Nanoaggregates of biodegradable amphiphilic random polycations for delivering water-insoluble drugs

Article abstract of DOI:10.1021/bm300251j

Cationic amphiphilic random copolyesters were obtained by copolymerization of 5-Z-amino-δ-valerolactone and ε-caprolactone. The amino content of the final copolymers was controlled by the polymerization feed ratio and was in the range 10 to 100%. Copolymers solubility and aggregation behavior was assessed by conductometric and zeta potential analyses. A critical aggregation concentration of ca. 0.05% (w/v) was found for all water-soluble copolymers that formed nanoaggregates. Two populations were found to be present in equilibrium with hydrodynamic diameters in the range of 30-50 and 100-250 nm. The capacity to use the amphiphilic and cationic character of the nanoaggregates to encapsulate highly hydrophobic compounds was further investigated. Finally, copolymers hemo- and cytocompatibility were evaluated by hemagglutination, hemolysis, and cells proliferation tests. The results showed that the proposed cationic amphiphilic random copolyesters are biocompatible.

Full text of DOI:10.1021/bm300251j

Article
pubs.acs.org/Biomac
Nanoaggregates of Biodegradable Amphiphilic Random Polycations
for Delivering Water-Insoluble Drugs
Benjamin Nottelet,* Manuela Patterer, Benjamin Franco
̧ is, Marc-Alexandre Schott, Martine Domurado,
Xavier Garric, Dominique Domurado, and Jean Coudane
Max Mousseron Institute of Biomolecules (IBMM), Artificial Biopolymers Group, UMR CNRS 5247 University of Montpellier 1,
University of Montpellier 2, Faculty of Pharmacy, 15 Av. C. Flahault, Montpellier, 34093, France
S
* Supporting Information
ABSTRACT: Cationic amphiphilic random copolyesters were obtained by copolymerization of 5-Z-amino-δ-valerolactone and
ε-caprolactone. The amino content of the final copolymers was controlled by the polymerization feed ratio and was in the range
10 to 100%. Copolymers solubility and aggregation behavior was assessed by conductometric and zeta potential analyses. A
critical aggregation concentration of ca. 0.05% (w/v) was found for all water-soluble copolymers that formed nanoaggregates.
Two populations were found to be present in equilibrium with hydrodynamic diameters in the range of 30−50 and 100−250 nm.
The capacity to use the amphiphilic and cationic character of the nanoaggregates to encapsulate highly hydrophobic compounds
was further investigated. Finally, copolymers hemo- and cytocompatibility were evaluated by hemagglutination, hemolysis, and
cells proliferation tests. The results showed that the proposed cationic amphiphilic random copolyesters are biocompatible.
molecules into their hydrophobic core through both chemical
conjugation and physical entrapment, polymer micelles have
been widely used to solubilize and deliver poorly water-soluble
drugs. The various polymers proposed for the formation of
block polymeric micelles include the pharmaceutically most
promising poly(ethylene glycol)−poly(lactide) (PEG-b-PLA)
and poly(ethylene glycol)−poly(lactide-glycolide) (PEG-b-
PLGA) diblock copolymers that have been extensively studied
because of their biocompatibility and the biodegradability.5−8
However, some basic micelle properties can advantageously be
improved by using alternative polymer blocks. For example,
degradability and drug loading can be improved by using
poly(malic acid) and poly(hexyl-lactic acid) blocks, and micelle
stability can be increased by using graft structures instead of
block copolymers.9−11
INTRODUCTION
■
Given that almost one-third of newly discovered drugs are
highly insoluble in water, more compounds with poor aqueous
solubility have entered the development pipeline.1,2 A number
of drug delivery systems, including molecular complexation
systems based on cyclodextrins, nanosuspensions, lipid
formulations, prodrugs, and polymeric micellar systems, have
been developed to overcome this solubility limitation.3 Of all of
these different systems, polymeric micelles have attracted the
greatest interest as promising novel drug delivery carriers
because of their remarkable advantages that include small size
and a narrow size distribution, long circulation times, good
stability, and targeting ability. Polymeric micelles are macro-
molecular assemblies composed of an inner core and an outer
shell. They can be divided in two main categories depending on
the driving force responsible for their formation: hydrophobic
micelles and polyion complex (PIC) micelles.4 The former, that
have undergone the most development, usually consist of
amphiphilic block copolymers. Typically, micellar drug carriers
involve AB- or ABA-type block copolymers, which sponta-
neously form nanosized objects in aqueous medium as a result
of the hydrophobic and hydrophilic blocks interacting differ-
ently with the solvent. Given the possibility to load lipophilic
Whereas amphiphilic block copolymers are used to form
hydrophobic micelles, charged polymer blocks may also be used
in micelles. PIC micelles result from electrostatic interactions
between ionic segments and oppositely charged species; double
Received: February 16, 2012
Revised: March 28, 2012
Published: March 29, 2012
© 2012 American Chemical Society
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(BzOH), ethyl acetate (AcOEt), tetrahydrofuran (THF), toluene,
dimethylformamide (DMF), diethyl ether (Et2O), dichloromethane
(CH2Cl2), poly-L-lysine hydrobromide (PLL) (Mw = 4000−15 000 g/
mol and Mw = 80 000 g/mol), 17α-ethinylestradiol (EE), flufenamic
acid (FA), pyrene, 1-pyrenecarboxylic acid, and 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were pur-
chased from Sigma-Aldrich (St-Quentin Fallavier, France). Benzo-
triazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate
(BOP) and tris(hydroxymethyl)aminomethane (Tris) were purchased
from Merck (Strasbourg, France). Modified Eagle’s medium (MEM),
horse serum, penicillin, streptomycin, Glutamax, and Dulbecco’s
phosphate-buffered saline (DBPS) were purchased from Invitrogen
(Cergy Pontoise, France). Cellstar polystyrene tissue culture plates
(TCPS) were purchased from Greiner Bio-One (Courtaboeuf,
France). All chemicals and solvents were used without purification
except for BzOH, ε-CL, and the polymerization solvents that were
dried over calcium hydride for 24 h at room temperature and distilled
under reduced pressure.
