Pharmaceutical Development and Technology, 13:513–521, 2008Copyright © Informa UK, Ltd.ISSN: 1083-7450 print / 1097-9867 onlineDOI: 10.1080/10837450802288865

513

LPDT

Chitosan Ascorbate: A Chitosan Salt with Improved Penetration Enhancement Properties

Chitosan Ascorbate as Penetration EnhancerSilvia Rossi, Marzia Marciello, Giuseppina Sandri, Maria Cristina Bonferoni, Franca Ferrari, and Carla CaramellaDepartment of Pharmaceutical Chemistry, University of Pavia, Viale Taramelli, Pavia, Italy

The aim of the present work was to investigate if chitosansalification with ascorbic acid could produce an increase in chito-san penetration enhancement properties towards buccal mucosaand intestinal Caco-2 cell monolayer. Three different chitosangrades were considered. Chitosan hydrochloride and lactate wereused as references. Fluorescein isothiocyanate-dextran (FD4),a hydrophilic high MW molecule, was employed as model pene-trant. Chitosan ascorbate showed better penetration enhancementproperties towards both buccal porcine mucosa and Caco-2 cellmonolayer with respect to hydrochloride and lactate salts. Cytotox-icity of chitosan ascorbate assessed on Caco-2 cells was comparablewith those of chitosan hydrochloride and lactate.

Keywords chitosan ascorbate, penetration enhancer, buccalmucosa, Caco-2 cell line, fluorescein isothiocyanatedextran

INTRODUCTION

Chitosan, the N-deacetyl product of chitin, is a dietaryfiber with potent hypocholesterolemic effects and able tointeract with acidic and neutral steroids in the intestinallumen to increase their fecal excretion. Recently it has alsobeen reported that the addition of ascorbic acid to chitosancauses a large increase in fecal fat excretion.[1–3] In a studyperformed on rats Kanauchi el al. evaluated the actionof different chitosan salts (chitosan ascorbate, lactate andcitrate) on absorption and fat fecal excretion, using celluloseand unsalified chitosan as references.[3] The salificationof chitosan with ascorbic acid resulted very promising:

chitosan ascorbate produced a larger increase in fecalexcretion without affecting protein digestibility.

In a subsequent work, the same authors studied themechanism of chitosan inhibition of lipid digestion and ofsynergism with ascorbic acid. They concluded that chitosandissolves into gastric fluid and becomes a gel able toentrap lipids of the intestinal lumen.[4]. The synergic effectof ascorbic acid was not acid-dependent, but due to thespecificity of ascorbic acid: comparable results wereobtained employing chitosan preparations enriched withsodium ascorbate.

In a more recent work Tsujikawa et al. designed apilot trial to investigate the tolerability and the amount offecal excretion after the oral administration of a chitosanand ascorbic acid mixture for inactive Crohn’s disease.[2]

The results indicated that the oral administration of sucha mixture in patients with inactive Chron’s disease wastolerable and effectively increased fecal fat excretion.

The mechanism for increasing fecal fat excretion bycoexisting chitosan and ascorbic acid remains unclear.Some authors suggested that chitosan-ascorbic acid synergiceffect is due to an increase in chitosan capability to interactand entrap dietary lipids by an emulsifying process mediatedby ascorbic acid.[2]

In the last years, chitosan has been proved to possessdrug penetration enhancement properties towards buccalmucosa.[5–7] Senel et al. proved that a chitosan solubilizedin a diluted solution of lactic acid was able to increase theabsorption of a model peptide across porcine buccalmucosa.[5] Also chitosan hydrochloride have been demon-strated to possess penetration enhancement properties towardsbuccal mucosa.[6,7] Chitosan penetration enhancement prop-erties were affected by the polymer molecular weight.[7]

It is generally recognized that the main obstacle todrug penetration across buccal mucosa via paracellularroute is represented by extracellular lipids. Some authorssuggested that chitosan penetration enhancement propertiesare due to chitosan interaction with the intracellular lipid

Received 30 April 2008, Accepted 16 June 2008.Address correspondence to Prof. Carla Caramella, Department

of Pharmaceutical Chemistry, University of Pavia, Viale Taramelli12, 27100 Pavia, Italy; E-mail: carla.caramella@unipv.it

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Gue

lph

on 0

4/18

/13

For

pers

onal

use

onl

y.

