Nanostructured films for controlled release of drugs for glaucoma

treatment

Monica Araujo

Department of Bioengineering, Instituto Superior Tecnicoand Instituto de Telecomunicacoes, Lisbon, Portugal

May 2016

Abstract

Glaucoma is an ocular degenerative disease caused by optical nerve inflammation which leads toan intraocular pressure (IOP) increase which can lead to total loss of vision. Its treatment, at initialstage, is based on brimonidine administration which allows the decrease of IOP. However, this drug isapplied by means of ocular drops and the patients noncompliance causes the disease worsening. Thiswork allowed the development of biocompatible films that can work as coatings to an intraocular devicein order to release the brimonidine autonomously and in-situ. The films, obtained by layer-by-layertechnique, were composed of brimonidine monolayers encapsulated in poly (β-cyclodextrin) alternatingwith monolayers of a hydrossoluble polymer (poly (β-amino ester)) and/or graphene oxide monolayerthat allow a precise drug release. The films growth and the drug release kinetics were monitorized byultraviolet-visible spectroscopy, quartz crystal microbalance and atomic force microscopy. The obtainedresults showed that the films are stable and the drug release can be controlled by the presence of thehydrossoluble polymer and the graphene oxide. In particular, it was observed that the graphene oxidedelays significantly the brimonidine release enabling the precise control of the amount of drug deliveredover a specific interval of time.Keywords: Glaucoma, controlled drug delivery, layer-by-layer, biodegradable polymers, nanostruc-tured films, brimonidine, graphene oxide.

1. Introduction

The glaucoma is a degenerative ocular disease, con-sidered by the World Health Organization as thesecond leading cause of blindness, with nearly 70million cases worldwide [1]. It is related with highintraocular pressure (IOP), being its reduction thetreatment applied for all types of glaucoma throughthe prescription of ophthalmic drugs, such as bri-monidine [2, 3]. More severe cases of glaucoma re-quire invasive surgeries without dispensing the useof eyedrops for life [4]. However, this conventionaltreatment methods, consisting on eye drops admin-istration, reveal several inconvenients, such as thepatient non-compliance (half of glaucoma patientsdo not use their ophthalmic medication properly)[5], and also because only 20% of the active drugin one droplet achieves the ocular anterior cham-ber, the remaining being drained through the na-solacrimal duct or runing down the chin, and fromthese, only 1% to 3% of the topic dosage actuallyreaches the target site and penetrates the tissue [6].

The solution and objective of this research is thedevelopment of multilayers and biocompatible filmswith drug delivery (DD) function that can replace

todays therapeutics. The developed films can coatan intraocular device and release the brimonidinein situ without human intervention. More specifi-cally, the main goal was to prepare DD layer-by-layer (LbL) films composed by the drug alreadyused in glaucoma treatment – brimonidine – able tobe encapsulated in a poly β-cyclodextrin (poly-CD)that, for its turn, is alternated layer-by-layer witha monolayer that delay the drug release: poly (β-amino ester) (PBAE) and/or graphene oxide (GO).

The LbL method is a simple and versatile tool forthe controlled fabrication of thin films for a widerange of purposes [7, 8, 9]. The LbL techniqueenables the formation of complex multilayer filmsmerely through the sequential adsorption of poly-mers through hydrogen bonding. With this kind ofdeposition, it is possible to obtain ultra thin mono-,bi-, or multilayers precision at molecular scale.

The polymer used for drug encapsulation, poly-CD, is a cyclic oligosaccharid with seven α-D-glucopyranose units attached by α-(1, 4) glyco-sidic bonds. The conformation of the glucopyranoseunits originate a three-dimensional structure com-monly described as a wreath-shaped truncated cone

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[10]. Their spatial conformation confers a wide ca-pability of interaction with a large variety of guestmolecules (under the form of noncovalent inclusioncomplexes). Further, these interactions and the factthat poly-CD is a water soluble polymer provideuseful properties for pharmaceutical applications.The safety and toxicology was already studied [11]showing that it is a non toxic material and with ex-cellent ocular biocompatible properties. The poly-CD and brimonidine form a stable DD complexwhich makes the poly-CD a potential material forthe proposed multilayer DD films.

