Layer-by-layer Assembled Films for Ocular Drug Delivery

onica Ara

ujo

, Jorge Morgado

1,2

and Quirina Ferreira

Instituto de Telecomunicac¸

oes, Av. Rovisco Pais, 1049-001, Lisbon, Portugal

Bioengineering Department, Instituto Superior T

ecnico, University of Lisbon,

Av. Rovisco Pais, 1049-001, Lisbon, Portugal

Keywords:

Drug Delivery, Layer-by-layer Films, Glaucoma.

Abstract:

In this paper we describe a simple and versatile method to prepare drug delivery ﬁlms composed of an ocular

drug used in glaucoma treatment, brimonidine, which was encapsulated in a polymer-beta cyclodextrin. The

ﬁlms were developed in order to allow a controlled sequential release during long periods of time. Here we

show that by introducing barrier layers of graphene oxide between the drug delivery ones it is possible to

delay the brimonidine release for a few days. The time interval between two dosages of drug release will be

controlled by adjusting the number and/or thickness of the graphene layers.

1 INTRODUCTION

The layer-by-layer method is a simple and versatile

tool for the controlled fabrication of thin ﬁlms to a

wide range of purposes (Raposo and Oliveira, 2000;

Ferreira et al., 2014; Ferreira et al., 2012). This

method was primarily introduced by Decher in 1992

as an assembly technique based on complementary

chemical interaction (Decher et al., 1992). However,

in theory, hydrogen bonding, Van der Waals forces

and also biomolecular recognition (i.e. any comple-

mentary interaction) can be used (Seo et al., 2008;

Ferreira et al., 2012; Ferreira et al., 2014). This tech-

nique is independent of the substrate type and due to

its simplicity, versatility and robustness has been ap-

plied to biomolecules, biosensores, implantable mate-

rials and drug delivery systems. Several surfaces can

be used to adsorb multilayers ﬁlms such as metals,

polymers, glasses and any kind of biomaterial (Tang

et al., 2006; Ferreira et al., 2007; Ferreira et al., 2014).

The LbL technique enables the formation of com-

plex multilayer ﬁlms merely through the sequential

adsorption of oppositely charged polymers, ceram-

ics, nanoparticles and biological molecules. With this

kind of deposition, it is possible to obtain ultrathin

mono-, bi- or multilayers with precision at molecular

scale. Varying the process parameters, such as con-

centration of the components, pH, ionic strenght and

immersion time, it is possible to ﬁne-tune the ﬁlms

(Hal

asz et al., 2015; Oliveira, O.N.Jr.; He, J.-A.; Zu-

colotto, V.; Balasubramanian, S.; Li, L.; Nalwa, H.S.;

Kumar, J.; Tripathy, 2002). A wide range of technolo-

gies, with different standard tools and procedures, can

be applied for the production of LbL ﬁlms depending

on the intended application.

In this article we present drug delivery (DD) ﬁlms

fabricated by LbL method which can be used for glau-

coma treatment. Glaucoma is an ocular degenera-

tive disease caused by optical nerve inﬂammation and

leads to an intraocular pressure (IOP) increase which

can cause total loss of vision. Its treatment, at initial

stage, is based on the prescription of eye drops com-

posed of an α

adrenergic agonist such as brimoni-

dine. However the non-compliance of the patients

(Leit

ao et al., 2010; Nordstrom et al., 2005) for the

auto-administration of the eye drops, as well as the

low ocular bioavailability leads to the progress of the

disease (European Glaucoma Society, 2014). The use

of novel controlled drug delivery systems have proved

to be particularly interesting since it increases the res-

idence time of drugs in the eye.

In order to develop an autonomously system able to

release brimonidine during long periods we develop

multilayers and biocompatible ﬁlms with DD func-

tion. Brimonidine was encapsulated in a polymer-β-

cyclodextrin (PolyCD) (see Figure1) and the release

was controlled by the presence of barrier layers com-

posed of two materials: an hydrosoluble polymer -

poly beta aminoester (PBAE) and charged graphene

oxide(GO) layers (see Figure2). The PBAE was

used to control de brimonidine release. PBAE is a

cationic polymer, degradable by hydrolysis of the es-

AraÃžjo M., Morgado J. and Ferreira Q.

Layer-by-layer Assembled Films for Ocular Drug Delivery.

