Controlled synthesis of pH responsive cationic polymers containing side-chain peptide moieties via RAFT polymerization and their self-assembly

Article abstract of DOI:10.1039/c2tb00170e

A general and facile strategy was developed to prepare biocompatible peptide side-chain polymeric materials via reversible addition-fragmentation chain transfer (RAFT) polymerization. Three new dipeptide based monomers, Boc-Phe-Phe-oxyethyl methacrylate (Boc-FF-EMA), Boc-Ile-Phe-oxyethyl methacrylate (Boc-IF-EMA) and Boc-Val-Phe-oxyethyl methacrylate (Boc-VF-EMA), were synthesized and subsequently polymerized by RAFT process to afford well-defined peptide side-chain polymers, P(Boc-dipep-EMA), with controlled molecular weight, narrow polydispersity and precise chain end functionality. Further, a monomethoxy poly(ethylene glycol) (mPEG) based macro-chain transfer agent was employed for RAFT polymerization of these monomers to prepare well defined amphiphilic block copolymers, mPEG-b-P(Boc-dipep-EMA). Subsequent deprotection of side-chain Boc groups produced pH responsive homo- and block copolymers with primary amine moieties at the side chains. The cationic surface charge of various polymeric architectures was studied using dynamic light scattering (DLS) measurements. Atomic force microscopy (AFM) was employed to investigate the self-assembly of block copolymers. The in vitro biocompatibility to HeLa cells was investigated with these polymers to confirm their minimum cytotoxicity. These polymers have great potential for the pH-sensitive delivery of small interfering RNA (siRNA) owing to their interesting phase transition behaviour and biocompatibility. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2tb00170e

Journal of
Materials Chemistry B
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PAPER
Controlled synthesis of pH responsive cationic polymers
containing side-chain peptide moieties via RAFT
polymerization and their self-assembly†
Cite this: J. Mater. Chem. B, 2013, 1,
946
a
b
b
a
*
Sonu Kumar, Rituparna Acharya, Urmi Chatterji and Priyadarsi De
A general and facile strategy was developed to prepare biocompatible peptide side-chain polymeric
materials via reversible addition–fragmentation chain transfer (RAFT) polymerization. Three new
dipeptide based monomers, Boc-Phe-Phe-oxyethyl methacrylate (Boc-FF-EMA), Boc-Ile-Phe-oxyethyl
methacrylate (Boc-IF-EMA) and Boc-Val-Phe-oxyethyl methacrylate (Boc-VF-EMA), were synthesized and
subsequently polymerized by RAFT process to afford well-defined peptide side-chain polymers, P(Boc-
dipep-EMA), with controlled molecular weight, narrow polydispersity and precise chain end
functionality. Further, a monomethoxy poly(ethylene glycol) (mPEG) based macro-chain transfer agent
was employed for RAFT polymerization of these monomers to prepare well defined amphiphilic block
copolymers, mPEG-b-P(Boc-dipep-EMA). Subsequent deprotection of side-chain Boc groups produced
pH responsive homo- and block copolymers with primary amine moieties at the side chains. The cationic
surface charge of various polymeric architectures was studied using dynamic light scattering (DLS)
measurements. Atomic force microscopy (AFM) was employed to investigate the self-assembly of block
copolymers. The in vitro biocompatibility to HeLa cells was investigated with these polymers to confirm
their minimum cytotoxicity. These polymers have great potential for the pH-sensitive delivery of small
interfering RNA (siRNA) owing to their interesting phase transition behaviour and biocompatibility.
Received 27th September 2012
Accepted 18th November 2012
DOI: 10.1039/c2tb00170e
www.rsc.org/MaterialsB
unmatched compositions with monomer feed ratios and
involves signicant homopolymer contaminants due to
Introduction
possible side reactions which are very difficult to eliminate.3
The advantage of peptide sequence incorporation in the side-
chains of polymers is that it offers facile integration of a
signicantly higher concentration of peptides per polymer. In
addition, the side-chain peptide segment can have reactive
functionalities which can be expediently modied further, or
can have stimuli responsive properties such as with pH,
temperature, solvent, or ionic strength. These synthetic poly-
mers with pendant peptides can be synthesized via direct
polymerization of peptide based monomers or by covalent
coupling of peptides to a pre-formed polymer backbone (post-
polymerization modication). The strategy of covalent coupling
of peptides is tricky and it is difficult to achieve quantitative
functionalization, which lowers peptide concentrations in the
nal polymer precursor. On the other hand, polymerization of
peptide based vinyl monomers provides polymers with inher-
ently peptide functionalized side-chains. However, non-trivial
synthesis of peptide functionalized vinyl monomers and
compatibility issues between the polymerization method and
peptide moiety limits this approach.
Conjugation of peptides to the synthetic polymers enables the
foundation of novel biocompatible materials with unique
opportunities to combine the best of both worlds for their
applications in drug and gene delivery, tissue engineering,
biosensing, etc.1 Incorporation of peptide motifs into polymers
has generated considerable interest over recent years and these
peptide–polymer conjugates can be classied into two groups:
peptide-based main-chain polymers having peptide as back-
bone segments (polypeptides), and peptide in the side-chain of
the polymers. Main-chain peptide–polymers are commonly
prepared by ring-opening polymerization (ROP) of a-amino acid
N-carboxyanhydrides (NCAs),2 which generally results in
aPolymer Research Centre, Department of Chemical Sciences, Indian Institute of
Science Education and Research, Kolkata, PO: BCKV Campus Main Office, Nadia,
Mohanpur – 741252, West Bengal, India. E-mail: p_de@iiserkol.ac.in; Fax: +91-33-
25873020
bDepartment of Zoology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata
– 700019, West Bengal, India
† Electronic supplementary information (ESI) available: ESI-MS spectra of
monomers, FT-IR spectra of monomers and polymers, 1H NMR spectra of CTA,
monomers and polymers, 13C NMR spectra of monomers, AFM images of block
copolymers, pH response curves of polymers, TGA thermograms of polymers,
DLS image and spectroscopic analysis of u-trithiocarbonate moiety of polymers.