To perform a structural role and to delay te poly-CD degradation it was used, poly (β-amino ester)(PBAE), a cationic polymer degradable by hydroly-sis of the ester bonds of the backbone at physiolog-ical relevant pH [12]. Plenty of studies [13, 14, 15]confirm that cells incubated with PBAE remain vi-able, that it degrades gradually under physiologicalconditions, and also that it retains its biocompati-bility and biodegradability properties, offering thetunability of polymer degradation.

The ability to control the timing of drug releaseis a crucial advance in order to improve the abilityto define and regulate the patient dose, and conse-quently improving the patient care and the treat-ment regime. The sequential controlled release ofthe drug, intercallated with a nanomaterial with de-sired thickness, offers a simple way to provide con-trolled doses in vivo. Due to the fact that GO ishighly hydrophilic, planar, chemically stable, bio-compatible and non-toxic it is possible to use thesenanosheets as temporary protective layer coatingsfor the PBAE and poly-CD delaying their hydroly-sis [16].

With this work we obtained a multilayer and DDfilms that can release controlled drug amounts infunction of time. Namely, films with GO can de-lay consecutive drug amounts up to 5 days. This isan promising result for the study of time-controlleddrug delivery where the thickness of GO layerscan be adjusted in accordance with the numbersof hours that it is necessary to delay consecutiveamounts of drug.

2. Methods and characterization tech-niques

2.1. Materials2.1.1 Poly (β-amino ester) synthesis

PBAE was synthetized in the Organic Electron-ics laboratory of the Instituto de Telecomunicacoesfollowing the protocol described by Lynn, et al.[17].3.28 g of 4,4′-trimethylenedipiperidine (97 %purity, Sigma Aldrich, CAS number 16898-52-5)was added to 2.87 mL of 1,4-butanediol diacrylate(99% purity, Alfa Aesar, CAS number 1070-70-8).The polymerization of these monomers proceeds

in tetrahydrofuran, under a temperature of 50 ◦Cand during 48 h. The final polymer was purifiedthrough repeated precipitation into diethyl ether.The precipitated polymer was vacuum filtrated witha Buchner funnel and left to dry in vacuum environ-ment over night. As for the structure of the yieldproduct was confirmed by nuclear magnetic reso-nance spectroscopy and gel permeation chromatog-raphy.

2.1.2 Brimonidine encapsulation

Complexes of brimonidine encapsulated in poly-CD(poly-CD+Brim) were prepared by dissolving thedrug into a solution of poly-CD at the proportion0.8 mg/mL. The solution was stirred during about17 hours until total brimonidine encapsulation, be-ing ceased when no drug precipitation was seen inthe bottom of the flask.

2.1.3 Preparation of graphene oxide positivelycharged

The negatively charged graphene oxide nanosheets(GO−) were purchased from Graphenea, asan aqueous dispersion with a concentration of0.5 mg/mL. A method developed by Hwang,et al. [18] was used to prepare positivelycharged graphene oxide (GO+) by mixing 50mL of (GO−) with 0.625 g of N-ethyl-N ′-3-dimethylaminopropyl)carbodiimide methiodide(EDC)(Sigma Aldrich) and with 5 mL of ethylene-diamine (Sigma Aldrich). The solution was leftstirring for 12 hours. EDC reacted with carboxylicgroups activating the coupling of ethylenediamine.

To separate the GO+ from the by-products a cel-lulose membrane (12 000-14 000 Da, Sigma Aldrich)was used to perform the dialysis.

