chitosan nano particles as delivery systems for doxorubicin

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Journal of Controlled Release 73 (2001) 255–267 www.elsevier.com / locate / jconrel Chitosan nanoparticles as delivery systems for doxorubicin a a b b Kevin A. Janes , Marie P. Fresneau , Ana Marazuela , Angels Fabra , a, * ´ ´ Marıa Jose Alonso a Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, The University of Santiago de Compostela, 15706 Santiago de Compostela, Spain b ` Institut de Recerca Oncologica, Hospital Duran i Reynals, 08907 Barcelona, Spain Received 17 August 2000; accepted 13 March 2001 Abstract The aim of this paper was to evaluate the potential of chitosan nanoparticles as carriers for the anthracycline drug, doxorubicin (DOX). The challenge was to entrap a cationic, hydrophilic molecule into nanoparticles formed by ionic gelation of the positively charged polysaccharide chitosan. To achieve this objective, we attempted to mask the positive charge of DOX by complexing it with the polyanion, dextran sulfate. This modification doubled DOX encapsulation efficiency relative to controls and enabled real loadings up to 4.0 wt.% DOX. Separately, we investigated the possibility of forming a complex between chitosan and DOX prior to the formation of the particles. Despite the low complexation efficiency, no dissociation of the complex was observed upon formation of the nanoparticles. Fluorimetric analysis of the drug released in vitro showed an initial release phase, the intensity of which was dependent on the association mode, followed by a very slow release. The evaluation of the activity of DOX-loaded nanoparticles in cell cultures indicated that those containing dextran sulfate were able to maintain cytostatic activity relative to free DOX, while DOX complexed to chitosan before nanoparticle formation showed slightly decreased activity. Additionally, confocal studies showed that DOX was not released in the cell culture medium but entered the cells while remaining associated to the nanoparticles. In conclusion, these preliminary studies showed the feasibility of chitosan nanoparticles to entrap the basic drug DOX and to deliver it into the cells in its active form. 2001 Elsevier Science B.V. All rights reserved. Keywords: Chitosan; Dextran sulfate; Nanoparticles; Doxorubicin; Adriamycin 1. Introduction related to the development of multidrug resistance [2] and acute cardiotoxicity [3] have led researchers Doxorubicin (DOX) and its bioactive derivatives to investigate alternative forms of administering are among the most widely used anticancer drugs in DOX for the treatment of cancer, with both prodrug chemotherapy treatment [1]. However, problems [4] and particulate [5] methods involved as active fields of DOX research for the past two decades. DOX microencapsulation has shown some appli- *Corresponding author. Tel.: 134-981-594-627; fax: 134-981- cations for the controlled release of DOX over 547-148. E-mail address: [email protected] (M.J. Alonso). extended periods of time [6]. Though relevant for 0168-3659 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0168-3659(01)00294-2

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Page 1: Chitosan Nano Particles as Delivery Systems for Doxorubicin

Journal of Controlled Release 73 (2001) 255–267www.elsevier.com/ locate / jconrel

Chitosan nanoparticles as delivery systems for doxorubicina a b bKevin A. Janes , Marie P. Fresneau , Ana Marazuela , Angels Fabra ,

a ,*´ ´Marıa Jose AlonsoaDepartment of Pharmacy and Pharmaceutical Technology, School of Pharmacy, The University of Santiago de Compostela,

15706 Santiago de Compostela, Spainb `Institut de Recerca Oncologica, Hospital Duran i Reynals, 08907 Barcelona, Spain

Received 17 August 2000; accepted 13 March 2001

Abstract

The aim of this paper was to evaluate the potential of chitosan nanoparticles as carriers for the anthracycline drug,doxorubicin (DOX). The challenge was to entrap a cationic, hydrophilic molecule into nanoparticles formed by ionicgelation of the positively charged polysaccharide chitosan. To achieve this objective, we attempted to mask the positivecharge of DOX by complexing it with the polyanion, dextran sulfate. This modification doubled DOX encapsulationefficiency relative to controls and enabled real loadings up to 4.0 wt.% DOX. Separately, we investigated the possibility offorming a complex between chitosan and DOX prior to the formation of the particles. Despite the low complexationefficiency, no dissociation of the complex was observed upon formation of the nanoparticles. Fluorimetric analysis of thedrug released in vitro showed an initial release phase, the intensity of which was dependent on the association mode,followed by a very slow release. The evaluation of the activity of DOX-loaded nanoparticles in cell cultures indicated thatthose containing dextran sulfate were able to maintain cytostatic activity relative to free DOX, while DOX complexed tochitosan before nanoparticle formation showed slightly decreased activity. Additionally, confocal studies showed that DOXwas not released in the cell culture medium but entered the cells while remaining associated to the nanoparticles. Inconclusion, these preliminary studies showed the feasibility of chitosan nanoparticles to entrap the basic drug DOX and todeliver it into the cells in its active form. 2001 Elsevier Science B.V. All rights reserved.

