Chitosan-Alginate Blended Nanoparticles as Carriers for the

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Biomacromolecules 2009, 10, 1736–1743

Chitosan-Alginate Blended Nanoparticles as Carriers for the Transmucosal Delivery of Macromolecules Francisco M. Goycoolea,†,‡ Giovanna Lollo,§ Carmen Remunãń-Lo´pez,† Fabiana Quaglia,§ and Marıá J. Alonso*,† Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Santiago de Compostela, Spain, CIAD, Laboratory of Biopolymers, P.O. Box 1735, Hermosillo, 83000 Mexico, and Department of Pharmaceutical and Toxicological Chemistry, University of Napoli Federico II, Via Domenico Montesano 49, 80131 Napoli, Italy Received January 31, 2009; Revised Manuscript Received May 28, 2009

Nanoparticles intended for use in the transmucosal delivery of macromolecules were prepared by the ionic gelation of chitosan (CS) hydrochloride with pentasodium tripolyphosphate (TPP) and concomitant complexation with sodium alginate (ALG). The incorporation of a small proportion of ALG of increasing molecular weight (Mw; from 4 to 74 kDa) into the nanoparticles led to a monotonic increase in colloidal size from ∼260 to ∼525 nm. This increase in size was regarded as a consequence of the formation of gradually more expanded structures. Insulin, taken as a model peptide, was associated to CS-TPP-ALG nanoparticles with efficiencies in the range of ∼41 to ∼52%, irrespective of the Mw of the ALG incorporated in the formulation. These CS-TPP-ALG nanoparticles exhibited a capacity to enhance the systemic absorption of insulin after nasal administration to conscious rabbits. Interestingly, it was observed that the duration of the hypoglycaemic response was affected by the ALG’s Mw. Briefly, this work describes a new nanoparticulate composition of potential value for increasing nasal insulin absorption.

Introduction Polysaccharide-based nanoparticles nowadays represent a very promising drug delivery platform, particularly for the transmucosal delivery of bioactive macromolecules. Their usefulness relies on a number of interesting properties, namely, muco- and bioadhesiveness, a high capacity to associate and release therapeutic macromolecules in their bioactive form, as well as their ability to enhance the transport of bioactive compounds across well organized epithelial barriers, such as the ocular, nasal and intestinal routes.1 Several proof-of-concept studies have consistently demonstrated the efficacy of these nanoparticles in various animal models.2-9 Among the polysaccharides used to this end, chitosan (CS) and alginate (ALG) are known because of their biocompatibility, low toxicity, biodegradability, and muco- and bioadhesiveness.10-18 CS is a polycationic polysaccharide obtained on industrial scales by the thermo-alkaline N-deacetylation of chitin isolated from crustacean waste. Chemically, CS is composed of a family of linear polysaccharides that are predominantly (1f4)-linked 2-amino-2-deoxy-β-D-glucose and residual 2-acetamido-2-deoxyβ-D-glucose. CS is known to be biodegraded by several enzymes, among them, chitinases, which are secreted by intestinal microorganisms, lysozyme, which is highly concentrated in mucosal surfaces and by human chitotriosidase.10-12 CS is firmly established as a biocompatible nontoxic mucoadhesive that exhibits the capacity to promote the absorption of poorly absorbed macromolecules across epithelial barriers by transient widening of cell tight junctions.13-15 * To whom correspondence should be addressed. Tel.: +34981563100. Fax: +34981547148. E-mail: [email protected]. † University of Santiago de Compostela. ‡ CIAD. § University of Napoli Federico II.

