2005 Bioadhesive Properties of a Polyaminoacidic Hydrogel Evaluation by ATR FT-IR Spectroscopy

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Author ProofAuthor ProofBioadhesive Properties of aPolyaminoacidic Hydrogel: Evaluationby ATR FT-IR Spectroscopy

F. Saiano, G. Pitarresi, D. Mandracchia,G. Giammona*

Macromol. Biosci. 2005, 5, 653–661

mabi.200400223C

Full Paper: In this work, the bioadhesiveproperties of a novel hydrogel based on apolyaspartamide derivative, have been inves-tigated by evaluating its interaction withmucin by means of dynamic swelling and

ATR FT-IR measurements. The bioadhesivebehaviour of the hydrogel, prepared withoutany photoinitiator, allows to widen its ap-plication in biomedical and pharmaceuticalfields.

PHG-UV

gel

Mucin

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Author ProofAuthor Proof

Bioadhesive Properties of a Polyaminoacidic Hydrogel:

Evaluation by ATR FT-IR Spectroscopy

Filippo Saiano,1 Giovanna Pitarresi,2 Delia Mandracchia,2 Gaetano Giammona*2

1Dipartimento di Ingegneria e Tecnologie Agro-Forestali, Universita degli Studi di Palermo, Viale delle Scienze 13, 90128,Palermo, Italy

2Dipartimento di Chimica e Tecnologie Farmaceutiche, Universita degli Studi di Palermo, Via Archirafi 32, 90123, Palermo, ItalyFax: 0039 0916236150; E-mail: [email protected]

Received: December 15, 2004; Revised: May 11, 2005; Accepted: May 17, 2005; DOI: 10.1002/mabi.200400223

Keywords: ATR FT-IR; bioadhesion; hydrogels; photopolymerization; swelling

Introduction

In the last few years, several researchers have focused their

attention on the design of novel pharmaceutical systems

able to overcome some disadvantages of conventional

dosage forms. In this context, hydrogels, i.e. networks of

hydrophilic polymers able to swell in an aqueous medium,

are a very interesting class of materials because of their

peculiar properties, such as biocompatibility, versatility in

application, easy preparation and low production costs. In

addition, due to their swelling ability in aqueous medium

and their soft and rubbery nature, hydrogels are compatible

with biological tissues. Therefore, they have been employ-

ed to prepare prostheses and artificial organs, as well as

systems for the modified release of drugs.[1–3] Hydrogels

are called ‘‘reversible’’ or ‘‘physical’’ gels when the net-

works are held together by molecular entanglements and/or

secondary forces including ionic, H-bonding or hydro-

phobic interactions. All these interactions are reversible

and they can be disrupted by changes in physical condi-

tions or application of stress. In contrast, they are called

‘‘permanent’’ or ‘‘chemical’’ gels when they are covalently

crosslinked networks; then they attain an equilibrium

swelling state without dissolving, even at high temperature.

Polymers employed to prepare hydrogels can exhibit

bioadhesive properties, including poly(acrylic acid), hydro-

xyalkyl cellulose, polymethacrylate, hyaluronic acid and

chitosan.[4–6] A bioadhesive behavior opens the oppor-

tunity to develop drug delivery systems that can be

administered by different routes (e.g. ocular, buccal, nasal,

rectal, vaginal) either for topical or systemic therapy. Bio-

adhesive drug delivery systems can increase the drug bio-

availability by prolonging the residence time of the dosage

form on the site of absorption and they can improve

Summary: The bioadhesive properties of a novel chemicalhydrogel based on a polymer of protein-like structure, havebeen investigated by using ATR FT-IR spectroscopy. Inparticular, the copolymer PHG obtained by partial derivati-zation of PHEA with GMA was chemically crosslinked byUV irradiation at 313 nm. Crosslinked PHGwas treated withwater to obtain a swelled sample, named PHG-UV gel, thatwas brought into contact with a phosphate buffer/citric acidsolution at pH7.0 in the absence or in the presence ofmucin atvarious concentrations (0.01, 0.1 and 1 wt.-%). Preliminary

dynamic swelling studies have evidenced the occurrence ofan interaction between the PHG-UVgel and the glycoprotein.This result was confirmed by ATR FT-IR measurements. Adiffusion model using a solution of Ficks’ second law wasemployed to determine the diffusion coefficient of water intoPHG-UV gel as a consequence of adsorption and/or inter-diffusion which occur at the PHG-UV gel/mucin solutioninterface. Experimental results suggest a potential use ofPHG-UV gel to prepare bioadhesive devices.

