The synthetic tuning of clickable pH responsive cationic polypeptides and block copolypeptides

Article abstract of DOI:10.1039/c1sm05064h

A series of pH responsive synthetic polypeptides has been developed based on an N-carboxyanhydride ring opening polymerization combined with a facile and versatile click chemistry. Poly(γ-propargyl l-glutamate) (PPLG) homopolymers and poly(ethylene glycol-b-γ-propargyl l-glutamate) (PEG-b-PPLG) block copolymers were substituted with various amine moieties that range in pK_a and hydrophobicity, providing the basis for a library of new synthetic structures that can be tuned for specific interactions and responsive behaviors. These amine-functionalized polypeptides have the ability to change solubility, or reversibly self-assemble into micelles with changes in the degree of ionization; they also adopt an α-helical structure at biologically relevant pHs. Here we characterize the pH responsive behavior of the new polypeptides and the hydrolysis of the ester containing amine side chains. We examine the reversible micellization with block copolymers of the polypeptides and nucleic acid encapsulation that demonstrate the potential use of these materials for systemic drug and gene delivery.

Full text of DOI:10.1039/c1sm05064h

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PAPER
The synthetic tuning of clickable pH responsive cationic polypeptides and
block copolypeptides†
a
ab
a
a
a
Amanda C. Engler, Daniel K. Bonner, Hilda G. Buss, Eva Y. Cheung and Paula T. Hammond*
Received 15th January 2011, Accepted 20th April 2011
DOI: 10.1039/c1sm05064h
A series of pH responsive synthetic polypeptides has been developed based on an N-carboxyanhydride
ring opening polymerization combined with a facile and versatile click chemistry. Poly(g-propargyl L-
glutamate) (PPLG) homopolymers and poly(ethylene glycol-b-g-propargyl L-glutamate) (PEG-b-
a
PPLG) block copolymers were substituted with various amine moieties that range in pK and
hydrophobicity, providing the basis for a library of new synthetic structures that can be tuned for
specific interactions and responsive behaviors. These amine-functionalized polypeptides have the
ability to change solubility, or reversibly self-assemble into micelles with changes in the degree of
ionization; they also adopt an a-helical structure at biologically relevant pHs. Here we characterize the
pH responsive behavior of the new polypeptides and the hydrolysis of the ester containing amine side
chains. We examine the reversible micellization with block copolymers of the polypeptides and nucleic
acid encapsulation that demonstrate the potential use of these materials for systemic drug and gene
delivery.
Introduction
Recently, we reported a new approach to the manipulation of
synthetic polypeptide composition and function through the
introduction of a new NCA polymer, poly(g-propargyl L-gluta-
Synthetic polypeptides have received attention because of their
1–4
unique structural properties and biocompatibility. Like their
naturally occurring analogs, these molecules have a poly(amino
acid) backbone and possess the ability to fold into stable secondary
structures. Helical structures, in particular, allow for proteins to
optimally display surface moieties that dictate cell signaling and
11
mate) (PPLG), which contains a pendant alkyne group that can
be reacted with an azide by the alkyne–azide cycloaddition click
11,12
reaction.
This synthetic strategy allows for the convenient and
efficient functionalization of a polypeptide without the need for
protection and deprotection steps. To demonstrate the efficiency
of polymer modification, we used a model PPLG-g-PEG system,
for which we were able to attain a ‘‘grafting onto’’ efficiency of
5
molecular docking. This property gives synthetic polypeptides an
advantage over most traditional polymers that can only adopt
a random coil structure. A considerable amount of research has
been performed on synthetic polypeptides to better understand the
complex features of proteins and to gain insight into their
11
over 96%. Since our initial report, other research groups have
extended this platform methodology of combining NCA poly-
merization and click chemistry side chain modification. Chen et al.
used PPLG to click on several different azide functionalized
6–10
secondarystructures. Syntheticpolypeptidescanbesynthesized
on a large scale by the ring opening polymerization (ROP) of N-
carboxyanhydrides (NCA) formed from naturally occurring
amino acids. These simple homopolypeptides are able to arrange
into or change their secondary structure based on solution condi-
13
monosaccharides to form glycopolypeptides. Tang and Zhang
reported the synthesis of poly(g-azidopropyl-L-glutamate), which
was functionalized with alkyne containing mannose moieties via
14
the alkyne–azide cycloaddition click reaction. Sun and Schlaad
7–10
tions. Although these macromolecules’ secondary structure can
be controlled to some extent, we are limited by the given side chain,
which dictates polymer function, structure, and responsive
behavior to temperature or pH among many other properties.
developed thiol–ene clickable polypeptides, where they synthe-
sized poly(D,L-allylglycine) and clicked on thiol functionalized
15
sugars. Huang et al. synthesized poly(D,L-propargylglycine) and
clicked on azide containing protected galactose, using alkyne–
16
azide cycloaddition click chemistry.
a
By employing our PPLG platform for the click chemistry of
amino-functional groups, we have developed several new pH
responsive macromolecules. A unique aspect of these new amine-
functionalized polypeptides is the ability to buffer and in some
cases, undergo a solubility phase transition with degree of ioniza-
tion, while adopting ana-helicalstructure over biologicallyrelevant
pHs. These polymers include both poly(g-propargyl L-glutamate)
Department of Chemical Engineering, Massachusetts Institute of
Technology, 77 Massachusetts Ave., Cambridge, MA, USA. E-mail:
hammond@mit.edu; Fax: +1 617-258-8991; Tel: +1 617-253-4562
b
Division of Health Science and Technology, Massachusetts Institute of
Technology, 77 Massachusetts Ave., Cambridge, MA, USA
†
Electronic supplementary information (ESI) available: Representative
1
H-NMR, additional titrations, reverse titrations and CD spectra. See
DOI: 10.1039/c1sm05064h
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Soft Matter, 2011, 7, 5627–5637 | 5627

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(
PPLG) based homopolymers and poly(ethylene glycol-b-g-prop-
argyl L-glutamate) (PEG-b-PPLG) block copolymers substituted
99%, was purchased from Arcos Organics. Sunbrightꢀ amine
terminated poly(ethylene glycol) was purchased from NOF
Corporation. siRNA was purchased from Dharmacon RNAi
Technologies and QuantiT Ribogreen RNA Reagent was
purchased from Invitrogen. All other chemicals were purchased
from Sigma Aldrich. All materials were used as received.
with various amine moieties that range in pK and hydrophobicity,
a
providing the basis for a library of new synthetic structures that can
be tuned for specific interactions and responsive behaviors.
The new PPLG based cationic polypeptides have the potential
to be used for many different applications. Polypeptides have been
17–19
investigated as smart molecules in lipid membranes,
4
crystals used in optical storage and display devices, vehicles for
liquid
Methods
3,20–29
30
anti-fouling coatings, components
1 13
H-NMR and C-NMR were recorded on a Bruker 400 MHz
drug and gene delivery,
for tissue engineering and biosensors, and synthetic mimics of
FT-NMR spectrophotometer. Gel permeation chromatography
measurements were carried out using a Waters Breeze 1525
HPLC system equipped with two Polypore columns operated at
1,3,31–34
naturally occurring molecules.
