Microfluidically fabricated pH-responsive anionic amphiphilic microgels for drug release

Article abstract of DOI:10.1039/c5tb02378e

Amphiphilic microgels of different composition based on the hydrophilic, pH-responsive acrylic acid (AA) and the hydrophobic, non-ionic n-butyl acrylate (BuA) were synthesised using a lab-on-a-chip device. Hydrophobic droplets were generated via a microfluidic platform that contained a protected form of AA, BuA, the hydrophobic crosslinker, ethylene glycol dimethacrylate (EGDMA), and a free radical initiator in an organic solvent. These hydrophobic droplets were photopolymerised within the microfluidic channels and subsequently hydrolysed, enabling an integrated platform for the rapid, automated, and in situ production of anionic amphiphilic microgels. The amphiphilic microgels did not feature the conventional core-shell structure but were instead based on random amphiphilic copolymers of AA and BuA and hydrophobic crosslinks. Due to their amphiphilic nature they were able to encapsulate and deliver both hydrophobic and hydrophilic moieties. The model drug delivery and the swelling ability of the microgels were influenced by the pH of the surrounding aqueous solution and the hydrophobic content of the microgels.

Full text of DOI:10.1039/c5tb02378e

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Microfluidically fabricated pH-responsive anionic
amphiphilic microgels for drug release
Cite this:DOI: 10.1039/c5tb02378e
B. Lu,a M. D. Tarn,a N. Pamme*a and T. K. Georgiou*b
Amphiphilic microgels of different composition based on the hydrophilic, pH-responsive acrylic acid (AA)
and the hydrophobic, non-ionic n-butyl acrylate (BuA) were synthesised using a lab-on-a-chip device.
Hydrophobic droplets were generated via a microfluidic platform that contained a protected form of AA,
BuA, the hydrophobic crosslinker, ethylene glycol dimethacrylate (EGDMA), and a free radical initiator in
an organic solvent. These hydrophobic droplets were photopolymerised within the microfluidic channels
and subsequently hydrolysed, enabling an integrated platform for the rapid, automated, and in situ
production of anionic amphiphilic microgels. The amphiphilic microgels did not feature the conventional
core–shell structure but were instead based on random amphiphilic copolymers of AA and BuA and
hydrophobic crosslinks. Due to their amphiphilic nature they were able to encapsulate and deliver
both hydrophobic and hydrophilic moieties. The model drug delivery and the swelling ability of the
microgels were influenced by the pH of the surrounding aqueous solution and the hydrophobic content
of the microgels.
Received 13th November 2015,
Accepted 26th December 2015
DOI: 10.1039/c5tb02378e
www.rsc.org/MaterialsB
Introduction
Three-dimensionally crosslinked polymer networks with sizes
ranging from 0.1 to 100 mm are often referred to as gel particles
or microgels. These particles, which are usually dispersed in a
solvent, have many exciting biomedical applications such as in
drug delivery,1–10 tissue engineering,2 and biosensing,11 as well
as applications in the oil industry,12 organic dye removal,13,14
coatings15 and the food industry.16 If the microgels are hydro-
philic then they can be dispersed in aqueous solvents, while if
they are hydrophobic they are usually called latex particles and
cannot be dispersed in aqueous media. Gel particles/microgels
that are amphiphilic, in other words containing both hydro-
philic and hydrophobic groups, offer the advantage that they
can be dispersed in both aqueous and non-aqueous media as
well as encapsulate and release both hydrophilic and hydro-
phobic moieties.
Fig. 1 Schematic illustration of the most common approach to amphi-
philic microgel production via emulsion templating and post-modification
of the microgel. The hydrophobic and hydrophilic moieties are coloured in
red and blue, respectively.
while the continuous phase (CP) is an organic solvent/oil in
Amphiphilic microgels, though, are challenging to fabricate which the reagents have reduced or no solubility. However,
because most of the production methods, such as conventional if the CP is aqueous then the hydrophilic reagent will diffuse
emulsion templating, involve the formation of droplets in an from the droplets to the surrounding CP, and vice versa. Likewise,
immiscible continuous phase, as shown in Fig. 1. The droplets if the CP is based on oil or an organic solvent, then hydro-
contain pre-gels or polymerisable reagents that are later reacted phobic reagents would diffuse from the droplets to the CP.