Keywords: Chitosan; Dextran sulfate; Nanoparticles; Doxorubicin; Adriamycin

1. Introduction related to the development of multidrug resistance[2] and acute cardiotoxicity [3] have led researchers

Doxorubicin (DOX) and its bioactive derivatives to investigate alternative forms of administeringare among the most widely used anticancer drugs in DOX for the treatment of cancer, with both prodrugchemotherapy treatment [1]. However, problems [4] and particulate [5] methods involved as active

fields of DOX research for the past two decades.DOX microencapsulation has shown some appli-*Corresponding author. Tel.: 134-981-594-627; fax: 134-981-

cations for the controlled release of DOX over547-148.E-mail address: [email protected] (M.J. Alonso). extended periods of time [6]. Though relevant for

0168-3659/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0168-3659( 01 )00294-2

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solid, accessible tumors, these particles are too large cell membranes, an attractive feature for the treat-to be endocytosed by most cells or circulate freely in ment of solid tumors. From the perspective ofthe bloodstream. Consequently, the association of intravenous administration, positively charged par-DOX to submicron carriers, such as liposomes [7], ticles would interact with different blood componentsnanoparticles [8], or micelles [9], has drawn greater as compared to negatively charged particles. Theseinterest. changes could potentially create a different biodistri-

The majority of attempts to associate DOX to bution and/or organ accumulation pattern followingnanoparticulate carriers have used anionic or neutral intravenous administration. Additionally, a positivelypolymers. Akiyoshi et al. [10] achieved DOX en- charged system that would be expected to interactcapsulation in cholesterol-bearing pullulan hydrogel with cells and/or membranes would be desirable fornanoparticles, though loading levels were very low testing alternative modes of administration of DOX,(,0.1 wt.%) and cytotoxic effects of the nanoparti- i.e. mucosal administration.cles were lower than that of free DOX. Combining We believed that an interesting candidate withprodrug and encapsulation strategies, Yoo et al. [11] which to test these hypotheses was the cationiccovalently linked DOX to the terminus of poly(D,L- polysaccharide, chitosan. This biopolymer has shownlactic-co-glycolic acid) (PLGA), then formed favorable biocompatibility characteristics [19] asnanoparticles with the conjugate by an emulsion- well as the ability to increase membrane permeabili-solvent diffusion method. The group was able to ty, both in vitro [20] and in vivo [21], and beobtain considerable loadings (3.45 wt.%), achieve a degraded by lysozyme in serum [22]. Consequently,controlled release of DOX over nearly 1 month, and the aim of this paper was to encapsulate appreciablemaintain antiproliferative activity relative to free quantities of DOX in chitosan nanoparticles made byDOX, though these results were possible only by ionotropic gelation with sodium tripolyphosphateforming the covalent linkage between the polymer (TPP) and test the effects of DOX encapsulationand the drug. and/or release on cytotoxic activity relative to free

The vast majority of work involving nanoparticu- DOX. To achieve this aim, we tried two approaches:late DOX association, however, has been with ionic bridging with a coincorporated polyanion andpolyacrylates, exploiting charge interactions of the polymer /drug complexation.polymer with the drug to achieve high associationefficiencies. Polymethacrylate nanoparticles with ad-sorbed DOX were administered intravenously tohepatoma patients and demonstrated prolonged plas- 2. Experimental sectionma levels, as well as reduced total clearance of DOXrelative to a control DOX solution [12]. DOXassociated to polyalkylcyanoacrylate nanoparticles 2.1. Materials[13] have demonstrated reduced cardiotoxicity fol-lowing intravenous administration in mice [14] as Chitosan hydrochloride salt, Protasan CL 110well as increased cytoxicity against multidrug resis- (M .100 kDa), was purchased from Pronova Bio-w

tant cell lines in vitro [15]. Later work showed that polymers (Oslo, Norway). Doxorubicin hydrochlo-coating of these particles with polysorbate 80 sig- ride was obtained as a 2 mg/ml solution in 0.9%nificantly increased DOX accumulation in brain (w/v) sodium chloride from Tedec-Meiji Farmatissue [16]. However, these DOX loaded particles (Madrid, Spain). TPP, type B gelatin (75 bloom),have demonstrated acute renal toxicity [17] as well polyphosphoric acid, and dextran sulfate (M 510w

as decreased permeability of the drug across artificial kDa) were all purchased from Sigma-Aldrich S.A.membranes with respect to free DOX [18]. (Madrid, Spain). Phosphorylated glucomannan was a

´An alternative approach would be to entrap DOX gift from Industrial Farmaceutica Cantabria (Madrid,into a positively charged carrier. Cell adhesion and Spain). Unless otherwise mentioned, water was

´potentially cell uptake of such particles should be ultrapure grade (Milli-Q plus, Millipore Iberica,favored due to their attraction to negatively charged Spain). All other chemicals were reagent grade.

