Glycopolymer-Grafted Nanoparticles: Synthesis Using RAFT Polymerization and Binding Study with Lectin

Article abstract of DOI:10.1021/acs.macromol.7b01265

The weak binding between carbohydrates and proteins is a major constraint toward the development of carbohydrate-based therapeutics. To address this, here we report the synthesis of glycopolymer (GP)-grafted silica nanoparticles (SiNP) by using reversible addition-fragmentation chain transfer (RAFT) polymerization through the grafting-from approach using a multistep process. GP chains of various lengths with controlled molecular weight and narrow polydispersities were grown on the RAFT agent anchored SiNP surface using mannosyloxyethyl methacrylate (MEMA) as a glycomonomer. Spectroscopic (FT-IR, NMR) and thermogravimetric studies confirmed the grafting of poly(MEMA) chains on the SiNP surface and also showed that the dry DMF is a better solvent as compared to water/ethanol mixture for carrying out the MEMA polymerization on SiNP surface. The mean diameter of the dry GP-grafted SiNPs (GP-g-SiNPs) obtained from microscopic studies was in the range 50-60 nm, whereas the hydrodynamic diameter as obtained using light scattering measurements varied between 90 and 165 nm depending on the chain length of poly(MEMA). Hydrolysis of silica cores using aqueous HF enabled characterization of cleaved polymer using GPC, and the obtained unimodal chromatogram and narrow PDI confirmed that the polymerization proceeded through the RAFT mechanism. GP-g-SiNPs displayed stronger binding to the mannose specific lectin, Concanavalin A, owing to the larger positive binding entropic contribution which resulted in an association constant that is 800- and 400-fold stronger than that of monomeric mannose and GP chains, respectively.

Full text of DOI:10.1021/acs.macromol.7b01265

Article
pubs.acs.org/Macromolecules
Glycopolymer-Grafted Nanoparticles: Synthesis Using RAFT
Polymerization and Binding Study with Lectin
S. N. Raju Kutcherlapati, Rambabu Koyilapu, Uma Maheswara Rao Boddu, Debparna Datta,
Ramu Sridhar Perali,* Musti J. Swamy,* and Tushar Jana*
School of Chemistry, University of Hyderabad, Hyderabad, India
S
* Supporting Information
ABSTRACT: The weak binding between carbohydrates and proteins is a major constraint toward the development of
carbohydrate-based therapeutics. To address this, here we report the synthesis of glycopolymer (GP)-grafted silica nanoparticles
(SiNP) by using reversible addition−fragmentation chain transfer (RAFT) polymerization through the grafting-from approach
using a multistep process. GP chains of various lengths with controlled molecular weight and narrow polydispersities were grown
on the RAFT agent anchored SiNP surface using mannosyloxyethyl methacrylate (MEMA) as a glycomonomer. Spectroscopic
(FT-IR, NMR) and thermogravimetric studies confirmed the grafting of poly(MEMA) chains on the SiNP surface and also
showed that the dry DMF is a better solvent as compared to water/ethanol mixture for carrying out the MEMA polymerization
on SiNP surface. The mean diameter of the dry GP-grafted SiNPs (GP-g-SiNPs) obtained from microscopic studies was in the
range 50−60 nm, whereas the hydrodynamic diameter as obtained using light scattering measurements varied between 90 and
165 nm depending on the chain length of poly(MEMA). Hydrolysis of silica cores using aqueous HF enabled characterization of
cleaved polymer using GPC, and the obtained unimodal chromatogram and narrow PDI confirmed that the polymerization
proceeded through the RAFT mechanism. GP-g-SiNPs displayed stronger binding to the mannose specific lectin, Concanavalin
A, owing to the larger positive binding entropic contribution which resulted in an association constant that is 800- and 400-fold
stronger than that of monomeric mannose and GP chains, respectively.
proteins. One can hypothesize that GP chains grafted on the
surface of nanoparticles might be the right choice for easy and
strong conjugation with protein/lectin since a large number of
GP chains on the nanoparticle surface would be available for
binding with protein which will perhaps enhance the binding
capability. However, this objective can only be realized if one
can grow the GP chains on the nanoparticle surface.
Among several nanoparticle surfaces which can be used to
graft GP chain, silica nanoparticles (SiNP) in particular provide
several interesting advantages like biocompatibly, low toxicity,
and readily functionalizable surface when compared with heavy
metals and other types of metallic/semiconducting surfaces.9−12
In the literature, carbohydrate modified nanoparticles (includ-
INTRODUCTION
■
Often, the association between carbohydrates and proteins is
recognized as low-affinity binding, which can be enhanced by
creating multivalent interactions using multiple carbohydrate
ligands. This approach can lead to increase in the interactions
(in terms of binding constant of protein−carbohydrates) by
manifold than that of corresponding monovalent interac-
tion.1−5 A polymeric chain in which carbohydrate groups are
hanging from the main backbone can act as multiple
carbohydrate ligands and be termed as glycopolymer (GP).
The synthesis of glycopolymers (GPs) with different
architecture is an important step toward the understanding of
interactions between GPs and proteins and more importantly
for unraveling the influence of GPs architecture on the binding
with proteins.6−8 Hence, there is a need to develop a versatile
method for the preparation of GPs especially in such a
structural form (architecture) which eases the conjugation with
Received: June 14, 2017
Revised: August 2, 2017
© XXXX American Chemical Society
A
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a
Scheme 1. Synthesis of Mannose-Capped Hydroxyethyl Methacrylate (MEMA)
a
This molecule is used as a monomer to grow the GP chain.