Macromol. Biosci. 2005, 5, 653–661 DOI: 10.1002/mabi.200400223 � 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

mabi.200400223

Full Paper 653

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Author ProofAuthor Proofpharmacological effectiveness since they allow the locali-

zation and release of a drug to a target region.[7–9]

The evaluation of bioadhesive behavior of both water

soluble polymers and hydrogels, can be performed by

different techniques[10–12] Since each technique has its own

set of experimental conditions it is difficult to compare

experimental data among investigators. Even for a given

method, a small variation in experimental parameters such

as contact time, speed of testing, preparation of biological

substrates, applied force, rate of removal of bioadhesive

compounds and presence of impurities, results in very dif-

ferent values so that it is not possible to assign an absolute

value representing the bioadhesive properties for a parti-

cular system.[13] However, among the several techniques

employed, spectroscopic analysis results to be rather

sensitive and reproducible. In particular, attenuated total

reflection infrared spectroscopy (ATR FT-IR) has been

successfully applied to study the interaction occurring

between poly(acrylic acid) and mucin.[5,14,15]

On the other hand, in a previous work we have

investigated the mucoadhesive properties of a polymer

at protein-like structure, a,b-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) using ATR FT-IR analysis.[16] PHEA

is a water soluble and biocompatible polymer proposed as a

plasma expander and material to synthesize both soluble

macromolecular prodrugs and water swellable networks

(hydrogels).[17–22]

Now, the aim of the present paper is the evaluation of the

bioadhesive behavior of a novel chemical hydrogel based

on a PHEA derivative. In particular, in order to increase the

reactivity of PHEA towards radical reactions, its structure

has been partially modified by introducing groups contain-

ing double bonds, i.e. PHEA has been derivatized with

glycidylmethacrylate (GMA) thus obtaining the copolymer

PHG that, like PHEA, is water soluble.[23]

Previous studies have shown that PHG can be chemically

crosslinked by means of UV irradiation.[24,25] The obtained

hydrogel, named PHG-UV, showed a high swelling ability

and due to the presence of ester groups. It undergoes a

partial degradation in the presence of esterases, as reported

elsewhere.[24,25] PHG-UVhydrogel is also able to release in

a prolonged way an anticancer drug, such as 5-fluoro-

uracil.[26] Taking into account the protein-like structure of

PHG-UV hydrogel and the presence of several polar groups

in its structure, it is probably able to interact with glyco-

protein chains. In order to confirm this assumption, in the

present work, we have employed ATR FT-IR analysis to

study the interaction between PHG-UV hydrogel and

mucin, thus proving its bioadhesive properties.

Experimental Part

Materials

D,L-Aspartic acid, ethanolamine, hydrazine hydrate, N,N-dimethylformamide (DMF) and N,N-dimethylacetamide

(DMA) were from Fluka (Milano, Italy). Disodium hydrogenphosphate, citric acid, hydrochloric acid, sodium hydroxideand mucin type I-S from bovine submaxillary glands, werefrom Sigma-Aldrich (Milano, Italy). GMA and 4-dimethyl-aminopyridine (4-DMAP) 99.9%were fromAldrich ChemicalCo. (Milano, Italy). Water was freshly distilled (Milli-Q). Allreagents were of the best available commercial grades.

PHEA was prepared by aminolysis of a polysuccinimide(PSI) with ethanolamine, according to a procedure re-ported elsewhere.[17] The batch of PHEA used in the presentstudy had a weight-average molecular weight of 55 kDa(Mw=Mn ¼ 1.75), determined by SEC analysis.

