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Organic–inorganic nanocomposite gels as an in situ gelation biomaterialfor injectable accommodative intraocular lens

Masahiko Annaka,*ab Kell Mortensen,c Toyoaki Matsuura,d Masaya Ito,a Katsunori Nochiokad

and Nahoko Ogatad

Received 7th March 2012, Accepted 16th April 2012

DOI: 10.1039/c2sm25534k

We focus on the development of a novel injectable accommodative lens for intraocular applications,

which is based on a thermosensitive hydrophobically modified poly(ethylene glycol) (HM-PEG)

containing hydrophilized silica nanoparticles. We distinguished macroscopically, with changes in the

temperature or concentration, two regions in the phase diagram for aqueous solution of HM-PEG:

transparent sol and transparent gel. These changes occurred reversibly, without hysteresis, when the

temperature was decreased. The temperature and concentration regime in which the gel formed are

reduced by adding silica nanoparticles into the gel matrix. Small-angle neutron scattering

measurements for nanocomposite gels provide good proof of a gel phase where the high shear-modulus

is gained by a high inter-micellar correlation originating in the crystalline order. Under the condition of

uniform distribution of silica nanoparticles with small size (2–5 nm) in the gel matrix, an increase in the

refractive index up to 0.0667 was obtained for the nanocomposite gel compared with the native gel

matrix without an increase in turbidity. This composite system could be formulated to match the

modulus and the refractive index of the natural lens (�1.411), and was easily extruded through

a narrow-gauge needle. Rapid endocapsular gelation yielded an optically clear gel within the lens

capsular bag. This technique enables us to validate methods to determine the biomechanics of the lens

and its role in accommodation. The modification of the mechanical response and stability of the HM-

PEG network by addition of silica nanoparticles was also investigated in detail.

Introduction

Nature combines different types of macromolecules in order to

form gels with outstanding physical properties. One example is

the lens, which projects the optical image on the retina together

with the cornea. The lens is a biconvex spheroidal tissue encap-

sulated within an elastic collagenous outer membrane of varying

thickness called the lens capsule.1,2 The lens has only one-half of

the refractive power of the cornea due to the very small change in

the refractive index at its surface. In order to resolve this situa-

tion, two different types of macromolecules are combined: the

elasticity of the lens lies in the lens capsule and the crystalline

protein in the lens cell primarily provides the refractive index.3

With increasing age, the ability to accommodate to near

targets gradually diminishes, resulting in the need for reading

aDepartment of Chemistry, Kyushu University, Fukuoka 812-8581, Japan.E-mail: annaka@chem.kyushu-univ.jp; Fax: +81-92-642-2607; Tel: +81-92-642-2594bInternational Research Center for Molecular Systems (IRCMS), KyushuUniversity, Fukuoka 819-0395, JapancNano-Science Center and Niels Bohr Institute, University of Copenhagen,Copenhagen, DenmarkdDepartment of Ophthalmology, Nara Medical University, Kashihara,Nara 634-8522, Japan

This journal is ª The Royal Society of Chemistry 2012

glasses or bifocals – a condition known as presbyopia. The

mechanism underlying presbyopia is still unresolved. Cataracts,

possibly leading to blurred vision, generally develop in people

over 60 years of age. Cataracts are optical defects of the natural

lens, which can increase scattering of light and light absorption,

both effects leading to a clouded and increasingly opaque natural

lens.

To restore vision, an eye surgeon can replace the natural lens

with an artificial lens with the optical power adjusted for sharp

distant or near vision. For ophthalmic applications, the

requirements for an in situ gel-forming system are stringent,

including an optically clear material with a refractive index of

�1.41, very low toxicity, and long-term stability in a wet, oxygen-

and photon-rich environment. Restoration of accommodation

by using variable focal-power intraocular lenses is of vital

importance in modern ophthalmic practice.3,4 For a young

individual the accommodation range of a phakic eye may exceed

10 diopters.5,6 This range is reduced significantly with age and

most people become presbyopic due to lens hardening and

a resulting loss in accommodative power of the eye at �35 years.

Replacing the lens of the human eye with an artificial intra-

ocular lens (IOL) in cataract surgery or for other medical reasons

restores clear vision, but results in a significant loss of

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accommodations, i.e., the ability of the eye to focus on a near

object.5,7,8 Attempts to improve cataract surgery to preserve

accommodation are often unsatisfactory. Standard IOLs are

monofocal, patients usually have clear vision only at distance,

requiring additional optical correction to extend the range of

vision. The majority of reported IOLs are currently in the early

stages of development and have not become widely accepted in

ophthalmic practice.8 Some carry an increased risk of optical

distortions of the eye due to large surgical incisions, while others

show insufficient contrast over the range of accommodation,

reduced peripheral vision, and high sensitivity to misalignments.

The ideal intraocular lens (IOL) material, in addition to its

optical and mechanical properties, should be easily injectable

and dimensionally stable once it is formed within the lens

capsular bag. In this study we propose a new idea for the design

of the accommodative IOLs by mimicking the natural lens by

injection of elastic polymers with a necessary refractive index

into the capsular bag of the eye as a fluid.9,10 The body temper-

ature transforms the polymer into a gel that has the shape of

a full-sized biconvex and completely fills the capsular bag. The

gel is expected to be entirely cohesive, not to leak out of the

capsular bag, and to behave similarly to the natural lens and

allow focusing from far to near. The potential benefits of a full-

sized, flexible accommodating lens system go beyond providing

the accommodation lost with age.

Background

In the young phakic eye, the accommodation is accomplished by

contraction of the ciliary body which releases tension on the

zonular fibers holding the natural crystalline lens in dynamic

suspension.7,11–13 The anterior surface of the crystalline lens

moves forward whereas the posterior surface moves slightly

backward. Due to the natural elasticity, the lens reverts to a lens

of stronger curvature with a higher focal power. When the eye is

focused on infinity, the ciliary muscle is relaxed and regains

a larger diameter, the lens is flattened and its focal power drops

to a lower value.11,13 The accommodation range is reduced

significantly with age and most people become presbyopic due to

lens hardening and a resulting loss in accommodative power of

the eye.

