Synthesis of statistical copolymers containing multiple functional peptides for nucleic acid delivery

Article abstract of DOI:10.1021/bm100806h

Our report describes RAFT copolymerization of multiple species of active peptide monomers with N-(2-hydroxypropyl(methacrylamide) (HPMA) under aqueous conditions. Resulting statistical copolymers are narrowly disperse with highly controlled molecular weight and composition. Side-chain peptide copolymers were synthesized using a DNA condensing peptide (K₁₂), and an endosomal escape peptide (K₆H₅) that had been modified with an aminohexanoic linker and capped with methacrylamide vinyl on the NH ₂-terminus. Copolymers of HMPA-co-K₁₂ and HPMA-co-K ₁₂-co-K₆H₅ efficiently condensed DNA into small particles that maintain size stability even in 150 mM salt solutions. With increasing peptide content, the peptide-based polymers demonstrated gene delivery efficiencies to HeLa cells that were comparable to branched polyethylenimine.

Full text of DOI:10.1021/bm100806h

Biomacromolecules 2010, 11, 3007–3013
3007
Synthesis of Statistical Copolymers Containing Multiple
Functional Peptides for Nucleic Acid Delivery
Russell N. Johnson, Rob S. Burke, Anthony J. Convertine, Allan S. Hoffman,
Patrick S. Stayton, and Suzie H. Pun*
Department of Bioengineering, University of Washington, Seattle, Washington 98195, United States
Received July 16, 2010; Revised Manuscript Received September 13, 2010
Our report describes RAFT copolymerization of multiple species of active peptide monomers with N-(2-
hydroxypropyl(methacrylamide) (HPMA) under aqueous conditions. Resulting statistical copolymers are narrowly
disperse with highly controlled molecular weight and composition. Side-chain peptide copolymers were synthesized
using a DNA condensing peptide (K12), and an endosomal escape peptide (K6H5) that had been modified with an
aminohexanoic linker and capped with methacrylamide vinyl on the NH2-terminus. Copolymers of HMPA-co-
K12 and HPMA-co-K12-co-K6H5 efficiently condensed DNA into small particles that maintain size stability even
in 150 mM salt solutions. With increasing peptide content, the peptide-based polymers demonstrated gene delivery
efficiencies to HeLa cells that were comparable to branched polyethylenimine.
polyplexes. The modified polyplexes demonstrated enhanced
serum stability in vitro and extended circulatory half-life in
vivo.10 Also, in a previous report, we explored the use of small
oligomers of L-lysine copolymerized with HPMA through free
radical polymerization.2 Although the incorporation efficiency
of the peptides was poor and the copolymers disperse, the study
demonstrated the potential for these types of peptide-based
hybrid materials as nucleic acid carriers.
In our current study, we sought out a method for synthesis
of side-chain peptide polymers that efficiently incorporate
several components of varying molecular weight into a statistical
copolymer while maintaining tight control of the resulting
polymer’s molecular weight and composition. By doing so,
downstream purification steps could potentially be eliminated,
conserving resources such as time and starting materials. Peptide
compatibility with RAFT polymerization has been demonstrated
by Bo¨rner and colleagues.11 More recently, Apostolovic and
coworkers demonstrated RAFT copolymerization as a viable
method to incorporate a functional and structurally constrained
peptide into HPMA copolymers at quantitative yields.12 The
success of their study bodes well for the development of novel
materials based on multiple peptides copolymerized in a
synthetic copolymer. We hypothesized that well-defined side
chain peptide-polymers composed of multiple species of active
peptides could be synthesized by RAFT polymerization and that
these materials would have useful applications as gene transfer
agents.
Introduction
Biomimetic copolymers containing peptide motifs combine
the highly specific functions of peptides with the scalable
synthesis of synthetic polymers. Recently, these hybrid materials
have received increased interest because of their applications
in drug and gene delivery, tissue engineering, and biosensors,
to name a few.1 The architecture of these materials includes
peptides incorporated as backbone segments in block copoly-
mers or as pendant side chains. The latter offers a significantly
higher concentration of peptides incorporated per polymer and
also facile incorporation of multiple peptide sequences.