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2.2. Spectrophotometric analysis formed by photon correlation spectroscopy and laserDoppler anemometry, respectively, with a Zetasizer

Reagent concentrations were fixed for all spectro- 3000HS (Malvern Instruments, UK). For size mea-scopic studies. Chitosan was maintained constant at surements, samples were diluted in water and mea-400 mg/ml and polyphosphoric acid, dextran sulfate, sured for a minimum of 180 s. Raw data wereand DOX concentrations all at 40 mg/ml. Spectra subsequently correlated to mean hydrodynamic sizewere recorded from 350 to 600 nm using a UV-VIS by cumulants analysis. For zeta potential measure-spectrophotometer (Model UV-1603, Shimadzu, ments, samples were diluted in 0.1 mM KCl andColumbia, MD) with a 2 nm slit width and a 1 cm measured in automatic mode. All measurementspath length at intervals of 0.5 nm using water as the were performed in triplicate.baseline reference. Particle morphology was examined by transmis-

sion electron microscopy (CM12 Philips, Eindhoven,2.3. Formation of chitosan–DOX complex Netherlands). Samples were stained with 2% phos-

photungstic acid for 10 min, immobilized on copperDOX was added to a solution of 0.2% (w/v) grids, and dried overnight for viewing.

chitosan in water to a final concentration of 30%(w/w) with respect to chitosan at pH 5.5. The 2.6. Evaluation of DOX encapsulationsolution was left under magnetic stirring 24 h atroom temperature, dialyzed against distilled water Encapsulation efficiency and nanoparticle yield oflowered to pH 5 with 1 N HCl for 36 h, and the different formulations were determined by cen-lyophilized. To avoid photodegradation of DOX trifugation of the samples at 24,0003g for 30 min.during the complexation and purification, all pro- Pellets were incubated at 808C overnight andcedures were performed in the absence of light. weighed. Supernatant DOX concentrations wereDOX loading was calculated spectrophotometrically calculated by fluorimetry (Model LS-50B, Perkin-

25at 487 nm (´51.74310 l /g cm). Elmer, Norwalk, CT) with a slit width of 5 nm andexcitation and emission wavelengths at 480 nm and

2.4. Preparation of chitosan nanoparticles 590 nm, respectively. Dilutions of samples andcalibration curves were performed in water, and all

Chitosan particles incorporating polyanions and measurements were performed in triplicate. Encapsu-chitosan–DOX complexed particles were prepared as lation efficiency was calculated as follows:described previously [23]. Briefly, chitosan or

DOX encapsulationchitosan–DOX complex was dissolved at 0.175%(w/v) with 1% (v/v) acetic acid and then raised to total DOX 2 free DOX

]]]]]]]efficiency 5 .pH 4.7–4.8 with 10 N NaOH. For DOX-loaded total DOXnanoparticles incorporating polyanions, 88–363 mgDOX (10–30% loading) was first incubated for 20– 2.7. Evaluation of in vitro DOX release30 s with 88 mg of the polyanion. The mixture wasthen added to a chitosan solution giving a final The nanoparticles were collected by centrifugationchitosan concentration of 0.175% (w/v). To 500 ml at 90003g for 40 min on a glycerol bed. The pelletsof this polymer solution, 100 ml of 0.291% (w/v) were resuspended and incubated in 4 ml of 100 mMTPP in water were added under magnetic stirring, acetate buffer (pH 4) or phosphate buffered salineleading to the immediate formation of the nanoparti- (pH 7.4) at 378C under light agitation. The quantitycles. of nanoparticles was adjusted to obtain a maximum

DOX concentration of 1 mg/ml. At varying time2.5. Physicochemical characterization of points, supernatants were isolated by centrifugationnanoparticles at 24,0003g for 30 min and measured by

fluorimetry as described earlier. Following superna-Size and zeta potential measurements were per- tant extraction, pellets were discarded (destructive

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sampling). Calibration curves were made with the The number of active cells was estimated by measur-incubation medium, and all measurements were ing the absorbance at 540 nm (Titertek Multiscan,performed in triplicate. ICN, Costa Mesa, CA). The percentage of cytostasis

was calculated as follows:2.8. Fluorimetric analysis

A 2 B]]Cytostasis 5 ADOX emission spectra were recorded in water

from 500 to 750 nm at a fixed excitation of 480 nm where A is the absorbance of cells incubated withwith excitation and emission slit widths of 5 nm. culture medium and B is the absorbance of cellsSpectra were read at a scan speed of 200 nm/min incubated with the different nanoparticle formula-and normalized with respect to peak emission. tions. All samples were made in sextuplicate.

