Nanoconjugate Platforms Development Based in Poly(β,L-Malic Acid) Methyl Esters for Tumor Drug Delivery

Abstract

New copolyesters derived from poly(β,L-malic acid) have been designed to serve as nanoconjugate platforms in drug delivery. 25% and 50% methylated derivatives (coPMLA-Me(25)H(75) and coPMLA-Me(50)H(50)) with absolute molecular weights of 32 600 Da and 33 100 Da, hydrodynamic diameters of 3.0 nm and 5.2 nm and zeta potential of -15mV and -8.25mV, respectively, were found to destabilize membranes of liposomes at pH 5.0 and pH 7.5 at concentrations above 0.05mg/mL. The copolymers were soluble in PBS (half life of 40 hours) and in human plasma (half life of 15 hours) but they showed tendency to aggregate at high levels of methylation. Fluorescence-labeled copolymers were internalized into MDA-MB-231 breast cancer cells with increased efficiency for the higher methylated copolymer. Viability of cultured brain and breast cancer cell lines indicated moderate toxicity that increased with methylation. The conclusion of the present work is that partially methylated poly(β,L-malic acid) copolyesters are suitable as nanoconjugate platforms for drug delivery.

Full text

Hindawi Publishing Corporation
Journal of Nanomaterials
Volume 2010, Article ID 825363, 8pages
doi:10.1155/2010/825363
Research Article
Nanoconjugate Platforms Development Based in
Poly(β,L-Malic Acid) Methyl Esters for Tumor Drug Delivery
Jos ´
ePortilla-Arias,
1Rameshwar Patil,1Jinwei Hu,1Hui Ding,1
Keith L. Black,1Montserrat Garc´
ıa-Alvarez,2Sebasti´
an Mu˜
noz-Guerra,2
Julia Y. Ljubimova,1and Eggehard Holler1, 3
1Department of Neurosurgery, Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, 8631 W. Third Street, Suite 800E,
Los Angeles, CA 90048, USA
2Departament d’Enginyeria Qu´
ımica, Universitat Polit`
ecnica de Catalunya, ETSEIB, Diagonal 647, 08028 Barcelona, Spain
3Institut f¨
ur Biophysik und Physikalische Biochemie, Universit¨
at Regensburg, D-93040 Regensburg, Germany
Correspondence should be addressed to Jos´
ePortilla-Arias,portillaj@cshs.org
Received 29 October 2009; Accepted 21 January 2010
Academic Editor: Chao Lin
Copyright © 2010 Jos´
e Portilla-Arias et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
New copolyesters derived from poly(β,L-malic acid) have been designed to serve as nanoconjugate platforms in drug delivery.
25% and 50% methylated derivatives (coPMLA-Me25H75 and coPMLA-Me50H50) with absolute molecular weights of 32 600 Da
and 33 100 Da, hydrodynamic diameters of 3.0nm and 5.2nm and zeta potential of −15 mV and −8.25 mV, respectively, were
found to destabilize membranes of liposomes at pH 5.0 and pH 7.5 at concentrations above 0.05mg/mL. The copolymers were
soluble in PBS (half life of 40 hours) and in human plasma (half life of 15 hours) but they showed tendency to aggregate at high
levels of methylation. Fluorescence-labeled copolymers were internalized into MDA-MB-231 breast cancer cells with increased
efficiency for the higher methylated copolymer. Viability of cultured brain and breast cancer cell lines indicated moderate toxicity
that increased with methylation. The conclusion of the present work is that partially methylated poly(β,L-malic acid) copolyesters
are suitable as nanoconjugate platforms for drug delivery.
1. Introduction
Biodegradable polymers are suitable materials for the man-
ufacture of various devices which, because of their biocom-
patibility, are widely applied in medicine and pharmacology.
Examples are poly(lactic acid) and (PLA), poly(glycolic acid)
and their copolymers, with a successful 30 years history in
surgical settings [1].
