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Journal of Pharmacy
Research Paper
And Pharmacology

Microneedle-mediated intrascleral delivery of in situ

forming thermoresponsive implants for sustained ocular
drug delivery
Raghu Raj Singh Thakur, Steven J. Fallows, Hannah L. McMillan, Ryan F. Donnelly and David S. Jones
School of Pharmacy, Queen’s University Belfast, Medical Biology Centre, Belfast, UK

Keywords
microneedle; minimally invasive; optical
coherence tomography; poloxamers; sclera
Correspondence
Raghu Raj Singh Thakur, School of Pharmacy,
Queens University Belfast, Medical Biology
Centre, 97 Lisburn Road, Belfast BT9 7BL, UK.
E-mail: r.thakur@qub.ac.uk
Received May 24, 2013
Accepted August 25, 2013
doi: 10.1111/jphp.12152
Abstract
Objectives This paper describes use of minimally invasive hollow microneedle
(HMN) to deliver in situ forming thermoresponsive poloxamer-based implants
into the scleral tissue to provide sustained drug delivery.
Methods In situ forming poloxamer formulations were prepared and investigated
for their rheological properties. HMN devices 400, 500 and 600 μm in height were
fabricated from hypodermic needles (i.e. 27, 29 and 30 G) and tested for depth of
penetration into rabbit sclera. Maximum force and work required to expel differ-
ent volumes of poloxamer formulations was also investigated. Release of fluores-
cein sodium (FS) from intrasclerally injected implants was also investigated.
Optical coherence tomography (OCT) was used to examine implant localisation
and scleral pore-closure.
Key findings Poloxamer formulations showed Newtonian behaviour at 20°C and
pseudoplastic (shear-thinning) behaviour at 37°C. Maximum force and work
required to expel different volumes of poloxamer formulations with different
needles ranged from 0.158 to 2.021 N and 0.173 to 6.000 N, respectively. OCT
showed intrascleral localisation of implants and scleral pore-closure occurred
within 2–3 h. Sustain release of FS was noticed over 24 h and varied with depth of
implant delivery.
Conclusions This study shows that the minimally invasive HMN device can
localise in situ forming implants in the scleral tissue and provide sustained drug
delivery.

Introduction
Sight threatening eye diseases such as age-related macular
degeneration, diabetic retinopathy, diabetic macular oede-
ma and uveitis originate in the posterior segment of the eye.
These diseases are chronic in nature, and current practice
includes frequent intravitreal injections of therapeutic
agents (e.g. flucinolone acetonide, ranibizumab). Even
though intravitreal injections provide direct delivery of
therapeutic agents into the eye, this method is invasive and
associated with severe side effects, such as high therapeutic
dosage-induced ocular toxicity, pain and discomfort, vitre-
ous haemorrhage, retinal detachment, endophthalmitis, and
cataract development.[1–3] On the other hand, the periocular
(or transscleral) route that includes retrobulbar, peribulbar,
subtenon and subconjunctival routes is considered to be less
painful and most efficient route of drug delivery to the
posterior segment of the eye that poses reduced risk of
endophthalmitis and retinal damage.[4]
The human sclera (the white, structural sheath around
the circumference of the eye) covers 95% of the eye’s
surface area and equates to approximately 17 cm2. It is a
largely acellular tissue composed of interweaving collagen
fibres embedded in an aqueous, glycosaminoglycan
matrix.[5] The permeability of the sclera is comparable with
the corneal stroma, and drug lipophilicity does not appear
to affect scleral permeability markedly.[6] The transscleral or
periocular route of drug delivery has shown to permit large