2.9. In vitro cytostasis assays2.10. Confocal microscopy analysis

Human melanoma A375 cells (ATCC, Rockville,MD) and C26 murine colorectal carcinoma cells DOX accumulation in treated cells was localizedwere grown in Ham F-12 medium (GIBCO, Grand by confocal microscopy. Briefly, cells (HumanIsland, NY) supplemented with 10% fetal bovine melanoma A375 cells (ATCC)) from exponential

2serum, sodium pyruvate, non-essential amino acids, cultures were grown on 1.13-cm glass coverslips¨L-glutamine, and twofold vitamin solution (GIBCO). (Objekttrager, Menzol Glaser, Germany) at a density4The cultures were maintained in plastic flasks and of 5310 cells /coverslip. One day later, the cultures

incubated in 5% CO /95% air at 378C in a were washed and treated with serum-free, Ham F-122

humidified incubator. The cell lines were examined medium containing either DOX-loaded chitosanand found to be free of mycoplasma, as assayed by nanoparticles incorporating dextran sulfate (30 minthe Gen-Probe Mycoplasma T.C. (Gen-Probe Inc., to 24 h incubation) or free DOX (30 min incubation).San Diego, CA). All incubations were carried out at 378C with an

The antiproliferative effect of DOX was analyzed equivalent final concentration of DOX (5 mg/ml).by the MTT method [24]. Cells from exponential Alternatively, cells were incubated using a two-cultures were seeded onto 96-well tissue culture compartment Boyden chamber. Briefly, either DOX-

3plates (TPP, Switzerland) at a density of 5310 loaded chitosan nanoparticles containing dextran2cells /well for a 0.36-cm well (optimal seeding sulfate or free DOX solutions were diluted in serum-

density that avoids full confluency for the length of free culture medium and incorporated into the upperthe 4-day experiment). One day later, the cultures compartment of polycarbonate transwell filters (0.4were washed and incubated for 2 h with the different mm pore diameter, Costar). The cells seeded onsamples at various dilutions in serum-free, Ham F-12 coverslips for 24 h were placed in the lower com-medium. The different preparations were adjusted to partment, thereby receiving the DOX solution fil-maintain the same drug concentration, and experi- tering from the upper compartment but excluding thements were performed in parallel wells with increas- DOX associated to the nanoparticles (a controling DOX concentrations of 0.1 mg/ml to 100 mg/ml. experiment was performed demonstrating that theFollowing incubation, the cell monolayers were particles did not cross the filter). Following incuba-washed five times with PBS and left 3 days in tion, cells were washed five times with PBS, and thecomplete media. After this time, 50 ml /well of PBS coverslips were mounted on slides. Fluorescencecontaining 1 mg/ml MTT (tetrazolium salt, Sigma- observation was carried out with a confocal micro-Aldrich S.A., Madrid) was added, and the plates scope (TCS 4D, Leica Instruments) using an argon/were incubated an additional 4 h. The intracellular krypton laser (75 mW) at 488 nm for excitation andformazan crystals resulting from the reduction of the an LP filter of 590 nm for DOX detection. Contrasttetrazolium salts present only in metabolically active images were simultaneously observed using thecells were solubilized with DMSO (Sigma-Aldrich). inverted microscope equipment with a PL Apo 633 /

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1.4-oil objective (DMIRBE, Leitz). All experimentswere performed in sextuplicate.

2.11. Statistical analysis

The statistical significance of all results wasdetermined using the two-tailed Student’s t-test.

3. Results

The molecular structures of DOX and the com-plexing agents, polyphosphoric acid and dextransulfate, are shown in Fig. 1. The protonable groupsin the DOX molecule were expected to interact withthe deprotonable groups of polyphosphoric acid anddextran sulfate.