Side-chain peptide-polymer bioconjugates can be synthesized
by covalent coupling of peptides to polymer precursors or by
direct polymerization of peptide monomers. Disadvantages of
peptide coupling include unfunctionalized polymer side chains
and generally lower peptide concentrations in the final polymer.
Alternatively, synthetic copolymers with pendant peptides have
been synthesized by free radical polymerization; however,
resulting polymers were broadly disperse and poorly controlled.2-4
Living polymerization of peptide monomers has been demon-
strated by atom transfer radical polymerization (ATRP), reverse
addition-fragmentation polymerization (RAFT), and ring-
opening metathesis polymerization (ROMP) techniques with
peptides including elastin-based pentapeptides, integrin-binding
sequences, and the antibiotic gramicidin.5-9 Limitations of these
approaches included (a) restriction to short peptides (typically
<10 amino acids in length likely due to steric hindrance), (b)
relatively low degrees of polymerization, and (c) exclusion of
lysine-containing peptides from RAFT syntheses due to potential
aminolysis of the CTA.
Material and Methods
Materials. HPMA was purchased from Polysciences (Warrington,
PA). The initiator VA-044 was purchased from Wako Chemicals
(Richmond, VA). All reagents used in solid-phase chemical synthesis
including NovaPEG Rink amide resin, HBTU, and Fmoc-protected
amino acids were purchased from EMD Biosciences (Darmstad,
Germany). All other materials were reagent grade or better and were
purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.
Synthesis of Peptide Monomers. Peptides, KKKKKKKKKKKK
(K12), and KKKKKKHHHHH (K6H5) were synthesized on a solid
support of NovaPEG Rink amide using standard Fmoc/tBu chemistry.
Synthesis was carried out on an automated PS3 peptide synthesizer
Of particular relevance is the combination of a synthetic
comonomer such as N-(2-hydroxypropyl)methacrylamide (HPMA)
with peptide motifs that enable DNA condensation or elicit
endosomal escape. Previous studies have combined a cationic
material, poly-L-lysine (PLL) with HPMA. In one example, an
HPMA copolymer containing active esters was covalently
coupled in situ to available primary amines of PLL-DNA
* To whom correspondence should be addressed. E-mail: spun@
u.washington.edu.
10.1021/bm100806h
 2010 American Chemical Society
Published on Web 10/05/2010

3008 Biomacromolecules, Vol. 11, No. 11, 2010
Johnson et al.
(Protein Technologies, Pheonix, AZ). Prior to peptide cleavage from
resin, the amino terminal of the peptides was deprotected and modified
with Fmoc-protected aminocaproic acid (Ahx), which was subsequently
deprotected and capped with either methacryloyl chloride or acryloyl
chloride.2 These peptide monomers are, respectively, referred to as
MaK12, AcK12, and MaK6H5. Synthesized peptides were cleaved from
resin by treating the solid support with a solution of TFA/H2O/TIS
(95:2.5:2.5, v/v/v) for 3 h with gentle mixing. Each peptide monomer
was purified by RP-HPLC, and products were confirmed by MALDI-
TOF MS. MALDI-TOF MS calculated for MaK12 (MH+), 1736.26;
found, 1736.23. MALDI-TOF MS calculated for AcK12 (MH+),
1722.24; found, 1722.22. MALDI-TOF MS calculated for MaK6H5
(MH+), 1652.99; found, 1652.99.
oil bath and immediately quenched by submersing in liquid nitrogen.
Frozen aliquots were then thawed and analyzed by NMR.
HPMA-co-K6H5. The mol % of HPMA and MaK6H5 in feed was
95 and 5%, respectively. The total moles of monomers were 0.144
mmol, including 0.136 mmol (19.54 g) of HPMA and 7.20 × 10-3
mmol of MaK6H5. The molar ratio of CTA/I used was 10, and the
molar ratio of total monomers/CTA used was 190. On the basis of
these ratios, 0.0758 × 10-3 mmol (0.201 mg) of ECT and 0.00758 ×
10-3 mmol (0.0247 mg) of VA-044 were added to monomers. The
total volume of the RAFT copolymerization reaction was 206 µL in
deuterated acetate buffer (1 M in H2O, pH 5.1). The solution was then
distributed into 5 mL round-bottomed flasks and sealed with a rubber
septum. The solution was purged with high purity N2 gas for 15 min
prior to incubation in an oil bath (44 °C) for 48 h.