ALG is a structural polyanionic polysaccharide that occurs in the cell wall of brown seaweeds, but an acetylated form is also produced as an exocellular polymer by certain bacteria. Chemically, alginate describes a family of linear block copolymers of (1f4)-linked β-D-mannuronic acid (M residues) and its C5 epimer, R-L-guluronic acid (G residues), arranged in a nonregular pattern. As a result of their biocompatibility and their gelling capacity, alginates have been applied in the engineering of biomaterials.16 It has also been known for a long time that ALG exhibits bioadhesive properties.17,18 There are some recent reports on the production and characterization of nanoparticles based on CS and ALG. In addition, there are a few reports describing their potential use in drug delivery. For example, a two-step procedure has been described in which alginate is pregelled in the presence of calcium chloride under sonication and the nanogels thus obtained are further coated with chitosan.19,20 These nanoparticles had an average size of 650 ( 22 nm and exhibited a capacity for the association of insulin. Moreover, the effectiveness of these insulin-loaded nanoparticles was evaluated following oral administration to diabetic rats at dose in the range 50-100 IU/ kg and it was found that the plasmatic glucose levels were reduced by 40% with respect to the basal ones while the hypoglycaemic response was maintained for up to 18 h.9,21 On the other hand, ALG-coated CS nanoparticles especially designed to deliver vaccines into mucosal surfaces have also been produced by coacervation.22 With this method, the model antigen (ovalbumin) was never exposed to potentially harsh conditions, such as the contact with the organic solvents, mechanical agitation, or sonication. Hence, the structural integrity of the protein was not significantly affected by the entrapment procedure. The system addressed in this work belongs to a newly developed class of hybrid nanoparticles that, in addition to

10.1021/bm9001377 CCC: $40.75  2009 American Chemical Society Published on Web 06/22/2009

Transmucosal Delivery of Macromolecules

CS and TPP,23 contain other polysaccharides (e.g., hyaluronic acid, konjac glucomannan)24,25 or oligosaccharides (e.g., cyclodextrins).26-28 These hybrid systems have shown improved physical properties (i.e., stability in biological media) and better pharmacological performance than conventional nanoparticles comprised solely of CS-TPP.29 Hence, taking this information into account, the aim of this work was to obtain hybrid CS-TPP-ALG nanoparticles according to a simple single-step procedure and to evaluate their physicochemical properties and also their capacity to associate, transport, and release insulin across the nasal epithelium.

Experimental Section Materials. Ultrapure chitosan as its hydrochloride salt (CS) was purchased from FMC Biopolymers [Protasan CL113 Batch No. FP503-03; the degree of acetylation was certified by the supplier as ∼14%, with a Mw of ∼119 kDa]. Samples of depolymerised sodium alginate (ALG) of varying molecular weight (∼4 to 74 kDa) and D-mannuronate/ D-guluronate (M/G) molar ratio (1.22-1.42) were supplied and characterized by Danisco A/S (Denmark). High performance liquid chromatography (HPLC) gradient grade acetonitrile (g99.9%) was purchased from Merck (Darmstadt, Germany). HPLC grade trifluoroacetic acid (TFA; g99% GC), insulin from bovine pancreas [Prod. No. I5500, Mw 5.7 kDa, pI 5.3, 29 IU/mg, Zn traces ∼0.5%], sodium D-galacturonate (g98%), and all other chemicals were of analytical grade, except for tripolyphosphate pentasodium salt (TPP), which was of practical grade (90-95%). Unless otherwise stated, all reagents were from Sigma-Aldrich Chemie (Steinheim, Germany). Deionized Milli-Q water was used throughout. Nanoparticle Preparation. The general experimental protocol to prepare CS-TPP-ALG nanoparticles described by our group23 was modified slightly to account for the incorporation of ALG, as described below. CS was fully dissolved (2 mg/mL) in water or in dilute NaCl aqueous solutions under magnetic stirring for ∼1 h. A solution of TPP (1.0 mg/mL) was prepared in the same solvent. ALG was added (0.45 mg/mL) to the TPP solution and allowed to dissolve. For insulin-loaded nanoparticles, the peptide (3 mg/mL) was dissolved directly in the TPP solution under gentle magnetic stirring for ∼2 h prior to the addition of ALG. Nanoparticles were formed spontaneously upon the rapid mixing of 1.0 mL of the TPP-ALG (or TPP-ALG-insulin) solution into 3 mL of the CS solution in a test tube under magnetic stirring (∼200 rpm) at room temperature for ∼10 min. The nanoparticles were isolated by centrifugation (10000 × g, 40 min, 25 °C) in accurately weighed eppendorf vials on a bed of glycerol (∼20 µL) carefully deposited at the bottom of the vial. The supernatant was removed cautiously with a pipet and the precise weight of the recovered nanoparticles was calculated by accurately weighing the eppendorf vials after the removal of the supernatant. The processing yield was determined by centrifuging (10000 × g, 40 min, 25 °C) accurately weighed aliquots of the nanoparticles in eppendorf vials without added glycerol. The supernatants were carefully separated and the centrifuged pellets were freezedried for three days and subsequently weighed (n ) 3). The optimization of conditions for nanoparticle formation were conducted on unloaded particles according to the experimental layout that is summarized in the Supporting Information. For each series of experiments the protocol for the preparation of the nanoparticles was identical to that described above, while the only variations were in the ionic strength of the solvent (water or varying NaCl concentrations), alginate concentration, or alginate Mw. Determination of Alginate Concentration. The amount of ALG in the nanoparticles was determined indirectly by measuring the concentration of polysaccharide in the supernatant after centrifugation. To this end, the phenol-sulphuric acid test method commonly used to measure total carbohydrate content30 was adapted to a 96-well microplate assay in a total volume of 300 µL. To this end, a 42 µL aliquot of the supernatant of unknown alginate concentration was placed