See DOI: 10.1039/c2tb00170e
Peptide based vinyl monomers can be designed by modifying
the peptide either at the amine site or at the carboxylic acid site
to generate N-terminus vinyl monomers for achieving anionic
polyelectrolytes (APEs) or C-terminus vinyl monomers for
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obtaining cationic polyelectrolytes (CPEs), respectively.4 Murata 4-Dimethylaminopyridine (DMAP, 99%), 1-hydroxybenzo-
et al. reported synthesis and conventional free radical poly- triazole hydrate (HOBt hydrate, 97%), anhydrous N,N-dime-
merization of methacrylamides having dipeptide structures.5 thylformamide (DMF, 99.9%), dicyclohexylcarbodiimide (DCC,
van Hest and co-workers contributed signicantly to the 99%) and 2-hydroxyethyl methacrylate (HEMA, 97%) were
synthesis of several peptide based methacrylate monomers by purchased from Sigma and used without any further purica-
solution or solid phase peptide synthesis and their polymeri- tion. The initiator, 2,20-azobisisobutyronitrile (AIBN, Sigma,
zation by atom transfer radical polymerization (ATRP).4a,6 98%) was recrystallized twice from methanol. The NMR solvents
Recently, high molecular weight elastin-based side-chain poly- such as CDCl3 (99.8% D), D2O (99.9% D) and DMSO-d6 (99.9%
mers7 with stimuli responsiveness were prepared in a controlled D) were purchased from Cambridge Isotope, USA. The solvents
manner with low polydispersity indices (PDI) via reversible hexanes, acetone, ethyl acetate, tetrahydrofuran (THF) and
addition–fragmentation chain transfer (RAFT) polymerization. dichloromethane (DCM) were puried by standard procedures.
In ATRP, there is the possibility of peptide-induced deactivation 4-Cyano-4-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid
due to the complexation of the copper catalyst with amide (CDP) was synthesized as reported elsewhere.20
bonds in the peptide side chains, which is absent in RAFT.7
However, for preparing peptide based CPEs, there are also
limitations through RAFT, due to potential aminolysis of the
Instrumentation
chain transfer agent (CTA) by free amino groups in C-terminus The molecular weights and molecular weight distributions were
modied peptide based vinyl monomers.8 By employing pro- determined by gel permeation chromatography (GPC) in THF
tecting chemistry9 these monomers may be polymerized relative to poly(methyl methacrylate) standards at 35 C using
ꢀ
through RAFT polymerization and free primary amine can be 1.0 mL minꢁ1 ow rate. The system is equipped with a Waters
generated by post-polymerization deprotection of the protecting Model 515 HPLC pump and a Waters Model 2414 refractive
groups. Pendant peptide based polymers carrying primary index detector. The 1H NMR and 13C NMR spectroscopy was
amino groups are of particular interest for their potential utility carried out in a Bruker AVANCEIII 500 MHz spectrometer
to a wide range of post-polymerization modication reactions, operating at 500 MHz and 125 MHz, respectively. Positive mode
such as amide and imine formation,10 ring-opening reactions,11 electrospray ionization mass spectra (ESI-MS) were recorded on
Michael addition reactions,12 aldol reactions,9,13 etc.
We present a general and facile strategy to prepare a series of mass spectrometer. The FT-IR spectra were obtained on a FT-IR
biocompatible CPEs with dipeptide moieties in the side chains Perkin-Elmer spectrometer at a nominal resolution of 2 cmꢁ1
a Q-TOF Micro YA263 high resolution (Waters Corporation)
.
and also report the synthesis and self-organization of diblock Fluorescence emission spectra were recorded on a uorescence
copolymers composed of a side-chain peptide block and poly- spectrometer (Horiba Jobin Yvon, Fluromax-3, Xe-150 W, 250–
(ethylene glycol) (PEG). The PEG is a Food and Drug Adminis- 900 nm). The UV-visible spectroscopic measurements were
tration (FDA)-approved polymer, and PEG containing block carried out on a U-4100 spectrophotometer HITACHI UV-vis
copolymers are advantageous for possible future biomedical spectrometer, with a scan rate of 240 nm minꢁ1. Particle size
applications,14 because of their biocompatibility, availability, and zeta potential (x) were determined by dynamic light scat-
water solubility and well-known physicochemical properties.15 tering (DLS) using a Zetasizer Nano ZS (Malvern Instrument
Herein, we have adopted very short peptide fragments from the Ltd., Malvern, UK) equipped with a He–Ne laser beam at 658
central hydrophobic cluster (CHC) of the amyloid b-peptide nm. Zeta potentials were measured at room temperature at a
Ab1–42, which is critical for bril formation in Alzheimer's scattering angle of 173ꢀ for 1.0 mg mLꢁ1 solution in deionized
disease.16 The dipeptide Phe–Phe recognition motif of the Alz- (DI) water. For zeta potential measurements, samples were
heimer's Ab19–20 peptide is described as the smallest peptide ltered through a 0.45 mm syringe lter prior to analysis, but
able to assemble into higher-order structures.17 The amyloido- samples were not ltered for particle size measurements due to
genic b-peptide Ab18–19 residue Val–Phe,18 and another dipep- the rupturing of possible secondary aggregations. Each
tide analogue Ile–Phe,19 have been shown to self-assemble to measurement was repeated three times to obtain the average
produce amyloid-like brillar nanostructures. In this context, value. Atomic force microscopy (AFM) imaging was carried out
we were intrigued to prepare these dipeptide based well-dened at room temperature with a NT-MDT NTEGRA Prima Scanning
side-chain homopolymers and PEG based block copolymers due Probe Microscope operated in semicontact mode. For cytotox-
to their possible inhibition of bril formation by Ab to treat icity tests, HeLa cells were grown on Eagle's minimal essential
Alzheimer's disease and also to determine their macro-supra- medium (EMEM, HiMedia) containing 10% fetal bovine serum
molecular properties.
(HiMedia) and penicillin–streptomycin (HiMedia) solution.
Formazan crystals produced from 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) were dissolved in MTT
solubilization solution (In Vitro Toxicology Assay Kit MTT
based, Sigma-Aldrich). For microscopic imaging, ME-180 cells
Experimental section
Materials
Boc-L-phenylalanine (Boc-L-Phe-OH, 99%), Boc-L-isoleucine were xed with 4% paraformaldehyde (Merck) and cells were
(Boc-L-Ile-OH, 99%), Boc-L-valine (Boc-L-Val-OH, 99%) and tri- permeabilized with Triton-X-100 (Sigma). Then, the cells were
uoroacetic acid (TFA, 99.5%) were purchased from Sisco subsequently stained with 40,6-diamidino-2-phenylindole
Research Laboratories Pvt. Ltd., India and used as received. (DAPI, USB Corporation).
