A general method for functionalisation of microgel particles with primary amines using click chemistry

Article abstract of DOI:10.1016/j.polymer.2013.12.022

In this study we introduce a general method for functionalising microgel particles with primary amine groups using a one-step copper catalysed azide-alkyne cycloaddition (CuAAC) reaction. Three different families of microgels containing copolymerised propargyl acrylate (PA) were prepared and then reacted with 2-azido-1-ethylamine (AEA) using CuAAC. The microgels contained poly(ethyl acrylate) (PEA), poly(2-vinylpyridine) (PVP) or poly(N-isopropylacrylamide) (PNP). The functionalisation of the microgels containing PA (i.e., PX-PA) by AEA to give primary amine functionalised particles (PX-PA-AEA) was assessed by elemental analysis and FTIR. The reaction of AEA with PA was quantitative for each of the PX-PA-AEA microgels (X = EA, VP and NP). The PX-PA-AEA systems generally showed larger pH-triggered swelling and zeta potentials than the non-clicked PX-PA particles. The results also showed that PA restricted swelling of the PX-PA and PX-PA-AEA particles by acting as a crosslinker. Of the three microgel systems studied, PVP-PA-AEA had the best combination of high AEA incorporation and pH-triggered swelling.

Full text of DOI:10.1016/j.polymer.2013.12.022

Accepted Manuscript
A general method for functionalisation of microgel particles with primary amines using
click chemistry
Robert Farley, Brian R. Saunders
PII:
S0032-3861(13)01122-1
10.1016/j.polymer.2013.12.022
JPOL 16659
DOI:
Reference:
To appear in: Polymer
Received Date: 27 October 2013
Revised Date: 7 December 2013
Accepted Date: 10 December 2013
Please cite this article as: Farley R, Saunders BR, A general method for functionalisation of microgel
particles with primary amines using click chemistry, Polymer (2014), doi: 10.1016/j.polymer.2013.12.022.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Click
PVP-PA
PVP-PA-EA

ACCEPTED MANUSCRIPT
1
A general method for functionalisation of microgel
particles with primary amines using click chemistry
Robert Farleya and Brian R. Saundersa,*
aBiomaterials Research Group, Manchester Materials Science Centre, School of Materials,
The University of Manchester, Grosvenor Street, Manchester, M1 7HS, U.K.
ABSTRACT
In this study we introduce a general method for functionalising microgel particles with primary
amine groups using a one-step copper catalysed azide-alkyne cycloaddition (CuAAC) reaction.
Three different families of microgels containing copolymerised propargyl acrylate (PA) were
prepared and then reacted with 2-azido-1-ethylamine (AEA) using CuAAC. The microgels
contained poly(ethyl acrylate) (PEA), poly(2-vinylpyridine) (PVP) or poly(N-isopropylacrylamide)
(PNP). The functionalisation of the microgels containing PA (i.e., PX-PA) by AEA to give primary
amine functionalised particles (PX-PA-AEA) was assessed by elemental analysis and FTIR. The
reaction of AEA with PA was quantitative for each of the PX-PA-AEA microgels (X = EA, VP and
NP). The PX-PA-AEA systems generally showed larger pH-triggered swelling and zeta potentials
than the non-clicked PX-PA particles. The results also showed that PA restricted swelling of the
PX-PA and PX-PA-AEA particles by acting as a crosslinker. Of the three microgel systems studied,
PVP-PA-AEA had the best combination of high AEA incorporation and pH-triggered swelling.
* Brian.saunders@manchester.ac.uk, Tel: 44 161 306 5944

ACCEPTED MANUSCRIPT
2
INTRODUCTION
The long-standing interest in microgels in the literature continues to increase1-4. Microgels are
crosslinked polymer colloids that swell in a good solvent or when the pH approaches the pKa of the
polyacid or polybase chains from which they are composed. Microgels can be designed to be
thermally responsive5-9, pH-responsive10, 11 or light responsive12. They have excellent potential for
construction of advanced responsive materials and biomaterials13. However, the preparation of
future advanced materials from microgels requires new methods for functionalising the particles in
a controlled manner. Ideally, functionalisation should occur quantitatively and without sacrificing
colloidal stability of the dispersions. Of special interest is the challenge of preparing microgel
particles containing high concentrations of primary amines. Microgels containing high primary
amine contents would themselves provide useful building blocks for construction of complex
assemblies because of the versatile nature of primary amine reactions14. Unfortunately, microgels
containing high primary amine contents have been challenging to prepare as stable dispersions15.
Copper catalysed azide-alkyne cycloaddition (CuAAC)16, 17 has proven to be a very effective
functionalisation method for polymers and nanoparticles. CuAAC has high efficiency, high
tolerance to functional groups and solvents, requires moderate reaction temperatures and is effective
for a range of interfaces18-21. The reaction yields little or no by-products. We were inspired by the
work of Jung et al.22 which showed that it was possible functionalise linear alkyne-containing
polymers with primary amines using CuAAC. Here, we sought to establish a general method that
would allow a range of alkyne-containing microgels to be functionalised with an azide-
functionalised primary amine in one step. In doing so we test the general principle that CuAAC
reactions provide quantitative reaction for a range of interfaces20. In the present case the interfaces
consist of swollen polymer chains within microgel particles. In addition to demonstrating the
generality of CuAAC reactions for three different microgel types, this study establishes a new
general method for preparing microgels functionalised with primary amines.

ACCEPTED MANUSCRIPT
3
CuAAC has been successfully used to prepare a wide range of materials with precise design
requirements. Budhathoki-Uprety used CuAAC to construct perfectly alternating functional
polymers23. CuAAc has been used to prepare new functional monomers24, functionalise surfaces25
and construct novel delivery systems26. CuAAC is a spring-loaded reaction27 that is well suited to
nanoparticle functionalisation28. Gokmen et al.29 demonstrated post-functionalisation of
polymerised high-internal phase emulsion beads using CuAAC. A key aspect for the success of the
reaction in the later study was good accessibility to the grafted azide groups by the alkyne-based
chromophore. CuAAC has also been studied in the context of core-shell PS particles30. The latter
were prepared with an uncrosslinked poly(methyl methacrylate-co-propargyl acrylate) shell and
functionalised with azide-terminated PEGs. That study noted that high levels of propargyl acrylate
(PA) could cause shell crosslinking. Evanoff et al. prepared poly(PA) particles by emulsion
polymerisation and used CuAAC to functionalise crystalline colloidal arrays31. CuAAC has also
been used to functionalise shell-crosslinked nanoparticles32.
CuAAC offers excellent potential to functionalise microgels by bespoke placement of azide or
alkyne groups. However, there have been very few publications involving CuAAC for microgels. In
a seminal study Meng et al.33 copolymerised PA within the shell of poly(N-isopropylacrylamide-co-
acrylic acid) (PNP-AA) microgels and then functionalised the microgels with azide dyes. The level
of PA incorporated within those microgel particles was low (nominal PA content was 5 mol.%
based on monomer) and the PA was confined to the shells because a seed-feed preparation method
was used. Kupal et al.34 prepared core-shell microgels using assembly of azido and alkyne
derivatives of poly(vinyl alcohol). They used residual surface alkyne groups to graft azido-
hyaluronic acid and showed that the microgels could target adenocarcinoma colon cells. Here, we
employ a PA-based microgel route to prepare three new families of PA-containing microgels. The
PA incorporation was between 6.3 and 28 mol.% and is relatively high compared to other work33.
Scheme 1 shows the PX, PX-PA and PX-PA-AEA systems investigated and their preparation. Here,
X = ethylacrylate (EA), 2-vinylpyridine (VP) or N-isopropylacrylamide (NP). The poly(ethyl

