Synthesis of alkyl-modified poly(sodium glutamate)s for preparation of polymer-protein nanoparticles in combination with N,N,N-trimethyl chitosan

Article abstract of DOI:10.1002/pola.27341

The negatively charged, water-soluble, hydrophobically modified poly(sodium glutamate)s containing different amounts of alkyl grafts were synthesized. First, poly(γ-benzyl-L-glutamate) was prepared by ring-opening polymerization of the corresponding N-carboxyanhydride, which was in the next step aminolysed with octylamine. After removal of the remaining benzyl protective groups, the alkyl-modified poly(sodium glutamate)s [P(Glu-oa)] were obtained and, together with the oppositely charged N,N,N-trimethyl chitosan (TMC), used for the preparation of nanoparticles (NPs) of a recombinant granulocyte colony-stimulating factor (GCSF) protein by polyelectrolyte complexation method. It is observed that, beside electrostatic interaction, the hydrophobic grafts on poly(sodium glutamate)s significantly contribute to association efficiency (AE) with GCSF protein. The addition of TMC solution to the dispersion of GCSF/P(Glu-oa) complexes results in formation of much more defined NPs with high AE and final protein loading.

Full text of DOI:10.1002/pola.27341

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Synthesis of Alkyl-Modified Poly(sodium glutamate)s for Preparation of
Polymer-Protein Nanoparticles in Combination with N,N,N-Trimethyl
Chitosan
1
1
2
2,3
1
ꢀ
David Pahovnik, Milijana Gruji c´ , Mateja Cegnar, Janez Ker cˇ , Ema Zagar
1
National Institute of Chemistry, Laboratory for Polymer Chemistry and Technology, Hajdrihova 19, SI-1001 Ljubljana, Slovenia
2
Lek Pharmaceuticals d.d., Sandoz Development Center Slovenia, Verov ꢀs kova 57, SI-1526 Ljubljana, Slovenia
3
University of Ljubljana, Faculty of Pharmacy, A ꢀs ker cˇ eva 7, SI-1000 Ljubljana, Slovenia
ꢀ
Correspondence to: E. Zagar (E-mail: ema.zagar@ki.si)
Received 5 June 2014; accepted 23 July 2014; published online 6 August 2014
DOI: 10.1002/pola.27341
ABSTRACT: The negatively charged, water-soluble, hydrophobi-
cally modified poly(sodium glutamate)s containing different
amounts of alkyl grafts were synthesized. First, poly(c-benzyl-L-
glutamate) was prepared by ring-opening polymerization of
the corresponding N-carboxyanhydride, which was in the next
step aminolysed with octylamine. After removal of the remain-
ing benzyl protective groups, the alkyl-modified poly(sodium
glutamate)s [P(Glu-oa)] were obtained and, together with the
oppositely charged N,N,N-trimethyl chitosan (TMC), used for
the preparation of nanoparticles (NPs) of a recombinant granu-
locyte colony-stimulating factor (GCSF) protein by polyelectro-
lyte complexation method. It is observed that, beside
electrostatic interaction, the hydrophobic grafts on poly(so-
dium glutamate)s significantly contribute to association effi-
ciency (AE) with GCSF protein. The addition of TMC solution
to the dispersion of GCSF/P(Glu-oa) complexes results in for-
mation of much more defined NPs with high AE and final pro-
tein loading. V
2014 Wiley Periodicals, Inc. J. Polym. Sci., Part
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A: Polym. Chem. 2014, 52, 2976–2985
KEYWORDS: drug delivery systems; nanoparticles; peptides; pro-
teins; polyelectrolytes
1
6,17
INTRODUCTION Polymeric nanoparticles (NPs) have been
example, chitosan/dextrane sulfate,
polyethyleneimine/
1
7,18
17
widely investigated for drug delivery, including oral-delivery
dextran sulfate,
poly-L-lysine/dextrane sulfate, chito-
san/alginate, chitosan/poly(c-glutamic acid),
1
–6
19
20,21
of pharmaceutically bioactive peptides and proteins.
A lot
poly(vinyl
2
2
of attention has been given to NPs prepared from synthetic
biodegradable and biocompatible polymers such as polycap-
pyrrolidone)/poly(acrylic acid), etc. Chitosan is an ideal
candidate for peroral drug-delivery due to its biocompatibil-
ity, biodegradability, muco-adhesion, and ability to transi-
ently open the tight junctions between the intestinal
epithelial cells, which results in facilitate transport of macro-
molecules through epithelia. Recently, the quaternized chi-
tosan derivatives were reported to have even better
permeation-enhancing properties than chitosan itself. Addi-
tionally, NPs based on quaternized chitosan derivatives show
greater stability due to enhanced electrostatic interaction,
originating from the pH-independent positive charge,
7
8
rolactone,
polylactide,
and poly-D,L-lactide-co-glycolide
9
,10
copolymer.
Since these polymers are hydrophobic they
are not ideal carriers for hydrophilic drugs like peptides and
proteins. In such cases the NPs preparation procedures
involve the use of organic solvents which can result in irre-
versible change of protein therapeutic activity unless special
2
3
1
1,12
emulsion formulation procedures are employed.
In addi-
tion, a lack of functionality in these polymers makes their
further modifications limited and, thus, an improvement of
the interaction between the polymeric carrier and the pro-
tein/peptide drug is difficult. However, the NPs of bioactive
macromolecules can be prepared under mild conditions by
mixing them with an oppositely charged, water-soluble poly-
mer (polyelectrolyte) in an aqueous medium to form the pol-
yelectrolyte complexes particularly via noncovalent
24–26
whereas the muco-adhesion of such NPs is preserved.