584 © 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 584–595
Raghu Raj Singh Thakur et al.
range of molecules including corticosteroids,[7,8] antisense
oligonucleotides,[9] immunoglobulins[10] and DNA[11] to the
posterior segment of the eye. For example, rabbit sclera is
permeable to drugs up to a MW of 150 kDa, but permeabil-
ity declines exponentially with increasing MW and molecu-
lar radius.[12] However, transscleral delivery is associated
with low bioavailability of drug in the vitreous, which is
attributed to loss of drug from the periocular space (via
reflux), presence of the blood retinal barrier, choroidal cir-
culation currents, binding of drugs to tissue proteins, pres-
ence of efflux transporters and prolonged time for diffusion
of drugs across sclera (e.g. transscleral route).[13]
To overcome transscleral permeation of drug molecules,
a number of studies have shown surgical administration of
drug-loaded solid implants within the scleral tissue (i.e.
intrascleral delivery) that can provide sustained posterior
drug delivery.[14–16] However, surgical method of administra-
tion is highly invasive and causes higher tissue trauma,
taking longer healing time, retinal detachment and
increased costs after implantation, such as the need for anti-
inflammatory eye drops to avoid any chances of post-
surgical infections. Furthermore, precise localisation of
implants within the scleral tissue is not possible by using
traditional hypodermic needles or by surgical interventions
because of relatively thin scleral tissue.
Microneedles that are third-generation minimally inva-
sive devices have been developed to enhance transdermal
drug delivery for a wide range of therapeutic molecules.[17]
Microneedles are typically 25–2000 μm in height and have
been fabricated from a wide range of materials and in dif-
ferent shapes. Microneedles can be fabricated either into
hollow or solid needles. To date, limited work has been
done in the use of microneedles for ocular drug delivery
applications. Using hollow microneedle (HMN) allows a
minimally invasive means of ocular drug delivery, where the
drug delivery system can be precisely localised within ocular
tissues such as in the suprachoroidal space, sclera tissue or
other ocular tissues.[18,19] However, to date, no studies have
been reported on the use of microneedles to inject in situ
implant-forming drug delivery systems into the scleral
tissue, which could potentially provide a minimally invasive
means of sustained intrascleral drug delivery.
Therefore, in this study, for the first time, we have inves-
tigated the application of HMNs to precisely deliver a
thermoresponsive-based in situ implant-forming poloxo-
mer formulations into the scleral tissue. The poloxomer
formulations were engineered to be free flowing at ambient
conditions to allow administering using HMNs but form
an implant at the physiological temperature of the eye and
provide sustained delivery. In so doing, we have studied
the rheological properties of the poloxamer formulations
with and without model drug (i.e. fluorescein sodium,
FS). Fabricated HMN devices from four different types of
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 584–595
Microneedle-mediated intrascleral delivery
hypodermic needles with different microneedle heights.
Investigated forces required for syringeability of poloxamer
solutions through HMN devices. HMN device was used to
administer the implant-forming FS-loaded poloxamer solu-
tions within the rabbit scleral tissue, and the release was
examined overtime. Furthermore, for the first time, in this
study, we have used optical coherence tomography (OCT)
to visualise the implant formation in the sclera and also
to characterise the scleral pore-closure following HMN
application.
Materials and Methods
Materials
FS (MW 376.27 Da) was purchased from Sigma-Aldrich,
Dorset, UK, and Pluronic® F-127 (poloxamer 407) (MW
12 500 Da) and Pluronic® F-87 (poloxamer 237) (MW
7700 Da) were purchased from BASF Chemical Company,
Ludwigshafen, Germany. Hypodermic needles 26 G
(Terumo Neolus®, 0.45 × 12 mm), 29 G (Terumo Myjector®
1.0 ml, 0.33 × 12 mm) and 30 G (BD Micro-Fine™ 0.3 ml,
0.3 × 8 mm) needles were used as supplied. A 1 ml Terumo
(Terumo Medical Corporation, Murray Hill, NJ, USA)
syringes were used as supplied.
Preparation of poloxamer solutions
A series of thermoresponsive poloxamer formulations were
prepared using the cold method, as shown in Tables 1 and 2.
Distilled water was cooled to 4°C, then poloxamers 407 and
237 were slowly added to the distilled water in the required
proportions with continuous agitation. The polymeric dis-
persions were then left at 4°C until a clear solution was
obtained.[20] Table 2 represents poloxamer formulations
F1–F6 containing 0.5% w/w of FS were prepared as dis-
cussed earlier.
Fabrication of microneedle adapters
Three microneedle adapters were fabricated by removal of
the syringe head of 16 G intravenous catheters (BD
Angiocath™, BD, Franklin Lakes, NJ, USA) and subse-
quently machined of the syringe head (Figure 1). The
adapters were attached to the head of needles and
imaged with a digital light microscope (GXMGE-5 digital
microscope, Laboratory Analysis Ltd, Devon, UK). The
microneedle adapters were machined to give a protrusion
distance (i.e. needle heights) of 400, 500 and 600 μm,
respectively, from the syringe head. Microneedle heights
were measured using the ruler function of the microscope
software.
585
Microneedle-mediated intrascleral delivery Raghu Raj Singh Thakur et al.

Table 1 Gelation temperatures of poloxamer mixtures, n = 3 (a)

Concentration Mean gelation temperature 16G A IV Catheter (BD AngiocathTM)
Poloxamer (% w/w) (±standard deviation) (°C)

407 10 >40 Cut positions

15 >40
20 23.9 ± 0.2
25 15.9 ± 0.3
30 11.7 ± 0.2
237 15 >40 Microneedle adapter
20 >40
25 >40 Hollow microneedle
(b)
30 39.6 ± 0.3
407/237 10/2.5 >40
10/5 >40
10/10 >40 Syringe with hypodermic needle
10/15 >40
Figure 1 Schematic representation of (a) adapter fabrication from
10/20 >40
intravenous catheters and (b) attaching the adapter to hypodermic
10/25 29.3 ± 0.2
needle and syringe assembly to create hollow microneedles of desired
12/2.5 >40
heights.
12/5 >40
12/10 39.0 ± 0.2
12/15 34.3 ± 0.3
12/20 22.5 ± 0.2 of viscosity (η), and the transition point was taken to be
15/2.5 36.3 ± 0.2 where the viscosity was halfway between the values for
15/5 34.8 ± 0.3 the solution and gel.[20] Measurements were performed in
15/10 17.3 ± 0.4 triplicate.