This journal is ª The Royal Society of Chemistry 2013
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Also, Boc-VF-EMA was synthesized from Boc-Val-OH and
H2N-F-EMA (yield 85%). 1H NMR (Fig. 1, CDCl3, d, ppm): 7.24–
7.04 (C6H5, 5H, m), 6.86–6.6 (CONHCH, 1H, d), 6.08 and 5.55
(C]CH2, 2H, s), 5.23–5.13 (NHCOO, 1H, d), 4.9–4.84
(C6H5CH2CH, 1H, m), 4.34–4.18 (OCH2CH2O, 4H, m), 4.1–3.9
(COCHNH, 1H, m), 3.1–2.97 (C6H5CH2, 2H, m), 2.01–1.94
(CH(CH3)2, 1H, m), 1.89 (C]CCH3, 3H, s), 1.39 (C(CH3)3, 9H, s),
0.89–0.70 (CH(CH3)2, 6H, m). 13C NMR (Fig. S9 in the ESI,†
CDCl3, d, ppm): 171.52, 170.96, 166.83, 135.91, 135.32, 129.49,
128.35, 127.16, 125.7, 79.85, 62.93, 62.0, 59.67, 52.95, 37.8,
30.78, 28.14, 19.08, 18.23. ESI-MS (Fig. S10 in the ESI†): [M +
Na]+ ¼ 499.11 m/z.
Monomer synthesis
The amino acid based vinyl monomer, Boc-Phe-EMA, was
synthesized by esterication of HEMA and Boc-L-Phe-OH,
according to the procedure reported by our group.21 The Boc-
Phe-EMA (4.0 g, 10.6 mmol) was dissolved in dry DCM (30 mL)
and 10 mL of TFA was added to this solution in an ice-water
bath and le to stir overnight at room temperature. The reac-
tion mixture was washed several times with saturated NaHCO3
solution and the organic layer was dried over anhydrous
Na2SO4. The solvent was removed by rotary evaporation and
further puried by silica gel column chromatography using 3%
methanol/DCM, resulting in pure colorless liquid compound
H2N-Phe-EMA, with a yield of 85%. 1H NMR (Fig. S1 in the ESI,†
CDCl3, d, ppm): 7.31–7.16 (C6H5, 5H, m), 6.11 and 5.59 (C]CH2,
2H, s), 4.4–4.25 (OCH2CH2O, 1H, m), 3.87–3.74 (C6H5CH2CH,
1H, m), 3.07 and 2.89 (C6H5CH2, 2H, m), 1.93 (C]CCH3, 3H, s).
13C NMR (Fig. S2 in the ESI,† CDCl3, d, ppm): 174.58, 167.22,
136.94, 135.8, 129.28, 128.44, 127.29, 125.87, 62.72, 61.89,
55.67, 40.66, 18.28.
Taking the synthesis of Boc-Phe-Phe-oxyethyl mathacrylate
(Boc-FF-EMA) as an example, Boc-Phe-OH (2.30 g, 8.65 mmol)
and H2N-Phe-EMA (2.00 g, 7.21 mmol) were dissolved in dry
DCM (40 mL) and the solution was purged with dry N2. Then, a
solution of DCC (1.79 g, 8.65 mmol) and DMAP (0.88 g, 0.72
mmol) in 10 mL of dry DCM was added dropwise to the reaction
mixture in an ice-water bath under stirring and was allowed to
react at room temperature for 24 h. Aer removing insoluble
N,N0-dicyclohexylurea (DCU) by suction ltration, the organic
layer was further washed with 1 N HCl, saturated NaHCO3 and
brine solution and dried over anhydrous Na2SO4. The organic
solvent was removed by rotary evaporation and the crude
product was puried by silica gel column chromatography
using 3% methanol/DCM as mobile phase (Rf ¼ 0.58), to get a
white solid compound Boc-FF-EMA, with a yield of 83%. 1H
NMR (Fig. S3 in the ESI,† CDCl3, d, ppm): 7.29–6.93 (C6H5, 10H,
m), 6.58–6.41 (CONHCH, 1H, d), 6.12 and 5.59 (C]CH2, 2H, s),
5.16–5.05 (NHCOO, 1H, d), 4.87–4.78 (C6H5CH2CH, 1H, m), 4.4–
4.22 (OCH2CH2O and COCHNH, 5H, m), 3.14–2.91 (C6H5CH2,
4H, m), 1.93 (C]CCH3, 3H, s), 1.38 (C(CH3)3, 9H, s). 13C NMR
(Fig. S4 in the ESI,† CDCl3, d, ppm): 170.95, 167.16, 155.18,
136.78, 135.65, 129.49, 128.41, 127.46, 125.99, 80.13, 62.91,
61.74, 53.55, 52.96, 38.12, 37.5, 28.14, 18.22. ESI-MS (Fig. S5 in
the ESI†): [M + Na]+ ¼ 547.17 m/z.
General method for the RAFT polymerization
To Boc-FF-EMA (0.20 g, 0.38 mmol) in a 20 mL septa sealed vial
were introduced CDP (10.26 mg, 2.54 ꢂ 10ꢁ2 mmol), AIBN
(0.417 mg, 2.54 mmol; 0.5 mL solution of 8.34 mg AIBN in 10 mL
DMF) and DMF (0.5 mL). The vial was purged with dry N2 for 20
min and placed in a preheated reaction block at 70 ꢀC. Around
0.1 mL reaction mixture was removed periodically by a N2
purged syringe to determine number average molecular weight
(Mn,GPC) and PDI by GPC and monomer conversion by 1H NMR
spectroscopy by comparing the integration of the monomer
vinyl protons with the DMF protons at 8.02 ppm. Polymeriza-
tion reaction was quenched by cooling in an ice-water bath and
exposing the solution to air, diluted with acetone and precipi-
tated into cold hexanes. The corresponding polymer, P(Boc-FF-
EMA), was reprecipitated four times from acetone/hexanes and
dried under high vacuum. Similarly, Boc-IF-EMA and Boc-VF-
EMA were polymerized to obtain the corresponding polymers,
P(Boc-IF-EMA) and P(Boc-VF-EMA), respectively. The puried
polymers were isolated as yellowish white powders.
Synthesis of mPEG-based macro-CTA
The mPEG macro-CTA was prepared22 as follows: mPEG (Mn ¼
2000 g molꢁ1, 1.0 g, 0.5 mmol) was dissolved in 50 mL of CH2Cl2
containing CDP (0.4 g, 1.0 mmol) and a catalytic amount of
DMAP (61.0 mg, 0.50 mmol). Aer the solution was homoge-
nized by stirring, DCC (0.2 g, 1.0 mmol) in DCM (15 mL) was
added dropwise while cooling in an ice-water bath. Then, at
room temperature the reaction mixture was le to stir for 18 h.
The precipitated DCU was ltered off, and the ltrate was
precipitated into an excess of diethyl ether and dried under high
vacuum for 10 h to obtain mPEG-CDP macro-CTA as a yellow
solid (yield: 90%, see Fig. S11 in the ESI† for the 1H NMR
spectrum and peak assignments).