We have characterized the
pH responsive behavior of the new polypeptides, the pH-depen-
dent hydrolysis rate of the ester containing amine side chains, and
have performed preliminary experiments that demonstrate the
potential use of these new materials for systemic drug and gene
delivery. More specifically, for pH responsive drug delivery, one
could design a micellar system that forms stable micelle drug
carriers in the blood stream and normal tissue at extracellular
ꢀ
75 C, a series 2414 refractive index detector, a series 1525 binary
HPLC pump, and a 717 plus autosampler. Waters’ Breeze
Chromatography Software Version 3.30 was used for data
collection as well as data processing. DMF with 0.01 M LiBr was
the eluent for analysis, and samples were dissolved at 4–6 mg
ꢁ1
mL in DMF. The average molecular weight of the sample was
calibrated against narrow molecular weight poly(methyl meth-
acrylate) standards.
35
conditions (pH 7.00–7.45) but destabilizes in the endosome
3
6
(
early endosome pH 5.5–6.3 and late endosome pH < 5.5) or in
35
hypoxic regions of tumors (pH approaching 6.0), to release the
drug. To achieve this behavior, a pH responsive polypeptide is
needed that is fully soluble at endosomal or tumor pH and
insoluble at extracellular pH. We have determined the solubility
behavior of the amine functionalized PPLG and the self-assembly
behavior of the amine functionalized PEG-b-PPLG as a function
of pH. For gene delivery, it is critical that the polymers complex
with siRNA or DNA to form protective polymer–gene complexes
Acid–base titrations were performed on all amine functional-
ized PPLG. 3 mL of 10 mM amine in 125 mM NaCl was adjusted
to a pH of 3 using 1 M HCl. The solution was titrated with 10 mL
aliquots of 0.1 M NaOH, measuring the pH with each addition.
For polymers where precipitation was observed, UV/Vis
measurements were obtained at 600 nm to monitor the solution
turbidity. UV/Visible measurements were carried out on an
Agilent Technologies G3172A spectrometer.
(
polyplexes); these polyplexes must escape the endosomal
Critical micelle concentration measurements of the diblock
polymers in aqueous solutions at different pH values were per-
formed by fluorescence spectroscopy using a pyrene probe.
Fluorescence peak intensity emission ratios (373 nm/384 nm) were
plotted against the logarithm of polymer concentrations to
compartments into which they are initially trafficked upon
37,38
internalization.
One such mode of endosomal escape is
through the so-called ‘‘proton sponge effect’’, in which the basic
polymer buffers the endosome during acidification, leading to
37,39
osmotic swelling and rupture.
43
The buffering capacity of the
determine CMC as the onset of micellization. Fluorescence
new polypeptides has been explored using titrations to determine
the pH range at which these polymers buffer. In addition, siRNA
complexation studies have been performed to determine if these
polymers complex with siRNA to form polyplexes.
spectroscopy was carried out on a Horiba FluoroLogꢀ-3 spec-
ꢀ
ꢁ7
trofluorometer at 25 C. Stock solutions of pyrene at 5 ꢂ 10 M in
water orpHbufferwereprepared. Polymer samples weredissolved
in the stock pyrene solution and diluted to specific concentrations.
Tapping-mode atomic force microscopy (AFM) measure-
ments were conducted in air with a Dimension 3100 system
(Digital Instruments, Santa Barbara, CA) operated under
ambient conditions. The samples were prepared for AFM anal-
ysis by spin coating a silicon wafer with a polymer solution at
A unique aspect of these new polypeptides is that there is an
ester linkage between the amine and the polymer backbone. These
ester side chains can be hydrolyzed, leaving behind a carboxylic
acid moiety, thus creating a charge shifting polymer (shifting with
hydrolysis from positive to negative net charge). We have exam-
ined the rate of hydrolysis of the ester side chain at various pH
conditions and the role of the shift in overall polymer charge plays
on disrupting the secondary structure. This hydrolysis and overall
shift in charge from positive to negative could play a role in
improving the safety and biodegradability of these substituted
ꢁ1
a concentration of 1 mg mL in Milli-Q water with the pH
adjusted using 0.1 M NaOH.
Circular dichroism (CD) spectroscopy of polymer solutions
was carried out using an Aviv model 202 CD spectrometer.
ꢀ
Measurements were performed at 25 ꢃ 0.1 C, sampling ever nm
4
0–42
poly(g-glutamic acid) based polymers,
delivery and subsequent unpackaging and release of nucleic acid
based cargos that are delivered using these systems.
and may also aid in the
with a 3–5 s average time over the range of 195–260 nm (band-
width ¼ 1.0 nm). Measurements were taken using a cell with a 1
mm path length. Samples were prepared at a concentration of
ꢁ1
0
.5–1 mg mL in either buffer solutions or Milli-Q water with
pH adjusted using 0.1 M NaOH and 0.1 M HCl solutions.
Ester hydrolysis samples were prepared by dissolving polymer
Experimental
ꢁ
1
in a stock solution at 10 mg mL for homopolymer and 20 mg
Materials
ꢁ1
mL for diblock copolymer. The stock solutions were then
ꢁ1
L-(+)-Glutamic acid, 99% minimum, was purchased from EMD
Chemicals. 3-Dimethylaminopropylchloride hydrochloride,
diluted with pH buffer to a concentration of 0.5 mg mL for
ꢁ1
homopolymer and 1 mg mL for diblock copolymer. At various
5
628 | Soft Matter, 2011, 7, 5627–5637
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time points, samples were analyzed by CD. Samples were also
To a flame dried Schlenk flask, heptylamine (14.5 mL, 0.0980
mmol) and DMF (8 mL) were combined under Ar. In a separate
vial, PLG–NCA (1.552, 7.35 mmol) was dissolved in dime-
thylformamide (DMF) (8 mL) and added to the reaction flask.
The reaction mixture was stirred for three days at room
temperature. The polymer was precipitated into diethyl ether and
ꢁ
1
freeze dried, reconcentrated in D O to 2.5 mg mL for homo-
2
ꢁ1
polymers and 3.75 mg mL for diblock copolymers, acidified
1
with trifluoroacetic acid to stop hydrolysis, and analyzed by H-
NMR.
Ribogreen assays (QuantiT Ribogreen RNA Quantification
Reagent, Invitrogen) were performed to determine the
complexation efficiency of the polymers with siRNA. Ribogreen
is a cyanine dye that is nearly non-fluorescent when unbound to
RNA, but exhibits a >1000 fold enhancement in fluorescence
when bound to RNA. When siRNA is complexed (e.g. by
a polymer), it is unavailable to bind to Ribogreen and thus the
1
removed by centrifugation (0.823 g, 67.0% recovered, by H-
1
NMR n ¼ 75, by DMF GPC M
NMR (400 MHz, [D
w
¼ 14 100, PDI ¼ 1.09). H-
6
] DMF) d ¼ 2.28 (br m, 2H, CH
2
), 2.55 (br
m, 2H, CH–CO), 3.38 (br m, 1H, C^CH), 4.09 (br m, 1H, CH),
1
4.76 (br m, 2H, CH CO), 8.5 (br m, 1H, NH). COSY H-NMR
2
shown in the ESI†.