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in the well, followed by a 42 µL aliquot of a freshly prepared aqueous phenol solution (5%) and 216 µL of concentrated sulphuric acid. The plate was left to stand for 20 min at room temperature and the absorbance at 490 nm was read on a BioRad microplate reader (model 680). Blanks were made with water. A standard curve of sodium D-galacturonate was prepared in water in the range of 10-150 µg/mL (n ) 3). Chitosan does not react with phenol and sulphuric acid to yield furfural, hence, it does not interfere with the ALG determination. Physical Characterization. The size of the nanoparticles was determined by dynamic light scattering with a noninvasive back scattering (DLS/NIBS) technology and the value of the surface zeta potential (ζ) was determined by mixed laser Doppler electrophoresis and phase analysis light scattering (M3-PALS) using a Malvern Zetasizer NanoZS (ZEN 3600, Malvern Instruments, Worcestershire, U.K.) fitted with a red laser light beam (λ ) 632.8 nm). The ultrastructure of the insulin-loaded nanoparticles was investigated by transmission electron microscopy (TEM) on a Philips CM12 instrument (Eindhoven, The Netherlands). To this end, 10 µL of a 2% solution of phosphotungstic acid (pH adjusted to 4.0 with 1 M NaOH) was mixed with an equal volume of the isolated nanoparticles previously diluted 1:10 with water. Immediately afterward, an aliquot of 10 µL was immobilized on a copper grid coated with a Formvar membrane, allowed to stand for 1 min, dried, washed with 5 µL of water for 10 s, and dried again. Stability Studies. The colloidal stability of the nanoparticles was evaluated in acetate buffer (pH ∼4.3) as the evolution of particle size with time during incubation at 37 °C. Size measurements at given time intervals were recorded as described above. Insulin Association Efficiency and Loading Capacity. The insulin association efficiency and loading capacity of the insulin-loaded nanoparticles was determined by measuring the amount of unbound insulin in the supernatant after centrifugation as described above and using the conventional definitions of each concept.25 The insulin concentration was analyzed by HPLC (Agilent 1100 Series, U.S.A.) using the method previously described.26 In Vitro Release Studies. Insulin release studies were performed by incubating aliquots of the isolated nanoparticles in 3 mL of acetate buffer (pH 4.3) in test tubes supported on 50 mL Erlenmeyer flasks immersed in a circulating water bath at 37 °C and stirred while in the water bath. The concentration of the nanoparticles in the release media was adjusted in order to assess sink conditions for insulin. At appropriate time intervals (10, 20, 60, and 120 min), aliquots of 500 µL were taken out of each tube and the same amount of buffer was immediately added back. Aliquots were filtered through Millipore Millex-GV (low protein binding Durapore PVDF) filters with a 0.22 µm pore size. The amount of insulin released was determined also by HPLC.26 Circular Dichroism Spectroscopy Studies. Circular dichroic spectra were obtained at room temperature on a Jasco Spectrometer J-710 (Jasco Inc., Easton, MD). Cells with 0.1 and 1.0 cm path lengths for far-UV CD and near-UV CD spectroscopy were used, respectively. Data are expressed as mean residue molar ellipticity [θ] according to the expression

[θ] ) (θM)/(Cl)