Synthetic polypeptides are biodegradable and biocompatible
polymers that can be used as an alternative to nondegrad-
able synthetic polymers and as a substitute for some highly-
functional natural polymers, which modification to prepare
carriers with desired and reproducible properties, is diffi-
1
3–15
electrostatic interactions.
Various polymer combinations
2
7
for protein NPs preparation have been reported with chito-
san being one of the most commonly applied polymers, for
cult. Synthetic homopolypeptides and copolypeptides of
well-defined structure, narrow molecular-weight distribution
V
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and various molecular architectures have been prepared
under well-controlled experimental conditions by ring-
opening polymerization of the N-carboxyanhydrides (NCA) of
dium glutamate) backbone on the AE with the GCSF protein
and subsequent NPs formation after addition of an oppositely
charged N,N,N-trimethyl chitosan (TMC) to a suspension of
GCSF/P(Glu-oa) complexes.
2
8
a-amino acids using primary amines as an initiator. Since
selection of a-amino acids, bearing various side groups, is
very broad, the NCA monomers of different functionalities
can be prepared. Together with the orthogonally protected
amino acids’ side groups and selective postpolymerization
modification it is possible to prepare the polypeptides with
versatile functionality. New approaches towards the design
and synthesis of multifunctional polypeptides have been con-
EXPERIMENTAL
Materials
Chemicals c-benzyl-L-glutamate (BLG; 99%, Acros Organics),
triphosgene (98%, Aldrich), hexylamine (99%, Sigma-
Aldrich), octylamine (98%, Merck), 2-hydroxypyridine (2-HP;
97%, Aldrich), HBr (33 wt % in acetic acid, Acros Organics),
2
9
tinuously developed.
w
HCl (37%, Merck), NaOH (p.a., Merck), chitosan (M 5 121
Synthetic polypeptides have been successfully applied in var-
ious drug delivery systems, mostly as amphiphilic hybrid
kDa, 85% deacetylation, Sigma Aldrich), formaldehyde (37%
water solution, Merck), MeI (99.5%, Sigma-Aldrich), formic
acid (p.a., Kemika), NaCl (99%, Sigma-Aldrich), and solvents
N,N-dimethylformamide (DMF, 99.8%, anhydrous Aldrich), N-
methyl-2-pyrrolidon (NMP, 99,5%, Merck), ethanol (99,8%,
Sigma-Aldrich), diethyl ether (99.7%, Merck), n-hexane
3
0–32
copolymers with poly(ethylene glycol) (PEG).
Due to the
amphiphilic nature of such copolymers, that is, PEG-b-
3
3
poly(b-benzyl-L-aspartate),
PEG-b-poly(N-hexyl stearate-L-
3
4
aspartamide),
PEG-b-poly(aspartic acid)-b-poly(D-leucine-
3
5
co-tyrosine), they form micelles in aqueous solutions into
which low molecular-weight active pharmaceutical ingre-
dients can be incorporated. Synthetic glycopolypeptides also
show a great potential for drug delivery applications due to
self-assembly behavior in solution and ability of carbohy-
(
99%, Merck), trifluoroacetic acid (TFA, 99%, Aldrich), and
tetrahydrofuran (THF, 99.9%, anhydrous, Sigma-Aldrich),
were used as received. The GCSF protein was kindly pro-
vided by Lek Pharmaceuticals d.d.
3
6
drate for specific recognition of biological targets. In addi-
tion, various combinations of aminoacids in the form of
block or random copolymers have been synthesized, for
example, poly(L-glutamic acid)-co-poly(L-lysine), which in
dependence of their molecular structure self-assemble
Synthesis of Water-Soluble Alkyl-Modified Sodium
Poly(glutamate)s
Synthesis of BLG N-Carboxyanhydride (NCA)
A BLG (5.00 g, 21.1 mmol) was suspended in dry THF
(
5
90 mL) and the suspension was heated up in an oil bath to
5 C. A solution of triphosgene (3.38 g, 11.4 mmol) in dry
3
7,38
into pH-sensitive vesicles or micelles.
The most
ꢀ
important parameters, defining the copolymer ability to
form self-assembled structures are the copolymer chemi-
cal composition, architecture, and molecular weight of
THF (15 mL) was then added drop-wise. The reaction mix-
ture was stirred for 75 min to obtain clear solution. Then,
the reaction mixture was concentrated by vacuum evapora-
tion and then hexane was slowly added. Afterwards, the
reaction mixture was left to stand in the freezer overnight to
ensure complete precipitation. The crude product was fil-
tered, washed with hexane, and crystalized three more times
from THF/hexane. (4.84 g, Y 5 87%).
3
9
both blocks.
The PEG-poly(amino acid) micelles have been applied for
4
0
delivery of proteins as well. Heffernan and Murthy pre-
pared micelles with cross-linked core from a block copoly-
mer of PEG and poly(L-lysine dithiopyridine) for delivery of
4
1
negatively charged proteins. Harada et al. prepared block
copolymer of PEG and poly(glutamic acid) modified with
octyl alcohol for intravenous delivery of a recombinant gran-
ulocyte colony-stimulating factor (GCSF) protein. The authors
observed an immediate release of a part of protein, which
was not incorporated into the micellar core but simply
adsorbed on the outer PEG corona. The amount of initial
protein release was smaller at lower initial protein loading,
indicating limited final loading (FL) of these polymeric
micelles.
Synthesis of Poly(c-benzyl-L-glutamate) (PBLG)
The BLG NCA (4.00 g, 15.2 mmol) was dissolved in dry DMF
(
72 mL) and cooled down in an ice bath. A solution of hexyl-
amine (0.335 mmol) in 4 mL of DMF was slowly added, and
the reaction mixture was stirred for 24 h in an ice bath.