514 S. Rossi et al.

domain.[5] Given these premises, primary aim of thepresent work was to investigate if chitosan salificationwith ascorbic acid could produce an increase in chitosanpenetration enhancement properties towards buccal mucosa.In particular, three different chitosan grades having differ-ent molecular weights were considered. They were salifiedwith ascorbic, hydrochloric and lactic acids. Chitosan hydro-chloride and lactate, well-known as buccal penetrationenhancers, were used as references. Fluorescein isothiocy-anate dextran (FD4), a hydrophilic molecule having a highmolecular weight (4400 kD), similar to that of peptidicdrugs, was used as model molecule.

In the past chitosan has been also proved to possesspenetration enhancement properties towards intestinalepithelium.[8–11] The mechanism suggested is completelydifferent from that supposed for buccal penetration enhance-ment. In fact, the main barrier to the passage of hydro-philic high molecular weight drugs in intestinal epitheliumis represented by tight junctions (TJs), absent from buccalmucosa. Experimental evidences have been given thatchitosan was able to open TJs.[10,11] In the present workthe eventual capability of the association of chitosan withascorbic acid to improve polymer penetration enhancementproperties towards Caco-2 cell monolayer was also inves-tigated. Moreover, cytotoxicity of chitosan ascorbate wasassessed on Caco-2 cells and compared with those ofchitosan hydrochloride and lactate.

EXPERIMENTAL PART

Polymers

The following Chitosan (CS) grades were considered:hMW (1568): deacetylation degree (DD) 91%; mMW(1961): DD 92%: lMW (1504): DD 98% (Giusto Faravelli,Milan, Italy) CS hMW was salified with: ascorbic acid(Carlo Erba Reagenti, Milan, Italy), lactic acid (80%,A.C.E.F., Piacenza, I), hydrochloric acid (Carlo ErbaReagenti, Milan, Italy). CS mMW and lMW were salifiedwith ascorbic acid and hydrochloric acid.

Model Molecule

Fluorescein isothiocyanate dextran (MW 4400) (FD4)(Sigma Aldrich, Milan, Italy) was used as model molecule.

Determination of MWv of the Three Chitosan Grades

CS solutions in 0.1 mol l−1 CH3COONa – 0.2 mol l−1

CH3COOH were prepared. The following chitosan

concentrations were considered: 0.1 mg/mL, 0.2 mg/mL,0.3 mg/mL, 0. 4 mg/mL e 0.5 mg/mL for hMW grade;0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.0005 g/mL and0.6 mg/mL for mMW; 1 mg/mL, 1.4 g/mL, 1.6 g/mL,1.8 g/mL and 2 mg/mL for lMW. An Ostwald viscometer,thermostated at 30 ± 0.5°C, was used. The efflux times ofeach polymer solution and of hydration medium weremeasured. For each polymer solution the reduced viscosity(ηrid) was calculated.[12] For each CS grade, intrinsic viscosityvalue was calculated as intercept on x axis of the straightline ηrid vs. concentration. The mean viscosimetric molecularweight (MWv) was calculated on the basis of Mark Houwinkequation as reported in [12]:

where:

Preparation of Chitosan Solutions

An exact amount of each acid (to obtain a 1:1 molarratio acid: CS deacetylated amine groups) was dissolved/diluted in/with distilled water. For rheological, mucoadhe-sive and buccal permeation measurements, CS was added toobtain 2.5% (w/w) polymer solutions. pH values of CS solu-tions range into interval 4.5–5.5. For permeation measure-ments across Caco-2 cell monolayer and toxicity studies,HBSS (Hanks’ balanced salt solution: CaCl2 anhydrous140 mg/l, MgCl2.6H2O 100 mg/L, MgSO4.7H2O 100 mg/L,KCl 400 mg/L, KH2PO4 60 mg/L, NaHCO3 350 mg/L,NaCl 8000 mg/L, Na2HPO4 48 mg/L, D-glucose 1000 mg/L,Phenol Red 10 mg/l, Gibco-BRL, NY, USA) buffered at pH5.5 with HCl 1 N was used instead of distilled water and CSwas added at 0.4% (w/w) concentration. FD4 was added ateach solution at 0.2% (w/w) concentration.

Rheological Analysis

Each polymer solution was subjected to a completerheological characterization by means of a rotational rhe-ometer (Rheostress 600, Haake, Spinea, Italy). A cone platecombination (C35) was used as a measuring system. Allmeasurements were carried out at 37°C, after a rest time of3 min. The apparent viscosity was measured on increasingshear rate values ranging into the interval 20–300 s−1.

Dynamic oscillatory tests were performed in the linearviscoelastic range. The viscoelastic parameters, storagemodulus (G′) and loss modulus (G′′), were measured atfrequency values ranging from 0.1–10 Hz. Loss tangent(tgδ) was calculated as the ratio between G′′ and G′ values.