The relatively new biopolymer, poly (β,L-malic acid)
(PMLA), is a carboxylic-functionalized polyester that can be
produced by either chemical synthesis or biological fermen-
tation from the slime mold Physarum polycephalum.Both
α-andβ-structures, either racemic or optically pure, may
be obtained by chemical methods whereas microorganisms
exclusively generate PMLA of extremely high optical purity
[2, and references therein]. The attractive properties in
nanobiotechnology and biomedicine are the lack of in vitro
and in vivo toxicity and non-immunogenicity. PMLA is
a completely biodegradable polymer that is metabolized
to water and carbon dioxide in the citric acid cycle. It is
biocompatible with regard to its limited stability in the
bloodstream thus prohibiting physiologically adverse host
responses after injection. It is chemically convenient to
handle due to its solubility in water and certain organic
solvents and to the reactivity of its pendant carboxylic groups
[2–9].
So far, the most advanced application reported for
PMLA, is the development of “Polycefin”, a nanoconjugate
prototype vehicle for the cellular delivery of antisense
oligonucleotides. It consists of PMLA that harbors antisense
morpholino oligonucleotides and several biochemically
functional units which are chemically conjugated with the
carboxyl groups. These functional units are tumor-specific
antibodies that promote receptor-mediated endosomal

2Journal of Nanomaterials
uptake by the tumor cells, a unit that allows the vehicle
to escape from the endosomal vesicles into the cytoplasm
and another unit that, after cleavage, releases free oligonu-
cleotides. This nanoconjugate can carry several antisense
oligonucleotides or other drugs at the same time and also
simultaneously several antibodies, which can bind specifi-
cally to cell surface antigens during drug delivery [5–9].
Both synthetic and biosynthetic polymalic acids are
spontaneously or enzymatically degraded in aqueous envi-
ronment (see review [2]). Regardless chirality, degradation is
moderate at physiological pH and proceeds rapidly in acidic
(pH <5) and basic (pH >9) solutions [2] by random scission
of the main chain ester bonds to yield malic acid as the
final degradation product [10]. By chemical blocking of the
pendant carboxylic groups, the properties of PMLA could
be changed to slow down its hydrolysis rate. Methylation
with diazomethane has proven to be an efficient method
that produced poly(α-methyl β,L-malic acid) (PMLA-Me)
without significant cleavage of the polyester backbone [11,
12]. While low degrees of methylation resulted in water sol-
uble products, 75% methylation of pendant carboxyl groups
allowed formulation of stable, water insoluble nanoparticles
that could be loaded with proteins for delivery applications
[13].
The soluble PMLA methyl esters containing 25% and
50% of methylated carboxylic side groups (coPMLA-
Me25H75 and coPMLA-Me50H50 )arecharacterizedhereby
investigating their light scattering properties, their degra-
dation in human plasma and their cytotoxicity for several
human cancer cell lines to be used as nanoplatforms for drug
delivery.
2. Materials and Methods
2.1. PMLA Production. PMLA of microbial origin was used
in this work. It was obtained by cultivation of Physarum
polycephalum and subsequent purification as described in
detail elsewhere [14]. The polyacid of NMR purity had
aMw=34 300 Da and a polydispersity Mw/Mn=1.1.
All chemicals of highest purity, including human plasma,
were bought from Merck (Germany) and Sigma-Aldrich
(Germany). Organic solvents were of analytical grade and
used without further purification. Water used for buffer
preparation was double distilled and deionized in a “Milli-
Q” system.
2.2. Esterification. Partial esterification of PMLA was per-
formed as described recently by Portilla-Arias et al. [11]. In
brief, a solution of diazomethane in ether (12.5 meq) was
added to a solution of PMLA in dry acetone (4.3 meq with
regard to malic acid units) in different ratios according to
the esterification degree to be obtained, and the mixture
was left under stirring at room temperature for 1 hour.
Thereactionmixturewasthenevaporatedundervacuum
and the residue was dissolved in a small amount of N-
methyl-2-pyrrolidone and precipitated with cold diethyl
ether. The copolymer was recovered by filtration as a white
powder. Yields for coPMLA-Me25H75 and coPMLA-Me50H50
were 97% and 92%, respectively. The 1H NMR analysis
in deuterated water revealed that the conversion actually
attained in the copolyesters was 20.2, 46.5, which are pretty
close to the nominal values.