ACCEPTED MANUSCRIPT
4
acrylate)/PA microgel (PEA-PA) was not intrinsically pH-responsive. PEA is hydrophobic and the
o
linear polymer has a glass transition temperature of about -24 C35. The poly(2-vinylpyridine)/PA
(PVP-PA) microgel is pH-responsive. Linear PVP is a weak polybase and is soluble in the
protonated form at pH values greater than about 4.036. The poly(N-isopropylacrylamide)/PA (PNP-
PA) microgel was temperature-responsive and PNP has a lower critical solution temperature of
o
about 32 C37. Those particles were mostly hydrophilic at room temperature. Consequently, the
three families of microgels have very different swelling behaviours. However, they were all
prepared using the same preparation conditions. The crosslinking monomers selected for each
microgel (Scheme 1) are well known to provide efficient crosslinking for each system. For the
CuAAC reactions, a single (common) method was used which incorporated 2-azido-1-ethylamine
(AEA) via the PA groups and formed PX-PA-AEA (Scheme 1).

ACCEPTED MANUSCRIPT
5
Scheme 1. Preparation of primary amine functionalised microgels using CuAAC chemistry. The tubes in (a) reveal that
the turbidity decreased due to microgel particle swelling in DMF. (Colour reproduction on web, B & W in print.)
There are several differences for our method for CuAAC of microgels compared to other reports33.
Firstly, the PX-PA microgels were prepared in a single step. Furthermore, in contrast to the

ACCEPTED MANUSCRIPT
6
approach by Meng et al.33 we used a solvent exchange method (via centrifugation) to transfer the
particles from water to DMF for the CuAAC reaction (Scheme 1). The PX-PA-AEA particles were
then dialysed against water to transfer them back to that phase. This process prevented the particles
becoming dry at any stage and preserved dispersion stability. PEA-PA and PVP-PA as well as all of
the PX-PA-AEA microgels have not been reported before to the best of our knowledge.
The study begins with the characterisation and analysis of the compositions of PX (X = EA, VP and
NP), PX-PA and PX-PA-AEA. We show that incorporation of AEA (and hence primary amines)
into the latter was quantitative and determined only by the extent of PA incorporation. The study
also investigates the pH-responsive behaviours of the PX-PA-AEA microgels as well as the
temperature responsive behaviour of PNP-PA-AEA. It is noted that the PX-PA-AEA systems are
well suited to further functionalisation reactions, as shown for other primary amine-rich
microgels15. This work introduces six microgels (PX-PA and PX-PA-AEA, where X = EA, VP and
NP) that have higher PA contents than other microgels33, were prepared using simple methods and
have versatile functionalisation potential.
EXPERIMENTAL
Reagents
EA (99%), NIPAm (97%) and 2VP (97%) were purchased from Aldrich and inhibitor removed by
passing chloroform solutions through a neutral alumina column followed by removal of the solvent
at reduced pressure. Aliquat 336 and α,α’-azodiisobutyramidine dihydrochloride (AIBA, 97%), 1,
4-butanediol diacrylate (BDD, 90%), N-N’-methylenebis(acrylamide) (BA, 99%) and p-
divinylbenzene (DVB, 85%) were also purchased from Aldrich and used as received.
Polyethyleneglycol methacrylate (PEGMA) with a number-average molecular weight of 2000
g/mol, NaN3 (> 99.5%), and 2-chloroethylamine hydrochloride (99%) were purchased from Aldrich
and used as received. The following linear polymers were purchased from Aldrich and used for
solubility tests: poly(2-vinylpyridine) (PVPL, Mn = 35,000 g/mol, PD = 1.07), poly(ethyl acrylate)

ACCEPTED MANUSCRIPT
7
(PEAL, Mw = 95,000 g/mol) and poly(N-isopropylacrylamide) (PNPL, 19,000 – 30,000 g/mol). For
PEAL, the toluene was removed by rotary evaporation prior to use. High purity water that was
distilled and deionised was used.
Synthesis of 2-azido-1-ethylamine
AEA was synthesised according to the method described by Jung et al.22. 2-chloroethylamine
hydrochloride (5 g, 43 mmol) was ground and dissolved in deionised water (50 ml). Sodium azide
(8 g, 123 mmol) was added and the mixture was heated at 80 oC for 18 hours. The solution was then
made alkaline with potassium hydroxide (15 g), extracted using diethyl ether and dried over
magnesium sulphate. Finally the solution was concentrated using rotary evaporation to give an
1
amber coloured, viscous oil. AEA was characterised using H NMR and FTIR spectroscopy (See
Fig. S1, Supporting information). The integrations for the peaks gave the correct proton ratios and
the FTIR spectra (Fig. 2(a)) showed a strong azide band at 2100 cm-1.
Microgel synthesis
The PX and PX-PA microgels were prepared by a batch emulsion (X = EA and VP) or precipitation
(X = NP) process and followed the method of Dupin et al.10 The masses of the monomers used are
given in Table S1. In all cases the mass of PEGMA, Aliquat and AIBA corresponded to,
respectively, 10, 1.0 and 1.0 wt.% with respect to the total monomer mass. Briefly, Aliquat 336
(1.50 g) and PEGMA (3.0 g of 50 wt.% aqueous solution) were dissolved in water (120 ml) in a
round bottomed flask and the co-monomer mixture added. For example, for preparation of PEA-PA,
EA (10.35 g), PA (4.5 g) and BDD (0.15 g) were used. The solution was degassed with argon and
o
then heated with overhead, mechanical, stirring to 60 C. AIBA (0.15 g) was added to begin the
polymerisation. This was continued for 24h, after which time the dispersion was extensively
dialysed against pH = 3 water. The crosslinking monomers were present at nominal concentration
of 1 mol.% with respect to monomer and are not identified in the nomenclature used. Here, PX (X =
EA, VP or NP) refer to poly(EA-co-BDD), poly(VP-co-DVB) or poly(NP-co-BA) microgels,
respectively. Furthermore, PX-PA (X = EA, VP or NP) refer to poly(EA-co-PA-co-BDD),

