Water soluble stimuli-responsive star copolymers with multiple encapsulation and release properties

Article abstract of DOI:10.1039/c5ra26144a

A series of three arm star shaped (random/block) and linear water soluble copolymers are synthesised by atom transfer radical polymerization (ATRP) using di(ethylene glycol) methyl ether methacrylate (DEGMA) and 2-(dimethylamino) ethyl methacrylate (DMAEM

Full text of DOI:10.1039/c5ra26144a

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Water soluble stimuli-responsive star copolymers
with multiple encapsulation and release
properties†
Cite this: RSC Adv., 2016, 6, 8773
Sandip Das, Dhruba P. Chatterjee,‡ Radhakanta Ghosh, Pradip Das§
*
and Arun K. Nandi
A series of three arm star shaped (random/block) and linear water soluble copolymers are synthesised by
atom transfer radical polymerization (ATRP) using di(ethylene glycol) methyl ether methacrylate (DEGMA)
and 2-(dimethylamino) ethyl methacrylate (DMAEMA). The structure and composition of the block and
random copolymers are characterized by 1H NMR spectra and gel permeation chromatography (GPC).
The self-assembly of these copolymers, investigated by dynamic light scattering (DLS), exhibits that
below the lower critical solution temperature (LCST) of pDEGMA all the copolymers are soluble in water
and possess lower particle size but above its LCST particle size increases particularly in a basic medium.
On addition of 8-anilino-1-naphthalenesulfonic acid (ANS) the particle size increases by ꢀ10 times
below the LCST. Both the DLS and fluorescence studies using a hydrophobic fluorescent dye, exhibit
temperature triggered encapsulation and pH triggered release. All the copolymers exhibit highly
reversible multiple aggregation at different temperature and pH conditions and require increased
temperature for the aggregation with decrease in pH of the medium. The random star copolymer
[3-arm-p(DEGMA40-co-DMAEMA18)] exhibits aggregate formation under physiological conditions (37 ꢁC
and pH 7.5) and with decreasing the pH to 6.5 the aggregates dissociate. The MTT assay and cell
morphology indicate that the three arm star and linear random copolymers have lower cytotoxicity
against normal CHO-K1 cells having lower positive zeta potential values.
Received 8th December 2015
Accepted 7th January 2016
DOI: 10.1039/c5ra26144a
www.rsc.org/advances
responsive systems in aqueous medium. Poly(N-iso-
propylacrylamide) (pNIPAM) is the most studied thermo-
1. Introduction
responsive water soluble polymer for biological applications
having a LCST near the physiological range at around 32 ꢁC (ref.
21 and 22) and the LCST of pNIPAM remains unchanged by
slight variation of pH and other chemical environments.22–25
Lutz et al. have made the application of poly(ethylene glycol)
based polymethacrylate systems as a very useful alternative of
pNIPAM.25–27 Short poly(ethylene glycol) substituted polymer
chain, e.g. poly[di(ethylene glycol) methyl ether methacrylate]
(pDEGMA), exhibits LCST around 21 ꢁC in aqueous solution.26,28
However, its LCST may be tuned at higher temperature by
copolymerizing with oligo(ethylene glycol) methyl ether meth-
acrylate (OEGMA) in suitable feed ratios of the monomers.29
Similarly, the LCST type phase transition temperatures of
a thermo responsive polymer system may also be modied by
copolymerization with pH sensitive polymer systems such as
N,N-dialkyl aminoethyl methacrylates having N,N dimethyl or
N,N-diethyl substituents.30,31 In comparison to the pKa of
another basic monomer vinyl pyridine (pKa ꢀ 4.5),32 2-(dime-
thylamino) ethyl methacrylate (DMAEMA) has a higher pKa
(ꢀ7),31 and therefore pDMAEMA gives the opportunity of
reversible protonation and deprotonation of the –NMe2 groups
to have aggregation and disaggregation within the pH window
Stimuli responsive polymers are extensively studied for their
potential applications as ‘smart materials’,1–7 and polymers with
the propensity of conformational/polarity change of the entire
chain or any of its constituting block, on the inuence of any
external trigger like temperature,2,5 pH,2,5,7 light,8–13 specic
molecules,2,14,15 redox2,16,17 or its combinations, are classied as
stimuli responsive polymers. Water soluble polymers showing
stimuli response with temperature and/or pH have received
special attention for potential applications in the eld of health
care and nano-biotechnology.18–20 Polymeric systems showing
a phase transition of lower critical solution temperature (LCST)
or upper critical solution temperature (UCST) types and/or
having a block copolymer segment with pH sensitive func-
tional group may function as thermo, pH or thermo-pH dual
Polymer Science Unit, Indian Association for the Cultivation of Science, Jadavpur,
Kolkata-700 032, India. E-mail: psuakn@iacs.res.in
† Electronic supplementary information (ESI) available: 1H NMR of TIBB and P1,
GPC, DLS and uorescence data of P1, and bar diagram of zeta potential values.
See DOI: 10.1039/c5ra26144a
‡ Dept of Chemistry, Presidency University, Kolkata-700 073.
§ Centre for Advanced Materials, IACS, Jadavpur, Kolkata-700 032.
