (FLD) and the
container (micelle), the solution was freeze-dried and taken back
1
in a dissociating solvent (DMSO-d
6
). The H NMR spectrum
showed, this time, well-resolved and sharp peaks of FLD. This
underlines the strong interaction between FLD and the micelle
when in aqueous medium (Fig. 4c).
1
Fig. 4 H NMR spectra of (a) FLD in DMSO-d
6
, (b) FLD/micelle
complex in D
2
O and (c) FLD/micelle complex in DMSO-d
6
.
Fig. 5 (a) Radioluminograms and (b) tissue distribution (% dose/g) of
To determine the effect of the polymerization step on drug
loading capacity, we carried out comparative measurements of the
inclusion of CPTD, in polymerized and non-polymerized micelles.
While mass balance measurements indicatedadrugloadingof 47%
for the polymerized formulation, a value of 3.3% was found for the
total radioactivity at different time in Wistar rats after single intravenous
administration of 4 MBq kg- of C-polymerized micelles.
1
14
The influence of the micelle on the pharmacokinetics of a thera-
peutic molecule was then investigated. Two different formulations
3
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were prepared: (i) formulation A consisting of a nanosuspension
Conclusions
1
4
of the free C-CPTD (reference formulation), and (ii) formulation
1
4
In conclusion, we report here a new drug carrier system based
on the self-assembly and polymerization of specifically designed
amphiphiles. The polydiacetylenic micelles provide effective hy-
drophobic drugs loading and solubility enhancement. The distri-
bution/clearance studies are promising as the body residence time
of drugs is increased. No specific organ seems to be targeted and
the primary pathway of excretion of both the drug and the micelles
is biliary.
B which was obtained by loading C-CPTD in non-labeled
polymerized micelles. While formulation A is a stable suspension,
B is a homogeneous solution. Each formulation was injected bolus
via the caudal vein to a series of five C57 black male mice at a dose
-
1
-1
corresponding to 5 mg kg of CPTD (4 MBq kg ). The mice were
sacrificed at variable times, from 5 min to 48 h, and saggital cuts
were analyzed by radioluminography (Fig. 6a).
Experimental procedures
Materials and methods
Chemicals were purchased from Aldrich. Common organic sol-
vents were used without further purification. CH
2
Cl
2
was distilled
over CaH prior to use. NMR spectra were recorded on a Bruker
2
1
13
Advanced 400 at 400 MHz ( H) and 100 MHz ( C). Chemical
shifts are given in ppm relative to the NMR solvent residual
peak. Mass spectra were recorded using a MarinerTM ESI-TOF
spectrometer. IR-spectra were recorded using a Perkin-Elmer 2000
-
1
FT-IR. Wavenumbers are given in cm at their maximum intensity.
Size distribution of micelles was measured using a Zetasizer Nano
series (Malvern).
Synthesis of amphiphile 1 (Scheme 2)
Fig. 6 (a) Radioluminograms and (b) tissue distribution (% dose/g) of
total radioactivity at different times in the C57 black male mice after single
-1
14
intravenous administration of 4 MBq kg of C-CPTD in polydiacetylenic
micelles.
Five minutes after administration of formulation A, accu-
mulation of CPTD in the liver, spleen, kidney cortex and
lachrymal glands was observed, leaving only residual amounts of
radioactivity in the blood compartment. In addition, the overall
radioactivity decreased rapidly in the body to be virtually nil at
2
4 h.
It appears as striking evidence that the residence time of C-
1
4
CPTD in the body is significantly improved when associated to
the polymerized micelle as, this time, the radioactivity decreased
slowly and steadily over a period of time greater than 48 h after
dosing of the formulation B. The TM is mainly distributed to
the liver, kidney cortex and, in a lesser proportion, to adrenal
glands and spleen (Fig. 6b). Distribution and tissue elimination
half-life increased compared to reference formulation A. Micelle
encapsulation significantly slowed down migration of the drug
from blood to tissues and decreased the elimination rate from the
body.
Scheme 2 Synthesis of photo-polymerizable amphiphile 1.
6
2
2
N -Carboxybenzyloxy-N ,N -bis(carboxymethyl)lysine
(2).