Rheological characterisation at 20 and 37°C

Table 2 Effect of 0.5% w/w fluorescein sodium (FS) loading on gela-
tion temperatures of selected poloxamer mixtures containing 12% w/w The rheological behaviour of F1–F6 formulations at
poloxamer 407 and variable concentrations of poloxamer 237, n = 3 20 ± 0.1°C and 37 ± 0.1°C was investigated using an AR
Mean gelation temperature 2000 rheometer in flow mode using a 4 cm diameter stain-
(±standard deviation) (in °C) less steel parallel plate geometry. Formulations under a con-
Concentration
stant force of 5.0 N were exposed to a continuous shear rate
of FS (% w/w) ranging from 0 to 50/s. Viscosity measurements for each
Poloxamer
formulation were performed in triplicates.
Formulation 237 (% w/w) 0 0.5

F1 15 34.3 ± 0.3 30.7 ± 0.2 Determination of maximum force and

F2 16 30.4 ± 0.2 27.3 ± 0.2
work required for injecting poloxamer
F3 17 27.1 ± 0.1 25.6 ± 0.2
F4 18 24.8 ± 0.2 24.1 ± 0.3
formulations through hollow
F5 19 23.1 ± 0.3 22.2 ± 0.3 microneedle device
F6 20 22.5 ± 0.2 20.8 ± 0.6
A Texture Analyser (TA-TX2, Stable Micro Systems, Surrey,
UK), set in compression mode, was used to simulate the
maximum forces and work (i.e. syringeability) required to
Rheological studies of poloxamer solutions inject the poloxamer formulations into scleral tissue at
20°C. The HMN devices tested for injections were consisted
Measurement of sol-gel transition temperatures
of three different types of needles, namely 26, 29 and 30 G,
of poloxamer solutions
as these are commonly used for intravitreal injection. Ini-
Sol-gel transition temperatures of the poloxamer formula- tially, the maximum force and work required to expel differ-
tions were determined using an AR 2000 rheometer (T.A. ent volumes, for example, 30, 50 and 100 μl of air, were
Instruments, Surrey, UK) in flow mode with 4 cm diameter determined as these represent the frictional component of
stainless steel parallel plate geometry. The formulations the force and work required to inject the poloxamer formu-
were heated between 5 and 40°C at a rate of 1°C/min under lations from the HMN device. A concentric cylinder probe
a constant shear rate of 5/s. Sol-gel transition temperatures (diameter 40 mm and depth 150 mm) was used to apply a
were determined by plotting temperature as a function force to the plunger of a syringe at a rate of 2 mm/s. Then