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of 5.5 and 7.4, which are typical for malignant tissues and solubilisation of the aggregate is made by lowering the pH of the
human blood system respectively. Also pDMAEMA shows LCST medium to a small extent. We have been able to optimize
near 50 ꢁC (ref. 30) at pH ¼ 7, however this is somewhat higher a composition for the 3-arm star random copolymer exhibiting
than the normal physiological temperature range of applica- aggregation at pH 7 under physiological temperature and
tions therefore in the release or encapsulation mechanism when pH is lowered to a value similar to malignant cells
LCST of pDMAEMA would have an insignicant effect.
dissociation of aggregate occurs. We have efficiently monitored
uorescent dye 8-anilino-1-
pDEGMA based polymers offer non-toxicity, anti- these changes using
a
immunogenicity and biocompatibility25–27 coupled with its naphthalenesulfonic acid (ANS) by both dynamic light scat-
facile synthesis technique by ATRP under simple cost effective tering (DLS) and uorescence methods with appreciable
and environmentally benign conditions.33–35 pDEGMA shows reversibility. Another hydrophobic dye, Nile red is also encap-
LCST-type phase transition in aqueous solution due to the sulated by the three arm star copolymer and has been moni-
differential solvation of water soluble di(ethylene glycol) moie- tored through uorescence spectroscopy. The cell viability and
ties and water insoluble polymethacrylate backbone segment. biocompatibility of the copolymers are investigated and
At a temperature lower than 21 ꢁC solvation of di(ethylene possible reason for the relative cell viability has been discussed
glycol) segment is sufficient to keep it soluble in water and from the zeta potential values.
above it de-solvation causes aggregation of the chains resulting
insolubility of the polymer. Therefore, copolymer of pDEGMA
with some other water soluble polymer, having both block and
2. Experimental section
2.1. Materials and purication
random architecture, may offer a micellar system in aqueous
solution above LCST of pDEGMA.
The monomers di(ethylene glycol) methyl ether methacrylate
(DEGMA) and 2-(dimethylamino) ethyl methacrylate (DMAEMA)
(Aldrich, USA) were puried by passing through a neutral and
basic alumina column, respectively. The ligands 1,1,4,7,10,10-
hexamethyl triethylene tetramine (HMTETA) and 4,40-dimethyl-
2,20-dipyridyl (DMDP) (Aldrich, USA) were used as received.
Phloroglucinol dehydrate, 8-anilino-1-naphthalenesulfonic acid
(ANS) and the initiator 2-bromoisobutyryl bromide (2BIB)
(Aldrich, USA) were used as received. The catalyst cuprous
chloride (CuCl) (Loba Chemicals, Mumbai) was puried under
nitrogen atmosphere by washing with 10% HCl followed by
CH3OH and diethyl ether in a Schlenk tube. The solvents, ani-
sole, chloroform (CHCl3), dichloromethane (DCM) and tetra-
hydrofuran (THF) were purchased from Loba Chemicals,
Mumbai and were puried by distillation.
In many applications, reversible dissociation/micellization
transitions triggered by external stimuli are desired, as the
reformation of micelles allows the re-encapsulation of the
released substances.8,12 Jiang and Zhao developed dual
responsive water soluble block copolymer [polyethyleneoxide-b-
p(DEGMA-co-methacrylic acid)] using thermo and pH sensitive
component blocks showing multiple micellization and disso-
ciation transition by coupled application of both of the trig-
gers.34 The carboxylic acid groups of polymethacrylic acid has
been used as pH sensitive block and increasing pH of the
medium helps in the process of micellar dissociation whereas
increased temperature causes remicellization.
So copolymers with pDEGMA as hydrophobic core in
aqueous solution may be designed for encapsulation and
release of hydrophobic dye molecules. In order to use these
micelles for delivery and diagnostic purposes, they must remain
in the aggregated state at physiological temperature (37 ꢁC) and
at typical blood pH (7.4) followed by its dissociation at the
2.2. Synthesis
2.2.1. Preparation
of
1,3,5-tri(2-bromo-isobutyryloxy)
environment of malignant tumour tissues having pH at the benzene (TIBB). Under nitrogen atmosphere phloroglucinol
acidic range (pH 5.5–6.5).36 Therefore, pDEGMA based copoly- dehydrate (2 g, 12.3 mmol) and triethylamine (5.1 ml, 32.9
mers having another component of basic functionalities would mmol) (Et3N) were dissolved in 200 ml anhydrous THF at 0 ꢁC.
be prospective for such application. In order to develop poly- 2BIB (5.25 ml, 42.5 mmol) was then injected drop wise into the
meric micelle based carrier systems, the polymer molecules reaction vessel and the reaction mixture was then allowed to
used should have appreciably lower value of critical aggregation stand for 24 h. Aer solvent evaporation by rotary evaporator the
concentration (CAC) such that during application, micellar dried sample was collected and dissolved in 60 ml ethyl acetate.
aggregate remains intact irrespective of dilution. In this regard The reaction mixture was then consecutively washed with water
multi-arm polymer systems are more acceptable than linear and saturated sodium bicarbonate solution. The organic layer
polymer molecules having comparable molar mass or molar was then separated and was passed through the anhydrous
ratios.37 In addition, these multi-arm or branched polymer Na2SO4 followed by solvent evaporation and re-crystallization at
systems are further attractive than linear polymeric systems due very low temperature to obtain a white crystalline solid as a nal
to their lower values of viscosity owing to the lower value of product (yield 80%).39
exponent (a) in Mark–Howink–Sakurada equation.38
1H NMR (CDCl3): d ¼ 6.95 (3H, s), 2.1 (18H, s); 13C NMR
Here we report the synthesis of 3-arm star shaped random & (CDCl3) d ¼ 169.6, 151.5, 112.6, 54.9 and 30.7.
