Chitosan Nano Particles As Delivery Systems For Doxorubicin

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

Chitosan nanoparticles as delivery systems for doxorubicin Kevin A. Janes a , Marie P. Fresneau a , Ana Marazuela b , Angels Fabra b , ´ Jose´ Alonso a , * Marıa a

Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, The University of Santiago de Compostela, 15706 Santiago de Compostela, Spain b ` , Hospital Duran i Reynals, 08907 Barcelona, Spain Institut de Recerca Oncologica 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 Doxorubicin (DOX) and its bioactive derivatives are among the most widely used anticancer drugs in chemotherapy treatment [1]. However, problems *Corresponding author. Tel.: 134-981-594-627; fax: 134-981547-148. E-mail address: [email protected] (M.J. Alonso).

related to the development of multidrug resistance [2] and acute cardiotoxicity [3] have led researchers to investigate alternative forms of administering DOX for the treatment of cancer, with both prodrug [4] and particulate [5] methods involved as active fields of DOX research for the past two decades. DOX microencapsulation has shown some applications for the controlled release of DOX over 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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solid, accessible tumors, these particles are too large to be endocytosed by most cells or circulate freely in the bloodstream. Consequently, the association of DOX to submicron carriers, such as liposomes [7], nanoparticles [8], or micelles [9], has drawn greater interest. The majority of attempts to associate DOX to nanoparticulate carriers have used anionic or neutral polymers. Akiyoshi et al. [10] achieved DOX encapsulation in cholesterol-bearing pullulan hydrogel nanoparticles, though loading levels were very low (,0.1 wt.%) and cytotoxic effects of the nanoparticles were lower than that of free DOX. Combining prodrug and encapsulation strategies, Yoo et al. [11] covalently linked DOX to the terminus of poly( D,Llactic-co-glycolic acid) (PLGA), then formed nanoparticles with the conjugate by an emulsionsolvent diffusion method. The group was able to obtain considerable loadings (3.45 wt.%), achieve a controlled release of DOX over nearly 1 month, and maintain antiproliferative activity relative to free DOX, though these results were possible only by forming the covalent linkage between the polymer and the drug. The vast majority of work involving nanoparticulate DOX association, however, has been with polyacrylates, exploiting charge interactions of the polymer with the drug to achieve high association efficiencies. Polymethacrylate nanoparticles with adsorbed DOX were administered intravenously to hepatoma patients and demonstrated prolonged plasma levels, as well as reduced total clearance of DOX relative to a control DOX solution [12]. DOX associated to polyalkylcyanoacrylate nanoparticles [13] have demonstrated reduced cardiotoxicity following intravenous administration in mice [14] as well as increased cytoxicity against multidrug resistant cell lines in vitro [15]. Later work showed that coating of these particles with polysorbate 80 significantly increased DOX accumulation in brain tissue [16]. However, these DOX loaded particles have demonstrated acute renal toxicity [17] as well as decreased permeability of the drug across artificial membranes with respect to free DOX [18]. An alternative approach would be to entrap DOX into a positively charged carrier. Cell adhesion and potentially cell uptake of such particles should be favored due to their attraction to negatively charged

cell membranes, an attractive feature for the treatment of solid tumors. From the perspective of intravenous administration, positively charged particles would interact with different blood components as compared to negatively charged particles. These changes could potentially create a different biodistribution and / or organ accumulation pattern following intravenous administration. Additionally, a positively charged system that would be expected to interact with cells and / or membranes would be desirable for testing alternative modes of administration of DOX, i.e. mucosal administration. We believed that an interesting candidate with which to test these hypotheses was the cationic polysaccharide, chitosan. This biopolymer has shown favorable biocompatibility characteristics [19] as well as the ability to increase membrane permeability, both in vitro [20] and in vivo [21], and be degraded by lysozyme in serum [22]. Consequently, the aim of this paper was to encapsulate appreciable quantities of DOX in chitosan nanoparticles made by ionotropic gelation with sodium tripolyphosphate (TPP) and test the effects of DOX encapsulation and / or release on cytotoxic activity relative to free DOX. To achieve this aim, we tried two approaches: ionic bridging with a coincorporated polyanion and polymer / drug complexation.