2.3. Absolute Molecular Weight, Hydrodynamic Diameter and
Zeta Potential Measurements. The copolymers were charac-
terized with respect to their absolute molecular weight (Mw),
size and ζpotential using a Malvern Zetasizer Nano (Malvern
Instruments, UK). Absolute weight average molecular weight
was calculated with a modification of the Rayleigh equation
that can be used to generate a Debye plot, which is a linear
fit of KC/Rθversus concentration according to the equation
Kc/Rθ=1/Mw+2A2cRθis the Rayleigh ratio of scattered to
incident light intensity, Kis a constant defined by the solvent
and analyte dependent refractive index increment (dn/dc=
0.169 mL/g for PMLA), Avogadro’s number and the solvent
refractive index. cis the particle concentration and A2is the
second virial coefficient [15]. The intercept obtained from
the Debye plot is equal to the inverse of the molecular weight
and the slope is twice the second virial coefficient.
The size was calculated on the basis of noninvasive back-
scattering (NIBS) measurements using the Stokes-Einstein
equation, d(H) =kT/3πηD.d(H) is the hydrodynamic diam-
eter, Dthe translational diffusion coefficient, kBoltzmann’s
constant, Tabsolute temperature and ηthe viscosity. The
diameter that is measured in DLS (Dynamic Light Scattering)
refers to the particle diffusion within a fluid and is referred
to as the hydrodynamic diameter corresponding to the
diameter of a sphere that has the same translational diffusion
coefficient as the particle [16]. The ζpotential was calculated
from the electrophoretic mobility based on the Helmholtz-
Smoluchowski formula, using electrophoresis M3-PALS. All
calculations were carried out by the Zetasizer 6.0 software.
For the molecular weight determination, 5 solutions of the
copolymers in phosphate buffered saline (PBS, pH 7.4) were
generated by serial dilution starting with 4 mg/mL. For the
measurement of the ζpotential, the concentration of the
sample was 2 mg/mL dissolved in water containing 10 mM
NaCl, and the voltage applied was 150V. For the particle
size measurements, the solutions were prepared in PBS
at a concentration of 2 mg/mL, filtered through a 0.2 μm
pore membrane. All the copolymer solutions were prepared
immediately before analysis at 25◦C. Data represent the
mean ±standard deviation obtained for three measure-
ments.
2.4. Copolyesters Stability in PBS and Human Plasma. The
degradation essays in human plasma were carried out at
37◦C with a polymer concentration of 1 mg/mL. The sample
vials were sealed to avoid evaporation and stored at 37◦Cin
an incubator. For the isolation from the plasma, aliquots of
1 mL were extracted with 5 mL of chloroform/ethyl acetate
(1 : 1 v/v). The copolyester contained in the organic phase
was dried and redissolved in PBS and the Mwmeasured by
sec-HPLC (Calibrated with polystyrene sulphonate-sodium
salt standards). Sample preparation with the polymers of
known Mwverified that the isolation had no effect on

Journal of Nanomaterials 3
O
O
O
O
OO
O
O
Me H
xy
coPMLA-Me25H75
coPMLA-Me50H50
x=20.2; y=79.8
x=46.5; y=53.5
Figure 1: Chemical structure of copolyesters studied in the present
work with indication of their contents in methylated and free-
carboxyl malic units.
molecular weights. For comparison, the degradation study
was performed in PBS (pH 7.4) at a concentration of
1 mg/mL for each copolymer. Chromatography was per-
formed on a Hitachi analytical Elite LaChrom HPLC-
UV system and size exclusion column BioSep-SEC-S 3000
column (300 ×7.80 mm) following the elution at 220 nm
wavelength. Molecular weights Mw(t) were plotted as a
function of degradation time with reference to Mw(t=0) at
zero incubation time.