586 © 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 584–595
Raghu Raj Singh Thakur et al.
the same process was carried out for syringes containing 30,
50 and 100 μl of F1–F6 formulations. Four replicate meas-
urements were made in each case. The force and work were
derived from the force-time plots, produced by Stable
Micro Systems’ Exponent software (v6.0.2.0, Stable Micro
Systems). The maximum force and work were determined
by measuring the peak maxima and the area under curve
values, respectively, from the force-time plots. To calculate
the maximum force and work directly attributable to the
poloxamer formulations, the maximum force and work
values for air (i.e. without formulations) were subtracted
from the values obtained with formulations.
Measurement of penetration force of HMN
into rabbit sclera
Rabbit eyeballs were obtained a day before the experiment
from a local abattoir and were stored at −20°C. Before dis-
section, the rabbit eyeballs were defrosted in distilled water
at 37°C for 6 h. Then, using a scapula, the intraocular and
extraocular muscles, and connective tissue attached to the
sclera of the rabbits’ eyes were carefully removed. The thick-
ness of the sclera in three areas, namely anterior, equator
and posterior, were measured by using OCT (high-
resolution EX1301 OCT Microscope, Michelson Diagnos-
tics Ltd, Kent, UK) (Figure 2). The swept-source Fourier
domain OCT system has a laser centre wavelength of
1305.0 ± 15.0 nm, facilitating real-time high-resolution
imaging of the scleral and other ocular tissue layers (7.5 μm
lateral and 10.0 μm vertical resolution). The sclera was
(a)
(b)
Lens
Pupil
Cornea
Iris
Figure 2 Representative optical coherence tomography images of scleral tissue at different positions of the eye (a) equator, lateral to the middle of
the lens; (b) anterior, at the edge of the orbit; and (c) posterior, lateral to the optic nerve. Scale bar = 400 μm.
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 584–595
Microneedle-mediated intrascleral delivery
scanned at a rate of up to 15 B-scans (two-dimensional
(2D) cross-sectional scans) per second (scan width =
2.0 mm). 2D images were analysed using the imaging soft-
ware ImageJ® (National Institutes of Health; http://
rsbweb.nih.gov/ij/list.html). The scale of the image files
obtained was 1 pixel = 4.2 μm. From this, the recorded
average equatorial, anterior and posterior scleral thickness
was 422 ± 57, 531 ± 82 and 675 ± 47 μm, respectively. Five
measurements were made for each scleral position.
It is important to note that only eyes that had sufficiently
thick sclera of at least 400 μm at the equator, at least
500 μm at the anterior and at least 600 μm at the posterior
were used for penetrative forces studies to prevent full
puncture of the sclera and give erroneous results. Following
thickness measurement an incision made behind the lens
allowed for drainage of the vitreous humour and a 9 mm
biopsy punch was used to punch out the scleral tissue at the
three respective positions. The tissue was kept in distilled
water at 37°C for a maximum of 1 h before use.
A Texture Analyser set to compression mode was used to
determine the scleral penetration forces of the HMN
(Figure 3). The syringe barrel of the HMN were attached to
a concentric cylinder probe using cyanoacrylate adhesive,
and the scleral tissue was placed onto the surface of a poly-
styrene hemisphere to mimic the curvature of the eye. It
was made sure that the tips of the HMN were just above the
surface of the sclera but did not contact with scleral tissue.
Then, the HMN penetrated the sclera tissue to a depth of
400 μm for equatorial sclera, 500 μm for anterior sclera and
600 μm for posterior sclera at a rate of 0.1 mm/s. Five meas-
(c)
Choroid
Retina
587
Microneedle-mediated intrascleral delivery
Concentric cylinder probe
attached to the texture
analyzer
HMN assembly attached to the
probe
Rabbit sclera
Polystyrene ball
hemisphere
Figure 3 Schematic representation of Texture Analyser setup for
determination of penetration forces of hollow microneedle into rabbit’s
scleral tissue.
urements were made for each scleral position. The penetra-
tion force was derived as the maximum force from the
force-time plots.
Visualisation of implant formation using
optical coherence tomography
In this study, whole rabbit eyeballs were defrosted in dis-
tilled water at 37°C for 6 h. The eyes where then placed into
a small water-jacketed cylindrical container (diameter
18 mm) so that the rabbit eyes could be kept stationary at
37°C. For visualising the implant formation, a 50 μl of F6
formulation was injected into the sclera using the 400, 500
and 600 μm HMN device fitted with 30 G needles into the
equatorial, anterior and posterior regions of the scleral
tissue, respectively. The injection sites and injected formula-
tion were visualised using OCT to confirm that the HMNs
penetrated to the required depth and that formulation had
been successfully injected and had formed a gel implant at
37°C (body temperature). High-resolution OCT images
were taken at regular intervals to assess implant behaviour
within the sclera and recovery of the scleral tissue at the
injection site. To allow differentiation between the implants
and different ocular tissues, false colours were applied using
Ability Photopaint® Version 4.14 (Ability Plus Software Ltd,
Crawley, UK). The experiment was setup so that the
implant could be visualised at 37°C in the cylindrical con-
588 © 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 584–595
Raghu Raj Singh Thakur et al.
tainer, thereby negating the chance of the poloxamer gel
implant from turning back into a liquid state.
Ex vivo release studies through rabbit sclera
As detailed in Section ‘Measurement of penetration force of
needles into rabbit sclera’, a 9 mm diameter scleral tissue
samples were excised from the three positions (equatorial,
anterior and posterior) of freshly defrosted rabbits’ eyes and
then positioned onto the receptor compartment of 5 ml
Franz Diffusion Cells (PermeGear, Hellertown, PA, USA)
such that there was intimate contact with the receptor fluid,