hydrophilic copolymers composed of a polyionic segment and a
hydrophilic segment are therefore generally used.4 Interpolye-
lectrolyte complexes (IPECs) are another example; they form
spontaneously in solution because of strong cooperative
electrostatic interactions between positively and negatively
charged polyions.12 IPECs are, for example, developed for gene
delivery applications, where they are known as polyplexes based
on the complexation of DNA with various polycations.13−16 In
addition to these purely hydrophilic compounds, amphiphilic
copolyelectrolytes undergo hydrophobic associations that
compete with electrostatic repulsions to form various types of
micelle-like nanostructures.17−20 Although these systems have
so far drawn little attention, they are of particular interest:
nanosized aggregates formed by random amphiphilic poly-
electrolytes can serve as carriers and are an interesting
alternative to hydrophobic micelles and polyionic complexes
for drug delivery.21 Morishima et al. were among the first to
conduct systematic studies on random anionic copolymers with
amphiphilic character and the effects of various parameters,
including charge density, hydrophobicity, charge location, and
molecular weight on their self-organization.22−24 Our group
recently capitalized on the advantage of the hydrophobic
nanodomains formed by such systems to encapsulate
clofazimine in hydrophobized poly(methyl vinyl ether-alt-
maleic acid) (PMVEMAc). We demonstrated that this
polymeric system is able to promote the solubilization of
clofazimine at blood pH and that solubilization results from
drug−polymer interactions that combine electrostatic and
hydrophobic interactions. Synergy between these two physical
phenomena resulted in the entrapment of large amounts of
water-insoluble clofazimine with up to one molecule of drug for
two mononeric units, corresponding to 1 g of drug for 1 g of
polymer.21
We were interested in broadening this strategy to random
amphiphilic polycations while at the same time adding
degradability to the polymer system. Because aliphatic
polyesters are well-known for their degradability, we focused
our efforts on amino-functionalized lactones with the intention
to generate a family of cationic copolymers. Only a few
examples of such monomers can be found in the literature,
mainly serine-based butyrolactones and one lysine-based
lactide.25−29 More recently, Lang’s group proposed an aminated
caprolactone that was polymerized with PEG to generate
double hydrophilic block copolymers.30,31 Our group also
reported the synthesis of 5-Z-amino-δ-valerolactone (5-NHZ-
VL), which is easily obtained in two steps from a glutamic acid
derivative and can be polymerized to yield cationic aliphatic
polyesters.32 In the work reported herein, 5-NHZ-VL and ε-
caprolactone (ε-CL) were copolymerized to generate a family
of random amphiphilic polycations with a predictable amino
content. Their ability to self-assemble as nanoaggregates and
generate hydrophobic domains was evaluated. Finally, poorly
water-soluble model compounds with various log P and log D
values were encapsulated in the copolymers to evaluate their
potential for future drug delivery applications.
Buffers. pH 7.4 phosphate-buffered and 2-amino-2-
(hydroxymethyl)propane-1,3-diol-buffered isoosmolar media were
prepared as previously described34 and are referred to as PBS and
Tris-buffer, respectively, in the following.
1
NMR Spectroscopy. H NMR and 13C spectra were recorded on
an AMX300 Brucker spectrometer operating at 300 and 75 MHz,
̈
respectively. Deuterated dimethyl sulfoxide or chloroform was used as
solvent.
Size Exclusion Chromatography. Polymer molecular weights
were determined by size exclusion chromatography (SEC) on a
Waters 600 controller system fitted with a Waters In-Line degasser and
two PLgel 5 μm MIXED-C (300 × 7.5 mm) columns. A Waters 2410
RI detector and a Waters 2489 UV/vis detector were used. The mobile
phase was DMF at 1 mL/min flow and 80 °C. Typically, the polymer
(10 mg) was dissolved in DMF (2 mL), the resulting solution was
filtered through a 0.45 μm Millipore filter, and 20 μL of filtrate was
injected. M and M were expressed according to calibration using
̅
̅
n
w
poly(methyl methacrylate) standards.
Differential Scanning Calorimetry. Differential scanning calo-
rimetry (DSC) measurements were made under nitrogen at a 10 °C/
min heating rate on a Perkin-Elmer DSC 6000 thermal analyzer.
Dynamic Light Scattering. Polymer solutions were prepared in
Milli-Q water and filtered through a 0.45 μm filter. Nanoaggregate
hydrodynamic diameter (<Dh>), zeta potential, and solutions
conductivity were evaluated by laser doppler velocytometry (NanoZS,
Malvern Instrument, U.K.). Measurements were made at 25 °C using a
633 nm laser. Zeta potential (ζ) was calculated from the electro-
phoretic mobility value, using Smoluchowski’s equation: ζ = (4πηu)/ε,
where u is electrophoretic mobility, η is viscosity, and ε is the dielectric
constant of the solvent.
Microscopy. Light microscopy examinations (40× magnification)
were carried out using a TE300 microscope (Nikon, Tokyo, Japan)
fitted with a Canon Power Shot A620 camera. Photomicrographs were
taken from two observation fields. The microscope stage was at room
temperature. Transmission electron microscope (TEM) micrographs
were obtained with a JEOL 1200 EXII (working voltage of 120 kV). A
drop of the solution was placed onto a carbon-supported copper grid
for 5 min. The excess liquid was sucked away by filter paper.