[ ]inth = KMa (1)

K DD a DD= × × = − × × +− −1 64 10 1 02 10 1 8230 14 2. , . .

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Gue

lph

on 0

4/18

/13

For

pers

onal

use

onl

y.

Chitosan Ascorbate as Penetration Enhancer 515

Release Measurements

In vitro FD4 release was assessed by means of a Franzdiffusion cell (Permegear, Bethlehem, PA, USA) with a 20mm diameter orifice (3.14 cm2 area)). The donor and thereceptor chambers were separated by a filter membrane(HA 0.45 μm, Millipore, Milan, Italy). Each polymer solu-tion (100 mg) was spread on a circular portion (2 cm2) ofthe filter membrane. 500 μl of pH 6.4 phosphate buffer(USP 31) were added in the donor chamber, over the polymersolution, to simulate the buccal environment. pH 7.4 salineisotonic solution (KH2PO4 1.90 g/L; Na2HPO4 8.10 g/L;NaCl 4.11 g/L) was used as receptor phase.

After 5 h, 500 μl of the acceptor phase were withdrawn.The FD4 released amount was assayed by means of aspectrofluorimetric method (LS50B spectrofluorimeter,Perkin Elmer, Milan, I, λex: 490 nm; λem: 515 nm).

Permeation Measurements Across Porcine Buccal Mucosa

The penetration enhancement properties of each polymersolution were evaluated at 37°C using porcine buccalmucosa as biological substrate. A circular epitheliummembrane of 5 cm2 area, obtained as described in [6], wasplaced between the donor and the receptor chamber of aFranz diffusion cell (Permegear, Bethlehem, PA, USA)with a 20 mm diameter orifice (3.14 cm2 area).

Each polymer solution (100 mg) was applied on acircular portion (2 cm2) of the epithelium membrane.500 μl of pH 6.4 phosphate buffer (USP 31) were addedover the polymer solution in the donor chamber, to simulatethe buccal environment, whereas pH 7.4 saline isotonicsolution was used as acceptor phase.

At fixed time intervals, 500 μl of the acceptor phasewere withdrawn and replaced with fresh buffer. The drugpermeated was assayed by means of the spectrofluorimetricmethod, above mentioned. The permeation test was alsoperformed using 100 μl of a 0.20% (w/w) FD4 solutionprepared in distilled water (reference).

To take into account the different release propertiesof the polymer solutions, the drug amount permeated orpenetrated at the end of the experiment (5 h) was normalizedwith respect to the drug amount released at the same time.The value obtained was expressed as percentage (% drugpermeated/released).

Penetration Measurements into Porcine Buccal Mucosa

By means of Confocal Laser Scanning Microscopy(CLSM) (Leica TCS SP2, Leica Microsystems, Milan,

Italy) FD4 penetration into porcine buccal mucosa wasalso evaluated (13). In particular an apparatus consistedof a circular plexiglas holder and of a Franz cell cup wasused.

100 mg of each polymer solution and of FD4 solutionwere layered on a circular portion of a freshly excisedporcine buccal epithelium (surface area: 3.14 cm2). Thebuccal epithelium was placed on a filter paper disc, laid ona circular holder. A Franz cell cup was put on the epithe-lium and then 500 μl of pH 6.4 phosphate buffer (USP 31)were placed on the sample. The apparatus was sealed withparafilm membrane and placed in an oven at 37°C for 5 h.At the end of the experiment, samples were removed fromthe mucosa which was rinsed twice with physiologicalsolution. Then mucosa was included in the OTC com-pound (Leica Microsystem, Milan, Italy), frozen in liquidnitrogen and stored at −80°C.

Slices perpendicular to the mucosa surface, 25 μm inthickness, were cut using a cryostat (Leica CM1510, LeicaMicrosystem, Milan, Italy) at −20°C. Each slice was placedon a microscope slide, dehydrated for 12 h and subse-quently fixed by dipping the microscope slides in acetone.The nuclei of the tissue slice were stained by dipping thebiological substrates into a solution (1:100000) of PropidiumIodide (Sigma Aldrich, Milan, Italy).

Each microscope slide, with mucosa slice, was mountedusing PVA-DABCO (polyvinyl alcohol mounting mediumwith DABCO antifading, BioChemika, Fluka, Milan, I)and covered with a cover glass.

The slides were observed using a Confocal LaserScanning Microscope (Leica TCS SP2, Leica Microsys-tems, Milan, I) using λex = 485 nm and λem = 515 nm forthe visualization of FD4 and λex = 520 nm and λem = 625 nmfor the visualization of Propidium Iodide. The acquiredimages were processed by means of a software (LeikaMicrosystem, Milan, Italy).