to form the microgel. Thus, when fabricating hydrophilic micro- Thus, amphiphilic microgels are usually prepared using post-
gels, the hydrophilic reagents are dispersed in aqueous droplets modifications1,4,7,17–21 and/or multi-step procedures.6,22 Typically,
hydrophilic microgels are initially fabricated and then modified to
produce amphiphilic microgels by covalently or electrostatically
a Department of Chemistry, University of Hull, HU6 7RX, Hull, UK.
binding amphiphilic or hydrophobic moieties.1,4,7,18–21 However
E-mail: n.pamme@hull.ac.uk
b Department of Materials, Imperial College London, SW7 2AZ, London, UK.
this results in a ‘‘core–shell’’ microgel structure since the attach-
ment happens mostly on the outer area/surface of the microgel
E-mail: t.georgiou@imperial.ac.uk
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and the hydrophobic groups are never within the elastic chain for drug delivery, the encapsulation and release of both hydro-
(the chain between the crosslinks) but instead grafted on phobic and hydrophilic moieties was investigated using Sudan I
the chain.
The application of microfluidic platforms23 for the production
and Trypan Blue, respectively, as model drugs.
of gel particles offers the advantages of (i) generating droplets,24,25
and consequently microgels, of narrower size distribution and
(ii) the ability to tailor the size of the droplets/microgels by
varying the applied flow rates of the droplet dispersed phase
(DP) and the CP.16,26–29 Furthermore, when using a micro-
fluidic device, reactions can be performed on the droplets
directly within the microchannels, or immediately following
off-chip collection of the droplets, thereby enabling the poly-
merisation of some relatively unstable and easily hydrolysed
reagents. For example, (meth)acrylic monomers tend to hydro-
lyse to (meth)acrylic acid when in contact with water,30 so they
cannot easily be dispersed in water or be polymerised in water
to produce amphiphilic microgels.
Experimental
Materials and methods
Acrylic acid (AA, 99%), n-butyl acrylate (BuA, 99%), 2,3-dihydro-
2H-pyran (DHP, 99%), ethylene glycol dimethacrylate (EGDMA,
crosslinker), sodium dodecyl sulfate (SDS), 2,2-diphenyl-1-picryl-
hydrazyl hydrate (DPPH, free radical inhibitor, 99%), 1-hydroxy-
cyclohexyl phenyl ketone (HCPK, 99%, free radical initiator),
Sudan I, Trypan Blue, phenothiazine (98%, free radical inhibitor),
sulphuric acid, sodium bicarbonate (NaHCO3), sodium sulphate
(Na2SO4), hydrochloric acid, sodium hydroxide and basic alumina
were purchased from Sigma-Aldrich (Dorset, UK). BuA and EGDMA
were passed through a basic alumina column to remove the free
radical initiator prior to use. The organic solvent used to prepare
the hydrophobic droplets was chloroform (99%), and was pur-
chased from Fisher Scientific (Loughborough, UK). The chemical
structures of the main reagents used for microgel fabrication (AA,
BuA and EGDMA), including the in-house synthesised monomer
(THPA, synthesis described below) are shown in Fig. 2.
Synthesis of protected acrylic acid monomer, tetrahydropyranyl
acrylate (THPA). The synthesis of THPA was similar to the pre-
viously described synthesis of tetrahydropyranyl (meth)acrylate.31–36
In a 500 mL three neck round bottom flask that contained 1 g of
phenothiazine (free radical inhibitor) and 125 mL of DHP (116.25 g,
1.38 mol), 20 drops of a 50% v/v sulphuric acid solution in water
were added drop-wise. The solution should change from colourless
to brown. Following this, a mixture of 125 mL DHP (116.25 g,
1.38 mol), 94.6 mL AA (99.4 g, 1.38 mol) and 1 g phenothiazine
(using a dropping funnel) was added and the round bottom
flask was placed in an oil bath. The mixture was then left to react
overnight at 50 1C.