2. Experimental section

2.1. Materials Chitosan hydrochloride salt, Protasan CL 110 (Mw .100 kDa), was purchased from Pronova Biopolymers (Oslo, Norway). Doxorubicin hydrochloride was obtained as a 2 mg / ml solution in 0.9% (w / v) sodium chloride from Tedec-Meiji Farma (Madrid, Spain). TPP, type B gelatin (75 bloom), polyphosphoric acid, and dextran sulfate (Mw 510 kDa) were all purchased from Sigma-Aldrich S.A. (Madrid, Spain). Phosphorylated glucomannan was a ´ gift from Industrial Farmaceutica Cantabria (Madrid, Spain). Unless otherwise mentioned, water was ´ ultrapure grade (Milli-Q plus, Millipore Iberica, Spain). All other chemicals were reagent grade.

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2.2. Spectrophotometric analysis Reagent concentrations were fixed for all spectroscopic studies. Chitosan was maintained constant at 400 mg / ml and polyphosphoric acid, dextran sulfate, and DOX concentrations all at 40 mg / ml. Spectra were recorded from 350 to 600 nm using a UV-VIS spectrophotometer (Model UV-1603, Shimadzu, Columbia, MD) with a 2 nm slit width and a 1 cm path length at intervals of 0.5 nm using water as the baseline reference.

2.3. Formation of chitosan–DOX complex DOX was added to a solution of 0.2% (w / v) chitosan in water to a final concentration of 30% (w / w) with respect to chitosan at pH 5.5. The solution was left under magnetic stirring 24 h at room temperature, dialyzed against distilled water lowered to pH 5 with 1 N HCl for 36 h, and lyophilized. To avoid photodegradation of DOX during the complexation and purification, all procedures were performed in the absence of light. DOX loading was calculated spectrophotometrically at 487 nm (´ 51.74310 25 l / g cm).

2.4. Preparation of chitosan nanoparticles Chitosan particles incorporating polyanions and chitosan–DOX complexed particles were prepared as described previously [23]. Briefly, chitosan or chitosan–DOX complex was dissolved at 0.175% (w / v) with 1% (v / v) acetic acid and then raised to pH 4.7–4.8 with 10 N NaOH. For DOX-loaded nanoparticles incorporating polyanions, 88–363 mg DOX (10–30% loading) was first incubated for 20– 30 s with 88 mg of the polyanion. The mixture was then added to a chitosan solution giving a final chitosan concentration of 0.175% (w / v). To 500 ml of this polymer solution, 100 ml of 0.291% (w / v) TPP in water were added under magnetic stirring, leading to the immediate formation of the nanoparticles.

2.5. Physicochemical characterization of nanoparticles Size and zeta potential measurements were per-

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formed by photon correlation spectroscopy and laser Doppler anemometry, respectively, with a Zetasizer  3000HS (Malvern Instruments, UK). For size measurements, samples were diluted in water and measured for a minimum of 180 s. Raw data were subsequently correlated to mean hydrodynamic size by cumulants analysis. For zeta potential measurements, samples were diluted in 0.1 mM KCl and measured in automatic mode. All measurements were performed in triplicate. Particle morphology was examined by transmission electron microscopy (CM12 Philips, Eindhoven, Netherlands). Samples were stained with 2% phosphotungstic acid for 10 min, immobilized on copper grids, and dried overnight for viewing.

2.6. Evaluation of DOX encapsulation Encapsulation efficiency and nanoparticle yield of the different formulations were determined by centrifugation of the samples at 24,0003g for 30 min. Pellets were incubated at 808C overnight and weighed. Supernatant DOX concentrations were calculated by fluorimetry (Model LS-50B, PerkinElmer, Norwalk, CT) with a slit width of 5 nm and excitation and emission wavelengths at 480 nm and 590 nm, respectively. Dilutions of samples and calibration curves were performed in water, and all measurements were performed in triplicate. Encapsulation efficiency was calculated as follows: DOX encapsulation total DOX 2 free DOX efficiency 5 ]]]]]]]. total DOX

