Maleimide-bearing nanogels as novel mucoadhesive materials for drug delivery

Article abstract of DOI:10.1039/c6tb02124g

Novel maleimide-functionalised nanogels have been synthesised via the polymerisation of 2,5-dimethylfuran-protected 3-maleimidoethyl butylacrylate in the presence of presynthesised poly(N-vinylpyrrolidone) (PVP) nanogel scaffolds using surfactant-free emulsion polymerisation techniques. The protected maleimide nanogels were subsequently deprotected to generate the reactive maleimide group via a retro-Diels-Alder reaction. These activated nanogels were found to exhibit excellent mucoadhesive properties on ex vivo conjunctival tissue when compared to the known mucoadhesive chitosan. In order to determine the viability of the materials as drug carriers, nanogels were loaded with a model drug compound and the in vitro release kinetics were analysed. The nanogels could sustain the release of a model drug compound over several hours owing to the swellable hydrophilic nanogel structure, exhibiting first order release kinetics. As a consequence, these findings support the potential of these maleimide-bearing nanogels as a novel platform for sustained drug delivery.

Full text of DOI:10.1039/c6tb02124g

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Materials Chemistry B
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Maleimide-bearing nanogels as novel
mucoadhesive materials for drug delivery†
Cite this:DOI: 10.1039/c6tb02124g
Prasopchai Tonglairoum,ab Ruairı P. Brannigan,a Praneet Opanasopitb and
´
Vitaliy V. Khutoryanskiy*a
Novel maleimide-functionalised nanogels have been synthesised via the polymerisation of 2,5-
dimethylfuran-protected 3-maleimidoethyl butylacrylate in the presence of presynthesised poly(N-
vinylpyrrolidone) (PVP) nanogel scaffolds using surfactant-free emulsion polymerisation techniques. The
protected maleimide nanogels were subsequently deprotected to generate the reactive maleimide
group via a retro-Diels–Alder reaction. These activated nanogels were found to exhibit excellent
mucoadhesive properties on ex vivo conjunctival tissue when compared to the known mucoadhesive
chitosan. In order to determine the viability of the materials as drug carriers, nanogels were loaded with
a model drug compound and the in vitro release kinetics were analysed. The nanogels could sustain
the release of a model drug compound over several hours owing to the swellable hydrophilic nanogel
structure, exhibiting first order release kinetics. As a consequence, these findings support the potential
of these maleimide-bearing nanogels as a novel platform for sustained drug delivery.
Received 20th August 2016,
Accepted 20th September 2016
DOI: 10.1039/c6tb02124g
www.rsc.org/MaterialsB
investigated as a means for prolonging residence times of
delivery vehicles at the site of application or absorption, there-
Introduction
Nanogels are defined as nanosized hydrogel materials which fore, providing an improvement in drug bioavailability and
consist of networks of chemically or physically cross-linked therapeutic effects.13,14 Recently, numerous mucoadhesive drug
polymers with sizes up to a few hundred nanometres which can delivery systems have been established for systemic and local
be swollen in aqueous environments.1,2 Nanogels combine the delivery through various mucosal tissues such as buccal, nasal,
desirable characteristics of both hydrogel and nano-sized materials, ocular, genitourinary, etc.15–18
which enables them to exhibit a large surface area, high water
A number of polymers have been shown to possess muco-
content, high aqueous dispersibility, a well-defined structure, adhesive capabilities as a consequence of their ability to generate
tunable chemical and physical structures, good mechanical interactions with mucin glycoproteins through physical and/or
properties and biocompatibility.3,4 Moreover, nanogels are capable non-covalent bonds such as hydrogen bonds, van der Waals
of encapsulating and releasing high dosages of therapeutic forces, ionic interactions and chain entanglement.19 However,
agents.4,5 As a result, nanogels are ideal candidates for biomedical in recent years, many studies have attempted to enhance the
applications in areas such as sustained drug delivery, imaging and mucoadhesive properties of materials by modifying polymers to
sensing, etc.5–7
bear specific covalent bond forming functional groups.20–22
The concept of mucoadhesion has attracted considerable Examples of such functionalities are acrylates,19 thiols20 and
attention in pharmaceutical science in recent decades.8–12 catechols.23
Mucoadhesion is defined as the ability of a material to adhere
Recently, Mantovani et al.24 reported the use of maleimide
to the mucus gel layer, which is an essential criterion in the functional groups as reactive chain-ends owing to their high
development of a mucoadhesive drug delivery system.12 Adhesion reactivity and selectivity towards cysteine residues present at
between polymeric materials and the mucosal layer has been protein surfaces via a Michael-type addition reaction.25 Maleimides
exhibit greater reactivity, in terms of CQC bond reactivity, in
comparison to fumarates, maleates or acrylates.26 The withdrawing
effects of two activating carbonyls, as well as the discharge of ring
strain upon product formation, provide a massive driving force
for thiol–maleimide reactions.27 Thiol–maleimide reactions
have been extensively used for bioconjugation owing to their
reliability, efficiency and selectivity.28 Furthermore, maleimide-
bearing materials have been found to exhibit a good degree of
a School of Pharmacy, University of Reading, Reading, RG6 6UR, UK.