To obtain depth information from specific sections(xz- and yz-sections), the confocal images of xy planeswere first acquired (parallel to the plane of the slicesurface). To generate the xz- and the yz-sections, twohorizontal lines were drawn across the regions of interestin the z = 0 μm-xy- plane, and the digitalized image dataof the successive xy-sections were optically sliced throughalong the z-axis. The results were the xz- and yz planaroptical cross-sections.[14]

Mucoadhesion Measurements

Mucoadhesive properties of polymer solutions weredetermined by means of a tensile stress tester previouslydescribed.[7] Porcine gastric mucin (Mucin type II, SigmaAldrich, Milan, Italy) was employed as biological substrate.

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Gue

lph

on 0

4/18

/13

For

pers

onal

use

onl

y.

516 S. Rossi et al.

100 mg of each polymer solution was layered on a filterpaper disc (area = 2 cm2) and fixed on the movablecarriage of the apparatus. 50 μl of 8% (w/w) mucin disper-sion in pH 6.4 phosphate buffer (USP 31) was layered onthe filter disc placed on the sample holder. A preload of2500 mN was applied in order to allow the formation ofthe mucoadhesive joints. After 3 min rest, the preload wasremoved and the movable carriage was moved at a constantspeed (4 mm/min) up to the complete separation ofthe two surfaces. Both the displacement of the movablecarriage and force of detachment data were recorded andsimultaneously collected on a personal computer.

The parameter work of adhesion (AUC) was calculatedas the area under the force of detachment vs. displacementcurve by means of the trapezoidal rule.

Permeation Measurements Across Caco-2 Cell Monolayer

Caco-2 cells (passage 37) were seeded on tissue-culture-treated polycarbonate filters (culture surface: 33.6 mm2) in24-well culture plates (Greineger Bio-one, PBInterna-tional, Milan, Italy) at a seeding density of 105 cells/cm2.Dulbecco’s modified Eagle’s medium (DMEM, pH 7.4,Bioindustries, Israel) supplemented with 10% foetalbovine serum, benzyl penicillin G (160 U/mL) and strep-tomycin sulphate (100 μg/mL (Bioindustries, Israel) andalso with 1% nonessential aminoacids (Sigma Aldrich,Milan, Italy) was used as culture medium. Cell cultureswere kept at 37°C in an atmosphere of 95% air and 5%CO2.

Filters were used for transepithelial electrical resis-tance (TEER) and FD4 transport experiments 23 days afterseeding. 250 μl of each sample were added on the apicalside of the monolayer. At fixed time intervals, the receptorphase was taken from the basolateral side and replacedwith fresh HBSS. FD4 was dosed in the receptor phaseby means of the above mentioned spectrofluorimetricmethod.

At the end of the experiment, the apical phase wasreplaced by 250 μl of 25 μg/mL Lucifer Yellow (LY)(Sigma Aldrich, Milan, Italy) solution in HBSS. After 1hcontact, LY permeated was assayed by means of a spec-trofluorimetric method ((LS50B spectrofluorimeter, PerkinElmer, Milan, Italy) λex: 428 nm; λem: 521 nm).

In-vitro Cytotoxicity Study

Caco-2 cells (passage 37) were seeded in 96-wellplates with area of 0.34 cm2 at density 105 cells/cm2. Sup-plemented DMEM, pH 7.4 was used as culture medium.

Cell cultures were kept at 37°C in an atmosphere of 95%air and 5% CO2. After seven days, the growing cellsattached to the well bottom and composed a monolayer.On the 8th day, experiments were performed. The toxicitystudy was performed using the neutral red (NR) assay(Tox Kit 4, Sigma Aldrich, Milano Italy) that provides todetermine the accumulation of the NR supravital dye inthe lysosomes of viable, uninjured cells.[15,16] Damagedcell membranes or lysosomes cause a poor capability or nocapability to pick up NR.

Each well was washed with saline phosphate buffer(PBS) and 280 μl of each polymer solution 0.4% (w/w)prepared in HBSS at pH 5.5 were put in contact with thecells. After 1 and 2 h the samples were removed and thecell substrates washed with PBS. 200 μl of NR solution(0.33 mg/mL in DMEM) were put in each well with 2 hof contact time. Cell substrates were then washed withPBS and then with the fixing medium (1% w/v CaCl2 and0.5% w/v formaldehyde aqueous solution) in order toremove NR not entrapped in the cells and to fix thesubstrate. The fixing solution was then removed and asolubilizing solution (1% v/v of acetic acid in ethanol)was added to each cell substrate to cause cell disruptionand, consequently, to produce the release of NR capturedby viable cells. The NR solution absorbance was deter-mined by means of ELISA plate reader (Perkin Elmer,Milan, Italy) at a wavelength of 490 nm with 650 nmwavelength filter. The absorbance for each sample wascompared with that of HBSS, negative control (nottoxic), that was considered to coincide with the maximumviability (100%).