Our previous study31 was, to the best of our knowledge, the
first in which amphiphilic microgels were produced using a
lab-on-a-chip device. Specifically, amphiphilic microgels based
on a hydrophilic monomer and a hydrophobic crosslinker were
fabricated by the formation of precursor droplets within the
microchannels and their subsequent collection and off-chip
polymerisation. In this study, we have designed a new micro-
fluidic chip with a long and deep serpentine channel that
allowed the droplets to travel within the chip itself for a longer
period of time, enabling their rapid and in situ polymerisation
in an automated fashion. This new platform offers the advan-
tage of ensuring even narrower size distributions of the droplets
without risking their coalescence or getting destabilisation. In
our previous study, anionic amphiphilic microgels of varying
crosslinking densities, which was proportional to the hydro-
phobic content, were fabricated and the effect on the ability of the
microgels to swell was investigated, in addition to their capacity to
encapsulate and release hydrophobic and hydrophilic moieties
used as models drugs.31
In the present study the crosslinking density was kept con-
stant and a hydrophobic monomer (n-butyl acrylate, BuA) was
introduced on the elastic chain (the chain between the crosslinks)
in order to allow the hydrophobic content to be systematically
varied without changing crosslinking density. The crosslinker,
ethylene glycol dimethacrylate (EGDMA), was kept the same as in
our previous work, as was the hydrophilic monomer, acrylic acid
(AA). Since acrylic acid is not soluble in the chloroform dispersed
phase into which the BuA and EGDMA were dissolved, the same
approach as used in the previous study was employed by which a
protected form of AA, specifically tetrahydropyranyl acrylate
(THPA), was synthesised in-house and incorporated into the DP
for polymerisation. The resulting THPA–BuA–EGDMA microgels
were then hydrolysed to deprotect the AA units and thus form
anionic amphiphilic AA–BuA–EGDMA microgels. Microgels of
varying hydrophobic/hydrophilic (BuA/AA) ratio but with the
same crosslinking density were fabricated. The pH responsive-
ness of the AA based microgels was evaluated by studying their
swelling in different aqueous pH solutions. Finally, to demon-
The next day, once the mixture had cooled, 10 g of NaHCO3
and 40 g of Na2SO4 were added and allowed to stir for 2–3 h.
The solids were then filtered out and the monomer mixture was
passed through two basic alumina columns (to remove the
un-reacted AA, the THPMA monomer yield was B60%). The
removal of AA was confirmed by 1H NMR. A free radical inhibitor,
DPPH, was added into the THPA monomer, which was then
stored in the fridge. The THPA was distilled under vacuum
prior to use.
strate the amphiphilic nature of the microgels and their potential Fig. 2 Chemical structures of the monomers and crosslinker.
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Fabrication of microgels
were used to control the flow rates of the CP and DP between
0.5 mL minÀ1 and 5 mL minÀ1. The syringes and the droplet
generation section on the chip were covered with aluminium
foil in order to prevent the reagents from polymerising prior to
droplet formation. Photopolymerisation was achieved using a
12 W, 365 nm UV light source (XX-15S, Ultra-Violet Products
Ltd., UK). The entire setup was covered with a thick black cloth
to in order to protect users from UV light.
Videos and images of on-chip droplet generation were
obtained using a colour CCD camera (MTV-63V1N, Mintron,
Taiwan) attached to an inverted microscope (Eclipse Ti, Nikon,
UK), with images captured via WinDVD Creator 2 (Corel Ltd,
UK) software. Collected microgels were observed either on the
same microscope setup, or using a second setup consisting of
an upright microscope (BH-2, Olympus, UK), a CCD camera
(INFINITYlite, Lumenera) and capture software (Studio Capture,
Mettler-Toledo, Inc.). ImageJ freeware was used for the analysis
of droplet size and colour intensity.