Encapsulation of Hydrophobic Compounds. The encapsula-
tion capability of the different copolymers was evaluated by adding
hydrophobic compounds (HCs) to a 1% (w/v) copolymer aqueous
solution. In a typical experiment, 3 mg of HC was added to 3 mL of a
1% (w/v) copolymer solution in distilled water. The mixture was
stirred for 3 h before centrifugation. The supernatant was then
carefully collected before dilution at 50% with acetone or ethanol
(depending on the HC) to release the HC entrapped in the copolymer
hydrophobic domains. The resulting solutions were analyzed on a
Perkin-Elmer Lambda 35 UV/vis spectrophotometer. The total
amount of dissolved HC was calculated by comparing the resulting
absorbance with standard plots. The standard plot for each HC was
constructed as the absorbance measured at the isosbestic point of the
spectra obtained for decreasing concentrations of HC. HC chemical
EXPERIMENTAL SECTION
■
Materials. N-Benzyloxycarbonyl-glutamic acid γ-tert-butyl ester
(ZGlu(OtBu)OH, 99%), N-N′-diisopropylethylamine (DIPEA), so-
dium borohydride powder (NaBH4, 99%), sodium bicarbonate
(NaHCO3), magnesium sulfate (MgSO4), ε-CL, tin(II) 2-ethyl-
hexanoate (Sn(Oct)2, ∼95%), trifluoroacetic acid (TFA), solution of
hydrobromic acid in acetic acid (HBr/AcOH, 33 wt %), benzyl alcohol
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Table 1. Characteristics of Synthesized Poly(5-Z-amino-δ-valerolactone-co-ε-caprolactone) and Poly(5-NH3 -δ-valerolactone-
co-ε-caprolactone)
a
composition NH-VL/ε-CL (%)
molecular weight (g/mol)
thermal properties (°C)
b
c
+d
3
copolymer
f NHZVL
FNHZVL
FNH
M
̅
before dep.
M /M
M after dep.
M /M
n
Tm
Tg
̅
̅
̅
̅
̅
w
w
n
n
w
P1
P2
P3
P4
P5
100
75
100
70
40
30
8
100
68
38
28
12
7000
1.20
1.15
1.19
1.20
1.20
5600
6200
6000
7500
5000
1.07
1.16
1.12
1.19
1.69
74
−22
−28
−28
−30
9500
none
none
none
50
11 000
12 300
8600
25
10
a
b
c
+
Thermal properties of poly(5-NH3 -δ-valerolactone-co-ε-caprolactone) with Br− as counterion. Calculated using eq 1. Calculated using 1H NMR
d
1
spectra before deprotection. Calculated using H NMR spectra after deprotection.
formulas, ethanol, and water ratios used for the analyses, isosbestic
points, water solubility, log P, and log D values are given in the
Supporting Information.
proliferation was evaluated after 2, 4, and 7 days by MTT assay.
Nonadhesive cells were removed from the TCPS at scheduled time
points. The TCPS was washed with DPBS, and an MTT solution (250
μL, 1 mg/mL in DPBS) was added. After 3 h of MTT incubation, the
reagent was removed and washed with DPBS, and isopropyl alcohol
was added to solubilize the formazan. The formazan was measured at
570 nm on a spectrophotometer multiplate reader (Victor X3
multilabel plate reader, PerkinElmer). All data points and standard
deviations were derived from triplicates.
Hemocompatibility. Hemocompatibilty was evaluated by assess-
ing whether red blood cells (RBCs) in contact with polymer solutions
underwent any hemolysis, hemagglutination, or morphological
changes. All tests were conducted in accordance with previously
described protocols.33,34 Wistar rats were housed in the Nımes
̂
University experimental research center. The drawing of blood
samples was accepted by the University’s Animal Research Ethics
Committee (CEEA-LR-1010). Polymer solutions were prepared in
Tris-buffer at concentrations of 2 and 10 mM with regards to the
amino groups. Rat venous blood was drawn onto disodium
ethylenediaminetetraacetic acid salt (EDTA) for anticoagulation, and
all RBCs were used within 5 h of blood collection. After centrifugation
at 1980g, autologous anticoagulated plasma was collected, the buffy
coat was removed, and the RBCs were washed three times with Tris-
buffer. After the last wash, 0.4 mL of packed RBCs was suspended in
0.6 mL of the selected experimental medium, namely, autologous
anticoagulated plasma, or Tris-buffer, to yield a 40% hematocrit. The
suspensions were incubated at 37 °C for 10 min, and 0.1 mL aliquots
of the different polymer solutions were then added. Controls were
prepared in a similar manner with Tris-buffer alone or PLL (Mn = 80
000 g/mol, 10 mM of amino groups) as negative and positive controls,
respectively. The final mixtures were further incubated for 15 min at
37 °C.
Synthesis of Tertiobutyl-4-(benzyloxycarbonylamino)-5-hy-
droxypentanoate (2). Selective reduction of the main chain
carboxylic acid group is based on the formation of an activated ester
using BOP as reagent, as previously described in the literature.36
Typically, DIPEA (1.17 mL, 7.1 mmol) and a BOP solution (2.87 g,
6.5 mmol) in 10 mL of THF were slowly added to a stirred suspension
of Z-Glu(OtBu)OH 1 (2.02 g, 6 mmol) in 20 mL of THF at room
temperature. After 10 min of stirring, NaBH4 (1.1 g, 30 mmol) was
slowly added to the reaction medium, which was then stirred for 2 h at
room temperature. The resulting mixture was then diluted in CH2Cl2
(150 mL) and washed with a 5% HCl solution (5 × 100 mL), followed
by an NaHCO3 saturated solution (3 × 100 mL) and brine (3 × 100
mL). The organic phase was then dried over MgSO4 and concentrated
under reduced pressure to yield a clear oil. Final purification of
compound 2 was obtained by flash column chromatography using
first-step CH2Cl2 to elute byproduct and second-step AcOEt to
1
recover pure 2 with a 90% overall yield. H NMR for 2: (300 MHz;
Possible hemagglutination and morphological changes were
evaluated by observing the RBCs under a microscope after 15 min
of incubation. Possible hemolysis was evaluated by centrifuging the
RBC suspensions for 15 min and separating the supernatant. We then
prepared 1 % solutions of the supernatant in Tris-buffer, and the
hemoglobin content was calculated from spectrophotometer measure-
ments at λ1 = 576 nm, λ2 = 560 nm, and λ3 = 592 nm.35 The samples
containing Tris-buffer as medium were measured against Tris-buffer,
and the samples containing plasma as medium were measured against
a 1% solution of pure plasma in Tris-buffer. The total amount of
hemoglobin initially present in the RBC suspension was determined by
preparing a 40% hematocrit solution in distilled water and processing
this as previously described. After centrifugation, the supernatant was
diluted for values to fall within the linear range of the
spectrophotometer standard plot. The hemoglobin released (rHb)
was defined as the percentage of hemoglobin present in the
supernatant compared with the total hemoglobin initially present in
the RBC suspension.