Scheme 2. Synthesis of 4-Cyanopentanoic Acid Dithiobenzoate (CPDB)-Modified Silica Nanoparticles (SiNP-CPDB)
ing SiNPs) have recently attracted much attention for
understanding lectin−carbohydrate interactions and find
applications in a variety of biological processes including
fertilization, cell migration, cancer therapy, and host−pathogen
interactions.7,8,13−24
Grafting of carbohydrates on the surface of SiNPs has been
carried out using a variety of conjugation techniques, including
copper-catalyzed azide−alkyne cycloaddition (CuAAC), amide
coupling, nucleophilic substitution, and photocoupling.13,25
The majority of these methods typically involve laborious
multistep procedures as well as costly and potentially toxic
metal catalysts. Also, these methodologies cannot be used to
graft GP chains since they are synthesized with protected
glycomonomers to avoid unnecessary hydrolysis during
polymerization, and deprotection is carried out after polymer-
ization. Few unprotected glycomonomers have been poly-
merized on the silica surface through surface-initiated polymer-
ization.26−28 The whole process of protection−deprotection on
the SiNP surface can become very tedious, and hence, there is a
need to develop an alternative and novel method for grafting of
GP chains on the SiNP surface. Two methods namely grafting-
to and grafting-from are mostly used for modification of
nanoparticle surfaces with polymers.29−34 In the case of the
grafting-to method, polymer chains are grafted to the
nanoparticle surface whereas the grafting reaction is conducted
by polymerizing monomers from the nanoparticle surface in the
case of the grafting-from method. Both these methods have
certain advantages along with few limitations and have been
used in the literature to produce a thin polymer layer on the
nanoparticle surface. Although the grafting-to method is simple
to be performed, the obtained graft density is fairly low since
the diffusion of polymer chains to the particle surface is
B
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and amine modification of silica are presented in the Supporting
Information. SiNP-NH2 (2.75 g) was dispersed in dry THF (70 mL)
and ultrasonication for 30 min followed by nitrogen purging for 20
min while stirring. To this, activated CPDB (CPDB-NHS, 0.5 g, 1.3
mmol) was added under stirring, and the mixture was stirred for 24 h
at room temperature. After the reaction, the reaction mixture was
precipitated from 4:1 mixture of cyclohexane and diethyl ether (400
mL) and isolated by using centrifugation at 8000 rpm for 20 min. This
purification cycle was repeated three times, and the obtained RAFT-
functionalized silica nanoparticles (SiNP-CPDB) were dried under
vacuum at 50 °C for 48 h. The yield obtained was 2.45 g (89%). The
whole process of SiNP-CPDB synthesis is shown in Scheme 2.
Polymerization of MEMA Using SiNP-CPDB. We have carried
out the polymerizations in two different solvents, namely a mixture of
water:ethanol (7:3) and DMF, to check the suitability of solvent so as
to get a good amount of glycopolymer (GP) loading on the SiNP
surface. We have also executed many control reactions to check the
role of free CPDB (RAFT agent) in the reaction mixture.
Water-Based Reactions. Into a series of Schlenk tubes containing a
7:3 mixture of water and ethanol (1.4 mL), SiNP-CPDB (0.1 g, which
has 7.607 μmol of CPDB), MEMA (calculated amount, we have varied
this amount to obtain different chain lengths), NaHCO3 (2 mg), free
CPDB (2.13 mg, 7.607 μmol), and ACP (0.426 mg, 1.521 μmol) were
added by keeping the ratio of [SiNP-CPDB]:[free CPDB]:[ACP] =
1:1:0.2 constant for all the reactions; only the amount of MEMA was
altered. These reaction vials were then sealed and subjected to three
freeze−pump−thaw cycles followed by sonication for 10 min and
stirred for 16 h in a preheated oil bath at 70 °C. After completion of
the reaction, the reaction mixture was quenched using liquid nitrogen.
The glycopolymer-grafted silica nanoparticles (GP-g-SiNP) were
precipitated from a large excess of hexane and isolated by using
centrifugation at 7000 rpm for 30 min. The precipitate obtained was
redispersed in methanol (40 mL) and centrifuged at 7000 rpm for 30
min. This step was repeated thrice, and the obtained GP-g-SiNP was
dried under vacuum at 70 °C for 48 h. We have altered the amount of
glycomonomer (MEMA) in the reaction medium by keeping all the
other reagents constant so as to obtain different chain length of GP
poly(MEMA) chain on the SiNP surface.
sterically hindered. Therefore, we believe the grafting-from
method to grow the GP chain onto the SiNP surface would be
the better choice for obtaining high graft density of GP chains.
Among the controlled radical polymerizations (CRPs),
reversible addition−fragmentation chain transfer polymer-
ization (RAFT) is highly popular for grafting polymers on
particle surfaces owing to the advantages of being compatible
with a wide range of monomers including functional
monomers, free of transition metal ion contamination and
mild reaction conditions.35−41
With this background as discussed above and to the best of
our knowledge no report has appeared until now on the
grafting of GP chains on the SiNP surface using the grafting-
from RAFT polymerization method which will allow control
over molecular weights, chain length, and grafting density and
hence the morphology of resulting materials, which in turn can
have a significant influence on protein binding. Therefore, in
this work, we report the synthesis of structurally well-defined,
glycopolymer-grafted SiNP (GP-g-SiNP) hybrid materials by
modifying the surface of SiNP with suitable RAFT agent and
then chain extended with glycomonomer to produce GP-
grafted SiNPs. The monomer used in this study is the mannose
derivative (mannosyloxyethyl methacrylate, MEMA) which has
strong lectin binding activity. In addition to synthesis,
characterization, and structural elucidation of GP-g-SiNP, we
have studied the lectin binding of this nanomaterial with
Concanavalin A (Con A) using isothermal titration calorimetry
(ITC).