Ideally, an accommodative IOL should provide excellent on-

axis optical performance of the eye, compared with that of an

emmetropic eye, weak dependence of the retinal image quality on

viewing field angle and wavelength, and sufficient accommoda-

tion for near work, e.g., reading. After implantation of a mono-

focal IOL, however, the accommodative power of the eye is

largely lost. Multifocal and bifocal IOLs were designed to

overcome the lack of accommodation in pseudophakic patients,

providing useful distance and near vision.14–20 Several studies

have shown that these IOLs allow good functional vision without

the use of corrective lenses, but they are also known to cause

reduced contrast sensitivity21–23 and tend to produce pronounced

halos, flare and glare.21,24

Both physical and chemical cross-linking techniques have been

attempted to fill the capsular bag or to form gels in situ. Kessler25

used Carquille’s immersion oil, silicone fluids, damar gum, and

Silastics as refilling materials to form the physical gels under

physiological temperature. Parel et al.26 utilized filler-free

7186 | Soft Matter, 2012, 8, 7185–7196

divinylmethylcyclo-siloxane as chemical cross-linker to form the

silicone elastomer. Nishi and Nishi27 used poly-

dimethyldisiloxane liquid containing a siloxane cross-linking

agent with a silicone plug for sealing the capsular opening to

prevent leakage of the injected material. The body-temperature

vulcanizing systems, however, suffer from a disadvantage in the

context of refill lens formation in that they cure slowly. Up to

12 h may be needed to complete their setting, and their slow

setting may result in material leakage out of the capsular bag

through the surgical incision. Furthermore, the used silicones

showed severe posterior capsular opacification. Hettlich et al.28

reported endocapsular polymerization in which a monomer

mixture was injected and photopolymerized in situ to form the

gel. The toxicity of unreacted monomers and exothermic nature

of the polymerization reaction were indicated as potential risks

to living tissues.29 Ravi et al.30 reported on an injectable hydrogel

prepared by oxidation of aqueous solution of acrylamide

copolymers containing pendant thiol (–SH) groups. Endocap-

sular gelation was achieved through a thiol–disulfide exchange

reaction by adding 3,30-dithiodipropionic acid. Although this

reversible hydrogel system could circumvent monomer toxicity

and heat of polymerization, it is still difficult to control endo-

capsular cross-linking, and its refractive index is too low to be of

value as an injectable IOL.

The ideal IOL material, in addition to dimensional stability in

the lens capsular bag, should satisfy both mechanical and optical

properties. Along this line of reasoning, several in situ forming

hydrogels for IOL have been developed. For most common

hydrogels formed from water-soluble polymer, however, the

refractive index is close to that of water (1.3333). Thus far we

could not satisfy both the required modulus and the refractive

index of the gel; if the modulus of the gel was in the appropriate

range, the refractive index was too low, or if the refractive index

was equivalent to the natural lens, the modulus was too high.

Individually, few pure materials embody all of the physical and

mechanical properties required for a given application; thus,

most such materials are used as composites. Organic–inorganic

hybrid materials have attracted attention since they can be

tailored to combine the advantages of organic polymers with

those of inorganic components yielding materials, which possess

enhanced mechanical properties, chemical resistance, optical

quality, and other useful properties which arise from the syner-

getic interaction of the individual organic and inorganic

constituents. In this study, we embed inorganic nanoparticles in

the polymer matrix to manipulate the refractive index while

maintaining the elastic performance of the polymer. Under the

condition of uniform distribution of nanoparticles with small size

(2–5 nm) in the gel matrix, we could expect the nanoparticles not

to scatter the visible light, and the transparency of the gel remains

excellent. The set of properties of this mixture is determined by

both components, namely polymer and nanoparticles, and by the

ratio of concentrations of them. At a rather high concentration

of nanoparticles with small size in the polymer matrix, the

nanocomposite gel is expected to effectively become a homoge-

neous medium, having an increased refractive index with low

scattering.

In this study, therefore, we focus on the development of

a novel injectable accommodative lens for intraocular applica-

tions, which is based on a thermosensitive hydrophobically

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modified poly(ethylene glycol) (HM-PEG) containing silica

nanoparticles. Poly(ethylene glycol) monomethyl ether end-

capped with an octadecyl group (E5KMC18) has the advantage

to form a gel that completely fills the capsular bag and provides

the elastic modulus, and octa(3-chloroammoniumpropyl)silses-

quioxane (OCAPS) nanoparticles have a high refractive index.31

Furthermore, the small distance between OCAPS nanoparticles

(diameter z 2 nm) provides an increase in the refractive index.

This composite system could be formulated to match the

modulus and the refractive index of the natural lens.

In filling the capsule, it might more closely resemble the action

of the young, natural lens, eliminating the possibility of any

intracapsular space for cell growth. Since a full-sized lens may

resist the fibrotic effect of the capsule, decentration and edge

glare may not be factors. In addition, a lens that has the

dimensions of the natural lens may overcome the problem of

spherical aberration induced by other artificial lens designs.

Furthermore, by refilling the capsular bag, the gel allows a lower

rate of retinal detachment seen more frequently with high

myopes. Another benefit is the relatively high refractive index of

the material. This may tend to increase the accommodative effect

with just small changes in shape or position, giving large changes

in accommodation with small, incremental changes in the

lens itself.32

From the structural viewpoint, the crystalline lens is a fasci-

nating result of natural evolution, and its structure is totally

different from the one of the E5KMC18 gel matrix. The goal of

this study, however, is to develop the accommodative IOL by

mimicking the physicochemical properties (e.g. viscoelasticity)

and optical properties (e.g. refractive index) of the natural lens. It

is not necessary to match the structure, composition and physi-

ology of the natural lens as well. Therefore, homogeneous

synthetic hydrogels are being evaluated as IOLs because they are

capable of matching the physicochemical and optical properties

of a natural lens.

Experimental section

Materials

Poly(ethylene glycol) monomethoxy ether (mPEG, Mw ¼ 5000,

Mw/Mn ¼ 1.15, Aldrich) was purified by chromatography on

activated alumina and lyophilized. 1,4-Dioxane (Wako) was

purified using standard methods. Octadecyl bromide (Wako),

sodium hydride (Wako) and 3-aminopropyltrimethoxysilane

(Gelest) were used as received.