Microfluidic chip fabrication and setup. The microfluidic
chip used for droplet formation and polymerisation consisted of
three distinct regions: (i) a flow focusing section for the generation
of THPA–BuA–EGDMA containing droplets, (ii) a short, shallow
serpentine mixing channel that allowed rapid mixing of the
droplet components, and (iii) a long, wide and deep serpentine
channel for UV irradiation of the droplets (Fig. 3a). The chip was
composed of a top layer and a bottom layer, and was fabricated
from glass (B270 glass, Telic, CA, USA) using conventional
photolithography and wet etching techniques.37 The design in
the top layer was etched to a depth of 10 mm and featured the
flow focusing droplet generation and short serpentine mixing
section that had channel widths of 30 mm, and a long, wide
serpentine channel that had a channel width of 720 mm and a
length of 188.35 cm. The bottom layer featured only the long,
wide serpentine channel, as a mirror image to the design on the
top later, that was etched to a width of 800 mm and a depth of
50 mm. The two layers were aligned and thermally bonded in a
furnace at 585 1C for 3 h, yielding a final depth for the long and
wide serpentine channel of 60 mm (Fig. 3b). Fused silica capil-
laries (150 mm i.d., 363 mm o.d., CM Scientific, UK) were glued
into the inlet and outlet holes and connected to 500 mL glass
syringes (SGE, Sigma-Aldrich, UK) via syringe adaptors (Kinesis, UK)
(Fig. 3b). Two syringe pumps (PHD2000, Harvard Apparatus, UK)
Formation of droplets and polymerisation of microgels. The
DP was based on chloroform containing THPA, BuA and EGDMA
with varying molar ratios of THPA and BuA (though the total
concentration of the reagents was always 30 wt% and thus the
monomer crosslinker ratio was always 70 :4 THPA/BuA : EGDMA),
and 4 wt% of HCPK (free radical photoinitiator). The CP was an
aqueous solution of 0.1 wt% SDS, with the concentration of SDS
being below the critical micelle concentration.38 During droplet
generation optimisation studies, the flow rate of the CP was
varied from 0.5 mL minÀ1 to 5 mL minÀ1, while the DP flow rate
was held at 0.5 mL minÀ1. Later microgel fabrication was per-
formed at CP and DP flow rates of 2 mL minÀ1 and 0.5 mL minÀ1
,
respectively. These flow rates allowed the droplets to be UV
irradiated for 30 min as they passed through the serpentine
channel, forming the microgels. One THPA–EGDMA microgel
and three THPA–BuA–EGDMA microgels of differing composi-
tions were formed, with molar ratios of 75 : 0, 49 : 21, 35 : 35,
and 21 : 49 THPA : BuA, respectively. The polymerised microgels
were collected off-chip and then hydrolysed in a solution of
0.01 M HCl solution (pH 2) overnight to produce pH responsive
AA–BuA–EGDMA microgels. The hydrolysis was confirmed
with FTIR.
Swelling studies in different pH environments
Aqueous solutions of NaOH (1 M) and HCl (1 M) were used to
vary the pH of the solution that the AA–BuA–EGDMA microgels
were suspended in, from pH 1 to pH 14. These tests were
performed in order to study the responsiveness of the microgel
particles. The microgels were photographed using the colour
CCD camera and microscope setups described previously, with
ImageJ used for image analysis. The extent of swelling and
shrinking (relative size) was calculated by dividing the microgel
Fig. 3 (a) Exploded schematic of the microfluidic device, featuring a flow diameter at a given pH by their original diameter measured
focusing junction and a long serpentine channel in the top layer (10 mm
deep, 30 mm wide), and the serpentine channel (50 mm deep, 800 mm wide,
at pH 7.
188.35 cm long) mirrored in the bottom layer. (b) Photograph of the
microfluidic chip fabricated in glass, with blue dye used to visualise the
pKa determination
The pKa values of the three synthesised AA–BuA–EGDMA micro-
microchannels. (c) Schematic showing the principle of droplet generation
via in situ UV polymerisation to form microgels.
gels were determined by potentiometric titration. Specifically,
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0.02 g of each microgel was dispersed in water, and 1 M NaOH
added to increase the pH above 11. The titration was performed
by adding 0.05 mL aliquots of 0.1 M HCl and measuring the pH
after each addition.