In order to obtain a similar pH of the polymersused to prepare the DD films, the pH of GO+ andGO− solutions was adjusted to pH=5 using a HClsolution (1.4 M) and NaOH (0.17 M) respectively.

2.1.4 Layer-by-layer technique

The DD films were prepared by immersive LbLtechnique. The adsorption was made at thesolid/liquid interface sequentially immersing acharged substrate in different polymer solution. Af-ter each polymer submersion (PBAE and poly-CDin figure 1), the sample was washed in order toremove the physically adsorbed particles, ensuringhomogeneous layers [19]. This procedure can be re-peated as many times as necessary until the desirednumber of layers is achieved. The formation of onebilayer is composed by four steps schematized infigure 1.

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Figure 1: Schematic representation of the LbL method.

2.2. Characterization techniques2.2.1 Quartz Crystal Microbalance

The Quartz Crystal Microbalance (QCM) used,a QCM200 (Stanford Research Systems), has aresolution of 5 MHz and it is a simple, cost-effective, real-time and nanogram-sensitive masssensing technique that uses the piezoelectric effectof a quartz crystal as the sensing element [20].

This method is based on the Sauerbrey model,which is described by a linear relationship, betweenfrequency and mass variation per unit area at thecrystal’s electrode surface. This variation is ob-served in the oscillation frequency of the crystal,described by equation 1 [21],

∆f = − 2f20 ∆m

A · √ρq · µq(1)

where ∆f is the shift in frequency (Hz), f0 is theunperturbed resonant frequency, i.e., without massadsorption (Hz), A is the piezoelectrically activearea of the excitation electrodes (cm2), ρq is thedensity of quartz (2,65 g.cm2) and µq is the shearmodulus of quartz (2,95x1011 dyn/cm2).

The frequency shift of the crystal is related withthe amount of mass adsorbed and/or dessorbedfrom the sensor surface in real-time [22].

QCM was used to monitor carried out for thefilms prepared by the LbL method, in order to con-trol the adsorption of the DD films step-by-step.

2.2.2 Ultraviolet-visible spectroscopy

Ultraviolet-visible (UV-Vis) absorption was used tocharacterize the solutions and the DD films. A CecilAquarius CE 7200 spectophotometer was used inall experiments. With this technique, absorptionspectra of the DD film were obtained to follow thefilm growth after the formation of each bilayer andalso to quantify the brimonidine release.

2.2.3 Atomic Force Microscopy

The surface morphology of DD films was charac-terized by AFM using Nano-Observer AFM fromCSInstruments.

All measurements were performed in non-contactmode with 256x256 pixels and with silicon probes.The main statistical amplitude parameter used tocharacterize the samples surface was the Rms andit is expressed by the equation (2)[23]. This param-eter was used to quantify spatial differences of eachmonolayer. The image is constituted by a matrixwith z(x,y) of height, with N lines and M columns.

Rms(N,M) =

√√√√ 1

NM

N∑x=1

M∑y=1

[z(x, y)− zmed(N,M)]2

(2)

2.2.4 Pharmacokinetics characterization

To analyse the drug release, the Korsemeyer-Peppasequation (see equation 3) was used, by which thedissolution rate of the drug from the film was de-termined [24]: (

Mt

M∞

)= Ktn (3)

where Mt is the amount of drug released at timet, M∞ corresponds to the total amount of drugpresent, K is the kinetics constant; and n is thediffusion value. In this model, the kinetics is de-termined by the diffusion exponent (n). Values ofn=0.5 imply classic Fickian diffusion, i.e. the mainmechanism that controls the release of the drug inthe system is diffusion. Values of n in the rangeof 0.5 <n <1, indicate that Fickian diffusion andCase II transport occur simultaneously, and in thiscase the drug release is diffusion-controlled, but alsoerosion-controlled, respectively. If the diffusion ex-ponent is n=1, it suggests Case II transport (orzero-order release) with constant release rate andcontrolled by polymer relaxation. At last, caseswith n >1, indicate Super Case II transport (orrelease that is erosion-controlled) [25, 26, 27].