RESULTS AND DISCUSSION

The mean MWv values of the three CS grades consideredwere: 251000 Da for lMW grade, 1163000 Da for mMWand 1855000 Da for hMW.

Rheological Analysis

In Figure 1 viscosity and loss tangent values observedfor the solutions of CS hMW grades salified with ascorbic,hydrochloric and lactic acids are reported. CS lactate solutionshows the highest viscosity profile, while CS hydrochlorideand CS ascorbate solutions are characterized by comparableprofiles. CS ascorbate solution shows loss tangent valueshigher than 1. Since such a parameter is calculated fromthe ratio between the viscoelastic parameters G′′ (lossmodulus) and G′ (storage modulus), it means that CSascorbate is characterized by a prevalence of the viscouscomponent on the elastic one. CS hydrochloride and

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Gue

lph

on 0

4/18

/13

For

pers

onal

use

onl

y.

Chitosan Ascorbate as Penetration Enhancer 517

lactate show comparable loss tangent values, close to 1; itindicates an equilibrium between the two viscoelasticcomponents. These results mean that CS lactate and hydro-chloride are characterized by a more entangled structurewith respect to CS ascorbate.

Permeation Measurements Across Porcine Buccal Mucosa

In Figure 2 the FD4 permeated amount vs. time profilesobserved for the aqueous solutions of all the CS gradesand salts and of a solution of FD4 alone (reference) arereported. lMW CS ascorbate solution shows a profile com-parable to that observed for the solution of FD4 alone, itindicates the lack of penetration enhancement properties.On the contrary, mMW CS ascorbate solution is characterizedby penetration enhancement properties, evidenced by aFD4 permeation profile higher than that observed for FD4solution. Higher penetration enhancement properties areshown by hMW CS ascorbate which causes the perme-ation of a FD4 amount 13 times higher than that observedfor the solution of FD4 alone.

The solutions of lMW and mMW CS hydrochlorideare capable to promote FD4 permeation across buccalmucosa. hMW CS hydrochloride is characterized by apermeation profile comparable to that observed for thesolution of FD4 alone. The higher profile is observed formMW grade which is able to increase FD4 permeated amountsix times with respect to the FD4 solution without CS.

Among CS hMW grades, the higher profile isobserved for CS salified with ascorbic acid. The pooror absent penetration enhancement properties observedfor hMW CS grades salified with lactic and hydrochloricacid could be due to the high entanglement of hydratedpolymer chains, evidenced by the greater elastic behav-iour of the CS lactate and hydrochloride solutions withrespect to the CS ascorbate one. A high polymer chainentanglement can cause a shielding of the functionalgroups (protonated amine groups) responsible for theinteraction of the macromolecular chains with themucosa.

Figure 1. Viscosity and loss tangent profiles observed for theaqueous solutions of hMW CS salts (mean values ± SE; n = 3).

0

3

6

9

12

15

18

0 100 200 300Shear rate (1/s)

Vis

cosi

ty (P

a.s)

CS ascorbate

CS hydrocloride

CS lactate

0

5

10

15

20

25

30

0 2 4 6 8 10Frequency (Hz)

loss

tang

ent

hMW ascorbate

hMW hydrochloride

hMW lactate

Figure 2. FD4 permeation profiles observed for the solutions of the all CS salts and of a solution of FD4 (mean values ± SE; n = 6).

0

5

10

15

20

25

30

0 1 2 3 4 5Time (h)

FD4

perm

eate

d am

ount

(µg)

FD4 solutionhMW CS ascorbatemMW CS ascorbatelMW CS ascorbatehMW CS HClmMW CS HCllMW CS HClhMW CS lactate

0

5

10

0 1 2 3 4 5

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Gue

lph

on 0

4/18

/13

For

pers

onal

use

onl

y.

518 S. Rossi et al.

In Figure 3, % FD4 permeated/released valuesof hMW CS salts are compared. Such a parameterexpresses FD4 amount able to permeate mucosa withrespect to FD4 amount released from polymer solutionand then actually available on mucosal surface. Theseresults confirm the best penetration enhancement prop-erties of hMW CS ascorbate with respect to the othersalts.