2.5. Cell Lines and Culture Conditions. Primary glioma cell
lines—U-87 MG and T98G—and invasive breast carci-
noma cell lines—MDA-MB-231 and MDA-MB-468—were
obtained from American Type Culture Collection (ATCC)
USA. U-87 MG and T98G cells were cultured in MEM
supplemented with the following ingredients (final concen-
trations): 10% fetal bovine serum, 1% MEM NEAA, 1 mM
sodium pyruvate and 2 mM L-glutamine. For MDA-MB-231
and MDA-MB-468, Leibovitz’s L-15 medium with 10% final
concentration fetal bovine serum was used. Cells were seeded
at 10,000 cells per well (0.1 mL) in 96-well flat-bottomed
plates and incubated overnight at 37◦C in humid atmosphere
with 5% CO2(breast cancer cell lines MDA-MB-231 and
MDA-MB-468 were incubated without CO2).
2.6. Cytotoxicity Test. The copolymers (1 mg/mL and serial
dilutions) were dissolved in culture media and incubated
with cells for 24 hours. Cell viability was measured using the
CellTiter 96 Aqueous One Solution Cell Proliferation Assay
kit (Promega Corporation, Cat. No.PR-G3580). Yellow [3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (MTS) is
bioreduced by cells into formazan that is soluble in the tissue
culture medium. The absorbance reading at 490 nm from
the 96-well plates was directly proportional to the number
of living cells [17]. The viability of the untreated cells was
referenced as 100%. The results shown are the means and
deviations standard of three independent measurements,
calculated with the statistical software GraphPad PRISM 3.0.
2.7. Liposome Leakage Assay. The capability of the copoly-
mers to escape from endosome was measured by the
liposome leakage assay. This method generally assumed to
represent main features of the endosome membrane and
Tab le 1: Physical properties of copolymers.
coPMLA-Me25 coPMLA-Me50
H75 H50
Methylation(a) (%) 20.2 46.5
Mw(b) 32,600 33,100
Mn(b) 26,100 24,300
Tm(c) (◦C) 174 172
Td(d) (◦C) 186 198
Microstructure(e)
Contiguous patches nfMe 3.9 11.8
Contiguous patches ngCOOH 11.5 14.5
R0.3 0.2
(a)Copolymer composition determined by 1H NMR; (b)Weight- and
number-average molecular weight measured by sec-HPLC; (c)Melting tem-
perature measured by differential scanning calorimetry; (d) Onset decom-
position temperature measured at 5% of loss of initial weight; (e)nfand
ngrefer to averaged numbers of methylated and free carboxyl group
within homogeneous sequences, respectively, and (R) refers the randomness
determined by 13C NMR analysis [11].
0 20 40 60 80 100 120 140 160
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
PMLA
coPMLA-(Me25H75)coPMLA-(Me50 H50)coPMLA-(Me25H75)
coPMLA-(Me50H50)
Time (h)
Mw(t)/Mw(0)
Human plasma, 37◦C
PBS (pH 7.4), 37◦C
Figure 2: Degradation at 37◦CinPBS(pH7.4)andhumanplasma
for PMLA and the copolyesters.
gives similar results as the red blood cell lysis method,
because it is less biased by adverse effects of proteins con-
tained in erythrocytes/membranes. Liposome suspensions
were prepared by the extrusion method. Briefly, the mixture
of egg phosphatidylcholine and cholesterol (molar ratio 2 : 1)
dissolved in CHCl3/MeOH (v/v, 2 : 1) was dried under a
stream of nitrogen. The lipid mixture was hydrated with HBS
buffer (5 mM HEPES, 150 mM NaCl, pH 7.4) containing
90 mM calcein, followed by 19 extrusions through 0.1 μm
polycarbonate membrane using mini-extruder (Avanti Polar
Lipids). Serial dilutions were carried out using 95 μLoftwo
buffers of different pH, 137 mM HEPES buffer pH 7.4 and
137 mM citrate buffer pH 5.0. Liposome 5 μL (lipid concen-
tration 200 μM) was added to each sample and the plate was
incubated at room temperature for 1hour. Complete leakage
of calcein (100% reference) was achieved with the addition
of 0.25% (v/v) Triton-X100 solution of respective buffers.