Derivatization of PHEA with GMA to obtain PHG co-polymer was carried out in an organic phase (anhydrousDMA),using 4-DMAP as reported elsewhere.[23] &

Q1authors:sentence ok now? ‘‘DMAP as’’ was probably missing&The degree of derivatization (DD) of the prepared PHG,determined by 1H NMR resulted to be 28� 1 mol-%. Theweight-average molecular weight of the PHG copolymeras determined by SEC analysis was 70 kDa (Mw=Mn ¼ 1.82).FT-IR analysis: 3293 br, 3078 m (nas (OH)þ nas (NH)þ nas(NH2)); 1712 m (nas (C O)); 1656 vs (amide I); 1541 s (amideII), 1437 m (d (C–H)), 1405 m-w (scissoring –C C–), 1180 m(ns (CO)þ ether COC), 951 m-w (wagging –C C–) cm�1.

Scheme 1 reports the synthesis of PHEA and PHGcopolymer.

Apparatus

Molecular weights of PHEA and PHG were determined by aSEC system equipped with a pump system, two Phenogelcolumns fromPhenomenex (5mmparticle size, 103 A and104 Aof pores size) and a 410 differential refractometer (DRI) as aconcentration detector, all fromWaters (Mildford, MA, USA).The following conditions were employed: DMFþ 0.01 M LiBras a mobile phase; 50 8C; 0.8 mL �min�1. The molecularweights were estimated based on PEO/PEG standards (range4000–318 000 Da).

1H NMR spectra were obtained with a Bruker AC-250instrument. Samples were solubilized in D2O.

FT-IR spectra were obtained with a Bruker Vector 22 instru-ment in the range 4000–700 cm�1 with 1 cm�1 of resolution.Samples were in KBr pellets.

UV irradiation was performed by using a Rayonet reactorequipped with a Rayonet Carousel motor assembly and 16mercury lamps of 8 Wat medium pressure with an emission at313 nm.

Centrifugations were performed with an InternationalEquipment Company Centra MP4R equipped with an 854rotor and temperature control.

Photocrosslinking of PHG (PHG-UV)

Photocrosslinking of PHG was performed, in the absence ofphotoinitiator, by using a procedure reported elsewhere.[25]

In particular, a solution of PHG (60 mg �mL�1) in doubly-distilled water, was placed in a Pyrex tube equipped with aninternal Pyrex piston in order to have a sample of about 2mm inthickness, then irradiated for 3.5 h under argon at 313 nm.

654 F. Saiano, G. Pitarresi, D. Mandracchia, G. Giammona

Macromol. Biosci. 2005, 5, 653–661 www.mbs-journal.de � 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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After irradiation, the obtained chemical hydrogel was puri-fied by several washes with doubly-distilled water, andcentrifuging, from time to time, at 12 000 rpm, and 4 8C for

20 min. Finally, the sample was lyophilized and recovered in ayield of 85 wt.-% based on the starting PHG. The chemicalhydrogel thus obtained was named in the text as PHG-UV.

Scheme 1. Synthesis of PHEA and PHG copolymer.&authors: please check again verycarefully your formulas in Scheme 1 and 2, especially for CH2, H2C , –CH2–, –NH–etc.&

Bioadhesive Properties of a Polyaminoacidic Hydrogel: Evaluation by ATR FT-IR Spectroscopy 655


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Scheme 2 reports a schematic representation of thepolymeric chemical network of PHG-UV obtained by UV-irradiation of PHG copolymer.

FT-IR analysis of PHG-UV hydrogel showed principal bandsat3389br,3102m(nas (OH)þ nas (NH)þ nas (NH2)),1728m(nas(C O)), 1658 vs (amide I), 1538 s (amide II), 1437m (d (C–H))and 1181m (ns (CO)þ ether COC) cm�1.

Lyophilized PHG-UV was treated with a suitable volumeof doubly-distilled water, in order to obtain an aqueous gelcontaining 20 wt.-% of PHG-UV (named in the text as PHG-UV 20 wt.-% gel) which was employed for the swelling andATR FT-IR measurements.

Swelling Measurements of PHG-UV 20 wt.-% Gel

Exactly weighed aliquots of PHG-UV 20 wt.-% gel wereplaced on a 5 ml sintered glass filter (Ø 10 mm; porosity: G3)

and left to swell by immersing the filter plus support in a beakercontaining 5 ml of the liquid medium, i.e. phosphate buffer/citric acid solution at pH 7.0 in the absence or in the presence ofmucin at various concentrations (0.01, 0.1 and 1wt.-%).After afixed time (range 15 min–24 h), the excess of liquid wasremoved by percolation at atmospheric pressure. The filter wasplaced in a properly sized centrifuge test tube, then centrifugedat 6000 rpm for 15 min and weighed.