(1)

where θ is the observed ellipticity (mdeg), M is the mean residue molecular weight (g/mol), l is the optical path length (cm), and C is the protein concentration (g/mL). CD spectra of insulin in HCl (pH 2.5), NaOH (pH 8.5), TPP (pH 8.5, obtained by dissolving TPP (1 mg/mL) in a solution of NaCl 0.5%), and acetate buffer (pH 4.3) at 0.1 mg/mL were collected at 25 °C. For CD spectra of CS-TPP insulin-loaded nanoparticles, 30 mg nanoparticles corresponding to 1 mg/mL of insulin were suspended in acetate buffer (pH 4.3). Spectra were collected at 37 °C after 40 and 60 min, reproducing the experimental conditions of the release studies. After 60 min, the nanoparticles were filtered through a 0.22 µm filter and the filtrate containing the released insulin was further analyzed by CD.

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Insulin concentration in the different samples was assessed by HPLC as previously described. In Vivo Studies in Rabbits. The protocol for the animal experiments was approved by the Ethics Committee of Universidad de Santiago de Compostela and was similar to that previously reported.2 Briefly, male New Zealand albino rabbits (∼2 kg) were initially fed for ∼48 h ad libitum and then fasted for 16 h prior to the experiment. Water was available ad libitum during the whole experiment. The following preparations were administered to each group: (1) control insulin solution; (2) insulin-loaded CS-TPP nanoparticles; and (3) insulinloaded CS-TPP-ALG nanoparticles. The total dose of insulin was 5 IU/kg in a volume of ∼150 µL of acetate buffer pH 4.3 (75 µL on each nostril) and was instilled using a polyethylene tubing inserted about 3 cm into the nostril. Blood samples were collected from the ear vein 30 min before the nasal administration to establish the baseline glucose levels and at different times after dosing for up to 5 h. Glucose plasma levels were determined by the glucose-oxidase method (Glucose-TR, Spinreact S.A., Spain). Results are presented as mean average values (% of basal level ( SE) for n ) 6. Glycaemia data were analyzed by two-sample t-test comparisons with respect to the free insulin group using Aspin-Welch and the Mann-Whitney U-test for unequal and equal variances (p < 0.05), respectively. Statistical analyses were conducted using the NCSS 2000 software package.

Results and Discussion In this work we have aimed to develop a new hybrid nanoparticle system that consists of two polysaccharides, namely, CS and ALG. We have previously reported the development of CS nanoparticles based on the ionic crosslinking of CS with a counterion such as TPP.23 These nanoparticles have shown a potential for the nasal administration of proteins.2 In the present work, the introduction of ALG as a second component in the formulation was expected to open new possibilities for the interaction with biological surfaces. As a first step toward this end, the preparation conditions of this system have been optimized in terms of the concentration of added NaCl, the ALG/CS molar ratio, and ALG’s Mw. Subsequently, insulin has been loaded to the prototype formulations that had optimal properties and we have studied the efficacy of association and in vitro release profile of the peptide by these systems as well as the pharmacological response after their intranasal administration to rabbits, as discussed in detail below. Formation of CS-ALG-TPP Nanoparticles. Effect of NaCl Concentration on the Nanoparticles Properties. CS-ALGTPP colloidal nanoparticles were formed spontaneously upon the addition of the TPP-ALG solution (pH ∼ 8.9) to the CS solution (pH 5.9). It is known that CS-TPP nanoparticles are formed by ionic gelation of CS, a mechanism that is driven by the cross-linking of the CS’s -NH3+ groups with the P3O5-10 and HP3O4-10 ionic species of TPP. The intra- and intermolecular linkages created between the negatively charged groups of TPP and a fraction of the positively charged amino groups of CS are responsible for the success of the process. In the presence of ALG, it is expected that the ionic gelation process occurs concomitantly with the complexation of the polyelectrolyte with ALG’s -COO- groups (final pH ∼ 6.1).31 In other CS-based hybrid nanoparticle systems composed of either neutral or negatively charged oligo- and polysaccharides such as cyclodextrin (CD) derivatives (e.g., hydroxypropyl-β-CD, sulfobutylether-β-CD, and carboximethyl-β-CD),26-28 hyaluronic acid,24 or konjac glucomannan,25 the interaction with CS has been suggested to be mediated by the concomitant contributions of these forces. Moreover, it has also been recognized that in these systems TPP acts cooperatively, favoring the incorporation of CS and the other component.27 FTIR spectroscopy