0.1 M phosphate buffered saline (PBS) at pH 7.4, which was
thermostatically controlled at 37°C by a surrounding water
jacket and stirred continuously at 600 rpm by a magnetic
stirrer, as shown in Figure 4. Upturned 5 ml Franz-cell
donor compartment lids were then used in conjunction
with stainless steel clamps supplied with the 5 ml Franz-
cells to hold the setup in place. Finally, a glass lid was placed
on top to prevent desiccation of the scleral tissue. Volumes
of 50 μl of formulations were injected into the scleral tissue
to a depth of 400 μm for equatorial sclera, 500 μm for ante-
rior sclera and 600 μm for posterior sclera using HMN
device equipped with the appropriate adapter; a slight
retraction of needle was done before injection of formula-
tion to create a void space before applying pressure on the
plunger to aid implant delivery in the sclera. Samples of
300 μl were then removed at defined time periods from the
receptor compartments and replaced with 300 μl of fresh
0.1 M pH 7.4 PBS. The samples were then analysed for FS
content using UV spectroscopy (PowerWave XS, Bio-Tek
Instruments, Inc., Winooski, VT, USA) at 450 nm.
Statistical analysis
The effects of the various experimental parameters on the
experimental outcomes were performed with a one-way
analysis of variance, where P < 0.05 was taken to represent a
statistically significant difference. When there was a statisti-
cally significant difference, post-hoc Tukey’s HSD tests
were used. Drug release data were analysed using paired
t-tests. In all cases, P < 0.05 was used to denote statistical
significance.
Results and Discussion
In situ implant-forming systems are those that exist as a
solution (i.e. liquid) under ambient conditions yet undergo
a phase transition to a gel when exposed to a physiological
environment or external stimulus. Factors that can trigger
implant formation include a change in temperature (as in
the case of poloxamers), pH, the presence of ions (usually
divalent ions, e.g. Mg2+, Ca2+), phase inversion (unfavour-
able mixing of polar and organic phases), enzymatic and
photo-cross-linking.[21]
Raghu Raj Singh Thakur et al.
30 G needle
Microneedle adapter
Stainless steel support
0.1 M pH 7.4
phosphate buffered
saline (37°C)
5 ml Franz cell
Magnetic stirrer
Figure 4 Schematic representation of Franz cell setup used for ex vivo FS release studies.
Several commercial products for anterior ocular use
based upon in situ gelling systems exist, showing the viabil-
ity of such systems. These include Timoptic-XE® (Merck &
Co., Whitehouse Station, NJ, USA) containing gellan gum
which has a temperature- and cation-dependent mecha-
nism of gel formation[22] and Virgan® (Spectrum Thea
Pharmaceuticals Ltd, Macclesfield, Cheshire, UK) that
contains carbomer 974 that has both temperature- and
pH-dependent mechanism of gel formation.[23]
In this study, triblock co-polymers (poly(ethylene
oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide)),
commercially called as poloxamers, were used as
thermoresponsive-based in situ implant-forming drug
delivery system. poloxamers are fluid below a characteristic
gelation temperature but convert to a gel above this tem-
perature.[24] At low concentrations of poloxamer, in cold-
water, solvation of the polymer chains occurs. With
increasing temperature, desolvation of the hydrophilic
chains occurs because of hydrogen bond breakage, and
hydrophobic interactions between PPO domains dominate,
thus results in gel formation.[25] The gel is micellar in nature
and the liquid micellar phase, which is stable at low tem-
peratures, converts to a cubic structure as temperature
increases.[25] Another form consisting of hexagonally packed
cylinders occurs at even higher temperatures. Gel formation
occurs only when poloxamer concentration is above the
critical micellar concentration.[26] Because of their good
solubilising capacity, opacity and low toxicity, they have
been used in ocular delivery of number of drugs to the
anterior eye including pilocarpine,[27] tropacamide,[28]
fluconazole[30] and antibiotics.[30,31] To the author’s knowl-
edge, this is the first time poloxamers have been injected
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 584–595
Microneedle-mediated intrascleral delivery
Sodium fluorescein -
poloxamer gel depot
Rabbit sclera
Sampling port
intrasclerally to form an implant and assessed for sustained
drug delivery.
High concentrations of poloxamers are usually needed
(e.g. 20–30% w/w) to induce gelation at body temperature
and the gelation itself occurs over a very narrow tempe-
rature range. To overcome this rapid transition and
to improve mechanical/rheological properties, a mixture
of two poloxamers with different MWs and relative
hydrophilicities/hydrophobicities may be used.[21] Ideally, a
gelation temperature of 20–37°C ensures complete gelation
when injected into the eye, that is, at 37°C, yet it is free-
flowing and easily injected at room temperature, that is, at
20°C. In this study, two poloxamer types were used to this
affect namely poloxamer 407 and poloxamer 237.
The gelation temperature of 10%, 15%, 25% and 30%
w/w of poloxamer 407 gels was >40, >40, 15.9 and 11.7°C,
respectively (Table 1). However, a concentration of 20%
w/w produced a mean gelation temperature of 23.9°C that
was within the desired range, that is, 20–37°C. Poloxamer
237 formulations did not form a gel within the desired
range at all concentration studied. On the other hand,
different poloxamer mixtures containing 12% w/w of
poloxamer 407 and variable concentrations of poloxamer
237 (15 and 20% w/w) showed gelling temperatures ranging
from 22.5 to 34.3°C and were chosen for further investiga-
tions. A series of formulations were prepared that contains
12% w/w of poloxamer 407 and a range of poloxamer 237
(15–20% w/w) loaded with 0.5% w/w FS; these formula-
tions were denoted as F1–F6, as shown in Table 2. It was
seen that addition of FS resulted in the reduction of the
gelation temperatures (Table 2). This may have been to
ionic bond, hydrogen bond or salting-out effect, which
589
Microneedle-mediated intrascleral delivery Raghu Raj Singh Thakur et al.