Preparation of mPEG end-capped with an octadecyl group

(E5KMC18)

mPEG end-capped with an octadecyl group was obtained via

Williamson reaction of octadecyl bromide on metalated mPEG.

mPEG (5.0 g; 0.50 mmol) was dissolved in 100 mL 1,4-dioxane.

Sodium hydride (purity 70%, 0.18 g; 5.0 mmol) was added por-

tionwise and the mixture was stirred for 1 h under nitrogen

atmosphere. 1-Bromooctadecane (2.6 mL; 7.5 mmol) was added

dropwise for 1 h. The solution was stirred for 24 h at room

temperature under nitrogen atmosphere. The crude product was

precipitated by the dropwise addition to 2000 mL of diethylether.

This precipitation process was repeated twice. The degree of

This journal is ª The Royal Society of Chemistry 2012

substitution of the hydroxyl groups was determined by 1H-NMR

and was found to be 100%.

Preparation of octa(3-chloroammoniumpropyl) silsesquioxane

(OCAPS)

OCAPS was prepared by acidic condensation of the 3-amino-

propyltrimethoxysilane according to the procedure described in

the literature.33,34 To 3-aminopropyltrimethoxysilane (100 mL,

0.427 mol) dissolved in methanol (800 mL) was added conc.

hydrochloric acid (135 mL) under stirring. The reaction mixture

was stirred for one week until the OCAPS precipitated as

a white powder at ambient temperature. The resulting

OCAPS was then washed with cold methanol and dried under

vacuum.

Preparation of an E5KMC18/OCAPS nanocomposite gel

The OCAPS nanoparticles are well dispersed and behave like

a dissolved molecule in water. The E5KMC18/OCAPS nano-

composite gel was prepared by suspending the E5KMC18 in

different concentrations of aqueous solutions of OCAPS. In this

study, the concentration of E5KMC18 was kept at 30 wt%, and

the concentration of OCAPS was varied from 0 to 40 wt%.

Nanocomposite gels are identified further as EmSn, where m

and n, respectively, represent the concentrations in weight of

E5KMC18 and OCAPS.

Rheology

The viscoelastic properties were investigated by oscillatory

measurements on a controlled ARES instrument (TA Instru-

ments, New Castle, USA) equipped with a cone-plate geometry

(diameter: 20 mm) and solvent trap. A frequency sweep

experiment was conducted in the linear viscoelastic regime of

the samples determined by strain sweep experiments.

Frequency varied from 0.001 to 100 s�1. Strain sweep experi-

ments were carried out with a fixed frequency of 1 s�1.

Temperature sweep experiments were performed at a heating

rate of 1 �C min�1.

Small-angle neutron scattering

The small-angle neutron scattering (SANS) experiments were

performed using a SANS-II spectrometer at the Swiss spallation

neutron source SINQ, Paul Scherrer Institute, in Switzerland.35

The data shown in this report were all obtained using neutrons

with a wavelength equal to 6.0 �A and a wavelength spread of 9%.

The collimation length and the sample-to-detector distance were

both 2.0 m. The temperature of the sample was kept constant

within �0.5 �C accuracy in a quartz cell with an optical length of

2 mm. The observed scattered intensity was corrected for cell and

solvent scattering, incoherent scattering, and transmission by

conventional procedures. The two-dimensional isotropic scat-

tering spectra were azimuthally averaged, converted to an

absolute scale, and corrected for detector efficiency by dividing

by the incoherent scattering spectrum of pure water, which was

measured with a 2 mm cell.

Soft Matter, 2012, 8, 7185–7196 | 7187

Fig. 1 (a) Phase diagram for aqueous solutions of EmS30 (m¼ 0–35 wt%,

COCAPS ¼ 30 wt%) as a function of E5KMC18 concentration Cp. Sol–gel

transition temperature for a pure E5KMC18 gel (EmS0, m ¼ 0–35 wt%,

COCAPS ¼ 0 wt%) is plotted for comparison. Sol–gel transition temper-

ature was determined from the intersection temperature between storage

modulus G0 and loss modulus G0 0 during heating at a rate of 1 �C min�1

under the condition of g ¼ 1% and u ¼ 1 s�1. Lines are guide to the eye.

(b) Sol–gel transition temperature Tc and storage modulus G0 at 35 �C(g ¼ 1%, u ¼ 1 s�1) for nanocomposite gel E30Sn (Cp ¼ 30 wt%, n ¼0–40 wt%) as a function of OCAPS concentration COCAPS.

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Determination of the phase diagram

The sol–gel transition temperature Tc was determined from the

intersection temperature between storage modulus G0 and loss

modulus G0 0 during heating at a rate of 1 �C min�1 under the

condition of g ¼ 1% and u ¼ 1 s�1.

Transmission

Transmission of light was measured using a JASCO V-660

spectrophotometer within the range of 200 nm to 800 nm in

a quartz cell with an optical length of 10 mm at 35 �C.

Refractive index

The refractive index was measured with an ATAGO DR-M2/

1550 refractometer. For all measurements, the temperature was

kept at 35 �C with an accuracy of �0.1 �C.

Endocapsular gelation

The E5KMC18/OCAPS nanocomposite gel, composed of 30 wt

% E5KMC18 and 30 wt% OCAPS, was evaluated for endocap-

sular gelation. Freshly dissected pig eyes were purchased from

a local abattoir shortly after slaughter. After a capsulotomy

(continuous circular capsulorhexis) of �2.0 mm diameter near

the equator of the anterior capsular bag, phacoemulsification

and aspiration were performed by using the INFINITI� Vision

System (Alcon Laboratories, Inc. Fort Worth, Texas). The

E5KMC18/OCAPS nanocomposite gel, which is in a soft gel

state at 40 �C, was injected carefully and quickly into the bottom

of the capsular bag and allowed to fill without bubbles by using

a syringe with a 27-gauge needle. The E5KMC18/OCAPS

nanocomposite gel reached equilibrium within 1–2 min.