Cytocompatibility. Mouse L929 fibroblasts (L929) were cultured
in MEM containing 10% horse serum, penicillin (100 μg/mL),
streptomycin (100 μg/mL), and Glutamax (1%). Polymers were
disinfected by dissolution and incubation in ethanol for 24 h. The
ethanol was then evaporated off at room temperature, and stock
solutions of 1 mg/mL were prepared in culture medium. Other
concentrations were obtained by diluting the stock solutions in culture
medium. The in vitro cytocompatibility of the polymers was assessed
by following the proliferation of L929 fibroblasts grown in 24-well
TCPS in culture medium containing polymer concentrations of 1 μg/
mL to1 mg/mL. In all, 3000 cells were seeded for each test, and
CDCl3): δ = 7.3 (m, 5H, Ph), 5.1 (m, 1H, NH), 5.0 (s, 2H, OCH2Ph),
3.8−3.7 (m, 1H, CH-NHZ), 3.6−3.5 (m, 2H, CH2−OH), 2.3−2.2 (m,
2H, CH2−CH2-CH), 1.9−1.7 (m, 2H, CH2-COOtBu), 1.4 (s, 9H,
tBu). HPLC for 2: 1.52 min. Calculated monoisotopic mass for 2
(C17H25NO5): 323.17 g/mol; ES-MS (70 eV, m/z) found for 2: 324.3
[M+H]+, 346.3 [M+Na]+.
Synthesis of Benzyl 6-Oxotetrahydro-2H-pyran-3-ylcarba-
mate (5-Z-Amino-δ-valerolactone) (3). Compound 2 was
lactonized via simultaneous removal of the tertiobutyl group and
cyclization of the resulting intermediate to yield 3. In a typical
experiment, 2 (2 g) was dissolved in a mixture of TFA (10 mL) and
CH2Cl2 (10 mL) and stirred for 3 h at room temperature. After
reaction completion, cold water (80 mL) and CH2Cl2 (80 mL) were
added. The organic layer was washed with water (5 × 50 mL), dried
over MgSO4, then filtered and concentrated under reduced pressure.
Column chromatography with AcOEt/Et2O (5:5) yielded compound
3 (0.85 g, 55%) as white crystals of at least 98% purity. mp 65 °C
(from DSC). 1H NMR for 3: (300 MHz; DMSO-d6): δ = 7.6 (m, 1H,
NH), 7.4−7.3 (m, 5H, Ph), 5.05 (s, 2H, OCH2Ph), 4.3−4.2 (m, 1Ha,
CH2−O), 4.1−4.0 (m, 1Hb, CH2−O), 3.9−3.8 (m, 1H, CH−NHZ),
2.6−2.4 (m, 2H, CH2−CO), 2.1−2.0 (m, 1Ha, CH2−CH2−CH), 1.8−
1.7 (m, 1Hb, CH2−CH2−CH). 13C NMR for 3: (75 MHz, DMSO-
d6): δ = 171 (s, C(O)O), 156.2 (s, NHC(O)O), 137.5 (s, CH2CCH),
128.8−127.6 (m, CH), 70.6 (s, CH2O), 65.9 (s, CH2C(O)ONH), 44.3
(s, CHNH), 27.5 (s, CH2CHNH), 24.5 (s, CH2C(O)). HPLC for 3:
1.23 min. Calculated monoisotopic mass for 3 (C13H15NO4): 249.10
g/mol; ES-MS (70 eV, m/z) found for 3: 250.2 [M+ + H], 499.4 [2M+
+ H].
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+
+
Scheme 1. Synthesis of 5-Z-Amino-δ-valerolactone, Poly(5-NH3 -δ-valerolactone), and Poly(5-NH3 -δ-valerolactone-co-ε-
caprolactone)
a
a
(i) DIPEA, BOP, THF, 30 min, RT and NaBH4, 2 h, RT; (ii) TFA/CH2Cl2 (1:1), 3 h, RT; (iii) BzOH, Sn(Oct)2, 24h, 110°C; and (iv) CH2Cl2/
HBr/AcOH, <1 h, RT.
+
+
+
Synthesis of Poly(5-Z-amino-δ-valerolactone-co-ε-caprolac-
tone) (4′). In a typical procedure, 3 (500 mg, 2 mmol) and ε-CL (76
mg, 0.67 mmol) were added to a Schlenk flask under nitrogen
blanketing (Table 1, entry 2). Catalyst (Sn(Oct)2 at 0.64 M in THF;
210 μL, 0.13 mmol) and initiator (BzOH at 2,6 M in THF; 5 μL, 1.34
× 10−5 mol) were added through the septum. The reaction was carried
out for 24 h at 110 °C under nitrogen blanketing and atmospheric
pressure. The reaction was stopped by adding 3 mL of THF, followed
by precipitation into cold methanol. The polymer obtained was filtered
and dried at room temperature under vacuum. Monomer conversions
were determined by 1H NMR analysis via the signal shift of the
methylene protons of the benzyl group from 5.05 to 4.95 ppm in 3 and
in the copolyesters, respectively, and the shift of the methylene
protons from 4.20 to 4.00 ppm in ε-CL and in the copolyesters,
respectively. Comonomer ratios in the copolyesters were determined
by 1H NMR analysis using the intensities of the signals at 4.95 (s, 2H,
OCH2Ph, 3) and 4.00 ppm (t, 2H, OCH2−CH2, ε-CL and m, 1Ha,
CH2−O, NHZVL). 1H NMR (300 MHz; DMSO-d 6): δ = 7.40−7.30
(m, 5H, Ph, NHZVL), 7.20 (m, 1H, NH, NHZVL), 4.95 (s, 2H,
OCH2Ph, NHZVL), 3.95−4.00 (m, 1Ha, CH2−O, NHZVL and 2H,
CH2−O, ε-CL), 3.85 (m, 1Hb, CH2−O, NHZVL), 3.65 (m, 1H, CH−
NHZ, NHZVL), 2.2−2.3 (m, 2H, CH2−CO, NHZVL and 2H, CH2−
CO, ε-CL), 1.65 (m, 1Ha, CH2 −C H2−CH, NHZVL), 1.50 (m, 1Hb,
CH−NH3 , NH3 VL), 2.60 (m, 2H, CH2−CO, NH3 VL), 2,30 (m,
+
2H, CH2−CO, ε-CL), 1.90 (m, 2H, CH2 −C H2−CH, NH3 VL), 1.55
(m, 4H, CH2−CH −CH2, ε-CL), 1.30 (m, 2H, CH2−C H2−CH2, ε-
2
CL).