Similarly, Boc-IF-EMA was synthesized from Boc-Ile-OH and
H2N-F-EMA (yield 85%). 1H NMR (Fig. S6 in the ESI,† CDCl3, d,
ppm): 7.25–7.07 (C6H5, 5H, m), 6.7–6.5 (CONHCH, 1H, d), 6.1
and 5.57 (C]CH2, 2H, s), 5.13–5.08 (NHCOO, 1H, d), 4.89–4.83
(C6H5CH2CH, 1H, m), 4.37–4.24 (OCH2CH2O, 4H, m), 4.12–3.91
(COCHNH, 1H, m), 3.13–3.01 (C6H5CH2, 2H, m), 1.91 (C]
CCH3, 3H, s), 1.79–1.71 (COCHCHCH3, 1H, m), 1.4 (C(CH3)3,
9H, s), 1.08–0.96 (CH3CH2, 2H, m), 0.87–0.77 (CH3CH2-
CH(CH3), 6H, m). 13C NMR (Fig. S7 in the ESI,† CDCl3, d, ppm):
171.55, 170.94, 166.86, 155.77, 135.91, 135.04, 129.49, 128.6,
127.47, 125.99, 79.86, 62.96, 62.01, 59.4, 52.92, 37.78, 36.62,
28.14, 24.38, 18.24, 15.3, 11.21. ESI-MS (Fig. S8 in the ESI†): [M
+ Na]+ ¼ 513.14 m/z.
Synthesis of block copolymers from mPEG-CDP macro-CTA
A typical block copolymerization procedure is described as
follows: Boc-FF-EMA (0.2 g, 0.38 mmol), mPEG-macroCTA (2400
g molꢁ1, 18.3 mg, 7.62 mmol), AIBN (0.125 mg, 0.762 mmol; 0.1
mL solution of 12.5 mg AIBN in 10 mL DMF) and DMF (0.5 mL)
were sealed in a 20 mL vial equipped with a magnetic stir bar.
Aer N2 purging (20 min), the vial was placed in a preheated
reaction block at 70 ꢀC. Aer a predetermined time, the vial was
cooled in an ice-water bath, and a portion of the reaction
948 | J. Mater. Chem. B, 2013, 1, 946–957
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mixture was analyzed by NMR to determine the conversion. and untreated control, ME-180 cells were seeded at a density of
Remaining solution was diluted with acetone and the polymer 2 ꢂ 104 cells per well in a 96 well plate and incubated for 24 h.
was precipitated into cold hexanes and allowed to stand, and Aer incubation, these cells were xed with 4% para-
then the solvent was decanted off. Finally, the block copolymer, formaldehyde and then stained with DAPI. Finally, it was used
mPEG-b-P(Boc-FF-EMA), was reprecipitated four times from for microscopy (BD Pathway855) at 20ꢂ magnication. The
acetone/hexane and dried under vacuum at room temperature lter set used for DAPI imaging was: excitation lter 380/10 nm,
for 10 h.
epiuorescence dichroic lter 400DCLP, emission at 430LP with
the exposure time of 6 milliseconds. Images were acquired
using BD Attovision soware.
Preparation of polymeric micelles and characterization
Typically, 10.0 mg of block copolymer was dissolved in 5 mL of
acetone, and the solution was transferred to a dialysis bag
(spectra/pore dialysis membrane, molecular weight cut-off
(MWCO): 6000–8000 Da) and dialysed against DI water for 48 h
(water was replaced aer every 2 to 6 h) to remove organic
solvents. The prepared micellar solutions were further diluted
for DLS and AFM studies.
Results and discussion
Monomer synthesis
The dipeptide based monomers, Boc-FF-EMA, Boc-IF-EMA and
Boc-VF-EMA, were prepared by the coupling reactions of H2N-
Phe-EMA with Boc-L-Phe-OH, Boc-L-Ile-OH and Boc-L-Val-OH,
respectively (Scheme 1). First, Boc-Phe-EMA was prepared21 and
subsequently treated with TFA in CH2Cl2 at room temperature
for deprotection of Boc-groups. Successful deprotection was
demonstrated by the disappearance of Boc-group signals in the
1H NMR and 13C NMR spectrum (see in the ESI, Fig. S1 and S2†).
The obtained deprotected vinyl monomer, H2N-Phe-EMA, was
further subjected for amide coupling reaction with the Boc-
amino acids in the presence of DCC and DMAP for producing
epimers of dipeptide based vinyl monomers, Boc-dipep-EMA,
with 80–90% yield. The structures of the monomers were
Determination of critical micelle concentration (CMC)
A predetermined amount of pyrene in acetone solution was
added to volumetric asks and acetone was then evaporated
completely. Different concentrations of polymer micellar solu-
tions were added to pyrene and le to equilibrate with pyrene
overnight, the pyrene concentration in the nal solution was
6.0 ꢂ 10ꢁ7 mol Lꢁ1. The uorescence intensities of prepared
solutions were measured with the excitation wavelength set at
339 nm and the ratios of pyrene probe emission uorescence
intensities at 395 and 376 nm (I395/I376) were plotted as a
function of the logarithm of polymer concentrations. The CMC
was obtained from the intersection of two tangent plots of
intensity ratio I395/I376 versus the logarithm of polymer
concentration.23,24
1
conrmed by FT-IR, H and 13C NMR spectra and mass spec-
troscopy measurements. As shown in Fig. 1, the vinyl protons in
Boc-VF-EMA were observed in the 1H NMR spectrum as two
distinct peaks at d 6.08 and 5.55 ppm, the amide proton of Boc-
amino acid at 5.23–5.13 ppm remained intact during the
coupling reaction. The amide proton peak of the dipeptide
linkage appeared at 6.86–6.6 ppm indicating the successful
amide coupling reaction. The characteristic peaks at 7.24–7.04
(phenyl group protons), 4.34–4.18 (methylene protons in
HEMA), 3.1–2.97 (benzyl peaks), 1.89 (methyl protons), 1.39
(methyl protons of the Boc group), 0.89–0.7 (dimethyl peaks of
valine) were clearly seen. In the 13C NMR spectrum of Boc-VF-
EMA, the signals of vinyl carbon and methyl carbons of Boc-
group are seen at 125.7 and 28.1 ppm, respectively. Other
carbon signals are also clearly classied as depicted in Fig. S9
Deprotection of Boc-protected polymers
In a typical example,21 P(Boc-FF-EMA) (50.0 mg) was taken in
1.0 mL DCM and 0.5 mL TFA was added dropwise while cooling
in an ice-water bath and was stirred at room temperature for
2 h. The resulting Boc-deprotected polymer, P(FF-EMA), was
isolated by precipitation from diethyl ether, and dried under
high vacuum.