EXPERIMENTAL SECTION
■
Synthesis of 2-(α-D-Mannosyloxy)ethyl Methacrylate
(MEMA, 5) Monomer. MEMA was synthesized by following the
reaction scheme shown in Scheme 1. Detailed synthetic protocols and
characterization data of compounds 2, 3, and 4 are described in the
Supporting Information. Conversion of compound 4 to the final
monomer MEMA (compound 5) was carried out as described below.
A solution of 2-O-(2′,3′,4′,6′-tetra-O-acetyl-α-D-mannosyl)ethyl meth-
acrylate 4 (11 g, 23.89 mmol) and freshly prepared sodium methoxide
(22 mL, 0.01 M) in anhydrous methanol (110 mL, 0.1 g/mL) were
stirred under a nitrogen atmosphere for 15 min at room temperature.
After this, the reaction mixture was neutralized to pH ∼ 7 with
DOWEX8W50 ion-exchange resin. The solution was filtered, and the
solvent was removed under vacuum. The obtained crude compound
was subjected to silica gel column chromatography (20% MeOH in
chloroform) to give MEMA 5 (5.76 g, 80%) as colorless solid. Rf 0.5
DMF-Based Reactions. The polymerization reactions were also
conducted using the identical protocols as described above in DMF
solvent.
Homopolymerization of MEMA Monomer Using CPDB as
RAFT Agent. In a 10 mL Schlenk tube containing DMF (3 mL),
MEMA (300 mg, 1.0273 mmol), CPDB (4.189 mg, 15 μmol), and
ACP (0.42 mg, 1.5 μmol) were added. The reaction tube was sealed
and subjected to three freeze−thaw cycles followed by sonication for
10 min and stirred for 16 h in a preheated oil bath at 70 °C. The
reaction mixture was subsequently quenched using liquid nitrogen; the
GP was precipitated from a large excess of cold hexane/diethyl ether
mixture and isolated by filtration. This precipitation step was repeated
twice, and the obtained GP was dried under vacuum at 60 °C for 24 h.
Cleaving of the GP Chains from the GP-g-SiNP. The GP was
cleaved from the GP-g-SiNP according to the procedure described
elsewhere.42,43 Briefly, the process is as follows: in a polyethylene tube,
the GP-g-SiNP (30 mg) was dissolved in 6 mL of DMF. Aqueous
hydrofluoric acid (HF, 48 wt %, 0.6 mL) was added to this solution,
and the reaction mixture was stirred at room temperature for 6 h. The
polymer was precipitated by adding the polymer solution into 20-fold
of excess cold hexane/diethyl ether mixture in a polyethylene beaker,
and the precipitate was collected by filtration. The GP obtained was
dried in a vacuum oven at 80 °C for 24 h. The recovered GP chains
were then subjected to molecular weight analysis and protein binding
studies as glycopolymer without any SiNP.
1
(MeOH:chloroform, 2:8). H NMR (500 MHz, MeOD): δ = 6.13
(dd, 1H, J = 1.0 Hz, J = 1.5 Hz), 5.65 (t, 1H, J = 1.5 Hz), 4.83 (d, 1H, J
= 1.5 Hz), 4.35 (ddd, 1H, J = 3.0 Hz, J = 6.0 Hz, J = 12.0 Hz), 4.32
(ddd, 1H, J = 3.0 Hz, J = 6.0 Hz, J = 12.0 Hz), 3.96 (ddd, 1H, J = 3.0
Hz, J = 6.0 Hz, J = 11.5 Hz), 3.81−3.84 (m, 2H), 3.74 (ddd, 1H, J =
2.5 Hz, J = 6.0 Hz, J = 11.5 Hz), 3.73 (t, 1H, J = 5.5 Hz), 3.69 (dd, 1H,
J = 3.5 Hz, J = 10.0 Hz), 3.64 (t, 1H, J = 9.5 Hz), 3.55−3.59 (m, 1H),
1.95 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3): δ = 167.3, 136.2,
125.0, 100.2, 73.3, 71.1, 70.7, 67.0, 64.9, 63.4, 61.4, 17.0. IR (neat): v =
̃
3375, 2920, 1711, 1634, 1453 cm−1. HRMS (ESI): calcd for
C12H20O8NH4 [M + NH4]+ 310.1502; found 310.1494.
1H and 13C NMR spectra of all the compounds (1−5) are presented
in Supporting Information Figures S1−S10.
Synthesis of RAFT-Modified Silica Nanoparticles (SiNP-
CPDB). Grafting of RAFT to the SiNP surface was carried out in
multiple steps as shown in Scheme 2.42 In the first step, synthesized
SiNP was modified with amine functionality (SiNP-NH2) which on
further reaction with activated RAFT agent produced RAFT-modified
SiNP. RAFT agent 4-cyanopentanoic acid dithiobenzoate (CPDB) was
activated using N-hydroxysuccinamide (NHS) to obtain activated
CPDB which is named as CPDB-NHS. The detailed procedure for the
preparation and activation of RAFT agent, synthesis of silica particles,
Characterization Techniques. All the details of various character-
ization techniques which were used to characterize GP-g-SiNP are
presented in the Supporting Information.