ACCEPTED MANUSCRIPT
8
poly(VP-co-PA-co-DVB) or poly(NP-co-PA-co-BA) microgels, respectively.
CuAAC Click reaction of microgels with AEA
The PX-PA microgel (0.2 g of particles) was transferred to DMF from water using centrifugation
and re-dispersion. The number of moles of PA present was typically 0.55 mmole. After degassing
the
dispersion
thoroughly,
AEA
(0.163
g,
1.33
mmole),
N,N,N′,N′′,N′′-
pentamethyldiethylenetriamine (PMDETA, 0.0165 g, 0.095 mmole) and CuBr (0.0136 g, 0.095
mmole) were added with stirring. The dispersion remained colloidally stable and the reaction was
allowed to proceed for 24 h at room temperature. The product was then extensively dialysed against
pH = 3 water to replace the DMF solvent with water. PX-PA-AEA (X = EA, VP or NP) refer to
poly(EA-co-(PA-AEA)-co-BDD), poly(VP-co-(PA-AEA)-co-DVB) or poly(NP-co-(PA-AEA)-co-
BA) microgels, respectively.
Physical Measurements
Elemental analysis (C, H, and N) was performed at the School of Chemistry, University of
Manchester using a Thermo Scientific Flash 2000 Organic Elemental Analyzer with the calibration
standards acetanilide and cyclohexanone 2,4 dinitrophenylhydrazone. The samples were freeze-
dried prior to measurement and the data are from single measurements. We have found elemental
analysis to be a very reproducible technique and the errors estimated from multiple analysis of
related polymers within our group are shown in Table S2. Fourier transform infrared (FTIR)
measurements were conducted on a Nicolet 5700 ATR FTIR instrument. Microgel samples were
freeze-dried to allow analysis, while PA and AEA were measured as pure liquids. The number of
scans per sample was 64 and the resolution 2.0 cm-1. Photon correlation spectroscopy (PCS)
measurements were performed using a BI-9000 Brookhaven light scattering apparatus (Brookhaven
Instrument Cooperation), fitted with a 20 mW HeNe laser and the detector was set at a scattering
angle of 90o. The extent of particle swelling was characterised in terms of the particle volume
swelling ratio, Qp. SEM images were obtained using a Philips XL30 FEG-SEM with an accelerating
voltage of 5kV. Samples were dried onto glass slides and coated with Au/Pd.

ACCEPTED MANUSCRIPT
9
(1)
For equation (1) the parameters dh and dh(coll) are the hydrodynamic diameters measured in the
swollen and collapsed states, respectively. SEM measurements were obtained using a Philips
FEGSEM instrument. A Malvern Zetasizer was used to measure the electrophoretic mobilities of
the particles in the presence of aqueous 0.001 M NaNO3. The mobilities were converted to zeta
potentials (ζ
) using the Smoluchowski equation38.
RESULTS AND DISCUSSION
Microgel Composition
The microgel compositions were characterised using elemental analysis and FTIR. The
experimental ratio of the % nitrogen to % carbon values (RNC(exp)) was a useful gauge for the extent
of PA inclusion (for PX-PA) or AEA functionalisation (for PX-PA-AEA). Because AEA is nitrogen
rich, its inclusion within PX-PA-AEA should increase RNC(exp) for each system (Scheme 1). (All the
elemental data obtained appear in Table S2.) Fig. 1 and Table 1 show the values for RNC(exp) for each
microgel. Compared to the respective RNC(exp) values for PVP and PNP, those for PVP-PA and PNP-
PA decreased because PA does not contain nitrogen. (PEA-PA and PEA monomers did not contain
nitrogen.) Moreover, the RNC(exp) value increased upon AEA functionalisation for each microgel
(Fig. 1). The increase of RNC(exp) was strongest for PEA-PA-AEA. These data qualitatively support
the view that (a) PA was incorporated within the PX-PA microgels as a consequence of emulsion
polymerisation and (b) AEA was subsequently incorporated into the PX-PA microgels (to give PX-
PA-AEA) as a result of CuAAC.

ACCEPTED MANUSCRIPT
10
Fig. 1. Changes in composition for the microgels. The values for RNC(exp) (= %N/%C) decreased or increased,
respectively, for incorporation of PA or AEA for the microgels based on VP and NP. The RNC(exp) values were zero for
PEA and PEA-PA because PEA and PA did not contain nitrogen. (Colour reproduction on web, B & W in print.)
The values for RNC(exp) were used to calculate the compositions of the microgels. A description of
the method used is given in the Supporting information. For PNP-PA and PVP-PA the compositions
were determined from elemental analysis data (Table S2). For the PEA-PA microgel a nominal
composition of PEA-PA0.28 based on the masses of co-monomers used for preparation was assumed
(Table 1). This appears to have been a good assumption 100% inclusion of AEA (i.e., PNP-PA-
AEA0.28) was determined - see Supplementary Information. Furthermore, quantitative inclusion of
AEA into the other two microgels was apparent from the respective analyses. Our analyses did not
consider competing reactions involving PA, such as crosslinking (discussed below). However, the
extent of PA crosslinking seems to have been relatively low compared to the total PA contents
because quantitative AEA inclusion was determined. We note that less than 1 mol.% of crosslinks
can strongly restrict microgel swelling2. PA crosslinking is considered further below. All microgel
compositions are shown in Table 1. We conclude that the AEA incorporation within PX-PA-AEA
particles was determined by the PA content. This is consistent with the high efficiency reported for
CuAAC click reactions20, 21, 27
.