RESULTS AND DISCUSSION
■
Copolymers Synthesis. Although cationic aliphatic
copolyesters are potentially of interest in a range of biomedical
applications including drug and gene delivery, almost none can
be found in the literature. This is mainly due to the difficulties
encountered during the synthesis and purification of the
corresponding amino-functional lactones and in some cases to
their low polymerizability. Until very recently, only two
examples of polyserinate and poly(aminated lactide) had been
described.27,29 Moreover, these cationic polyesters gave rise to
only very limited applications, that is, mainly physicochemical
model study on IPEC stability.37 Our group recently reported
the convenient synthesis of a new amino-functional lactone,
namely, 5-Z-amino-δ-valerolactone (5-NHZVL), which can be
obtained in only two steps and can be easily purified, giving an
overall yield of 50% (Scheme 1).32
Homopolymerizations were carried out, and the feasability of
copolymerizations with ε-CL was demonstrated. Because we
were interested in using these degradable cationic copolyesters
for drug or gene delivery, we undertook a more systematic
study for purposes of controlling the composition of the
copolymers. Copolymerizations of 5-NHZVL were carried out
with ε-CL contents ranging from 0 to 70% (Table 1). Ring-
opening polymerizations were run in bulk with Sn(Oct)2 as
catalyst and BzOH as initiator. In accordance with our previous
report, all polymerizations were conducted for 24 h with
temperature held at 110 °C to avoid side reactions. More than
90% conversions were obtained for all copolymers. Exper-
imental molar compositions calculated from NMR measure-
ments were in good agreement with theoretical compositions.
Copolymer compositions were therefore close to target (Table
1). Molecular weights of about 10 000 g/mol and low
polydispersity indexes (1.15 to 1.20) were obtained for all
compositions. This narrow distribution indicates faster
CH2 −C H2−CH, NHZVL and 4H, CH −CH2−CH2, ε-CL), 1.30
2
(m, 2H, CH2 −CH2−CH , ε-CL).
2
Theoretical compositions were calculated using the following
equation
[NHZVL]
[ε‐CL] + [NHZVL]
fNHZVL
=
(1)
Synthesis of Poly(5-NH3+-δ-valerolactone-co-ε-caprolac-
tone) (5′). Z-amino protected polymer was deprotected under acidic
conditions to yield the corresponding polyester with free ammonium
pendent groups. Typically, a solution of hydrobromic acid (3 equiv to
Z group) in acetic acid (33 wt %) was slowly added to a stirred
suspension of copolymer (10% w/v) in CH2Cl2. Deprotection was
carried out at room temperature and was stopped after ca. 20 min. The
reaction medium was precipitated and washed with cold Et2O. The
polymer obtained was then dissolved in distilled water and dialyzed
against water before freeze-drying to yield the final copolymer as a
1
white to slightly orange powder depending on composition. H NMR
+
(300 MHz; DMSO-d6): δ = 4.20 (m, 1Ha, CH2−O, NH3 VL), 4.10
+
(m, 1Hb, CH2−O, NH3 VL), 4.00 (2H, CH2−O, ε-CL), 3.5 (m, 1H,
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initiation than propagation and faster propagation than transfer
or termination reactions. The statistical nature of the
copolymers was confirmed by 13C NMR analyses by
comparison of the signals corresponding to the carbonyl
groups in PCL and P(NHZ-VL) homopolymers and the
present copolymers (Supporting Information).
with a melting temperature (Tm) of ∼60 °C and a glass-
transition temperature (Tg) of about −60 °C.39 We previously
reported that P(NHZVL) is also semicrystalline with a Tm of
180 °C and a Tg of ∼45 °C.32 By contrast, all deprotected
+
polymers, including poly(NH3 -VL), were found to be
amorphous. Homopolymer P1 has a Tg of −22 °C and
copolymers P2−P4 have a Tg of about −30 °C. A transition
such as this from semicrystalline to amorphous copolymers has
already been described by Yan et al. for copolymers of ε-CL
and γ-amino-ε-CL (γAεCL).31 Once the aminated ester units
content of the copolymer exceeds 20%, no melting temperature
can be obtained because of the statistical distribution of the two
monomer types. This observation corroborates our results
obtained in copolymers containing between 28 and 100%
functionalized units. Additionally, because our copolymers were
in the bromhydrate form, steric hindrance of the bromide ion
would be expected to hinder the crystallization process. It is
interesting to note that the Tg values found for the copolymers
are close to the ones calculated based on the Fox law in
accordance with the expected random structure of the
copolymers (Supporting Information).
The Z protecting group was removed under acidic conditions
using HBr/acetic acid solutions. Quantitative deprotection was
confirmed by 1H NMR, with no protecting group being
detected in the final copolymers, as shown by the absence of a
signal at 7.4 to 7.3 ppm and 4.95 ppm. Given that our aim was
to prepare cationic copolyesters of controlled composition, a
critical point was to avoid degradation and any molar
composition drift during the deprotection step. Table 1 gives
the characteristics of the deprotected copolymers. Ratios
between the two monomers were the same before and after
deprotection. A negligible decrease corresponding to 2% of
aminated-units content was observed for all compositions
(Table 1). The only exception was copolymer 5, where a slight
increase was observed. This variation may be ascribed to the
low aminated units content that resulted in less well-defined
peaks and lower NMR signals intensities than with ε-CL units,
making precise signal integration more difficult. The molecular
weight of the deprotected copolymers was also determined in
addition to their molar composition. All copolymers had a
molecular weight of 5000 to 7500 g/mol. Figure 1 shows
Water Solubility and Aggregation Behavior. A draw-
back of conventional, degradable aliphatic polyesters, for
instance, PLA and PCL, is their lack of functionality and
hydrophilicity. Chemical modification of these polyesters and
copolymerization are two strategies that can be used to
overcome this limitation and generate water-soluble PCL
copolymers.9,11,40 In the study reported here, we were
interested in evaluating the water solubility of cationic
+
poly(ε-CL-co-NH3 -VL) in relation to their composition.