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Figure 1. Effect of the variation in NaCl concentration on Z-average diameter size of CS-TPP-ALG nanoparticles (mass ratio 6:1:1; mean ( SD, n ) 3) comprising alginate of Mw of 4 (Q), 18 (]), 32 (2), and 39 (1) kDa.

analysis of the isolated nanoparticles (results not shown) indicates that there are not free TPP molecules entrapped into the nanoparticles, thus suggesting that all of them are involved in the ionic interaction with CS. The role of pH and ionic strength on the size of the CS-TPP nanoparticles has been previously described.32 Hence, the particle size of the system was optimized by controlling the solution’s ionic strength with various concentrations of added NaCl (a table with the experimental layout utilized is available in Supporting Information). Figure 1 shows the variation of the Z-average particle size (at a fixed CS-ALG-TPP mass ratio of 6.0:0.56:1.0) with increasing concentrations of NaCl for nanoparticles comprised of ALG of varying Mw. At low ionic strength, between 0 and 85 mM NaCl, the nanoparticle size remains unchanged. Increases in the NaCl concentration beyond ∼85 mM NaCl, lead invariably to the formation of larger nanoparticles and this effect is progressively pronounced for systems involving ALG with high Mw. These results suggest that, as expected, at low NaCl concentrations ( ∼0.1 a discontinuity is clearly discernible. The fact that the critical value (R ∼ 0.1) where this discontinuity is observed matches exactly the value for the onset of an increase in nanoparticle size (Figure 2) is consistent with the notion that a change in the molecular organization of the system occurs at this charge ratio. Based on these experiments, we decided to fix the composition of the nanoparticles at an ALG/CS molar ratio of 0.1 for the formulation of insulin-loaded nanoparticles. Effect of Alginate Mw on the Nanoparticles Properties. Figure 4 illustrates the variation in the Z-average diameter for ALG Mws ranging from 4 to 74 kDa. For comparison, a sample of CS-TPP was also included in the experiment. Inspection of the curves reveals that even though ALG is present at a very low proportion in the system, the incorporation of this polysaccharide with increasing Mw was accompanied by a monotonic increase in size. This may be a consequence of the formation of gradually expanding structures produced by the longer ALG chains due


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Figure 3. Effect of the variation in the alginate/chitosan molar charge ratio on the effective amount of alginate incorporated to CS-TPPALG nanoparticles (ALG Mw ∼ 39 kDa; NaCl 85 mM; mean ( SD,n ) 3).

Figure 4. Effect of the variation of alginate Mw on the Z-average diameter size of CS-TPP-ALG nanoparticles (mean ( SD, n ) 3).

to the uneven charge compensation between the oppositely charged CS and ALG residues. This interpretation is in line with the argument given above to account for the effect of increasing amounts of added electrolyte. Also, it is well-known that the average size of polysaccharide-based colloidal particles and their structural organization are highly influenced by both the molar mixing ratio and the molecular weight of the initial components.15,23,25 In general, systems composed of low Mw ALG are believed to undergo complexation in a guest-host or “ladder-like” residue-to-residue interaction pattern,33,34,36-38 where the short ALG stretches condense themselves at the periphery of the CS chains in random coil conformations “frozen” by their cross-linking with TPP. Conversely, in high Mw ALG the interactions between both polyelectrolytes may occur via the formation of a “scrambled egg” pattern. Based on these results, it was decided to formulate insulin-loaded CSTPP-ALG nanoparticles with ALGs ranging in Mw from ∼4 to ∼32 kDa so as to obtain nanoparticles smaller than ∼450 nm. Development of Insulin-Loaded CS-ALG-TPP Nanoparticle. Insulin could be associated with the nanoparticles without the need to substantially modify the preparation protocol adopted for nonloaded (blank) nanoparticles, apart from dissolving the polypeptide in the TPP solution prior to the addition of ALG (pH ∼ 7.9). Based on the previous results with nonloaded nanoparticles, we selected three ALGs ranging in Mw from ∼4 to ∼32 kDa for the formulation of insulin-loaded CS-TPP-ALG nanoparticles. The materials without alginate are denoted by CS-TPP, while CS-TPP-ALG4, CS-TPP-ALG18,