3. Results and discussionThe multilayer films were prepared using the LbLtechnique, where layer adsorption was assessed byQCM and UV-Vis spectroscopy. The following sec-tions describe the results obtained for the preparedmultilayer drug delivery films.

3.1. Quartz crystal microbalance measure-ments

3.1.1 Adsorption time determination

Multilayer films of (PBAE/poly−CD)n were pre-pared by the LbL technique using reported ad-sorption times of about 3 and 10 minutes [28, 29].However, the results suggested a random mass ad-sorption as function of the number of layers and

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films presented visible salt agglomerates that sug-gest mass accumulation in some regions.

In order to overcome these unsuccessful results,several experiments were carried out to determinethe optimum adsorption time of PBAE and poly-CD. Each polymer was studied individually, wheresmall amounts of mass were adsorbed with an accu-rate control and optimization of the assembly pro-cess (figure 1), periodic checkpoints were performedto follow the progress of the process. The sub-strate was left in the polymer solution for one andhalf minutes, followed by washing with sodium ac-etate and dry with nitrogen gas. The mass addedto the substrate was inferred from the frequencyvalue obtained by the QCM through the Sauerbreyequation (equation 1). After immersion of the sub-strate in the polymer during short intervals of time,the frequency value was registered. The same pro-cess was repeated until the frequency obtained bythe QCM showed some stability, evidencing thatno more molecules of polymer could be adsorbed atthe substrate, suggesting that the surface was fullycovered by the polymer.

From these experiments it was possible to con-clude that 6 minutes are required for PBAE to fullycover the substrates surface. AFM technique wasused to analyze the surface of PBAE in order toconfirm if indeed the polymer covers all substrate.The topography and phase images of figure showthat the PBAE has a granular morphology and fullycovers the substrate, once the phase image only re-veals one type of material (figure 3).

Figure 3: Topography (a), and phase (b) images of PBAE with1.3 µm scanning length.

The same method was used to determine the ad-sorption time of poly-CD on PBAE and for PBAEon poly-CD. Figure 1 shows the results obtained foreach monolayer and it is possible to observe that thepoly-CD needs 11 minutes to cover the first PBAEmonolayer (figure 1b) and the PBAE needs about7 minutes to cover the poly-CD surface (figure 1c).

After the determination of the optimal adsorp-tion time for each polymer it was possible to pro-ceed to the self-assembly of the monolayers usingQCM for the process characterization. The mul-tilayer films were prepared with and without bri-monidine in order to compare both kinetics. Thefollowing sections describe the obtained results.

3.1.2 Growth of layer-by-layer films

A multilayer film with six (PBAE/poly-CD) alter-nating layers without brimonidine was prepared us-ing the LbL technique. Figure 4 shows the variationof mass of each PBAE and poly-CD layer obtainedby QCM.

Figure 4: Mass as a function of the number of layers of PBAEand poly-CD without brimonidine obtained by QCM. The redline is the linear fit of the average values.

As for the mass values, arithmetic average fromthree different experiments were taken into consid-eration and the error bars represent the standarddeviation of the average values. It was possible toconclude that standard deviation is very significant,

Figure 2: a) Mass of PBAE adsorbed on the substrate as a function of time. b) Mass of poly-CD adsorbed on PBAE as functionof the immersion time. c)Mass of PBAE adsorbed on poly-CD as function of the immersion time.

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that is due to QCM sensitivity. This technique isvery sensitive for instance to the floor vibrationsthat can influence the measurements. In spite ofthe large error bars, the growth is linear as indi-cated by the linear fit (red dotted line of the figure)with an average adsorbed mass approximately con-stant between layers.