40 (a)
35

Shear stress (Pa)

30
25
20
15
10
5
0
0 5 10 15 20 25 30 35 40 45 50
Shear strain (s–1)

2000 (b)
1800
1600
Shear stress (Pa)

1400
1200
1000
800 F1 F2
600
400 F3 F4
200 F5 F6
0
0 5 10 15 20 25 30 35 40 45 50
Shear strain (s–1)

Figure 5 Effect of 0.5% w/w fluorescein sodium loading on rheological behaviour of poloxamer mixtures containing 12% w/w poloxamer 407
and variable concentrations of poloxamer 237 (i.e. (F1) 15% w/w, (F2) 16% w/w, (F3) 17% w/w, (F4) 18% w/w, (F5) 19% w/w and (F6) 20% w/w
of poloxamer 237) at (a) 20°C and (b) 37°C. n = 3.

could be due to interaction of FS with the poloxamer
chains.
Below their gelation temperature, poloxamer solutions
exhibit Newtonian behaviour, whereby the shear stress is
directly proportional to the shear rate.[28] F1–F6 exhibited
this behaviour at 20°C (Figure 5a) as they have gelation
temperatures greater than 20°C. Viscosity increased with
increase in poloxamer 237 concentration as shown by the
steeper plots. This was likely due to increased hydrophobic
interactions between poloxamer 407 and poloxamer 237
molecules. Previously, the gelation temperature poloxamer
solutions exhibit pseudoplastic (shear thinning) behaviour,
where viscosity decreases exponentially as a function of
increasing shear stress and shear rate.[29] These temperature
dependent rheological changes ensure that the FS-
poloxamer solutions remain as a free-flowing solutions
making them easy for injection at room temperature, but
once it is injected into the sclera (at physiological tempera-
ture), a more vicious gel phase is formed and remains at the
site of injection, providing sustained drug release.
It is essential to minimise the pain experienced by the
patient upon injection, especially for invasive procedures
such as intravitreal or periocular injections.[32] Therefore, it
is a common practice for eye drops containing local anaes-
thetics (often tetracaine or lidocaine in combination with
adrenaline) to be instilled onto the surface of the eye before
intravitreal injection to numb the injection site. The pain is
due to two mechanical processes: the initial piercing and
penetration of the scleral tissue, and then the injection of
the solution into the vitreous humour that also raises
intraocular pressure.
To overcome the pain and tissue trauma associated with
ocular injections or surgical implantation, we have pro-
posed a minimally invasive HMN device that can selec-
tively localise the thermosensitive poloxamer polymers
within the scleral tissue and result in forming intrascleral
implants.
Before performing minimally invasive intrascleral injec-
tions of F1–F6 poloxamer formulations the maximum force
and work (i.e. syringeability forces) required for injecting
the formulations through the HMN device were investi-
gated. The HMN gauge (i.e. 26, 29 and 30 G) and volumes
(i.e. 30, 50 and 100 μl) tested in this study are commonly
used for intravitreal injections. Initially, maximum forces
required to overcome the frictional forces of the syringe to
expel air from the HMN device into water was determined
at 20°C. As shown in Tables 3–5, the maximum force
required to expel air increased with volume of air and
decreasing needle diameter (26–30 G). Similar results
were observed for F1–F6 formulations when injected from

590 © 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 584–595
Raghu Raj Singh Thakur et al. Microneedle-mediated intrascleral delivery

Table 3 Maximum forces required to expel different volumes of air Table 6 Work (i.e. syringeability) required to overcome the frictional
(i.e. to overcome the frictional forces) and formulations from hollow forces (i.e. expel of air) and for expelling different volumes of formula-
microneedle devices fabricated from 26 G needle into water at 20°C, tions from hollow microneedle devices fabricated from 26 G needle
n=5 into water at 20°C, n = 5

Volume of formulation (μl) Volume of poloxamer formulation (μl)

30 50 100 30 50 100

Formulation Mean maximum force (±standard deviation) (in N) Formulation Mean syringeability (±standard deviation) (Ns)

Expel air 1.394 ± 0.048 2.125 ± 0.054 2.448 ± 0.033 Expel air 4.042 ± 0.048 7.257 ± 0.054 11.371 ± 0.083
F1 0.158 ± 0.026 0.325 ± 0.102 0.445 ± 0.060 F1 0.173 ± 0.047 0.268 ± 0.094 0.378 ± 0.095
F2 0.248 ± 0.028 0.449 ± 0.056 0.626 ± 0.059 F2 0.322 ± 0.054 0.493 ± 0.078 0.634 ± 0.061
F3 0.359 ± 0.050 0.600 ± 0.059 0.841 ± 0.068 F3 0.877 ± 0.034 0.974 ± 0.050 1.226 ± 0.055
F4 0.475 ± 0.034 0.823 ± 0.039 1.035 ± 0.078 F4 1.367 ± 0.042 1.476 ± 0.064 1.782 ± 0.066
F5 0.571 ± 0.044 1.099 ± 0.076 1.272 ± 0.047 F5 1.580 ± 0.033 1.817 ± 0.114 2.477 ± 0.093
F6 0.681 ± 0.024 1.329 ± 0.031 1.480 ± 0.078 F6 1.920 ± 0.067 2.342 ± 0.070 3.398 ± 0.024

Table 4 Maximum forces required to expel different volumes of air Table 7 Work (i.e. syringeability) required to overcome the frictional
(i.e. to overcome the frictional forces) and formulations from hollow forces (i.e. expel of air) and for expelling different volumes of formula-
microneedle devices fabricated from 29 G needle into water at 20°C, tions from hollow microneedle devices fabricated from 29 G needle
n=5 into water at 20°C, n = 5

Volume of formulation (μl) Volume of poloxamer formulation (μl)

30 50 100 30 50 100

Formulation Mean maximum force (±standard deviation) (in N) Formulation Mean syringeability (±standard deviation) (Ns)

Expel air 1.805 ± 0.029 2.963 ± 0.107 3.493 ± 0.044 Expel air 5.640 ± 0.042 11.469 ± 0.082 15.207 ± 0.025
F1 0.266 ± 0.049 0.394 ± 0.085 0.492 ± 0.076 F1 0.443 ± 0.042 0.660 ± 0.081 1.223 ± 0.049
F2 0.425 ± 0.053 0.567 ± 0.075 0.715 ± 0.036 F2 0.816 ± 0.041 1.086 ± 0.056 1.772 ± 0.138
F3 0.570 ± 0.027 0.764 ± 0.140 0.941 ± 0.141 F3 1.394 ± 0.081 1.611 ± 0.088 2.404 ± 0.053
F4 0.749 ± 0.047 0.993 ± 0.067 1.213 ± 0.080 F4 1.883 ± 0.034 2.180 ± 0.052 3.216 ± 0.081
F5 0.887 ± 0.043 1.048 ± 0.070 1.345 ± 0.060 F5 2.679 ± 0.057 2.918 ± 0.066 4.051 ± 0.110
F6 1.083 ± 0.055 1.215 ± 0.056 1.399 ± 0.061 F6 3.360 ± 0.114 3.613 ± 0.047 4.741 ± 0.104