2.7. Evaluation of in vitro DOX release The nanoparticles were collected by centrifugation at 90003g for 40 min on a glycerol bed. The pellets were resuspended and incubated in 4 ml of 100 mM acetate buffer (pH 4) or phosphate buffered saline (pH 7.4) at 378C under light agitation. The quantity of nanoparticles was adjusted to obtain a maximum DOX concentration of 1 mg / ml. At varying time points, supernatants were isolated by centrifugation at 24,0003g for 30 min and measured by fluorimetry as described earlier. Following supernatant extraction, pellets were discarded (destructive

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sampling). Calibration curves were made with the incubation medium, and all measurements were performed in triplicate.

2.8. Fluorimetric analysis DOX emission spectra were recorded in water from 500 to 750 nm at a fixed excitation of 480 nm with excitation and emission slit widths of 5 nm. Spectra were read at a scan speed of 200 nm / min and normalized with respect to peak emission.

The number of active cells was estimated by measuring the absorbance at 540 nm (Titertek Multiscan, ICN, Costa Mesa, CA). The percentage of cytostasis was calculated as follows: A2B Cytostasis 5 ]] A where A is the absorbance of cells incubated with culture medium and B is the absorbance of cells incubated with the different nanoparticle formulations. All samples were made in sextuplicate.

2.9. In vitro cytostasis assays 2.10. Confocal microscopy analysis Human melanoma A375 cells (ATCC, Rockville, MD) and C26 murine colorectal carcinoma cells were grown in Ham F-12 medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum, sodium pyruvate, non-essential amino acids, L-glutamine, and twofold vitamin solution (GIBCO). The cultures were maintained in plastic flasks and incubated in 5% CO 2 / 95% air at 378C in a humidified incubator. The cell lines were examined and found to be free of mycoplasma, as assayed by the Gen-Probe Mycoplasma T.C. (Gen-Probe Inc., San Diego, CA). The antiproliferative effect of DOX was analyzed by the MTT method [24]. Cells from exponential cultures were seeded onto 96-well tissue culture plates (TPP, Switzerland) at a density of 5310 3 cells / well for a 0.36-cm 2 well (optimal seeding density that avoids full confluency for the length of the 4-day experiment). One day later, the cultures were washed and incubated for 2 h with the different samples at various dilutions in serum-free, Ham F-12 medium. The different preparations were adjusted to maintain the same drug concentration, and experiments were performed in parallel wells with increasing DOX concentrations of 0.1 mg / ml to 100 mg / ml. Following incubation, the cell monolayers were washed five times with PBS and left 3 days in complete media. After this time, 50 ml / well of PBS containing 1 mg / ml MTT (tetrazolium salt, SigmaAldrich S.A., Madrid) was added, and the plates were incubated an additional 4 h. The intracellular formazan crystals resulting from the reduction of the tetrazolium salts present only in metabolically active cells were solubilized with DMSO (Sigma-Aldrich).

DOX accumulation in treated cells was localized by confocal microscopy. Briefly, cells (Human melanoma A375 cells (ATCC)) from exponential cultures were grown on 1.13-cm 2 glass coverslips ¨ (Objekttrager, Menzol Glaser, Germany) at a density of 5310 4 cells / coverslip. One day later, the cultures were washed and treated with serum-free, Ham F-12 medium containing either DOX-loaded chitosan nanoparticles incorporating dextran sulfate (30 min to 24 h incubation) or free DOX (30 min incubation). All incubations were carried out at 378C with an equivalent final concentration of DOX (5 mg / ml). Alternatively, cells were incubated using a twocompartment Boyden chamber. Briefly, either DOXloaded chitosan nanoparticles containing dextran sulfate or free DOX solutions were diluted in serumfree culture medium and incorporated into the upper compartment of polycarbonate transwell filters (0.4 mm pore diameter, Costar). The cells seeded on coverslips for 24 h were placed in the lower compartment, thereby receiving the DOX solution filtering from the upper compartment but excluding the DOX associated to the nanoparticles (a control experiment was performed demonstrating that the particles did not cross the filter). Following incubation, cells were washed five times with PBS, and the coverslips were mounted on slides. Fluorescence observation was carried out with a confocal microscope (TCS 4D, Leica Instruments) using an argon / krypton laser (75 mW) at 488 nm for excitation and an LP filter of 590 nm for DOX detection. Contrast images were simultaneously observed using the inverted microscope equipment with a PL Apo 633 /

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

2.11. Statistical analysis The statistical significance of all results was determined using the two-tailed Student’s t-test.