E-mail: v.khutoryanskiy@reading.ac.uk
b Pharmaceutical Development of Green Innovations Group (PDGIG),
Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand
† Electronic supplementary information (ESI) available: FT-IR and 1H NMR
spectra and TGA thermograms of monomers and nanogels, equations for
maleimide content and drug loading calculations, release kinetics and statistical
analysis. See DOI: 10.1039/c6tb02124g
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biocompatibility with a minimal effect on physical properties.29 methanol and cooled using an ice-bath for 30 min before the
Recently, there has been increasing interest in exploiting thiol– addition of triethylamine (13 mL, 98.47 mmol) with stirring. To
maleimide reactions in materials synthesis.30 Maleimide-bearing the cooled reaction mixture, ethanolamine (6.2 mL, 102.52 mmol)
nanogels offer a unique platform to improve the mucoadhesive was added dropwise with rigorous stirring and was allowed to
properties of these delivery vehicles.
stir for 10 min before subsequently being warmed to room
Herein, we describe the utilisation of a previously synthe- temperature for 30 min, and finally, heated to reflux (75 1C) for
sised monomer bearing a pendant furan-protected maleimide 18 h. In order to maximise conversion, after the reaction was
group to yield nanogel particles using surfactant-free emulsion complete, a further 1 mL of ethanolamine was added and the
polymerisation techniques in the presence of a presynthesised reaction was allowed to proceed for a further 2 h. The reaction
poly(N-vinylpyrrolidone) (PVP) nanogel ‘scaffold’. The nanogels mixture was then cooled to room temperature and the product
were subsequently deprotected to obtain the active maleimide precipitated as white crystalline powder which was collected by
functional groups on their surface, which were found to exhibit filtration and washed with isopropyl alcohol (IPA). Yield: 34%.
excellent mucoadhesive properties on the ocular mucosal tissues. 1H NMR (400 MHz, DMSO-d6) d 2.93 (s, 2H, CH), 3.42 (m, 4H,
To the best of our knowledge, the use of maleimide functional (CO)2NCH2–CH2OH), 4.79 (s, 1H, OH), 5.12 (s, 2H, CH), 6.55 (s,
groups to enhance the mucoadhesive properties of polymers has 2H, CH) (Fig. S2, ESI†).
not previously been reported.
Synthesis of 2,5-dimethylfuran-protected 3-maleimidoethyl
butylacrylate (3)
Experimental
Materials
In a clean dry round-bottom flask fitted with a stirrer bar and
flushed with N2, furan-protected maleimide alcohol (2) (5.0 g,
24 mmol) was suspended in 100 mL of dichloromethane (DCM)
Maleic anhydride, ethanolamine and acryloyl chloride were
before the addition of triethanolamine (4.2 mL, 31.8 mmol).