Cytotoxicity

A murine catecholaminergic cell line derived from the B6/D2 F1

mouse, CATH.a, was seeded in six-well culture dishes at

a density of 1 � 104 cells per well in Dulbecco’s modified Eagle’s

medium (DMEM, SIGMA) supplemented with 10% fetal bovine

serum (FBS, GIBCO/BRL), 50 U mL�1 penicillin and 50 mg

mL�1 streptomycin (GIBCO). The cells were incubated at 37 �Cin a humidified 5% CO2 atmosphere. After 7 days of culture, the

medium in the wells was replaced with the fresh medium con-

taining a varying concentration of the E5KMC18 polymer (5, 10,

and 30 wt%). After 2 days, cell viability was assessed by trypan

blue exclusion.

Results and discussion

Phase behavior of the E5KMC18/OCAPS nanocomposite gel

Hydrophobically end-capped poly(ethylene oxide) methyl ether

(mPEG) with an aliphatic end group (a-PEG) belongs to the

family of the associative polymers36–44 and was selected as the

main element for the intraocular lens material. a-PEG was

prepared by functionalization of the terminal hydroxyl group of

mPEG (Mw ¼ 5000) with an octadecyl group, E5KMC18. The

degree of substitution of the hydroxyl groups was determined by1H-NMR and was found to be 100%. Above a certain

7188 | Soft Matter, 2012, 8, 7185–7196

concentration the polymers associate into well-defined small

aggregates to formmicelles which is due to the fact that octadecyl

groups have a tendency to form hydrophobic aggregates in

water. At higher concentration, they aggregate through hydro-

phobic interaction and percolate through the whole system to

form the network-like structure of the transparent gel.45

The time constant sdiss for dissociation of the sticker from

a micelle can be related to the activation barrier energy Dm for

dissociation by

sdiss ¼ U0�1eDm=kBT (1)

where Dm is the free energy of micellization per sticker, and U0 is

a fundamental vibrational frequency, U�10 � 10�10 s.46 For an

alkyl chain, Dm increases by roughly 1.5 kBT per CH2 unit.

Roughly consistent with this, Annable et al.47 found that the

relaxation time s and the zero-shear viscosity h0 of a typical

telechelic hydrophobically modified ethoxylated urethane

(HEUR) solution increase exponentially with the number of CH2

units in the sticker, with an increment of around 0.9 kBT in Dm

per methylene unit for stickers containing 12–22 CH2 units.

When Dm/kBT [ 1, there will be few free stickers, and we can

expect structural stability of the network with a long end-group.

Therefore we selected the octadecyl end-group as a sticker by

taking the molecular weight of mPEG into consideration.

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Fig. 1a shows the sol–gel state diagram for the E5KMC18/

OCAPS nanocomposite gel with fixed COCAPS ¼ 30 wt% (EmS30,

m ¼ 15–35) as a function of temperature and concentration of

E5KMC18, CP. We distinguished macroscopically, with changes

in the temperature or concentration, two regions in the phase

diagram for an aqueous solution of E5KMC18 in weight:

transparent sol and transparent gel. The sol–gel transition

temperature Tc was determined from the intersection tempera-

ture between storage modulus G0 and loss modulus G0 0 duringheating at a rate of 1 �Cmin�1 under the condition of g¼ 1% and

u ¼ 1 s�1. Tc for aqueous solution of pure E5KMC18 is also

plotted for comparison. The temperature and concentration

regime in which the gel formed are reduced by adding OCAPS

nanoparticles in the gel matrix. The effect of adding OCAPS

nanoparticles was studied in more detail for a constant

E5KMC18 of 30 wt% (E30Sn). The concentration of OCAPS,

COCAPS, was varied from 0 to 40 wt%. As shown in Fig. 1b, both

the sol–gel transition temperature Tc and the storage modulus G0

at 35 �C (g ¼ 1%, u ¼ 1 s�1) decreased with increasing COCAPS.

This may be attributed to the osmotic pressure due to the addi-

tive OCAPS nanoparticles. OCAPS nanoparticles can penetrate

the corona of the micelles and swell them, which leads to loose

corona–corona connectivity. In other words, OCAPS solution

acts effectively as a solvent of enhanced quality for the PEG

chains.48–51

Microscopic structure of the E5KMC18/OCAPS nanocomposite

gel

The self-assembly of E5KMC18/OCAPS polymers suspended in

heavy water was examined by small-angle neutron scattering

(SANS). We have studied the effect of increasing OCAPS

concentration, as obtained at a fixed E5KMC18 concentration of

30 wt% (E30Sn, n ¼ 0–30) and fixed temperature (35 �C), andversus the temperature for E30S30. Fig. 2 shows examples of

experimental SANS data of E30Sn (n ¼ 0–30) polymers, as

measured in heavy water suspensions. Fig. 2a–c show SANS data

for E30Sn (n ¼ 0, 10, and 30) at T ¼ 25–65 �C. Fig. 2d–f show the

corresponding presentation of data for E30Sn (n¼ 0, 5, 10, 20 and

30) as obtained at temperatures of 25, 45, and 65 �C.Most of the SANS data are characterized by a pronounced

correlation peak close to q ¼ 0.040–0.045 �A�1. Many of the

patterns exhibit clear higher-order reflections signifying real

crystalline order of the micelles. These characteristics are

observed in the part of the phase-diagram that corresponds to the

gel-phase (see Fig. 1). The patterns obtained at intermediate

temperatures and concentrations, on the other hand, show the

characteristics of liquid micellar systems. The scattering patterns

obtained in the high-temperature and high OCAPS concentra-

tion regime reflect a two-phase system. Fig. 3 shows the phase

diagram according to the classification of micellar crystal,

micellar liquid, and two-phase regime.