4Journal of Nanomaterials
8 9 10 11 12 13 14 15 16
ab
Retention time (min)
Absorbance
Figure 3: Elution profiles obtained by sec-HPLC for coPMLA-
Me50H50 at time zero (a) and after 24 hours of incubation
(b) in human plasma at 37◦C. Samples were extracted with
chloroform/ethylacetate before chromatography (see Section 2).
Fluorescence of released calcein release was measured using
excitation wavelength 488nm and an emission wavelength
535 nm. The results shown are the means and deviations
standard of three independent measurements, calculated
with the statistical software GraphPad PRISM 3.0.
2.8. Method of Fluorescence Labeling. The copolymers were
fluorescence labeled with rhodamine as follows: Pendant
carboxyl groups of copolymer (1 mmol malyl residues) in
1 mL of dimethyl formamide (DMF) were activated with
amixtureofN-hydroxysuccinimide (NHS, 1 mmol) and
dicyclohexylcarbodiimide (DCC, 1 mmol) dissolved in 2 ml
ofDMFatroomtemperaturefor3-4hoursundervigorous
stirring. 2-Mercapto-1-ethylamine (0.1 mmol) and 0.1 mmol
of dithiothreitol (DTT). were added and the reaction was
completed in 30–40 minutes. After 30 minutes of stirring
in 6 mL of water at room temperature, the mixture was
centrifuged and the clear supernatant passed over Sephadex
G10 columns in water. The product containing fractions
were freeze dryed yielding a white powder. Rhodamine Red
C2maleimide (40 μLof1mg/mLsolutioninDMF)was
added to 2 mg of activated copolymer dissolved in 2 mL of
PBS of pH 5.5 and stirred for 3 hours at room temperature.
The fluorescent polymers were purified over Sephadex PD-
10 columns pre-equilibrated with PBS (pH 7.4).
2.9. Fluorescence Microscopy. MDA-MB-231 cells were
seeded into the microscopic chamber slide. For fluorescence
microscopy study, cells were incubated with rhodamine-
labeled-polymer (1 mg/mL) in fetal bovine serum (FBS) free
medium for 3 hours. The stained cells were washed with PBS
and fixed in 4% paraformaldehyde (PFA) for 15 minutes.
Then the cells were counterstaining with DAPI to visualize
the nuclei and observed under a DM6000 Leica fluorescence
microscope (Wetzlar, Germany). The amount of polymer
0.00001 0.0001 0.001 0.01 0.1 1 10
0
5
10
15
20
25
30
coPMLA-(Me25H75)
coPMLA-(Me50H50)
Concentration (mg/mL)
Leakage (%)
pH 7.5
pH 5
Figure 4: Liposome leakage at pH 5.0 and 7.5 and 37◦C. 100%
leakage is obtained after the addition of 0.25% Triton X100.
uptake was calculated from the rhodamine fluorescence
intensity, with subtracted background and divided by the
area of the cell under study using the Image J1.43c software
from NIH (values averaged over >50 cells). The experiment
was done by triplicate, with three independent preparations
of cells. Mean and deviation standard were calculated with
the statistical software GraphPad PRISM 3.0.
3. Results and Discussion
3.1. Chemical Characterization. Partially methylated poly(β-
l-malic acid) copolymers were synthesized in high yield and
purity by the diazomethane in acetone method. The two
products are denoted as coPMLA-Me25H75 and coPMLA-
Me50H50 , their chemical formulae are depicted in Figure 1
and their properties are shown in Tabl e 1 .Thedegreeof
methylation attained in the two cases (20.2 and 46.5%) in
the reaction was close to the input ratio of diazomethane
to total carboxylic acid (25 and 50%). Molecular weights
of Mw(weight-average) =32,600 Da and 33,100 Da with
polydispersity indexes (Mw/Mn) of 1.3 and 1.1, respectively,
were determined by sec-HPLC for the two copolyesters which
are values comparable to those measured for the original
polymalic acid (Mw=34 300, Mw/Mn=1.1). The slightly
decrease in Mwand increase in polydispersity observed after
methylation suggest partial cleavage during the chemical syn-
thesis. The 13C-NMR analysis revealed that the distribution
of methyl groups along the copolyesters chain is not random.