DOI: 10.5220/0006320503950401

ter bonds of the backbone at physiological relevant

pH (Zugates et al., 2007; Macdonald et al., 2008).

It was designed by Langer and co-workers, produced

through Michael addition polymerization of acrylate

and amine monomers (Lynn and Langer, 2000; Smith,

2010). Studies in vivo showed that the hydrolysis

of the polyester backbone of the polymer persists for

several hours to a few days, but this property is largely

affected by the polymer structure as well as the sur-

rounding cellular condition (Deng et al., 2014). The

GO is a single layer of two-dimensional carbon lat-

tice tightly packed (Hong et al., 2012). GO is a

graphene sheet funcionalized with oxygen-rich func-

tional groups in the form of ether, hydroxyl, carboxyl,

and epoxy groups (Choi et al., 2013). Graphene and

GO layers have become an appelative ﬁeld of study

due to the promising biomedical applications revealed

by these nanomaterials, such as in enzime adsorption,

cell imaging, biosensors and drug delivery. Due to the

fact that GO is highly hydrophilic, planar and chem-

ically stable, it is possible to use these nanosheets as

a temporary protective layer coating for the PBAE

and poly-CD, delaying hydrolysis (Choi et al., 2013;

Bosch-Navarro et al., 2012).

Results on the growth of ﬁlms composed of brimoni-

dine encapsulated in PolyCD and intercalated with

layers of PBAE and GO are discussed in this article.

The brimonidine release was followed under physio-

logical conditions.

2 EXPERIMENTAL SECTION

2.1 Materials

The poly-β-cyclodextrin (polyCD) and the brimoni-

dine (see ﬁgure 1) were purchase from Sigma Aldrich

and used as received.

The poly-β-aminoester (PBAE) (see ﬁgure 2)

was synthetized using the protocol described by

Lynn et al. (Lynn and Langer, 2000), adding 3.28

g of 4,4

-trimethylenedipiperidine (S1) (97% purity,

Sigma Aldrich, CAS number 16898-52-5) added

to 2.87 mL of 1,4-butanediol diacrylate (S2) (99%

purity, Alfa Aesar, CAS number 1070-70-8). The

copolymerization of these monomers was carried

out in THF (that was previously distilled) at a

temperature of 50

◦

C, during 48 h. The ﬁnal polymer

PBAE, (Figure 2) was puriﬁed through repeated

precipitation into diethyl ether. The precipitated

polymer was vacuum ﬁltrated with a Buchner funnel

and left to dry in vacuum over night. The structure of

the ﬁnal product was conﬁrmed by nuclear magnetic

Figure 1: Chemical structure of a) β-cyclodextrin polymer

and b) brimonidine. The polymer is composed of n rings of

cyclodextrin. R corresponds to the hydroxyl group present

in the molecule.

resonance spectroscopy. It was further characterized

by gel permeation chromatography.

The negatively charged graphene (GO-COO

−

)

was purchased from Graphenea, as an aqueous

dispersion with a concentration of 0.5 mg/mL (see

Figure 2). The negative charges are due to the

presence of carboxylic acids that deprotonate in low

pH.

The positive graphene (GO-NH

) was pre-

pared in laboratory reducing the negative GO

and linking amine groups to the carboxylic acids

(Hwang et al., 2012). A method developed

by Hwang, et al. was used to prepare posi-

tively charged GO. 50 mL of negative GO solu-

tion were mixed with 0.625 g of N-ethyl-N’-(3-

dimethylaminopropyl)carbodiimede (EDC) (Sigma

Aldrich) and with 5 mL of ethylenediamine (Sigma

Aldrich). The solution was left stirring for 12 hours.

EDC reacted with carboxylic groups activating the

coupling of ethylenediamine. A dialysis of the ﬁnal

solution was performed in order to separate the func-

tionalized graphene from the secondary products.