The scattering functions observed for the 10% OCAPS sample

at 62 �C, the 20% OCAPS sample at 55 �C (not shown), and the

30% OCAPS sample at 45 �C SANS show significant low-q

scattering indicating the vicinity of a two-phase boundary. Visual

inspection of the samples at 65 �C confirms the separation into

two macroscopic phases at this temperature, showing a meniscus

between the two phases (Fig. 4). The refractive index difference

This journal is ª The Royal Society of Chemistry 2012

of the two phases, and thereby the visibility of the meniscus,

seems most pronounced in the high OCAPS-concentration

samples. Fig. 4 shows the volume fraction of the upper phase

versus the concentration of OCAPS nanoparticles as obtained at

a temperature close to 65 �C. The SANS patterns obtained for

the phase-separated samples are dominated by the properties of

the upper phase (see Fig. 4b), since the neutron beam passed

through this part of the quartz cuvette. The scattering pattern of

the material shows an ordered structure of micelles within the

gel-phase. The peak-positions correspond to that of bcc. Fig. 5

shows two examples where the scattering function of a model of

spherical micelles approached as simple solid spheres ordered on

a bcc-lattice is fitted to the experimental SANS data. The

smearing of the experimental data due to finite resolution is

taken into account in the fitting routines by convoluting the

model scattering function with the appropriate smearing func-

tion.52 The figure shows the pristine pure polymer suspension:

30% E5KMC18 polymer in D2O (E30S0) and the equivalent

sample with 20% E5KMC18 polymer and 30 wt% OCAPS

nanoparticles (E20S30). Both samples are measured at 25 �C (see

also the phase diagram Fig. 3). The fitting in all cases is very

satisfactory, giving good proof of a gel phase where the high

shear-modulus is gained by a high inter-micellar correlation

originating in the crystalline order. The micellar size changes

from approximately 51 �A for the system with no silica to 57 �A for

the system with 30% OCAPS. The crystalline phase is charac-

terized by a correlation length of the order of 750 �A and 590 �A

for the same two systems.

Further evidence of the bcc-ordered micellar structure of the

gel-phase is obtained from the scattering pattern of a sample that

has been exposed to a slight shear. The 2D-pattern shown in

Fig. 6a is SANS data of the 30% E5KMC18 polymer after being

‘‘hand-sheared’’ slightly between two alumina plates. The inten-

sity is shown in the logarithmic scale to include the weaker high-

order Bragg reflections. The pattern shows a pronounced texture

corresponding to a bcc-twin structure with the [111]-axis in the

(vertical) shear direction, and two [110]-axes ([�1�10] and [1�10]) in

the horizontal neutral direction. Fig. 6b shows the calculated

scattering pattern of such a twin domain structure, in perfect

agreement with the experimentally obtained pattern. Corre-

sponding shear-alignment was obtained in the E30S30 composite

gel even though we apparently lost some water during that

treatment due to the lack of environmental control. These data

are therefore not shown here. The change in concentration was

easily seen from the position of the Bragg-reflections.

The SANS structural data of the intermediate temperature and

concentration regime are well fitted to the model of hard-sphere

interacting polymeric micelles,

I(q) ¼ KPmic(q)Shs(q) (2)

where q ¼ (4p/l)sin(q/2) is the length of the scattering vector, l

being the neutron wavelength and q the scattering angle. Pmic(q)

is the form factor of polymeric micelles as described in ref. 53,

and Shs(q) is the form factor given by the Percus–Yevic

approximation using a hard-sphere interaction potential.54 The

model fits the data very well, as shown in the examples displayed

in Fig. 7, showing results of the pristine E5KMC18-polymer, and

the E30S30 composite gel with 30% OCAPS nanoparticles, as

Soft Matter, 2012, 8, 7185–7196 | 7189

Fig. 2 Experimental small-angle neutron scattering data, intensity versus scattering vector, of EmSn (m concentration of E5KMC18 polymer, nOCAPS

concentration). (a), (b) and (c) highlight the temperature dependence of I(q) for E30S0, E30S10, and E30S30, respectively, plotted for temperatures equal to

25, 35, 45, 55, 62 and 65 �C. (d), (e) and (f) highlight the OCAPS concentration dependence of I(q) for 25, 45 and 65 �C, respectively, plotted for OCAPS

concentrations equal to 0, 5, 10, 20 and 30 wt%.

Fig. 3 Phase-diagram of EmSn (m concentration in weight of the

E5KMC18 polymer, n concentration in weight of the OCAPS nano-

particle) versusOCAPS concentration and temperature, as obtained from

scattering experiments and visual inspection. C: bcc-ordered gel-phase,

B: liquid micellar phase, and >: demixed into two phases.

Fig. 4 (a) Fraction of the upper phase of the demixed state of EmSn, as

obtained from (b) the position of the meniscus in the sample.

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obtained at different temperatures. A slight deviation between

the fit and experimental data in the E30S30 sample at 45 �Creflects that this sample is slightly demixed into two phases, as

discussed above (see also the phase diagram shown in Fig. 3).

The resulting parameters are the micellar volume fraction, f, the

micellar core size, Rc, the size of the polymer chains of the

corona, Rg, and the micellar interaction distance, Rhs.55,56

Fits to the liquid scattering function, eqn (1), were extended to

experimental data obtained within the bcc-ordered gel phase as

7190 | Soft Matter, 2012, 8, 7185–7196

well as in the demixed regime. Fig. 8a and b show the resulting

volume fraction plotted versus the temperature and concentra-

tion of OCAPS nanoparticles. The closed symbols represent

fit-results from samples that obey a single-phase liquid structure

and give rise to good fits within the whole measured q-range. The

open symbols represent fits where the sample is either in the

ordered bcc phase or in the demixed two-phase regime. These

latter fits are typically good only near the main correlation-peak,

and the resulting parameters should be accordingly taken with

some reservations.

The samples obey ordered structures for volume fractions f

beyond approximately 0.47, as verified from the scattering

This journal is ª The Royal Society of Chemistry 2012

Fig. 5 Small-angle neutron scattering data E30S0, E20S30, as obtained at

25 �C. Solid lines represent the best fit to solid spheres (micelles) ordered

on a bcc-lattice.

Fig. 6 (a) Experimental scattering pattern of the shear-oriented polymer

gel of 30% E5KMC18 polymer in D2O obtained at 25 �C. (b) Theoreti-cally calculated scattering pattern of the twin-bcc structure with common

(110)-planes ([�1�10] and [1�10] axes horizontal) and [111]-axis vertical. The

scattering pattern of the two twin domains is highlighted in red and blue

color, respectively.

Fig. 7 Experimental SANS scattering function, I(q), of EmSn composites

within the liquid micellar regime. (a) I(q) of pristine 30% E5KMC18

suspension obtained at 62 and 65 �C. (b) SANS I(q) of E30S30 composites

obtained at 25, 35 and 45 �C, respectively. The solid lines represent best

fits to the hard-sphere interacting micellar scattering function.