Synthesis of Ethyl Cyanovaleric Trithiocarbonate. The synthesis
of the RAFT chain transfer agent (CTA) ethyl cyanovaleric trithiocar-
bonate (ECT) has been previously described by Convertine et al.13 In
brief, over a 10 min period, ethanethiol (4.72 g, 76 mmol) was added
to a stirred suspension of sodium hydride (60% in oil) (3.15 g, 79 mmol)
in 150 mL of diethyl ether at 0 °C. The solution was stirred for a further
10 min before carbon disulfide (6.0 g, 79 mmol) was added. Crude
sodium S-ethyl trithiocarbonate (7.85 g, 0.049 mol) was collected by
filtration, suspended in 100 mL of diethyl ether, and reacted with iodine
(6.3 g, 0.025 mol). After 1 h, the solution was filtered, washed with
aqueous sodium thiosulfate, and thoroughly dried over sodium sulfate;
the crude bis(ethylsulfanylthiocarbonyl) disulfide was then isolated by
rotary evaporation. A solution of bis(ethylsulfanylthiocarbonyl) disulfide
(1.37 g, 0.005 mol) and 4,4′-azobis(4-cyanopentanoic acid) (2.10 g,
0.0075 mol) in 50 mL of ethyl acetate was heated at reflux for 18 h.
Following rotary evaporation of the solvent, the crude ECT was isolated
by column chromatography using silica gel as the stationary phase and
HPMA-co-MaK12-co-MaK6H5. Kinetic studies were done using
total mole amount of 0.653 mmol. This included 0.587 mmol (84.2
mg) of HPMA, 0.0326 mmol (58.1 mg) of MaK12, and 0.0326 mmol
(54.0 mg) of MaK6H5. The molar ratio of CTA/I used was 10, and the
molar ratio of total monomers/CTA used was 190. On the basis of
these ratios, 3.43 × 10-3 mmol (0.904 mg) of ECT and 0.343 × 10-3
mmol (0.111 mg) of VA-044 were added to monomers. The total
volume of the RAFT copolymerization reaction was 936 µL in
deuterated acetate buffer (1 M in deuterated H2O, pH 5.1). The solution
was aliquotted into equivalent volumes in nine 5 mL round-bottomed
flasks and sealed with a rubber septum. The aliquots were purged with
high-purity N2 gas for 15 min prior to incubation in an oil bath (44
°C). At designated time points, aliquots were removed from the oil
bath and immediately quenched by submersing in liquid nitrogen.
Frozen aliquots were then thawed and analyzed by NMR.
1H Nuclear Magnetic Resonance. Polymerization kinetics for
copolymers of HPMA-co-MaK12, and HPMA-co-MaK12-co-MaK6H5
were assessed by 1H NMR to determine the conversion of vinyl-coupled
hydrogens. As described above, copolymerizations were carried out in
acetate-buffered deuterated water. Each time point of polymerization
was quenched by immersion in liquid nitrogen. After the samples had
been thawed, 0.5 mL of deuterated water was added to the solution
(0.144 mmol of monomer), making the final volume 0.604 mL (final
concentration of monomer: 0.238 mol/L). The samples were then
applied to NMR using a Bruker AV300 running at 300 Hz. Conversion
of monomers was determined on the basis of the ration of vinyl protons
(δ 5.4 and 5.6) relative to 3-methyl protons (δ 1.1) of HPMA. These
peaks on the NMR spectrum were well-separated from peaks generated
1
50:50 ethyl acetate-hexane as the eluent. H NMR(CDCl3, δ): 1.36 t
(SCH2CH3); 1.88 s (CCNCH3); 2.3-2.65 m (CH2CH2); 3.35 q
(SCH2CH3).