3.1.3 Growth of layer-by-layer films with brimoni-dine

Following the study of the system without encap-sulated drugs it was used the same strategy, butthis time, with poly-CD carrying brimonidine in-side of the cavity. Figure 5 shows the evolution ofmass measured with QCM of a film composed by 16alternating layers of PBAE and poly-CD with bri-monidine (poly-CD+Brim). The mass values areaveraged from two different experiments and theerror bars represent the standard deviation. Thegrowth of the films is linear as it was obtained forthe film without brimonidine. The presence of thedrug does not influence the integrity of the film asshown in figure 5.

Figure 5: Average mass of the film with alternating layers ofPBAE and poly-CD with brimonidine as a function of the num-ber of layers, obtained by QCM.

The surface of the first and fourth (PBAE/poly-CD+Brim) bilayers was analyzed by AFM, as it isshown in figure 6, and 7, respectively.

Figure 6: Topography (a), and phase (b) images of (PBAE/poly-CD+Brim) with 1.3 µm scanning length.

The (poly-CD+Brim) surface shows a Rms of4.19 nm, revealing to be smoother than the PBAElayer. These results suggest a good interaction be-tween layers, since the (poly-CD+Brim) layer was

able to bind and cover the PBAE layer. The phaseimage does not show two phases in the sample be-cause the film background and the grains have thesame color contrast. This suggests that only poly-CD is present at this layer surface. This indicatesinterdiffusion between PBAE and poly-CD duringpoly-CD adsorption does not occur.

Figure 7: Topography (a), and phase (b) images of(PBAE/poly − CD + Brim)4 with 1.0 µm scanning length.

The topographic image in figure 7a correspondsto a surface with a Rms of 3.149 nm that is lowercompared with the sample of (poly-CD+Brim)(shown in figure 6). This data suggests that the sur-face becomes smoother upon increase of the numberof bilayers adsorbed.

3.2. Ultraviolet-visible spectroscopy charac-terization

Ultraviolet-visible spectrosocopy was used to sup-port the results obtained by QCM, where an ab-sorption spectra was obtained for each bilayer of(PBAE/poly-CD+Brim). The figure 8 shows theevolution of absorption spectra for a film with 14bilayers. The films have an almost linear growth,established by an increase in the absorbance of bri-monidine, which means that more molecules of bri-monidine were bonded in the film.

Figure 8: Absorption spectra of each (PBAE/poly − CD +Brim)14 bilayers.

3.3. Drug Release KineticsThe brimonidine release kinetics was followed byUV-Vis, recording the absorption spectra of thesubstrate after its immersion in a PBS solution at37 ◦C, (where a glass beaker with PBS was main-tained inside an oven at this temperature) in orderto mimic the physiological conditions (where a glass

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beaker with PBS was maintained inside an oven atthis temperature). The PBS solution was changedafter each immersion. After a specific period of timethe substrate was removed, dried with a nitrogenflux and its adsorption spectrum was recorded. Fig-ure 9 shows the absorption spectrum of a film with4 bilayers of (PBAE/poly-CD+Brim) and the ab-sorption spectra of the same film, after immersion ina PBS solution at 37 ◦C, during determined periodsof time up to a maximum of 14 minutes and 30 sec-onds. It is possible to see that the absorbance of thefilm after exposure to the PBS solution decreases intime, demonstrating the brimonidine desorption.

Figure 9: Absorption spectra of the 4 (PBAE/poly-CD+Brim)layers (symbols). and the spectra obtained after the film immer-sion in PBS, during 14 minutes and 30 seconds (blue spectra).

In particular, it is possible to observe in figure 10that after 30 seconds of immersion in PBS solution,the film has lost the (PBAE/poly −CD+Brim)4

layer because its absorption spectrum is similar tospectrum of the film with 3 bilayers. This meansthat it takes 30 seconds for the 4th bilayer to bereleased to the PBS solution. It was also observedthat after 1 minute and 30 seconds in PBS solu-tion the same film shows a spectrum similar to thespectrum obtained with the film with two bilayersadsorbed ((PBAE/poly−CD+Brim)2), revealingthat the (PBAE/poly−CD+Brim)3 layer was re-leased to the medium.