ACCEPTED MANUSCRIPT
11
Table 1. Characterisation data for the microgels.
RNC(exp) dSEM / nm dh / nm dh / nm in Qp
in Qp in water
ζ
/
Code
Compositiona
waterb
DMF
mVc
in DMF
PEA
PEA
0
107 [16]
125 [12]
166 [10]
213 ± 11
157 ± 8
-
102 ± 5
133 ± 7
160 ± 8
9.1 ± 1.4
1.6 ± 0.2
-
1.0 ± 0.15 17.1
1.0 ± 0.15 12.9
1.7 ± 0.3e 23.6
d
PEA-PA
PEA-PA-AEA
0
PEA-PA0.28
0.222
PEA-(PA-EA)0.28
PVP
PVP
0.148
0.121
0.235
372 [24]
339 [11]
403 [24]
664 ± 33 1225 ± 61f 4.5 ± 0.7 28 ± 4
18.8
3.3 ± 0.5g 20.4
PVP-PA
PVP-PA-AEA
512 ± 26
-
537 ± 27f 2.8 ± 0.4
PVP-PA0.18
PVP-(PA-AEA)0.18
1020 ± 51f
-
23 ± 3g
20.6
PNP
PNP
0.178
0.166
0.211
245 [7.5] 417 ± 21
200 [7.6] 505 ± 25
338 ± 17
333 ± 17
375 ± 19
37 ± 6 h
18 ± 3 h
-
20 ± 3h
0.2
6.8
PNP-PA
PNP-PA-AEA
5.1 ± 0.8 h
7.7 ± 1 h
PNP-PA0.063
PNP-(PA-AEA)0.063
249 [7.8]
-
10.8
a
The compositions were calculated from the RNC(exp) values unless otherwise stated – see Supplementary Information for
method used. b Values measured at pH = 4. c Zeta potential measured at pH = 4. d Compositions assumed based on conditions
used for preparation. e Estimated using the PEA-PA dh value for collapsed diameter. f Measured at pH = 2. g Calculated using
h
dh values measured at pH = 2.0 and 12.0. Estimated at pH = 4. For these calculations the hydrodynamic diameter for each
respective microgel measured at 50 oC was used as the collapsed diameter.
The compositions for PX-PA (Table 1) show that PA incorporation decreased in the order PEA >
PVP > PNP. This trend was unexpected. To the best of our knowledge the Alfrey and Price Q and E
values (which are measures of reactivity and polarity, respectively35) for PA have not been reported.
However, butyl acrylate has a similar structure to PA. The Q and E values for butyl acrylate (0.40
and 0.3535) and NP (0.40 and 0.4739) are very similar implying similar reactivity ratios and a
statistical copolymer would be expected if all other factors were equal. It follows that differences in
co-monomer reactivity ratios are not responsible for the low inclusion of PA within the PNP-PA
microgel. A different explanation is required to account for the compositions in Table 1.
Swelling of primary particles by monomer is an important part of incorporation of co-monomers by
emulsion polymerisation40. We tested the solubility of the linear polymers PNPL, PVPL and PEAL
in PA. It was found that PEAL was readily soluble in PA. For PVPL, the polymer also dissolved in
PA, although this was slower than for PEAL. Interestingly, PNPL was insoluble in PA. We propose
that the reason for the low PA incorporation within PNP microgels (and hence compositional
difference) is incompatibility between PNP and PA. PA would not have been able to swell PNP-

ACCEPTED MANUSCRIPT
12
rich particles during growth. This contrasts to PEA where good solubility of PA enhanced swelling
of the PEA particles and incorporation. The less facile dissolution of PVPL in PA is also consistent
with the differences in PA incorporation evident (Table 1).
FTIR spectra for the microgels are shown in Fig. 2. The PEA-PA and PEA-PA-AEA microgels
contained the highest PA and AEA contents based on elemental analysis data (28 mol.%, Table 1).
The alkyne band at31 3280 cm-1 is clearly evident in the spectrum for PEA-PA (Fig. 2(a)) supporting
a high extent of PA incorporation. The alkyne band, which was clearly evident in the spectrum for
PA, was not apparent in the spectrum for PEA-PA-AEA, i.e., after CuAAC reaction of PEA-PA
with AEA. Furthermore, the azide band (2100 cm-1) from AEA (Fig. 2(a)) was not present in the
spectrum for PEA-PA-AEA. These spectral changes for PEA-PA-AEA are indicative of a
successful CuAAC reaction29. There is also evidence of a broad RNH2 band15, 41 at about 1590 cm-1.
This feature is also present in the FTIR spectrum for AEA. It can also be seen that the broadening of
the features in the 3300 cm-1 region of the FTIR for PEA-PA-AEA match those of AEA and are
absent from the spectrum for PEA-PA. The latter is also indicative of RNH241-43. Consequently, the
FTIR spectra confirm that primary amine groups were incorporated (Scheme 1(a)) and are in
qualitative agreement with the compositions for the EA-based microgels given in Table 1.

ACCEPTED MANUSCRIPT
13
Fig. 2. FTIR spectra of various microgels. Spectra for AEA and PA are also shown in (a) for comparisons. Expanded
spectra for the 3000 – 3400 cm-1 region are shown for the microgels. (Colour reproduction on web, B & W in print.)
The spectrum for PNP-PA (Fig. 2(b)) shows a weak alkyne band at 3280 cm-1, which is consistent
with the lower extent of incorporation (18 mol.%) compared to that for PEA-PA (28 mol.%). The
band was superimposed on a broad RNH2 band. For this microgel support for incorporation of PA
can be found from the strong band due to the ester groups present at 1725 cm-1. The latter band was
not present in the spectrum for PVP. The band was also present in the spectrum for PVP-PA-AEA
(Fig. 2(b)). The absence of the alkyne and azide bands in the spectrum for PVP-PA-AEA confirm

ACCEPTED MANUSCRIPT
14
that efficient CuAAC reaction occurred. There is also evidence of a broad band at about 1590 cm-1
due to RNH2 with sharper bands due to PVP superimposed on it. Furthermore, the broad absorption
due to RNH2 groups is also present in the spectrum in the 3300 cm-1 region. The spectrum for PVP-
PA-AEA is also consistent with significant primary amine group incorporation.
Fig. 2(c) shows the spectra for the PNP microgel systems. For PNP-PA the alkyne band was
obscured by a broad band due to N-H groups at about 3300 cm-1. However, the ester band at 1725
cm-1 was clearly present; although, at a lower relative absorbance compared to the same band in the
spectrum for PVP-PA (Fig. 2(b)). This confirms a lower PA incorporation within this microgel (6.3
mol.%). The spectrum for PNP-PA-AEA does not show evidence of an azide band, as expected.
However, the FTIR spectrum does not show any clear RNH2 bands because of the strong amide I
band44 in the 1650 cm-1 region and low AEA content.
The FTIR spectra for all of the microgels are consistent with the compositions determined from
elemental analysis (Table 1). The CuAAC reactions for each of the microgels shown in Scheme 1
was quantitative. This shows that our simple approach to functionalise the microgels using CuAAC
is generally applicable and only limited by the ability to incorporate PA into the PX-PA microgel
particles during particle growth.
Microgel particle characterisation and swelling
SEM images for the microgels are shown in Fig. 3. The particles were well dispersed in each case
as evidenced by separated particles. There was some evidence of a nodular surface morphology for
the PX-PA and PX-PA-AEA (X = EA and VP) particles. The number-average diameters measured
from SEM (dSEM) for the PEA-based microgels were all close to the respective dh values measured
in water (Table 1), showing that the particles existed as isolated particles in dispersion. For the
PVP-based microgels the dSEM values are in the range of 339 to 403 nm and are in the same range as
the dh values measured for these microgels in water at pH = 12 (Fig. 4(c)), i.e., in the collapsed
state. This also supports the view that the particles were well dispersed. A comparison between the

ACCEPTED MANUSCRIPT
15
dSEM and dh values for the PNP-based microgels is more difficult because PNP microgels are well
known to deform greatly upon deposition onto SEM stubs45. Values for dh at 50 oC were measured
(Fig. 6) and the values for PNP-PA and PNP-PA-AEA were about 195 nm. The latter value is close
to the respective dSEM values and confirms that particles existed as isolated particles in dispersion.
Thus, the data strongly support the view that our CuAAC method (Scheme 1) did not result in
significant aggregation, which is essential for a microgel functionalisation strategy to be useful.
Fig. 3. SEM images for various microgel particles. The scale bars for the main images and insets represent 500 and
100 nm, respectively.
The high CuAAC efficiencies for the microgels established above imply complete access of the
click reagents (PMDETA, CuBr and AEA) to all of the PA within the PX-PA particles. This implies
that the microgel particles were swollen. We selected DMF as the dispersion solvent for the click
reactions because (a) it has been used for CuAAC of AEA22, (b) it was miscible with water and (c)
the PX-PA microgel particles were swollen by that solvent. The latter is evident from the dh values
for PEA-PA, PNP-PA and PVP-PA measured in DMF (Table 1). It can be seen that the dh values