Statistical RAFT Copolymerization of HPMA with Peptide
Monomers. HPMA-co-K12 Copolymers. Two sets of HPMA-co-K12
copolymers were synthesized. One set of copolymers varied the number
of K12 monomers in the feed for both MaK12 and AcK12 monomers
from 1 to 20 mol %. In these experiments, the total moles of monomers
was kept at 0.144 mmol, whereas the amount of K12 monomer was 1,
5, 10, and 20 mol %, and HPMA amounts varied, respectively. To
carry out these polymerizations, we added 1.43 × 10-3, 7.18 × 10-3
,
14.36 × 10-3, and 28.72 × 10-3 mmol of either MaK12 or AcK12 to
separate vials. To these vials, we added 0.142 (20.35 mg), 0.136 (19.54
mg), 0.129 (18.51 mg), and 0.115 mmol (16.45 mg) of HPMA to
respective vials, making the total moles of monomer equal to 0.144
mmol. The molar ratio of CTA/I used was 10, and the molar ratio of
total monomers/CTA used was 190. On the basis of these ratios, 0.0758
× 10-3 mmol (0. 201 mg) of ECT and 0.00758 × 10-3 mmol (0.0247
mg) of VA-044 were added to monomers. The total volume of the
RAFT copolymerization reaction was 206 µL in acetate buffer (1 M
in H2O, pH 5.1). The solutions were then distributed into 5 mL round-
bottomed flasks and sealed with a rubber septum. The aliquots were
then purged with high purity N2 gas for 15 min prior to incubation in
an oil bath (44 °C) for 48 h.
1
from the addition of MaK12. H NMR (300 Hz, deuterated H2O, δ):
1.1 (d, 3H), 1.9 (d, 3H), 2.6 (s, 1H), 2.9-3.2 (m, 2H), 3.9 (m, 1H), 5.4
(s, 1H), 5.6(s, 1H), 8.03 (s, 1H).
Molecular Weight and Compositional Characterization. Size
Exclusion Chromatography (SEC). Molecular weight analysis was
carried out by size exclusion chromatography. To prepare materials
for analysis, copolymers were extensively dialyzed against distilled H2O,
removing any unreacted monomers and residual buffer salts, and then
lyophilized to recover them as a fluffy while solid. Then, the copolymers
were dissolved at 5 mg/mL in running buffer (0.3 M sodium acetate
buffered to pH 4.4 with acetic acid) for analysis by SEC, as described
by Hennink and coworkers.14 This was done by applying samples to a
OHpak SB-804 HQ column (Shodex) in line with a miniDAWN
TREOS light scattering detector (Wyatt) and a Optilab rEX refractive
index detector (Wyatt). Absolute molecular weight averages (Mn and
Mw), and dn/dc were calculated using ASTRA software (Wyatt).
Ninhydrin Assay. Content of K12 and K6H5 peptides within HPMA
copolymers were determined through ninhydrin assay of primary
amines. In brief, a stock solution of reagent was made by dissolving
0.1340 g of ninhydrin and 0.0234 g hydrindantin in 5 mL of
2-methoxyethanol; then, 3.325 mL of 4 N acetate buffer (pH 5.5) was
added. Standards of glycine at a concentration of 200 to 0 µM were
then made through serial dilutions. Samples were then prepared at an
approximate concentration of 40 µM of primary amines in double-
distilled and deionized water. To 0.5 mL of sample, 0.25 mL of
Kinetic studies of HPMA-co-K12 copolymerization were done using
total mole amount of 0.653 mmol. This included 0.620 mmol (88.8
mg) of HPMA and 0.0326 mmol (58.1 mg) of MaK12. The molar ratio
of CTA/I used was 10, and the molar ratio of total monomers/CTA
used was 190. On the basis of these ratios, 3.43 × 10-3 mmol (0.904
mg) of ECT and 0.343 × 10-3 mmol (0.111 mg) of VA-044 were added
to monomers. The total volume of the RAFT copolymerization reaction
was 936 µL in deuterated acetate buffer (1 M in D2O, pH 5.1). The
solution was aliquotted into equivalent volumes into nine 5 mL round-
bottomed flasks and sealed with a rubber septum. The aliquots were
then purged with N2 gas for 15 min prior to placing in an oil bath set
at 44 °C. At designated time points, aliquots were removed from the

Synthesis of Statistical Copolymers
Biomacromolecules, Vol. 11, No. 11, 2010 3009
Scheme 1. RAFT Polymerization of Peptide-HPMA Copolymers
ninhydrin reagent solution was added and then mixed well. The reaction
mixture was boiled for 15 min and allowed to cool. After the solution
had reached room temperature, 0.75 mL of ethanol was added. Then,
100 µL of the final solution was transferred to wells on a UV-transparent
96-well plate, and the absorbance was measured at 440 nm on a Tecan
Safire2 96-well plate reader.