The weight swelling ratio (q) was calculated as:

q ¼ Wf=Wi

whereWf andWi are the weights of gel after (final) and before(initial) the experiment, respectively. Each experiment wasperformed in triplicate and the results were in agreementwithin� 2% error.

Scheme 2. Schematic representation of the polymeric chemical network of PHG-UVobtained by UVirradiation of PHG copolymer.



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ATR FT-IR Spectroscopy

A FT-IR spectrometer (Vector 22 Bruker) with a horizontalATR accessory (Specac), with a cover to prevent solventevaporation, was used in the configuration shown in Figure 1.

This arrangement permits the IR beam to enter the layer ofgel to a small fixed depth and to be specifically attenuatedaccording to the molecules present in this region. The ATRcrystal was ZnSe with an angle of incidence of 458, 50 mmlong, 10 mm wide and 2 mm thick. The cell and ZnSe crystalwere sealed together using a petroleum gel and the jointsmonitored for leaks. An aliquot of PHG-UV 20 wt.-% gel wasplaced on the crystal in order to have a layer whose thicknesswasmeasured atmultiple points using a digitalmicrometer andhad been found to be 1 mm thick. The gel was contacted with5 mL of phosphate buffer/citric acid solution at pH 7.0 in theabsence or in the presence of mucin at various concentrations(0.01, 0.1 and 1 wt.-%). The measurement range was 4000–700 cm�1 and the spectra were collected in situ, every 300 s,with 16 averaged scans and a resolution of 4 cm�1. In order toacquire useful spectra evidencing the water incoming frommucin solution, a buffer solution in contact with ZnSe crystalwas always used as background for all experiments. Obviously,in this way, a negative band appears in the range 3000–3500 cm�1 of the spectra. Therefore, we considered the morenegative area of PHG-UV gel, before the addition of bufferor mucin solutions, as the reference to which to compare thesuccessively less negative integrated area; in this manner wehad positive and growing values. We have considered thismethod analogous to others reported in the literature wherethe bands are positive, using as reference spectrum the ATRcrystal/air or ATR crystal/lyophilized polymer couple.Although frequently, and also in this case, the integrated areain the same range is analogously very small (due to the strongabsorbance value of water), a quantitative determination isperformed all the same.[27–33]

The spectrometer was linked to a PC equippedwith a BrukerOpus 2 software which allows for the continuous automatedcollection and subsequent manipulation of spectra, includingthe deconvolution and fit routines.

Curve Fitting

Curve fitting was used for the calculation of single componentsin the system of overlapping bands in the range 1750–

1450 cm�1. A model consisting of an estimated number ofbands and a baseline should be generated before the fittingcalculation is started. Since the result of the calculation ishighly dependent on the model chosen, care must be taken thatthe model is reasonable from the chemical point of view. Ourmodel considered the presence of Amide I and II band with aresidual component due to water.

The curve fitting routine provided in the Opus softwarepackage uses the Levenberg-Marquardt algorithm for the opti-mization of the fit model with Gaussian or Lorentzian peaks.This type of manipulation is typically left to Opus to perform.The statistical parameters defined in the software manual wereused as a guide to ‘‘best fit’’. The best fit was obtained with a100%Gaussian peaks for Amide I and II and 100% Lorentzianpeak for residual water.

Chemical Hydrolysis Study of PHG-UV 20 wt.-% Gel

Chemical hydrolysis of PHG-UV 20 wt.-% gel was investi-gated in phosphate buffer/citric acid solution at pH 7.0 in theabsence or in the presence of mucin at various concentrations(0.01, 0.1 and 1 wt.-%). Samples of PHG-UV 20 wt.-% gel(25 mg) were dispersed in 10 ml of the aqueous medium, thenkept in a water bath at 37� 0.1 8C with continuous stirring(100 rpm) for 24 h. After this time, samples were centrifuged at12 000 rpm at 10 8C for 15 min and the supernatant wasseparated. For each sample, the centrifuged residue was wash-ed several times with continuously stirred doubly-distilledwater at 37 8C to extract mucin, electrolytes and possibledegradation products. Finally, each sample was lyophilized.The solid residue was weighed and treated with doubly-distilled water to obtain again PHG-UV 20 wt.-% gel that hadbeen employed to perform swelling and ATR FT-IR measure-ments. No difference was found in swelling and ATR FT-IRspectroscopy data for PHG-UV 20 wt.-% gel before and afterhydrolytic treatment. This behavior, in accordance with datareported elsewhere,[26] demonstrates that in the conditionsemployed for swelling and ATR FT-IR measurements(phosphate buffer/citric acid solution at pH 7.0 in the absenceor in the presence of mucin and until 24 h) no degradationoccurs in the polymeric network of PHG-UV. Since PHG-UV20wt.-%gel does not undergo any degradation in the employedconditions, no change occurs in the pH of the medium or in thebioadhesive properties of PHG-UV gel.