2.2 Methods

The ﬁlms were prepared by layer-by-layer technique

(Ferreira et al., 2014). Quartz substrates were used

to adsorb the ﬁlms that were previously submitted to

oxygen plasma and immersed in a piranha solution

in order to clean all organic residues and to nega-

tively charge the surfaces. A quartz crystal lamella

was immersed into a solution of PBAE. After re-

maining in this solution during 5 minutes, the sub-

strate was rinsed with sodium acetate (with pH ad-

justed to 5.0) in order to remove all the molecules

that are not adsorbed, or only physically adsorbed

and then dried with nitrogen gas. After this sequence

of steps a monolayer of PBAE formed. Then the

substrate was immersed, one more time, but now in

Figure 2: Schematic representation of chemically modiﬁed

graphene oxide a) GO-COO

−

and b) GO-NH

. c) Chemi-

cal structure of poly(β-amino ester) (PBAE).

the polymeric solution of polyCD. The quartz sub-

strate was left in the polyCD solution during 5 min-

utes and then was washed in sodium acetate (pH=5.0)

and dried with nitrogen. This process completes one

cycle of the LbL assembly, forming one bilayer of

(PBAE/polyCD). The deposition cycle was repeated

the number of times equivalent to the number of bi-

layers intended.

3 RESULTS

The DD LbL ﬁlms were prepared using the LbL

technique, where each layer adsorption was followed

by UV-Vis spectroscopy. Two types of ﬁlms were

performed: ﬁlms with polymeric bilayers of PBAE

and PolyCD+Brim and ﬁlms with charged GO lay-

ers intercalated between the polymeric ones, see the

schematic illustration of Figure 3.

Figure 4 shows the absorption spectra of each

(PBAE/PolyCD) bilayer of a ﬁlm with 4 bilayers.

The ﬁlm has an almost linear growth, established by

an increase in the absorbance of brimonidine, which

means that more molecules are being added to the

ﬁlm. The brimonidine release was also followed

by UV-Vis spectroscopy. The LbL ﬁlms were im-

mersed in a Phosphate Buffer Saline (PBS) solution

that has properties similar to those of biological ﬂu-

ids, in terms of pH (pH=7.4, equal to the physiologic

pH) and concentration of salts. A phosphate buffered

saline solution consists on a phosphate buffer with a

concentration of 0.01M and a sodium chloride con-

centration of 0.154 M. The experiments were done at

◦

C in order to mimic the physiological conditions

where a glass beaker with PBS was maintained in-

Figure 3: Schematic representation of drug delivery layer-

by-layer (DD LBL) ﬁlms. a)DD LBL ﬁlm composed

of 4 bilayers of (PBAE/PolyCD + Brim)

. b)DD LBL

ﬁlm composed of a graphene bilayer of charged graphene

between DD bilayers - ((PBAE/PolyCD + Brim)

/GO −

COO

−

/GO − NH

/(PBAE/PolyCD + Brim)

side an oven at this temperature. The PBS solution is

changed at the end of each immersion. After a spe-

ciﬁc period of time the substrate was removed, dried

with a nitrogen ﬂux and its absorption spectrum was

recorded. The Figure 5 shows the absorption spec-

trum of (PBAE/polyCD + Brim)

and the absorption

spectra of the same ﬁlm, after immersion into a PBS

solution at 37

◦

C, after determined periods of time up

to a maximum of 14 minutes and 30 seconds. It is pos-

sible to see that the absorbance of the ﬁlm immersed

in to the PBS solution decreases in time demonstrat-

ing the brimonidine desorption. In particular, it is

possible to observe that after 30 seconds of immer-

sion, the ﬁlm has lost one bilayer because its absorp-

tion spectrum is similar to that of the ﬁlm with 3 bi-

layers. This means that it takes 30 seconds for the

bilayer to be released to the PBS solution. It was

also observed that after 1 minute and 30 seconds in

PBS solution the same ﬁlm has the same spectrum as

that obtained for two bilayers revealing that the third

bilayer was released. The immersion of the ﬁlm in

PBS solution continued up to 14 minutes and 30 sec-

onds. However, after the 10 minutes of immersion,

salt deposition was observed on the top of the ﬁlm

that affected the absorption spectra. The kinetics of

brimonidine was only quantiﬁed up to 10 minutes of

ﬁlm immersion.

Figure 6 represents the percentage of brimonidine

released to the PBS solution as function of time that

was calculated subtracting the absorbance at 220 nm

after the ﬁlm immersion to the absorbance at the same

wavelength before ﬁlm immersion. The brimonidine

kinetic shows that after 9 minutes of immersion time

in PBS, 30% of the drug was released. That could

correspond to the two outer (PBAE/polyCD+Brim)

bilayers. The kinetics of brimonidine released, repre-

Figure 4: Absorption spectrum of each of the 4

(PBAE/PolyCD + Brim) bilayers.