ACCEPTED MANUSCRIPT
16
for PEA-PA and PNP-PA dispersed in DMF were larger than the respective values measured in
water. This could be seen visually for the PEA-PA dispersions in water and DMF at the same
particle concentration (See Scheme 1(a)) where the turbidity was less for the DMF dispersion – due
to swollen particles scattering less light. In the case of PVP-PA the dh value of 512 nm measured for
the particles in DMF (Table 1) was much higher than the collapsed value in water at pH = 12 (of
362 nm from Fig. 4(c)) and those microgel particles were also swollen in DMF.
A noticeable consequence of incorporating PA into the microgels was that the extent of particle
swelling decreased. This can be seen from comparing the Qp values for the PX or PX-PA (X = EA
and NP) particles in DMF (Table 1). In each case the Qp values were less for the PX-PA particles.
The Qp values for the PNP and PNP-PA microgels also follow this trend. However, those values are
o
approximate because the diameters measured for each respectively microgel at 50 C was used for
dh(coll) in equation (1). PNP is known to retain water at 50 oC46, 47. Indeed, Destribats et al.48 assumed
o
that their PNP microgels contained 29 wt.% of water at 50 C. This means that the true Qp values
will be larger than those shown in Table 1. The Qp values used here for those systems are minimum
values. Nevertheless, our comparison of the Qp values calculated in this way within the PNP
microgel series (PNP, PNP-PA and PNP-PA-AEA) is valid because the relatively low levels of PA
and PA-AEA incorporated (ca. 6.3 mol.%) are unlikely to have significantly altered the water
contents at 50 oC.
PA is soluble in DMF which suggests that the decreased swelling for the microgels containing PA
discussed above is not due to PA-solvent incompatibility. PA is known to act as a crosslinking
during emulsion polymerisation30. Accordingly, the decreased swelling for the PX-PA particles is
attributed to crosslinking by PA units during particle formation. This is supported by a comparison
of the Qp values measured in DMF (Table 1) for PEA (9.1 ± 1.4) and PEA-PA microgel (1.6 ± 0.2).
We tested this further by preparing a PEA-PA microgel without any added BDD crosslinker, i.e.,
BDD-free PEA-PA. The hydrodynamic diameters for the latter particles in water and DMF were,
respectively, 144 ± 7 and 163 ± 8 nm. The value for Qp was 1.5 ± 0.2. This Qp value is not

ACCEPTED MANUSCRIPT
17
significantly different to the value obtained for PEA-PA microgels prepared using BDD (Table 1).
This result demonstrates that the crosslinking for the PEA-PA microgels prepared using BDD was
dominated by PA. It follows that PA can be used to generate highly functionalised microgels where
PA has two roles, i.e., as a crosslinker and a CuAAC site. This is a new observation for microgels.
It is instructive to compare the Qp values for the PX-PA-AEA microgels at pH = 4 to those for the
parent PX-PA microgels. The Qp values increased in each case due to inclusion of AEA. Given the
pKa of primary amines should be about49 10 we can conclude that the increase in Qp is because of
electrostatic repulsion between neighbouring AEA groups within the microgel particles. The
ζ
values in Table 1 support this view because at low pH the values were highest for the PX-PA-AEA
microgels. These data support primary amine group incorporation within PX-PA-AEA.
pH-dependent microgel particle properties
The trends for the dh values described above at pH = 4 were maintained for the PEA-based
microgels across the entire pH range (Fig. 4(a) and (b)). The Qp values for the PEA-PA-AEA
microgel particles in the pH range of 4 to 10 had an average value of 1.7 which is not significantly
different to the Qp value for the PEA-PA particles dispersed in DMF (1.6, Table 1). Although
+
repulsion between RNH3 groups was able to swell the particles in water, the maximum Qp values
are much lower than would normally be expected for microgel particles containing 28 mol.% of
charged groups. For example anionic EA-based microgels containing about 30 mol.% of
deprotonated RCOO- groups had Qp values of around50 30. It is likely that PA crosslinks contribute
to the strongly restricted swelling for PEA-PA-AEA. There was some evidence of a decrease in dh
(and Qp) at a pH value of 12 (Fig. 4(a) and (b)), which would be expected due to deprotonation of
the primary amine groups. However, complete collapse of the particles at a pH of 12 is not expected
because the nitrogen-rich structure of AEA is hydrophilic.

ACCEPTED MANUSCRIPT
18
Fig. 4 pH-dependent swelling for various microgels. The figures on the top and bottom rows show the variations of
hydrodynamic diameter and swelling ratio, respectively. In some cases the error bars are smaller than the symbols. The
legends are the same for (a) and (b), (c) and (d) as well as (e) and (f). (Colour reproduction on web, B & W in print.)
The PVP-PA and PVP-PA-AEA microgels contained lower PA and PA-AEA contents (cf. PEA-PA
and PEA-PA-AEA). They also contained high proportions of a pH-responsive monomer (VP). PVP
has a pKa of about10 4.9. The PVP microgel showed strong pH-triggered swelling (Fig. 4(c) and (d))
when the pH decreased to 4 or less, and the data are consistent with previous reports10. Importantly,
the PVP-PA microgel showed a much lower swelling response (cf. PVP) which is attributed to the
relatively high PA content (18 mol.%). Interestingly, after functionalisation with AEA the strong
low pH swelling behaviour of PVP-PA-AEA returned and was comparable to that for the parent
PVP particles. However, there was also evidence of increased swelling at pH = 7 and 10. We
speculate that this ability to restore the original particle swelling behaviour is due to a relatively low
level of additional crosslinking provided by the PA groups (cf. PEA-PA). The suppressed swelling
for PVP-PA compared to PVP at low pH would have been the result of a dilution of the positive
charged groups with non-charged PA. The loss of positive charge was restored upon
functionalisation of the PA groups with AEA. This interpretation is supported by the higher Qp
values for PVP-PA-AEA at pH = 4 to 10 (Fig. 4(d)) compared to PVP-PA. However, the swelling
from the AEA groups alone was not sufficient to strongly swell the PVP-PA-AEA particles at pH