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pattern which better fits the bcc-ordered micellar phase in this

regime. This critical value of f is in perfect agreement with hard-

sphere induced ordering, as seen in a variety of equivalent

micellar systems. The effective volume fraction shows a clear

tendency to decrease with both temperature and OCAPS

concentration. The parameters describe the micellar size that is

rather correlated, and the scattering data do not allow inde-

pendent values of micellar core, Rc, and corona polymer char-

acteristics, Rg. We found values for Rc of the order of 8 �A and Rg

of the order of 25–30 �A, in reasonable agreement with the results

of simple solid micelles (50 �A) obtained from fits in the ordered

This journal is ª The Royal Society of Chemistry 2012

regime. We found no systematic variation in micellar dimensions

versus temperature or OCAPS concentration. A model of

micelles represented by simple solid spheres does not fit the data

in the liquid-micellar regime.

Fig. 8c and d show the hard-sphere interaction distance plotted

versus the temperature and concentration of OCAPS nano-

particles. As in the plot of the volume fraction shown in Fig. 8a

and b, only the closed symbols represent fit-results from samples

that obey a single-phase liquid structure. The open symbols

represent fits where the sample is either in the ordered bcc

phase or obey two-phases. The characteristic distance is rather

independent of the OCAPS concentration, but decreases

systematically with temperature.

Optical properties of the E5KMC18/OCAPS nanocomposite gel

Fig. 9a shows the dependence of the refractive indices of the

30 wt% E5KMC18 gel on the COCAPS. The refractive index of

the gel increases with increasing COCAPS. At COCAPS ¼ 30 wt%,

the refractive index was 1.411, compared with a refractive index

of 1.366 for the pure 30 wt% E5KMC18 gel. Thus, the increase in

the refractive index is 0.045. The nanocomposite gel is clear and

colorless even at COCAPS ¼ 30 wt%. This transparency of the gel

is considered to be a result of uniform distribution of OCAPS

nanoparticles inside the structure of a nanocomposite at optical

wavelength. At a rather high concentration of nanoparticles with

diameters much smaller than the wavelength of visible light, the

nanocomposite effectively becomes a homogeneous medium,

having an increased refractive index and OCAPS does not scatter

the visible light. As shown in Fig. 9b, the E30S30 nanocomposite

gel has a reasonable transmission (>90% ranging from 400 nm to

800 nm), which is comparable to the human lens and filters out

most of the UV light.

Soft Matter, 2012, 8, 7185–7196 | 7191

Fig. 8 Micellar volume fraction f and hard-sphere micellar interaction distance Rhs resulting from fits to the experimental scattering function are

plotted versus temperature and OCAPS concentration. The solid symbols represent scattering data obtained within the liquid phase of micelles. Open

symbols are data from the bcc-ordered gel-phase, or the demixed phase.

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Mechanical properties of the E5KMC18/OCAPS composite gel

To determine the dynamic moduli as a function of applied

frequency, oscillatory frequency sweep experiments were per-

formed. Fig. 10a shows the storage modulus G0 and the loss

modulusG0 0 for the different concentrations of the E5KMC18 gel

Fig. 9 (a) Dependence of the refractive index for the 30 wt% E5KMC18

gel on the OCAPS concentration. (b) The transmission of light for the

E30S30 nanocomposite gel as a function of wavelength ranging from

250 nm to 800 nm.

7192 | Soft Matter, 2012, 8, 7185–7196

as a function of applied frequency at 35 �C. The moduli show the

behavior typical of the gel samples in that G0 was substantiallygreater than G0 0 over the full range of frequency measured. As

a function of E5KMC18 polymer concentration, the G0/G0 0 ratio

Fig. 10 (a) Storage modulus (G0) and loss modulus (G0 0) as a function of

frequency for different concentrations of the pure E5KMC18 gel at

35 �C. (b) Comparison of dynamic moduli (G0 and G0 0) versus frequencyfor the pure 30 wt% E5KMC18 (E30S0) gel and E30S30 nanocomposite gel

at 35 �C.

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varied between 3 and 10 in the gel phase. In Fig. 10b, G0 and G0 0

for the E30S30 nanocomposite gel are compared with those for

pure 30 wt% E5KMC18 gel as a function of applied frequency.

Upon adding OCAPS nanoparticles, the gel persists and becomes

slightly weaker at the same temperature and the same E5KMC18

concentration CP. It is likely that the swelling of the PEG chains

of the micellar corona due to the osmotic pressure generated by

the penetration of OCAPS loosen the corona–corona

connectivity.

During injection of the E30S30 nanocomposite gel into the

capsular bag, a proper flow of the material through a syringe is

crucial. Therefore we investigated the rheological behavior of the

E5KMC18 gel upon stress and deformation. Fig. 11a shows G0

and G0 0 at u¼ 1 s�1 as a function of the amplitude of shear strain,

g, for the E30S30 nanocomposite gel at 35 �C. The gel exhibits

a linear viscoelastic response below a critical shear strain

gc � 1.2%, where G0 > G0 0. Above this narrow linear viscoelastic

regime, G0 decreases as the shear strain increases whereas G0 0

increases, reaches a maximum for g � 12%, and then decreases.

Such aG00 feature at intermediate strain amplitudes was observed

with other hydrophobically modified polymers and was ascribed

to a strain-induced imbalance between the junction destruction

rate and the junction creation rate within the network.57,58

At g � 12%, G00 exceeds G0, and the gel starts to flow and

exhibits sol–gel transition. The relatively low shear strain

amplitude g � 12% required for sol–gel transition allows the

composite gel to form extrudable materials that can be delivered

to the capsular bag by injection. However, only the smooth flow

through a syringe is not enough for the application of an

injectable gel. After being delivered into the capsular bag, the

Fig. 11 (a) Storage modulus (G0) and loss modulus (G0 0) as a function of

shear strain for the E30S30 nanocomposite gel at 35 �C. (b) Evolution of

storage modulus (G0) and loss modulus (G0 0) for the E30S30 nano-

composite gel with time following two successive pulses of high defor-

mation (solid line) at 35 �C.