block copolymers and linear copolymer of pDEGMA and
2.2.2. Preparation of 1,2-bis(bromoisobutyryloxy)ethane
pDMAEMA and compare its multistep micellar aggregation at (EGBIB). Under nitrogen atmosphere 1.5 ml ethylene glycol was
varied temperature and pH conditions. All these systems dissolved in 55 ml anhydrous DCM and then 10 ml Et3N was
require increased temperature for aggregation and subsequent added into the reaction mixture. 10 ml 2BIB was injected drop
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ꢁ
wise into the reaction vessel at 0 ꢁC. The reaction mixture, 18 h at 30 C. Aer the desired time the reaction mixture was
stirred for overnight, was ltered and was washed with satu- precipitated into excess petroleum ether (60–80 ꢁC) and the
rated sodium bicarbonate and NaCl solution, respectively. The resultant polymer was separated, re-dissolved in THF and was
organic layer was collected, passed through anhydrous Na2SO4 re-precipitated with petroleum ether for repeated times to
and was evaporated by rotary evaporator. The residue was dis- eliminate the small amount of entrapped monomer. To remove
solved in methanol at 50 ꢁC and was re-crystallized at ꢂ5 ꢁC to copper the THF solution of the polymer was then passed
obtain white needle like crystal of the product (yield 75%).40
through silica column and was followed by solvent evaporation
1H NMR (CDCl3): d ¼ 1.95 (12H, s), 4.45 (4H, s): 13C NMR to obtain pure polymer (yield-70%).
(CDCl3) d ¼ 172, 63, 54.5 and 30.5.
2.2.6. Synthesis of linear-random poly[di(ethylene glycol)
2.2.3. Synthesis of 3-arm-poly[di(ethylene glycol) methyl methyl ether methacrylate]-co-poly[2-(dimethylamino) ethyl
ether methacrylate]3-Cl3 macro-initiator {3-arm-p(DEGMA)3- methacrylate] copolymer {L-p(DEGMA32-R-DMAEMA23)}. Also
Cl3}. 30 mg TIBB, 2 ml anisole, 5 mg CuCl were taken in the linear copolymer L-p(DEGMA32-R-DMAEMA23
) (P5) is
a nitrogen purged reaction vessel (8 ꢃ 2.5 cm) and DEGMA synthesized by ATRP using EGBIB as the initiator and CuCl/
(1 ml) was injected into the reaction vessel under nitrogen HMTETA (catalyst/ligand) system at 25 ꢁC. The mole ratio of
atmosphere. Then 16 mg DMDP was added into the reaction DEGMEM and DMAEMA are taken as 60 : 40.
vessel and the closed reaction vessel was then kept at 55 ꢁC for
10 h under stirring condition. The reaction mixture was poured
into excess petroleum ether (boiling range 60–80 ꢁC) and the
separated polymer was isolated, re-dissolved in THF and was re-
precipitated into excess petroleum ether. The process was
repeated for three times to remove any trace amount of mono-
mer entrapped within the polymer. Then the THF solution of
the polymer was passed through silica column to remove copper
and was followed by solvent evaporation to obtain the polymer
(Scheme 1) (yield-70%).
2.2.4. Synthesis of 3-arm-poly[di(ethylene glycol) methyl
ether methacrylate]-block-poly[2-(dimethylamino) ethyl meth-
acrylate] {3-arm-p(DEGMA34-B-DMAEMA21)}(P1). In another
nitrogen purged reaction tube 0.6 g 3-arm-p(DEGMA)3-Cl3, 1 ml
anisole, 5 mg CuCl were taken and the monomer DMAEMA
(1 ml) was injected and nitrogen purging was continued for
20 min. The reaction tube was closed with a rubber septum and
10 mL HMTETA was injected, stirred for 10 h at 5ꢁ0 ꢁC, and was
precipitated into excess petroleum ether (60–80 C). The poly-
mer was separated, re-precipitated from THF with excess
petroleum ether. The trace amount of monomer entrapped
within the polymer was removed by repeated dissolution and re-
precipitation. The polymer was nally dissolved in THF, passed
through silica column, followed by solvent evaporation to
obtain copper free 3-arm-p(DEGMA34-B-DMAEMA21) (Scheme 1,
2.3. Characterization techniques of the polymers
2.3.1. 1H NMR spectra. The 1H NMR and 13C NMR spectra
were recorded in CDCl3 with a 500 MHz Bruker NMR
spectrometer.
2.3.2. Gel permeation chromatography. The gel perme-
ation chromatography (GPC) experiments were performed on
a Waters instrument equipped with Waters 1515 pump, Waters
2414 differential refractive index detector and three m-styragel
column using poly(methyl methacrylate) (pMMA) as the stan-
dard and dimethylformamide (DMF) as the solvent.
2.3.3. TEM microscopy. The morphology of the P3 copol-
ymer samples (with and without ANS) were monitored by
transmission electron microscopy (TEM JEOL, 2010EX) oper-
ated at an acceleration voltage of 200 kV and tted with a CCD
camera. A specimen for TEM study was made by spreading
a small drop of the above transparent solution on a carbon-
coated copper grid and allowing the drops to dry in air at low
ꢁ
ꢁ
temperature (5 C) and nally in vacuum at 30 C for about 1
day. Another TEM grid is also prepared by drying the TEM grids
ꢁ
at high temperature (above LCST, 40 C).