Then, the reaction mixture was poured into an ice-cold water
to precipitate the product. The precipitate was isolated by
centrifugation (8000 rpm, 5 min), washed with water several
times and freeze-dried (3.21 g, Y 5 96%).
In this work, we synthesized negatively charged, water-soluble,
to a different degree hydrophobically modified poly(sodium
glutamate)s (P(Glu-oa)) with randomly distributed octyl chains
along the polypeptide backbone to study their ability for multi-
ple type of interactions (electrostatic, hydrophobic interactions,
and hydrogen bonds) with positively charged GCSF protein
drug. Our goal was to prepare NPs with high association effi-
ciency (AE) and high final protein loading per NP mass. Thus,
we studied the influence of degree of octyl grafting on poly(so-
Aminolysis of P(BLG)
Synthesis of P(BLG-oa) 4%. P(BLG; 1.00 g) and 2-HP
(
0.43 g, 4.57 mmol) were dissolved in dry DMF (15.2 mL).
Then, octylamine (375 mL, 2.26 mmol) was added and the
reaction mixture was stirred for 24 h at room temperature.
Afterwards, the reaction mixture was poured into cold dis-
tilled water to precipitate the product. The precipitate was
collected by filtration, washed with water, and dried in vac-
uum. Thus obtained [P(BLG-oa)] 4% product was washed
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with hexane to remove the remaining octylamine (0.80 g,
Y 5 73%).
TMC was purified by dialysis for 3 days (dialysis membrane
with MWcutoff 5 1 kDa) and freeze dried to yield a white
product (0.49 g, Y 5 80%).
Synthesis of P(BLG-oa) 13%. Similar procedure as above:
P(BLG; 1.00 g), 2-HP (0.43 g, 4,57 mmol), octylamine
Characterization
(
1.51 mL, 9.1 mmol), DMF (15.2 mL; 0.93 g, Y 5 85%).
NMR Spectroscopy
The H NMR spectra of samples were recorded, depending
on their solubility, in DMSO-d or D O on a 300-MHz Agilent
Technologies DD2 spectrometer in the pulse Fourier Trans-
form mode with both a relaxation delay and an acquisition
time of 5 s. Tetramethylsilane (TMS, d 5 0) and sodium
1
Synthesis of P(BLG-oa) 24%. Similar procedure as above:
P(BLG) (1.00 g), 2-HP (0.43 g, 4.57 mmol), octylamine
(
6
2
3.78 mL, 22.8 mmol), DMF (15.2 mL; 0.94 g, Y 5 86%).
Deprotection of P(BLG-oa) Samples
3-(trimethyl silyl) propionate-d4 (TSP-d , d 5 0) were used
4
Synthesis of P(Glu-oa) 4%. P(BLG-oa) (0.81 g) was dissolved
in TFA (20 mL). Then, HBr/acetic acid (33% w/w) was
added (3.5 mL) and the reaction mixture stirred for 1 h.
Afterwards, the reaction mixture was poured into diethyl
ether to precipitate the product, which was collected by fil-
tration. The product was neutralized with 0.1 M NaOH to
pH 5 8.5, to convert it into water-soluble alkyl-modified pol-
y(sodium glutamate) [P(Glu-oa) 4%], which was then dia-
lyzed (dialysis membrane with MWcutoff 5 500 Da) and
finally freeze-dried (0.40 g, 70%).
as the internal chemical-shift standards in DMSO-d and D O,
respectively.
6
2
Size-Exclusion Chromatography Coupled to a Multiangle
Light-Scattering Photometer (SEC-MALS)
The SEC-MALS measurements were performed at room
temperature using a Hewlett-Packard pump series 1100
coupled to a DAWN HELEOS laser photometer with a
GaAs linearly polarized laser (k 5 658 nm) and to an Opti-
lab rEX interferometric refractometer (RI) operating at
the same wavelength as the photometer (both instruments
are from Wyatt Technology Corp). The separation of chito-
san and TMC was carried out on a Novema linear column
Synthesis of P(Glu-oa) 13%. Similar procedure as above:
P(BLG-oa) (0.85 g), TFA (21.2 mL), HBr/acetic acid (3.6 mL;
0.45 g, Y 5 71%).
2
with a pre-column (8 3 300 mm , Polymer Standards
6
Service, molar mass range: up to 2 3 10 Da) in 0.2 M
Synthesis of P(Glu-oa) 24%. Similar procedure as above:
solution of sodium acetate/acetic acid in miliQ water at
pH 4.4. The separation of PBLG sample was carried out
on a PolarGel-L 8 lm column with a pre-column (300 mm
length and 7.5 mm i.d., Polymer Laboratories, molar mass
range: up to 30 kDa) in 0.05 M LiBr in N,N-dimethylaceta-
mide at a flow rate of 1 mL/min. The masses of the
samples injected onto the column were typically 1 3
P(BLG-oa) (0.91 g), TFA (22.8 mL), HBr/acetic acid (3.9 mL;
0.49 g, Y 5 69%).
Synthesis of TMC
Synthesis of N,N-Dimethyl Chitosan (DMC)
Chitosan (5.0 g) was suspended in formic acid (15 mL), fol-
lowed by addition of 37% aqueous formaldehyde solution
24
1
1
0
0
g, whereas the solution concentrations were 1 3
g/mL.