This journal is ª The Royal Society of Chemistry 2012

nanocomposite gel is required to show the rapid recovery in

a robust gel. We, therefore, applied the large oscillatory shear in

a stepwise manner, as shown in Fig. 11b, to the E30S30 nano-

composite gel for the shear frequency u ¼ 1 s�1 at 35 �C. Theshear strain amplitude g was increased from 1% to 100% or from

1% to 300%, and then the sample was allowed to relax back to

a quiescent state with measurements of the dynamic moduli at

g ¼ 1%. The results are plotted as the dynamic shear moduli

(G0 and G0 0) in response to stepwise changes in shear strain

amplitude g as a function of time: g ¼ 1%, 100%, 1%, 300%, and

1% in Fig. 11b. The onset of nonlinear response is apparent at

100% and 300% strain: the loss modulus G0 0 was larger than the

storage modulus G0. Both G0 and G0 0 decreased as the strain was

increased, showing that the gel was shear thinning. The cessation

of the large-amplitude oscillatory shear led to the rapid recovery

of the original values of G0 and G0 0, which indicates that the

physical junctions of the E5KMC18 micelles through the corona

chain entanglements rapidly formed and percolated through the

whole system to form the network-like structure of the gel.

Temperature sweep measurement of G0 and G00 was performed

at the heating and cooling rates of 1 �C min�1 and a constant

frequency of 1 s�1. The sample was heated from 25 �C to 60 �C.The temperature sweep of G0 and G0 0 for the E30S30 nano-

composite gel is presented in Fig. 12a. With rising temperature,

the moduli start to decrease sharply at 40 �C, and G0 and G00

values at 45 �C are two orders of magnitude smaller than those

for ambient temperature. Upon further increase in temperature,

Fig. 12 (a) Temperature dependence of dynamic moduli (G0 and G0 0) ofthe E30S30 nanocomposite gel during isochronal dynamic temperature

sweep experiments in the heating and cooling process with 1 �C min�1 for

temperatures ranging from 25 �C to 60 �C. (b) Comparison of the

dynamic elastic moduli for the E30S30 nanocomposite gel before and after

extrusion through a 27-gauge needle as a function of frequency

(measured with a cone and plate geometry) at g ¼ 1% and 37 �C.

Soft Matter, 2012, 8, 7185–7196 | 7193

Fig. 13 (a) Normalized stress relaxation modulus G(g, t)/G0 of the

E30S30 nanocomposite gel for strain amplitudes g ranging from 0.4% to

4% at g ¼ 1%, u ¼ 1 s�1, and 35 �C. The continuous lines through the

data represent the best-fitting curves obtained from eqn (3). (b) Variation

of the viscoelastic relaxation time s(g) as a function of applied strain. s(g)is deduced from fitting the stress relaxation in (a).

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the crossover between G0 and G0 0 is observed at approximately

50 �C and marks the transition from the gel state to the sol state.

Decreasing the temperature after an annealing period of 5 min at

60 �C, despite small hysteresis, the rheological behavior of the

E5KMC18/OCAPS nanocomposite gel is essentially thermor-

eversible. The moduli at 25 �C are of the same magnitude before

and after heating the sample to 60 �C.It is preferred that the mechanical properties of the gel remain

unchanged in the capsular bag after injection. In Fig. 12b, the

storage modulus G0 and the loss modulus G0 0 of the E30S30nanocomposite gel before and after extruding through

a 27-gauge needle at 37 �C are compared as a function of applied

frequency. The gel shows the same rheological behavior before

and after extrusion, indicating that the injection does not affect

the network structure of the E30S30 nanocomposite gel. These

features of the polymer indicate that the E30S30 nanocomposite

gel demonstrates the feasibility of the development of an

injectable in situ gelation biomaterial.

E5KMC18 is a mono-functionalized polymer and forms the

micelles similar to starlike polymers. The difference with micelles

formed by double end-capped polymers is the absence of back-

folding and bridging. Therefore, the E5KMC18 gel is formed

when micelles aggregate under an inter-micelle attraction to form

a space-spanning percolated network. All the features displayed

in Fig. 11a, including the G0-dominant linear viscoelastic regime

followed by a pronounced loss maximum prior to the onset of

strain softening, are consistent with expectations for the soft

glasses,59–70 for which this behavior is attributed to strain-

induced breakage of collections of structural units termed

‘‘cages’’, which constrain the motion of individual particles.

The measurement of the relaxation modulus G(t) ¼ s(t)/g

following the application of a step strain g(t) ¼ gH(t), where s is

the stress and H(t) is a step function, provides a straightforward

method for extending the dynamic range of the oscillatory shear

experiment and provides the most direct comparison of experi-

ment data with soft glassy rheology model predictions.69,70 Prior

to each measurement, the E30S30 nanocomposite gel was pre-

sheared using large-amplitude oscillatory shear and reset for

a period of 3600 seconds. The E30S30 nanocomposite gel at 35 �Cwas, therefore, submitted to stress relaxation measurements

in the linear (g ¼ 0.4%, 0.6% and 1.0% < gc) and non-linear

(g ¼ 2.0%, 3.0% and 4.0% > gc) viscoelastic regime. Stress

relaxation functions G(t) at multiple shear strains following the

imposition of the step strain are plotted in Fig. 13a. It is apparent

from Fig. 13a that the decay of G(t) speeds up with increasing

shear strain. The continuous lines in the figure indicate that the

stress relaxation is of the stretched exponential function or

Kohlrausch–William–Watts (KWW) form:67–70

Gðt;gÞ ¼ G0 exp

"��

t

sðgÞ�a

#(3)

where G0 is the instantaneous modulus, t is the elapsed time after

the step, and s is the characteristic relaxation time. The coupling

parameter a (0 < a < 1) is the stretched exponent that quantifies

the departure from the mono-exponential function; it measures

the broadness of the time relaxation distribution, the smaller

a-values corresponding to broader distribution. Stretched

exponentials provided as eqn (3) have been suggested to describe

7194 | Soft Matter, 2012, 8, 7185–7196

the phenomenology of relaxations in complex, slowly relaxing

and strongly interacting materials.57 The characteristic relaxation

time s and coupling parameter a deduced from the fits are

provided in Fig. 13b. It can be seen from the figure that s is

constant at low strains, indicative of the linear viscoelastic regime

and that it decreases at high strains. The exponent a decreases

with shear strain, however, the change is much more modest than

the reports for polymeric glasses.71,72 This is ascribed to the fact

that the system studied here is not in a glassy state, but mesoscale

crystalline ordered micelles where the correlated state is the basis

for a high modulus.