ACCEPTED MANUSCRIPT
19
values greater than the pKa of PVP (ca. 4.9 for linear PVP10). This is probably due to the
hydrophobicity of the non-charged VP units that opposed particle swelling in that pH range.
The variable pH data for the PNP, PNP-PA and PNP-PA-AEA microgels (Fig. 4(e) and (f)) were
different to the other systems because there were few differences between the three microgels for
this series. This is attributed to the content of PA and PA-AEA being the lowest of the three
microgel systems (6.3 mol.%). However, the dh and Qp values at pH = 4 and 7 were higher for PNP-
PA-AEA compared to PNP-PA, supporting the view that electrostatic swelling occurred.
Surprisingly there was a significant increase in swelling at pH = 12 for all three microgels. This is
proposed to be due to hydrolysis and is discussed further below.
The effect of pH on the
ζ
values were also measured (Fig. 5). For all three microgel systems there
with its value being highest at low pH. Moreover, the values at
was a strong pH dependence of
ζ
ζ
pH = 4 and 7 for all of the PX-PA-AEA microgels were significantly larger than those for the
respective PX-PA microgels. This suggests that there was a higher positive charge density for the
AEA-containing microgels in that pH range. This finding suggests that each of the PX-PA-AEA
microgels had comparable outer shell structures which contained primary amine groups. However,
it is also possible that protonated 1,2,3-triazole species also contributed to the positive charge in the
above pH range because these species are known to have a pKa around51 9.
Fig. 5 pH-dependent zeta potentials for various microgels. In all cases the error bars are smaller than the symbols.
(Colour reproduction on web, B & W in print.)
An important observation concerning the
ζ
data is that relative changes for these values did not
values for PVP-PA and PVP/PEA-
always match the respective changes for dh. For example, the
ζ

ACCEPTED MANUSCRIPT
20
AEA (Fig. 4(b)) show similar strong decreases with increasing pH and have comparable values.
However, the changes in dh (and Qp) for PVP-PA-AEA are more pronounced than those for PVP-
PA (Fig. 4(c) and (d)). This is because
ζ and dh values measure outer layer and whole particle
properties, respectively. The charge density changes that occur within outer layer of microgels do
not always map onto changes in charge density occurring within the particle interior11. We consider
the
ζ values as a guide to the changes in swelling and charge densities that occur in the outer shell
of the microgel particles or surface of the microgels in the collapsed, latex, form.
The
ζ data for all of the systems show an isoelectric point (iep). The PEGMa provided steric
stabilisation and prevented aggregation of these dispersions even when the pH passed through the
ieps. The ieps indicate that negatively charged species were present. Amidines have a pKa in the
vicinity of52 7. Furthermore, they are known to undergo hydrolysis, along with esters, under alkaline
conditions. This is a common explanation for the appearance of ieps within amidine-stabilised
latexes and microgels52, 53. It is the acquisition of negative charge in the shell of the PNP-based
microgels that may explain the moderate swelling observed for these microgels at pH = 12 (Fig.
4(e) and (f)). We note that there was no evidence of aggregation for any of these dispersions.
To further probe the effect of incorporating PA and PA-AEA within the PNP microgels variable
temperature dh and
ζ measurements were conducted (Fig. 6). The data show that the similarity of
the hydrodynamic diameters for PEA-PA and PEA-PA-AEA remained over the entire temperature
o
range studied (25 – 50 C). By contrast, the particle size changes were much higher for PNP. This
can be seen from Fig. S2 where Qp values are plotted as a function of temperature. This is further
support that PA acted as a crosslinker. PA is hydrophobic and would be expected to suppress
swelling and this would have contributed to the low Qp values. However, the fact that there was
only a small increase in Qp occurred upon AEA incorporation suggests that the primary cause for
lack of swelling was the covalent crosslinking involving PA.

ACCEPTED MANUSCRIPT
21
Fig. 6. Variable temperature diameter (a) and zeta potential data (b) for NP-based microgels. The data were
obtained at pH = 7. (Colour reproduction on web, B & W in print.)
The variable temperature
ζ data (Fig. 6(b)) show clear differences between the three PNP-based
microgel systems. The
ζ
values at temperatures of 25 – 35 oC increased in the order PNP < PNP-PA
< PNP-PA-AEA. This implies that there were significant differences in the volume surface charge
density in the outer layer of these particles. The charge density was highest for PNP-PA-AEA. The
o
ζ
values were similar at 50 C because the particles were mostly collapsed and had similar surface
charge densities. These three microgels would be expected to have possessed similar charge
densities when prepared because they were prepared using similar monomer-to-initiator ratios.
A final point can be made from a comparison of the dh and
ζ
data (Fig. 6) for PNP-PA and PNP-
data
PA-AEA. The equivalence of the dh data for these two microgels and the dissimilarity of the
ζ
at lower temperatures, coupled with the relatively low levels for their incorporation (6.3 mol.%),
imply that the PA and PA-AEA groups were preferentially located in the outer shell of the
microgels. This is potentially useful for future microgel functionalisation using CuAAC chemistry
because it facilitates their accessibility to reactants.
CONCLUSIONS
In this study we have investigated the ability to functionalise three different types of microgels with
primary amine groups using CuAAC chemistry. Incorporation of AEA into the microgels was
quantitative for each microgel system which confirms the principle of achieving high yield CuAAC
reactions applies to swollen microgel particles. The limiting factor controlling AEA incorporation

ACCEPTED MANUSCRIPT
22
was the PA content in the precursor microgel particles. The highest AEA content incorporated was
for PEA-PA-AEA which contained 28 mol.% of AEA. Using this approach we prepared three new
examples of microgels containing primary amine groups; PX-PA-AEA (X = EA, VP and NP). The
extent of incorporation of AEA into PNP-PA-AEA was the lowest (6.3 mol.%) and this was
attributed to the inability of PA to swell PNP. The PCS and zeta potential data for that system imply
that the PA-AEA units were located mostly in the outer shell of the PNP/microgel-AEA particles.
PA was found to act as a crosslinker and it was possible to use that co-monomer as the only
crosslinker for PEA microgel preparation. All of the PX-PA-AEA microgels exhibited pH-triggered
swelling in water due to the primary amine groups at pH values less than or equal to 7. The pH-
responsive swelling was strongest for PVP-PA-AEA and this system appeared to provide a good
compromise between crosslinking due to PA and swelling by AEA incorporation. This study has
established a general method that can be used to efficiently functionalise microgels with azide-
containing molecules by CuAAC. Our CuAAC method for microgels did not cause aggregation and
appears versatile. In addition, the three new PX-PA-AEA microgels (X = EA, VP and NP) studied
here offer additional functionalisation opportunities through the wide range of reactions available
for primary amines14.
Acknowledgements
BRS and RF would like to thank the EPSRC for funding.