Interaction of GP-g-SiNP with Con A: ITC Studies. Con A was
purified from jack beans (Canavalia ensiformis) as described earlier.44,45
A VP-ITC isothermal titration calorimeter from MicroCal (North-
ampton, MA) equipped with a 1.445 mL sample cell was used for all
C
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Figure 1. FT-IR spectra (A) and TGA profiles (B) of bare SiNPs and RAFT-modified SiNPs.
the experiments. Before titrations Con A samples were dialyzed
extensively against 10 mM Tris buffer, pH 7.6, containing 0.15 M
NaCl, 1 mM CaCl2, and 1 mM MnCl2 (TBS) and degassed properly
using a ThemoVac degassing unit. The GP-g-SiNP and the
glycopolymer (GP chains after removal of SiNP by HF treatment as
described in the previous section) solutions were prepared in TBS
buffer. Calorimetric titrations were carried out essentially as described
previously46 with minor modification. Briefly, 7 μL aliquots of 5 mM
glycopolymer solution were added to a 100 μM Con A (monomer
concentration) solution taken in the sample cell at 7 min intervals via a
rotating stirrer−syringe. For investigating GP-g-SiNP-Con A inter-
action, titrations were performed by injecting 8 μL aliquots of 1 mM
Con A (monomer concentration) into 2.5 mM GP-g-SiNP solution
taken in the cell. A blank titration in each case was carried out in order
to estimate heat changes arising due to ligand−buffer interactions, and
the resulting heat changes were subtracted from the changes observed
in the main titration. The titration data could be analyzed satisfactorily
by MicroCal Origin ITC software using “one set of sites” binding
model as described elsewhere,46,47 which yielded the following
thermodynamic parameters associated with the binding reaction:
association constant (Ka), binding stoichiometry (n), and enthalpy of
binding (ΔH). After that free energy (ΔG) and entropy (ΔS) of
binding were calculated according to the following equations:
SiNP-NH2 is 47 2 nm (FESEM and TEM Figure S1I) and 65
nm (light scattering, Figure S12).
It is well-known that dithiobenzoate-based RAFT agents can
control the radical polymerization of a wide range of monomers
including methacrylates, acrylates, styrene, etc.,49,50 to provide
polymers with predictable molecular weight and narrow
polydispersities. The carboxylic functionality of RAFT agent
CPDB can be readily utilized to immobilize CPDB on particle
surface through a covalent amide bond with SiNP-NH2.
Accordingly, in the third step of GP-g-SiNP preparation, N-
hydroxysuccinamide (NHS) activated CPDB (CPDB-NHS), as
shown in Scheme 2, was successfully attached to silica
nanoparticles by reacting with amine-modified SiNP (SiNP-
NH2).42,51 The FESEM and TEM images of CPDB-modified
silica nanoparticles (SiNP-CPDB) are presented in Figure S11
along with bare SiNP and SiNP-NH2. The measured particle
size is 48 3 nm (FESEM and TEM, Figure S11) and 59 nm
(light scattering, Figure S12).
FT-IR and TGA analyses provide clear evidence for the step-
by-step surface modification of SiNPs. Figure 1A compares the
FT-IR spectra of the SiNP-CPDB with the bare SiNP. The
sharp bands at 1645 and 1552 cm−1 are due to CO and N−
H stretching of amide group, respectively. The IR band at 2254
cm−1 is due to the CN stretching frequency of CPDB, and
peaks at 2948 and 1448 cm−1 appear because of the saturated
C−H vibrations of CPDB. This indicates that the dithioben-
zoate group of CPDB RAFT agent is covalently attached to the
surface of the SiNPs. The TGA profiles shown in Figure 1B
display that the final weight loss increases from bare SiNP to
SiNP-NH2 to SiNP-CPDB due to the attachment of organic
groups (propylamine or CPDB) to the surface of the SiNPs.
The amount of RAFT agent (CPDB) which is covalently
attached to the SiNPs was calculated from the TGA plots and
found to be equal to 21.3 mg/g (76 μmol/g).30,52 The
calculated RAFT agent (CPDB) surface density from the
loaded amount of CPDB on the 48 nm SiNP particles is found
to be equal to 1.798 RAFT agents/nm2. 13C NMR (Figure S12)
also gives the clear evidence of the successful surface grafting of
CPDB. The peaks at 168, 144, and 120 ppm are due to the
amide carbonyl carbon, anomeric carbon of phenyl ring in
CPDB, and the cyano group in CPDB, respectively. The peaks
around 124−130 ppm and between 18 and 40 ppm are due to
the phenyl ring carbon atoms and the alkyl carbons from CPDB
RAFT agent and SiNP-NH2, respectively.
ΔG = −RT ln Ka
(1)
(2)
ΔG = ΔH − TΔS
RESULTS AND DISCUSSION
■
Polymerizable SiNP Surface. In this article, we report the
successful grafting of mannose-based glycopolymers (GP) on
silica nanoparticles (SiNP) surface using the grafting-from
RAFT polymerization technique. A multistep process has been
developed to prepare glycopolymer-grafted SiNP (GP-g-SiNP).
First, the well-known Stober method was followed to prepare
̈
monodisperse SiNPs from tetraethyl orthosilicate [Si(OEt)4]
through a hydrolysis and condensation reaction.48 The
measured particle size obtained from FE-SEM and TEM is
47 2 nm in diameter as shown in Figure S11, and the particle
size measured using light scattering (Figure S12) is 54 nm.