(
20 mL) and distilled water (90 mL). The solution was
23
ꢀ
heated to 70 C and stirred for 118 h under reflux con-
denser. Then, the reaction mixture was partially evaporated
to obtain viscous solution to which 1 M NaOH solution was
added to set the pH of the medium to 12, at which the gel
was formed. The gel was washed with water over a glass fil-
ter several times. DMC was dissolved in water at pH 4, that
was adjusted with 1 M HCl. Solution was purified by dialysis
for 3 days (dialysis membrane with MWcutoff 5 12 kDa). The
remaining solution was freeze-dried to yield a white product
The GCSF protein content in supernatants was determined
by SEC coupled to a MALS-UV-RI multidetection system
using a PROTEEMA column with a pre-column (100 Å, 8 3
2
3
00 mm , Polymer Standards Service, PSS, Germany, molar
5
mass range: up to 1 3 10 Da) in 50 mM solution of NaNO
3
in miliQ water with 0.02% sodium azide. For the data acqui-
sition and evaluation, the Astra 5.3.4 software (Wyatt Tech-
nology) was used. By knowing the protein specific UV
extinction coefficient at 280 nm (e 5 0.815 mL/g cm) or its
(
4.0 g, Y 5 70%).
4
2
refractive index increment (dn/dc 5 0.186 mL/g), we calcu-
lated, from the concentration traces (UV or RI detector
responses), the exact mass of the uncomplexed protein in
the injected volume of the supernatant and, consequently, in
the total volume of the supernatant/suspension (volume of
all the solutions mixed for NP preparation). Based on these
results the AE and the FL of GCSF in NPs were calculated
according to eqs 1 and 2.
Synthesis of TMC from DMC
DMC was dissolved in water (160 mL) and the pH was
adjusted with 1 M NaOH to 11, at which the gel was formed.
This step is performed to ensure deprotonation of DMC terti-
ary amino groups. Then, the gel was washed with water and
acetone several times. Thus, prepared DMC (0.50 g) was sus-
pended in NMP (140 mL) to which the iodomethane was
ꢀ
added (4 mL). Suspension was heated to 40 C and stirred.
The precipitate (TMC) was isolated from ethanol/diethyl
ether mixture (50/50) by filtration on a glass filter. After
drying overnight, TMC was dissolved in an aqueous NaCl
solution (10 wt %, 200 mL) and stirred for 18 h. Finally,
MALDI-TOF MS
MALDI-TOF mass spectra were recorded on a Bruker Ultra-
fleXtreme MALDI-TOF-TOF mass spectrometer (Bruker Dalto-
nik, Bremen, Germany). The protected polypeptides were
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recorded in a positive reflectron mode, while the deprotected
polypeptides were recorded in a negative reflectron mode.
The calibration was made externally with a Peptide calibra-
tion standard II (Bruker Daltonics) using nearest-neighbor
positions. Matrix used was super DHB (mixture of 2,5-dihy-
droxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid)
and sodium trifluoroacetate was used as a cationizer in the
case of protected polypeptides.
amount of free GCSF protein present in aqueous phase after
centrifugation was divided with the total amount of GCSF
protein added.
total amount of GCSF2unassociated GCSF
AE%5
3100 (1)
total amount of GCSF
The proportion of associated GCSF protein within com-
plexes/NPs is defined as a FL by eq. 2. The FL represents a
difference between the total amount of GCSF protein used
for complexes/NPs preparation and the amount of non-asso-
ciated GCSF protein, which was divided by the total amount
of complexes/NPs.
Scanning Electron Microscopy (SEM)
SEM microscopy was used for morphological evaluation of
NPs using a JSM-7001F Jeol (Japan) instrument with an
acceleration voltage of 5.0 kV and a secondary electron
detector. Freshly prepared NP suspensions were centrifuged
at 15,000 rpm for 15 min, followed by resuspension of NPs
in water to remove buffer salt. Such prepared suspensions
were deposited on a double-sided carbon tape (diameter
total amount of GCSF2unassociated GCSF
FL%5
3100 (2)
total amount of NPs
1
2 mm, Oxford instruments, Oxon, UK), dried in a vacuum
RESULTS AND DISCUSSION
ꢀ
dryer at 25 C for 5 h, and then the SEM images were
taken.
Synthesis of Water-Soluble Alkyl-Modified Sodium
Poly(glutamate)s
For preparation of hydrophobically modified poly(sodium
glutamate)s [P(Glu-oa)] (Scheme 1) the BLG N-carboxyanhy-
dride (NCA) was first prepared from the BLG and triphos-
gene. The BLG NCA was extensively purified by
crystallization before it was polymerized since pure NCA is
essential for good control over the polypeptide molar mass,
Polyelectrolyte Complexation/NPs Formation and Their
Characterization
The protein solution (4.4 mg/mL in 10 mM acetic buffer
with 5% sorbitol, pH 4.5) was added drop-wise to the P(Glu-
oa) polymer solution (2 or 3 mg/mL in 10 mM acetic buffer,
pH 4.5) to form polyelectrolyte complexes. After stirring for
a defined time, a solution of TMC (3 mg/mL) with opposite
charge to the P(Glu-oa) polymer was added to GCSF/P(Glu-
oa) complexes, which resulted in the formation of GCSF/
P(Glu-oa)/TMC NPs.
2
8
the molar mass dispersity and the type of end groups.
Polymerization of BLG NCA was initiated with primary hexyl-
ꢀ
amine and performed at 0 C to limit side-reactions, which
are known to take place to a higher extent at room tempera-
4
3
ture.