Cellular cytotoxicity of polymers
The MTT reduction assay was used for investigating the cyto-
toxicity of the prepared polymers.25 HeLa cells were seeded at a
density of 4 ꢂ 104 cells per well in a 96 well plate and incubated
for 24 h to reach ꢃ80% conuency. Cells were treated with
polymers having different concentrations (20 mg mLꢁ1 to 250 mg
mLꢁ1) and incubated for 3 days. A set of untreated control cells
were also incubated for the same time period. The untreated
control and polymer treatment was performed in triplicate. To
each well, 10% culture volume MTT solution was added and was
incubated for 3 h. Aer incubation, the cell culture medium was
disposed of, and MTT solubilization solution was added equally
to the culture media volume. Aer 10 min of incubation, the
absorbance at 570 nm was measured. For uorescence
microscopy, two polymer concentrations (20 and 60 mg mLꢁ1
)
Scheme 1 Synthesis of dipeptide based monomers.
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observed at 1657 cmꢁ1 suggested the formation of peptide
bond. The band present at 1160 cmꢁ1 is assigned to the
stretching vibrations of C–O. Two strong and sharp peaks at
1754 and 1725 cmꢁ1 are attributed to the stretching vibrations
of carbonyl (C]O) groups, demonstrating the presence of C]O
(ester) linkages.26
RAFT polymerization
Recently, we reported RAFT polymerization of Boc-Phe-EMA at
70 ꢀC in DMF using AIBN as initiator and CDP as RAFT agent led
to narrow-disperse amino acid based polymers with high
conversion and good molecular weight control.21 The controlled
character of the RAFT polymerization of dipeptide based
monomers, Boc-FF-EMA, Boc-IF-EMA and Boc-VF-EMA, was
studied under similar conditions (Scheme 2) in terms of the
time-dependent change in the molecular weights and PDI of the
resulting polymers. Linear pseudo-rst-order kinetic plots for
the homopolymerizations of those monomers (Fig. 2A) were
observed. Also, Mn increased linearly with monomer conversion
and agreement between Mn,GPC and number average theoretical
molecular weight (Mn,theo) was reasonably good throughout the
conversion ranges (Fig. 2B). The unimodal refractive index (RI)
traces in every homopolymerization shied smoothly toward
lower elution volume with increasing monomer conversion with
no evidence of high molecular weight species from bimolecular
termination (Fig. 2C–E). The relative linearity of Fig. 2A and B
indicated a constant concentration of macro-growing radicals
during each polymerization.27 A range of polymerization
conditions was used to verify the effect of reaction stoichiom-
etry on polymerization control and we noticed, as expected, that
increasing [monomer]/[CDP] gives polymers of higher molec-
ular weight (Fig. 3 and Table 1).
Fig. 1 1H NMR spectra of Boc-VF-EMA in CDCl3 (A), homopolymer P(Boc-VF-
EMA) in CDCl3 (B), and corresponding deprotected polymer P(VF-EMA) in DMSO-
d6 (C) (* denotes the solvent resonances).
(see in the ESI†). Furthermore, the structures of Boc-FF-EMA,
Boc-IF-EMA and Boc-VF-EMA were conrmed by ESI-MS with
observed molecular masses [M + Na]+ ¼ 547.17 m/z, [M + Na]+ ¼
513.14 m/z and [M + Na]+ ¼ 499.11 m/z, respectively (see in the
ESI, Fig. S5, S8 and S10†). The FT-IR spectra also gave evidence
for the formation of dipeptide based vinyl monomers. As an
example, for Boc-VF-EMA, aer condensation reaction of Boc-
Val-OH with NH2-Phe-EMA, the characteristic absorption peaks
of amide groups of Boc-Val-OH can still be observed at 3316 and
1522 cm (Fig. S12 in the ESI†). The strong amide I band
The homopolymers, P(Boc-FF-EMA), P(Boc-IF-EMA) and
P(Boc-VF-EMA), were characterized by 1H NMR spectroscopy
(Fig. 1B for P(Boc-VF-EMA), and in the ESI, Fig. S3B and S6B† for
Scheme 2 Synthesis of dipeptide based homopolymers and block-copolymers by RAFT polymerization, followed by deprotection of the Boc-group.
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Fig. 2 Pseudo-first-order kinetics plot for the RAFT polymerization of dipeptide based monomers (A), the corresponding Mn versus monomer conversion plot (B), GPC
refractive index (RI) traces as a function of elution volume for the RAFT polymerization of Boc-FF-EMA (C), Boc-IF-EMA (D) and Boc-VF-EMA (E).
P(Boc-FF-EMA) and P(Boc-IF-EMA), respectively). The typical molecular weight (Mn,UV-vis) determined by this method (see in
resonance signals for the different protons in the repeating the ESI†). These results indicate the formation of P(Boc-dipep-
units of the polymers are assigned on the spectra. Comparison EMA) with the CTA fragments at the polymer chain ends.
of the integration areas from the terminal –CH2–CH2– protons
(from the HOOC–CH2–CH2–C(CN)(CH3)– chain end) at 2.4–2.6
ppm and the repeating unit protons at 3.8–4.5 ppm (for –O–
CH2–CH2–O– and chiral proton) allowed calculation of the
mPEG-CDP macro-CTA derived peptide based block
copolymers
number-average molecular weight (Mn,NMR) from NMR spec-
troscopy. Good agreement between the Mn,theo, Mn,GPC and
Mn,NMR molecular weights suggests controlled RAFT polymeri-
zation (Table 1). Also, UV-vis spectroscopy was used to study the
end groups. All homopolymers demonstrated the expected
absorption for the trithiocarbonate moiety at lmax ꢃ 309 nm,
indicating retention of the u-trithiocarbonate group during
RAFT polymerization. We observed >95% retention of trithio-
carbonate by comparing the Mn,GPC and number-average
To prepare diblock copolymers consisting of nonionic PEG and
peptide containing cationic blocks (aer Boc-deprotection, vide
infra), we performed RAFT polymerization of dipeptide mono-
mers using mPEG macro-CTA. The mPEG derived macro-CTA
was synthesized by esterication reaction of the terminal
hydroxyl group of mPEG with the carboxyl group of CDP in DCM
in the presence of DCC and DMAP.22,28 Characteristic signals of
both mPEG and CDP moieties were clearly seen in the 1H NMR
spectrum of mPEG-CDP (Fig. S11 in the ESI†). The resonance at
Fig. 3 GPC RI traces for the RAFT polymerization of (A) Boc-FF-EMA, (B) Boc-IF-EMA and (C) Boc-VF-EMA at different [monomer]/[CTA] ratios varying from 15 : 1 to
50 : 1 keeping [CTA]/[I] ratio 1 : 0.1 constant in DMF at 70 ꢀC.