These particles are highly monodisperse, and the size of these
nanoparticles can be readily controlled by altering the mole
ratio of the reactants used in the synthesis. In the second step
of GP-g-SiNP preparation, the amine-functionalized silica
nanoparticles (SiNP-NH2) were prepared by refluxing (3-
aminopropyl)triethoxysilane (APTES) with silica nanoparticles
(SiNP) as shown in Scheme 2, and the measured particle size of
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Scheme 3. Synthesis of GP (pMEMA)-Grafted SiNPs (PMEMA-g-SiNP) from RAFT-Modified Polymerizable SiNP
a
Table 1. Summary of Various Physical Data of pMEMA-g-SiNP
sample
identity
amount of GP grafting
targeted
size (TEM)
size (light scattering)
b
c
d
n
d
e
f
sample type
(wt %/GP-g-SiNP)
M
M
̅
PDI
(nm)
(nm)
̅
n
SiNP-CPDB
48
49
50
49
51
53
57
58
105
122
117
91
P1
P2
pMEMA18-g-SiNP
pMEMA24-g-SiNP
pMEMA16-g-SiNP
pMEMA9-g-SiNP
pMEMA32-g-SiNP
pMEMA138-g-SiNP
pMEMA74 (GP)
2.7
4.2
5000
10000
10000
2500
5500
7100
1.22
1.19
1.19
1.17
1.19
1.37
1.06
g
P3
2.8
4900
P4
P5
P6
P7
9.5
2700
19.4
38.1
10000
50000
20000
9400
141
164
40400
21700
a
The polymerization of MEMA was carried out in two different solvents: P1, P2, and P3 are carried out in a water/ethanol (7:3) mixture, 2 mg of
b
c
NaHCO is added to dissolve ACP initiator, and P4−P7 are carried out in dry DMF. Estimated from the TGA analysis. Targeted M is calculated
based on the equation which is generally used for RAFT polymerization. Determined by gel permeation chromatography. With a standard
̅
3
n
d
e
f
deviation of 2 until P4 and 4 for P5 and P6. Measured in water medium, and size polydispersity obtained from light scattering is less than 0.1.
g
Reaction was carried out without the addition of free CPDB.
The particle size and size distribution of SiNPs were
measured in water by light scattering measurements, and the
results obtained are presented in Figure S12B. The mean
diameter of bare SiNP is 53 nm and increased to 64 nm upon
the amine modification which is due to the formation of
hydrophilic amine shell and then decreased to 58 nm after
attaching CPDB on SiNP particle surface. The decrease in the
diameter may be due to a decrease in hydration shell of the
particle after CPDB attachment owing to the hydrophobic
nature of attached CPDB. Hence, the above discussions clearly
prove that the SiNP surface is now ready for further grafting of
polymer (glycopolymer) chain.
Growth of Mannose-Based Glycopolymer Chain on
the SiNP Surface. Polymer chains can be grown on any solid
(particle) surface using the RAFT-mediated grafting-from
approach. There are two methods, namely “arm-first” and
“core-first”, by which polymer chain be grown using the
grafting-from RAFT method.39,53,54 The relatively lower
grafting density on the particle surface is usually achieved in
the arm-first way,39 where the Z-group of the RAFT agent is
attached to the particle surface whereas the R-group of the
RAFT agent is attached to the particle surface in the case of the
“core-first” method. The SiNP-CPDB has an R-group on the
SiNP surface; therefore, any growth of chain in the current
surface is expected to yield higher grafting density of the
polymer chain.
The MEMA monomer was synthesized using the protocol
described in the Experimental Section (Scheme 1) in multiple
steps. All the characterization data are also included in the
1
Experimental Section. The H and 13C NMR spectra of all the
compounds synthesized in each step are presented in
Supporting Information (Figures S1−S10), and the data
confirm the structure of MEMA. The polymerizable SiNP-
CPDB surface is used to grow the poly(mannosyloxyethyl
methacrylate) (pMEMA) chain to produce the GP-g-SiNP
where GP is pMEMA, and hence this point onward we refer to
it as pMEMA-g-SiNP. The reaction scheme for the pMEMA
grafting is pictorially presented in Scheme 3. The polymer-
ization was carried out in two different solvents: water/ethanol
mixture (7:3) and DMF. The polymerization of MEMA in the
presence of SiNP-CPDB was performed using an additional
amount of CPDB as a free chain transfer agent and ACP as a
thermal initiator at 70 °C by keeping the reactant ratio as
[SiNP-CPDB]:[free CPDB]:[ACP] = 1:1:0.2, and this ratio
was kept constant for various sets of the reaction while varying
the amount of MEMA to get different chain lengths of pMEMA
on the SiNP surface. Table 1 summarizes physical details of all
the synthesized polymers. To check the suitability of solvents,
we have synthesized a series of polymers with variable chain
lengths of pMEMA, and this has been achieved by varying the
feed of MEMA monomer in the polymerization. P1, P2, and P3
samples (refer Table 1) were obtained in the water/ethanol
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Figure 2. TGA analysis of pMEMA-g-SiNP samples of different chain lengths obtained from two different solvents: water/ethanol (A) and DMF
(B). Inset shows the difference in weight loss in the case of P2 and P3.
(7:3) mixture, and P4, P5, and P6 (refer Table 1) were
obtained using DMF as the solvent. P7 is also obtained using
DMF solvent without any SiNP core. The number of repeat
units (as presented in the subscript of sample type in Table 1)
is calculated from the molecular weight data which is obtained
from GPC measurement and will be discussed in detail in a
later section.
The relative amount of pMEMA (GP) grafted to SiNPs in
the final products can be estimated from the TGA results.
Figure 2 shows the TGA curves for the SiNP-CPDB and
SiNPs-g-pMEMA-g-SiNP prepared using two different solvents
and pMEMA (homopolymer without grafting on to the SiNP
surface). The decomposition temperature range for all the
samples is 250−600 °C as shown in Figure 2. The pure
pMEMA (without SiNP, P7) decomposed completely as can be
seen from the TGA graphs (Figure 2B). The TGA data clearly
show that with increase in pMEMA chain length (refer to Table
1) the final weight loss increases (P4 to P6 weight loss
increases significantly), which in turn attributes a higher
amount of GP loading on the SiNP surface. Similarly, a
comparison of P2 and P3 GPC data (Table 1) confirms the
higher chain length of P2 than P3, which is also proved from
the higher weight loss in the case of P2 as shown in the inset of
Figure 2A.