The average number of contiguous methylated carboxylic
groups indicated hydrophobic patches as opposed to regions
with contiguous free carboxyl groups and interdispersed
free/methylated carboxyl groups. As expected, the length of
hydrophobic sequences was larger for the polymer with the
higher degree of methylation.
3.2. Absolute Molecular Weight, Hydrodynamic Diameter and
Zeta Potential. The values for absolute weight averaged
molecular weight, particle size and zeta potential of the
resulting copolyesters together with PMLA are shown in
Tabl e 2 . The values of molecular weight and polydispersity

Journal of Nanomaterials 5
Tab le 2: Light scattering and zeta potential measurements.
PMLA coPMLA-Me25H75 coPMLA-Me50H50
Molecular weight (Da) 34,200 30,100 31,900
Polydispersity index 1.1 1.22 1.41
2nd virial coefficient A2(mL ·mol/g2) 6.50E-05 2.85E-05 3.50E-08
Hydrodynamic diameter (nm) 3.4 (±0.1) 3.0 (±0.1) 5.2 (±0.1)
Zeta potential (mV) −22.9 (±1.7) −15 (±1.1) −8.25 (±1.3)
0 200 400 600 800 1000
40
50
60
70
80
90
100
110
PMLA
coPMLA-(Me25H75)
coPMLA-(Me50H50)
Concentration (mg/mL)
U-87 MG
Viability (%)
(a)
0 200 400 600 800 1000 1200
40
50
60
70
80
90
100
110
PMLA
coPMLA-
(Me25H75 )
coPMLA-(Me50H50)
Concentration (mg/mL)
T98G
Viability (%)
(b)
0 200 400 600 800 1000
40
50
60
70
80
90
100
110 PMLA
coPMLA-(Me25H75)
coPMLA-(Me50H50)
Concentration (mg/mL)
MDA-MB-231
Viability (%)
(c)
0 200 400 600 800 1000 1200
40
50
60
70
80
90
100
110
PMLA
coPMLA-(Me25H75)
coPMLA-(Me50H50)
Concentration (mg/mL)
MDA-MB-468
Viability (%)
(d)
Figure 5: Cell viability of cultured cells after 24 hours incubation at 37◦Cfordifferent cell lines in the presence of the indicated polyesters.
found by DLS are similar to those obtained by GPC (see
Tabl e 1 ) and corroborate the notion that some cleavage
occurred during synthesis. While the hydrodynamic diam-
eter does not follow a clear trend upon methylation, the
second virial coefficient A2and the ζ-potential correlate well
with the degree of methylation. The second virial coefficient
is a parameter describing the interaction strength between
the molecule and the solvent [16]. The relatively high values
obtained for PMLA and coPMLA-Me25H75 indicate the
ability of these polymers to stay in solution, whereas the
much lower value obtained for coPMLA-Me50H50 indicates
that this copolymer has some tendency for aggregation. The
conclusion is corroborated by the progressive decrease of
negative ζ-potential observed for increasing methylation,
that is, less repulsion and thus higher tendency for aggrega-
tion.
The effects of esterification on solubility are both an
increase in hydrophobicity and a decrease in electrostatic
mutual repulsion between polymers at higher degrees of
methylation. The blocky microstructure of the copolymer
harboring highly hydrophobic domains of contiguously
methylated units could contribute to aggregation by favoring
intermolecular hydrophobic contacts. The polyacid is highly
soluble in water and acetone, coPMLA-Me50H50 is less
soluble in water than coPMLA-Me25H75,andcoPMLA-
Me75H25 (not studied here) has been reported to be water
insoluble [11].