The interaction of DOX with different polyanionsand chitosan was first investigated spectrophotomet-rically. As seen in Fig. 2A and B, the DOX peak at480 nm was reduced by |53% upon incubation witheither polyphosphoric acid (Fig. 2A) or dextransulfate (Fig. 2B). Spectral changes in the DOX peakwere also observed for the samples, with polyphos-phoric acid and dextran sulfate inducing red shifts of9 and 14 nm for DOX solutions, respectively.Subsequent addition of chitosan increased the in-tensity of the 480 nm peak with polyphosphoric acidand dextran sulfate to 110 and 83% of the originalDOX absorbance. Spectral peaks for both samplesreturned to 480 nm. In control studies, no detectableabsorbance was noted for individual chitosan, poly-phosphoric acid, or dextran sulfate solutions over thechosen wavelength range at the concentrations tested(data not shown). No significant differences in pH

Fig. 1. Chemical structure of: (A) DOX, (B) polyphosphoric acid,were noted among any of the formulations shown(C) dextran sulfate. *Deprotonable functional group, **protonable(DOX, DOX1polyanion, DOX1polyanion1functional group.

chitosan).A comparison of the DOX encapsulation efficien-

cies for different chitosan–TPP nanoparticle formu- polyanion concentration, forming visible agglomer-lations is shown in Table 1. At 10% (w/w) polyanion ates. Incorporation of dextran sulfate increased DOXwith respect to chitosan, no significant differences in encapsulation efficiency approximately twofold withDOX encapsulation efficiencies were observed with respect to the control formulation.the coincorporation of gelatin, glucomannan, or The effect of initial DOX loading on encapsulationpolyphosphoric acid relative to the control formula- efficiency for chitosan–TPP nanoparticles incor-tion, with all encapsulation efficiencies between 8 porating dextran sulfate can be seen in Table 2, withand 13%. Also, the addition of polyphosphoric acid values ranging from 19 to 23%. Mean encapsulationcaused a destabilization of the suspension at this efficiencies decreased only slightly with increases in

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Table 2Effect of DOX loading on encapsulation efficiency for chitosannanoparticles incorporating dextran sulfate (n53)

Theoretical Encapsulationloading (%) efficiency (%)

5 22.561.210 21.962.520 19.361.8

particles showed a dense, spherical structure, whichwas consistent with previous observations [25],though size dispersion did appear to be greater withthe addition of dextran sulfate. Particle size and zetapotential values for the unloaded nanoparticles were259615 nm and 133.460.8 mV, respectively. DOXloading (10% theoretical) did not significantly alterthese values (292642 nm and 133.260.1 mV).Blank nanoparticle yield was 5161% which wasunchanged following 10% DOX loading (5262%),resulting in a real DOX loading of 4 wt.% withrespect to the polymer.

A similar morphology was observed for DOX–chitosan complexed nanoparticles (data not shown).DOX complexation efficiency to chitosan was de-termined to be 1.4% (w/w). The total amount of

Fig. 2. (A) UV-VIS spectra of: (———) DOX solution,DOX complexed to chitosan was incorporated in the(— — —) DOX1polyphosphoric acid solution, (- - -) DOX1nanoparticles structure, resulting in a real loading ofpolyphosphoric acid1chitosan solution. (B) UV-VIS spectra of:0.43% (w/w). DOX–chitosan complexed nanoparti-(———) DOX solution, (— — —) DOX1dextran sulfate solu-

tion, (- - -) DOX1dextran sulfate1chitosan solution. cles possessed a mean nanoparticle size of 21363nm and zeta potential of 133.760.6 mV. The low

DOX loading, and differences were only marginally degree of complexation was not expected to sig-significant (P,0.1) between the highest and lowest nificantly alter the characteristics of the formulation,loading levels. and the yield was assumed to be |100%, in accord-

TEM images of chitosan–TPP nanoparticles incor- ance with previously reported yields [23] for thisporating dextran sulfate are shown in Fig. 3. The chitosan–TPP formulation.

The in vitro release profile of chitosan–TPPnanoparticles in acetate buffer (pH 4) is shown in

Table 1 Fig. 4. The particles incorporating dextran sulfateEncapsulation efficiencies for different chitosan nanoparticle

showed a burst release of 17% at 2 h, followed by anformulations. All polyanions were incorporated at 10% (w/w)additional release of 4.5% over the next 2 days. Forwith respect to chitosan. Theoretical DOX loading: 10% (n53)nanoparticles containing DOX complexed withPolyanion Encapsulationchitosan, a small release of 4.5% was noted at 2 h,incorporated efficiency (%)with negligible DOX increases in release detected

No polyanion 9.162.2over the proceeding 5 days.Type B gelatin 8.461.5

Fluorescence profiles of DOX emission spectra areGlucomannan 9.363.3Polyphosphoric acid 12.264.1 shown in Fig. 5. Relative fluorescence was nearlyDextran sulfate 21.962.5* identical for the DOX solution and DOX released

* P,0.01. from nanoparticles, with an average difference of

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Fig. 3. TEM images of chitosan–TPP nanoparticles incorporating dextran sulfate.