purchased from Alfa Aesar (UK). N-Vinyl pyrrolidone (NVP),
The solution was cooled using an ice bath for 30 min, after
triethylamine, N,N-methylenebis(acrylamide) (MBA), FITC-dextran
which acryloyl chloride (4.3 mL, 53.2 mmol) was added drop-
(MW = 10 kDa), fluorescein sodium 2,20-azobis(2-methylpropion-
wise with vigorous stirring. The reaction was allowed to proceed
amidine)hydrochloride (V-50), 2,20-azobis(2-methylpropionitrile)
for 2 h before the reaction mixture was warmed to room
(AIBN), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), chitosan
temperature for 24 h. The reaction mixture was filtered to
(62 kDa, 70% deacetylation) and cysteine hydrochloride were
remove any salt product produced during the reaction and
purchased from Sigma-Aldrich (UK). All other reagents and
the filtrate was extracted with 1 M NH4Cl (500 mL), deionised
solvents were of analytical grade and were used without further
water (500 mL) and brine (500 mL). The organic phases were
purification unless otherwise stated. Simulated tear fluid was
combined and the solvent was removed via rotary evaporation
composed of NaCl (0.67 g), NaHCO3 (0.20 g), and CaCl2ꢀ2H2O
1
to yield the product as a pale yellow oil. Yield: 67%. H NMR
(0.008 g) dissolved in deionized water (100 mL).1 Whole bovine
3
(400 MHz, DMSO-d6) d 2.95 (s, 1H, CH), 3.66 (t, JH–H = 5.5 Hz,
eyeballs were sourced from a local abattoir on the day of slaughter,
and transported in phosphate buffer (pH 7.4) maintained at 4–8 1C.
The conjunctival tissues were removed and washed using phos-
2H, (CO)2NCH2–), 4.18 (t, 3JH–H = 5.5 Hz, 2H, –CH2OCO), 5.12 (s, 2H,
CH), 6.93–6.30 (m, 3H, CHQCH2), 6.55 (s, 2H, CH) (Fig. S3, ESI†).
phate buffer. All tissues were used within 24 h of retrieval.
Synthesis of poly(vinylpyrrolidone) (PVP) nanogels
PVP nanogels were prepared using a modified procedure from
the study of Yang et al.31 Briefly, 200 mL of deionised water was
added to a round-bottom flask and deoxygenated by stirring
under nitrogen bubbling for 20 min. After purging, the monomer
mixture, N-vinylpyrrolidone (NVP) (2.5 g, 22.5 mmol) and
methylene bis-acrylamide (MBA) (0.25 g, 1.6 mmol) in 5 mL
of chloroform, was added followed by the initiator V-50 (2.5 mg,
9.2 mmol). The polymerisation mixture was heated to 70 1C and
polymerisation allowed to proceed for 18 h at 400 rpm stirring.
The PVP nanogels were subsequently purified by dialysis
against deionised H2O before lyophilisation. The nanogels were
stored at 4–8 1C under nitrogen before use.
Synthesis of 2,5-dimethylfuran-protected anhydride (1)
The following procedures were modified from previous studies
of Mantovani et al.24 and Syrett et al.30 Briefly, in a clean two-
necked round-bottom flask fitted with a condenser, a stirrer bar
and a septum, maleic anhydride (30 g, 306 mmol) was dissolved
in toluene (150 mL) and heated to reflux. Using a syringe, furan
(33.4 mL, 459 mmol) was slowly added and reaction was
allowed to proceed overnight. Subsequently, the reaction mixture
was allowed to cool to ambient temperature (25 1C) to yield the
product as a white solid which was retrieved by filtration and
washed with petroleum ether. Yield: 79%. 1H NMR (400 MHz,
DMSO-d6) d 3.29 (s, 2H, CH), 5.33 (s, 2H, CH), 6.56 (s, 2H, CH)
(Fig. S1, ESI†).