ACCEPTED MANUSCRIPT
23
References
1.
2.
L. Hu, A. K. Sarker, M. R. Islam, X. Li, Z. Lu, and M. J. Serpe. J. Polym. Sci. , A; Polym. Chem.
2013, 51, 3004.
B. R. Saunders, N. Laajam, E. Daly, S. Teow, X. Hu, and R. Stepto. Adv. Coll. Interf. Sci. 2009, 147-
148, 251.
3.
4.
N. M. B. Smeets, and T. Hoare. J. Polym. Sci. , A; Polym. Chem. 2013, 51, 3027.
A. Fernandez-Barbero, I. J. Suarez, B. Sierra-Martin, A. Fernandez-Nieves, F. J. de las Nieves, M.
Marquez, J. Rubio-Retama, and E. Lopez-Cabarcos. Adv. Coll. Interf. Sci. 2009, 147-148, 88.
R. Pelton. Adv. Coll. Interf. Sci. 2000, 85, 1.
5.
6.
7.
P. A. FitzGerald, D. Dupin, S. P. Armes, and E. J. Wanless. Soft Matter 2007, 3, 580.
J. D. Debord, and L. A. Lyon. Langmuir 2003, 19, 7662.
8.
S. Fujii, and S. P. Armes. Langmuir 2006, 22, 6818.
9.
Y. Wang, J. Nie, B. Chang, Y. Sun, and W. Yang. Biomacromolecules 2013, 14, 3034.
D. Dupin, S. Fujii, S. P. Armes, P. Reeve, and S. M. Baxter. Langmuir 2006, 22, 3381.
H. Dalmont, O. Pinprayoon, and B. R. Saunders. Langmuir 2008, 24, 2834.
D. Klinger, and K. Landfester. Macromolecules 2011, 44, 9758.
A. H. Milani, T. J. Freemont, J. A. Hoyland, D. J. Adlam, and B. R. Saunders. Biomacromolecules
2012, 13, 2793.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
R. K. Pinschmidt. J. Polym. Sci. A., Polym. Chem. 2010, 48, 2257.
S. Thaiboonrod, C. Berkland, A. H. Milani, R. Ulijn, and B. R. Saunders. Soft Matter 2013, 9, 3920.
Z. P. Demko, and K. B. Sharpless. Angew. Chem. Int. Ed. 2002, 41, 2110.
Z. P. Demko, and K. B. Sharpless. Angew. Chem. Int. Ed. 2002, 41, 2113.
N.-T. Huynh, Y.-S. Jeon, M. Zrinyi, and J.-H. Kim. Polym. Int. 2013, 62, 266.
X. Jiang, E. B. Vogel, M. R. Smith III, and G. L. Baker. Macromolecules 2008, 41, 1937.
W. H. Binder, and R. Sachsenhofer. Macromol. Rapid Commun. 2007, 28, 15.
R. K. Iha, K. L. Wooley, A. M. Nystrom, D. J. Burke, M. J. Kade, and C. J. Hawker. Chem. Rev.
2009, 109, 5620.
22.
23.
24.
25.
S.-H. Jung, H.-Y. Song, Y. Lee, H. M. Jeong, and H. Lee. Macromolecules 2011, 44, 1628.
J. Budhathoki-Uperty, J. F. Reuther, and B. M. Novak. Macromolecules 2012, 45, 8155.
H. Nulwala, D. J. Burke, A. Khan, A. Serrano, and C. J. Hawker. Macromolecules 2010, 43, 5474.
X. Liu, H.-N. Zheng, Y.-Z. Ma, Y.-Z. Ma, Q. Yan, and S.-J. Xiao. J. Coll. Interf. Sci. 2011, 358,
116.
26.
Y. Yu, C.-K. Chen, W.-C. Law, J. Mok, J. Zou, P. N. Prasad, and C. Cheng. Mol. Pharmaceutics
2013, 10, 867.
27.
28.
29.
H. C. Kolb, M. G. Finn, and K. B. Sharpless. Angew. Chem. Int. Ed. 2001, 40, 2004.
N. Li, and W. H. Binder. J. Mater. Chem. 2011, 21, 16717.
M. T. Gokmen, W. V. Camp, P. J. Colver, S. A. F. Bon, and F. E. Du Prez. Macromolecules 2009,
42, 9289.
30.
31.
R. D. Roeder, P. Rungta, V. Tsyalkovskyy, Y. Bandera, and S. H. Foulger. Soft Matter 2012, 8,
5493.
D. D. Evanoff, S. E. Hayes, Y. Ying, G. H. Shim, J. R. Lawrence, J. B. Carroll, R. D. Roeder, J. M.
Houchins, C. F. Huebner, and S. H. Foulger. Adv. Mater. 2007, 19, 3507.
R. K. O'Reilly, M. J. Joralemon, K. L. Wooley, and C. J. Hawker. Chem. Mater. 2005, 17, 5976.
Z. Meng, G. R. Hendrickson, and L. A. Lyon. Macromolecules 2009, 42, 7664.
S. G. Kupal, B. Cerroni, S. V. Ghugare, E. Chiessi, and G. Paradossi. Biomacromolecules 2012, 13,
3592.
32.
33.
34.
35.
36.
J. Brandrup, E. H. Immergut, E. A. Grulke, A. Abe, and D. R. Bloch (1999) CRC Polymer
Handbook, 4 ed., John Wiley & Sons.
J. McParlane, D. Dupin, J. M. Saunders, S. Lally, S. P. Armes, and B. R. Saunders. Soft Matter
2012, 8, 6239.
37.
38.
H. G. Schild, and D. A. Tirrell. Langmuir 1991, 7, 665.
D. J. Shaw. Introduction to colloid and surface chemistry, Butterworth-Heinemann Ltd, Oxford
1992.
39.
40.
C. K. Chiklis, and J. M. Grasshoff. J. Polym. Sci. (A2) 1970, 8, 1617.
P. A. Lovell, and M. S. El-Aasser. Emulsion polymerization and emulsion polymers, Wiley 1997.

ACCEPTED MANUSCRIPT
24
41.
42.
L. Shi, and C. Berkland. Macromolecules 2007, 40, 4635.
R. C. Weast, M. J. Astle, and W. H. Beyer (1985) CRC Handbook of Chemistry and Physics, 65 ed.,
CRC, Boca Raton.
43.
S. Seifert, F. Simon, G. Baumann, M. Hietschold, A. Seifert, and A. Spange. Langmuir 2011, 27,
14279.
44.
45.
46.
47.
48.
Y. Maeda, T. Higuchi, and I. Ikeda. Langmuir 2000, 16, 7503.
B. R. Saunders, and B. Vincent. J. Chem. Soc., Faraday Trans. 1996, 92, 3385.
H. M. Crowther, and B. Vincent. Coll. Polym. Sci. 1998, 276, 46.
A. K. Lele, M. M. Hirve, M. V. Badiger, and R. A. Mashelkar. Macromolecules 1997, 30, 157.
M. Destribats, M. Wolfs, F. Pinaud, V. Lapeyre, E. Sellier, V. Schmitt, and V. Ravaine. Langmuir
2013, 29, 12367.
49.
50.
51.
K. Samaru, H. Matsuoka, and H. Yamaoka. J. Phys. Chem. 1996, 100, 9000.
R. Liu, A. H. Milani, T. J. Freemont, and B. R. Saunders. Soft Matter 2011, 7, 4696.
S. Granados-Focil, R. C. Woudenberg, O. Yavuzcetin, M. T. Tuominen, and E. B. Coughlin.
Macromolecules 2007, 40, 8708.
52.
53.
F. Meunier, A. Elaissari, and C. Pichot. Polym. Adv. Tech. 1994, 6, 489.
F. Sauzedde, F. Ganachaud, A. Elaissari, and C. Pichot. J. Appl. Polym. Sci. 1997, 65, 2331.