Table 5 Maximum forces required to expel different volumes of air Table 8 Work (i.e. syringeability) required to overcome the frictional
(i.e. to overcome the frictional forces) and formulations from hollow forces (i.e. expel of air) and for expelling different volumes of formula-
microneedle devices fabricated from 30 G needles into water at 20°C, tions from hollow microneedle devices fabricated from 30 G needles
n=5 into water at 20°C, n = 5

Volume of formulation (μl) Volume of poloxamer formulation (μl)

30 50 100 30 50 100

Formulation Mean maximum force (±standard deviation) (in N) Formulation Mean syringeability (±standard deviation) (Ns)

Expel air 2.000 ± 0.026 3.067 ± 0.062 3.601 ± 0.071 Expel air 6.009 ± 0.064 11.854 ± 0.098 15.696 ± 0.055
F1 0.401 ± 0.083 0.567 ± 0.047 1.011 ± 0.124 F1 0.496 ± 0.147 0.788 ± 0.084 1.714 ± 0.075
F2 0.529 ± 0.057 0.735 ± 0.064 1.252 ± 0.097 F2 1.054 ± 0.191 1.392 ± 0.097 2.236 ± 0.063
F3 0.694 ± 0.033 0.915 ± 0.056 1.436 ± 0.047 F3 1.684 ± 0.154 1.984 ± 0.066 2.798 ± 0.077
F4 0.910 ± 0.045 1.195 ± 0.091 1.654 ± 0.107 F4 2.250 ± 0.070 2.814 ± 0.103 3.767 ± 0.063
F5 1.131 ± 0.049 1.477 ± 0.044 1.836 ± 0.086 F5 3.263 ± 0.084 3.722 ± 0.084 4.835 ± 0.059
F6 1.301 ± 0.025 1.824 ± 0.012 2.021 ± 0.091 F6 4.519 ± 0.129 4.907 ± 0.110 6.000 ± 0.007

26, 29 and 30 G HMN devices, as shown in Table 3–5.
Increasing formulation viscosity also increased the
maximum force requirement. In parallel to the maximum
force, the work required to inject FS-loaded formulations
also increased with the volume of formulation and decreas-
ing the needle diameter (Tables 6–8). The maximum force
and work required to inject air were significantly higher
than those for any formulation; this is due to the frictional
forces associated with the syringes in injecting different
volumes.

© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 584–595 591
Microneedle-mediated intrascleral delivery Raghu Raj Singh Thakur et al.

Table 9 Mean penetration forces required to pierce rabbit scleral OCT, a non-invasive optical imaging technique, is often
tissue using hollow microneedle devices with different needle sizes, highlighted as the optical analogue to ultrasound. OCT
n=5
maps the variation of reflected light rather than sound from
Scleral location a biological sample as a function of depth.[33] Because of
Equator Anterior Posterior extensive light scattering of skin tissue, the typical penetra-
(400 μma) (500 μma) (600 μma) tion depth of optical techniques is low. However, OCT is
Needle
capable of penetrating to depths of approximately 2.0 mm
gauge Mean penetration force (±standard deviation) (in N)
in comparison with confocal microscopy, which can only
26G 1.159 ± 0.084 1.346 ± 0.065 1.589 ± 0.101 reach depths of approximately 250 μm,[34] therefore, is the
29G 0.703 ± 0.034 0.886 ± 0.063 1.101 ± 0.098
only optical method for cross-sectional imaging of the
30G 0.549 ± 0.077 0.710 ± 0.087 0.905 ± 0.058
sclera and other ocular tissue layers in vivo and ex vivo.
a
Penetration depth. Thus, it has been used extensively for ophthalmological

(A) (a) (b) (c)

300 µm 300 µm 300 µm

(a) (b) (c) (d)

(B)

300 µm 300 µm 300 µm 300 µm

(C) (a) (b) (c) (d) (e)

300 µm 300 µm 300 µm 300 µm 300 µm

Figure 6 Optical coherence tomography images showing 30 G hollow microneedle injection of 50 μl F6 formulation (coloured in red) injected into
equatorial sclera to a depth of (A) 400 μm at (a) 0, (b) 1 and (c) 2 h; (B) 500 μm at (a) 0, (b) 1, (c) 2 and (d) 2.5 h; and (C) 600 μm at (a) 0, (b) 1, (c)
2, (d) 2.5 and (e) 3 h. The arrow indicates empty space in sclera created following hollow microneedle application and its subsequent closure over
time.