Synthesis of furan-protected maleimide-PVP (pMal-PVP) nanogels
In a clean round-bottom flask, 200 mL of deionised H2O was
bubbled with nitrogen for 20 min before the addition of the
azo-initiator AIBN (27.5 mg). PVP nanogels (250 mg), MBA
Synthesis of 2,5-dimethylfuran-protected 3-maleimido ethyl
alcohol (2)
Employing the same apparatus previously used, furan protected (275 mg) and the furan-protected maleimide acrylate monomer
anhydride (1) (15.5 g, 93 mmol) was suspended in 400 mL of (3) (2.50 g) were dissolved in chloroform (5 mL) before being
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added to the deoxygenated initiator solution. The polymerisation using a Varian Cary Eclipse fluorescence spectrometer at the
mixture was heated to 70 1C and the polymerisation was allowed excitation and emission wavelengths of l = 460 and 512 nm,
to proceed for 6 h under N2 at 400 rpm stirring. The opaque respectively, and the encapsulation efficiency (eqn (S2), ESI†) and
solution of nanogels was then cooled and purified by dialysis loading capacity (eqn (S3), ESI†) were determined.
against deionized H2O before lyophilisation. The nanogels were
Mucoadhesion of nanogels on ex vivo bovine conjunctival mucosa
stored at 4–8 1C under nitrogen before use.
The mucoadhesive properties of the nanogels on bovine con-
junctival mucosa were determined using a method modified
from previous work done within the group.33 Aqueous solutions
of deprotected nanogels (1 mg mLꢁ1 in deionised H2O),
protected nanogels (1 mg mLꢁ1 in deionised H2O), chitosan
(1 mg mLꢁ1 in 0.5% acetic acid) and dextran (1 mg mLꢁ1 in
deionised H2O) were prepared and loaded with fluorescein
sodium (0.5 mg mLꢁ1, procedure for nanogel loading later
described) in simulated tear fluid. Free fluorescein sodium
was dissolved in deionised water and added to viscous solutions
of chitosan and dextran in simulated tear fluid with stirring
until a homogeneous gel was achieved. The pH of chitosan
solution was adjusted to 6 using 1% NaOH. An aliquot (20 mL)
was pipetted onto a 1 ꢂ 1 cm2 piece of ex vivo conjunctival
mucosa, which was then placed onto a sloped channel, and washed
with simulated tear fluid using a syringe pump (200 mL minꢁ1). At
pre-determined intervals, fluorescence images of the whole tissue
were taken using a Leica MZ10F fluorescence stereomicroscope
fitted with a GFP filter. The fluorescence images were then analysed
using ImageJ software by measuring the pixel intensity of the
images, which was linearly correlated with the concentration of
nanogels present. The pixel intensity of the blank samples
(conjunctival mucosa without nanogels present) was deducted
from each measurement. The data were presented as a percent of
nanogels remaining on the mucosa surface with elution time. The
experiments were conducted in triplicate.
Synthesis of free maleimide-PVP (Mal-PVP) nanogels
The deprotection of the furan-protected maleimide groups was
performed using a retro-Diels–Alder reaction to yield free
maleimide groups. Briefly, 50 mg of protected pMal-PVP nanogels
were dispersed in toluene (50 mL) and heated to reflux for 18 h.
The mixture was subsequently cooled and toluene was removed
in vacuo. Deprotected Mal-PVP nanogels were dialysed against
DMSO followed by deionised H2O before lyophilisation, in order
to remove trace toluene and furan. The Mal-PVP nanogels were
stored at 4–8 1C before use.
Determination of maleimide group content
To determine the maleimide content of the nanogels, Mal-PVP
nanogels were first reacted with a known amount of excess thiol
and then the remaining unreacted thiol was quantified using
Ellman’s assay.32 Briefly, the deprotected nanogels (30 mg) were
placed in a 5 mL glass vial containing a solution of cysteineꢀHCl
(3 mg, 19 mmol) dissolved in a phosphate buffer solution (0.5 M,
pH 8). The nanogels were allowed to swell and react for 1 h after
which the nanogels were separated from the aqueous solutions
by centrifugation at 13 000 rpm for 10 min. The supernatant
liquid was determined for the remaining thiol using Ellman’s
reagent, 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). Ellman’s
reagent or DTNB (3 mg, 7.6 mmol) was dissolved in 10 mL of
phosphate buffer solution (0.5 M, pH 8). Then 500 mL of DTNB
stock solution was added to 500 mL of the supernatant liquid
and incubated in the dark for 2 h. The absorbance of the
mixture was determined using an Epoch Microplate Spectro-
photometer microplate reader at l = 420 nm. The unreacted
thiol was quantified using a calibration curve of cysteine hydro-
chloride prepared as a series of solutions, with a concentration
range of 0.018–0.797 mmol mLꢁ1, prepared under the same
conditions. The cysteine solution without being mixed with
the nanogels was used as a control. The quantity of free
maleimide present in the Mal-PVP nanogels is calculated as
the difference between the initial amount of thiol and the
amount of unreacted thiol after the complete reaction of all
maleimide groups (eqn (S1), ESI†).