3. Results The molecular structures of DOX and the complexing agents, polyphosphoric acid and dextran sulfate, are shown in Fig. 1. The protonable groups in the DOX molecule were expected to interact with the deprotonable groups of polyphosphoric acid and dextran sulfate. The interaction of DOX with different polyanions and chitosan was first investigated spectrophotometrically. As seen in Fig. 2A and B, the DOX peak at 480 nm was reduced by |53% upon incubation with either polyphosphoric acid (Fig. 2A) or dextran sulfate (Fig. 2B). Spectral changes in the DOX peak were also observed for the samples, with polyphosphoric acid and dextran sulfate inducing red shifts of 9 and 14 nm for DOX solutions, respectively. Subsequent addition of chitosan increased the intensity of the 480 nm peak with polyphosphoric acid and dextran sulfate to 110 and 83% of the original DOX absorbance. Spectral peaks for both samples returned to 480 nm. In control studies, no detectable absorbance was noted for individual chitosan, polyphosphoric acid, or dextran sulfate solutions over the chosen wavelength range at the concentrations tested (data not shown). No significant differences in pH were noted among any of the formulations shown (DOX, DOX1polyanion, DOX1polyanion1 chitosan). A comparison of the DOX encapsulation efficiencies for different chitosan–TPP nanoparticle formulations is shown in Table 1. At 10% (w / w) polyanion with respect to chitosan, no significant differences in DOX encapsulation efficiencies were observed with the coincorporation of gelatin, glucomannan, or polyphosphoric acid relative to the control formulation, with all encapsulation efficiencies between 8 and 13%. Also, the addition of polyphosphoric acid caused a destabilization of the suspension at this

Fig. 1. Chemical structure of: (A) DOX, (B) polyphosphoric acid, (C) dextran sulfate. *Deprotonable functional group, **protonable functional group.

polyanion concentration, forming visible agglomerates. Incorporation of dextran sulfate increased DOX encapsulation efficiency approximately twofold with respect to the control formulation. The effect of initial DOX loading on encapsulation efficiency for chitosan–TPP nanoparticles incorporating dextran sulfate can be seen in Table 2, with values ranging from 19 to 23%. Mean encapsulation efficiencies decreased only slightly with increases in

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K. A. Janes et al. / Journal of Controlled Release 73 (2001) 255 – 267 Table 2 Effect of DOX loading on encapsulation efficiency for chitosan nanoparticles incorporating dextran sulfate (n53)

Fig. 2. (A) UV-VIS spectra of: (———) DOX solution, (— — —) DOX1polyphosphoric acid solution, (- - -) DOX1 polyphosphoric acid1chitosan solution. (B) UV-VIS spectra of: (———) DOX solution, (— — —) DOX1dextran sulfate solution, (- - -) DOX1dextran sulfate1chitosan solution.

DOX loading, and differences were only marginally significant (P,0.1) between the highest and lowest loading levels. TEM images of chitosan–TPP nanoparticles incorporating dextran sulfate are shown in Fig. 3. The

Table 1 Encapsulation efficiencies for different chitosan nanoparticle formulations. All polyanions were incorporated at 10% (w / w) with respect to chitosan. Theoretical DOX loading: 10% (n53) Polyanion incorporated

Encapsulation efficiency (%)

No polyanion Type B gelatin Glucomannan Polyphosphoric acid Dextran sulfate

9.162.2 8.461.5 9.363.3 12.264.1 21.962.5*

* P,0.01.