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Table 1 Results from synthesis of homopolymers at 70 ꢀC in DMF
[M]/[CTA]/[I] Time (min) Conv.a (%) Mn,GPC (g molꢁ1
)
PDIb Mn,theo (g molꢁ1
)
Mn,NMRd (g molꢁ1
)
Mn,UV-vise (g molꢁ1
)
b
c
Polymer
P(Boc-FF-EMA) 15/1/0.1
P(Boc-FF-EMA) 25/1/0.1
P(Boc-FF-EMA) 50/1/0.1
P(Boc-IF-EMA) 15/1/0.1
P(Boc-IF-EMA) 25/1/0.1
P(Boc-IF-EMA) 50/1/0.1
P(Boc-VF-EMA) 15/1/0.1
P(Boc-VF-EMA) 25/1/0.1
P(Boc-VF-EMA) 50/1/0.1
P(Boc-VF-EMA) 100/1/0.1
210
240
260
210
240
260
210
240
260
270
48
40
57
51
43
55
48
38
48
58
4200
4900
13 860
4800
5900
13 625
4400
5200
10 890
25 510
1.13
1.16
1.38 15 350
1.16
1.21
1.32 13 900
1.13
1.17
1.28 11 840
1.44 28 044
4180
5600
4790
4520
NDf
4350
4270
NDf
4900
5580
NDf
4160
5680
6160
NDf
NDf
3835
4930
3870
5150
NDf
4560
5070
NDf
NDf
NDf
Calculated by 1H NMR spectroscopy in CDCl3. Measured by GPC. The theoretical molecular weight (Mn,theo) ¼ ([Boc-dipep-EMA]0/[CTA]0
ꢂ
a
b
c
molecular weight (MW) of Boc-dipep-EMA) ꢂ conversion + (MW of CTA), Boc-dipep-EMA ¼ Boc-FF-EMA or Boc-IF-EMA or Boc-VF-EMA.
d
Determined by 1H NMR from the integration areas of the terminal –CH2–CH2– protons (from the HOOC–CH2–CH2–C(CN)(CH3)– moiety) at
e
f
2.4–2.6 ppm and the repeating unit protons at 3.8–4.5 ppm (for –O–CH2–CH2–O– and chiral proton). Determined by UV-vis analysis. Not
determined.
3.3 ppm is attributed to the trithiocarbonate methylene protons
(–SCH2–) and resonance at 4.24 ppm is assigned to the mPEG
ester methylene protons, conrms the formation of the esteri-
ed product. The capping efficiency of mPEG with CDP was
estimated to be 97% by comparing the NMR peak area of these
two peaks indicating that the mPEG-CDP macro-CTA can be
successfully synthesized with quantitatively high yield.
The mPEG-CDP macro-CTA (2400 g molꢁ1) was further used
for the synthesis of block copolymers by the polymerization of
Boc-dipep-EMA at [Boc-dipep-EMA]/[mPEG-CDP macroCTA]/
[AIBN] ¼ 50/1/0.1. These block copolymerizations were conve-
niently carried out with the conversions in the range of 54–66%
and their corresponding Mn's were determined from GPC (Table
2). As depicted by Fig. 4, GPC chromatograms have shied
toward the higher molecular weight region aer the block
copolymerization using mPEG-CDP macro-CTA suggesting the
chain extension was effective although unreacted macro-CTA
remained present in all block copolymer samples. Similar
observations were already reported using mPEG macro-CTA.29
In the GPC trace of the block copolymers, the elution volume of
the low molecular weight shoulder corresponded to that of the
mPEG macro-CTA and the Mn,GPC determined for the block
copolymers (Table 2) are a little lower than the theoretical
Fig. 4 GPC traces of the mPEG-CDP based macro-CTA and block copolymers.
mPEG-b-P(Boc-FF-EMA) and mPEG-b-P(Boc-IF-EMA), respec-
tively). These results suggest that synthesized mPEG-CDP
macro-CTA can be successfully employed for the synthesis of
various peptide based block copolymers with a relatively narrow
molecular weight distribution and controlled molecular
weights.
Deprotection of Boc-group
1
molecular weight determined based on conversion. In the H
To obtain polymers with free primary amine groups in the side
chains, Boc-protecting groups were removed by TFA/DCM
(0.5 : 1, v/v) at room temperature (Scheme 2). Successful
deprotection of Boc-groups for the homopolymers and block
copolymers was conrmed by the disappearance of Boc-proton
NMR spectrum of the block copolymer measured in CDCl3, the
peaks corresponding to the components of both blocks are
clearly detected, conrming the targeted structures (Fig. 5A for
mPEG-b-P(Boc-VF-EMA) and Fig. S13 and S14 in the ESI† for
Table 2 Block copolymerization with mPEG-based macro-CTA at 70 ꢀC in DMF for 4 h
Block copolymera
Conv.b (%)
Mn,GPC (g molꢁ1
)
PDIc
Mn,theod (g molꢁ1
)
c
mPEG-b-P(Boc-FF-EMA)
mPEG-b-P(Boc-IF-EMA)
mPEG-b-P(Boc-VF-EMA)
66
54
63
13 710
12 120
12 790
1.27
1.23
1.24
19 710
15 650
17 410
a
Molar ratio of [monomer (M)]/[macro chain transfer agent (macro-CTA)]/[initiator (I)] ¼ 50/1/0.1. b Determined by 1H NMR spectroscopy in CDCl3.
c
Measured by GPC. d The theoretical molecular weight (Mn,theo) ¼ ([Boc-dipep-EMA]0/[mPEG-macro CTA]0 ꢂ molecular weight (MW) of Boc-dipep-
EMA) ꢂ conversion + (MW of mPEG-macro CTA), Boc-dipep-EMA ¼ Boc-FF-EMA or Boc-IF-EMA or Boc-VF-EMA.
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Fig. 6 (A) Effect of pH on the absorbance of Boc-deprotected homopolymer
P(VF-EMA) at 550 nm, and (B) CMC determination of amphiphilic mPEG-b-P-
(Boc-dipep-EMA) block copolymers.
determined by UV-vis spectroscopy and the turbidity curve
measured at 550 nm for P(VF-EMA) is shown in Fig. 6A (Fig. S17
in the ESI† for the homopolymer P(IF-EMA) and corresponding
block copolymer mPEG-b-P(IF-EMA)). At lower pH, the primary
amine moiety is entirely protonated and the polymer solution
was transparent. However, the solution was transformed from
transparent to opaque with increasing pH and gradually
precipitation occurred from the solution as the pH changed
above a particular value due to the deprotonation of amino
groups, indicating the obvious pH-responsive property of these
polymers. The pH-responsiveness for P(IF-EMA) and P(VF-EMA)
was observed at pH 6.1 and 6.5, respectively. However, pH-
responsiveness for block copolymers mPEG-b-P(FF-EMA),
mPEG-b-P(IF-EMA) and mPEG-b-P(VF-EMA) was observed at pH
5.5, 6.2 and 6.5, respectively. These pH-responsive phase tran-
sitions were reversible and attributed to the protonation/
deprotonation of the primary amine groups of the peptide
moieties in the side chains.