PHG-UV gel Mucin

steel cell

ZnSe crystal

Layer of PHG-UV gel

Mucin solution

IR Beam IR Beam out to detector in

Figure 1. Scheme of the ATR FT-IR experimental arrangement.



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Author ProofAuthor ProofResults and Discussion

PHG copolymer, obtained by partial derivatization of

PHEA with GMA,[23] was irradiated in aqueous solution

at 313 nm for 3.5 h in order to obtain a chemical crosslinked

hydrogel having a high swelling ability as previously

reported.[25] PHG-UV hydrogel was thus obtained without

the use of photoinitiators. Since it is well known that

photoinitiators are reactive molecules (such as benzophe-

none, acetophenone and 2,2-dimethoxy-2-phenylacetophe-

none) whose traces can cause toxic effects on human,

the possibility to obtain a hydrogel in the absence of

chemical initiators offers the opportunity to obtain a bio-

compatible material.

PHG-UV hydrogel is insoluble in water and in common

organic solvents, such as dichloromethane, acetone, ethanol,

dimethylsulfoxide, dimethylacetamide and dimethyl-

formamide. This confirms the crosslinked (‘‘chemical’’)

structure of the PHG-UV hydrogel. & Author: Pleasecheck edited sentence&On the other hand, FT-IR analysis

has confirmed that the crosslinking reaction is completed.

The disappearance of peaks related to double bonds, i.e.

1405 (scissoring –C C–) and 951 cm�1 (wagging –C C–)

assigned to the vinyl group of the methacrylate residue in

PHG, confirms that the crosslinking involves the complete

opening of the double bonds, probably through the forma-

tion of free radicals which give rise to inter- and intra-

polymeric chemical bonds.

Chemically crosslinked PHG-UVwas swollen in doubly-

distilled water in order to obtain an aqueous chemical gel

containing 20 wt.-% of the polymeric matrix. The sample

(PHG-UV 20 wt.-%) thus obtained was employed for the

swelling and ATR FT-IR measurements.

Swelling Measurements of PHG-UV 20 wt.-% Gel

Swelling measurements were performed in order to obtain

preliminary information about a potential interaction

between PHG-UV gel and mucin, chosen as a representa-

tive component of the physiological mucus. In particular,

we have evaluated the ability of PHG-UV 20 wt.-% gel to

uptake a further amount of water when it is brought into

contactwith a phosphate buffer/citric acid solution at pH7.0

in the absence or in the presence of mucin at various

concentrations (0.01, 0.1 and 1 wt.-%). For this reason,

dynamic swelling measurements were performed in the

range 15 min–24 h by using the procedure reported in

the experimental section. Experiments were performed at

pH7.0 inorder to simulate a physiologicalmedium.Figure2

reports the experimental results of swelling studies.

As it can be observed, in the absence of mucin, PHG-UV

gel undergoes a rapid swelling and it reaches an equilibrium

swelling after 3 h. On the contrary, the presence of mucin in

the swelling medium reduces the rate and the amount of

water uptake. In particular, in the presence of 0.01 wt.-% of

mucin, a slight lag in the rate of water uptake was found.

The equilibrium swelling is reached after 4 h and theweight

swelling ratio (q) resulted to be lower. This result suggests

that, in the presence of mucin, the interaction glycoprotein/

water reduces the amount of free water molecules able

to penetrate into the PHG-UV gel. The slowdown in the rate

of water uptake is more evident in the presence of 0.1 wt.-%

and, especially, 1 wt.-% of mucin (see Figure 2). The swel-

ling equilibrium is reached after 8 and 15 h, respectively.