The molar mass characteristics of thus prepared
The GCSF/P(Glu-oa) complexes and GCSF/P(Glu-oa)/TMC
NPs were characterized by dynamic light-scattering (DLS)
using a Zetasizer Nano ZS ZEN 3600 (4 mW He-Ne laser,
P(BLG) are: M 5 7.6 kDa, M 5 8.0 kDa, and D- 5 1.05 as
n
w
determined by SEC-MALS [Fig. 1(A)]. Comparable molar
mass with the peak apex at 8.2 kDa is obtained by MALDI-
TOF MS. The mass spectrum shows a set of peaks with a dif-
ference of 219 Da between them, which corresponds to a
benzyl glutamate repeating unit. The peak masses indicate
macromolecules which are initiated with the hexylamine and
terminated by the amine group as expected for a normal
6
33 nm) from Malvern instruments, UK. Scattering light
ꢀ
was detected at 173 by automatically adjusted laser
attenuation filters and measurement position within the
cell at temperature of 25 C. For data analysis, the viscosity
ꢀ
(
0.8863 mPa) and refractive index (1.330 at 633 nm) of the
ꢀ
distilled water at 25 C were used. By DLS measurements
4
4,45
amine polymerization mechanism [Fig. 1(B)].
h
we obtained the average hydrodynamic radius (D ) of NPs
and the size distribution that is described by the polydis-
persity index (PDI), which is a dimensionless number
extrapolated from the autocorrelation function and ranges
from the values close to zero for the uniform particles’ size
distribution and up to the values close to 1 for the broad
size distribution of particles.
Alkyl-modified P(BLG) samples with different content of
alkyl grafts were synthesized by post-polymerization modifi-
cation using partial aminolysis of P(BLG) with octylamine.
Thus modified polypeptides consist of most probably ran-
domly distributed alkyl chains along the backbone, which
thus prevent supramolecular organization into well-defined
micellar structures, typical for the block copolymers. Low
content of alkyl side chains provides water-solubility of
P(Glu-oa) copolymers. In addition, the formation of amide
bond by aminolysis reaction avoids the use of carbodiimide
coupling reagents, usually employed in postpolymerization
modification of carboxyl groups, together with the associated
side reactions (N-acylurea formation, etc.) and the issue with
AE and FL of GCSF protein in complexes/NPs were deter-
mined indirectly after separating GCSF/P(Glu-oa) complexes
or GCSF/P(Glu-oa)/TMC NPs from the dispersion media by
centrifugation at 40,000 rpm for 20 min. Thus, obtained
supernatants were analyzed for free protein content with
SEC-MALS technique (see Experimental part).
4
6
The AE of GCSF protein with polymer(s) was calculated
according to eq. 1. The difference between the total amount
of GCSF protein used to prepare the complexes/NPs and the
removal of urea as a side product. The degree of P(BLG)
aminolysis was controlled by changing the mole ratio of
octylamine to benzyl glutamate repeat units. The polypeptide
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SCHEME 1 Synthetic pathway for preparation of alkyl-modified poly(sodium glutamate)s, [P(Glu-oa)].
degradation during aminolysis was to a large extent pre-
vented by the addition of 2-HP as a bifunctional catalyst.
zation is most likely additionally catalyzed by 2-
hydroxypyrdine. A mass difference of 219 Da between the
peaks of the main distribution corresponds to benzyl gluta-
mate repeating unit, whereas a mass difference of 240 Da
between the peaks of less intense peak distribution is due to
octyl glutamide repeating unit. Thus, each peak of the main
distribution shows sub-distributed peaks with the mass dif-
ference of 21 Da, which equals the difference between the
benzyl ester and octyl glutamide units. Sub-distributed peaks
indicate the polymer chains with the same degree of poly-
merization but different number of alkyl side chains. With
increasing degree of alkylation the distributions of benzyl
ester and octyl glutamide units become broader with accom-
panying signal overlapping, which make a detailed interpre-
tation of MALDI-TOF mass spectra more difficult.
Additionally, the mass spectra of P(BLG-oa) samples show
very low intensity peak distribution with a mass difference
of 129 Da, which reveals the presence of macromolecules
with one deprotected carboxyl group. A peak distribution
indicating macromolecules with two deprotected carboxyl
groups was also observed, however these signals are of even
lower intensity and strongly overlap with other signals.
Thus, MALDI-TOF MS results reveal partial deprotection of
benzyl protected carboxyl groups during aminolysis, which
in our case is not a problem since the next synthetic step
involves the removal of all benzyl protective groups. No deg-
radation products originating from aminolysis and/or hydro-
lysis of the polypeptide backbone are observed in MALDI-
TOF mass spectra of the P(BLG-oa) samples.
4
7
MALDI-TOF mass spectra of P(BLG-oa) samples reveal that
all macromolecules contain the hexyl group at one chain end
that originates from the initiator, whereas the amino end
group presented in original P(BLG) samples transformed
during aminolysis into a pyroglutamic group via intramolecu-
lar cyclization reaction with the adjacent benzyl ester group
(
Fig. 2). The intramolecular cyclization reaction to pyrogluta-
4
3
mic end group is favored at elevated temperature, whereas
during aminolysis performed at room temperature, the cycli-
The benzyl protected carboxyl groups of partially alkylated
P(BLG-oa) were deprotected under acidic conditions and,
then, neutralized with NaOH to yield the water-soluble alkyl-
modified poly(sodium glutamate)s, (P(Glu-oa)). The MALDI-
TOF mass spectra of all three octyl modified and deprotected
P(Glu-oa) polypeptides show peak apex at the expected
molar masses. A slight increase in peak apex is noticed with
increasing degree of P(Glu-oa) alkylation (Fig. 3). The sub-
distribution of peaks due to the different chemical
FIGURE 1 A: SEC-MALS chromatogram of P(BLG), black solid
curve: refractive index detector response, red dotted line: light-
ꢀ
scattering detector response at 90 angle, black squares: molar
mass as a function of elution volume; (B) MALDI-TOF mass
spectrum of P(BLG). [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
2
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FIGURE 2 MALDI-TOF mass spectrum of P(BLG-oa) 13% (top) and magnified MALDI-TOF mass spectra of P(BLG-oa) samples with
different degree of alkylation (bottom). The mass difference between the peaks, that is, 219, 240, and 129 Da belong to benzyl glu-
tamate, octyl glutamide, and glutamic acid repeating units, respectively. The mass difference of 21 Da is due to different ratio
between benzyl glutamate (A) and octyl glutamide (B) repeating units in the chains with the same degree of polymerization. [Color
figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
composition of the chains with equal degree of polymeriza-
tion is much more difficult to identify in P(Glu-oa) mass
spectra since a difference between the octyl glutamide (240
Da) and the glutamic acid (129 Da) repeating units is 111
Da, which causes broadening of the peak distribution over
several hundred Da and significant peak overlapping.