2.4. Instrumental techniques
2.4.1. Dynamic light scattering (DLS). The DLS experi-
yield-40%). From the NMR data the mole ratio of DEGMA (62%) ments were performed using a Malvern instrument with a He–
and DMAEMA (38%) are observed in the 3-arm star block Ne laser at an angle 173ꢁ. For temperature dependent experi-
copolymer.
ment, the aqueous solution of the polymer was heated from low
2.2.5. Synthesis of 3-arm random poly[di(ethylene glycol) to higher temperature. Aer reaching the desired temperature
methyl ether methacrylate]-co-poly[2-(dimethylamino) ethyl the sample was allowed to equilibrate at that temperature for
methacrylate] copolymer {3-arm-p(DEGMAi-R-DMAEMAj)}. 30 5 min before both the heating and cooling experiments. The
mg TIBB, 3 ml anisole and 10 mg CuCl were taken in nitrogen zeta potential value of the aqueous solution of the copolymers
purged similar reaction vessel. The monomers DEGMA and [1 mg mlꢂ1; 0.1% (w/v)] were measured using the same DLS
ꢁ
DMAEMA were next injected and nitrogen purging was instrument at 25 C.
continued for 25 min. For the synthesis of different random
copolymers 3-arm-p(DEGMA33-R-DMAEMA23 (P2), 3-arm- photoluminescence (PL) spectra of all the copolymer solutions
2.4.2. Fluorescence study. The temperature dependent
)
p(DEGMA40-R-DMAEMA18) (P3) and 3-arm-p(DEGMA16-R- (0.4% w/v) containing 17 mM ANS probe were recorded using
DMAEMA35) (P4), the two monomers DEGMA and DMAEMA a Fluoromax-3 (Horiva Jovin Yvon) instrument. The sample was
were taken in 3 : 2, 2 : 1 & 1 : 2 (v/v) ratios (mole ratios are taken in a quartz cell with 1 cm path length and was excited at
60 : 40; 66 : 33; 33 : 66) respectively. The reaction vessel was 370 nm. Emission scans were recorded from 390 to 620 nm
then closed with a rubber septum and 40 mL HMTETA was using a slit width of 3 nm with an increment of 1 nm wavelength
injected and was sealed with para lm followed by stirring for having an integration time of 0.1 s. Samples were permitted to
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Scheme 1 Synthetic procedure adopted for the preparation of 3-arm-p(DEGMA)3-Cl3 macro-initiator and P1 copolymer (B means block copolymer).
stand for 10 minute at each temperature for equilibrating the CHO-K1 cells were seeded in 24-well plate in presence of 500 mL
temperature.
Dulbecco's modied Eagle medium (DMEM) medium supple-
mented with 10% fetal bovine serum (FBS) and 1% penicillin/
streptomycin at 37 ꢁC and 5% CO2. Aer overnight growth,
the cells were incubated with different polymer at nal
concentration of 200 mg mlꢂ1 for 24 hours. Then cells were
washed with PBS buffer solution to remove the unbound poly-
mer micelle. Then fresh 500 mL of supplemented DMEM
medium was added. The cells morphology was observed by
using Olympus IX81 optical microscopy.
2.5. Measurement of pKa values
pKa values of the different copolymers were measured by acid–
base titration method from 0.3% (w/v) solutions of the polymers
using pH meter (Oakton).
2.6. Cytotoxicity
The effect of different copolymer on normal cells morphology
The cell viability of different copolymer against normal CHO-
was observed by optical microscope. Chinese hamster ovary K1 cells was evaluated by conventional 3-(4,5-dimethylthiazol-2-
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yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The cells present in the polymer. Signals at the d values 0.8–1.1 ppm and
were seeded in 24-well plates at high density in 500 mL of DMEM 1.7–2.0 ppm are due to ‘t’ and ‘r’ protons, respectively and
medium supplanted with 10% FBS and 1% penicillin/ splitting is observed for different tacticities of the methacrylate
streptomycin at 37 ꢁC and 5% CO2. Aer overnight of cell polymer chains. Protons ‘j’, ‘i’, and ‘h’ present in pDMAEMA
attachment, the cells were incubated with different copolymer units resonate at d values 2.25, 2.6, and 4.05 ppm, respectively
at nal concentrations (10–200 mg mlꢂ1 and upto 1mg mlꢂ1 only and the signals at 3.35, (3.55–3.75) and 4.15 ppm corresponds to
for P3 copolymer) for 24 hours. Then cells were washed with PBS ‘g’, ‘f’, and ‘e’ protons, respectively, present in the pDEGMA
buffer solution and fresh 500 mL of supplanted DMEM medium units. It is important to note that signal at about d values of 7
were added to each well. Aer that 50 mL of aqueous solution of ppm corresponds to three aromatic ring protons present in the
MTT (5 mg mlꢂ1) were added and allowed to stand for 4 hours. TIBB initiator of the polymer and it is also found in the 1H NMR
The violet formazan was dissolved in 500 mL of sodium dodecyl spectrum of TIBB (Fig. S1†). Similar 1H NMR spectrum is
sulfate solution in water/DMF mixture. Absorbance of formazan observed for P1 block copolymer (Fig. S2†). The NMR spectrum
solution was measured at 570 nm using a BioTek SynergyMx of P5 copolymer is presented in Fig. 2. Mole ratios of DEGMA
microplate reader.
and DMAEMA monomer units in different copolymeric systems
are calculated from the signal area ratios of proton ‘g’ and ‘j’
with respect to ‘b’ and ‘b0’ protons, respectively.
3. Results and discussion
Fig. S3a† shows gel permeation chromatography (GPC) trace
corresponding to 3-arm star shaped random copolymer (P2) of
pDEGMA and pDMAEMA having 59 and 41 mol% of their cor-
responding monomeric units (Table 1). GPC trace in Fig. S3a†
shows a relatively sharp signal but having a small shoulder at
the high molecular end of the distribution curve. However, no
trailing is observed at the lower molar mass side of the GPC
trace, which indicates that the bimolecular termination occurs
at the advanced stages of the reaction leading to the generation
of high molar mass dead polymers. Fig. S3b† shows the overlay
of GPC traces corresponding to the macro initiator 3-arm-
p(DEGMA)3-Cl3 and 3-arm block copolymer (P1). In Fig. S3b†
prominent bimodality is noticed in case of 3-arm-p(DEGMA)3-
Cl3 macro-initiator and also in the P1 block copolymer. Devel-
opment of the shoulder at high molar mass side of the GPC
traces during P2 synthesis may be as a result of inefficient
deactivation from Cu/HMTETA catalyst.33 Similar bimodality is
also observed during homo polymerization of DEGMA with
bipyridyl based copper catalyst in anisole medium. This may be
further attributed to the lesser activity of Cu/bpy complex in
polar aprotic medium than in polar protic medium. Also some
star–star coupling product is inevitable during this type of
radical polymerization.42
3.1. Synthesis and characterization
Thermo and pH responsive linear, 3-arm star shaped block and
random co-polymeric (pDEGMA-B/R-pDMAEMA) systems are
synthesized by ATRP technique under amiable conditions.