Fig. 5 1H NMR spectra of block copolymers (A) mPEG-b-P(Boc-VF-EMA) in CDCl3,
(B) mPEG-b-P(Boc-VF-EMA) in D2O, and (C) mPEG-b-P(VF-HEMA) in D2O
(* denotes the solvent resonances).
signals at about 1.4 ppm in the 1H NMR spectrum shown in
Fig. 1 for P(VF-EMA) and Fig. 5 for mPEG-b-P(VF-EMA) (also, see
Fig. S3 for P(FF-EMA), Fig. S6 for P(IF-EMA), Fig. S13 for mPEG-
b-P(FF-EMA) and Fig. S14 for mPEG-b-P(IF-EMA) in the ESI†). In
addition, Boc-deprotection was further conrmed by FT-IR
study. As an example, the absorption peak at 1514 cmꢁ1
attributed to the N–H (amide II band) in the FT-IR spectra of
P(Boc-VF-EMA) and mPEG-b-P(Boc-VF-EMA) disappears aer
deprotection (Fig. S12 and S15 in the ESI†) due to the conversion
Zeta potential
of Boc-group into –NH2 functionality. Aer Boc-deprotection, The surface charges (zeta potential) of various Boc-deprotected
P(IF-EMA) and P(VF-EMA) homopolymers were soluble in polymers in DI water (1.0 mg mLꢁ1) at pH 4.5 were measured by
aqueous media while P(FF-EMA) was still insoluble in water due DLS analysis excluding P(FF-EMA) homopolymer due to its
to the high hydrophobicity from two phenyl groups in each insolubility in water. The zeta potential of P(IF-EMA) and P(VF-
repeating unit. Furthermore, Boc-deprotection of all three EMA) in DI water were found to be +78.5 and +34.0 mV, respec-
amphiphilic block copolymers produced double hydrophilic tively. The zeta potential of mPEG-based double hydrophilic
block copolymers.
block copolymers mPEG-b-P(FF-EMA), mPEG-b-P(IF-EMA) and
The thermal stability of the homopolymers was studied by mPEG-b-P(VF-EMA) were determined as +45.6, +37.1 and +36.2
TGA with a heating rate of 10 ꢀC minꢁ1 under nitrogen ow mV, respectively. These positive zeta potentials are due to the
(Fig. S16 in the ESI† for the TGA analysis of P(Boc-FF-EMA) and cationic primary amine groups in the side-chains. Since the
P(FF-EMA)). It was observed that both Boc-protected and Boc- homopolymers and block copolymers contain peptide moieties
ꢀ
deprotected homopolymers were thermally stable up to 175 C with amide bonds and also phenyl rings, so inter- and intra-
and then started degrading above that temperature.30
molecular hydrogen bonding as well as p–p interaction associ-
ated, which can affect their orientations and may alter the overall
surface charge values. Further, since phenylalanine is the most
hydrophobic amino acid according to the hydrophilicity values,
which may favour hydrophobic interactions,32 so the decrease of
surface charge values was observed for those polymers.
pH responsiveness
The free primary amine groups in the side-chain of homo- and
block copolymers can be reversibly protonated by adjusting the
pH of their aqueous solution.31 In order to determine the pH
responsiveness of hydrophilic homopolymers (P(IF-EMA) and
P(VF-EMA)) as well as double hydrophilic block copolymers,
Self-assembly of block copolymers
aqueous solutions (0.1 wt.%) of these polymers were analyzed as The 1H NMR spectrum of mPEG-b-P(Boc-VF-EMA) block
ꢀ
a function of pH at 27 C. The pH responsiveness values were copolymer in CDCl3 (Fig. 5A) displays all NMR resonances from
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1
the mPEG and Boc-VF-EMA units. However, the H NMR spec- (0.1 mg mLꢁ1) and investigated by AFM. As illustrated by
trum of mPEG-b-P(Boc-VF-EMA) in D2O showed peaks only from Fig. 7A, a spherical morphology for the mPEG-b-P(Boc-FF-EMA)
the hydrophilic mPEG block and there was a complete loss of was observed. The average diameter of the micelle was about
proton resonances from the Boc-VF-EMA block, due to possible 140 nm while the calculated height of the micelle was about 27
suppressed molecular motion of the aggregated hydrophobic nm and the height prole indicates rough micellar surface
P(Boc-VF-EMA) segments surrounded by the hydrophilic mPEG (Fig. S18 in the ESI†). The size obtained by DLS studies was 148
(Fig. 5B),23 strongly indicative of
a stable higher-order nm, which matches well with the AFM data. Fig. 7B also shows
morphology formation with a hydrophobic inner core.33 The the formation of well-dened structures from self-assembly of
self-association of amphiphilic mPEG-b-P(Boc-dipep-EMA) mPEG-b-P(Boc-IF-EMA) in aqueous solution and a few units of
block copolymers towards micellar formation was achieved micelles gave this denite uniform shape having an average
using dialysis of the polymer solutions in acetone against DI diameter of about 235 nm while the height was around 53 nm
water and investigated by uorescence spectroscopy, DLS and (Fig. S20 in the ESI†). In the case of mPEG-b-P(Boc-VF-EMA),
AFM. First, to check micelle formation from mPEG-b-P(Boc- large multimolecular composite structures were observed by
dipep-EMA) block copolymers, their aqueous solution was AFM, formed by the secondary aggregation of primary micelles
studied by uorescence spectroscopy technique using pyrene as in aqueous solution, having well dened morphology with
a hydrophobic uorescent probe because of its high sensitivity smooth surfaces (Fig. 7C and S21 in the ESI†). The formation of
to the local polarity of the medium.34 With the increase of such large composite structures by the secondary aggregation
polymer concentration, a red shi from 376 to 395 nm of pyrene mechanism of initially formed small micelles in aqueous
uorescence spectrum is observed, indicates that pyrene solution has been reported in the literature.37–40 Eisenberg and
molecules are partitioned into a less polar environment.34a The co-workers have earlier explained the formation of micro/
ratios of pyrene probe uorescence intensities at 395 and 376 nanospheres which are actually large compound micelles
nm (I395/I376) were plotted against the concentration logarithm formed by secondary aggregation of primary micelles.40 The
of all the three block copolymers. As depicted in Fig. 6B, the formation of large micelles named as supermicelles has also
intensity ratio remained almost unchanged at low concentra- been reported by Muller et al.41 We believe that secondary
tions of copolymer and increased drastically once the copol- micellar aggregation in our case is obtained due to the presence
ymer concentration reached the CMC, indicating the formation of peptide moieties in the side-chain of the hydrophobic block
of micelles. The CMC values were determined by the intersec- which induce secondary interactions.