Figure 10: Absorption spectra of the film with (PBAE/poly −CD + Brim)4 and (PBAE/poly − CD + Brim)3 layers andthe spectrum obtained after the film immersion in PBS solutionduring 30 seconds.

The graphic of figure 11 shows the spectra of thefilm with 2 and 3 bilayers and the spectrum afterimmersion in PBS solution. It is possible to observethat the third bilayer is released after 1 minute and30 seconds.

Figure 11: Absorption spectra of the film with (PBAE/poly −CD+Brim)3 and (PBAE/poly−CD+Brim)2 layers and thespectrum of the film after immersed in PBS solution during 1and half minutes.

Figure 12 represents the percentage of brimoni-dine released to the PBS solution as function of timethat was calculated subtracting the absorbance at220 nm after the film immersion to the absorbanceat the same wavelength before film immersion. Thebrimonidine kinetics shows that after 9 minutesimmersion time in PBS, 30% of the drug was re-leased to the medium. That could correspond to thetwo outer (PBAE/poly-CD+Brim) bilayers. Thekinetics represented in figure 12 was fitted withKorsemeyer-Peppas equation (eq. 3). The value ofdiffusion exponent for this system is n = 0.49±0.04with K = 12.0±0.1, which means that, in this case,the mechanism of drug release follows the Fickiandiffusion (the driving force behind the brimonidinerelease in this film is diffusion).

Figure 12: Percentage of released brimonidine from(PBAE/poly − CD + Brim)4 up to 8 minutes in immer-sion time in PBS solution.

The easy release of the two outer bilayers com-

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pared with the first ones can be due to the mor-phology of the surface. AFM results showed thatthe first layers present a granular morphology (seefigure 3) while the surface of the 4th bilayer issmoother. These differences in morphology areinfluenced by the conformation of the polymericchains, having the last bilayers a possible confor-mation that allows the desorption of these layers.

Another sample, with fourteen layers was alsostudied using the same method, aiming to inves-tigate how the number of layers influences the des-orption kinetics from the films. After 1 minute ofimmersion it was observed that the last 3 bilayerswere released from the film, as shown in figure 13,where the absorption spectrum of the film after thistime is almost coincident with the spectrum of thefilm with 11 bilayers (PBAE/poly−CD+Brim)11.

Figure 13: Absorption spectra of the film with 11 and 14 bilayersand after immersed on PBS solution for 30 seconds and 1 minute.

For the film with 14 bilayers of figure 13 it wasconcluded that the last 3 bilayers are released dur-ing 1 minute as the spectrum is almost coincidentwith the film with 11 bilayers. This result is similarwto that observed for the film with 4 bilayers.

The desorption proceeded until 11 minutes and30 seconds of immersion in PBS solution. The ab-sorption spectrum of the film has an intensity be-tween that of the 9th and of the 10th bilayers, asshown in figure 14.

Figure 14: Absorption spectra of the film with 9 and 10 bilayersand the film with 14 bilayers after immersion in PBS solutionduring 11 minutes and 30 seconds.

In accordance with figure 14, the film needs about10 minutes to release the 10th bilayer. This is a sig-nificant difference compared with the time observedfor the last 3 bilayers. This behavior could be ex-plained by the polymeric chains arrangement in thefilm, whose interaction of the last bilayers could beweaker and influencing the brimonidine release.

The film was maintained in solution for more 56minutes. However, it was impossible to monitoringthe film absorbance from the 15 minutes onwards-due to salt accumulation that occurred on the top ofthe film. This effect can be explained by the scatter-ing effect: salts lead to light dispersion that reducesthe light transmission and appears as absorption.