With the notable exception of copolymer P5 that contains
12% of ammonium groups, all of the copolymers were found to
be soluble in distilled water at the tested concentration of 1%
w/v. The pH of the resulting copolymer solutions was 2.5 to 3.
Phosphate and Tris-buffer solutions were prepared to evaluate
+
the possibility of using poly(ε-CL-co-NH3 -VL) at physiological
pH. Only homopolymer P1 was found to be soluble at pH 7.4.
Copolymer P2, containing 68% of ammonium groups, was only
dispersible (Supporting Information), whereas the other
copolymers with lower ammonium group contents were
insoluble in buffered solutions. Partial screening of the cationic
charges by the salts present in the buffers explains the
insolubility of copolymers having less hydrophilic functions.41
Additional solubility studies were therefore conducted in water.
Considering block copolymers, self-assembly occurs when
the critical micellar concentration (CMC) is reached. However,
random amphiphilic copolymers may behave in three different
manners. They may remain solubilized either as isolated
macromolecules with an extended shape or in the form of small
intrapolymer aggregate, or they may be water-soluble in the
form of larger interpolymer aggregates. These morphologies
depend on polymer properties, composition (hydrophilic/
hydrophobic balance), architecture (random or gradient,
pending groups or not, etc.), and external conditions (pH,
presence of salts, etc.). Solutions of copolymers P2 to P4 were
prepared in Milli-Q water, and their conductivity was measured.
Such conductometric methods have been used in the past to
determine critical aggregation concentrations (CACs) by
detecting the slope break in the conductivity versus
concentration plot.42 CACs for all tested copolymers were ca.
0.05% (w/v) in water (Supporting Information). This CAC
value was further confirmed by zeta potential measurements
with stable values obtained above the same concentration of
Figure 1. Molecular weights (M ) of copolymers before and after
̅
n
deprotection and comparison between the theoretical (theo) and
+
experimental (exp) final molecular weights of poly(5-NH3 -δ-
valerolactone-co-ε-caprolactone).
copolymer molecular weights before deprotection and after
deprotection and the expected molecular weight calculated
considering the loss of the protecting group and addition of the
bromide counterion (eq 2). This comparison clearly demon-
strates that limited degradation of the copolymer backbones
occurred during acid treatment. Degradation was nevertheless
observed, corresponding to ca. 0.7 to 1 ester bond hydrolysis
per chain for all initial compositions. This is acceptable
compared with the previously reported degradation sensitivity
of polyserinate to deprotection.26,38
M
̅
n before dep.
M
̅
=
n after dep. theo.
(x × MNHZVL + (1 − x) × MCL
)
+Br−)VL
× (x × M(NH
+ (1 − x) × MCL
)
(2)
3
.
The thermal behavior of the different copolymers post-
deprotection was studied by DSC analysis (Table 1).
Homopolymer PCL is known to be a semicrystalline material
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0.05% (w/v), indicating that aggregates were formed
(Supporting Information). In addition, the very existence of a
CAC shows that our random copolymers are prone to
intermolecular rather than intramolecular aggregation. This
result is in good agreement with literature data: random PAA-
co-DodPMAm copolymers behave differently depending on the
distance (d) between the charged groups and the backbone.19
More precisely, preferential interpolymer aggregation takes
place for charges located near the backbone (d ≈ 1.5 to 2 Å),
whereas intrapolymer aggregation is found if the charges are
located farther from the polymer chain (d ≈ 8 to 9 Å). With
only one covalent bond between the ammonium group and the
surrounded by a corona of swollen loops formed by hydrophilic
parts of the polymer. The fact that our nanoaggregates were 30
to 50 nm in size and that two populations were found to coexist
in equilibrium is in agreement with the results reported for
other systems. For example 80:20 copolymers of N-(2-
hydroxypropyl) methacrylamide and lauryl methacrylate
exhibited mean diameters of ca. 60−70 nm, whereas more
hydrophobic poly(hydroxyethyl methacrylate) derivatives con-
taining 25% esterified units exhibited mean diameters of 35
nm.20,43 The presence of larger aggregates may be explained as
described in Scheme 2. Depending on their amphiphilicity,
dispersed random amphiphilic copolymers can rearrange to
form “shuttlecock” morphologies with a corklike hydrophobic
head and a cone-shaped hydrophilic tail.20 These unstable
structures assemble to generate spherical micelle-like aggregates
with diameter ≈ 30−50 nm. Finally, these aggregates can
themselves further aggregate to generate larger aggregates with
diameters ≈ 100−250 nm. The coexistence of these aggregates
was confirmed by TEM analysis of copolymer solutions as
shown in Figure 3. Noncondensed aggregates can be seen in
equilibrium with small aggregates of <Dh> ≈ 80 nm and larger
ones exhibiting a nonspherical shape and of <Dh> ≈ 250 nm.
Encapsulation of Hydrophobic Compounds. Our group
recently reported the use of nondegradable, random
amphiphilic anionic polyelectrolytes to encapsulate clofazimine,
a highly hydrophobic drug which is insoluble in water except in
a narrow range of pH values close to pH 3. Poly(methy vinyl
ether-alt-maleic acid) (PMVEMAc) hydrophobized with 35%
dodecyl groups was used. Interestingly, a strong synergetic
effect stemming from both electrostatic and hydrophobic
interactions between the polymer and the drug led to the
solubilization of up to one molecule of drug for two mononeric
units.21 Bearing this result in mind, we decided to test our
copolymers for their capacity to encapsulate various HCs. All of
the compounds chosen are very hydrophobic with log P and log
D values of more than 4. Figure 4 shows the solubility of the
chosen HCs in water containing 1% (w/v) copolymer. The
amino groups on the copolymers were protonated under these
conditions (pH 2.5). We first studied the incorporation of
pyrene (Py), a well-known neutral hydrophobic probe
considered to be water-insoluble (solubility of 0.135 mg/L).