Penetration Measurements into Porcine Buccal Mucosa

In Figures 4 CLSM photographs of porcine buccalmucosa placed in contact for 5 h with FD4 solution andhMW CS salts are reported. Green zones indicate thepenetration depht of FD4, while colour intensity is corre-lated to the FD4 amount penetrated; red points are thecell nuclei coloured by propidium iodide. To better pointout FD4 penetration into mucosa, CLSM and opticalmicroscopic photographs of the same mucosa portionhave been superimposed. FD4 solution produces a drugpenetration into mucosa up to 40 μm depth; the lowintensity of green colour indicates the presence of a pooramount of FD4 into mucosa (Figure 4a). In presence ofCS ascorbate (Figure 4b), FD4 penetrates up to 100 μmdepth; the intense green colour means the penetration ofa higher amount of FD4 with respect to the solution ofFD4 alone. CS hydrochloride and lactate show interme-diate behaviours (Figures 4c, 4d), showing a penetrationdepth of 25 and 45 μm, respectively, accompanied byhigher amounts of FD4 penetrated with respect to FD4solution. The results obtained confirm the best penetra-tion enhancement properties of CS ascorbate with respectto the other salts.

Figure 3. % FD4 permeated/released values of C salts (meanvalues ± SE {calculated according to the error propagation theory]).

0

2

4

6

8

10

12

14

lMW mMW hMW lMWCS ascorbate CS hydrochloride CS lactate

mMW hMW hMW

% F

D4

perm

eate

d/re

leas

ed

Figure 4. CLSM photograph of porcine buccal mucosa placed in contact for 5 h at 37°C with the solution of FD4 alone or of hMWCS salt. (a) Solution of FD4 alone; (b) CS ascorbate; (c) CS hydrochloride; (d) CS lactate. The image on the right results fromsuperimposition of CLSM and optical microscopy photographs.

(a) (b)

(c) (d)

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Gue

lph

on 0

4/18

/13

For

pers

onal

use

onl

y.

Chitosan Ascorbate as Penetration Enhancer 519

Mucoadhesion measurements

In Figure 5 work of adhesion (AUC) values obtainedfor the three hMW CS salts are reported. CS ascorbate ischaracterized by the highest AUC values, followed indecreasing order, by CS lactate and hydrochloride. Thisindicates a higher capability of CS when salified withascorbic acid to interact with the biological substrate.

Permeation measurements across Caco-2 cell monolayer

In Figure 6 TEER% values as a function of time arereported for the three hMW CS salts and for the solutionof FD4 alone. All the CS solutions are characterized bya decrease in TEER % values on increasing time. Itindicates the interaction of all the three salts with celltight junctions (TJ). Such a decrease is higher for CSascorbate solution with respect to CS hydrochloride andlactate solutions.

In Figure 7, FD4 permeated amount vs. time profilesobserved for the three CS salts and FD4 solution arereported. The salification with ascorbic acid produces anincrease in FD4 amount permeated after 2 h with respectto the other two salts. Such an increase is lower withrespect to that observed using buccal mucosa as biologicalsubstrate. CS lactate does not seem to possess penetrationenhancement properties, showing a FD4 permeation profilecomparable to those of FD4 solution.

In Figure 8 Papp values of Lucifer Yellow (LY)calculated for all the three CS salts and FD4 solution arereported. Papp values of LY, low MW hydrophilic mole-cule, confirm the results obtained for CS ascorbate andhydrochloride solutions employing FD4 as penetrant mol-ecule. Also CS lactate solution is able to promote LYabsorption; it is, in fact, characterized by a Papp valuehigher than that observed in presence of FD4 solution(reference). This can be explained by the low molecularweight of LY with respect to FD4: CS lactate at theconcentration employed is able to disturb TJ integrity(as evidenced by the decrease in TEER% [Figure 6]) pro-ducing a TJ opening sufficient to facilitate the permeationof LY but not of a high molecular weight molecule suchas FD4.

In-vitro cytotoxicity study

In Figure 9 cell viability % values observed for thethree hMW CS salts are reported. Such values have beencalculated by normalizing the cell viability observed inpresence of the polymer solutions with respect to thatobserved in presence of HBSS alone. The test durationdoes not affect the results obtained: for each CS salt, notsignificant different cell viability % values are observedon increasing time. CS ascorbate solution is characterizedat 1 and 2 h by a cell viability % comparable to thoseobserved for the other two salts.

Figure 5. Work of adhesion (AUC) values observed for the threehMW salts (mean values ± SE; n = 6) vs. P < 0.001 Fisher test.