Figure 5: Absorption spectra of a)(PBAE/polyCD +

Brim)

and (PBAE/polyCD + Brim)

layers and the spec-

trum obtained after immersion in PBS solution of the ﬁlm

with 4 bilayers during 30 seconds. b)(PBAE/polyCD +

Brim)

and (PBAE/polyCD + Brim)

layers and the spec-

trum of the ﬁlm obtained after immersion in PBS solution

during 1 minute and 30 seconds.

sented in Figure 6 was ﬁtted with Korsemeyer-Peppas

model.

The majority of drugs reveal a ﬁrst-order release

(or “burst release”) from the substrate followed by a

continuous decrease in drug concentration in the PBS

solution. The ideal pharmacokinetic system is rep-

resented by a zero-order kinetic response over time,

since it minimizes the variation of drug concentration,

allowing a constant release rate of drug. To analyse

the release, the Korsemeyer-Peppas equation (equa-

tion 1) (Holowka and Bhatia, 2014) was used, by

which the dissolution rate of the drug from the ma-

trix was determined:



∞



= Kt

(1)

where M

is the amount of drug released at time t,

∞

corresponds to the total amount of drug present,

K is the kinetic constant; and n is the diffusion value.

In this model, the kinetics is determined by the diffu-

sion expoent value (n). Values of n=0.5 imply clas-

sic Fickian diffusion, i.e. the main mechanism that

controls the release of the drug in the system is pure

diffusion. In diffusion-controlled systems, the drug

release process occurs due to aqueous stimuli through

polymer swelling, causing an uniform volume expan-

Figure 6: Percentage of released brimonidine for the

(PBAE/polyCD + Brim)

after 8 minutes of immersion

time in PBS solution. The ﬁtting curve was calculated using

equation (1).

sion of the bulk material. Ultimately, this will lead to

pore opening of the matrix structure. Values of n in

the range of 0.5 <n <1, indicate that the drug release

occurs by Fickian diffusion and Case II transport,

i.e., in this regime the drug release is both diffusion-

controlled, and erosion-controlled, respectively. In

erosion-control systems the mechanism of drug re-

lease relies on the attack of the covalent bonds in the

polymer matrix by the components present in the re-

lease solution, allowing the drug to escape. It can

occur due to volume decrease of the matrix, where

its density remains constant; or due to decrease in

the matrix density, while the volume remains con-

stant. In these cases the diffusion obeys the Fick’s

law (Fick, 1995). If the diffusion exponent is n=1,

it suggests Case II transport (or zero-order release)

with constant release rate and controlled by polymer

relaxation. At last, cases with n >1, indicate Super

Case II transport (or release that is erosion-controlled)

(Holowka and Bhatia, 2014; Siegel and Rathbone,

2012). The value of diffusion exponent for this sys-

tem is n = 0.49 ± 0.04 with K = 12.0 ± 0.1, which

means that, in this case, the mechanism of drug re-

lease follows the Fickian diffusion (the driving force

behind the brimonidine release in this ﬁlm is diffu-

sion).

Due to the fast brimonidine release to the biolog-

ical medium, layers of graphene oxide were intro-

duced between the polymeric (PBAE/PolyCD+Brim)

bilayers (see schema of Figure 3). The multilayer ﬁlm

growth and subsequent release kinetics were monitor-

ized by UV-Vis absorption. The ﬁlms were also pre-

pared by LbL method. Figure 7 shows the absorbance

spectra of all layers of a ﬁlm composed of 4 bilayers

of (PBAE/PolyCD + Brim)

followed by one bilayer

of charged graphene (GO

/GO

−

) and with more four

outer polymeric bilayers of PBAE/PolyCD + Brim)

Figure 7: Absorption spectra of LbL ﬁlm as

a function of the growth step, up to the ﬁ-

nal structure composed by (PBAE/polyCD +

Brim)

/(GO

/GO

−

)/(PBAE/polyCD + Brim)

Figure 8: Percentage of released bri-

monidine for the (PBAE/polyCD +

Brim)

/(GO

/GO

−

)/(PBAE/polyCD + Brim)

after

15 minutes immersion ﬁlm in PBS solution.