4
4
fluorescence signal decreases relative to uncomplexed siRNA.
ꢁ
1
2
5 mL of siRNA at 0.006 mg mL was aliquoted into wells of
Synthesis of poly(ethylene glycol)-b-poly(g-propargyl L-gluta-
mate). A typical procedure for the polymerization is as follows. A
round bottom flask was rinsed with acetone and oven dried. In
a 96 well plate and the appropriate amount of polymer was
added to attain the desired polymer : siRNA ratio (N/P) in
a total volume of 50 mL. After allowing 10 minutes for
complexation, 20 mL of the complex solution was added to
a black, flat-bottomed, polypropylene 96-well plate containing
2
a glove box, PEG–NH (0.900 g, 0.180 mmol) was dissolved in
DMF (9 mL) in a round bottom flask. PLG–NCA (0.950 g, 4.50
mmol) was dissolved in dry DMF (9 mL) added to the reaction
flask. The reaction mixture was stirred for three days at room
temperature. The reaction solution was rotovaped and dried
under high vacuum to remove the DMF. To remove any residual
PLG–NCA and DMF, the polymer was redissolved in
dichloromethane precipitated into diethyl ether and removed by
1
00 mL of Ribogreen (diluted 1 : 200 in water per manufacturer
instructions). The fluorescence of each well was measured on
a Perkin Elmer Plate 1420 Multilabel plate reader and the frac-
tion of uncomplexed siRNA was determined by comparing the
fluorescence of the polymer complexes with the fluorescence of
a free siRNA control. For the heparin destabilization titrations,
1
centrifugation (1.45 g, 87.9% recovered, by H-NMR n ¼ 23, by
ꢁ1
1
heparin (167 IU mg ) was dissolved in a stock solution at 0.5 IU
GPC PDI ¼ 1.09). H-NMR (400 MHz, [D
6
] DMF) d ¼ 2.28
ꢁ1
mL and added to polyplex/ribogreen solutions.
(dm, 2H, CH
2
PPLG), 2.55 (dm, 2H, CH–CO PPLG), 3.38 (m,
CH PEG), 4.09 (m, 1H,
CO PPLG), 8.5 (m, 1H, NH
1
H, C^CH PPLG), 3.59 (s, 4H, CH
2
2
Synthesis of g-propargyl L-glutamate hydrochloride. L-Gluta-
CH PPLG), 4.76 (m, 2H, CH
PPLG).
2
mic acid (15 g, 102 mmol) was suspended in propargyl alcohol
(
550 mL) under argon. Chlorotrimethylsilane (28.5 mL, 224
mmol) was added dropwise to the suspension over 1 hour. The
resulting solution was stirred at room temperature for two days
until there was no undissolved L-glutamic acid. The reaction
solution was precipitated into diethyl ether giving a white solid.
The crude product was removed by filtration, dissolved in boiling
isopropanol, and precipitated into diethyl ether. The product was
Synthesis of 2-bromo-N-methylethanamine hydrobromide. 2-
Bromo-N-methylethanamine hydrobromide was synthesized
45
following the protocol presented by Schutte et al. Briefly, in
a round bottom flask, 48% w/w HBr (30 mL) was cooled in an ice
ꢀ
bath to 4 C and 2-(methylamino)ethanol (10 mL, 125 mmol)
was added dropwise. H
2
O and HBr were distilled off and the
ꢀ
filtered, washed with diethyl ether, and dried under vacuum to
1
yield 19.13 g (84.5%). H-NMR (400 MHz, D
crude product solution was cooled to 60 C. The solution was
slowly added to a solution of cold acetone, where it precipitated
out to form a white solid. The precipitant was removed, washed
2
O) d ¼ 2.20 (m,
2
H, CH
2
), 2.63 (dt, 2H, CH–CO), 2.86 (t, 1H, C^CH), 4.05 (t,
CO).
1
H, CH), 4.69 (d, 2H, CH
2
with cold acetone, and dried under high vacuum (16.46 g, 60.4%
1
yield). H-NMR d (400 MHz, D
2
O) 3.69 (t, 2H, BrCH
2
), 3.50 (t,
Synthesis of N-carboxyanhydride of g-propargyl L-glutamate
PLG–NCA). g-Propargyl L-glutamate hydrochloride (6 g, 27
2 3
2H, CH N), 2.75 (s, 3H, CH ).
(
mmol) was suspended in dry ethyl acetate (190 mL). The solution
was heated to reflux and triphosgene (2.67 g, 9 mmol) was added.
The reaction solution was refluxed for 6 hours under nitrogen. The
reaction solution was cooled to room temperature and any
unreacted g-propargyl L-glutamate hydrochloride was removed
General synthesis of amino azides. Organic azides can be
EXPLOSIVE! A guide to safe handling and storage of organic
azides can be found in ‘‘Click Chemistry: Diverse chemical function
12
from a few good reactions’’ by Kolb et al. The shorthand nota-
tion for each side group used in this article is provided in
parentheses after the specific chemical name. Amino azides were
ꢀ
by filtration. The reaction solution was then cooled to 5 C and
46
washed with 190 mL of water, 190 mL of saturated sodium
ꢀ
synthesized using the protocol presented by Carboni et al.
A
bicarbonate, and 190 mL of brine all at 5 C. The solution was then
representative example, 3-dimethylamino-1-propylchloride
hydrochloride (10 g, 63 mmol) and sodium azide (8.22 g, 126
dried with magnesium sulfate, filtered, and concentrated down to
1
viscous oil (4.53 g, 79.2% yield). H-NMR (400 MHz, CDCl
ꢁ1
3
) d ¼
mmol) were dissolved in water (1 mL mmol ) and heated at 75
ꢀ
2
4
.20 (dm, 2H, CH ), 2.49 (t, 1H, C^CH), 2.58 (t, 2H, CH–CO),
2
C for 15 h. The reaction mixture was cooled in an ice bath and
.39 (t, 1H, CH), 4.68 (d, 2H, CH CO), 6.5 (s, 1H, NH).