Dye encapsulation and release studies
Two types of dye, acting as model drugs, were encapsulated
in the microgels via two different methodologies to determine
the capability of the microgels to encapsulate and release both
hydrophilic and hydrophobic moieties. 1 wt% hydrophobic
Sudan I was mixed into the DP prior to droplet formation,
hence the dye was already present inside the microgel following
droplet generation and polymerisation. 1 wt% hydrophilic
Trypan Blue was loaded into already polymerised AA containing
microgels in a pH 14 solution by diffusion with sonication for
30 min, before removing the microgels by pipette, adding them
to a microscope slide and removing any excess solution. The
chemical structures of both dyes are shown in Fig. 4. The model
drug release was monitored using the colour camera of the micro-
scopes. In particular, the change in microgel colour intensity over
time. Images were taken at regular time intervals (every 1 min or
5 min depending on the rate of release). ImageJ freeware was used
for the analysis of the colour intensity.
Fig. 5 Control of droplet size by varying the flow rate of the aqueous
continuous phase, with the dispersed phase flow rate held constant at
0.5 mL minÀ1
.
Results and discussion
Droplet generation and polymerisation
The influence of flow rate on droplet size in the new micro-
fluidic chip design was first investigated by increasing the flow
rate of the CP from 0.5 mL minÀ1 to 5 mL minÀ1, while the DP
flow rate was held at 0.5 mL minÀ1, allowing droplet diameters
to be controlled between 8 mm (volume of 0.3 pL) and 18 mm
(3.1 pL) (CV typical B9%) (Fig. 5). This enabled far smaller
monodisperse droplets to be produced compared to the micro-
fluidic chip design used in the previous study,31 in which
droplets of B130 mm could be generated with similar CVs to
those shown here, while smaller droplets yielded much larger
CVs. The smaller droplet sizes in this study were achieved as a
result of the much shallower and thinner channel dimensions
in the flow focusing section of the chip (Fig. 6a). For amphi-
philic microgel fabrication, the flow rates of the CP and DP
Fig. 6 Photographs of droplets (a) being generated at the flow focusing
junction, (b) in the 10 mm deep channel, (c) entering the 60 mm deep
serpentine channel, and (d) inside the serpentine channel where they
would undergo UV polymerisation.
were 2 mL minÀ1 and 0.5 mL minÀ1, respectively, yielding
droplets with a volume of 3.1 pL (CV 7%). The droplets then
passed through the short serpentine mixing section, with the
same dimensions as the flow focusing channels due to their
being in the same layer of the fabricated chip, which allowed
rapid mixing of the monomers (THPA and BuA) and crosslinker
(EGDMA) inside the droplets. Following this, the droplets
passed from the 10 mm deep channel (Fig. 6b) to the 60 mm deep
serpentine channel (Fig. 6c), where they went from being pancake
shaped in the narrow channels to spherical in the deeper
channel. Interestingly, as the droplets traversed this shallow
serpentine channel they were observed to experience inertial
forces,39,40 which focused them to the outer edges of the channel
before they entered the deeper channel region (Fig. 6c).
The dimensions of the large serpentine channel meant that,
at the applied flow rates, the droplets were polymerised to
Fig. 4 Chemical structures of (a) hydrophobic Sudan I dye, and (b) hydro-
philic Trypan Blue dye.
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THPA–BuA–EGDMA microgels for 30 min as they passed through
it (Fig. 6d). These features offered further improvement over the
previous chip design31 in which the generated droplets could not
be polymerised on-chip due to the extremely short residence
time, while the droplets also remained flattened in the chip and
only became spherical when collected in a dish for off-chip
polymerisation (30 min). Thus, the new chip design and setup
enabled integrated, in situ polymerisation of small, mono-
disperse, spherical microgels, in an essentially automated
fashion; a scenario that was not possible with the previous
chip setup. This also allowed better microgel size distribution
and more accurate polymerisation times without risking the
droplets being destabilised and any monomer leaking out of
the droplets prior to polymerisation.
The polymerised microgels were collected from the chip
outlet, and the THPA groups were deprotected by adding the
microgels to a solution of HCl (pH 2) to form AA groups by
hydrolysis. The resulting amphiphilic AA–BuA–EGDMA micro-
gels consisted of a hydrophilic AA, hydrophobic BuA and hydro-
phobic EGDMA crosslinked internal network, whose ratios could
Fig. 7 Swelling ratios of the AA–BuA–EGDMA microgels and the AA–EGDMA
microgel at different pH values. Microgels formed with AA : BuA : EGDMA molar
ratios of 70 : 0 : 4, 21 : 49 : 4, 35 : 35 : 4, 49 : 21 : 4 are represented as green stars,
blue triangles, red circles and black squares, respectively.
be altered by varying the relative amounts of each component in at pH 7 the actual size/diameters of all microgels ranged from
the precursor DP solution.