The above discussion clearly demonstrated that we were able
to control and alter the pMEMA chain length on the SiNP
surface by choosing the appropriate reaction recipe. However, it
must be noted that the number of pMEMA chains grafted on
the SiNP surface (can be called as grafting density) remains
identical for all the pMEMA-g-SiNP samples since we have
used the same surface-activated SiNPs (SiNP-CPDB) for
carrying out all the polymerization. The growth of pMEMA
chains starts from the CPDB, and therefore the grafting density
can be altered only by changing the amount of CPDB loaded
on the SiNP surface. The variation of grafting density on the
SiNP surface may influence the lectin binding properties of this
class of materials, and further investigation on this aspect is in
progress in our laboratory.
ethanol mixture and DMF) and identical targeted molecular
weight (10 000 g/mol) by keeping all other reaction conditions
unchanged were conducted. The relative amount of GP that is
grafted to SiNP is analyzed from the TGA data (Figure S13).
As seen from the TGA graph (Figure S13), the final weight loss
is significantly higher when the polymer is prepared from DMF
solvent (P5) than in water solvent (P2), and this clearly
indicates the growth of bigger chain in the case of DMF than
aqueous solvent. This may be due to the higher reinitiating
capacity of the formed oligomeric radicals in DMF compared to
water solvent. The calculated loading presented in Table 1 also
proves higher GP loading in the case of P5 than P2. Although
the targeted molecular weight for both P5 and P2 is 10 000 g/
mol, the measured (using GPC) M values of P5 is higher than
̅
n
P2, which also confirms that the DMF is better than the
aqueous solvent. Therefore, we can conclude that DMF is a
better solvent for MEMA polymerization and results in the
grafting of higher amount of pMEMA on the SiNP surface.
Further, we want to check the role of free CPDB (RAFT
agent) in the polymerization of MEMA using the grafting-from
RAFT approach. For this reason, we have conducted a set of
same reactions with identical targeted MW (P2 and P3, details
are presented in Table 1) where we have taken all the
ingredients in the same proportion except that free CPDB is
not added in the case of P3 and to maintain the [M]:[R]:[I]
ratio, we have also halved the monomer as well as initiator in
the case of P3. The TGA data of isolated P2 and P3 samples
(inset of Figure 2A) clearly show less weight loss in the case of
P3 compared to P2. This is attributed to the higher grafting of
pMEMA on SiNP in the presence of free CPDB as also can be
verified from the GP grafting amount (calculated from TGA) as
shown in Table 1. The excess CPDB in the polymerization
mixture helps in exchanging oligomeric radicals, and therefore
the polymerization goes more steady which leads to high GP
grafting. Hence, we have conducted remaining reactions (P4−
P6) with the addition of a similar amount of free CPDB and in
DMF so that we can achieve better GP grafting.
Spectroscopic Evidence of Poly(MEMA) Grafting on
SiNP. The grafting of pMEMA on the particle surfaces is
confirmed by comparing the FT-IR spectra of SiNP-CPDB,
pMEMA-g-SiNP, and pMEMA (Figure 3). The spectrum of
pMEMA-grafted SiNP shows a peak at around 1217 cm−1
which corresponds to C−O bond stretching of the mannose
Role of Solvent and Free RAFT Agent in the
Polymerization. We have carried out the polymerization in
two different solvents to check the role of solvent in the
polymerization of MEMA in the grafting-from RAFT approach.
Similar reactions (P2 and P5) in two different solvents (water/
F
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Figure 3. FT-IR spectra of (a) SiNP-CPDB, (b) pMEMA-g-SiNP, and
(c) pMEMA.
moiety, which is apparent in the pMEMA spectrum at 1210
cm−1. The bands observed at 1379 and 1735 cm−1 are assigned
to the C−O−C group and CO ester double bond of the
mannose sugar, respectively, and these are also seen in
pMEMA. The presence of characteristic bands of SiNP-
CPDB on the pMEMA-g-SiNP spectrum proves that
pMEMA is grafted on the SiNP-CPDB surface.
The grafting of GP on SiNP surface was further confirmed by
13C NMR spectroscopy. Figure 4 represents the 13C NMR
spectra of GP-g-SiNP recorded in D2O solvent. A peak at 170.9
ppm corresponds to the carbonyl carbon (CO) of the ester
from the pMEMA. The peak at 105.8 ppm is due to the
anomeric carbon of mannose in MEMA. Several peaks between
64 and 77 ppm are attributed to the five remaining carbons
from the mannose ring, and the remaining two peaks are due to
the ethylene part of MEMA. Two peaks at 48.6 and 29.2 ppm
are assigned to the polymer backbone, indicating the successful
grafting of pMEMA on the SiNP nanoparticle surface.
Particle Size and Dispersity. Figure 5 and Figure S14
show the FE-SEM and TEM images of pMEMA-g-SiNP
prepared from both DMF and water/ethanol solvents. In an
earlier section, we have observed that the surface of SiNP-
CPDB (Figure S11c) is smooth and without much roughness,
but GP-g-SiNP samples show a rough surface (FESEM images)
and also agglomeration of silica particles is seen as shown in
TEM images (Figure 5, right panel). This observation is
Figure 5. FE-SEM images (left panel) and TEM images (right panel)
of pMEMA-g-SiNP nanoparticles obtained after purification for
different chain lengths prepared using DMF as a solvent. The sample
identification (P4, P5, and P6) can be found in Table 1.
attributed to the presence of pMEMA on the surface of SiNP.