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Table 1. Characteristics of Insulin-Loaded and Blank Chitosan-TPP and Chitosan-TPP-ALGinate Blended Nanoparticles (Mean Values ( SD; n ) 3) alginate Mw (kDa) d e

4f 4g 18f 18g 32f 32g

size (nm)

P.I.a

ζ (mV)

insulin association efficiencyb (A.E., %)

275.4 ( 2.7 270.6 ( 4.2 295.7 ( 3.2 247.5 ( 4.9 388.6 ( 7.0 286.5 ( 5.0 297.4 ( 3.6 306.8 ( 5.3

0.23 0.21 0.31 0.17 0.36 0.19 0.25 0.22

+46.7 ( 1.0 +48.7 ( 1.2 +42.1 ( 0.1 n.d.h +43.2 ( 0.8 +44.9 ( 0.1 +41.7 ( 1.2 +41.9 ( 0.1

44.1 ( 3.6 no insulin 50.7 ( 1.5 no insulin 49.2 ( 4.0 no insulin 46.3 ( 1.2 no insulin

insulin loading efficiencyc (L.E., %) 46.9 ( 1.7 no insulin 52.6 ( 0.8 no insulin 41.2 ( 0.4 no insulin 47.9 ( 1.2 no insulin

yield (%) 27.6 ( 1.0 21.3 ( 1.5 26.7 ( 0.4 19.4 ( 0.6 34.3 ( 0.3 24.6 ( 1.5 36.0 ( 1.0 23.1 ( 0.5

a Polydispersity index. b Insulin A.E. (%) ) (insulintotal - insulinunloaded)/insulintotal × 100. c Insulin L.E. (%) ) (insulintotal - insulinunloaded)/nanoparticles weight × 100. d Insulin-loaded nanoparticles; no added alginate (CS/TPP/insulin mass ratio 6:1:3). e Blank nanoparticles; no added alginate (CS/TPP mass ratio 6:1). f Insulin-loaded nanoparticles (CS/TPP/ALG/insulin mass ratio 6:1:0.5:3). g Blank nanoparticles (CS/TPP/ALG mass ratio 6:1:0.5). h Not determined.

and CS-TPP-ALG32 denote the materials fabricated from alginate of Mw ∼4, 18, and 32 kDa, respectively. The physicochemical properties displayed by the insulinloaded and blank nanoparticles are depicted in Table 1. The average size of the insulin-loaded nanoparticles remained in the range from ∼273 to ∼396 nm. These values were somewhat above the range observed with the blank nanoparticles (∼266 to ∼312 nm). However, in contrast to their blank counterparts, the average of particle size of the insulin-loaded formulations did not show a systematic relationship with the ALG’s Mw, as the largest and most polydisperse system was the CS-TPPALG18. Additionally, the yield of the blank nanoparticles was ∼30% lower than that of the insulin-loaded systems. One possible underlying mechanism to account for the differences in the pattern of behavior between the blank and the insulinloaded nanoparticles is that the electrostatic association of insulin with CS occurs at the expense of chitosan-alginate interactions and, thus, the ALG’s effect on the colloidal particle size is no longer the same as in the blank nanoparticles. Also, it is possible that ALG associates with insulin via metal-ligand interactions, which may alter the way CS and ALG associate this possibility is further explained below. In addition, the increased yields observed with the insulin-loaded nanoparticles, relative to the blank materials, are consistent with a greater association between the polysaccharides and the insulin. Figure 5’s TEM micrographs of insulin-loaded nanoparticles indicate that a spherical morphology was invariably attained irrespective of the ALG’s Mw. The positive sign of the ζ values reflect the excess of charged -NH3+ groups from CS exposed on the nanoparticles’ surface. Even though the inclusion of ALG in the CS-TPP-ALG systems leads to a very subtle reduction in ζ with respect to the CSTPP materials, the system retains a high positively charged ζ potential. A positive surface charge is considered to be an advantage from the biopharmaceutical viewpoint, as it favors the mucoand bioadhesion of the nanoparticles to negatively charged sites on cell surfaces and tight junctions.39-41 The other parameter evaluated was our hybrid system’s efficiency in associating with insulin. The results in Table 1 show that the CS-TPP-ALG nanoparticles were able to associate insulin with efficiencies of between ∼41 to ∼52%. The presence of ALG had a slight but consistent effect in favoring insulin association relative to the CS-TPP nanoparticles, which resulted in somewhat greater (5-12%) amounts of the peptide being associated. Even though electrostatic forces between insulin and CS may account for the dominant interaction in this system, other interaction mechanisms, such as hydrophobic association, may also contribute to the association of insulin. Furthermore, the