Amino Acid Analysis. To confirm K12 or K6H5 content within HPMA
copolymers, amino acid analysis was carried out at UC Davis’s
proteomics core facility with an L-8800 Hitachi analyzer that utilizes
a sodium citrate buffer system. HPMA content was calculated by
subtracting amino acid content in milligrams from the total weight of
the copolymers used for analysis.
described.2 Branched poly(ethylene imine) (PEI, MW 25 000) and poly-
L-lysine hydrobromide (PLL, MW 15 000-30 000) were used as
positive controls. After mixing, the polyplexes were allowed to form
for 5 min at room temperature. Each polyplex solution (20 µL,
containing 1 µg DNA) was mixed with either 80 µL of double-distilled
H2O or PBS (where PBS was used to dilute polyplex solutions, the
final ionic strength of the solution was 150 mM) and then used to
determine the particle size of the polyplexes by dynamic light scattering
(DLS) measured on a Brookhaven Instruments Corp ZetaPALS
instrument. Particle sizing measurements were performed at a wave-
length of 659.0 nm with a detection angle of 90° at RT.
In Vitro Transfection Efficiency. Human cervix epithelial adeno-
carcinoma cells (HeLa, passage 25, ATCC no. CCL-2) were seeded in
complete cell culture medium (MEM + 10% FBS + 1% antibiotic/
antimicrobial) at a density of 5 × 104 cells/well in each well of a 24-
well plate. The 24-well plates were placed in a 37 °C incubator with
5% CO2 for 24 h to allow the HeLa cells to attach. Polyplexes were
formed as described above at N/P ) 5 using 1 µg of gWiz-Luc plasmid
DNA in 20 µL total volume. Each sample was brought to 200 µL with
OptiMEM I (Invitrogen). The cells were washed twice with PBS, and
the transfection solutions were added. The 24-well plates were returned
to the incubator for 4 h, at which time the polyplex solution was
replaced with complete cell culture media after washing the cells with
PBS. The cells were returned to the incubator for a further 44 h. After
a total incubation of 48 h, the luciferase expression was quantified with
a luciferase assay kit (Promega) according to the manufacturer’s
instructions, except that a freeze-thaw cycle at -20 °C was included
after the addition of the lysis buffer to ensure complete cell lysis.
Luminescence intensity was measured on a Tecan Safire2 plate reader
with integration for 1 s. The total protein content in each well was
measured by a BCA Protein Assay Kit (Pierce) according to the
manufacturer’s instructions so that the luciferase activity measured in
each well could be normalized by the total protein content in each well.
Polyplex Formulation and Characterization. The gWiz-Luc
plasmid (endotoxin free, purchased from Aldevron, Fargo, ND) was
diluted in double-distilled H2O to a concentration of 0.1 mg/mL and
mixed with an equal volume of polymer (also diluted in double-distilled
H2O) at a lysine-to-DNA phosphate ratio (N/P) of 5, as previously
Figure 1. Correlation between MaK12 monomer feed concentration
in mol % and concentration of peptides in the HPMA copolymers.

3010 Biomacromolecules, Vol. 11, No. 11, 2010
Johnson et al.