Synthesis of linear and star shaped pDEGMA-co-pDMAEMA
[p(DEGMA-R/B-DMAEMA)] polymer systems is conducted using
EGBIB and 1,3,5-tri(20-bromo-isobutyryloxy) benzene (TIBB) as
initiators, respectively. For the synthesis of three arm star sha-
ped P1 block copolymers, at rst 3-arm-p(DEGMA)3-Cl3 is
synthesized with CuCl/DMDP catalyst at 55 ꢁC and in subse-
quent step, polymerization of DMAEMA is made using the
above macro-initiator with CuCl/HMTETA catalyst/ligand
ꢁ
system at 50 C in anisole (Scheme 1) and the macroinitiator
3-arm-p(DEGMA)3-Cl3 is produced instead of 3-arm-p(DEGMA)3-
Br3 due to halogen exchange.41 Also the linear copolymer
L-p(DEGMA32-R-DMAEMA23) (P5) is synthesized by ATRP using
ꢁ
CuCl/HMTETA (catalyst/ligand) system at 25 C (Scheme 2).
1
The H NMR spectrum of P2 copolymer (Fig. 1) exhibits all
the characteristic signals of pDEGMA and pDMAEMA segments
3.2. Phase separation
In Fig. 3 the plots of Z-average sizes of the polymers P2, P3 and P5
obtained from DLS study at different pH of the medium are
presented. It is evident from the plots that all the polymers
exhibit small size (ꢀ30 nm) at lower temperatures probably due
to the micelle formation as pDEGMA segments are hydrophobic
and pDMAEMA is hydrophilic in nature producing a self orga-
nized micellar structure with former segments at the core. But
aer attaining a certain temperature increase of size occurs
particularly for pH 9.2. The onset temperature of increase of size
may be called as LCST-type phase transition temperature of the
respective polymers and it arises due to breaking of H-bonding
interactions between pDEGMA segments of the copolymers
with the water molecules causing aggregation of the co-polymer
chains. But pDMAEMA chains (LCST ꢀ50 ꢁC) though tend to
remain soluble become entrapped within the aggregate of
Scheme 2 Synthetic procedure adopted for the preparation of the
linear P5 copolymer (R means random copolymer).
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Fig. 1 1H NMR spectrum of P2 copolymer in CDCl3 along with their peak assignments.
Fig. 2 1H NMR spectrum of P5 copolymer in CDCl3 along with their peak assignments.
pDEGMA part. This causes an increase of the Z-average size with attributed to the solvated nature of the copolymers in these pH
rise of temperature above the LCST. However, this type of values because the protonation of basic –NMe2 group (pKa
increase of size is not observed at pH 7 and 4 and it has been ꢀ7)31,43 causes increased solubility (Scheme 3b). The pKa values
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Table 1 Compositions of the various constituents present in the synthesized star and linear copolymers
Monomer feed
ratio used
Molar ratio obtained
from 1H NMR
Molar mass
DEGMA from
(mol%) GPC (Da)
Dispersity 1H NMR (Da) per arm
Molar
mass from
No. of DEGMA &
DMAEMA unit
DMAEMA DEGMA DMAEMA
Polymer
Entry (mol%)
(mol%) (mol%)
3-arm-p(DEGMA34-B-DMAEMA21
)
P1
P2
P3
P4
P5
38
62
59
63
30
61
25 500
28 300
26 700
21 900
15 100
1.19
1.26
1.23
1.25
1.28
30 000
30 100
31 300
26 500
19 700
34 & 21
33 & 23
40 & 18
16 & 35
32 & 23
3-arm-p(DEGMA33-R-DMAEMA23
3-arm-p(DEGMA40-R-DMAEMA18
3-arm-p(DEGMA16-R-DMAEMA35
)
)
)
40
33
66
40
60
66
33
60
41
36
69
39
L-p(DEGMA32-R-DMAEMA23
)
the onset temperatures of P2 and P5 systems, having similar
chain composition but differing in architecture, indicates that
the 3-arm random copolymer has lower LCST phase transition
than that in the linear copolymer.
Fig. 4 shows the DLS study of 3-arm random copolymer (P2)
in aqueous medium in the presence of ANS molecule with
variation of temperature in different pH values. In Fig. 4a it is
observed that below 26 ꢁC relatively bigger sized particles (ꢀ400
nm) than that in pure P2 is noticed at pH-9.2. No denite reason
for the large size of the copolymer – ANS system below 26 ꢁC is
known and it may be attributed to the hydrophobic ANS mole-
cules, some of which may remain entrapped at the pDEGMA
core of the copolymer micelle. Even the possibility of increasing
multiple chain aggregation of the copolymer via supramolec-
ular interaction with the ANS molecule is inevitable. Upon
increasing temperature, above 26 ꢁC, the particles size of the
polymer shows a similar signicant hike which is attributed to
the fact that at this pH above 26 ꢁC LCST of pDEGMA segment is
reached. Hence, pDEGMA chains become insoluble in water
producing large aggregated structures (Scheme 3a). At lower pH
(4 & 7) on increase of temperature no sharp increase of hydro-
dynamic size is noticed (Fig. 3a) due to the solvated nature of
pDMAEMA chains as discussed above.