The drug release kinetics study was developed

with the immersion of the ﬁlm with DD layers into a

PBS solution (diluted in milli-Q water 1/10, pH=7.4)

at 37

◦

C as described previously. During the ﬁrst 15

minutes, spectra were recorded every 30 seconds of

immersion in PBS, and the PBS solution was changed

after each measurement. After that, the spectra were

obtained with 30 minutes interval up to a total of

3 hours and 15 minutes of immersion (changing the

PBS solution after each 15 minutes). Afterwards the

immersion time was extended to 1 hour, until the total

desorption time reached 6 hours and 15 minutes (with

fresh PBS solution after each half hour). Concluding

this period, the desorption time has been extended to

an average of 12 hours of continuous desorption, fol-

lowed by spectral analysis.

Between the beginning of the experiment until

the 15

minute the outer polymer bilayers were des-

Figure 9: Absorption spectra of a

ﬁlm with all layers: (PBAE/polyCD +

Brim)

/(GO

/GO

−

)/(PBAE/polyCD + Brim)

. Af-

ter more than 4 and a half days, the outer polymeric layers

and the GO

layer were desorbed remaining only the layer

of GO

. The absorbance spectrum of this desorption data

is almost coincident to the GO

layer.

orbed from the substrate. The release of the brimoni-

dine from the outer bilayers was quantiﬁed using the

Korsemeyer-Peppas equation (described in equation

1). It is possible to observe that about 80% of bri-

monidine presented in the outer two bilayers is re-

lease during 14 minutes. The red line is the result

of the ﬁtting, where it was obtained a n greater than

0.5 (n = 0.71 ± 0.15) and K = 12.0 ± 4.9, leading to

the conclusion that this system exhibits a drug re-

lease that is both diffusion-controlled and erosion-

controlled. By deﬁnition, in controlled released sys-

tems with 0.5 ≤ n ≤ 1.0, the drug release is a com-

bination of Fikian diffusion and Case II transport of

drug molecules through the polymeric ﬁlm (Enscore

et al., 1977; Ritger and Peppas, 1987). The monitor-

ization of the kinetics continued during more than 5

days. At t=110 h (more than 4 and a half days after

the desorption began) the adsorption has undergone

an impressive decrease in its intensity. The spectrum

of the ﬁlm was almost coincident with the spectrum of

the GO

layer as we can see in (ﬁgure 9). After this

immersion time, the absorbance spectrum indicated

that only the layers (PBAE/polyCD + Brim)

/(GO

)

remained in the ﬁlm.

After 5 days in immersion (approximately

t=121 h) the last GO layer was desorbed. The ob-

tained absorbance spectrum is almost coincident with

the (PBAE/polyCD + Brim)

ﬁlm, as it is possible to

conclude from the spectra of Figure 10.

Figure 10: Absorption spectra of a

ﬁlm with all layers: (PBAE/polyCD +

Brim)

/(GO

/GO

−

)/(PBAE/polyCD + Brim)

and

the spectrum obtained after more than 5 days immersion

time.

4 CONCLUSIONS

A new drug delivery system based on multilayer ﬁlms

fabricated by the layer-by-layer technique was pre-

sented. This versatile method allowed the fabrication

of a time controlled system introducing in the com-

position of the ﬁlm an hydrosoluble polymer - poly

(β-amino ester) - and charged graphene oxyde layers.

Both materials are able to control the release of the

studied drug (brimonidine) encapsulated in a poly β-

cyclodextrin but the presence of graphene oxide can

delay the brimonidine release up to 5 days. This is

an important result for the study of time-controlled

drug delivery systems since it allows adaptation of the

number of layers and the ﬁlm architecture in order to

delay the ﬁlm desorption, stopping the release of bri-

monidine in the eye, in the way that only the needed

dose will be administrated.

This work developed the ﬁrst DD LbL ﬁlms

for glaucoma treatment using biocompatible and

biodegradable materials for the release of precise

amounts of an anti-IOP drug, at determined periods

of time. The high non-compliance level in glau-

coma treatment leads to thousands of individuals to go

blind every year. However, the latest developments on

drug delivery of drugs, with the most varied carriers

have revolutionized the ophthalmic treatments offer-

ing new, improved systems that can control the glau-

coma condition but also substitute the current treat-

ments with daily eye drop application.

ACKNOWLEDGEMENTS

The authors thank Fundac¸ao para a Ci

encia e

Tecnologia-Portugal for ﬁnancial support under the

project UID/EEA/50008/2013 and Post-Doc grant

SFRH/BPD/75338/2010.

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