FIGURE 3 MALDI-TOF mass spectra of P(Glu-oa) samples after carboxyl group deprotection.
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TABLE 1 Experimental Conditions for Complex Preparation,
AE, and FL of GCSF Protein in GCSF/P(Glu-oa) Complexes
P(Glu-oa)
V (1 mL)
GCSF
c (4.4 mg/mL)
V (mL)
Initial
GCSF
AE FL
c (mg/mL)
load (%) (%) (%)
P(Glu-oa) 4%
P(Glu-oa) 13%
P(Glu-oa) 24%
2
3
2
3
2
3
100
18.0
30.6
12.8
22.7
18.0
30.6
12.8
22.7
18.0
30.6
12.8
22.7
35
6
2
00
100
00
100
00
100
00
100
00
37 11
31
41
4
9
2
75 14
84 26
2
52
7
2
67 15
79 14
88 27
1
FIGURE 4 H NMR spectra of alkyl-modified poly(sodium gluta-
mate)s with different degree of randomly distributed alkyl
chains: red spectrum: P(Glu-oa) 4%; green spectrum: P(Glu-oa)
2
1
3% and blue spectrum: P(Glu-oa) 24%. [Color figure can be
100
200
65
8
viewed in the online issue, which is available at wileyonlineli-
brary.com.]
79 18
4
8
Clark reaction (Scheme 2). Advantage of the described
TMC synthetic procedure over direct chitosan quaternization
using MeI and NaOH is in higher degree of quaternization
and in absence of chitosan O-methylation as a side reaction,
4
9
which allows further chitosan modification.
SCHEME 2 Synthetic pathway for preparation of TMC.
The degree of alkylation of P(Glu-oa) samples was calculated
1
from the H NMR spectra of products (Fig. 4) from the inte-
gral of the signals for the methyl group of the alkyl chain
(
(
signal 1) and the methyne group of the polymer backbone
signal 5) and is determined to be 4, 13 and 24% (% refers
to alkyl chains per glutamate repeating units).
Synthesis of TMC
The TMC was prepared by quaternization of amine groups of
DMC using MeI. The DMC was synthesized by Eschweiler-
1
FIGURE 5 H NMR spectrum of TMC with assignation of the
FIGURE 6 Histograms of 1 mL solutions of P(Glu-oa) 13%
(3 mg/mL) with 100 lL of GCSF (4.4 mg/mL) without TMC (A)
and with 10 lL (B), 30 lL (C), and 60 lL (D) of TMC (3 mg/mL).
main signals. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
2
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TABLE 2 Experimental Conditions for NPs Preparation, AE, FL of GCSF Protein in GCSF/P(Glu-oa)/TMC NPs Together with the Size
and Polydispersity of NPs
P(Glu-oa) V (1 mL)
c (mg/mL)
Initial GCSF
load (%)
a
TMC V (lL)
D
h
(nm)
PDI
AE (%)
FL (%)
P(Glu-oa) 4%
P(Glu-oa) 13%
P(Glu-oa) 24%
2
3
10
17.8
17.4
12.7
12.5
12.2
17.8
17.4
12.7
12.5
12.2
17.8
17.4
12.7
12.5
12.2
267
0.198
0.347
0.243
0.404
0.427
0.486
0.456
0.648
0.392
0.400
0.574
0.523
0.510
0.471
0.299
45
47
54
42
37
70
85
69
69
73
93
93
78
76
77
8.9
7.0
3
0
354
282
696
1107
325
366
332
404
409
570
474
284
422
292
10
7.3
3
6
0
0
5.6
4.9
2
3
10
13.2
15.2
9.1
3
0
10
3
6
0
0
8.9
9.2
2
3
10
16.8
16.4
10.2
9.8
3
0
10
3
6
0
0
9.6
a
Concentration of TMC was 3 mg/mL.
For all experiments 100 mL of 4.4 mg/mL GCSF protein solution was
used.
The molar mass characteristics of TMC we have applied for
4% polymer has negligible effect on AE of this particular
polymer with GCSF, however, in the case of the P(Glu-oa)
13% and the P(Glu-oa) 24% the AE somewhat deteriorate,
most probably due to the polymer self-association which
results in lower probability of polymer to interact with the
protein. However, higher amount of added GCSF solution
NPs preparation are M 5 58 kDa, M 5 114 kDa, and
n
w
D- 5 1.9 as determined by SEC-MALS. The ratio between the
trimethylamine and dimethylamine groups is 5 to 1 as was
calculated from the integral of the signals for the respective
1
groups in H NMR spectrum of TMC (Fig. 5).
(
200 instead of 100 mL, c 5 4.4 mg/mL) to P(Glu-oa) solution
NPs Formation from GCSF, P(Glu-oa) Polymers, and TMC
The ability of polymer to associate with protein is crucial for
successful NPs preparation. In addition to efficient protein
association, its FL in NPs should be high enough to obtain
pharmaceutically acceptable system that is applicable for fur-
ther dosage form formulation, which usually increases for-
mulation overall mass.
results in an improved AE between protein and polymer.