0

1000

2000

3000

4000

5000

CS ascorbate CS hydrochloride CS lactate

Wor

k of

adh

esio

n (A

UC

) (m

N.m

m)

°

°°

Figure 6. TEER % vs. time profiles observed for the solutions of the three hMW CS salts (mean values ± SE; n = 6).

0

20

40

60

80

100

120

140

0,0 0,5 1,0 1,5 2,0Time (h)

TE

ER%

FD4 solution

CS ascorbate

CS hydrochloride

CS lactate

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Gue

lph

on 0

4/18

/13

For

pers

onal

use

onl

y.

520 S. Rossi et al.

CONCLUSIONS

Depending on salt type, CS solutions are characterizedby different viscosity and viscoelastic properties. In partic-ular, hMW CS lactate solution is characterized by thehighest viscosity profile, while CS ascorbate and hydro-chloride solutions show comparable viscosity profiles. Asfor the elastic properties, the rank order between CS saltsis: lactate > hydrochloride > ascorbate. These results meana different degree of polymer chain entanglement betweenCS salts: lactate and hydrochloride salts are characterizedby a more entangled structure whit respect to ascorbateone.

FD4 permeation measurements evidenced that CSpenetration enhancement properties are affected by salttype and polymer molecular weight.

In particular, mMW and hMW CS ascorbate saltsare able to promote the absorption of the model moleculeFD4 (high MW hydrophilic molecule) across porcinebuccal mucosa; the penetration enhancement propertiesare particularly marked for hMW grade, that permits thepermeation of a FD4 amount 13 times higher with respectto that observed for the solution of FD4 alone. Theseresults are confirmed by the values of the normalizedparameter % FD4 permeated/released, which take intoaccount the different release properties of CS solutions,and by CLSM analysis. Such analysis allowed us to evalu-ate the penetration depth of FD4 into porcine buccalmucosa.

Mucoadhesion measurements have proved that hMWCS ascorbate is characterized by higher mucoadhesionproperties with respect to the other salts.

The permeation test performed on Caco-2 cell mono-layer proved that hMW CS ascorbate, hydrochloride andlactate are able to interact with tight junctions: all the threeCS solutions produce a decrease in TEER. hMW CSascorbate is characterized after 2h by the highest FD4permeated amount. Also CS hydrochloride promotes FD4permeation, but to a lower extent; on the contrary CS lactatesolutions does not possess penetration enhancement prop-erties towards FD4. The increase in CS penetrationenhancement properties in presence of ascorbic acid areless evident with respect to that observed when buccalmucosa was used as biological substrate. This is probablydue to the fact that two different interaction mechanismsare involved: an interaction with extracellular lipids forbuccal mucosa (poor in TJs) and an interaction with TJsfor the intestinal one.

The apparent coefficient values calculated for LY(hydrophilic low MW molecule) and TEER values indi-cate that also CS lactate is able to disturb TJ integrity;it causes a TJ opening, sufficient only to facilitate thepermeation of LY but not of FD4 (high MW molecule).

Figure 7. Profiles of FD4 permeation across Caco-2 monolayerobserved for the solutions of the three hMW CS salts (meanvalues ± SE; n = 6).

0

2

4

6

8

10

12

0,0 0,5 1,0 1,5 2,0Time (h)

µg F

D4

perm

eate

d

FD4 solution

CS ascorbate

CS hydrochloride

CS lactate

Figure 8. Papp values of LY calculated after 2 h for thesolutions of the three hMW CS salts (mean values ± SE; n = 6).

0

2

4

6

8

10

12

14

FD4 solution hMWascorbate

hMWhydrochloride

hMW lactate

Pap

p.10

5 (cm

/s)

Figure 9. % Cell viability observed after 1 and 2 h for thesolutions of the three hMW CS salts (mean values ± SE; n = 6).

0

20

40

60

80

100

CS ascorbate CS hydrochloride CS lactate

cell

viab

ility

%

1h 2h

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Gue

lph

on 0

4/18

/13

For

pers

onal

use

onl

y.

Chitosan Ascorbate as Penetration Enhancer 521

Cytotoxicity tests proved CS ascorbate biocompatibility:such salt shows a cell biocompatibility comparable tothose of CS hydrochloride and lactate, well-known in liter-ature as salts characterized by low toxicity.