In vitro release from Mal-PVP nanogels
The in vitro release of fluorescein sodium from loaded nanogels
was studied using a dialysis method modified from previously
reported work.34 Briefly, 2 mL of fluorescein sodium-loaded
nanogels in simulated tear fluid (7.5 mg mLꢁ1) was contained
in a Pur-A-Lyzert Maxi 3500 dialysis kit. The dialysis kit was
submerged in 30 mL of simulated tear fluid (pH 7.4) that was
incubated at 35 1C and shaken at 80 rpm for 24 h. At given
intervals, 5.0 mL aliquots of the release medium were withdrawn
and replaced with fresh medium to maintain a constant volume.
The released fluorescein sodium in each aliquot was determined
by fluorescence spectroscopy at excitation and emission wave-
lengths of l = 460 and 512 nm, respectively. The experiments were
conducted in triplicate and the release kinetics of fluorescein
sodium from the nanogels were also calculated using the zero-
order model, the first-order model and the Higuchi model.
Model drug loading of Mal-PVP nanogels
In order to demonstrate the applicability of the nanogels for
drug delivery, fluorescein sodium was employed as a model
compound to load into the nanogel using an adsorption
method. Briefly, lyophilised Mal-PVP nanogels (30 mg) were
added to a 2 mL aqueous solution of fluorescein sodium
(2 mg mLꢁ1 in deionised H2O) and subsequently mixed using a
rotary mixer. After 48 h, the nanogels were removed from solution
Results and discussion
Synthesis of pMal-PVP and Mal-PVP nanogels
by centrifugation at 13 000 rpm for 10 minutes. The amount of In agreement with previous studies, 2,5-dimethylfuran-protected
free fluorescein sodium in the supernatant was measured by 3-maleimidoethyl butylacrylate (3) was synthesised in reasonable
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Fig. 1 (a) Synthetic route of the protected maleimide monomer;
(i) toluene, reflux, 18 h. (ii) Ethanolamine, triethanolamine, MeOH, 0 1C
to 75 1C, 18 h. (iii) Acryloyl chloride, triethanolamine, DCM, 0 1C to 25 1C,
24 h. (b) Nanogel synthesis and deprotection to activate maleimide
groups: (iv) MBA, NVP, deionised H2O, 70 1C, 18 h. (v) Monomer (3), PVP
nanogels, MBA, deionised H2O, 70 1C, 6 h. (vi) Protected maleimide
nanogels, toluene, 110 1C, 18 h.
Fig. 2 Hydrodynamic diameter of PVP nanogels, protected nanogels and
deprotected nanogels as determined by dynamic light scattering. Inset:
TEM image of the Mal-PVP nanogels (20 000ꢂ). The scale bar is 100 nm.
yields with minimal purification needed (Fig. 1(a)). The 1H NMR
Furthermore, the retention of the characteristic PVP peaks
spectrum of the protected monomer exhibited a multiplet previously described is indicative of the di-polymeric system.
between 5.93 and 6.30 ppm which is highly characteristic of This is corroborated by the FT-IR spectrum through the
acrylate formation. Furthermore, the complete loss of the triplet presence of absorption bands that are attributable to both the
at 4.79 ppm, attributed to the free alcohol of the pre-monomer protected maleimide and PVP components, namely, the absorption
(2), and the appearance of a triplet at 4.18 ppm, attributed to the bands at 1170 and 1695 cmꢁ1 attributed to CQO stretching
CH2 adjacent to the acrylate group, are indicative of complete vibration of maleimide (Amide I), bands at 1186 and 1019 cmꢁ1
acrylation (Fig. S2 and S3, ESI†). In addition, the presence of attributed to the C–O–C and CQC stretching vibration of the
singlets at 2.95, 5.12 and 6.55 ppm indicates the retention of the furan ring respectively, and a band at 1647 cmꢁ1 corresponding
furan protecting group.
to the CQO group of PVP (Fig. S5, ESI†).