The increase in particle agglomeration with increase in chain
length of the grafted pMEMA nanoparticles when compared
with SiNP-CPDB (Figure.S11c) is very clear in all the cases as
shown in Figure 5 and Figure S14. This kind of crowding of
particles may be due to the interaction between the pMEMA
chain within the particle and with the neighboring particles.
The increase in particle size (Table 1) from 48 2 nm (SiNP-
CPDB) to 57 4 nm (P6) after grafting the pMEMA chains
reveals that the chains are chemically bound to the silica
nanoparticles. FE-SEM and TEM analyses of the SiNP before
and after grafting show that the shape and size distribution of
the particles remain unchanged, indicating that the nano-
particles are stable under the current polymerization conditions.
The particle size and size distribution of pMEMA-g-SiNP
samples were also estimated in water by light scattering
Figure 4. 13C NMR spectrum of pMEMA-g-SiNP. Spectrum was recorded in D2O by dispersing the solid particles of the sample.
G
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Figure 6. Light scattering results of pMEMA-g-SiNP obtained from the polymerization conducted in (A) water/ethanol (7:3) and (B) DMF
solvents.
Figure 7. GPC curves of pMEMA chains cleaved from pMEMA-g-SiNP by using aqueous HF. Polymers obtained using water/ethanol (A) and DMF
(B) as a solvent for the polymerization.
measurements (Figure 6), and the results obtained are listed in
Table 1. Even though the core (SiNP-CPDB) diameters of all
the particles are the same (as prepared from the same sample of
SiNP-CPDB), the hydrodynamic diameter of pMEMA-g-SiNP
has increased by 2−3-fold (Table 1), indicating that the
pMEMA-g-SiNP possesses the graft pMEMA chains which are
highly hydrophilic and are in the swollen state. The size from
light scattering measurement is proportional to the GP chain
length on the particle surface; in other words, the particle size
increases with increasing chain length of pMEMA (Table 1).
The core reason for this increase in the hydrodynamic diameter
is a result of the surface grafting of pMEMA chains on SiNP,
and similar results are obtained by others when particles are
coated with a hydrophilic polymer chain.55 These results
indicate that the grafting of pMEMA on the SiNP surface was
successfully achieved by RAFT-mediated controlled radical
polymerization of MEMA using SiNP-CPDB. A comparison of
size measured from microscopic and light scattering techniques
reveals that pMEMA chain is in the swollen state in the solvent
but in collapsed state in dry conditions. It must be noted from
the data presented in Table 1 that the changes in particle size
are almost insignificant with increasing GP chain length in the
case of data obtained from the microscope technique; however,
it is a very significant increase with increasing GP chain length
when measured in an aqueous medium. This observation once
again reiterates the swelling nature of the hydrophilic pMEMA
chain. It should be mentioned that all the particles are highly
monodisperse in nature as seen from both microscopic and
light scattering data.
Molecular Weight of Grafted pMEMA on SiNP
Surface. The molecular weight and molecular weight
distributions of grafted polymer chains, the pMEMA chains,
were measured using GPC after cleaving the polymer from
SiNP by treating the pMEMA-g-SiNP with aqueous HF
solution. Figure 7 shows the GPC chromatograms of all the
samples which are obtained after cleaving from SiNP surface.
The GPC curves are all symmetric, unimodal, and narrow,
attributing that the polymerization proceeded in a controlled
manner. The number-average molecular weight (M ), poly-
̅
n
dispersity index (PDI), and the number of pMEMA chain
H
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Figure 8. Calorimetric titration of Con A with (A) P7 [pMEMA74 (GP)] and (B) P5 [pMEMA32-g-SiNP] at 25 °C. Upper panel of A displays the
raw ITC data obtained from 40 automatic injections of 7 μL aliquots of 5 mM P7 into 100 μM Con A while upper panel of B displays 35 automatic
injections of 8 μL aliquots of 1 mM Con A into 2.5 mM P5. Lower panels show the integrated data obtained from the raw data shown in the upper
panels.
Table 2. Binding Constants (Ka), Stoichiometry (n), Binding Enthalpy (ΔH), and Entropy (ΔS) for the Interaction of Con A
a
with pMEMA74 (GP) and pMEMA32-g-SiNP Obtained from “One Set of Sites” Binding Model
sample
MeαMan
n
Ka (M−1
)
ΔH (kcal mol−1
)
ΔS (cal mol−1 K−1
)
1.0
(7.0 0.3) × 103
(1.45 0.05) × 104
(5.77 1.99) × 106
−6.3 1.4
−2.7 0.1
−5.2 0.1
−3.6 4.5
9.9 0.2
P7 [pMEMA74 (GP)]
P5 [pMEMA32-g-SiNP]
2.79 0.13
30.19 3.24
13.5 1.1
a
Data for MeαMan binding were taken from ref 56.
(calculated from M ) of all the polymers are tabulated in Table
squares fit of the data to “one set of sites” model are shown as
solid lines. The close agreement of the solid lines with the
experimental data indicates that the binding model satisfactorily
fits the data for pMEMA as well as pMEMA-g-SiNP. All the
experiments were performed in duplicate, and the average
thermodynamic parameters obtained by analyzing the raw data
are listed in Table 2. For comparison, corresponding values for
the binding of methyl-α-D-mannopyranoside (MeαMan), taken
from the literature,56 are also listed in Table 2.