Figure 5. Transmission electron microscopy (TEM) images of insulinloaded nanoparticles made from (a) CS-TPP-ALG, (b) CS-TPP-ALG4, (c) CS-TPP-ALG18, and (d) CS-TPP-ALG32. Magnification as shown in bars.

possibility that insulin associates to ALG via an electrostatic interaction with traces of Zn found in insulin cannot be ruled out. It is well-known that alginate interacts specifically with divalent metal ions by means of electrostatic, covalent, and chelation binding mechanisms.42 Zn is added during the purification of insulin to aid its crystallization and Zn is known to form complexes with the amino acids histidine and glutamic acid.43 Therefore, a fraction of the insulin may also be associated with ALG, possibly via ligand-metal interactions, in an manner



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Figure 6. Evolution of the Z-average diameter size with time of insulinloaded nanoparticles incubated in acetate buffer pH 4.3 at 37 °C made from CS-TPP (O); CS-TPP-ALG4 (Q); CS-TPP-ALG18 (]) and CSTPP-ALG32 (2).

Figure 7. Insulin release profiles from nanoparticles during incubation in acetate buffer pH 4.3 at 37 °C (CS/INS 6/3; mean ( SD, n ) 3). Nanoparticles composition: CS-TPP (O); CS-TPP-ALG4 (Q); CS-TPPALG18 (]); and CS-TPP-ALG32 (2).

analogous to that recently proposed for nanoparticles comprised of CS and poly(γ-glutamic acid).6 FTIR spectroscopy was utilized to glean insight into the chemical structure and interactions operating in the solid state among the various chemical species present in the insulin-loaded nanoparticles (see Supporting Information). The results were consistent with the notion that increases in ALG Mw prevent insulin from adopting its dimeric form, thus, effectively leading to a decrease in antiparallel β-sheet structure. An explanation of these results may be found in the stronger association of insulin to CS-TPP-ALG systems composed of ALG of Mw g 18 kDa. This could occur via a hydrophobic interaction between insulin and the polyionic complex formed by CS-ALG, and/or via a specific interaction mediated by the trace Zn found in insulin, as suggested above. Study of the Stability of the CS-TPP-ALG Nanoparticles in Acetate Buffer at pH 4.3. In previous studies conducted by our group on CS-based nanoparticles that addressed the development of nanoparticle formulations intended for the nasal delivery of insulin, it was demonstrated that acetate buffer at pH 4.3 was an adequate medium to administer insulinloaded nanoparticles intranasally to conscious rabbits and achieve enhanced reduction in glucose plasma levels.2,3 This pH is somewhat lower than that of nasal secretions (pH 5.5-6.5),44 but is adequate for the solubilization of insulin (pI 5.3). In light of these previous studies, this buffer was selected as the medium for the developed formulations. It was therefore important to know in advance the stability of our system in this buffered medium. As shown in Figure 6, the insulin-loaded formulations were stable for up to 80 min in the acetate buffer (pH 4.3) with no discernible signs of elevation in the Z-average diameter of the colloidal species. In Vitro Insulin Release from CS-TPP-ALG Nanoparticles. The in vitro release rate of insulin from the various formulations is depicted in Figure 7. Only small differences in the release behavior between the various formulations can be distinguished. In general, a burst release effect is observed in all the systems, and more than 80% of the polypeptide was released within the first 20 min and nearly all of it was released within 120 min. In general, the observed release performance of these systems essentially agrees with that previously recorded with CS-TPP nanoparticles.2,3 This behavior is understood to be a consequence of the weak ionic interaction of insulin with CS. Study of the Conformational Stability of Insulin. The conformation of the insulin in solution during the process of