2
NaOH (4 g) was added. The solution phase separated and the
organic phase was removed. The aqueous phase was extracted
with diethyl ether twice. The organic layers were combined, dried
Synthesis of poly(g-propargyl L-glutamate) initiated by heptyl-
amine. A typical procedure for the polymerization is as follows.
with MgSO
4
, and concentrated down to an oil (6.60 g, 80.8%
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yield). 3-Azido-N,N-dimethylpropan-1-amine (dimethylpropan-
1
Table 1 Summary of polymerization feed NCA-monomer/initiator,
1
degree of polymerization by H-NMR, molecular weight and poly-
dispersity determined by DMF GPC with PMMA standards
amine) H-NMR (400 MHz, CDCl ) d (ppm) ¼ 3.30 (t, 2H,
3
N CH ), 2.30 (t, 2H, CH N), 2.17 (s, 6H, N(CH ) ), 1.71 (m, 2H,
3
2
2
3 2
1
N
3
CH
2
CH
2
). 2-Azidoethanamine (primary amine) H-NMR
) d (ppm) ¼ 3.32 (t, 2H, N CH ), 2.83 (t, 2H,
), 1.45 (s, 2H, NH ). 2-Azido-N-methylethanamine
secondary amine) H-NMR (400 MHz, CDCl
) d (ppm) ¼ 3.45
t, 2H, N CH ), 2.72 (t, 2H, CH NH), 2.39 (s, 3H, CH ), 1.28 (s,
H, NH). 2-Azido-N,N-dimethylethanamine (dimethylethan-
DMF GPC with PMMA
standards
DP
(
400 MHz, CDCl
3
3
2
CH NH
2
2
2
Polymer
Feed ratio
by NMR
M
n
M
w
PDI
1
(
3
(
3
2
2
3
PPLG
PPLG
PPLG
PPLG
PEG–NH2
PEG-b-PPLG
25
50
75
150
—
25
30
56
75
140
—
23
6100
12 700
17 900
42 900
10 000
14 600
7600
14 100
19 400
50 000
11 500
15 800
1.25
1.11
1.09
1.17
1.14
1.08
1
1
amine) H-NMR (400 MHz, CDCl
3
) d (ppm) ¼ 3.32 (t, 2H,
N CH ), 2.47 (t, 2H, CH N), 2.24 (s, 6H, N(CH ) ). 2-Azido-N,
3
2
2
3 2
1
N-diethylethanamine (diethylamine) H-NMR (400 MHz,
CDCl ) d (ppm) ¼ 3.25 (t, 2H, N CH ), 2.62 (t, 2H, CH CH N),
3
3
2
2
2
2
.52 (q, 2.54, N(CH CH ) ), 1.00 (s, 6H, (CH CH ) ). N-(2-
2 3 2 2 3 2
1
propanamine, diethylamine, and diisopropylamine) were
attached to four different molecular weight PPLG backbones
and a PEG-b-PPLG diblock copolymer through the copper-
mediated 1,3 cycloaddition between the alkynes on the PPLG
backbone and the azide bearing amine groups, shown in Scheme
Azidoethyl)-N-isopropylpropan-2-amine (diisopropylamine) H-
NMR (400 MHz, CDCl CH ), 2.98
N), 0.99 (d, 12H,
3
) d (ppm) ¼ 3.01 (t, 2H, N
3
2
(
m, 2H, N(CH(CH
CH (CH ).
3
)
2
)
2
), 2.62 (t, 2.54, CH
2
(
2
3 2 2
) )
1
. The labels under each side group are used to refer to each
General synthesis of substituted PPLG. A typical procedure
polymer bearing that side group. The specific chemical name for
each side group can be found in the Experimental section. The
PPLG was coupled with azido amines using CuBr/PMDETA as
a catalyst in DMF with a molar ratio of alkyne/azide/CuBr/
PMDETA equal to 1/1.2/0.1/0.1. After the reaction was
complete, the polymer was purified by dialysis against water
acidified with HCl (pH # 4) to remove any unreacted amino
azides and the copper catalyst.
started with a feed ratio of alkyne/azide/CuBr/PMDETA equal
to 1/1.2/0.1/0.1. The PPLG (0.0750 g, 0.45 mmol alkyne repeat
units), amino azide (0.069 g, 0.54 mmol 3-azido-N,N-dimethyl-
propan-1-amine), and PMDETA (9.4 mL, 0.045 mmol) were all
dissolved in DMF (3 mL). The solution was degassed by
bubbling argon through the solution for 20 minutes. CuBr
catalyst (0.0064 g, 0.045 mmol) was added, and the reaction
solution was stirred at room temperature, under argon. Once the
reaction was complete, the reaction solution was purified by
dialysis against water acidified by HCl (pH < 4) for 2–3 days,
followed by dialysis with Milli-Q water to remove acid before
1
The polymer structures were confirmed using H-NMR.
1
Representative H-NMR spectra of the diethylamine and diiso-
1
propylamine substituted PPLG compared to the H-NMR
1
spectra of PPLG are shown in Fig. 1. For all amine groups, the
coupling efficiency was near quantitative, as indicated by the
disappearance of the PPLG alkyne peak (a, 3.4 ppm) and ester
peak (b, 4.7 ppm) and the appearance of a new ester peak (k, 5.2
ppm) and the triazole ring peak (m, 8.15 ppm). Furthermore, the
peak integration for all samples tested was as expected for near
quantitative substitution without hydrolysis of the ester group on
freeze drying. The final polymer was a white solid. For H-NMR
d (400 MHz, D O) see the ESI†.
2
Results and discussion
Polymer synthesis
11
The PPLG polymers were prepared as previously described.
Briefly, g-propargyl L-glutamate was reacted with triphosgene to
form the NCA. PPLG and PEG-b-PPLG were prepared by ring
opening polymerization in dimethylformamide (DMF) at room
temperature by initiation with heptylamine and PEG–NH (M ¼
2
w
5
000), respectively. Table 1 summarizes the stoichiometric feed
ratio of each polymerization, the degree of polymerization char-
1
acterized by H-NMR, and the molecular weight and molecular
weight distribution characterized by gel permeation chromatog-
raphy (GPC) with DMF with 0.01 M LiBr as the carrier solvent.
The narrow polydispersities (1.09–1.25) and reaction feed ratio
1
compared to the degree of polymerization by H-NMR indicate
that the polymerization is well controlled. Furthermore, this
polymerization route allows for high molecular weight polymers
with a degree of polymerization as high as 140. As indicated by
Poch ꢀe et al., a high degree of polymerization can be obtained if the
NCA monomer purity is high; the washing strategy employed in
this NCA monomer preparation does significantly improve the
47
monomer purity by removing residual HCl.
Six different amine moieties ranging in pK
amines) and hydrophobicity (dimethylethanamine, dimethyl-
ꢀ
ꢀ
ꢀ
a
(1 , 2 , and 3
Scheme 1 Functionalization of PPLG by the alkyne–azide cycloaddi-
tion click reaction and the pH responsive side groups.
5
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1
the polymer side chains. Representative H-NMR for the
35,36
range of typical extracellular tissue to late endosomal pH.
remaining amine functionalized PPLG and PEG-b-PPLG can be
The diisopropylamine polypeptide exhibits the sharpest buff-
ering transition at pH 5.25; the diethylamine polymer also
buffers in this range, but with a broader transition that has
a midpoint at the slightly higher pH of approximately 6.5. The
primary and secondary amine functional polymers interestingly
exhibit similar broad buffering behavior beginning at pH 5.5
with a midpoint at 7.25. One would typically expect buffering at
found in the ESI†.