17.9 to 18.6 mm. When exposed to high pHs the microgels with
lesser hydrophobic content (i.e. lower BuA/AA ratio) swelled
more since they were more anionic charges that forced the
Microgel fabrication and pH responsiveness studies
Three types of amphiphilic AA–BuA–EDGMA microgels of dif- elastic chains to extend more as well as to repel each other, and
ferent compositions, in addition to a AA–EGDMA homopolymer thus force the whole microgel structure to swell. For example
microgel, were successfully fabricated via the microfluidic at pH 9 the diameter of the microgels increased from 21.3 to
approach described above. Specifically, the four populations 31.0 mm as the AA content increased in the microgel. This was
contained different monomer ratios (AA : BuA) but the same expected and similarly observed in macrogels.34,35,45–50
crosslinking density (monomer : EGDMA 70 : 4), yielding final
pKas of amphiphilic microgels
AA : BuA : EGDMA ratios of 41 : 29 : 4, 35 : 35 : 4, 21 : 49 : 4, and
70 : 0 : 4 (i.e. 70 : 4 AA:EGDMA), were produced. Thus, the hydro- The hydrophobic content also influenced the pKa of the microgels,
phobic content was only varied by altering the hydrophilic which were determined by potentiometric titration. Specifically,
(AA) : hydrophobic (BuA) monomer molar ratio.
when the hydrophobic BuA content was varied between 0%
The pH responsiveness, in terms of swelling and shrinking, of (AA:BuA:EGDMA 70:0:4), 41% (21:49:4), 50% (35:35:4) and
the microgels was studied by measuring the size of the microgels 59% (49 : 21 : 4), based on the molar ratio of BuA (BuA/(BuA + AA) Â
at different pH values. As can be observed in Fig. 7, the size of 100%), the pKa values increased from 6.1 to 6.2, 6.4 and 6.6,
the microgels increased with the increase in the pH of the respectively. Thus, the effective pKa values decreased as the
solution, as was expected. The AA units at high pH become AA content of the amphiphilic microgels increased, or equiva-
ionised due to deprotonation and thus more hydrophilic. This lently, as the BuA content decreased. In other words, AA became
allows more water to enter the microgel, allowing it to swell. a stronger acid as the microgels became less hydrophobic. This
Furthermore, the anionic charges of the AA groups repel each is because a decrease in the BuA content caused an increase
other, forcing polymer chains to extend more and so cause in the hydrophilicity and in the dielectric constant of the
the microgel to swell. This has been observed previously microgels, rendering ionisation easier, which resulted in the
for (meth)acrylic acid containing microgels4,5,31,41 and macro- lowering of the effective pKa.51 This has been similarly observed
gels.33–35,42–44 Note that the size of the microgels at very basic in anionic (meth)acrylic acid based amphiphilic macrogels34,35,45
pH reduces because of increased ionic strength due to the and the reverse trend has been seen in amino (base) containing
increased NaOH concentration, as has also been observed in amphiphilic macrogels (pKa decreased with increasing hydro-
macrogels34,35 and microgels.31
phobic content).46,49,50
When comparing the four different microgels it can be
clearly observed that the size change, and thus the degree of
Dye encapsulation and release studies
swelling, was affected by the hydrophobic content but not at all Having established that the microfluidically fabricated micro-
pH values. At low pH, the AA units became protonated and non- gels were pH responsive, their ability to encapsulate and release
ionised, while the hydrophobic content did not have an effect on both hydrophilic and hydrophobic moieties was examined. The
the swelling ratio because the whole elastic chain (polymer chain intent was two-fold: (i) to prove the amphiphilic nature of the
between the crosslinks) was in a collapsed state. For example, anionic AA microgels, and (ii) to determine their potential as
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delivery vehicles for drugs of varying hydrophobicity/hydro-
philicity, similar to macrogels.52,53 A hydrophobic dye (Sudan I)
and a hydrophilic dye (Trypan Blue) were employed as model
drugs for these studies, with their release triggered by swelling
of the microgels.