Bromoacetic acid (10.82 g, 4 equiv.) was solubilized in 120 mL
◦
of 1 N NaOH. The solution was cooled to 0 C and Z-lysine
(5.39 g, 19 mmol, 1 equiv.) in 30 mL of 1 N NaOH was added
◦
It must be pointed out that, with both formulations, a large
quantity of radioactivity was observed at some point in the
intestine, evidence that biliary excretion is a major elimination
pathway for CPTD and/or its metabolites.
dropwise. The solution was stirred at r.t. for 2 h and at 50 C for
◦
12 h. The solution was cooled to 0 C and acidified with 37%
HCl. The white precipitate was collected by filtration and dried
1
under vacuum over P
2
O
5
. Yield: quant. H NMR (DMSO-d
6
):
This journal is © The Royal Society of Chemistry 2010
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7
(
(
.25–7.40 (m, 5H), 7.21 (t, 1H, J = 5.5 Hz), 4.98 (s, 2H), 3.46
m, 4H), 3.33 (t, 1H, J = 7.2 Hz), 2.85–3.05 (m, 2H), 1.15–1.65
m, 6H). C NMR (DMSO-d ): 174.5, 174.0 (2C), 156.5, 137.6,
28.7 (2C), 128.0 (3C), 65.4, 64.9, 59.8, 54.2, 29.5–29.6 (2C); 23.4.
produce monodisperse micelles. The solution was polymerized by
irradiation at 254 nm for 5 h (low pressure mercury UV lamp—
Heraeus). The pH was finally adjusted to 7.5 and osmolality to ca.
290 mOsm by addition of NaCl.
1
3
6
1
+
+
-1
MS (ESI /TOF) m/z: 397 [M + H] . IR (KBr, cm ): 3364, 3042,
935, 1721, 1523, 1278, 1131, 1013, 905, 742, 683.
2
Drug loading of hydrophobic therapeutic molecules (TM)
2
6
N -Bis(carboxymethyl)lysine
(3). N -Carboxybenzyloxy-
In a standard experiment, 40 mg of the drug was dispersed and
2
2
N ,N -bis(carboxymethyl)lysine 2 (7.6 g, 19.2 mmol, 1 equiv.) was
solubilized in 200 mL of MeOH. 300 mg of 10% Pd/C was added
and the flask was purged 4 times with N
solution was stirred under H at r.t. for 12 h. The precipitate was
collected by filtration and taken back into H O. Palladium was
filtered off, and the solution freeze dried. Yield: 50%. H NMR
◦
stirred for 12 h at 50 C in 2 mL of an aqueous solution of
-1
the micelles (10 mg mL ). The solution was then filtered (0.2
2
, and 4 times with H
2
. The
mm) to remove unsolublilized drug and to yield a clear solution
with no particles in suspension. The solution was freeze-dried and
weighed. In parallel, the same sequence was repeated, but with no
drug. The weight of solubilized therapeutic molecules can then be
determined by mass balance of the two experiments. From this
value, a drug loading was calculated by dividing the mass of the
drug by the mass of the micelles incorporating the drug (see ESI†).
2
2
1
(
(
5
2
2
DMSO-d
6
): 3.75–3.9 (m, 5H), 2.89 (t, 2H, J = 7.2 Hz), 1.4–1.9
1
3
m, 6H). C NMR (DMSO-d ): 171.9 (1C), 170.1 (2C), 67.6 (1C),
5.1 (2C), 38.9 (1C), 26.3 (2C), 22.9 (1C). MS (ESI /TOF) m/z:
63 [M + H] , 285 [M + Na] . IR (KBr, cm ): 3498, 3324, 2996,
975, 1750, 1634, 1401, 1263, 1159, 984, 876, 751.
6
+
+
+
-1
14
Distribution/elimination study of C-polymerized micelles
2
,5-Dioxo-pyrrolidin-1-yl
pentacosa-10,12-diynoate
(5).
All experiments were performed in accordance with European
regulations on care and use of laboratory animals. Polymerized
micelles were administered by bolus injection in the caudal vein
at 100 mg kg (4 MBq kg ) to two male Wistar rats. Rats were
sacrificed at 10 min and 24 h after administration for quantitative
tissue distribution of radioactivity.