Theoretical loading (%)

Encapsulation efficiency (%)

5 10 20

22.561.2 21.962.5 19.361.8

particles showed a dense, spherical structure, which was consistent with previous observations [25], though size dispersion did appear to be greater with the addition of dextran sulfate. Particle size and zeta potential values for the unloaded nanoparticles were 259615 nm and 133.460.8 mV, respectively. DOX loading (10% theoretical) did not significantly alter these values (292642 nm and 133.260.1 mV). Blank nanoparticle yield was 5161% which was unchanged following 10% DOX loading (5262%), resulting in a real DOX loading of 4 wt.% with respect to the polymer. A similar morphology was observed for DOX– chitosan complexed nanoparticles (data not shown). DOX complexation efficiency to chitosan was determined to be 1.4% (w / w). The total amount of DOX complexed to chitosan was incorporated in the nanoparticles structure, resulting in a real loading of 0.43% (w / w). DOX–chitosan complexed nanoparticles possessed a mean nanoparticle size of 21363 nm and zeta potential of 133.760.6 mV. The low degree of complexation was not expected to significantly alter the characteristics of the formulation, and the yield was assumed to be |100%, in accordance with previously reported yields [23] for this chitosan–TPP formulation. The in vitro release profile of chitosan–TPP nanoparticles in acetate buffer (pH 4) is shown in Fig. 4. The particles incorporating dextran sulfate showed a burst release of 17% at 2 h, followed by an additional release of 4.5% over the next 2 days. For nanoparticles containing DOX complexed with chitosan, a small release of 4.5% was noted at 2 h, with negligible DOX increases in release detected over the proceeding 5 days. Fluorescence profiles of DOX emission spectra are shown in Fig. 5. Relative fluorescence was nearly identical for the DOX solution and DOX released 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. Conversely, encapsulated DOX exhibited an additional emission band at 630 nm which was evident over the range of 600–700 nm. Fig. 6 shows the cytostasis of the different chitosan formulations in vitro for the C26 cell line. No significant differences were noted between the DOX loaded chitosan–TPP nanoparticles incorporating dextran sulfate and the control DOX solution over drug concentrations from 0.1 mg / ml to 100 mg / ml in any of the cell lines tested. Nanoparticles

made from DOX complexed to chitosan showed slightly decreased cytostatic activity relative to the DOX solution over this same concentration range. However, for most concentrations assayed, the differences were not statistically significant. The blank nanoparticle suspension showed no significant cytostasis. Very similar results were obtained using human melanoma A375 cells (results not shown). DOX localization of different nanoparticle / control formulations in human melanoma A375 cells is 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– TPP nanoparticles incorporating dextran sulfate (DOX loading: 4%) and (– j –) DOX complexed nanoparticles (DOX loading: 0.4%) (n53).

Fig. 5. Fluorescence emission spectra of: (———) DOX solution in water, (— — —) DOX encapsulated in chitosan–TPP nanoparticles, (- - -) DOX released from chitosan–TPP nanoparticles.

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Fig. 6. In vitro cytostasis in C60 cells for: (9) DOX loaded chitosan nanoparticles incorporating dextran sulfate, ( ) chitosan nanoparticles containing DOX complexed to chitosan, ( ) blank chitosan nanoparticles, (h) DOX solution (n56).

whereas intracellular localization of DOX loaded into chitosan nanoparticles containing dextran sulfate could be visualized only after 24 h incubation. At this point, no differences were seen in the intracellular localization of free DOX versus DOX previously associated to chitosan nanoparticles (Fig. 7A and B). To determine if DOX might be released from chitosan nanoparticles before entering the cells, we used a two compartment set-up where cells and DOX samples were separated by a 0.4 mm polycarbonate filter. As shown in Fig. 7C, no DOX was observed in cells incubated for 24 h with DOX-loaded nanoparticles in the upper compartment. A control mixture of free DOX and blank nanoparticles in the upper compartment showed an intracellular distribution comparable to free DOX in solution (Fig. 7D).