This trend suggests that mucin, depending on its concen-

tration, interacts with the gel surface to form an interfacial

film exhibiting a resistance to the diffusion of water. This

interaction was confirmed by ATR FT-IR studies.

ATR FT-IR Studies

Taking into account that ATR FT-IR spectroscopy has been

employed successful to study the diffusion of water mole-

cules in polymeric matrices, membranes or films[34–36] in

this paper the same technique has been used to evaluate the

diffusion of water in PHG-UV gel from a phosphate buffer/

citric acid solution at pH 7.0 in the absence or in the

presence of mucin at various concentrations (0.01, 0.1 and

1,00

1,25

1,50

1,75

2,00

2,25

2,50

2,75

0 4 8 12 16 20 24

Time (hours)

Wei

gh

t sw

elli

ng

ra

tio

, q buffer

buffer + mucin 0.01 wt.-%

buffer + mucin 0.1 wt.-%

buffer + mucin 1 wt.-%

Figure 2. Swelling of PHG-UV 20 wt.-% gel in phosphatebuffer/citric acid solution at pH 7.0 in the absence or in thepresence of mucin at various concentrations.

Figure 3. ATR FT-IR spectrum of PHG-UV 20 wt.-% gel in thefrequency region from 4000 to 700 cm�1. The inlet shows thepeaks obtained from the curve fitting in the 1750–1450 cm�1

frequency region to assign Amide I and II bands.



WILEY-VCH

Author ProofAuthor Proof1 wt.-%). This evaluation allows to have information

about the potential bioadhesive behavior of the prepared

hydrogel.

Figure 3 shows the ATR FT-IR spectrum of PHG-UV

20 wt.-% gel alone, in the frequency region from 4000 to

700 cm�1. Since the amount of water in buffer solution

(used as background) is higher than the amount of water in

PHG-UV gel, the expected broad and strong band in the

range 3500–3000 cm�1 (due to N–H andO–H stretching of

gel and water extensively involved in hydrogen bonds)

appears obviously negative. The peaks centred around

2850 cm�1 arise from the C–H stretching. The bands at

1656 and 1543 cm�1, the so-called Amide I and Amide II

bands, are due, respectively, to C O stretching and N–H

bendingofamidegroupsofPHG-UV.Thebandsat1181cm�1

are due to C–O stretching. In the inlet of Figure 3 we report

the peaks obtained from the curve fitting in the 1750–

1450 cm�1 frequency region to assign Amide I and II bands.

When PHG-UV gel is brought into contact with phos-

phate buffer/citric acid solution at pH 7.0 in the absence or

in the presence ofmucin at various concentrations (0.01, 0.1

and 1 wt.-%), a diffusion of water into the PHG-UV gel

occurs. Then there will be a steady concentration build-up

of the water at the crystal/PHG-UV gel interface.

When PHG-UV gel changes from the starting to the

equilibrium condition, a general decrease of all band in-

tensities occurs due to a further swelling of PHG-UVgel as a

consequence of water diffusion into the PHG-UV network.

Obviously, this process causes an increase of the intensity of

the water band. Since the ATR FT-IR technique detects

preferentially the molecules close to the crystal surface and

the glycoprotein is entangled on the upper surface of the

PHG-UV gel, the only band of mucin, at 1550 cm�1 due to

the dimeric C O stretching vibration, is not detectable.

As a consequence, by ATR FT-IR analysis we cannot

study the interaction PHG-UV gel/mucin in a direct way,

but the integrated area of OH stretching band of water

centred at 3400 cm�1 was used to monitor the diffusion of

water as an indirect measure of any resistance changes

resulting from adsorption and/or interpenetration processes

at the interface PHG-UVgel/mucin solution. As the layer of

PHG-UV gel placed on the crystal swells with time, it was

necessary to perform a correction for the change in the

dimension of PHG-UV thickness. In particular, the peak at

1181 cm�1 (C-O stretching) was used to monitor the swel-

ling of PHG-UV gel. Therefore, we have normalized the

area of the water peak with the area of the PHG-UV peak at

1181 cm�1 to obtain corrected areas.