Investigation of polymer buffering and solubility
To investigate pH responsiveness and the buffering behavior of
these polypeptide systems, titrations were performed on all
polymers. Polymers were dissolved in 125 mM NaCl at 10 mM
polypeptide–amine (molarity based on repeat unit), titrated with
increasing pH to a pH of 10–10.5 using 0.1 M NaOH, and
subsequently titrated with decreasing pH using 0.1 M HCl. After
higher pH for primary and secondary amines (pK
48,49
a
approxi-
mately 9–11),
although some buffering is observed in these
polymers from pH 8 to 10. Polyelectrolytes typically exhibit
broad buffering behavior and shifted pKa values due to
segmental charge repulsion. For the dimethyl substituted amines,
the dimethylethanamine exhibits buffering behavior starting at
the same pH as the primary and secondary amines with
a midpoint falling between 6.5 and 7.0. These values are
consistent with the series of polymers with ethylene linker groups
to the tetrazole ring; whereas, the dimethylpropanamine polymer
exhibits buffering at higher pHs. The additional carbon between
titrations were complete, representative samples were freeze
1
dried, dissolved in D O, and analyzed using H-NMR. From the
2
1
H-NMR, the spectra were nearly identical to those obtained
before titrations, indicating that hydrolysis did not occur during
the 2–3 hour titration process (see ESI†). Representative titra-
tions with increasing pH are shown in Fig. 2, where Fig. 2A
consists of the dimethylethanamine polymers at varying degrees
of polymerization, and Fig. 2B consists of titrations of each
polymer side functional group with a degree of polymerization of
NCA backbone of 75 (titrations of additional polymers can be
found in the ESI†). All polymers appear to have strong buffering
capacity in the pH range of 5–7.35, which scales with the pH
the amine group and the triazole ring results in a higher pK
a
for
the dimethylpropanamine. This shift in pK could be the result of
a
the amine group being further removed from the electron with-
drawing triazole ring or from the decreased crowding experi-
enced by the amine group. All of the polymers exhibit a small
amount of buffering at the start of the titration curve, at pH 3–4;
Fig. 2 Titrations of polymers at a concentration of 10 mM using 0.1 M
sodium hydroxide. (A) PPLG polymers functionalized with dimethyle-
thanamine with varying degrees of polymerization, and (B) PPLG
polymers with a degree of polymerization of 75.
7
Fig. 1 (A) PPLG in d DMF, (B) PPLG functionalized with diethyl-
1
amine in D
amine in D
2
O, and (C) H-NMR of PPLG functionalized with diethyl-
O. The PPLG backbone has a degree of polymerization of 75.
2
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the buffering in this region could be a result of the triazole ring
a micellar system. To determine if the precipitation pH could be
tuned, a 50 : 50 mixture of diethylamine and diisopropylamine
side groups was attached to PPLG (DP ¼ 140) to generate
a random copolymer. As shown in Fig. 3 (dotted-gray line), the
copolymer precipitation pH falls between the precipitation pH
values observed for the diethylamine and diisopropylamine
substituted PPLG (DP ¼ 140), indicating that the pH respon-
siveness of the amine substitute PPLG block can be fine tuned by
changing the ratio of side groups. One could also envision using
this strategy to incorporate side groups that will improve the
loading of a specific drug or increase polymer–gene complexation
efficiency. The buffering and the precipitation behavior was
found to be fully reversible, as indicated by reverse titrations that
were performed on all polymers (reverse titrations can be found
in the ESI†). For the completely water soluble primary,
secondary, and dimethyl polymers, the reverse titration curve has
the same shape as the original titration with no signs of hyster-
esis. For tertiary amine polymers that precipitated out of solu-
tion, hysteresis was often observed for the larger degrees of
polymerization, such that the pH value for which the polymers
re-dissolved was often lower than the value observed for
precipitation. For the shortest degree of polymerization (DP ¼
30), the polymers returned to solution at nearly the same pH as
when the precipitation was initially observed (Fig. 4).
generated during the click reaction; triazoles exhibit pK ’s of less
a
48,50
than 3.0.
The polymer buffering appears to have little
dependence on polymer molecular weight, as indicated in
Fig. 2A.
The primary, secondary, and dimethyl polypeptides remain
water soluble over the entire pH range; however, as the cationic
diethylamine and diisopropylamine functionalized polypeptides
are titrated from acidic to basic conditions, the amines become
deprotonated. The resulting uncharged polypeptide is no longer
soluble in water, leading to precipitation of the polypeptide from
aqueous solution. For the diethylamine and diisopropylamine
functionalized PPLG, polymer precipitation was observed at
various pH values depending on polymer molecular weight. To
determine the pH where precipitation occurs, turbidity
measurements were performed on the diethylamine and diiso-
propylamine functionalized PPLGs by monitoring polymer
solution transmission at 550 nm, shown in Fig. 3. When the
polymers begin to precipitate out of solution, there is a sharp
drop in transmission. For the diethylamine functionalized PPLG
(
Fig. 3, solid lines), precipitation occurred between 6.80 and 7.45
depending on the degree of polymerization and for the diiso-
propylamine functionalized PPLG (Fig. 3, dashed lines),
precipitation occurred between 5.23 and 5.59. These values are
consistent with the titration data shown in Fig. 2 and in the ESI†.
In general we see the anticipated trend that increased molecular
weight leads to precipitation at lower pH values and higher
degrees of ionization of the polymer functional group. It is
notable that the diethylamine series is more sensitive to molec-
ular weight than the diisopropylamine series, which seems to
approach a limiting minimum pH value for precipitation. This
result may be due to the greater hydrophobicity of the diiso-
propylamine group as opposed to the diethylamine, which would
lead to a lower degree of solubility of the amine side chain and
a decreased dependence on molecular weight.
Functionalized PEG-b-PPLG self-assembly
The self-assembly of PEG-b-PPLG functionalized with diethyl-
amine and diisopropylamine was studied as a function of pH.
The critical micelle concentration (CMC) was determined for
PEG-b-PPLG in water and amine functionalized PEG-b-PPLG
in buffer solutions at pH 9 and pH 5.5. The CMC was deter-
mined by fluorometry using a pyrene probe. A representative
example of the diisopropylamine functionalized PEG-b-PPLG is
shown in Fig. 5 (information for diethylamine substituted PEG-
b-PPLG can be found in the ESI†). As shown in Fig. 5A, there is
a clear break in the emission ratio indicating a CMC for the
amine functionalized PEG-b-PPLG in pH 9 buffer. In pH 5.5
buffer, no break in emission ratio was observed for the func-
tionalized polymer, indicating that these macromolecules do not
self-assemble at all at this acidic pH, but remain completely
The pH transition observed for both the diisopropylamine and
diethylamine functionalized polymers can be utilized for the
design of a pH responsive drug carrier in which the responsive
PPLG block would be the interior, pH responsive block of
Fig. 3 Transmission as a function of pH for all diethylamine and dii-
sopropylamine functionalized polymers. Diethylamine is abbreviated DE
and diisopropylamine is abbreviated DI.
Fig. 4 Transmission as a function of increasing and decreasing pH for
diethylamine and diisopropylamine with DP ¼ 30.