K6H5 (%)c
Table 1. Physical and Chemical Characterization of HPMA-Peptide Copolymers Copolymerized via RAFT
copolymera
CTAo/Mo
predicted Mn (kDa)
actual Mn (kDa)b
Mw/Mn
K12 (%)c
P-01-00
P-05-00
P-10-00
P-20-00
P-00-05
P-05-05
240
240
240
240
190
190
38.18
53.44
72.51
110.64
41.55
56.70
31.31
55.17
89.00
110.50
44.83
45.80
1.16
1.05
1.02
1.28
1.15
1.08
0.58
4.43
9.66
19.41
NA
NA
NA
NA
NA
4.15
4.41
3.94
a HPMA-peptide samples are named by using a P-xx-yy notation, where P indicates that the sample is a copolymer of HPMA with xx mol % of MaK12
peptide monomer and yy mol % of MaK6H5 peptide monomer in the polymerization reaction. b Number-average molecular weight was determined using
SEC coupled to light scattering and refractive index detectors. c Mol % of K12 were determined by both ninhydrin assay and amino acid analysis; reported
values are from amino acid analysis.
Copolymerization of peptide monomers with HPMA was
carried out under aqueous conditions at 44 °C using the RAFT
agent ECT and VA-044 as the initiator. ECT has previously
demonstrated excellent properties as a RAFT agent for HPMA
and other methacrylamido- and acrylamido-based monomers.18
An acetic acid buffer at pH 5.1 with a molar strength of 1 M
was used to ensure that ε-amines of L-lysine were fully
protonated, thereby protecting the trithiocarbonate from nucleo-
philic attack.
Initial studies focused on the copolymerization of MaK12
relative to AcK12 peptides. In these experiments, the amount of
peptide monomers in feed was varied from 1 to 20 mol %,
whereas the remainder of feed was HPMA. Copolymerizations
Figure 2. Pseudo first-order rate plot of the bulk copolymerization of
HPMA with 5 mol % of MaK12 in the feed.
of both methacrylamido- and acrylamido- modified peptides with
HPMA were carried out in parallel and demonstrated near
quantitative yields of peptide monomer incorporation into the
Furthermore, the BCA protein assay was also used to assess cytotoxicity
of the materials. Each sample was tested in replicates with n ) 6. The
final copolymer for the full range of peptide monomer percent-
statistical significance of differences in the samples was determined
ages tested. Interestingly, the MaK12 peptide monomer was
using the Student t test, where a p value of <0.05 was considered to be
copolymerized more efficiently at 20 mol % of peptide in feed
significant.
than its acrylamido counterpart (Figure 1). As shown in Table
1, the molecular weights of the resulting polymers were near
Results and Discussion
the predicted value with low polydispersities (Mw/Mn < 1.2).
We then studied the copolymerization kinetics of HPMA-co-
MaK12 copolymers with peptide feed amount of 5 mol %.
To test our main hypothesis, peptide monomers composed
of either 12 L-lysine residues (MaK12) or 6 L-lysine residues
and 5 L-histadine residues (MaK6H5) were copolymerized with
HPMA (Mw of comonomers: HPMA 143.2 Da, MaK12 1732.2
Da, and MaK6H5 1653.0 Da). Peptides were chosen for their
ability to either efficiently condense nucleic acids (K12)15 or elicit
endosomal escape (K6H5).16 HPMA was chosen as a model
comonomer because of its commercial availability, its use as a
comonomer in clinically tested drug delivery materials, and its
extensive use in peptide-polymer conjugates.17 Peptides were
synthesized by solid-phase peptide synthesis using standard
Fmoc/tBu chemistries. Prior to cleavage from resin, the amine
terminus of the peptide was modified with a linker (6-amino-
caproic acid) and then capped with either a methacrylamido-
(MaK12 and MaK6H5) or acrylamido-functional group (AcK12)
for polymerization (Scheme 1).