The overall results indicate that the salification ofhMW chitosan with ascorbic acid produces an increase inpenetration enhancement properties of the polymertowards both buccal mucosa and Caco-2 cell monolayer.Such an increase is higher when buccal mucosa is used asbiological substrate. A hypopthesis to explain such behav-iour could be that ascorbic acid increases chitosan capabil-ity to interact with extracellular lipids (main barrier todrug transport across buccal mucosa). Such a hypothesis issupported by relevant literature: as mentioned in the intro-duction, some authors have suggested that chitosan is ableto interact with buccal lipids and it has been also provedthat the polymer salification with ascorbic acid produces ahigher chitosan interaction with diet lipids. Work is inprogress to clarify the mechanisms by which the synergiceffect between chitosan and ascorbic acid acts.

ACKNOWLEDGMENTS

This study was supported by funding from the ItalianMinistry of University and Scientific Research (MIUR)(PRIN/COFIN 2005).

REFERENCES

1. Muzzarelli RAA. Chitosan-based dietary foods. Carbohydr.Polym. 1996; 29:309–316.

2. Tsujikawa T, Kanauchi O, Andoh A, Saotome T, Sasaki M,Fujiyama Y, Bamba T. Supplement of a chitosan and ascorbicacid mixture for Crohn’s disease: a pilot study. Appl. Nutrit.Investig. 2003;19:137–139.

3. Kanauchi O, Deuchi K, Imasato Y, Kobayashi E. Increasingeffect of a chitosan and ascorbic acid mixture on fecaldietary fat excretion. Biosci. Biotechnol. Biochem. 1994;58:1617–1620.

4. Kanauchi O, Deuchi K, Imasato Y, Shizukuishi M,Kobayashi E. Mechanism for the inhibition of fat digestion

by chitosan and for the synergistic effect of ascorbate.Biosci. Biotechnol. Biochem. 1995;595:786–790.

5. Senel S, Kremer MJ, Kas S, Wertz PW, Hincal AA, SquierCA. Enhancing effect of chitosan on peptide drug deliveryacross buccal mucosa. Biomaterials. 2000;21:2067–2071.

6. Rossi S, Sandri G, Ferrari F, Bonferoni MC, Caramella C.Buccal delivery of acyclovir from films based on chitosanand polyacrylic acid. Pharmaceut. Develop. Technol. 2003;8(2):199–208.

7. Sandri G, Rossi S, Ferrari F, Bonferoni MC, Muzzarelli C,Caramella C. Assessment of chitosan derivatives as buccaland vaginal penetration enhancers. Eur. J. Pharmaceut. Sci.2004;21:35–39.

8. Artursson P, Lindmark T, Davis SS, Illum L. Effect of chitosanon the permeability of monolayers of intestinal epithelialcells (Caco-2). Pharmaceut. Res. 1994;11:1358–1361.

9. Schipper NGM, Varurn KM, Artursson P. Chitosans asabsorption enhancers for poorly absorbable drugs: influenceof molecular weight and degree of acetylation on drugtransport across human intestinal epithelial (Caco-2) cells.Pharmaceut. Res. 1996;13(11):1686–1692.

10. Thanou M, Verhoef C, Junginger RE. Oral drugs absorptionenhancement by chitosan and its derivatives. Adv. DrugDeliv. Rev. 2001;25:117–126.

11. Smith J, Wood E, Dornish M. Effect of chitosan on epithelialcell tight junctions. Pharmaceut. Res. 2004;21:43–49.

12. Jia Z, Shen D. Effect of reaction temperature and reactiontime on the preparation of low-molecular-weight chito-san using phosphoric acid. Carbohydr. Polym. 2002;49:393–396.

13. Sandri G, Poggi P, Bonferoni M.C, Rossi S, Ferrari F,Caramella C. Histological evaluation of buccal penetrationenhancement properties of chitosan and trimethyl chitosan.J. Pharm. Pharmacol. 2006;58(10):1327–1336.

14. Alvarez-Roman R, Naik A, Kalia YN, Fessi H, Guy RH.Visualization of skin penetration using confocal laserscanning microscopy. Eur. J. Pharmaceut Biopharmaceut.2004;58:301–316.

15. Popiolkiewicz J, Polkowski K, Skierski JS, Mazurek AP.In vitro toxicity evaluation in the development of new anti-cancer drugs – genistein glycosides. Cancer Lett. 2005;229:67–75.

16. Fotakis G, Timbrell JA. In vitro cytotoxicity assays:Comparison of LDH, neutral red, MTT and protein assay inhepatoma cell lines following exposure to cadmium chloride.Toxicol. Lett. 2006;160:171–177.

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Gue

lph

on 0

4/18

/13

For

pers

onal

use

onl

y.

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Gue

lph

on 0

4/18

/13

For

pers

onal

use

onl

y.