Figure 4 reports the normalized integrated areas under

the peak ofwater plotted against the evolution time of PHG-

UV gel brought into contact with phosphate buffer/citric

acid solution at pH 7.0 in the absence and in the presence of

mucin at various concentrations.

As it can be observed in Figure 4, in the absence ofmucin,

the water penetration into PHG-UV gel is very fast and a

plateau is reached after about 80 min (plot A). In the

presence of mucin at 0.01 wt.-%, a little lag (about 50 min,

see plot B) in the water penetration is observed in

0

0.2

0.4

0.6

0.8

1

0 200 400 600 800 1000 1200 1400

Time (min)

D

0

0.2

0.4

0.6

0.8

1

0 200 400 600 800

Time (min)

C

0

0.2

0.4

0.6

0.8

1

0 200 400

Time (min)

B

0

0.2

0.4

0.6

0.8

1

0 100 200

Time (min)

A/A

0 no

rmal

ized

A/A

0 no

rmal

ized

A/A

0 no

rmal

ized

A

Figure 4. Integrated and normalized areas under the peak ofwater plotted against the evolution time of PHG-UV 20 wt.-% gelbrought into contact with phosphate buffer/citric acid solution atpH 7.0 alone (A) and in the presence of 0.01 wt.-% (B), 0.1 wt.-%(C) or 1 wt.-% (D) of mucin.



WILEY-VCH

Author ProofAuthor Proofpronounced contrast with the interfacial resistance evident

in the presence of mucin at 1 wt.-%. In fact, in this case the

water diffusion is hindered until 13 h from the beginning of

the experiment (see plot D). An intermediate behavior was

found when PHG-UV gel was brought into contact with a

solution containing 0.1 wt.-% of mucin, the lag being of 5 h

(see plot C).

The initial lag phase of water diffusion data, according to

swelling studies, confirms that mucin interacts with the

surface of PHG-UV gel thus causing the formation of an

interfacial film that hinders water penetration.

Various assumptions can bemade to explain the probable

mechanisms involved in this interaction. Since PHG-UV

gel is a non-ionic network, we can overlook the contribution

of the electrostatic effect. On the contrary, to explain the

interaction between PHG-UV gel and mucin, we can sup-

pose the occurrence of an adsorptionmechanism associated

to a partial interpenetration process at the interface PHG-

UV gel/mucin solution. The first process could result from

the formation of hydrogen and van der Waals bonds

between PHG-UV chains and mucin. The intimate and

prolonged contact between the adherents (PHG-UV gel and

mucin) could promote this mechanism. As a consequence

of the adhesion process, we can also suppose the occurrence

of a diffusion/interpenetration mechanism. Even if PHG-

UV is chemically crosslinked, a partial mobility of its

chains towards mucin solution could be possible as well

as a concomitant partial diffusion of mucin chains into

PHG-UV network. The swelling of PHG-UV gel allows the

relaxation of the polymer chains, thus promoting this

interpenetration. On the other hand, previous studies have

shown that the PHG-UV network results in an amorphous

structure;[25,26] then the absence of crystalline regions

suggests a chain segment mobility which could facilitate

this interpenetration. However, this process could only

concern the surface of PHG-UV gel in contact with mucin

solution. In fact, ATR FT-IR spectra show only small

changes in the position and intensity of bands of the PHG-

UV gel. On the contrary, in the case of deeper interpenetra-

tion, greater variations in the ATR FT-IR spectra would

have been observed.

Figure 5 depicts a schematic representation of the pro-

bable mechanisms involved during the interaction between

PHG-UV gel and mucin.

In order to study the diffusion ofwatermolecules through

PHG-UV gel which interacts with mucin, we have employ-

ed the following solution of Ficks’ second law that satisfies

both initial and subsequent boundary conditions:[14,37]

C=C0 ¼ A=A0 ¼ 1� 4=p�fð�1Þn=ð2nþ 1Þg� expfð�Dð2nþ 1Þ2p2tÞ=4h2g ð1Þ

whereC is the water concentration at the interface at time t;

C0 is the solubility of thewater in the PHG-UV gel;D is the

water diffusion coefficient (in cm2 � s�1); h is the PHG-UV

gel thickness (in centimeter). Concentration terms can be

replaced with experimental absorbances, i.e. C/C0¼A/A0,

where A is the normalized area under the water peak curve

and A0 is the normalized area under the water peak curve

corresponding to equilibrium.[15] The diffusion coefficient

was calculated by employing a non-linear curve fitting

package in order to fit the experimental data to Equation (1).