1
Conversion of monomers was followed by H NMR. Kinetics
closely followed a pseudo-first-order model characteristic of
living radical polymerization (Figure 2). Elution profiles from
size exclusion chromatography analysis demonstrated an initial
bimodal size distribution that quickly became unimodal, as seen
in Figure 3. Additionally, studies were included to determine
lysine content at each time point through both ninhydrin assay
and amino acid analysis. As demonstrated in Figure 4c, lysine
content of copolymers remained constant at near quantitative
yields throughout polymerization. Also, as the polymerization
experiment proceeded, the number molecular weight average,
Mn, remained close to predicted values. The polydispersity fell
from 1.8 to 1.1 through the course of the experiment, as can be
seen in Figure 4.
Figure 3. Time-point SEC of HPMA-MaK12 statistical copolymers. Initial distributions demonstrate a bimodal molecular weight distribution that
quickly resolves to single distribution peak with narrow polydispersity.

Synthesis of Statistical Copolymers
Biomacromolecules, Vol. 11, No. 11, 2010 3011
Figure 4. RAFT statistical copolymerization of HPMA-co-MaK12
copolymers using a feed of 5 mol % of MaK12 peptide monomer. (a)
Polydispersity versus conversion, (b) Mn growth versus conversion,
(c) and incorporation of K12 and copolymer’s number average (Mn)
versus time.
Figure 6. RAFT statistical copolymerization of HPMA-co-MaK12-co-
MaK6H5 copolymers using a feed of 5 mol % MaK12 and 5 mol % of
MaK6H5 peptide monomers. (a) Polydispersity versus conversion. (b)
Mn growth versus conversion and (c) incorporation of K12, K6H5, and the
copolymer’s number-average molecular weight (Mn) versus time.
weight distribution, which indicates that the growing copolymer
chains were reaching a steady equilibrium. The final copolymer
reached a conversion of 86% of monomers and had an Mn near
the predicted value with low polydispersity (final Mw/Mn ) 1.08).
A possible explanation for bimodal molecular weight distributions
in early time points is the formation of macroCTAs by the initial
addition of peptide monomers to ECT. This would cause hetero-
geneous RAFT efficiency, as has been described by Huang et al.19
This phenomenon could possibly result in some heterogeneity in
the copolymers in the early stages of RAFT polymerization;
however, the inclusion of peptide monomers remained consistently
close to the feed peptide monomer concentration. As polymerization
proceeded, RAFT efficiencies became homogeneous, as is indicated
by the copolymer presenting a single distribution of molecular
weights with a narrow polydispersity (Mw/Mn).
To determine the benefit of including multiple peptide
components into the copolymers, we tested materials for their
ability to function as nucleic acid carriers. This analysis included
the ability of the HPMA-peptide copolymers to form complexes
with plasmid DNA (termed “polyplexes”) and the copolymers’
ability to deliver plasmid to logarithmically growing HeLa cells
in culture. HPMA-peptide copolymers displayed the ability to
condense effectively the plasmid into small particles (<200 nm),
as displayed in Figure 8. Also, the particles formed by the
HPMA-peptide copolymers demonstrated salt stability when
in PBS buffers, whereas the size of polyplexes formed by
cationic peptides and homopolymers of linear PEI and poly(L-
lysine) (PLL) grew considerably in the presence of salt. The
salt stability of HPMA-peptide copolymers was consistent with
Figure 5. Pseudo-first-order rate plot of the bulk copolymerization of
HPMA with 5 mol % of MaK12 and 5 mol % of MaK6H5 in the feed.
To demonstrate RAFT polymerization’s usefulness in a synthesis
of copolymer that included multiple peptides, HPMA copolymers
were synthesized with 5 mol % MaK12 and 5 mol % MaK6H5
peptide monomers in the feed with the remainder of monomers
being HPMA. Copolymerization of the two-peptide HPMA
copolymer was monitored by 1H NMR, amino acid analysis, and
SEC. Conversion of monomers to polymers appeared to follow
pseudo-first-order kinetics (Figure 5), and the final K12 and K6H5
contents were 3.9 and 4.4 mol %, respectively. Again, copolymers
with molecular weights near predicted values and low polydisper-
sities were obtained, as shown in Table 1 and Figure 6a,b. Both
HPMA-MaK12 and HPMA-MaK12-MaK6H5 copolymers displayed
multimodal distributions of molecular weight in early time points
that quickly resolved into a single distribution of copolymers
(Figures 3 and 7). Also, the Mn of HPMA-co-MaK12-co-MaK6H5
initially deviated from predicted values with very broad polydis-
persity produced by the multimodal molecular weight distributions.