The glycopolymer (pMEMA) P7 with 74 mannose residues
per chain shows a stoichiometry close to three (2.79 0.13)
for its binding to Con A, indicating that approximately one
mannose residue out of three residues binds to a Con A
subunit. The association constant obtained for this binding
(1.45 × 104 M−1) is about 2-fold higher than the value reported
for the Con A−MeαMan interaction.56 On the other hand, the
grafted glycopolymer on SiNP (pMEMA32-g-SiNP, P5) bearing
32 mannose residues exhibits a stoichiometry of 30 for its
binding to Con A. It indicates that only one mannose residue
per chain can bind to Con A, but with a much higher affinity.
The association constant for this interaction is 5.77 × 106 M−1,
which is ∼400-fold higher than that obtained for the interaction
between the glycopolymer (P7) and Con A.
̅
n
1. The narrow PDI values (<1.4) indicated that the
polymerization proceeded in accordance with a controlled/
living radical polymerization mechanism as expected from
RAFT method. The number-average degree of polymerization
(DP) is calculated from M and presented in Table 1 with the
̅
n
sample identification. It must be noted that the M obtained
̅
n
from GPC is in excellent agreement with the M (Table 1)
̅
n
values theoretically calculated from monomer and RAFT agent
concentration in the polymerization feed. This indicates a high
degree of conversion and well-controlled reaction. Another
interesting observation from Table 1 and Figure 7A is the M
̅
n
value of P3. The calculated M (targeted) at P3 is 10 000 g/
̅
n
mol; however, M from GPC is only 4900 g/mol. This is simply
̅
n
because of the absence of free RAFT agent. This once again
reconfirms the necessity of addition of free RAFT agent
(CPDB) in the polymerization feed.
Interaction of pMEMA-g-SiNP and pMEMA with Con
A. Results of calorimetric titrations between the mannose-
specific lectin, Con A, and pMEMA-g-SiNP as well as pMEMA
are shown in Figure 8. The upper panels represent the
exothermic heat of binding at each injection which gradually
decreases until saturation is achieved. The lower panels
represent plots of incremental heat released as a function of
glycopolymer/Con A monomer ratio where nonlinear least-
It is instructive to compare the thermodynamic parameters
obtained for the binding of the glycopolymer P7 and the
I
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glyconanoparticle (glycopolymer grafted on SiNP) P5 with
those reported for the monosaccharide MeαMan. It is
interesting to note that while the enthalpy of binding for the
association of MeαMan is higher than the values obtained for
the binding of P7 and P5, a negative contribution associated
with the binding of MeαMan makes the overall binding weaker.
On the other hand, although the enthalpy of binding associated
with the glycopolymer is less than half of that for the
monosaccharide, a positive entropic contribution (ΔS = 9.9 cal
mol−1 K−1) associated with its binding results in the Ka value
for P7 being 2-fold higher. Finally, for the glyconanoparticle P5
interaction with Con A, although the enthalpy of binding is
marginally smaller than that for MeαMan, due to the relatively
larger positive binding entropy makes its binding ∼800- and
400-fold stronger than MeαMan and P7, respectively. The
positive entropic contribution for the binding of Con A to P5
and P7 may be attributed to the increased statistical probability
of the binding of the lectin to the GP and GP-g-SiNPs due to
the proximal presence of multiple mannose moieties in the
neighborhood.
AUTHOR INFORMATION
■
Corresponding Authors
*Tel (91) 40 23134808; Fax (91) 40 23012460; e-mail
tusharjana@uohyd.ac.in (T.J.).
*E-mail p_ramu_sridhar@uohyd.ac.in (R.S.P.).
*E-mail mjswamy@uohyd.ac.in (M.J.S.).
ORCID
S. N. Raju Kutcherlapati: 0000-0003-3958-9177
Tushar Jana: 0000-0002-3680-2959
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
S.N.R.K. thanks the CSIR-SRF for fellowship. Authors also
thank Mr. Durga Prasad for his cooperation with FESEM and
TEM analysis. We thank Council of Scientific and Industrial
Research (CSIR), Govt. of India, for the research funding
through a research grant [02(0175)/14/EMR-II dated 07-05-
2014]. Financial and infrastructure support from the University
Grants Commission, New Delhi (through the UPE and CAS
programs), and the Department of Science and Technology,
New Delhi (through the PURSE and FIST programs), are
gratefully acknowledged.
CONCLUSION
■
In summary, we have demonstrated a simple method to
covalently graft glycopolymers (carbohydrates) on the surface
of silica nanoparticles. Initially, CPDB RAFT agent anchored
SiNPs were prepared, and then a mannose-containing
glycopolymer chain was grown on the surface of SiNPs using
the grafting-from RAFT polymerization method. The chain
length of the GP on the SiNPs surface has been varied in order
to graft higher amount of GP. The resulting GP-g-SiNP
particles display a narrow size distributions and the size of the
GP chain on the particle surface can be readily controlled by
changing the monomer amount in the polymerization feed.
Molecular weights obtained from GPC analysis after cleaving
the GP from the SiNP confirm the formation of GP-g-SiNP.
The microscopic, spectroscopic, thermal, and light scattering
studies unequivocally confirm the formation of GP-g-SiNP
particles. The mannose-containing GP-g-SiNP shows very
strong binding affinity and selectivity toward lectin Con A.
Currently, we are in the process of extending this method to
graft multiple carbohydrates on the single nanoparticle surface
and study the binding capability with various lectins. The
present methodology delivers a controlled synthesis of well-
defined core−shell nanoparticles with protein binding glyco-
polymer on the surface of the particles which may be useful for
delivering new carbohydrate-based therapeutics.
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