3.3. Copolyesters Degradation in PBS and Human Plasma.
Copolyester degradation was followed by measuring molecu-
lar weights Mwby sec-HPLC as a function of incubation time
(Figure 2). During incubation in PBS (pH 7.4) at 37◦C the
polymers were slowly degraded as indicated by an increase in
the retention time [11]. The measurements for degradation
in serum at 37◦C were complicated by the presence of
proteins which co-eluted and interfered with the polymers
in sec-HPLC, rendering polymer detection impossible. The
degraded polyesters except PMLA could be separated from

6Journal of Nanomaterials
proteins by extraction of the plasma mixture with chloro-
form/ethyl acetate. As an example the chromatograms of
coPMLA-Me50H50 incubated in serum at time zero (a) and
after incubation (b) are compared in Figure 3. This case
once more demonstrates the degradability of PMLA and
its derivatives in physiological conditions. Specific PMLA
hydrolases have been reported for microorganisms [18,19].
As in the case of degradation in PBS (not shown), the elution
profile indicated a single peak. This suggested cleavage
from the ends and not fragmentation at internal cleavage
sites.
Comparison of kinetics in Figure 2 revealed that (i)
degradation in human plasma was faster than in PBS with
half lives of 13 h in plasma for coPMLA-Me25H75 and 20
hours for coPMLA-Me50H50 compared with 45 hours and
50 hours in PBS, respectively and (ii) Half life was lowest
for PMLA and increased with higher levels of methylation in
agreement with the notion that alkylation of the α-carboxyl
group stabilized the main chain ester bond. Hydrolysis was
significantly enhanced by constituents of human plasma,
either by general catalysis or by hydrolytic enzymes, most
likely esterases, such as plasma lipases or cholinesterases [20].
3.4. Membrane Disruption. The membrane disruption activ-
ity of coPMLA-Me25H75 and coPMLA-Me50H50 was mea-
sured by the phosphatidylcholine liposome leakage assay.
The liposomes are filled with calcein, which leaks out if
the liposome becomes destabilized or disrupted. Leakage
was measured at pH 7.5, resembling physiological pH, and
at pH 5.0, resembling pH of late endosomes/lysosomes.
While coPMLA-Me25H75 did not show leakage activity,
coPMLA-Me50H50 was active at concentrations above 0.1
mg/mL and the activity was pH independent (Figure 4).
The finding suggested that membrane leakage was inferred
by methylation of pendant carboxyl groups that increased
hydrophobicity and neutralized negative charges (decrease
in ζ-potential, Tabl e 2). The absence of effect of pH change
indicated that the carboxylic groups of the methylated PMLA
did not protonate in this pH range, or if they did, they had
no effect on the leakage activity.
The role of hydrophobicity together with charge neutral-
ization has been considered in drug delivery to be the mech-
anism for membrane disruption by a variety of molecular
devices [21–25]. In the case of our methylated PMLA we
think that the methylation dependent leakage refers mainly
to the formation of the contiguous, electrically neutral
hydrophobic patches (Tab le 2) which are prone to intrude
into the lipid bilayer and cause the membrane damage. The
membrane disruption activity especially coPMLA-Me50H50
is thought to be useful in the design of nanoconjugates that
can deliver drugs to intracellular targets.
3.5. Cytotoxicity. Partially methylated PMLA contains car-
boxylic groups that can be conjugated to several prodrugs
and in addition to a variety of biologically active units such as
antibodies for targeting or PEG for protection against enzy-
matic degradation and resorption by the reticuloendothelial
system (RES). The pro-drug is activated within the targeted
cell compartment and only then unfolds its cytotoxic or any
other activity.
It is desirable to know whether methylated PMLA as the
platform is itself not toxic. To this end, the in vitro toxicity
of the copolymers was tested. Figure 5 shows the viability
of cultured brain and breast cancer cells: T98G, U-87 MG,
MDA-MB-231 and MDA-MB-468 cells after 24 hours of
incubation as a function of copolymer concentrations.