This had a rather unique effect of maintaining Mn values of ∼19
kDa while conversion increased from 15 to 50%. During this same
period, the copolymer’s Mw/Mn ratio diminished from 3.1 to 1.5,
and the SEC elution of the copolymer resolved to a single molecular

3012 Biomacromolecules, Vol. 11, No. 11, 2010
Johnson et al.
Figure 7. SEC analysis of statistical copolymers of MaK12-MaK5H6-HPMA copolymers during various time points of compolymerization. Initial
samples displayed bimodal distributions that were broadly disperse. However, as polymerization proceeded, copolymers became more uniform
in molecular weight with a single molecular weight distribution and a narrow polydispersity (Mw/Mn 1.08).
Figure 8. Particle sizing of polyplexes formed with gWiz-Luc luciferase
plasmid with either cationic peptides or HPMA-peptide copolymers.
Figure10.ToxicityofthePLL,PEI,cationicpeptides,andHPMA-peptide
In all formulations, an N/P of 5 was used. HPMA-peptide samples
copolymers on HeLa cells. Protein content was determined through
are named by using a P-xx-yy notation, where P indicates that the
a BCA protein assay. The protein content was normalized to untreated
sample is a copolymer of HPMA with xx mol % of MaK12 peptide
cells.
monomer and yy mol % of MaK6H5 peptide monomer in the
polymerization reaction.
caused by PEGylating PEI.21 Although HPMA copolymers with
increased amounts of oligolysine transfected HeLa cells more
readily, the increased cationic character also increased the
cytotoxicity of the copolymers (Figure 10). However, the
HPMA-peptide copolymers demonstrated much less toxicity
than their PLL counterpart.
Conclusions
In conclusion, our study presented the synthesis of well-
defined statistical side-chain peptide-HPMA copolymers with
lysine- and histadine-based, long (>10 amino acids) peptides
by RAFT polymerization. Copolymerization reactions demon-
strated pseudo-first-order kinetics characteristic of living radical
polymerization. Controlled growth of copolymers, inclusion of
Figure 9. Transfection efficiency of PEI, PLL, cationic peptides, and
multiple peptides, and the narrow polydispersities of resulting
HPMA-peptide copolymers. Gene delivery efficiency of selected
copolymers suggest the robustness of this technique for polym-
carriers (formulated at N/P ) 5) to HeLa cells, as measured by
erization and synthesis of graft copolymers. These materials
proved to be effective nucleic acid delivery vehicles that can
potentially be used directly in vivo without further modification
because of their salt stability. Although our study focused on
two peptides with functionally distinct roles, this technique can
possibly be extended to other peptides with diverse functional
and structural characteristics. This may facilitate the develop-
ment of novel and multifunctional materials for self-assembly,
drug-delivery, and biotechnology.
luciferase activity 48 h after transfection. Results are reported as the
mean RLU/mg protein ( SD for replicate samples, with all samples
having n ) 6. Nomenclature follows the same convention as that
described in Figure 8. *P-20-00 demonstrated statistical significance
with all other materials except PEI (p < 0.05). **P-10-00 and P-05-05
demonstrated statistical significance to all materials except PEI and
P-20-00 (p < 0.05). ***P-05-00 demonstrated statistical significance
to K6H5, K12, P-01-00, and DNA (p < 0.05).
polyplexes formed by PEG-modified cationic polymers.20 Ad-
ditionally, the transfection efficiency of HPMA-K12 copolymers
approached that of PEI at high K12 content (20 mol % of K12),
reaching efficiencies well beyond that of PLL (Figure 9),
contrasting sharply with the diminished transfection efficiency
Acknowledgment. This work was supported by an NSF
CAREER Award (CBET-0448547) to S.H.P. and NIH/NINDS
1R01NS064404. We also appreciated helpful discussions with
Pavla Kopecˇkova´.

Synthesis of Statistical Copolymers
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