As shown in Figure 4, its solubility was greatly dependent on
+
backbone, poly(ε-CL-co-NH3 -VL) copolymers not surprisingly
belong to the first category. It should also be noted that the
CAC measured for our linear copolymers, 5 × 10−1 mg/mL, is
far higher than that determined for other random amphiphilic
copolymers with pendent hydrophobic groups (lauryl groups)
where CAC values of ca. 5 × 10−3 mg/mL are found.43 The
zeta potential value is also an important aggregation character-
istic because it can affect aggregate stability. In theory, more
pronounced (positive or negative) zeta potential values tend to
stabilize aggregates suspensions as a result of electrostatic
repulsion between aggregates with electric charge of the same
sign.44 We found a zeta potential of 30 mV, which may indicate
that the aggregates formed by copolymers P2−P4 will be
stable.
Aggregate sizes were determined by dynamic light scattering
experiments carried out on copolymer solutions above the
CAC (1 mg/mL). Comparisons between the hydrodynamic
diameters (<Dh>) obtained in the intensity and number modes
+
suggested that poly(ε-CL-co-NH3 -VL) formed two aggregate
populations in an equilibrium. The first population had a <Dh>
of 30−50 nm, as identified by the <Dh> in number mode, and
coexisted with larger aggregates that had a <Dh> of 100 to 250
nm, as identified by the <Dh> in intensity mode (Figure 2).
+
poly(ε-CL-co-NH3 -VL) composition. Whereas a low Py
+
concentration (3 mg/L) was found when poly(ε-CL-co-NH3 -
VL) containing 70% ammonium groups was used, this steadily
increased with copolymer hydrophobicity to reach 142 mg/L
+
for poly(ε-CL-co-NH3 -VL) containing 30% of ammonium
groups. This corresponds to a remarkable 1 000-fold increase
above the native water solubility. The same trend was obtained
for the other tested HCs, namely, EE and FA, with solubilities
increasing by a factor 140 and 155, respectively. Two
conclusions can be drawn from these results. First, it appears
Figure 2. Mean hydrodynamic diameters (<Dh> int: intensity mode;
<Dh> numb: number mode) of the poly(ε-CL-co-NH3 -VL)
copolymers in relation to their composition.
+
+
that the amphiphilic balance of the poly(ε-CL-co-NH3 -VL)
Interestingly, whereas the small aggregates did not vary
significantly in size with copolymer composition, the large
aggregates tended to increase in size as the proportion of
ammonium groups increased. As discussed elsewhere,19 this is
related to the balance between hydrophobic interactions of the
copolymers must be finely tuned if optimum solubilization is to
be obtained. This is particularly evident when considering the
solubility gap between the copolymers containing 30 and 40%
ammonium groups. This apparently small 10% composition
change had drastic effects, with solubilities increasing by a
factor 10 to 30. Second, it should be noted that because of the
linear nature of the ε-CL hydrophobic units, higher hydro-
phobization, of ∼70%, is necessary to generate hydrophobic
nanodomains able to solubilize HCs efficiently. By contrast,
only 35% hydrophobic pendent units were sufficient in the case
+
CL units and electrostatic repulsion of the NH3 -VL units.
When the hydrophobization level is low, charge repulsion
prevails over hydrophobic association, yielding larger aggre-
gates. Previous studies suggest that aggregates of random
amphiphilic copolymers have micelle-like shapes.20,43,45,46
These aggregates consist of a dense hydrophobic core
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Scheme 2. Schematic Illustration of the Morphological Transitions in Nanoaggregates Self-Assembled from Amphiphilic
+
Random Poly(ε-CL-co-NH3 -VL) Copolymers
+
Figure 3. TEM images of poly(ε-CL-co-NH3 -VL) (P3) water solutions at 0.2%. Scales bars are (a) 50 nm and (b,c) 500 nm.
Figure 4. Solubility of selected hydrophobic compounds in water
containing 1% (w/v) copolymers (solution pH 2.5) in relation to
copolymer composition.
Figure 5. Comparative solubilities of 1-pyrenecarboxylic acid in
relation to pH and copolymer composition.
contrary to our expectations, PyCa in its carboxylate form
(PyCa-O−) was less incorporated in the nanoaggregates than
Pyca in its carboxylic form (PyCa−OH). In more detail,
whereas PyCa−OH solubility increased by a factor 5 between
pure water and solution of copolymer containing 40%
ammonium groups, PyCa-O− solubility was increased by only
a factor 2. Under the same conditions, Py solubility was
increased by a factor 100. These results may indicate that the
driving force of the solubilization induced by our systems
mainly arises from hydrophobic associations and that, if
electrostatic interactions do exist, they play only a minor role
in the solubilization process. The relatively high water solubility
of PyCa (0.6 mg/mL) compared with that of Py (0.135 mg/L)
and the copolymer linearity may be responsible for these
findings. Further work on this specific point with other
ionizable non-water-soluble HCs should be undertaken.
of PMVEMAC derivatives.21 This first series of measurements
was carried out at pH 2.5, which is lower than the pKa of all of
the tested compounds. In particular, at this pH, FA (pKa = 3.7)
was in its carboxylic acid form and could not form electrostatic
interactions with the cationic copolymers.
In an attempt to determine whether a synergetic effect was
occurring between the hydrophobic and electrostatic inter-
actions in our copolymers, another compound, 1-pyrenecarbox-
ylic acid (PyCa, pKa = 3,5, solubility at pH 2 = 0.6 mg/L), was
tested. As previously mentioned, water solutions of our
copolymers had a pH of ca. 2.5 and could be increased to 4
without precipitation. Figure 5 illustrates the results obtained.
As expected, PyCa solubility increased by a factor 2 to 3 when
pH increased from 2.5 to 4 because of the more soluble
carboxylate groups. Like for the other tested compounds, PyCa
solubility increased with the ε-CL content in the copolymer
and the hydrophobicity of the copolymers. However, and
Hemo- and Cyto-Compatibilty. Cationic polymers tend
to be toxic, particularly due to their ability to agglutinate RBCs
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and to induce their lysis. This toxicity is the major obstacle to
the development and intravenous use of cationic polymeric
carriers, especially for gene delivery applications. The toxicity of
cationic polymers depends on their molecular weight, their
concentration, the pK of ionizable groups (tertiary or
quaternary ammoniums), and the chemical structure of the
polymer backbone.33 As a consequence, one strategy to
overcome this limitation consists of decreasing the molecular
weight of the polycations or associating them with degradable
spectrophotometric analysis. As shown in Figure 7, hemolysis
was in all cases lower in plasma than in Tris-buffer solutions.
structures.47,48 Because poly(NH3 -VL) has a novel chemical
+
structure, we were interested in testing the hemocompatibility
of this degradable polymer in accordance with a previously
described methodology that considers hemagglutination and
hemolysis.33 Figure 6 shows the hemagglutination of RBCs and
Figure 7. rHb expressed as percentage (%), measured in the
supernatant of RBC suspensions in Tris-buffer and in relation to
polymer solutions added to these suspensions. Values represent the
mean SEM of at least three experiments per polymer solution.