Pentacosa-10,12-diynoic acid 4 (1 g, 2.7 mmol, 1 equiv.),
N-(3-dimethylaminopropyl)-N¢-ethylcarbodiimide (0.78 g, 1.5
equiv.) and N-hydroxysuccinimide (0.5 g, 1.8 equiv.) were
solubilized in 50 mL of anhydrous CH
stirred at r.t. for 12 h under N and quenched with H
aqueous phase was extracted twice with CH Cl . The organic
phases were collected, dried and concentrated under vacuum
-
1
-1
2
Cl
2
. The solution was
2
2
O. The
2
2
1
4
Distribution study of C-CPTD
1
to give 5 as a white solid. Yield: quant. H NMR (DMSO-d
6
):
2
.7–2.9 (m, 4H), 2.58 (t, 2H, J = 7.2 Hz), 2.22 (t, 4H, J = 7.2
14
-1
Each C-CPTD formulation was injected at a dose of 5 mg kg
Hz), 1.72 (q, 2H, J = 7.2 Hz), 1.49 (t, 2H, J = 7.2 Hz), 1.2–1.42
-1
and 4 MBq kg by bolus injection in the caudal vein to a series
of five C57 black male mice. Mice were sacrificed at variable times
1
3
(
(
(
m, 28H), 0.86 (t, 3H, J = 7.2 Hz). C NMR (DMSO-d
6
): 169.2
2C), 168.5 (1C), 77.6 (1C), 77.3 (1C), 65.3 (1C), 65.2 (1C), 31.8
1C), 28.1–30.7 (16C), 24.4 (1C), 22.5 (1C), 18.9 (2C), 13.9 (1C).
(
5 min to 48 h) after administration for quantitative tissue
distribution of radioactivity.
+
+
+
MS (ESI /TOF) m/z: 472 [M + H] , 494 [M + Na] .
6
2
2
Quantitative tissue distribution of radioactivity
N -Pentacosa-10,12-diynoyl-N ,N -bis(carboxymethyl)lysine
2
2
(
3
1). N ,N -Bis(carboxymethyl)lysine 3 (1 g, 1.2 equiv.) and
.1 mL of NEt (7 equiv.) were dispersed in 100 mL of DMF. H
was added dropwise until complete solubilization. 2,5-dioxo-
pyrrolidin-1-yl pentacosa-10,12-diynoate 5 (1.44 g, 3.05 mmol, 1
equiv.) in 50 mL of DMF was then added. The solution was stirred
at r.t. for 12 h. The solution was concentrated under vacuum,
Frozen rats and mice were individually embedded in car-
boxymethylcellulose which was subsequently frozen. Using a
cryomicrotome (PMV 450 – LKB), 20 mm saggital sections
were cut from each animal to reveal the tissues of interest. The
quantification of the freeze dried saggital sections was obtained by
radioluminography (BAS 2000, FUJI PHOTO FILM Co. Ltd.),
using standard calibration sources prepared by mixing radioactive
compound with blood samples.
3
2
O
taken into H
2
O, and acidified with 37% HCl. The white solid was
filtered off, washed with water and dried overnight under vacuum
and P
1
2
O
5
. Yield: 76%. H NMR (DMSO-d
6
): 7.68 (t, 1H, J = 5.6
Hz), 3.39–3.50 (AB, 4H, JAB = 17.6 Hz), 3.35 (t, J = 7.3 Hz, 1H),
Acknowledgements
2
1
1
5
(
6
[
(
1
.97 (m, 2H), 2.24 (t, 4H, J = 6.8 Hz), 2.00 (t, 2H, J = 7.2 Hz),
1
3
.1–1.6 (m, 38H), 0.82 (t, 3H, J = 6.8 Hz). C NMR (DMSO-d
6
):
We thank A. Petit, N. Bongibault-Besnard and N. Masson-
Lancelot for NMR studies.
74.3 (1C), 173.6 (2C), 172.2 (1C), 78.0 (2C), 65.7 (1C), 64.7 (2C),
3.7 (2C), 38.6 (1C), 35.8 (1C), 28.0–31.7 (16C), 25.7 (1C), 23.5
1C), 22.5 (1C), 18.7 (2C), 14.3 (1C). MS (ESI/TOF) m/z: (ESI )
19 [M + H] , 641 [M + Na] , (ESI ) 617 [M - H] . ESI HRMS
M - H] calcd for C35
KBr, cm ): 3323, 2925, 2853, 1929, 1732, 1645, 1546, 1464, 1425,
256, 983, 892, 720.
+
+
+
-
-
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H
57
N
2
O : 617.4166; found: 617.4165. IR
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