tion of the lines drawn through the at region points at low
The self-assembly of the Boc-deprotected double hydrophilic
concentrations and the abruptly increasing regions at high block copolymers was also investigated by AFM. Their aqueous
concentrations. The observed CMC values of mPEG-b-P(Boc-FF- solutions (0.1 mg mLꢁ1) were sonicated for 1.0 h and drop-cast
EMA), mPEG-b-P(Boc-IF-EMA), and mPEG-b-P(Boc-VF-EMA) on microscopic glass coverslips and were further observed by
were 3.3 ꢂ 10ꢁ3 mg mLꢁ1, 1.4 ꢂ 10ꢁ3 mg mLꢁ1 and 3.2 ꢂ 10ꢁ3 AFM. Interestingly, mPEG-b-P(FF-EMA) and mPEG-b-P(IF-EMA)
mg mLꢁ1, respectively. The obtained CMC values indicated the self-assemble into spherical shapes (Fig. S22 and S23 in the
high tendency of the amphiphilic block copolymers to self- ESI†), due to the presence of hydrophobic segments of amino
assemble in aqueous solution due to the achieved high hydro- acid moieties in each dipeptide unit. However, mPEG-b-P(VF-
phobicity provided by the introduction of hydrophobic peptide EMA) self-assembles into uniform rod-like morphology as
units.35 In addition, possible intra- and intermolecular inter- depicted by Fig. 8 (also see Fig. S24 in the ESI†), possibly due to
actions between peptide groups (p–p interactions and acquired secondary structures possessed by the strong
hydrogen bonding) also favour the self-association.36
secondary interactions induced by the presence of the valine
The aqueous micellar solutions obtained aer dialysis were moiety having symmetric isopropyl groups in each
aged for 2 days and drop-cast on microscopic glass coverslips dipeptide unit.
Fig. 7 AFM height images of block copolymers: (A) mPEG-b-P(Boc-FF-EMA), (B) mPEG-b-P(Boc-IF-EMA) and (c) mPEG-b-P(Boc-VF-EMA) (prepared from 0.1 mg mLꢁ1
aqueous solution).
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Fig. 8 AFM height (A), magnitude (B) and 3D (C) images of mPEG-b-P(VF-EMA) block copolymer (prepared from 0.1 mg mLꢁ1 aqueous solution).
P(IF-EMA) and mPEG-b-P(VF-EMA) (Fig. S25B in the ESI†) and
untreated control at various concentrations demonstrates
negligible cytotoxicity proles of peptide based homopolymers
and block copolymers up to 250 mg mLꢁ1 concentrations.
Confocal microscopic study was also performed for
untreated, and peptide based polymers treated ME-180 cervical
cancer cells (Fig. 9). The obtained DAPI stained uorescence
images of cells showed no obvious change in nuclear structure
of the polymer treated cells compared to the control. DAPI
images of cells were overlaid on bright eld images (phase
contrast images) to conrm the morphological integrity of cells
treated with different polymers (Fig. 9 and S26 in the ESI†).
These results reveal that side-chain peptide containing poly-
meric architectures exhibit great potential as a drug delivery
vehicle due to their excellent biocompatibility prole.
Cytotoxicity
Cytotoxicity assessment is crucial before biological and
biomedical applications of polymers, and we have therefore
studied the in vitro cytotoxic effects of the side-chain peptide
based polymers at different concentrations, using the MTT
assay, on the HeLa (human cervical cancer) cell line. The assay
determines the viability of cells based on mitochondrial activity
of the cells. Depending on the metabolic activity of cells, addi-
tion of the MTT dye produces formazan crystals, which when
dissolved in MTT solubilization solution, gives rise to a col-
oured solution whose absorbance can be read at 570 nm for
determining the optical density (O.D.) value, thus giving the
approximate number of viable cells in each well. HeLa cells were
subsequently incubated with a set of untreated control and
various amounts of polymers for 72 h. Comparison of cytotox-
icity of P(IF-EMA) and P(VF-EMA) homopolymers (Fig. S25A in
the ESI†) and their corresponding block copolymers, mPEG-b- Conclusions
We successfully prepared a series of well-dened dipeptide side-
chain containing polymers by RAFT polymerization. Using
mPEG-macro CTA, we synthesized amphiphilic block copoly-
mers mPEG-b-P(Boc-dipep-EMA) with Boc-FF-EMA, Boc-IF-EMA
and Boc-VF-EMA as the second block repeating units. Boc-
deprotection resulted in pH-responsive hydrophilic homopoly-
mers and double hydrophilic block copolymers with a side
primary amine group at each repeating unit. Zeta potential
measurements demonstrated the electropositive surface charge
of these new peptide based CPEs, conrming their cationic
nature. The amphiphilic block copolymers, mPEG-b-P(Boc-
dipep-EMA), self-assemble in aqueous solution; AFM and DLS
studies show that mPEG-b-P(Boc-FF-EMA) aggregates into
spherical micelles. However, mPEG-b-P(Boc-IF-EMA) and
mPEG-b-P(Boc-VF-EMA) form well-dened large multimolecular
composite aggregates due to the secondary aggregation of
primary micelles in aqueous solution. The sonication induced
macro-supramolecular self-assembly of Boc-deprotected double
hydrophilic block copolymers in aqueous medium, mPEG-b-
P(NH2-dipep-EMA), was also successfully studied by AFM. Boc-
deprotected homopolymers and block copolymers exhibited a
minimal cytotoxicity to HeLa cells. The possibility of using these
polymers towards inhibition of bril formation by Ab is under
investigation to treat Alzheimer's disease. We are also currently
extending our studies to the design and synthesis of highly
Fig. 9 Fluorescence microscopic images of ME-180 cells taken at 72 h post-
incubation of polymer treatment with 20 mg mLꢁ1 concentrations.
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Acknowledgements
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This study was supported nancially by the Department of
Science and Technology (DST), New Delhi, India [Project no.:
SR/S1/OC-51/2010] and Indian Institute of Science Education
and Research – Kolkata. We thank the Centre for Research in
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