Hydrophobic dye – Sudan I. Sudan I (Fig. 4a) was encapsulated
into the microgels during the on-chip polymerisation step. The
Sudan I was mixed into the DP with the monomer and cross-
linker reagents (THPA, BuA, EGDMA), such that when the
precursor droplets were formed and polymerised they already
contained the dye. Fig. 8a shows a newly prepared AA49-co-
BuA21-co-EGDMA4 microgel with the Sudan I encapsulated
inside, suspended in a pH 7 solution. When a basic solution
(pH 14) was added the microgel immediately swelled and
started to release the dye. The progress could be observed as
a colour change over time, from immediately after addition of
the pH 14 solution (Fig. 8b) to 30 min later (Fig. 8c). As
described in the previous section, upon addition of the basic
solution the AA units became hydrophilic and prone to internal
electrostatic repulsion between deprotonated AA groups, thus
causing the polymer chains to extend and forcing the microgel
to swell to the extent that the microgel could no longer retain
the hydrophobic dye, resulting in a loss in colour intensity
over time.
Fig. 9 Hydrophobic drug release in basic (4pH 11) conditions. The plots
show the relative Sudan I colour intensity versus time for four types of
AA–BuA–EGDMA microgels with different AA/BuA molar ratios. Green
stars, black squares, red circles and blue triangles represent the microgels
with 70 : 0 : 4, 49 : 21 : 4, 35 : 35 : 4 and 21 : 49 : 4 AA : BuA : EGDMA molar
ratios, respectively.
The dye release rate was further investigated for each of the
four types of microgel, and the results are shown in Fig. 9. Each
of the microgels was able to encapsulate and release the dye,
but there was a clear influence of the hydrophobic content on the
release rate. In particular, by increasing the hydrophobic mono-
mer (BuA) molar ratio content from 0% (70 :0 :4 AA:Bu: EGDMA)
to 70% (21 : 49 : 4), the rate of release of the hydrophobic dye
decreased. This was expected since the hydrophobic components
of the microgel retained the hydrophobic dye in the microgel
Fig. 10 Hydrophilic Trypan Blue encapsulation in, and release from, a
AA49-co-BuA21-co-EGDMA4 microgel (a) immediately following encapsu-
lation and suspension in pH 7 solution, (b) 5 min later, and (c) 30 min after
suspension in pH 7 solution.
structure to a greater extent, as a result of hydrophobic inter- of the hydrophilic Trypan Blue in the surrounding water can be
actions. Furthermore, the fact that the microgels with a higher observed based on intensity of the blue colour inside the
hydrophobic content swelled less would also have an influence as microgel, which faded with time.
it would slow the diffusion of the dye through the microgel, thus
reducing the release rate.
The release of the hydrophilic dye was further investigated
over time for all four AA–BuA–EGDMA microgels in neutral
Hydrophilic dye – Trypan Blue. The encapsulation and (pH B7, Fig. 11) and basic (pH 11, Fig. 12) solutions. Two main
release of the hydrophilic dye, Trypan Blue (Fig. 4b), was also observations can be made from the acquired data. Firstly, that
investigated. Fig. 10a shows an AA49-co-BuA21-co-EGDMA4 by increasing the hydrophobic content (greater ratio of BuA),
microgel immediately following Trypan Blue encapsulation in the hydrophilic dye release rate was decreased, as was expected
pH 14 solution (as described in the Experimental section) and and as had also been observed for the hydrophobic dye. This was
subsequent washing and suspension in pH 7 solution. Fig. 10b as a result of the microgels with higher hydrophobic content
and c show the same microgel after 5 and 30 minutes following swelling to a lesser extent, the pore/mesh size being smaller, and
suspension in pH 7 solution, respectively. The gradual release the fact that the dye could not diffuse out of the microgel as fast,
compared to those with lower hydrophobic content. The second
observation was that the dye release was much faster at pH 11
(Fig. 12) than pH 7 (Fig. 11). This was observed for all four
varieties of microgels, and had similarly occurred in our previous
studies,31 since more basic conditions yielded greater swelling
(Fig. 7) due to the AA ionisation.