To analyse the percentage of released brimoni-dine, during the first 11 minutes and half, the filmabsorption at 220 nm, after each immersion wasplotted as function of the number of bilayers as itis shown in figure 15. The red line corresponds tothe fitting and it is possible to conclude that 25% ofthe brimonidine was released from the film after 11minutes and 30 seconds. The fitting is very poor,yeilds an n value obtain was n = 0.50 ± 0.05 , anda K value of 6.9 ± 0.1, which means that this filmdisplays a mechanism of drug release that followsthe Fickian diffusion, i.e., the brimonidine releaseis diffusion-controlled [24].

Figure 15: Percentage of brimonidine released for the(PBAE/poly − CD + Brim)14 as function of immersion timein PBS solution up to 12 minutes. The fitting curve was calcu-lated from the equation 3.

Comparing the brimonidine released from thefilm with 14 bilayers with the film with 4 bilay-ers, it is possible to conclude that in both cases theamount of brimonidine released in approximately12 minutes is about 25%. The number of bilayersseems not to influence the drug release. However,the kinetic study was only conducted up to 15 minimmersion time, which we attribute to the accumu-lation of salts.

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3.4. Drug delivery films with graphene oxidegrown by layer-by-layer

We found that it is possible to delay the brimonidinerelease introducing graphene oxide layers between(PBAE/poly-CD+Brim) layers.

The film growth followed by UV-Vis is shown infigure 16 where it is possible to observe that the filmpresents a stable, continuous growth of all layers.

Figure 16: Absorption spectra of a (PBAE/poly − CD +

Brim)4/(GO+/GO−)2 / (PBAE/poly − CD + Brim)2 film.

The surface of GO and the outer polymeric bi-layers were analyzed by AFM. Figure 17 shows atopographic and phase images of (PBAE/poly −CD+Brim)4/ (GO+/GO−)2 surface. The imagesdisplay straight domains that we attribute to GOnanosheets overlapping due to the π-π stacking in-teractions. The phase images do not show evidencesof the underlying polymeric layer, suggesting thatthe bilayer of graphene totally covers the surface ofthe film. Also, the surface seems to be substantialsmoother comparing with the samples without GOadsorbed, as proven by the Rms that decreases from3.149 to 2.766 nm.

Figure 17: Topography (a) and phase (b) images of

(PBAE/poly − CD + Brim)4/(GO+/GO−)2 with 1.0 µm ofscanning length.

Figure 18 shows the topographic and phaseimages of (PBAE/poly − CD + Brim)4 /(GO+/GO−)2/(PBAE/poly−CD+Brim)2. Someaggregates that have a significant contrast in phaseimage, are observed. It is not clear if the contrastof the grains is different from the contrast of the

background surface. For this reason we cannot con-clude if the bilayer (PBAE/poly-CD+Brim) totallycovers the GO surface.

Figure 18: Topography (a) and phase (b) images of

(PBAE/poly − CD + Brim)4 + (GO+/GO−)2 +(PBAE/poly − CD + Brim)2 with 1.0 µm scanninglength.

3.4.1 Drug release kinetics of films with grapheneoxide

The drug release kinetics study was developed withthe immersion of the film with DD layers into a PBSsolution (diluted in milli-Q water 1/10, pH=7.4) at37 ◦C as detailed previously.

Between the beginning of the experiment untilthe 15th minute the two outer polymer bilayers weredesorbed from the substrate as it is shown in thefigure 19. The spectra of the film recorded dur-ing the 15 minutes of immersion are similar to thespectrum of the film with (PBAE/poly − CD +Brim)4/(GO+/GO−)2 layers. Note that, the spec-tra are not coincident, but at t=10 min, the valueat 230 nm is almost coincident suggesting that thetwo outer bilayers were desorbed. This result is sim-ilar with the observed for the film of (PBAE/poly-CD+Brim) with 14 bilayers (figure 8) where the lasttwo bilayers needed about 10 minutes to be releasedfor the PBS solution.