PVP nanogels were prepared using a surfactant-free emulsion
Recently, thermogravimetric analysis (TGA) was employed
polymerisation technique previously reported in the literature.31 to further verify the presence of two polymeric species. The
The successful synthesis of the nanogels was verified by 1H NMR TGA thermograms verified that the nanogels consisted of two
and FT-IR spectroscopy, whilst the hydrodynamic diameter, size components, with two major thermal degradation temperatures
distribution and colloidal stability were determined by dynamic of 150 and 380 1C, corresponding to the decomposition of the
light scattering (DLS).
In agreement with previous studies, the H NMR spectrum
pMal layer and the PVP ‘scaffold’, respectively (Fig. S6, ESI†).
1
Interestingly, the DLS analysis revealed that after copoly
exhibited the characteristic PVP peaks attributed to the protons merisation of the PVP nanogel scaffolds with the 2,5-dimethyl-
of the carbon backbone (1.43–1.7 ppm and 3.8 ppm) in addition furan-protected 3-maleimidoethyl butylacrylate monomer, the
to peaks attributed to the pyrrolidone side group (2.0, 2.4 and hydrodynamic diameter increased substantially (d = 134 ꢃ
3.2 ppm) (Fig. S4, ESI†). Furthermore, the FT-IR spectrum 1 nm), with a large reduction in the PDI value (PDI = 0.09)
displayed absorption bands at 1647, 1427 and 1288 cmꢁ1 that (Table 1 and Fig. 2). It is proposed that this may be attributed to
correspond to the CQO group, C–H bending vibration and C–N the aggregation of the PVP nanogels during the polymerisation
stretching vibration, respectively, typical of PVP (Fig. S5, ESI†). in order to stabilise the relatively hydrophobic protected maleimide
The DLS analysis revealed that the synthesis yielded nanogels polymer. In addition to this, it was found that the zeta potential
which displayed a monodisperse size distribution (PDI = 0.21) of the pMal-PVP nanogels also decreased (z-potential = ꢁ19.43 ꢃ
with a mean hydrodynamic diameter of 45 ꢃ 1 nm. Furthermore, 3.53 mV), indicative of a higher degree of colloidal stability.
the PVP nanogels displayed a reasonable colloidal stability,
To ‘activate’ the maleimide functional groups on the nanogels,
exhibiting a z-potential = ꢁ15.08 ꢃ 3.36 mV (Fig. 2 and Table 1). the furan protecting group was removed via a retro-Diels–Alder
1
After synthesis, the PVP nanogels were utilised as a nanogel reaction (Fig. 1(b)). After deprotection, the H NMR spectrum of
‘scaffold’ for the polymerisation of the protected maleimide the Mal-PVP nanogels showed the complete loss of the singlets at
monomer, using MBA as a crosslinker, to produce ‘core–shell’ 5.12 and 6.55 ppm, characteristic of the Diels–Alder adduct, and
type protected maleimide-bearing (pMal-PVP) nanogels (Fig. 1(b)). the formation of a singlet at 7.01 ppm which is attributed to the
As with the PVP nanogel ‘scaffolds’, the core–shell type nanogels protons of the deprotected maleimide (Fig. 3). Additionally, the
1
were analysed via H NMR and FT-IR spectroscopy and DLS. The FT-IR spectrum reveals the disappearance of the absorption
1H NMR spectrum of the pMal-PVP nanogels displayed singlet bands at 1186 and 1019 cmꢁ1 that were assigned to the C–O–C
peaks at 6.51, 5.26 and 2.90 ppm characteristic of the protons of and CQC band of the furan ring in conjunction with the
the Diels–Alder adduct, confirming the retention of the furan appearance of a band at 833 cmꢁ1, corresponding to the QCH
protecting groups (Fig. 3(a)).