Fig. 3 Z average size vs. temperature plot of P2, P3, and P5 copolymer
in aqueous medium (0.4% w/v) at different pH.
Fig. 4b shows a thermo-reversible nature of the formation
and dissociation of the aggregates at pH 9.2 as similar variation
of particle size is noticed during both heating and cooling
processes. The same behaviour is observed during DLS analysis
of P1 system (Fig. S4†) which also has almost similar mole ratio
(Table 1) of DEGMA and DMAEMA units of P2. A keen obser-
vation of the phase transition data indicates that P1 solution has
2 ꢁC lower transition temperature than that of P2 solution at pH
9.2. It alerts us the architectural dependency of hydrophobic/
hydrophilic moiety among the random and block structures
of the 3-arm star copolymer. The block structure favour aggre-
gation at lower temperature than the random one causing
a lowering of LCST. This is because the randomly distributed
segments of DEGMA remain soluble due to the better inuence
of statistically distributed DMAEMA segments. But in case of P1
Scheme 3 (a) Schematic illustration of change in hydrated structure of
P
2 copolymer above LCST of pDEGMA chain at pH 9.2. (b) Schematic
illustration of change in hydrated structure of P2 copolymer above
LCST at low pH (4 & 7). [Sky colour spheres stand for water molecules,
green hairs correspond to pDEGMA chain, violet hairs stand for
pDMAEMA chain and red spheres represents ANS molecule. The green
shell surrounding the yellow sphere represents the collapsed structure
of pDEGMA chain above its LCST].
determined from tritrimetric method of the copolymers P1, P3, block copolymer this effect is less and pDEGMA block gets
P4, and P5 are 6.29, 6.30, 6.31 and 6.40, respectively. At lower pH aggregated more easily showing lower LCST.
the –NMe2 groups become protonated and columbic repulsion
Fig. 5a shows the TEM images of P3 dried from its aqueous
between the –NMe2H+ groups prevent the precipitation of solution at 5 ꢁC which is below the LCST of pDEGMA at pH 9.2.
pDEGMA chain even at higher temperatures. A comparison of As expected, below the LCST of pDEGMA, the TEM image
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Fig. 4 (a) Z average size vs. temperature plot of P2 copolymer containing 17 mM ANS at different pH obtained from DLS study in aqueous solution
(0.4% w/v) and (b) Z average size vs. temperature plot of P2 (0.4% w/v) containing 17 mM ANS at pH-9.2 with increasing and decreasing of
temperature.
segments facilitate to entrap water molecules at the centre. This
results in the formation vesicular aggregates showing a very
large dimension above the LCST.
The P3 solution has self-assembled pDEGMA chains at the
core and hydrophilic corona having dispersed pDMAEMA
chains producing micellar structure in the solution. This may
be evidenced from the observation of increasing uorescence
intensity of the uorescent probe (ANS) which itself exhibits
almost a non-detectable uorescence in aqueous solution but in
the P3 solution it exhibits ꢀ3 times higher uorescence inten-
sity (Fig. S5†) for its partial encapsulation at the hydrophobic
core of the micelle. The critical aggregation concentration (CAC)
of P3 and P5 with 17 mM ANS solution can also be obtained from
this method. Fig. 6a and b shows that 3-arm star polymeric
system (P3) in aqueous solution has shown a CAC value of 0.2
mg mlꢂ1, whereas the CAC value of linear P5 polymer is 0.53 mg
mꢂ1 Lꢂ1 at 45 ꢁC. This result indicates that the P3 has lower CAC
value than that of P5 both having comparable mole ratios of the
co-monomers. This gives us indirect evidence that P3 produced
is a three arm star copolymers which usually exhibit lower CAC
values due to lower viscosity than that of the linear copolymer.38
Fig. 5 TEM images of P3 copolymer at (a) low temperature (below
LCST) without ANS (b) low temperature (below LCST) with ANS (c)
Fig. S6a† shows the increase of uorescence intensity of
aqueous solution of 17 mM ANS solution taken in the pres-
ence of 0.4% (w/v) P2 solution with increasing temperature.
higher temperature (above LCST) with ANS.
(Fig. 5a) exhibit homogenously distributed micellar This increase in uorescence intensity of ANS at and above 26
morphology. The TEM grid produced from the same P3 sample ꢁC indicates greater degree of solvation of ANS from the water
with 17 mM ANS probe exhibit bigger size (ꢀ400 nm) and from molecules within the hydrophobic core of the polymer
the inset picture small black spots, possibly of aggregated ANS aggregate. Therefore, it may be argued that above LCST of
particles, are noticed inside the structure (Fig. 5b, inset). The pDEGMA, this aggregated structure may act as better scaf-
ꢀ10 times increase of size in the presence of ANS is interesting folds for encapsulating hydrophobic molecules like dyes,
and may be attributed to the ANS induced aggregation of P3 drugs and other organic pollutants. The uorescence inten-
chains through noncovalent interaction. But TEM grid dried at sities for both heating and cooling process of P2 (Fig. 7a)
40 ꢁC (above the LCST) exhibits a very large aggregated structure when plotted with temperature exhibits high degree of
(ꢀ2 mm) produced from the LCST type phase separation of self reversibility. The reversibility also indicates that such release
organized pDEGMA chains inducing the majority of pDMAEMA and recapturing of hydrophobic ANS molecule at the core by
chains to precipitate due to entrapment. The larger aggregates pDEGMA units would occur very efficiently with decrease and
are produced from the aggregation of small aggregates and increase of temperature, respectively. Another hydrophobic
a careful observation indicates a vesicular like structure (inset dye Nile red also exhibits similar increase of uorescence
Fig. 5c). Possibly above LCST during the collapsing of micellar intensity due to encapsulation of the dye within the hydro-
aggregates the pDMAEMA segments of the three arm structure phobic core of the P3 due to increase of temperature
of the polymer do not fully collapse and the outer remaining (Fig. S6b† and 7b).