4. Discussion The major goal of this work was to develop a chitosan nanoparticulate system as a novel, positively charged, colloidal carrier for DOX. The greatest challenge was to encapsulate appreciable quantities of DOX, overcoming the charge repulsion between the cationic polymer (pKa 56.5) [26] and the predominantly positively charged anthracycline drug (pKa 58.2) [27]. To begin, we selected a chitosan– TPP nanoparticle formulation [23] which could accommodate a large quantity of TPP (only |25%

molar excess of chitosan), thus minimizing the polymer–drug repulsion by pairing a large fraction of the positive amino groups with negatively charged phosphates. This formulation in itself did allow small quantities of DOX to be retained within the particles (Table 1), likely by physical entrapment. However, the efficiency was quite low (9.1%), and since the nanoparticles were still positively charged, we feared that there would be a repulsion between chitosan and DOX, owing to the positive charge of the drug. In an attempt to remedy these concerns, we tested a variety of polyanions which could simultaneously form a strong ionic interaction between both chitosan and DOX, increasing the encapsulation efficiency and binding the molecule tightly to the particles. Type B gelatin (pKa 54.7–5.0, as stated by the manufacturer) and phosphorylated glucomannan (763% phosphorylation, as stated by the manufacturer) fit these criteria as macromolecules, but no effect on the spectroscopic profile of DOX, potentially indicating intermolecular interactions, was observed (data not shown). We concluded that neither gelatin nor glucomannan possessed a sufficiently high negative charge density to complex appreciable quantities of both chitosan and DOX. These observations were corroborated by failed attempts to augment DOX encapsulation with the incorporation of these molecules into chitosan–TPP nanoparticles, shown in Table 1. From this point, we shifted our attention to polymers with higher anionic charge densities (at least one negative charge per monomer). We first tested polyphosphoric acid, which did appear to interact strongly with DOX in solution. This interaction, however, was completely eradicated by the addition of chitosan, as evidenced by the reversion of the spectroscopic profile in Fig. 2A. The overall absorption profile of this system was, in fact, higher than the original DOX spectrum, perhaps due to an increase in sample turbidity as polyphosphoric acid formed particulated complexes with chitosan. Nevertheless, a mixture of chitosan and polyphosphoric acid at the same concentrations but in the absence of DOX showed no significant absorption over the range tested (data not shown). Corroborating the absorption spectra, the incorporation of polyphosphoric acid did not increase DOX encapsulation. 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-loaded nanoparticles in the upper compartment of a Boyden chamber (overnight incubation), and (D) DOX1blank nanoparticle mixture in the upper compartment of a Boyden chamber (overnight incubation). All confocal studies were performed in A375 melanoma cells with 5 mg / ml equivalent DOX concentration. Magnifications: 3630 (A, B, D), 3400 (C).

chitosan solution led to the formation of aggregates, rather than nanoparticles, upon addition of TPP. Sulfonic acid groups have been shown to bind DOX in considerable quantities when incorporated in ion-exchange resins [28]. Additionally, dextran sulfate has been used successfully to augment the encapsulation of DOX in albumin microspheres [29]. With these reports in mind, we decided to test the feasibility of incorporating dextran sulfate into

chitosan nanoparticles for the encapsulation of DOX. As could be anticipated, effects of the polyanion on the UV-VIS spectrum of DOX were readily visible. More importantly, however, the DOX–dextran sulfate complex appeared to only be partially dissociated by the addition of chitosan (Fig. 2B), opening the possibility of using the complex to draw more DOX into the nanoparticles. Indeed, this entrapment did occur, as the formulation incorporating dextran