A better fit according to the experimental datawas observed

at shorter time periods that however are more critical to

the calculation of the diffusion coefficient. The best fit with

the experimental data gave the mean diffusion coefficients

reported in Table 1.

As it is possible to see in the Table 1, there is a

considerable effect of the mucin on water diffusion through

PHG-UV gel: the difference in the diffusion coefficients is

about three orders of magnitude in comparison with the

value found when PHG-UV gel is brought into contact with

phosphate buffer/citric acid solution in the absence of

mucin. It is also evident, as expected, that there is a pro-

nounced effect due to the different structure between PHEA

(whose mucoadhesive behavior has been investigated in

ref.[16]) and PHG-UV gel. In fact, the chemically cross-

linked structure of PHG-UV gel, slows down the diffusion

of water molecules through the gel (the value of D is

3.5� 10�8 cm2 s�1 when PHG-UV gel is brought into

contact with mucin 1 wt.-% solution), whereas when an

aqueous film of PHEA is brought into contact with mucin

1 wt.-% solution, being PHEA a water soluble polymer, a

complete interdiffusion occurs between PHEA film and

mucin solution, then the value of diffusion coefficient for

water inPHEAfilmismuchhigher (D is 7.1� 10�4 cm2s�1)

than that found for PHG-UV gel. For the uncrosslinked

PHG copolymer (that is, like PHEA, a water soluble

Figure 5. Schematic representation of the interaction between PHG-UV 20 wt.-% gel andmucin. A: Starting contact between PHG-UV 20 wt.-% gel and phosphate buffer/citric acidsolution at pH7.0 containingmucin. B:Adhesion between PHG-UV20wt.-%gel andmucin.C: Interpenetration between PHG-UV 20 wt.-% gel and mucin.



WILEY-VCH


polymer) we have obtained a behavior similar to that found

for PHEA film.

On the basis of the data obtained in our experiments, we

can conclude that PHG-UV gel is able to interact with

mucin; therefore it is reasonable to deduce that this gel has

bioadhesive properties.

Conclusion

The results obtained are a clear evidence for the occurrence

of an interaction between PHG-UV gel and mucin. This

interaction could be due to a combination of an adhesion

(adsorption) mechanism with a partial diffusion/interpene-

tration process at the interface hydrogel/mucin. The first

phenomenon presumably is the prevailing one because of

the easiness in the formation of hydrogen and van derWaals

bonds between PHG-UV chains and mucin. On the con-

trary, the second mechanism could be less prominent since

the presence of a chemical polymeric network and the high

molecular weight of mucin allows only the occurrence of a

local and partial interpenetration.

The ability of PHG-UV gel to interact with mucin repre-

sents an important property of this material and suggests its

potential use for bioadhesive drug delivery systems.

Acknowledgements: We thank &author: please write outMIUR.&MIUR for financial support.

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Q1: Please clarify throughout the article all editorial/technical

requests marked by black boxes.

Table 1. Values of diffusion coefficient (D) of water throughPHG-UV gel in contact with phosphate buffer/citric acid solutionat pH 7.0 in the absence and in the presence of mucin at variousconcentrations. For comparison D of an aqueous film of PHEA incontact with phosphate buffer/citric acid solution at pH 7.0 in thepresence of mucin is also reported. Values are the mean� s.d.(n¼ 3).

Samples D

cm2 � s�1

PHG-UV gel/buffer 1.5� 10�5 (�0.4� 10�5)PHG-UV gel/bufferþmucin

0.01 wt.-%5.0� 10�8 (�0.2� 10�8)

PHG-UV gel/bufferþmucin0.1 wt.-%

4.2� 10�8 (�0.3� 10�8)

PHG-UV gel/bufferþmucin1 wt.-%

3.5� 10�8 (�0.3� 10�8)

PHEAaqueous film/bufferþmucin1 wt.-%

7.1� 10�4 (�0.2� 10�4)a)

a) Value reported in ref. [16]



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2005 Bioadhesive Properties of a Polyaminoacidic Hydrogel Evaluation by ATR FT-IR Spectroscopy