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soluble in water. The observed CMC values for all diblock
polymers tested (Table 2) are of the same order of magnitude of
Table 2 CMC values for PEG-b-PPLG in water and amine function-
alized PEG-b-PPLG in pH 5 and pH 9 buffer
21
PEG-b-PBLA and are several orders of magnitude lower than
ꢁ
1
Solvent
CMC/mg mL
CMC/M
51
Pluronic micelle CMC values. To further verify that the self-
assembled structures were micelles, AFM was performed on
diethylamine and diisopropylamine substituted PEG-b-PPLG
cast from a water solution adjusted with 0.1 M NaOH to pH z
ꢁ
ꢁ
4
2
ꢁ8
ꢁ7
PEG-b-PPLG
Diethylamine
MQ water
3.75 ꢂ 10
1.11 ꢂ 10
—
3.74 ꢂ 10
7.49 ꢂ 10
—
pH 9 buffer
pH 5.5 buffer
pH 9 buffer
pH 5.5 buffer
ꢁ3
ꢁ8
Diisopropylamine
1—. 05 ꢂ 10
7— .38 ꢂ 10
9
. Spherical micelles were observed for the amine substituted
PEG-b-PPLG; Fig. 5B shows an AFM image of the diisopro-
pylamine functionalized diblock copolymer. The micelles are
thus able to form at moderate to high pH, but become completely
destabilized at low pH, making them of interest for drug release
in which a pH triggered rapid disassembly of drug carrier can be
designed to take place within acidic compartments to release
a drug.
polypeptide with DP ¼ 75 is shown in Fig. 6. When initially
brought down to a pH of 3, the sample adopts a mixture of a-
helix and random coil conformations, as indicated by the
minimum at 222 nm, which is characteristic of an a-helix and the
second, more negative minimum at 204 nm, which is indicative of
a combination of a-helix and random coil. As the sample pH is
increased, the sample adopts an all a-helical structure at high pH
values (pH > 6.36), as indicated by the minima at 208 nm and 222
Secondary structure
Circular dichroism (CD) was used to probe the secondary
structure of the various polymers as a function of pH. Polymer
52
nm. When the pH is decreased stepwise back down to acidic
pH, this a-helical structure transitions back to a mixture of a-
helix and random coil. The a-helix to random coil transition
ꢁ1
dissolved at 1 mg mL was brought down to a pH of 3, titrated
to a pH higher than 10, and then immediately titrated back to
a pH of 3. A sample CD titration of a secondary amine
a
correlates well with the pK observed in the polymer titrations. In
summary, the a-helix structure appears to correlate with the
uncharged polymer backbone; as the backbone becomes
charged, the helical structure becomes reversibly disrupted and
exhibits some random coil structure.
Fig. 5 (A) CMC determination by fluorometry using a pyrene probe for
the diisopropylamine substituted PEG-b-PPLG in pH 5.5 and 9 buffer
and (B) AFM images of diisopropylamine substituted PEG-b-PPLG at
pH 8.88. The AFM images are 2 by 2 mm with a height range from ꢁ30
mm to 30 mm.
Fig. 6 (A) Increasing pH CD titrations and (B) decreasing pH CD
titrations for secondary amine, DP ¼ 75.
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41
cytotoxicity. The introduction of a mechanism that eliminates
the multivalent positive charge and transforms the polymer into
the benign and naturally occurring negatively charged poly-
(
g-glutamic acid), which enhances the long-term biocompati-
bility of these polymers.
40,41
To determine the side chain ester hydrolysis rate and the
1
change in polymer secondary structure, H-NMR and CD
measurements were taken at various time points and pH condi-
tions. Polymer samples (PPLG DP ¼ 75 with secondary amine
and PEG-b-PPLG with diethylamine and diisopropylamine)
were dissolved in various pH buffers to a concentration of 0.5–1
ꢁ1
mg mL and left to hydrolyze at room temperature. From
1
H-NMR, the amount of ester hydrolyzed was determined by
comparing the peak integration of the triazole peak from the
ester side chain (8.15 ppm) to the integration of a new triazole
peak from the alcohol side chain byproduct (8.07 ppm). A
1
sample H-NMR for PEG-b-PPLG functionalized with diethyl-
amine hydrolyzed at pH 9 is shown in the ESI†. When the
polyamide backbone, which maintains an a-helical structure
when at equilibrium at all pH conditions investigated (pH 7.4, 9,
and 11), undergoes hydrolysis, a glutamic acid residue is gener-
ated. Poly(g-glutamic acid), like poly(L-lysine), maintains an
a-helix in the uncharged state, and is a random coil in the
55
charged state; thus as hydrolysis occurs at more basic condi-
tions we observe the loss of the a-helical polymer structure.
Circular dichroism at 222 nm was observed to determine the
change in secondary structure as a function of time. At 222 nm,
a shift from a strong negative value towards a small positive
value is indicative of a secondary structure shift, in this case,
a shift from an a-helix to a random coil (representative full
spectra and plots for PPLG functionalized with secondary amine
at pH 7.4 and pH 9 can be found in the ESI†).
The results of the ester hydrolysis study are shown in Fig. 7
and 8. Representative ester hydrolysis plots for PPLG (DP ¼ 75)
functionalized with secondary amine and PEG-b-PPLG func-
tionalized with diethyl and diisopropylamine are shown in
Fig. 7A–C, respectively. In Fig. 8, the CD value observed at
2
22 nm is plotted at various pH values as a function of time for
PPLG (DP ¼ 75) functionalized with secondary amine. For all
polymers, the rate of ester hydrolysis was highest at pH 11 and
was increasingly slower as the pH was decreased. For example, in
Fig. 7 (A) Percentage of ester side chains hydrolyzed as a function of
time for PPLG (DP ¼ 75) functionalized with secondary amine, (B) PEG-
b-PPLG functionalized with diethylamine, and (C) PEG-b-PPLG func-
tionalized with diisopropylamine.
Impact of pH on side chain hydrolysis
The functional groups introduced along the PPLG backbone are
esters that can undergo hydrolysis under basic conditions,
yielding the loss of the amino side group and the introduction of
the carboxylate anion, thus introducing negative charge to the
polyelectrolyte backbone. Slow or moderate changes in the
polypeptide backbone may be of interest for drug delivery, gene
30,42,53,54
delivery, tissue engineering, and coating applications.
Specifically, for systemic use, positively charged polymers such as
poly(L-lysine) and poly(ethylene imine) often exhibit significant
Fig. 8 CD value observed at 222 nm at various pH values as a function
of time for PPLG (DP ¼ 75) functionalized with secondary amine.