This is due to the presence of proteins in the plasma that
interact with the polymers.34,51 In plasma, rHb was 5% with
+
PLL and only 1.6 and 2.2% for poly(NH3 -VL) at 2 and 10
mM, respectively. These low values, like the aggregation
experiments, confirm that our cationic polyesters are
hemocompatible. It should be noted that PLL, used here as a
positive control, is known to induce moderate hemolysis and
that other polycations, such as poly[thio-1-(N,N-diethyl-
aminomethyl)ethylene] polycations, can induce up to 40%
hemolysis under these conditions, which confirms the low level
Figure 6. RBCs suspended in plasma: (a) without polymer, (b) in the
+
presence of 10 mM 80 kDa PLL, (c) in the presence of 2 mM 5 kDa
of in vitro hemolysis induced by our poly(NH3 -VL).
+
+
poly(NH3 -VL), and (d) in the presence of 10 mM 5 kDa poly(NH3 -
Finally, our structure might be compared with poly(ε-lysine)
VL). The scale bar represents 25 μm.
+
(PεL) which, like poly(NH3 -VL) but unlike PLL, possesses an
amino group close to the polymeric backbone. No significant
hemagglutination or hemolysis is observed with PεL.33 These
results, combined with those described in the study conducted
here on poly(NH3 -VL), tend to confirm that the chemical
nature and structure of polycations are key parameters that
drive their overall hemocompatibility.
In a move to characterize further the biocompatibility of
these cationic copolyesters, we tested their cytocompatibility
with L929 fibroblasts. The effect of both concentration and
composition was studied. Figure 8 shows the proliferation
their morphological changes in plasma containing cationic
copolymers. Hemagglutination is thought to be due to
electrostatic interactions between the positively charged
polycations and the negatively charged RBC membrane.
Whereas the positive control, consisting of high-molecular-
+
weight PLL (10 mM), induced very marked hemagglutination
+
with almost no visible isolated RBC, poly(NH -VL) with M =
̅
3
n
5000 g/mol was found to be very well-tolerated. More
specifically, no difference was seen between the polymer-free
negative control and the RBC suspension containing 2 mM
+
poly(NH3 -VL). All RBCs were well-separated, and no
hemagglutination or deformed erythrocytes were visible. At
the higher concentration used with the PLL positive control
(10 mM), a few RBCs started to agglutinate and form small
isolated islets. Some morphological changes were also visible
with a small proportion of the erythrocytes converting to
echinocytes. However, according to previous studies on cationic
polymers carried out by Moreau et al.,33,34 these results
correspond to a low level of hemotoxicity and confirm that
+
poly(NH3 -VL) presents no acute hemotoxicity and might be
considered for in vivo testing. Also, the polymer’s degradability
should guarantee decreasing hemotoxicity over time because
smaller macromolecules tend to aggregate less than the larger
ones.49,50 RBC hemolysis was also evaluated in Tris-buffer and
plasma. PLL was again used as positive control, and the
percentage of hemoglobin (rHb) released was measured by
Figure 8. Proliferation profiles of L929 fibroblasts in the presence of
increasing concentrations of poly(NH3 -VL).
+
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profiles of L929 fibroblasts in the presence of increasing
concentrations of poly(NH3 -VL). Two criteria were assessed,
These aggregates are able to solubilize highly hydrophobic
compounds, increasing their solubility 100 to 1000 fold
depending on the HC employed. Furthermore, solubilization
experiments demonstrated that hydrophobic interactions were
the main factor driving this solubilization. Contrary to what was
observed with anionic random copolymers bearing pendent
hydrophobic groups, no synergy between hydrophobic and
electrostatic interactions was found with our linear systems.
Future work focusing on the use of comonomers bearing
pendent hydrophobic groups should be undertaken to provide
more insight into this finding. Finally, positive hemo- and cyto-
compatibility tests showed that the polycationic copolyesters
may be considered to be suitable candidates for future
development in selected biomedical applications.
+
namely, the presence of cell proliferation and the extent of this
proliferation compared with the polymer-free control culture
medium. We considered that if our copolymer resulted in at
least 50% of the proliferation obtained with the control, then
this copolymer was deemed to be biocompatible. As shown in
Figure 8, fibroblasts proliferation after 7 days was not inhibited
+
by poly(NH3 -VL), and cell proliferation was observed at all
tested concentrations. Proliferation ratios of ∼60% were found
in all cases except for the highest 1 mg/mL concentration that
gave a proliferation ratio of ca. 40%. This showed that
+
poly(NH3 -VL) has a good biocompatibility if used at a
concentrations below 1 mg/mL. It should be noted that this
limit is far higher than the concentrations normally targeted for
drug delivery purposes because it corresponds to ca. 5 g of
polymer injected in an adult with a ∼5 L blood volume. Finally,
the effect of copolymer composition was also assessed. Figure 9
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional NMR spectra, zeta potential and conductometric
plots, and data. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: benjamin.nottelet@univ-montp1.fr.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the French Ministry of Education and Research for
Marc-Alexandre Schott’s fellowship, the Erasmus Mundus
Program for Manuela Paterer fellowship, Sylvie Hunger and
Figure 9. Proliferation profiles of L929 fibroblasts in relation to
polyelectrolyte nature at a 10 mM concentration.
́
Cedric Paniagua for the NMR analyses, and Frank Godiard for
the TEM analyses.
+
shows the results obtained with PLL, poly(NH3 -VL), and a
poly(ε-CL-co-NH3 -VL) containing 70% ammonium units used
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