By subsequent addition of the positively charged TMC polymer
solution to the dispersion of GCSF/P(Glu-oa) complexes the
particles of larger size are formed (Fig. 6). The lowest amount
The GCSF protein solution (c 5 4.4 mg/mL, V 5 100 or 200
lL) in a medium with pH 4.5, that is below the protein iso-
electric point (pI 6.1), was added stepwise to a solution of
P(Glu-oa) polymer (c 5 2 or 3 mg/mL, V 5 1 mL) with nega-
tive overall charge. After formation of the GCSF/P(Glu-oa)
complexes, the oppositely charged TMC (c 5 3 mg/mL,
V 5 10, 30 or 60 lL) was added to obtain well-defined NPs.
The initial GCSF load is thus the amount of GCSF used
divided by the total amount of all components.
The AE of GCSF protein with P(Glu-oa) polymers increases
with the amount of alkyl grafts in poly(glutamate) in the fol-
lowing order: P(Glu-oa) 24% > P(Glu-oa) 13% >> P(Glu-oa)
4% (Table 1). A large increase in AE from the P(Glu-oa) 4%
to the P(Glu-oa) 13% indicates significant contribution of
hydrophobic interaction to complex formation. Higher solu-
tion concentration (3 instead of 2 mg/mL) of the P(Glu-oa)
FIGURE 7 SEM of NPs prepared from 1 mL solution of P(Glu-
oa) 13% (3 mg/mL) with 100 lL of GCSF (4.4 mg/mL) and 60 lL
of TMC (3 mg/mL).
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of added TMC results in bimodal size distribution [Fig. 6(B)].
However, by increasing the amount of TMC added, more
defined particles are formed [Fig. 6(C,D)] without significant
deterioration in AE (Table 2). The formation of NPs of protein,
P(Glu-oa) and TMC was also confirmed by SEM (Fig. 7).
9 Y. S. Nam, J. Y. Park, S.-H. Han, I.-S. Chang, Biotechnol. Lett.
2
002, 24, 2093–2098.
1
0 A. Kumari, S. K. Yadav, S. C. Yadav, Colloid Surf. B 2010,
7
5, 1–18.
1
1 G. Liu, X. Hong, M. Jiang, W. Yuan, Int. J. Nanomed. 2012,
7, 4559–4569.
Higher amount of TMC can be added to the 3 mg/mL than
to the 2 mg/mL P(Glu-oa) polymer solution before the pre-
cipitation of particles occurs, which reveals the ability of
TMC to form stable NPs through electrostatic interaction and
fine-tuning of final NPs size. Excellent AE as well as rather
high final GCSF loading obtained with the P(Glu-oa) 13%
and the P(Glu-oa) 24% polymers in combination with TMC
demonstrate a potential for further investigation of these
NPs for application in oral delivery of biopharmaceuticals.
12 S. H. Choi, T. G. Park, Int. J. Pharm. 2006, 311, 223–228.
13 S. Boddohi, N. Moore, P. A. Johnson, M. J. Kipper, Bioma-
cromolecules 2009, 10, 1402–1409.
14 S. Mizrahy, D. Peer, Chem. Soc. Rev. 2012, 41, 2623–2640.
15 H.-W. Sung, K. Sonaje, Z.-X. Liao, L.-W. Hsu, E.-Y, Chuang,
Acc. Chem. Res. 2012, 45, 619–629.
1
6 Y. Chen, V. J. Mohanraj, J. E. Parkin, Lett. Pept. Sci. 2003,
1
0, 621–629.
1
7 M. Huang, S. N. Vitharana, L. J. Peek, T. Coop, C. Berkland,
Biomacromolecules 2007, 8, 1607–1614.
8 W. Tiyaboonchai, J. Woiszwillo, R. C. Sims, C. R. Middaugh,
Int. J. Pharm. 2003, 255, 139–151.
9 B. Sarmento, A. Ribeiro, F. Veiga, P. Sampaio, R. Neufeld,
D. Ferreira, Pharm. Res. 2007, 24, 2198–2206.
0 Y.-H. Lin, F.-L. Mi, C.-T. Chen, W.-C. Chang, S.-F. Peng, H.-F.
Liang, H.-W. Sung, Biomacromolecules 2007, 8, 146–152.
1 E.-Y. Chuang, K.-J. Lin, F.-Y. Su, F.-L. Mi, B. Maiti, C.-T.
1
CONCLUSIONS
1
Hydrophobically modified, negatively-charged, water-soluble
poly(sodium glutamates) with different content of randomly
distributed octyl chains in combination with TMC proved to
be very effective in formation of GCSF protein loaded NPs
using polyelectrolyte complexation method. The content of
octyl grafts on poly(glutamate) backbone plays an important
role in complexation efficiency of P(Glu-oa) polymers with
GCSF protein. By optimization the ratio between the GCSF
protein, P(Glu-oa, 13 or 24%) and TMC well-defined NPs
with high AE and FL were prepared. Thus, a combination of
P(Glu-oa) and TMC polymers for preparation of NPs loaded
with GCSF provides minimal loss of protein drug during NPs
preparation. This work demonstrates that polymers should
be carefully designed to tune their properties in a way to
efficiently interact with protein drug, which is a prerequisite
for successful preparation of well-defined NPs with high FL.
2
2
Chen, S.-P. Wey, T.-C. Yen, J.-H. Juang, H.-W. Sung, J. Control.
Release 2013, 172, 513–522.
2
2 B. Deutel, M. Greindl, M. Thaurer, A. Bernkop-Schn u€ rch,
Biomacromolecules 2008, 9, 278–285.
2
2
3 K.A. Janes, P. Calvo, M. J. Alonso, Adv. Drug Delivery Rev.
001, 47, 83–97.