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sulfate was the only one to achieve encapsulation efficiencies significantly above the control formulation. DOX encapsulation was also visibly apparent with these nanoparticles, which formed a dense red pellet upon centrifugation. The difference between DOX–dextran sulfate and DOX–polyphosphoric acid interactions is intriguing, in that one would expect coulombic forces to be stronger with the acid, owing to its higher charge density on a per weight basis. However, another important consideration is the interaction of these polyanions with chitosan. Using the same argument of charge density, polyphosphoric acid should exhibit far greater avidity to chitosan relative to dextran sulfate. Hence, more DOX would be displaced from polyphosphoric acid than from dextran sulfate upon addition of chitosan. Interestingly, DOX encapsulation efficiency in the formulation containing dextran sulfate appeared minimally dependent upon theoretical DOX loading over the range of 5–20% (w / w). At 10% DOX loading, there remains a 3.7-fold molar charge excess of negatively-charged sulfonic acid groups relative to DOX amino groups, so it is quite feasible that, due to this excess, the saturation level of DOX association is not reached. Under these conditions, therefore, it is likely that the formation of DOX–dextran sulfate complexes is favored by higher quantities of DOX incubated, with real loading increasing almost linearly with theoretical loading. An entirely different approach was taken with nanoparticles containing DOX complexed to chitosan. As an amphoteric drug (protonable amino group and deprotonable phenolic group), there continually exists an equilibrium between the positively charged, negatively charged, neutral, and zwitterionic species of DOX (Fig. 1). Additionally, there are other factors (hydrophobic / hydrophilic interactions, resonance effects, etc.) which could allow small quantities of DOX to complex with chitosan, despite the overwhelming charge repulsion between the two molecules, as has been noted previously between DOX and positively charged transition metal ions [30,31]. We tested the extent of this association by incubating DOX and chitosan in solution, dialyzing to remove non-associated DOX, and lyophilizing to promote polymer–drug interactions. We did not expect considerable association to occur, but that the DOX which did associate would

be tightly bound to chitosan. Indeed, this phenomenon did take place. While there was only a minimal yield for the complexation (0.43 wt.%), all of the DOX that was complexed remained incorporated within the nanoparticles. Therefore, it could be induced that the entrapment efficiency of DOX previously associated to chitosan was 100%. The possibility that the high entrapment efficiency is merely caused by low initial DOX loading was also considered. As a control study, we prepared chitosan nanoparticles with the same DOX initial 0.43 wt.% loading, but adding DOX to the chitosan solution prior to the nanoparticles formation. In this case the encapsulation efficiency was far lower (23.061.0%) than that observed for chitosan–DOX complexes. In vitro release studies were performed in acetate buffer (pH 4). We chose this medium because DOX is maximally stable at pH values between 3 and 5 and also because, at higher pHs we encountered problems of fluorescence quenching or interference for the quantification of released DOX. Obviously, in these experiments, we did not expect to predict the release behavior of these particles in vivo but to compare the formulations developed and to gain some insight about the mechanism of release. Nanoparticles incorporating dextran sulfate showed a burst release of 17% at 2 h, followed by an additional release of 4.5% over the next 2 days. This slow release was quite distinct from the profiles obtained from similar chitosan nanoparticles encapsulating insulin, where 100% release was observed within 15 min [21]. Even less DOX was detected in PBS at 5 days (data not shown), probably due to DOX degradation in the near neutral medium. The initial phase of release is logically attributed to the DOX located at the surface of the particles while the remainder of the unreleased DOX was assumed to be well entrapped within the chitosan nanoparticles and tightly associated to the chitosan molecules, probably as an ionic complex with dextran sulfate. Therefore, the degradation of chitosan would be required for accomplishing the release process. Unfortunately, direct confirmation of this hypothesis by enzymatic digestion of the nanoparticles was not possible since chitosanase treatment of the nanoparticle suspension also degraded DOX solutions and / or quenched fluorescence in control studies. Nevertheless, the effect of dextran sulfate on DOX release was apparent, as chitosan nanoparticles without the polyanion