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Fig. 6 PL intensity vs. log(C) plot for (a) P3 and (b) P5 copolymers where C indicates concentration of polymer solution at 45 ꢁC.
similar conditions they remain in well segregated state. It is
interesting to note that during cooling below the LCST the
uorescence intensity of ANS crosses that of the heating curve
and exhibits much lower value than that at the onset of heating.
A probable reason is that at the onset of heating the polymer
chains remain somewhat H-bonded with each other and with
water molecules, but during instant cooling due to the lack of
sufficient time for equilibration the inter-chain H-bonding is
much lesser, causing the ANS molecules more free exhibiting
lower uorescence intensity.27 Fig. S7a† shows that in case of P1
ꢁ
copolymer the uorescence hike has started from 22 C but in
ꢁ
case of DLS study the increase of particle size occurs at 24 C.
Both the increase of particle size and increase of uorescence
intensities of the ANS probe are indicative of starting of
molecular aggregation. However, the light scattering technique
requires some critical degree of aggregation prior being detec-
ted, hence DLS result shows LCST phase transition at higher
temperature than that found in uorescence study arising from
the decrease of local polarity around ANS molecules as soon as
pDEGMA chains start getting dehydrated with increase in
temperature.
Fig. 7 (a) PL intensity vs. temperature plot of P2 copolymer solution
(0.4% w/v) containing 17 mM ANS with increasing temperature at pH-
9.2. (b) PL intensity vs. temperature plot of P3 copolymer solution (0.4%
3.3. Multiple aggregation & release of hydrophobic molecule
The DLS data of 3-arm-p(DEGMAi-R-DMAEMAj) having 17 mM
ANS solution exhibit the phenomenon of multiple aggregation
with variation of temperature and pH (Fig. 8). The P2 system
shows increase in particle size due to aggregation at 27 ꢁC when
w/v) containing 17 mM Nile red at pH-9.2 with increasing temperature.
Fig. S7a and b† exhibits the uorescence data with temper- the medium pH is 9.2 (Fig. 8a). At this condition when 33 mL of 1
ature for both heating and cooling processes of aqueous solu- (M) HCl solution is added, suddenly the pH drops to 8.5 and
tion of P1 copolymer with same quantity of ANS probe. immediate dissociation of the micelles occurs causing the
ꢁ
Evidently, in this case the reversibility is relatively poor in spite particle size to drop to a lower value similar to that at 24 C of
of having almost similar mole ratio of pDEGMA and pDMAEMA pH 9.2.
units with P2. This may be due to the well segregated nature of
At this pH value of 8.5, when the system is further heated
pDEGMA and pDMAEMA in this block copolymer than their aggregate formation again starts and maximum increase of size
random counterpart. The presence of randomly distributed occurs at about 34 ꢁC. This once more changes by lowering the
hydrophilic pDMAEMA segments helps the pDEGMA chains to pH of the medium to 7.5 by the addition of 20 mL 1 (M) HCl and
attain the reversibility better than that of the block co-polymer it returns almost to the molecularly dissolved state of the
in encapsulating and releasing of ANS probe. This is because polymer. On further heating, this system again exhibits aggre-
ꢁ
the randomly distributed pDMAEMA chains within pDEGMA gation showing a maximum size at 43 C. Addition of 10 mL 1
domains entrap some water molecules (being associated with (M) HCl decreases the medium pH from 7.5 to 6.5 and the
pDMAEMA chains) within the pDEGMA domains above its aggregates further dissociate. Evidently at a particular pH value,
LCST. It is unlike the block copolymer systems where under the incidence of aggregate formation results from the
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Fig. 8 (a) Z average size vs. temperature plot of aqueous solution of P2 (b) P3 (c) P5 copolymers with 17 mM ANS solution for variation of pH and (d)
phase transition temperature (TPL) vs. pH plots of different 3-arm random copolymers.
development of hydrophobicity with increase of temperature of the 3-arm random copolymers with pH are presented and it is
causing aggregation of pDEGMA segments. At pH 6.5, proton- evident that the TPL of all the copolymers decreases almost
ation occurs at the basic –NMe2 groups developing poly-cations linearly with increase of pH of the medium. It is also important
and therefore at that particular temperature precipitation of the to note that for the 3-arm random copolymer at the same pH the
pDEGMA chains becomes restricted due to the repulsion TPL decreases with increase of DEGMA concentration for the
between the –NMe2H+ groups followed by its solvation and the increased hydrophobicity.
increased solubility of the polymer causes ‘aggregate-burst’.
Fig. 9 shows the variation in uorescence intensity of ANS in
Fig. 8a also shows an increase of particle size due to increase in P2 copolymer solution under varied temperature and pH
temperature or decrease in particle size due to pH lowering. It conditions. At pH 9.2 when the temperature of the system is
occurs almost vertically with respect to the temperature axis raised, maximum uorescence intensity signifying maximum
indicating that the process of aggregation or dissociation is amount of ANS encapsulation occurs at about 30 ꢁC and as soon
quite sharp and also very much reversible. Fig. 8b shows the as pH of the system is lowered to 8.5 by addition of HCl, uo-
DLS analysis of P3 system which is slightly richer in pDEGMA rescence intensity gets reduced to the initial value of molecu-
than the previous system P2. This system also shows similar larly dissolved state. This result signies the release of ANS
event of multiple aggregation and most important is that it molecules from the hydrophobic core of the aggregate following
shows aggregate formation in the physiological range of pH-7.5 a ‘aggregate-burst’.