0.260.8% between the normalized profiles. Con- made from DOX complexed to chitosan showedversely, encapsulated DOX exhibited an additional slightly decreased cytostatic activity relative to theemission band at 630 nm which was evident over the DOX solution over this same concentration range.range of 600–700 nm. However, for most concentrations assayed, the dif-

Fig. 6 shows the cytostasis of the different ferences were not statistically significant. The blankchitosan formulations in vitro for the C26 cell line. nanoparticle suspension showed no significant cyto-No significant differences were noted between the stasis. Very similar results were obtained usingDOX loaded chitosan–TPP nanoparticles incorporat- human melanoma A375 cells (results not shown).ing dextran sulfate and the control DOX solution DOX localization of different nanoparticle /controlover drug concentrations from 0.1 mg/ml to 100 formulations in human melanoma A375 cells ismg/ml in any of the cell lines tested. Nanoparticles shown in Fig. 7. Within 30 min incubation, free

DOX was localized within the cell nucleus (Fig. 7A),

Fig. 4. In vitro release profile for: (– + –) DOX loaded chitosan– Fig. 5. Fluorescence emission spectra of: (———) DOX solutionTPP nanoparticles incorporating dextran sulfate (DOX loading: in water, (— — —) DOX encapsulated in chitosan–TPP4%) and (– j –) DOX complexed nanoparticles (DOX loading: nanoparticles, (- - -) DOX released from chitosan–TPP nanoparti-0.4%) (n53). cles.

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molar excess of chitosan), thus minimizing thepolymer–drug repulsion by pairing a large fractionof the positive amino groups with negatively chargedphosphates.

This formulation in itself did allow small quan-tities of DOX to be retained within the particles(Table 1), likely by physical entrapment. However,the efficiency was quite low (9.1%), and since thenanoparticles were still positively charged, we fearedthat there would be a repulsion between chitosan andDOX, owing to the positive charge of the drug. In anattempt to remedy these concerns, we tested a variety

Fig. 6. In vitro cytostasis in C60 cells for: (9) DOX loaded of polyanions which could simultaneously form achitosan nanoparticles incorporating dextran sulfate, ( ) chitosan strong ionic interaction between both chitosan andnanoparticles containing DOX complexed to chitosan, ( ) blank

DOX, increasing the encapsulation efficiency andchitosan nanoparticles, (h) DOX solution (n56).binding the molecule tightly to the particles. Type Bgelatin (pK 54.7–5.0, as stated by the manufacturer)a

and phosphorylated glucomannan (763% phos-whereas intracellular localization of DOX loaded phorylation, as stated by the manufacturer) fit theseinto chitosan nanoparticles containing dextran sulfate criteria as macromolecules, but no effect on thecould be visualized only after 24 h incubation. At spectroscopic profile of DOX, potentially indicatingthis point, no differences were seen in the intracellu- intermolecular interactions, was observed (data notlar localization of free DOX versus DOX previously shown). We concluded that neither gelatin nor gluco-associated to chitosan nanoparticles (Fig. 7A and B). mannan possessed a sufficiently high negative chargeTo determine if DOX might be released from density to complex appreciable quantities of bothchitosan nanoparticles before entering the cells, we chitosan and DOX. These observations were cor-used a two compartment set-up where cells and DOX roborated by failed attempts to augment DOX en-samples were separated by a 0.4 mm polycarbonate capsulation with the incorporation of these moleculesfilter. As shown in Fig. 7C, no DOX was observed in into chitosan–TPP nanoparticles, shown in Table 1.cells incubated for 24 h with DOX-loaded nanoparti- From this point, we shifted our attention tocles in the upper compartment. A control mixture of polymers with higher anionic charge densities (atfree DOX and blank nanoparticles in the upper least one negative charge per monomer). We firstcompartment showed an intracellular distribution tested polyphosphoric acid, which did appear tocomparable to free DOX in solution (Fig. 7D). interact strongly with DOX in solution. This inter-

action, however, was completely eradicated by theaddition of chitosan, as evidenced by the reversion of

4. Discussion the spectroscopic profile in Fig. 2A. The overallabsorption profile of this system was, in fact, higher

The major goal of this work was to develop a than the original DOX spectrum, perhaps due to anchitosan nanoparticulate system as a novel, positive- increase in sample turbidity as polyphosphoric acidly charged, colloidal carrier for DOX. The greatest formed particulated complexes with chitosan. Never-challenge was to encapsulate appreciable quantities theless, a mixture of chitosan and polyphosphoricof DOX, overcoming the charge repulsion between acid at the same concentrations but in the absence ofthe cationic polymer (pK 56.5) [26] and the pre- DOX showed no significant absorption over thea

dominantly positively charged anthracycline drug range tested (data not shown). Corroborating the(pK 58.2) [27]. To begin, we selected a chitosan– absorption spectra, the incorporation of polyphos-a

TPP nanoparticle formulation [23] which could phoric acid did not increase DOX encapsulation.accommodate a large quantity of TPP (only |25% Moreover, the presence of this polymer in the