Endocapsular gelation

The suitability of the E5KMC18/OCAPS nanocomposite gel for

in situ endocapsular gelation was demonstrated in a pre-evacu-

ated pig lens capsular bag. As mentioned in a previous section,

the moduli at 45 �C are two orders of magnitude smaller than

those at ambient temperature, therefore we expect a proper flow

of the material during injection and the rapid endocapsular

gelation. The E30S30 nanocomposite gel preheated at 45 �C was

injected carefully and quickly into the bottom of the capsular

bag and allowed to fill without bubbles by using a syringe with

a 27-gauge needle (Fig. 14a). The material shows rapid gelation,

and did not leak out of the capsular bag. Fig. 14b depicts the

nanocomposite gel excised five minutes after injection. It is

confirmed that E5KMC18/OCAPS formed a robust gel rapidly

and kept a proper shape in the lens capsular bag. Objects viewed

through the E5KMC18/OCAPS gel lens appeared clear, undis-

torted and of the same magnification as for the dissected natural

pig lens as shown in Fig. 14c and d.

This journal is ª The Royal Society of Chemistry 2012

Fig. 14 (a) Injection of the E30S30 nanocomposite gel into the pig lens capsular bag by using a syringe with a 27-gauge needle. (b) Lens capsule filled with

the E30S30 nanocomposite gel (top view). (c) Lens capsule filled with the E30S30 nanocomposite gel (side view). The E30S30 nanocomposite gel is

confirmed to rapidly form a robust gel and keep a proper shape in the pig lens capsular bag. (d) Objects viewed through the in situ endocapsular gelled

E30S30 lens appeared clear and undistorted. The E30S30 lens provides the same magnification as for the dissected natural pig lens. A dissected natural pig

lens is shown on the downside for comparison. (e) Cell viability of a murine catecholaminergic cell line derived from the B6/D2 F1 mouse after 48-hour

incubation as measured with a trypan blue assay. Data represent mean � standard deviation (n ¼ 3 for each group).

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Cytotoxicity

Quantitative concentration–toxicity relationships were evaluated

with murine catecholaminergic cells. To assess cytotoxicity,

5 wt%, 10 wt%, and 30 wt% E5KMC18 hydrogels were evalu-

ated, and the cytotoxic effects of the hydrogels are presented in

Fig. 14e.73,74 The tested hydrogels show no adverse effects on the

viability of the cells, where the minimum viability was 96 � 4%.

Therefore it can be concluded that the cytotoxicity of the

E10KDC18 hydrogel is sufficiently low since there is no marked

decrease in the cells interacting with the E10KDC18 hydrogel for

prolonged periods.

The framework of polyhedral oligomeric silsesquioxane

(POSS), constituted by Si–O and Si–C bonds, is similar to sili-

cone, which is a favored option in biomaterials due to the inert

nature and low inflammatory response. Its biocompatibility can

be ascribed to the foci of silicon-rich areas with increased surface

energy.75 Unlike carbon nanotubes,76 POSS moieties have been

confirmed as non-toxic and cytocompatible.77–79 Therefore we

are motivated to incorporate into the PEG-based hydrogel,

E5KMC18, and to extend nanocomposites to the accommoda-

tive IOL.

In future experiments, the biocompatibility with ocular

tissue will be investigated using a perfusion cell culture model of

full-thickness porcine retina, an in vitro model suitable for the

evaluation of biomaterials in more organotypical

environments.80,81

Conclusions

Polymer solutions, capable of forming a gel in a body cavity or

tissues with latent space, have potential uses in ophthalmology as

intraocular lenses, vitreous substitutes, and drug-delivery

devices. Such in situ forming gels have the advantages to form

a gel that completely fills any irregular cavity and provides the

elastic modulus, and of being delivered using minimally invasive

techniques. In this work, we focus on the development of a novel

injectable accommodative lens for intraocular applications,

which is based on a thermosensitive HM-PEG, E5KMC18

containing hydrophilized silica nanoparticles, OCAPS. We

distinguished macroscopically, with changes in the temperature

This journal is ª The Royal Society of Chemistry 2012

or concentration, two regions in the phase diagram for an

aqueous solution of E5KMC18/OCAPS in weight: transparent

sol and transparent gel. These changes occurred reversibly,

without hysteresis, when the temperature was decreased. The

temperature and concentration regime in which the gel formed

were reduced by adding OCAPS in the gel matrix. Small-angle

neutron scattering measurements for nanocomposite gels

provide good proof of a gel phase where the high shear-modulus

is gained by a high inter-micellar correlation originating in the

crystalline order. By injection of elastic polymers into the

capsular bag of the eye as a fluid, body temperature transforms

the polymer into an optically clear gel that has the shape of

a full-sized biconvex and completely fills the capsular bag. The

gel is entirely cohesive and does not leak out of the capsular bag.

Under the condition of uniform distribution of silica nano-

particles with a small size (2–5 nm), the micellar ordered struc-

ture, giving rise to the gel-like matrix, is retained and an increase

in refractive index up to 0.0667 was obtained for a nano-

composite compared with a native gel matrix without an increase

in turbidity. This composite system could be formulated to

match the modulus and the refractive index of the natural lens

(�1.411). The potential benefits of a full-sized, flexible accom-

modating lens system go beyond providing the accommodation

lost with age. In filling the capsule, it might more closely resemble

the action of the young, natural lens, eliminating the possibility

of any intracapsular space for cell growth. Further in vivo study

on long-term biocompatibility is under way and will be reported

in future publication.

Acknowledgements

The work was partly supported by a grant-in-aid (no. 22350053

and 24655103) and a grant-in-aid for the Global COE Program,

‘‘Science for Future Molecular Systems’’ from the Ministry of

Education, Culture, Science, Sports and Technology of Japan,

and from the UNIK Synthetic Biology and DANSCATT

programs of the Danish Research Council.

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