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Fig. 9 Percentage of uncomplexed siRNA as a function of siRNA : polymer (N/P) ratio for each amine substituted PPLG for degree of polymerization
140 (A and B) and 75 (C and D). Polyplexes were formed in either sodium acetate buffer (A and C) or PBS (B and D). The DP140 diisopropylamine
sample was insoluble in PBS.
all cases complete hydrolysis was observed at pH 11 (at 2 days for
the secondary amine and diethylamine and 11 days for the dii-
sopropylamine), but at pH 5.5 after 15 days, all samples were less
than 2% hydrolyzed. When comparing the ester side chain
hydrolysis between polymers, the rate of hydrolysis at pH 7.4, 9,
and 11 was fastest for PPLG functionalized with secondary
amine and slowest for PEG-b-PPLG functionalized with diiso-
propylamine. For the diblock polymers, the polypeptide is
encapsulated as the inner core of a micelle, and is partially pro-
tected from hydrolysis, thus greatly slowing the rate of
hydrolysis.
siRNA complexation studies
Studies have been performed to determine if the amine func-
tionalized homopolymers complex siRNA into protective poly-
plexes. Polymers were mixed with siRNA at various PPLG
polymer to siRNA charge ratios (N/P) ranging from 1 : 1 to
5
0 : 1 in either sodium acetate buffer (pH 5.5) or PBS (pH 7.4).
Ribogreen was used to determine the complexation efficiency of
each polymer at the various ratios, shown in Fig. 9. As shown in
Fig. 9A and C, all amine functionalized PPLG homopolymers
prevent dye access to more than 90% of siRNA at charge ratios
above 4 : 1 in sodium acetate. Additionally PPLGs with primary
amine substituents are able to completely complex siRNA at
a charge ratio that is two-fold lower, indicating the strength of
primary amines for complexation. At the higher pH of PBS (7.4),
fewer amines are charged, particularly in the case of the dime-
thylethanamine, diethylamine, and diisopropylamine substitu-
ents, leading to looser complexes and greater dye access. This
manifests itself both at low polymer : siRNA ratios for all of the
polymers, and most notably for the dimethylethanamine, dieth-
ylamine, and diisopropylamine PPLGs (see Fig. 9B and D).
While these tertiary amine substituents may be useful for stim-
ulating endosomal escape, the copolymers with primary and
tertiary amines are more likely to exhibit properties that enable
full encapsulation of siRNA and buffering effects in vivo.
When looking at the secondary structure of PPLG (DP ¼ 75)
functionalized with secondary amine (Fig. 8), the polymer adopts
a random coil after 1 day (24 hours) in pH 11 buffer solution, at
pH 9, the polymer gradually adopts a random coil over several
days, and at pH 7.4 the polymer primarily maintains an a-helical
1
structure for multiple days. When compared to the H-NMR
data, at pH 11, the ester side chains have completely hydrolyzed
in two days, leaving poly(g-glutamic acid) which is in a random
coil conformation. For pH 9, at day 4, the polymer is 50%
hydrolyzed, and the polymer structure is nearly all random coil.
This observation indicates that not all the ester side chains need
to be hydrolyzed for the a-helix to be disrupted. Similar CD
trends were observed for all PPLG (DP ¼ 75) and PEG-b-PPLG
polymers tested and can be found in the ESI†. In summary, we
can control the rate of ester degradation and the rate of a-helix
disruption by changing the side chain functionality.
Polyplexes can be disrupted by the addition of a competing
polyanion, such as heparin. In Fig. 10A, PPLGs (DP ¼ 140) with
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change solubility, or self-assemble into micelles for the case of
diblock polymers, with degree of ionization and adopt an
a-helical structure at biologically relevant pHs. The impact of
side chain hydrolysis was also explored to determine the hydro-
lysis rate as a function of pH and the impact of hydrolysis on
polymer side chain conformation. These properties are of interest
for a number of applications, here we have performed prelimi-
nary experiments that demonstrate that these polymers are
strong candidates for drug and gene delivery.
Acknowledgements
This research was funded by the Polymers Program of the
National Science Foundation Division of Materials Research,
NSF DMR Grant Number 0705234, the MIT-Portugal
Program, the MIT-Singapore Alliance, and the United States
Environmental Protection Agency Science to Achieve Results
Graduate Fellowship. Use of equipment at the Institute for
Soldier Nanotechnologies, the Department of Chemistry
Instrumentation Facility, the Center for Materials Science and
Engineering Shared Experimental Facilities, and the Biophysical
Instrumentation Facility for the Study of Complex Macromo-
lecular Systems (NSF-0070319 and NIH GM68762) is greatly
acknowledged. We thank Prof. Hyung-il Lee, Jason Cox, and
Caroline Chopko for their helpful discussion and Dr Jeff Simp-
son for obtaining COSY NMR spectra.
Fig. 10 Percentage of uncomplexed siRNA as a function of added
heparin for various complexation conditions. (A) Complexes were
formed in pH 5.5 sodium acetate buffer (squares) or PBS (circles) at two
different polymer : siRNA ratios (N/P). (B) PPLGs with primary (circle),
secondary (square), or dimethylpropanamine (triangle) substitutions
were complexed in sodium acetate buffer prior to dissociation with
heparin.
primary amine substituents were complexed at low (5 : 1) and
high (25 : 1) polymer : siRNA ratios (N/P) in either sodium
acetate or PBS, along with PEI and Lipofectamine 2000 as
controls. As anticipated, relatively low levels of heparin were
required to dissociate PPLG complexes formed at 5 : 1 as
compared with those complexes formed at the 25 : 1 N/P ratio.
PPLG complexes formed in PBS were more easily disrupted than
those formed at low pH, most likely because those formed at low
pH contained more highly charged amines, and were thus more
tightly complexed. Fig. 10B demonstrates this concept with
different amine substituents. In the DP75 polymers (red), the
tertiary amine in the dimethylpropanamine group forms a looser
polyplex and is disrupted more readily than the secondary and
primary amines. However, for DP140, the dimethylpropanamine
polyplexes begin to dissociate with the same amount of added
heparin as the primary and secondary polyplexes, indicating that
molecular weight is also a factor in polyplex stability. In
summary, the siRNA complexation behavior of these systems is
tunable, and can be altered through the introduction of different
buffering amine functionalities, molecular weight and pH
conditions of complexation. Further studies of these systems as
siRNA and gene delivery vehicles are ongoing.
References
1 T. J. Deming, Prog. Polym. Sci., 2007, 32, 858–875.
2
T. J. Deming, in Peptide Hybrid Polymers, Springer, 2006, vol. 202,
pp. 1–18.
3
K. Osada and K. Kataoka, in Peptide Hybrid Polymers, Springer,
Berlin, 2006, vol. 202, pp. 113–153.
4 W. H. Daly, D. Poche and I. I. Negulescu, Prog. Polym. Sci., 1994, 19,
9–135.
7
E. H. C. Bromley, K. Channon, E. Moutevelis and D. N. Woolfson,
5
ACS Chem. Biol., 2008, 3, 38–50.
6 Progress in Molecular Biology and Translational Science, ed. P. M.
Conn, Elsevier Inc., London, 2008.
7
8
A. Harada, S. Cammas and K. Kataoka, Macromolecules, 1996, 29,
183–6188.
P. Appel and J. T. Yang, Biochemistry, 1965, 4, 1244–1249.
6
9 A. Ciferri, D. Puett and L. Rajagh, Biopolymers, 1968, 6, 1019–1036.
0 B. H. Zimm and J. K. Bragg, J. Chem. Phys., 1959, 31, 526–535.
1 A. C. Engler, H. I. Lee and P. T. Hammond, Angew. Chem., Int. Ed.,
1
1
2
009, 48, 9334–9338.