2
4 A. Bayat, F. A. Dorkoosh, A. R. Dehpour, L. Moezi, B.
Larijani, H. E. Junginger, M. Rafiee-Tehrani, Int. J. Pharm.
2008, 356, 259–266.
25 S. M. van der Merwe, J. C. Verhoef, J. H. M. Verheijden, A.
F. Kotze, H. E. Junginger, Eur. J. Pharm. Biopharm. 2004, 58,
2
25–235.
2
2
6 F. Qian, F. Cui, J. Ding, C. Tang, C. Yin, Biomacromolecules
006, 7, 2722–2727.
ACKNOWLEDGMENTS
2
7 T. J. Deming, Prog. Polym. Sci. 2007, 32, 858–875.
The authors gratefully acknowledge the financial support of the
Ministry of Higher Education, Science and Technology of the
Republic of Slovenia through the Slovenian Research Agency
28 N. Hadjichristidis, H. Iatrou, M. Pitsikalis, G. Sakellariou,
Chem. Rev. 2009, 109, 5528–5578.
2
2
9 E. Garanger, S. Lecommandoux, Angew. Chem. Int. Ed.
012, 51, 3060–3062.
(
Program P2-0145 and Projects L2-4166 and Z2-6755) and the
3
0 Y. Bae, K. Kataoka, Adv. Drug Delivery Rev. 2009, 61, 768–784.
1 A. Lalatsa, A. G. Sch a€ tzlein, M. Mazza, T. B. Hang Le , I. F.
Centre of Excellence, Polymer Materials and Technologies for
access to the MALDI-TOF MS.
3
Uchegbu, J. Control. Release 2012, 161, 523–536.
2 A. Lavasanifar, J. Samuel, G. S. Kwon, Adv. Drug Delivery
Rev. 2002, 54, 169–190.
3 K. Kataoka, T. Matsumoto, M. Yokoyama, T. Okano, Y.
3
REFERENCES AND NOTES
1
C. Pinto Reis, R. J. Neufeld, A. J. Ribeiro, F. Veiga, Nanome-
3
dicine 2006, 2, 8–21.
Sakurai, S. Fukushima, K. Okamoto, G. S. Kwon, J. Control.
Release 2000, 64, 143–153.
2
2
F. Chiellini, A. M. Piras, C. Errico, E. Chiellini, Nanomedicine
008, 3, 367–393.
3
4 A. Lavasanifar, J. Samuel, G. S. Kwon, J. Control. Release
3
R. Duncan, Nat. Rev. 2003, 2, 347–360.
2002, 79, 165–172.
4
D. Peer, J. M. Karp, S. Hong, O. C. Farokhzad, R. Margalit, R.
35 J. Rios-Doria, A. Carie, T. Costich, B. Burke, H. Skaff, R.
Panicucci, K. Sill, J. Drug Delivery, in press, DOI: 10.1155/2012/
951741.
Langer, Nat. Nanotechnol. 2007, 2, 751–760.
5
M. E. Davis, Z. Chen, D. M. Shin, Nat. Rev. 2008, 7, 771–782.
6
K. S. Soppimath, T. M. Aminabhavi, A. R. Kulkarni, W. E.
36 C. Bonduelle, S. Lecommandoux, Biomacromolecules 2013,
14, 2973–2983.
Rudzinski, J. Control. Release 2001, 70, 1–20.
7
C. Damge, P. Maincent, N. Ubrich, J. Control. Release 2007,
37 J. Rodrıguez-Hernandez, S. Lecommandoux, J. Am. Chem.
Soc. 2005, 127, 2026–2027.
117, 163–170.
8
2
H. Gao, Y. N. Wang, Y. G. Fan, J. B. Ma, J. Control. Release
005, 107, 158–173.
38 Y. Huang, Z. Tang, X. Zhang, H. Yu, H. Sun, X. Pang, X.
Chen, Biomacromolecules 2013, 14, 2023–2032.
2
984
JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY 2014, 52, 2976–2985

JOURNAL OF
POLYMER SCIENCE
WWW.POLYMERCHEMISTRY.ORG
ARTICLE
3
4
4
9 I. F. Uchegbu, Expert Opin. Drug Delivery 2006, 3, 629–640.
0 M. Heffernan, N. Murthy, Ann. Biomed. Eng. 2009, 37, 1993–2002.
1 M. Harada, M. Ohuchi, T. Hayashi, Y. Kato, J. Control.
45 M. Goodman, J. Hutchison, J. Am. Chem. Soc. 1966, 88,
3
627–3630.
4
6 C. A. G. N. Montalbetti, V. Falque, Tetrahedron 2005, 61,
1
0827–10852.
Release 2011, 156, 101–108.
ꢀ
47 A. De Marre, H. Soyez, E. Schacht, J. Pytela, Polymer 1994,
4
2 K. Rebolj, D. Pahovnik, E. Zagar, Anal. Chem. 2012, 84,
3
5, 2443–2446.
7374–7383.
4
8 R. J. Verheul, M. Amidi, S. van der Wal, E. van Riet, W.
43 G. J. M. Habraken, M. Peeters, C. H. J. T. Dietz, C. E.
Jiskoot, W. E. Hennink, Biomaterials 2008, 29, 3642–3649.
Koning, A. Heise, Polym. Chem. 2010, 1, 514–524.
4
9 N. M. Alves, J. F. Mano, Int. J. Biol. Macromol. 2008, 43,
4
4 E. Peggion, M. Terbjevich, A. Cosani, C. Colombini, J. Am.
4
01–414.
Chem. Soc. 1966, 88, 3630–3632.
WWW.MATERIALSVIEWS.COM
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