While PMLA only marginally decreased cell viability, the
copolymers showed toxicity that increased with the degree
of methylation, but the decrease in percentage cell viability
was moderate and above 50% at concentrations ≤1mg/mL.
Effects on cell viability depended also on the type of cell line,
glioma U-87 MG cells, and breast cancer MDA-MB-231 cells
being more affected than glioma T98G cells and breast cancer
MDA-MB-468 cells.
There are two main toxicity mechanisms commonly
considered: (i) toxicity due to physical damage such as
destabilization of membranes and (ii) toxicity resulting from
the degradation products after intracellular uptake. The
possibility of physical damage due to membrane disrupture
of the kind as seen by the liposome leakage assay (Figure 5)
may not be significant since the effect of copolymers on cell
viability is not manifested in the time scale of minutes or a
few hours (results not shown). It is highly likely that toxicity
was the result of methanol formation during degradation. It
is known that degradation in pure deuterated water generates
as main products methanol and L-malic acid [11]. While
l-malic acid is converted into water and carbon dioxide
in the tricarboxylic acid cycle, methanol is known to be
toxic for living cells. In the same line it has been reported
that benzylesters of PMLA were toxic due to release of
free benzylalcohol during degradation [26]. However, for
consideration of the copolymer application in drug delivery,
the short residence times of only a few hours before complete
clearance through the renal system relatives their toxicity in
vivo.
3.6. Cellular Uptake Study. Since we have found that
the copolymers coPMLA-Me25H75 and coPMLA-Me50H50
contained hydrophobic patches that could be active in
membrane disruption, it was important to test whether
the polymers could enter cells in vitro. Rhodamine labeled
copolymers were incubated with MDA-MB-231 cells for
3 hours. The cell’s uptake of the fluorescent polymers
was seen under the fluorescence microscope (Figure 6).
Rhodamine alone did not stain the cells (picture not
shown). The distribution of copolymers-rhodamine in all
cells, appeared to be homogeneous and probably involved
nuclei. The fluorescence intensity was semi quantitatively
measured by triplicate in >50cells,andappearedtobe
1.8 (Standard Deviation=±0.3) folds more intensive for
coPMLA-Me50H50 than for coPMLA-Me25H75 accordingly
to the Image J1.43c software, thus correlating with the
higher degree of hydrophobicity of the higher methylated
polymer. The results are evidences that the copolymers
are highly versatile materials and suitable for various
applications.

Journal of Nanomaterials 7
coPMLA-(Me50H50)coPMLA-(Me25H75)
(a) (b) (c)
Figure 6: Localization of rhodamine tagged copolymers in MDA-MB-231 cells. Panels (a) and (b) separated copolymer-rhodamine uptake
and DAPI staining. Panel (c) The copolymers are indicated by red fluorescence co-localizing with nuclei stained in blue with DAPI.
4. Conclusion
New designs of nanoconjugate drug delivery systems are
introduced here. The systems involve a polymer platform
containing pendant chemically reactive groups to be con-
jugated with molecular units that function in pro-drug
attachment, cell recognition, membrane penetration and
protection. The copolymers coPMLA-Me25H75 and coPMLA-
Me50H50 are biodegradable and biocompatible and its half-
life is limited, so they may minimize adverse host’s responses
and development of liver storage diseases. The copolymers
are endowed with membrane disrupting/penetrating activi-
ties which allow them to deliver drugs directly to intracellular
targets by passing the plasma membrane. By raising the
degree of methylation above 50%, the copolymers become
insoluble and can be used as drug delivering nanoparticles.
These results support the fact that poly (β,L-malic acid) is
a highly versatile material and can be used for the design
of a variety of nanoconjugate platforms for drug delivery
systems.
Acknowledgments
The project was funded by grants from NIH/NCI R01
CA123495 to JYL; MAT2006-13209-C02-02 from CICYT
(Comisi´
on Interministerial de Ciencia y Tecnolog´
ıa) of Spain
to SM and CONACYT-M´
exico (Concejo Nacional de Ciencia
y Tecnolog´
ıa) for fellow granted to J. Portilla-Arias.
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