12 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed.,
2001, 40, 2004–2021.
1
3 C. Xiao, C. Zhao, P. He, Z. Tang, X. Chen and X. Jing, Macromol.
Rapid Commun., 2010, 31, 991–997.
1
4 H. Y. Tang and D. H. Zhang, Biomacromolecules, 2010, 11, 1585–
1592.
1
1
5 J. Sun and H. Schlaad, Macromolecules, 2010, 43, 4445–4448.
6 J. Huang, G. Habraken, F. Audouin and A. Heise, Macromolecules,
2010, 43, 6050–6057.
7 Y. K. Reshetnyak, O. A. Andreev, M. Segala, V. S. Markin and
D. M. Engelman, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 15340–
1
Conclusion
1
5345.
8 M. Zoonens, Y. K. Reshetnyak and D. M. Engelman, Biophys. J.,
008, 95, 225–235.
We have developed a new library of pH responsive polypeptides
based on the combination of NCA polymerization and alkyne–
azide cycloaddition click chemistry. PPLG homopolymers and
PEG-b-PPLG block copolymers were substituted with various
1
2
19 Y. K. Reshetnyak, O. A. Andreev, U. Lehnert and D. M. Engelman,
Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 6460–6465.
0 M. Yokoyama, G. S. Kwon, T. Okano, Y. Sakurai, T. Seto and
K. Kataoka, Bioconjugate Chem., 1992, 3, 295–301.
1 G. Kwon, M. Naito, M. Yokoyama, T. Okano, Y. Sakurai and
K. Kataoka, Langmuir, 1993, 9, 945–949.
2
2
a
amine moieties that range in pK and hydrophobicity, that can be
tuned for specific interactions and responsive behaviors. We have
demonstrated that these new amine-functionalized polypeptides
5
636 | Soft Matter, 2011, 7, 5627–5637
This journal is ª The Royal Society of Chemistry 2011

View Article Online
2
2
2 S. Katayose and K. Kataoka, Adv. Biomater. Biomed. Eng. Drug
Delivery Syst., 1996, 319–320.
3 S. Takae, K. Miyata, M. Oba, T. Ishii, N. Nishiyama, K. Itaka,
Y. Yamasaki, H. Koyama and K. Kataoka, J. Am. Chem. Soc.,
38 K. A. Whitehead, R. Langer and D. G. Anderson, Nat. Rev. Drug
Discovery, 2009, 8, 129–138.
39 S. Boeckle, K. von Gersdorff, S. van der Piepen, C. Culmsee,
E. Wagner and M. Ogris, J. Gene Med., 2004, 6, 1102–1111.
40 M. L. Adams, A. Lavasanifar and G. S. Kwon, J. Pharm. Sci., 2003,
92, 1343–1355.
41 D. M. Lynn and R. Langer, J. Am. Chem. Soc., 2000, 122, 10761–
10768.
42 L. Veron, A. Ganee, M. T. Charreyre, C. Pichot and T. Delair,
Macromol. Biosci., 2004, 4, 431–444.
43 K. Kalyanasundaram and J. K. Thomas, J. Am. Chem. Soc., 1977, 99,
2039–2044.
44 L. J. Jones, S. T. Yue, C.-Y. Cheung and V. L. Singer, Anal. Biochem.,
1998, 265, 368–374.
45 E. Schutte, T. J. R. Weakley and D. R. Tyler, J. Am. Chem. Soc.,
2003, 125, 10319–10326.
46 B. Carboni, A. Benalil and M. Vaultier, J. Org. Chem., 1993, 58,
3736–3741.
2008, 130, 6001–6009.
2
2
2
2
2
2
3
3
3
4 K. Miyata, S. Fukushima, N. Nishiyama, Y. Yamasaki and
K. Kataoka, J. Controlled Release, 2007, 122, 252–260.
5 K. Masago, K. Itaka, N. Nishiyama, U. I. Chung and K. Kataoka,
Biomaterials, 2007, 28, 5169–5175.
6 P. Opanasopit, M. Yokoyama, M. Watanabe, K. Kawano,
Y. Maitani and T. Okano, Pharm. Res., 2004, 21, 2001–2008.
7 K. Itaka, T. Ishii, Y. Hasegawa and K. Kataoka, Biomaterials, 2010,
31, 3707–3714.
8 L. Dekie, V. Toncheva, P. Dubruel, E. H. Schacht, L. Barrett and
L. W. Seymour, J. Controlled Release, 2000, 65, 187–202.
9 P. Dubruel, L. Dekie and E. Schacht, Biomacromolecules, 2003, 4,
1
168–1176.
0 S. F. Chen, Z. Q. Cao and S. Y. Jiang, Biomaterials, 2009, 30, 5892–
896.
1 Q. Wan, J. Chen, G. Chen and S. J. Danishefsky, J. Org. Chem., 2006,
1, 8244–8249.
5
47 D. S. Poch ꢀe , M. J. Moore and J. L. Bowles, Synth. Commun., 1999, 29,
843–854.
48 J. Clayden, N. Greeves, S. Warren and P. Wothers, Organic
Chemistry, Oxford University Press, Oxford, 2001.
49 S. R. Bhatia, S. F. Khattak and S. C. Roberts, Curr. Opin. Colloid
Interface Sci., 2005, 10, 45–51.
7
2 C. Y. Yang, B. B. Song, Y. Ao, A. P. Nowak, R. B. Abelowitz,
R. A. Korsak, L. A. Havton, T. J. Deming and M. V. Sofroniew,
Biomaterials, 2009, 30, 2881–2898.
3
3
3
3 D. J. Pochan, L. Pakstis, B. Ozbas, A. P. Nowak and T. J. Deming,
Macromolecules, 2002, 35, 5358–5360.
4 A. P. Nowak, V. Breedveld, L. Pakstis, B. Ozbas, D. J. Pine,
D. Pochan and T. J. Deming, Nature, 2002, 417, 424–428.
5 P. Vaupel, F. Kallinowski and P. Okunieff, Cancer Res., 1989, 49,
50 T. Eicher, S. Hauptmann and A. Speicher, The Chemistry of
Heterocycles, Wiley-VCH, Weinheim, 2nd edn, 2003.
51 P. Alexandridis, J. F. Holzwarth and T. A. Hatton, Macromolecules,
1994, 27, 2414–2425.
52 W. C. Johnson, Proteins: Struct., Funct., Genet., 1990, 7, 205–214.
53 X. H. Liu, J. T. Zhang and D. M. Lynn, Adv. Mater., 2008, 20, 4148–
4153.
6449–6465.
36 I. Mellman, J. Exp. Biol., 1992, 172, 39–45.
3
7 N. D. Sonawane, F. C. Szoka and A. S. Verkman, J. Biol. Chem.,
54 J. T. Zhang and D. M. Lynn, Adv. Mater., 2007, 19, 4218–4223.
55 Y. P. Myer, Macromolecules, 1969, 2, 624–628.
2003, 278, 44826–44831.
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