Figure 19: Absorption spectra of the (PBAE/poly −CD + Brim)4/(GO+/GO−)2 film, (PBAE/poly − CD +

Brim)4/(GO+/GO−)2 / (PBAE/poly−CD+Brim)1 film and

(PBAE/poly − CD + Brim)4/(GO+/GO−)2/(PBAE/poly −CD+Brim)2 film and absorption spectra of the film immersedin PBS solution up to 15 minutes.

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The release of the brimonidine from the outerbilayers was analysed using the equation 3 (figure20). It is possible to observe that about 80% ofbrimonidine presented in the outer two bilayers isrelease during 14 minutes. The red line is the fit,yeilding n greater than 0.5 (n = 0.71 ± 0.15) andK = 12.0 ± 4.9, leading to the conclusion that thissystem exhibits a drug release that is both diffusion-controlled and erosion-controlled. By definition, incontrolled released systems with 0.5 ≤ n ≤ 1.0,the drug release is a combination between Fikiandiffusion and Case II transport of drug moleculesthrough the polymeric film [30].

Figure 20: Percentage of released brimonidine for the(PBAE/poly−CD+Brim)4/(GO+/GO−)2 / (PBAE/poly−CD + Brim)2 after 15 minutes immersed in PBS solution.

The monitoring of the kinetics continued but itwas observed that the film began to adsorb PBSsalts. However, more than 4 and half days after thedesorption began, the absorption has undergone animpressive decrease in its intensity. The spectrumof the film was almost coincident with the spec-trum of the first layer of GO+ as shown in figure21. After this time, the absorbance spectrum in-dicated that only the layers (PBAE/poly − CD +Brim)4/(GO+) remained in the film.

Figure 21: Absorption spectra of a film with all layers:(PBAE/poly −CD +Brim)4/(GO+/GO−)2/ (PBAE/poly −CD + Brim)2 and the spectrum of the film after immersion inPBS for more than 4 and half days.

After 5 days, all graphene oxide layers were des-orbed. The absorbance spectrum was almost coin-cident with that of (PBAE/poly − CD + Brim)4

film (figure 22).

Figure 22: Absorption spectra of a film with all layers up to(PBAE/poly − CD + Brim)4/(GO+/GO−)2/(PBAE/poly −CD+Brim)2 and the spectrum of the film obtained after 5 daysin immersion.

The absorption spectrum of the film after 5 daysin PBS (t=124 h 40 min) is similar to that ofthe film with (PBAE/poly − CD + Brim)2 lay-ers indicating that the film has only 2 bilayers of(PBAE/poly-CD+Brim), as shown in figure 23.

Figure 23: Absorption spectra of a film with (PBAE/poly −CD+Brim)2, (PBAE/poly−CD+Brim)3 and (PBAE/poly−CD+Brim)4 layers and the spectrum of the film obtained aftermore than 5 days in immersion.

4. ConclusionsNew therapeutics that can replace the daily appli-cation of eye drops are imperative, in order to pro-vide more life quality to the patients but also toovercome the limitations that cause the poor IOPcontrol leading to the advancement of the disease.

With this work it was possible to develop a newbiocompatible DD system with PBAE, poly-CDand charged graphene oxide layers able to carrydrugs and release them at controlled periods oftime. The developed films can coat ocular devices

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in order to release the drugs in situ and withouthuman intervention.

AcknowledgementsThe author would like to thank Prof. Jorge Mor-gado for all the guidance and support, Dr.a QuirinaFerreira for the tireless work and profound knowl-edge, and Dr.a Ana Charas for all the valuable helpand expertise shared.

All the support from the team and colleaguesat Organic Electronics laboratory, which made thiswork possible is acknowledged.

Lastly, the author wants to thank the Bioengi-neering department and the Instituto of Teleco-municacoes for the facilities, and for the finan-cial support of the project UID/EEA/50008/2013(STMImage).

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