When comparing the hydrophobic Sudan I release to that of
Trypan Blue from the amphiphilic microgels, the latter demon-
Fig. 8 (a) AA49-co-BuA21-co-EGDMA4 microgel with hydrophobic Sudan
strated much faster release characteristics for all four microgels
addition of pH 14 solution, and (c) 30 min after addition of pH 14 solution. at basic pH. For example, it took more than 40 min for the
I dye encapsulated inside at pH 7. (b) The same microgel immediately after
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and it has been demonstrated that the release rate can be tailored
by adjusting the hydrophobic content of the amphiphilic micro-
gels, which shows promise for their use in future drug delivery
applications.
Conclusions
Amphiphilic microgels based on hydrophilic, ionisable acrylic
acid (AA), hydrophobic, non-ionic n-butyl acrylate (BuA) and
the hydrophobic crosslinker ethylene glycol dimethacrylate
(EGDMA) were successfully fabricated in a lab-on-a-chip device
via in situ generation and direct UV polymerisation of spherical
precursor droplets in an expanded serpentine channel. The use of
such an integrated microfluidic platform enabled the systematic
variation of the amphiphilic nature of the microgels by varying
the hydrophilic/hydrophobic monomer ratio, while maintaining
the crosslinking density, producing microgels with narrower size
distributions than those prepared by conventional methods. The
microgels were pH responsive; by increasing the pH of the
surrounding environment the microgels swelled due to ionisation
of the AA units and the resultant electrostatic repulsion between
the polymer chains inside the gel particles. Furthermore, it was
demonstrated that the extent of swelling could be controlled by
tailoring the hydrophobic content, as the microgel swelling was
found to decrease by increasing the hydrophobic BuA content.
Finally, the encapsulation and release of two model drugs, one
hydrophobic dye and one hydrophilic dye, were investigated, and
highlighted the amphiphilic nature of the microgels. The release
was influenced by both the hydrophobic content of the microgels
and the pH of the surrounding solution. In particular, by increas-
ing the hydrophilic content of the microgels and suspending them
in a more basic solution, the release of the dyes was accelerated.
These properties and initial trials show great potential for the use
of on-chip generated amphiphilic microgels for the encapsulation
and triggered release of hydrophilic and hydrophobic payloads,
particularly with a view to drug delivery applications.
Fig. 11 Hydrophilic dye release in neutral pH solution (BpH 7). The plots
show the relative Trypan Blue colour intensity versus time for each variation
of the AA–BuA–EGDMA microgels. Green stars, black squares, red circles
and blue triangles represent microgels with 70 : 0 : 4, 49: 21: 4, 35: 35 : 4
and 21 : 49: 4 AA : BuA : EGDMA molar ratios, respectively.
Fig. 12 Hydrophilic dye release in basic pH solution (BpH 11). The plots
show the relative Trypan Blue colour intensity versus time for all AA–BuA–
EGDMA microgels with different AA–BuA ratios and the AA homopolymer,
AA–EGDMA. Green stars, black squares, red circles and blue triangles
represent the microgels with 70 : 4, 49 : 21 : 4, 35 : 35 : 4 and 21 : 49 : 4
AA : BuA : EGDMA molar ratios, respectively.
Acknowledgements
University of Hull is thanked for funding Mr Lu’s PhD student-
ship. Dr Stephen Clark is thanked for fabrication of the micro-
fluidic devices.
release of hydrophobic Sudan I from AA49-co-BuMA21-co-EGDMA4
microgels at basic pH to reach 50% of the relative colour intensity
(compared to the initial intensity), while this level was reached in
only 5 min during hydrophilic Trypan Blue release. This can be
attributed to two factors: (i) the difficulty of the hydrophobic dye
to diffuse into an aqueous environment compared to the hydro-
philic dye, and (ii) the fact that the hydrophobic domains of the
microgels would retain for longer the hydrophobic dye through
hydrophobic interactions. It should be noted that this was
observed in our previous study on off-chip polymerised AA–
EGDMA microgels as well.31
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