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Fig. 7. Confocal images of (A) free DOX (30 min incubation), (B) DOX-loaded nanoparticles (overnight incubation), (C) DOX-loadednanoparticles in the upper compartment of a Boyden chamber (overnight incubation), and (D) DOX1blank nanoparticle mixture in theupper compartment of a Boyden chamber (overnight incubation). All confocal studies were performed in A375 melanoma cells with 5mg/ml equivalent DOX concentration. Magnifications: 3630 (A, B, D), 3400 (C).

chitosan solution led to the formation of aggregates, chitosan nanoparticles for the encapsulation of DOX.rather than nanoparticles, upon addition of TPP. As could be anticipated, effects of the polyanion on

Sulfonic acid groups have been shown to bind the UV-VIS spectrum of DOX were readily visible.DOX in considerable quantities when incorporated in More importantly, however, the DOX–dextran sul-ion-exchange resins [28]. Additionally, dextran sul- fate complex appeared to only be partially disso-fate has been used successfully to augment the ciated by the addition of chitosan (Fig. 2B), openingencapsulation of DOX in albumin microspheres [29]. the possibility of using the complex to draw moreWith these reports in mind, we decided to test the DOX into the nanoparticles. Indeed, this entrapmentfeasibility of incorporating dextran sulfate into did occur, as the formulation incorporating dextran

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sulfate was the only one to achieve encapsulation be tightly bound to chitosan. Indeed, this phenom-efficiencies significantly above the control formula- enon did take place. While there was only a minimaltion. DOX encapsulation was also visibly apparent yield for the complexation (0.43 wt.%), all of thewith these nanoparticles, which formed a dense red DOX that was complexed remained incorporatedpellet upon centrifugation. within the nanoparticles. Therefore, it could be

The difference between DOX–dextran sulfate and induced that the entrapment efficiency of DOXDOX–polyphosphoric acid interactions is intriguing, previously associated to chitosan was 100%. Thein that one would expect coulombic forces to be possibility that the high entrapment efficiency isstronger with the acid, owing to its higher charge merely caused by low initial DOX loading was alsodensity on a per weight basis. However, another considered. As a control study, we prepared chitosanimportant consideration is the interaction of these nanoparticles with the same DOX initial 0.43 wt.%polyanions with chitosan. Using the same argument loading, but adding DOX to the chitosan solutionof charge density, polyphosphoric acid should ex- prior to the nanoparticles formation. In this case thehibit far greater avidity to chitosan relative to dextran encapsulation efficiency was far lower (23.061.0%)sulfate. Hence, more DOX would be displaced from than that observed for chitosan–DOX complexes.polyphosphoric acid than from dextran sulfate upon In vitro release studies were performed in acetateaddition of chitosan. buffer (pH 4). We chose this medium because DOX

Interestingly, DOX encapsulation efficiency in the is maximally stable at pH values between 3 and 5formulation containing dextran sulfate appeared and also because, at higher pHs we encounteredminimally dependent upon theoretical DOX loading problems of fluorescence quenching or interferenceover the range of 5–20% (w/w). At 10% DOX for the quantification of released DOX. Obviously, inloading, there remains a 3.7-fold molar charge excess these experiments, we did not expect to predict theof negatively-charged sulfonic acid groups relative to release behavior of these particles in vivo but toDOX amino groups, so it is quite feasible that, due to compare the formulations developed and to gainthis excess, the saturation level of DOX association some insight about the mechanism of release.is not reached. Under these conditions, therefore, it is Nanoparticles incorporating dextran sulfate showed alikely that the formation of DOX–dextran sulfate burst release of 17% at 2 h, followed by an addition-complexes is favored by higher quantities of DOX al release of 4.5% over the next 2 days. This slowincubated, with real loading increasing almost linear- release was quite distinct from the profiles obtainedly with theoretical loading. from similar chitosan nanoparticles encapsulating