ACCEPTED MANUSCRIPT
25
SUPPORTING INFORMATION
A general method for functionalisation of microgel
particles with primary amines using click chemistry
Robert Farleya and Brian R. Saundersa,*
aBiomaterials Research Group, Manchester Materials Science Centre, School of Materials,
The University of Manchester, Grosvenor Street, Manchester, M1 7HS, U.K.
Fig. S1. 1H NMR spectrum for AEA.
Table S1. Preparation and characterisation data for the microgels investigated in this work
PX or PX-PA
Nominal composition
X / g
Crosslinkera / g
PA / g
PEA
PVP
PNP
PEA0.99BDD0.01
PVP0.99DVB0.01
PNP0.99BA0.01
14.37
14.79
14.79
0.29 (BDD)
0.19 (DVB)
0.20 (BA)
0
0
0
PEA-PA
PVP-PA
PNP-PA
4.50
4.36
4.13
PEA071PA0.28BDD0.01
PVP0.71PA0.28DVB0.01
PNP071PA0.28BA0.01
10.35
10.54
10.73
0.15 (BDD)
0.096 (DVB)
0.11 (BA)
a The identity of the crosslinker is shown in brackets.

ACCEPTED MANUSCRIPT
26
Calculation of compositions for microgels using elemental analysis data
1. Introduction
In the following we assume for calculation purposes that the only contributions to % C, %N and
RNC (= %N/%C) values are the monomers shown in Scheme S1. The contributions from PEGMA,
crosslinker and added surfactant are assessed empirically using the control (homopolymer)
microgels (below). Our approach is described in the following where we first consider the PVP and
PNP-based microgels before finishing with the PEA-based microgels. In the following calculations
we ignore crosslinking of PA for the reasons discussed in the main text.
N3
+
y
O
(a)
x
O
O
1ꢀxꢀy
O
0.28
H2N
Azide
AEA
O
O
O
O
O
0.72
O
N
N
N
NH3
PEAꢀPA particles
PEAꢀPAꢀAEA particles
N3
(b)
y
+
x
O
O
1ꢀxꢀy
x
O
H2N
Azide
AEA
N
O
O
1ꢀx
O
N
N
N
N
NH3
PVPꢀPA particles
PVPꢀPAꢀAEA particles
N3
y
+
(c)
O
O
x
x
O
O
1ꢀxꢀy
H2N
Azide
AEA
O
O
O
1ꢀx
O
HN
HN
N
N
N
NH3
PNPꢀPA particles
PNPꢀPAꢀAEA particles
Scheme S1. CuAAC reaction schemes used to determine microgel compositions.
2. PVP-based microgels
Although we cannot be certain of the contributions of PEGMA and DVB to the experimental RNC

ACCEPTED MANUSCRIPT
27
value (RNC(exp)), this can be estimated using the theoretical value (RNC(thry) for the PVP microgel as
follows.
2.1 PVP microgel
The repeat unit formula for PVP is C7H7N with a molecular weight of MW. The value of RNC(thry) is:
= 0.167
(S1)
The value for RNC(exp) was 0.148. This difference is ascribed to incorporation of carbon-rich
PEGMA and DVB. The difference was 0.019 from the following equation:
∆
RNC = RNC(thry) - RNC(exp)
(S2)
We therefore added
∆
RNC to the RNC(exp) values for the PVP-PA and PVP-PA-AEA microgels in
order to compensate for the decrease due to PEGMA and DVB incorporation. These corrected
values (RNC(exp)Cor = RNC(exp)
+ ∆RNC) are shown in Table S2.
Table S2. Elemental analysis data used for composition calculationsa
Cor
RNC(exp)
0.000
RNC(exp)
∆
RNC
Code
PEA
% C
% H
8.46 ± 0.26
% N
0
0.0175
0.0175
0.0175
-
-
58.77 ± 0.26
7.38 ± 0.23
6.94 ± 0.22
0
0.000
PEA-PA
PEA-PA-AEA
59.48 ± 0.27
51.20 ± 0.23
11.39 ± 0.05
0.222 ± 0.001
0.240
0.148 ± 0.001
0.121 ± 0.001
0.235 ± 0.001
0.019
0.019
0.019
-
PVP
75.66 ± 0.34
7.63 ± 0.24
11.23 ± 0.05
0.140
0.255
PVP-PA
PVP-PA-AEA
73.33 ± 0.33
62.56 ± 0.28
6.85 ± 0.21
7.05 ± 0.22
8.90 ± 0.04
14.73 ± 0.07
0.178 ± 0.001
0.166 ± 0.001
0.211 ± 0.001
0.016
0.016
0.016
-
PNP
60.00 ± 0.27
9.97 ± 0.31
10.70 ± 0.05
0.182
0.227
PNP-PA
PNP-PA-AEA
61.00 ± 0.27
57.23 ± 0.26
9.90 ± 0.31
8.78 ± 0.27
10.13 ± 0.05
12.08 ± 0.06
a See text for meanings of the parameters. The errors are estimated from multiple analyses for related polymers prepared
in our laboratory and analysed by the same instrument and methods as used for the microgels studied here.
2.2 PVP-PA microgel
We next consider PVP-PA, which has a repeat unit formula of (C7H7N)1-x(C6H6O2)x from Scheme
S1(b). The following equations apply.

ACCEPTED MANUSCRIPT
28
(S3)
(S4)
Using these equations and the definition for RNC (= %N/%C)), we obtained:
(S5)
Cor
Inserting the value of RNC = RNC(exp) = 0.140 into equation (S5) gives x = 0.18. The estimated
composition of the microgel was PVP-PA0.18. The latter does not show the crosslinker (DVB)
because its nominal concentration was very small (1 mol.% with respect to total monomer used
from Table S1). For this reason the crosslinker is not shown in the other estimated compositions.
2.3 PVP-PA-AEA microgel
PVP-PA-AEA has the structural formula (Scheme S1(b)) of (C7H7N)1-x-y(C6H6O2)x(C8H12N4O2)y.
Using the constraint that x + y = 0.18, it can be shown that the following equation applies.
(S6)
Inserting the value of RNC = RNC(exp)Cor = 0.255 into equation (S6) gives x = -0.008, which is taken as
x = 0, and hence, y = 0.18. The composition of this microgel was PVP-(PA-AEA)0.18, i.e., 100%
efficiency for the CuAAC reaction.
3. PNP-based microgels
We follow a similar approach to that described above for these microgels.
3.1 PNP microgel
The repeat unit formula for PNP is C6H11NO with a molecular weight of MW. The value of RNC(thry)
comes from:
= 0.194
(S7)