ꢁ
and temperature ꢀ37 C, which may be very much interesting
with respect to its application in biological systems. Fig. S8†
shows a similar study with P4 copolymer, a system which is rich
in pDMAEMA. A closer look to the DLS analysis of this system
reveals that the onset of phase transition temperature of LCST
type (TPL) of the copolymer at a given pH gradually increases
probably due to the increasing proportion of more hydrophilic
pDMAEMA segment. At pH 9.ꢁ2, P2, P3 and P4 copolymer systems
exhibit TPL at 26, 22 and 30 C, respectively. Fig. 8c shows the
aggregation and dissociation transitions of the linear P5 system.
This system also has comparable mole ratios of DEGMA and
DMAEMA units as in P1 and P2 copolymers. Here also three
stages of aggregation and dissociation transition are observed
ꢁ
at the same temperature range (25–55 C) and pH range (9.2–
Fig. 9 Fluorescence intensity vs. temperature plot of aqueous solution
6.5). In Fig. 8d the variation of the transition temperature (TPL
)
of P2 copolymer containing ANS probe with variation of pH.
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The similar sequence of ANS entrapment within the aggre-
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gate core with increasing the temperature occurs at pH 8.5
showing maximum intensity at 36 ꢁC and the release of ANS
occurs as soon as the pH is lowered to 7.5 by addition of HCl. On
further heating at this pH value, maximum extent of ANS
encapsulation associated with maximum uorescence intensity
is observed at 42 ꢁC. Therefore, analysis of multiple aggregation
of P2 in aqueous solution using the ANS uorescence probe
shows very much similar result as observed during DLS
analysis.
Therefore, the different polymer systems used in this study
may be useful for reversible capturing and release of suitable
hydrophobic dye or drug molecules depending on the pH and
temperature of the system. In this respect P3 would be very
much successful as it shows dye encapsulation at pH 7.5 and at
37 ꢁC both of which are physiologically very much important as
37 ꢁC is normal physiological temperature and blood pH is
about 7.4. Therefore, in case of entrapment of any suitable
hydrophobic dye/drug molecule, this polymer system should
exhibit maximum loading in the range of 37 ꢁC at pH 7.5. This
reversible temperature/pH triggered uptake and release of dye
molecules might be useful for delivery in biological systems
particularly at lower pH.
3.4. Biocompatibility and cell viability
Fig. 10 In vitro cytotoxicity of different copolymers in normal CHO-K1
cells after 24 hours incubation at different concentrations.
All the copolymers P1, P2, P3 and P5 are temperature sensitive
and pH responsive, which are biologically very important
parameters to oblige with different biological applications. For
this purpose we have made cytotoxicity analysis of all four
different types of copolymers separately (Fig. 10a and b). It is
apparent from the gure that at a very low concentration (10–25
mg mlꢂ1) of substrates both P2 and P5 exhibit very good cell
viability (>90%). On the other hand the 3-arm block copolymer
(P1) (Fig. 10a) has some toxic behaviour (cell viability ꢀ60%)
because, the block copolymer have only DMAEMA units at their
periphery.44 With increasing the concentration of copolymers
the cell viability gradually decreases. It is important to note that
the cell viability of both the random copolymer (linear and star),
however, remains constant at ꢀ78–82% even at a concentration
of 200 mg mlꢂ1 although at this concentration the cell viability of
star block copolymer reduces to the value ꢀ38%. The highly
positive surface charged (zeta potential +28.1 ꢄ 5.8 mV) star
block copolymer (P1) shows high cytotoxicity due to signicant
amount of nonspecic interaction with cells causing increased
cellular uptake and hinder cell function compare with the lower
positively surface charged linear/star random copolymer (zeta
potential +11 ꢄ 6.8 mV for linear & +13 ꢄ 7.2 mV for star
random) (Fig. S9†).45 For the polymer P3 which is practically
important as a biological material, the cytotoxicity has been
tested and this data exhibits 70% cell viability even at 500 mg
mlꢂ1 concentration (Fig. 10b). In addition, Fig. 11 shows the
morphologies of normal CHO-K1 cells in absence and presence
of various copolymers to conrm the biocompatibility. The
morphology of the cells aer 24 hours of incubation at 37 ꢁC in
Fig. 11 Differential interference contrast (DIC) images of CHO-K1 cells (a) in
absence of copolymer and in presence of (b) P5, (c) P2 and (d) P1 copolymer
after 24 hour incubation in the copolymer solution (200 mg mlꢂ1).
good biocompatibility of the polymer. But, the change of
morphology of the cells in presence of the star block copolymer
(P1) indicates its prominent toxic effect.
4. Conclusion
presence of linear and star random copolymers is almost the ATRP technique is used to synthesise stimuli responsive 3-arm
same with the morphology in absence of polymer indicating star random/block and linear random copolymers of DEGMA
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and DMAEMA. DLS study indicates the self-assembly of these
copolymers with increase of temperature at a particular pH due
to LCST type phase separation and it persists even in the pres-
ence of the uorescent ANS molecule which enters into the
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Acknowledgements
We gratefully acknowledge SERB New Delhi (grant no. SB/SI/OC-
11/2013) for nancial support. S. Das, R. Ghosh and P. Das 19 D. Schmaljohann, Adv. Drug Delivery Rev., 2006, 58, 1655–
acknowledges DST “INSPIRE” program and CSIR for providing
1670.
the fellowship. We acknowledge Dr N. R. Jana of CAM, IACS for 20 R. Langer and D. A. Tirrell, Nature, 2004, 428, 487–492.
helping in MTT assay.
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