An entirely different approach was taken with insulin, where 100% release was observed within 15nanoparticles containing DOX complexed to min [21]. Even less DOX was detected in PBS at 5chitosan. As an amphoteric drug (protonable amino days (data not shown), probably due to DOX degra-group and deprotonable phenolic group), there con- dation in the near neutral medium. The initial phasetinually exists an equilibrium between the positively of release is logically attributed to the DOX locatedcharged, negatively charged, neutral, and zwit- at the surface of the particles while the remainder ofterionic species of DOX (Fig. 1). Additionally, there the unreleased DOX was assumed to be well en-are other factors (hydrophobic /hydrophilic interac- trapped within the chitosan nanoparticles and tightlytions, resonance effects, etc.) which could allow associated to the chitosan molecules, probably as ansmall quantities of DOX to complex with chitosan, ionic complex with dextran sulfate. Therefore, thedespite the overwhelming charge repulsion between degradation of chitosan would be required for ac-the two molecules, as has been noted previously complishing the release process. Unfortunately, di-between DOX and positively charged transition rect confirmation of this hypothesis by enzymaticmetal ions [30,31]. We tested the extent of this digestion of the nanoparticles was not possible sinceassociation by incubating DOX and chitosan in chitosanase treatment of the nanoparticle suspensionsolution, dialyzing to remove non-associated DOX, also degraded DOX solutions and/or quenchedand lyophilizing to promote polymer–drug interac- fluorescence in control studies. Nevertheless, thetions. We did not expect considerable association to effect of dextran sulfate on DOX release was appar-occur, but that the DOX which did associate would ent, as chitosan nanoparticles without the polyanion

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showed over twice the burst effect after 2 h under the than to the release of free drug in the cell culturesame conditions (36.760.3%). medium. To confirm this hypothesis we investigated

Nanoparticles containing DOX complexed with the mechanism of in vitro cytotoxicity for DOX-chitosan displayed an even smaller release over the loaded chitosan nanoparticles, using humansame period, an observation that is easily explained melanoma A375 cells, via confocal microscopy.by the aforementioned interactions binding the drug Comparable fluorescence localization, visualized asto the polymers. Indeed, since the interaction DOX– red, was seen for DOX-loaded nanoparticles relativechitosan seems to be quite stable, any drug released to free DOX (Fig. 7A and B), however, a sig-would be a result of degradation of chitosan or by nificantly longer incubation time was required for therelease of DOX complexed on the particle surface. nanoparticles to display the intracellular fluorescence

Notwithstanding, spectral analysis of the DOX signals. This suggests that these particles might enterreleased from chitosan nanoparticles incorporating the cells, and that this internalization process occursdextran sulfate showed that the released DOX was over a much longer time than the diffusion of thefluorimetrically identical to that of the native DOX free drug. Indeed, from these observations it could besolution, as seen in Fig. 5. This preservation of the inferred that, after short incubation times, thefluorescence signature supports the claim that DOX nanoparticles are not sufficiently associated with thestructure is retained following encapsulation in cells and are thus easily removed in the subsequentchitosan nanoparticles, though it is not a definitive washes.indication in itself. Conversely, the spectrum of An alternative explanation of these results, how-associated DOX showed an entirely new, longer ever, could be that the longer incubation time iswavelength fluorescence band. This same band has needed simply to allow the particles to release anbeen previously reported for DOX in environments amount of DOX in the culture medium comparablewith high dielectric constant [32], and suggests that to that of the control DOX solution. To exclude thisDOX is mostly associated with the nanoparticles (via possibility, we used a two-compartment in vitro set-encapsulation, adsorption, or both), rather than up where the DOX-loaded nanoparticles were placednanoprecipitated outside of the particles. in the donor compartment and the cells in a receptor

The retention of DOX bioactivity was best demon- compartment, separated via a polycarbonate mem-strated by the in vitro cytostasis assays, shown in brane. After 24 h incubation, no DOX could beFig. 6. Despite that only about one fifth of the detected in the cell culture compartment (Fig. 7C).encapsulated DOX was released from chitosan–TPP This led us to conclude that no significant amountsnanoparticles in vitro, this formulation was equally of DOX were released from the nanoparticles intoable to slow tumor cell proliferation relative to DOX the cell culture medium. This was also confirmed bysolutions for the C26 and human melanoma A375 the fact that a control mixture of free DOX and blankcell lines, indicating that DOX must maintain its nanoparticles under the same experimental condi-bioactivity within these nanoparticles. The same was tions showed significant DOX accumulation (Fig.the case with the nanoparticles using DOX–chitosan 7D), indicating that the drug can freely pass throughcomplexes, though the formulation did show lesser the membrane. The results of the in vitro release andcytostasis at certain drug concentrations relative to confocal studies combined strongly support ourthe control DOX solution. This could be due to an hypothesis that DOX-loaded chitosan nanoparticlesexcessively tight interaction between the drug and are internalized by cells and degraded intracellularlychitosan, which might impede its transit to the to release the drug. However, more thorough con-nucleus. However, one cannot discard the possibility focal studies are needed to ultimately prove thisthat partial damage to the molecular structure of mechanism.DOX occurred during its complexation withchitosan.

Given the limited DOX release exhibited by the 5. Conclusiontwo formulations over this period, we hypothesizedthat the cytotoxic action exhibited by these In this paper, we describe the feasibility of usingnanoparticles would be due to endocytosis, rather chitosan nanoparticles as colloidal carriers for the

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