wag vibration of the free maleimide (Fig. S5, ESI†). The appearance
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Table 1 Physicochemical features of PVP, pMal-PVP and Mal-PVP nanogels
Nanogel
Size (nm)
PDI
Zeta potential (mV)
Yield (%)
Maleimide content (mmol gꢁ1
)
PVP
Protected
Deprotected
45 ꢃ 1
134 ꢃ 1
152 ꢃ 1
0.21 ꢃ 0.01
0.09 ꢃ 0.01
0.15 ꢃ 0.02
ꢁ15.08 ꢃ 3.36
ꢁ19.43 ꢃ 3.53
ꢁ20.41 ꢃ 1.10
68
37
88
N/A
N/A
323.47 ꢃ 2.04
Model drug loading of Mal-PVP nanogels
In order to demonstrate the potential of the maleimide bearing
nanogels for drug delivery application, fluorescein sodium was
used as both a model drug compound and a fluorescent ‘tag’
for analysis of mucoadhesive properties. The fluorescein was
loaded into the nanogels using the adsorption method by
straightforwardly incubating the nanogels in an aqueous
solution of fluorescein sodium. It was found by fluorescence
spectrometry that the loading efficiency of the Mal-PVP nanogels
was E15%, with a maximum loading capacity = 21 mg mgꢁ1 of
Mal-PVP nanogels.
Fig. 3 1H NMR spectra of the nanogels (a) prior to deprotection and (b)
after deprotection in CHCl3 (400 MHz, 25 1C, CDCl3). Inset: The structure
of the nanogels before and after deprotection.
Mucoadhesive properties of the nanogels
In order to assess the mucoadhesive properties of the Mal-PVP
nanogels, the retention of fluorescein loaded on ex vivo bovine
conjunctival tissue was assessed using fluorescence microscopy
(Fig. 4). Chitosan and dextran were employed as a positive
control and a negative control, respectively, based on previous
studies, however, it is important to note that a direct comparison
between materials is not possible as a consequence of chitosan
and dextran being linear polymers.36,37 In addition, the muco-
adhesive properties of the protected pMal-PVP nanogels were also
assessed in order to demonstrate the effect of free maleimide
on the mucoadhesive capabilities of the nanogels. After pixel
analysis of the images obtained from fluorescence microscopy,
of new peaks together with changes in existing peaks in both the
1H NMR and FT-IR spectra is highly indicative of the successful
activation of the maleimide moiety.
As predicted, owing to an increase in hydrophilicity, it was
found by DLS that the hydrodynamic diameter of the nanogels
increased significantly upon deprotection (d = 152 ꢃ 1 nm)
(Fig. 2 and Table 1). Furthermore, an additional decrease in the
zeta potential is observed (z-potential = ꢁ20.41 ꢃ 1.10 mV),
suggesting an improvement in colloidal stability, typical of
these hydrophilic systems (Table 1). It is important to note that
only a small decrease in zeta potential was observed as a
consequence of the loss of the relatively neutral, in terms of
charge potential, furan protecting group.
Determination of maleimide group content
As a consequence of the high reactivity of the maleimide moiety
towards the cysteine subunit of mucins, the Mal-PVP nanogels
were expected to have strong mucoadhesive properties. In order
to determine the quantity of maleimide groups available for
mucosal binding, a reverse Ellman’s assay was conducted.35 In
this procedure, the nanogels were first reacted with a known
amount of excess thiol and then the remaining unreacted thiol
was determined via a traditional Ellman’s assay. The amount of
maleimide is computed as the difference between the initial
amount of thiol and the amount of unreacted thiol after the
complete reaction of all maleimide groups (eqn (S1), ESI†). The
result showed that the available maleimide content of the Mal-
PVP nanogels was 323.47 ꢃ 2.04 mmol gꢁ1 of nanogels (Table 1).