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showed over twice the burst effect after 2 h under the same conditions (36.760.3%). Nanoparticles containing DOX complexed with chitosan displayed an even smaller release over the same period, an observation that is easily explained by the aforementioned interactions binding the drug to the polymers. Indeed, since the interaction DOX– chitosan seems to be quite stable, any drug released would be a result of degradation of chitosan or by release of DOX complexed on the particle surface. Notwithstanding, spectral analysis of the DOX released from chitosan nanoparticles incorporating dextran sulfate showed that the released DOX was fluorimetrically identical to that of the native DOX solution, as seen in Fig. 5. This preservation of the fluorescence signature supports the claim that DOX structure is retained following encapsulation in chitosan nanoparticles, though it is not a definitive indication in itself. Conversely, the spectrum of associated DOX showed an entirely new, longer wavelength fluorescence band. This same band has been previously reported for DOX in environments with high dielectric constant [32], and suggests that DOX is mostly associated with the nanoparticles (via encapsulation, adsorption, or both), rather than nanoprecipitated outside of the particles. The retention of DOX bioactivity was best demonstrated by the in vitro cytostasis assays, shown in Fig. 6. Despite that only about one fifth of the encapsulated DOX was released from chitosan–TPP nanoparticles in vitro, this formulation was equally able to slow tumor cell proliferation relative to DOX solutions for the C26 and human melanoma A375 cell lines, indicating that DOX must maintain its bioactivity within these nanoparticles. The same was the case with the nanoparticles using DOX–chitosan complexes, though the formulation did show lesser cytostasis at certain drug concentrations relative to the control DOX solution. This could be due to an excessively tight interaction between the drug and chitosan, which might impede its transit to the nucleus. However, one cannot discard the possibility that partial damage to the molecular structure of DOX occurred during its complexation with chitosan. Given the limited DOX release exhibited by the two formulations over this period, we hypothesized that the cytotoxic action exhibited by these nanoparticles would be due to endocytosis, rather

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than to the release of free drug in the cell culture medium. To confirm this hypothesis we investigated the mechanism of in vitro cytotoxicity for DOXloaded chitosan nanoparticles, using human melanoma A375 cells, via confocal microscopy. Comparable fluorescence localization, visualized as red, was seen for DOX-loaded nanoparticles relative to free DOX (Fig. 7A and B), however, a significantly longer incubation time was required for the nanoparticles to display the intracellular fluorescence signals. This suggests that these particles might enter the cells, and that this internalization process occurs over a much longer time than the diffusion of the free drug. Indeed, from these observations it could be inferred that, after short incubation times, the nanoparticles are not sufficiently associated with the cells and are thus easily removed in the subsequent washes. An alternative explanation of these results, however, could be that the longer incubation time is needed simply to allow the particles to release an amount of DOX in the culture medium comparable to that of the control DOX solution. To exclude this possibility, we used a two-compartment in vitro setup where the DOX-loaded nanoparticles were placed in the donor compartment and the cells in a receptor compartment, separated via a polycarbonate membrane. After 24 h incubation, no DOX could be detected in the cell culture compartment (Fig. 7C). This led us to conclude that no significant amounts of DOX were released from the nanoparticles into the cell culture medium. This was also confirmed by the fact that a control mixture of free DOX and blank nanoparticles under the same experimental conditions showed significant DOX accumulation (Fig. 7D), indicating that the drug can freely pass through the membrane. The results of the in vitro release and confocal studies combined strongly support our hypothesis that DOX-loaded chitosan nanoparticles are internalized by cells and degraded intracellularly to release the drug. However, more thorough confocal studies are needed to ultimately prove this mechanism.

5. Conclusion In this paper, we describe the feasibility of using chitosan nanoparticles as colloidal carriers for the

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delivery of the small, cationic anthracycline drug, doxorubicin (DOX). By incorporating the polyanion, dextran sulfate, we were able to encapsulate considerably high quantities of DOX considering the inherent polymer–drug charge repulsion. These particles demonstrated a minimal burst release and retained the cytotoxic activity of DOX in vitro. Additionally, we showed that DOX can be complexed to chitosan before particle formation by incubation and separation of the complexes. This approach appeared to bind drug even more tightly to the particles, though the complexation reduced the anti-proliferative activity of DOX. Confocal images appeared to indicate that these particles enter the cells via an endocytic mechanism and release DOX intracellularly. Further experimentation, most notably the testing of these formulations in vitro with DOX resistant cell lines and in vivo, is necessary to clarify the potential of chitosan nanoparticles for the delivery of DOX.

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Acknowledgements ´ ´ We would like to thank Monica Hombreiro Perez for her valuable help in the preparation of the samples for the confocal studies. This work was supported by a grant from the Spanish government (SAF97-0169). K.A.J. and M.P.F. would like to additionally thank the USIA Fulbright Association and the UE Socrates Program, respectively, for financial support.

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