592 © 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 584–595
Raghu Raj Singh Thakur et al. Microneedle-mediated intrascleral delivery

100
90
90 (a) (b)
80
80
70
70
60
% FS release

% FS release
60
50
50
40 40

30 30
F1
20 F4 20
10 F6 10
0 0
0 200 400 600 800 1000 1200 1400 0 200 400 600 800 1000 1200 1400
Time (min) Time (min)

70
(c)
60

50
% FS release

40

30

20

10

0
0 200 400 600 800 1000 1200 1400
Time (min)

Figure 7 In vitro release of fluorescein sodium from F1, F4 and F6 formulations at 37°C following intrascleral injection into rabbit sclera at (a)
400 μm (equatorially), (b) 500 μm (anteriorly) and (c) 600 μm (posteriorly), using 30 G hollow microneedle device. Mean ± standard deviation, n = 3.

medical imaging of the retina and anterior segment of the
eye, and it has also been reported in the use of calculating
distances and permeation rates of solutes throughout the
cornea and scleral tissues.[35]
OCT showed an increase in scleral thickness around the
circumference of the eye. A mean thickness of 422 μm was
noted proximal to the lens, and near the orbit, the thickness
was 531 μm and reached its maximal thickness of 675 μm
proximal to the optic nerve. The penetration force required
to pierce the sclera increased with depth of penetration and
increasing needle’s diameter, as represented in Table 9.
In this study, for the first time, we have shown the ability
of HMN device to precisely localise intrascleral implant-
forming sustain release gel system and the scleral-pore
closure. Measurements were made using image analysis
software that confirmed the penetration of HMNs into the
sclera to the required depth, creating a temporary void
space in the sclera. When 50 μl of F6 formulation was
injected into this void space, a gel-based implant was
formed, as shown in Figure 6. The formed gel depot pro-
vided a sustained release of FS (Figure 7). F6 was chosen as
it was the most viscous and had the lowest gelation tem-
perature in the desired range, hence ensuring implant for-
mation and retention within the void space intrasclerally.
The most interesting observation was the reformation/
closure of the sclera tissue, which appeared to envelope the
implant encouraging retention in the injection site. Recov-
ery time was dependent upon the depth of needle penetra-
tion. For example, recovery appeared to be complete within
2, 2.5 and 3 h for injection at a depth of 400, 500 and
600 μm (Figure 6), respectively. Further studies are required
to assess the effect of long-term injection to the mechanical
properties and pore-closure ability of the sclera.
Permeation of FS from the intrasclerally implanted gels
was conducted using a modified Franz-cell setup (Figure 4).
Depth of implant delivery within the sclera significantly
affected %FS release over 24 h (Figure 7). The release data is
best interpreted as the remaining distance that FS must

© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 584–595 593
Microneedle-mediated intrascleral delivery Raghu Raj Singh Thakur et al.

diffuse through the scleral to reach the release media when
the average scleral thickness of a given position is used as
the maximum vertical distance. For example, when a 50 μl
of F6 formulation was injected into equatorial sclera to a
depth of 400 μm, then FS has to travel a mean distance of
22 μm before it can reach the receptor compartment of the
Franz-diffusion cells. Similarly, FS has to travel a mean dis-
tance of 31 and 75 μm of the sclera following injections to a
depth of 500 μm (anterior sclera) and 600 μm (posterior
sclera), respectively. After 24 h, %FS release ranged from
80.88% to 87.56% (Figure 7a), 70.33% to 75.01%
(Figure 7b) and 52.86% to 60.63% (Figure 7c) for an injec-
tion at 400 μm (equatorially), 500 μm (anteriorly) and
600 μm (posteriorly), respectively. Additionally, there was a
small but significant difference between the %FS release
between F1, F4 and F6 after 24 h. Clearly, these results indi-
cate that the rate and extent of drug release following
intrascleral delivery is dependent upon the region of
scleral injection, depth of implantation and composition of
formulation.
investigated the pore-closure property of scleral tissue by
using OCT, which was dependent upon the depth of HMN
penetration. Finally, this study demonstrated a promising
method of administering implant-forming gels in a mini-
mally invasive manner, which could be a potential alterna-
tive to invasive intravitreal injections. Furthermore,
sustained drug delivery can be achieved by varying the
depth of needle penetration, composition of implant-
forming gels and region of intrascleral injections. Further
studies are now needed to investigate the effect of multiple
intrascleral injections of implants by using HMN device
and study its effect on drug release and scleral recovery; in
addition, long-term implant retention should also be inves-
tigated. We also believe that HMN causes lesser scleral
damage when compared with intravitreal injections;
however, this needs further investigations.
Declarations
Conflict of interest

Conclusions The Author(s) declare(s) that they have no conflicts of

In conclusion, a minimally invasive HMN device was fabri-
cated, with varying needle heights that enable precise locali-
sation of in situ forming FS-loaded thermoresponsive
poloxamer formulations within the sclera. Sustained release
of FS was observed, and the percentage of FS release was
depended upon the depth of implant delivery and region of
intrascleral injection. Also, for the first time, this study
interest to disclose. None of the authors has a financial or
proprietary interest in any method or material mentioned.
Acknowledgement
The Authors would like to acknowledge and extend their
gratitude to Vacation Scholarship from the Wellcome Trust
awarded (Grant no. 099660) to Steven Fallows.

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