Furthermore, it was found that both the PVP and pMal-PVP
exhibited no free maleimide as expected. In comparison with
other mucoadhesive nanogels synthesised by the group, namely
thiolated nanogels, the content of mucoadhesive functionality
per gram of nanogel is relatively high and offers a suitable
platform for mucoadhesion while negating stability issues
associated with thiol-bearing materials, namely oxidation.34
Fig. 4 (a) Exemplar fluorescence images of conjunctival tissue after
washing. (b) Percentage retention of deprotected nanogels, protected
nanogels, chitosan and dextran on the surface of ex vivo bovine con-
junctival tissue. Data are expressed as mean ꢃ standard deviation (n = 3).
* Statistically significant difference (P o 0.05).
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it was found that the deprotected nanogels exhibited excellent (R2 4 0.99) (Fig. S7–S9, ESI†) which describes the drug dissolution
mucoadhesive properties which were comparable to the muco- in pharmaceutical dosage forms containing water-soluble
adhesive properties of chitosan. It was found that 33% of the drugs in hydrophilic matrices. This may be a consequence
Mal-PVP nanogels were retained on the conjunctival tissue after of the hydrophilic Mal-PVP nanogel swelling, resulting in
1 h of washing as compared to 28% of chitosan. Although their % an increased diffusion path length in combination with the
retention was found not to be statistically significantly, different reduction concentration dependent diffusion.38
from chitosan, the mucoadhesive properties of these maleimide
bearing materials show a major advancement in the field of
mucoadhesive polymers.
Conclusions
Furthermore, the control experiment investigating the muco-
In conclusion, we have reported the first example of maleimide-
adhesive properties of the protected pMal-PVP nanogels revealed
functionalised polymers being used as mucoadhesive materials.
that without the presence of free maleimide groups, the nanogels
Utilising cost efficient and facile synthetic routes, we have
displayed a statistically significant lower retention capability
developed a unique, and potentially scalable, platform for the
synthesis of maleimide-containing nanogels. Furthermore, the
mucoadhesive and loading and release capacities of these
compared to the Mal-PVP nanogels (p o 0.05) (Tables S1 and
S2, ESI†). It was found that only 17% of the pMal-PVP nanogels
remained on the tissue after 1 h of washing. These experiments
materials have been demonstrated and therefore, these nano-
confirm the desirable mucoadhesive properties of the deprotected
gels may be promising candidates for controlled drug delivery.
nanogels which can potentially be used as mucoadhesive drug
carriers. Moreover, these maleimide bearing nanogels may be
considered as a novel class of mucoadhesive materials.
Acknowledgements
Joint first co-authorship is attributed to Prasopchai Tonglairoum
In vitro release from Mal-PVP nanogels
´
and Dr Ruairı P. Brannigan. The authors gratefully acknowledge
The in vitro release studies of fluorescein sodium from loaded
Mal-PVP nanogels were performed in simulated tear fluid (pH
7.4), and the release characteristics were plotted as a function of
time (Fig. 5). The release experiment indicated that the fluorescein
sodium was gradually released from the nanogels in a time-
dependent manner. Consistent with a burst release typical for these
systems, the release was found to be faster in the first four hours of
the study. In order to improve the efficacy of the drug being
delivered, not only the retention of the drug carriers at the site of
action is important, but also the capability of the carrier to sustain
the release of the drug over a suitable period of time. The release
along with mucoadhesion studies indicated that the nanogels could
retain and release the drug for a period in excess of 24 h.
the Leverhulme Trust for funding (RPG-2013-017), the Commission
of Higher Education (Thailand), the Thailand Research Funds
through the Golden Jubilee PhD Program (Grant No. PHD/0092/
2554) and the Newton-TRF PhD Scholarship for financial support
and providing 12-month placement for PT at the University of
Reading. Special thanks to Dr Peter Harris for his help with TEM
analysis and Dr Samuel C. Bizley for his advice on this work. The
Chemical Analysis Facility at the University of Reading is
thanked for access to NMR, TGA, FT-IR and TEM.
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