nanoparticle formation and after its release into the acetate buffer from the CS-TPP nanoparticles was probed by circular dichroism (CD). In previous studies on CS-coated ALG-based nanoparticles, far-UV CD was utilized to probe the secondary structure of insulin and it was demonstrated that its integrity, and presumably its bioactivity, was preserved during its entrapment in the CS-ALG nanoparticles.45 In the present work, we first evaluated the effect of solubilizing insulin (1 mg/mL) in aqueous dilute solutions of TPP in NaCl 0.5% (pH 8.5), HCl (pH 2.5), or NaOH (pH 8.5) on its conformational stability. Figure 8 shows the far-UV CD spectra for the various insulin solutions tested. It is clear that in all of the spectra two minima invariably appear at λ ) 209 and 223 nm, which is diagnostic of an R-helical polypeptide conformation.46-48 The intensity of these negative bands in the spectra in Figure 8a was evaluated quantitatively and the ratio of the molar ellipticities [θ]209/[θ]223 was calculated. The value of this ratio was 1.10 for insulin dissolved in HCl at pH 2.5, which is rather close to the values recorded when the peptide was dissolved at the same pH in the TPP and NaOH solutions, which were 1.16 and 1.08, respectively. This result demonstrates that the dissolution of insulin in TPP during nanoparticle preparation does not alter its conformation and that it remains mainly in its monomeric form. It was also of interest to probe the conformation of the insulin associated with the CS-TPP nanoparticles during its release into acetate buffer at pH 4.3 and 37 °C. Figure 8b shows a CD spectrum of insulin-loaded nanoparticles collected at varying time intervals during the insulin’s release, while conducting the experiment directly in a CD cell. At time zero, the value of the [θ]209/[θ]223 ratio was 0.56, suggesting an increase in antiparallel β-sheet structure and hence, an increase in the prevalence of the dimeric form in the nanoparticles. However, this ratio increases to 0.62 and 0.80 at 40 and 60 min, respectively, indicating a progressive increase in the amount of the monomeric form. Figure 8c shows the CD spectrum of the release medium after 60 min and after filtration, that is, once the insulin release was complete, along with a reference spectrum of insulin in acetate buffer. A comparison of these spectra reveals that the insulin released was essentially monomeric ([θ]209/[θ]223 ) 0.98). These results suggest that, although insulin exists in a dimeric form in the nanoparticle, an idea that agrees with the FTIR results recorded in the solid state (see Supporting Information), once it is released it adopts a monomeric conformation.

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Figure 9. Plasma glucose levels achieved in rabbits after nasal instillation (at pH 4.3; mean ( SD,n ) 6) of insulin acetate solution pH 4.3 (0; initial plasma glucose level 110.5 ( 10.5 mg/dL); insulinloaded CS-TPP nanoparticles suspended in acetate buffer pH 4.3 (O; initial plasma glucose level 102.7 ( 9.0 mg/dL); insulin-loaded CS-TPP-ALG18 nanoparticles suspended in acetate buffer pH 4.3 (]; initial plasma glucose level 112.0 ( 11.1 mg/dL); and insulinloaded CS-TPP-ALG32 nanoparticles suspended in acetate buffer pH 4.3 (2; initial plasma glucose level 105.6 ( 7.0 mg/dL). *Statistical significant differences (p e 0.05) between insulin-loaded nanoparticle and insulin acetate solution control group.

Figure 8. Far-UV circular dichroism spectra of bovine insulin solution (1 mg/mL) at 25 °C in (a) TPP pH 8.5 (bordeaux), NaOH pH 8.5 (green), and HCl pH 2.0 (blue); (b) during its release from CS-TPP nanoparticles in acetate buffer pH 4.3 at 37 °C after 0 min (blue), 40 min (green), and 60 min (bordeaux); and (c) after complete release from nanoparticles in acetate buffer pH 4.3 at 37 °C (green) in comparison with a solution of insulin made in the same buffer (blue).

In Vivo Efficacy of Insulin-Loaded CS-TPP-ALG Nanoparticles. Insulin-loaded CS-TPP and CS-TPP-ALG nanoparticles were administered intranasally to conscious rabbits (dose of insulin: 5 IU/kg). For this purpose, we selected two nanoparticle formulations containing alginate, namely, CS-TPPALG18 and CS-TPP-ALG32, and we also used a solution of insulin as a control. The results in Figure 9 indicate that, as expected, insulin in solution is very poorly absorbed, exhibiting a maximum 14% decrease in blood glucose levels at 30 min postadministration. Besides, we have previously verified that the treatment of rabbits with acetate buffer (control) do not cause any changes in glucose levels which could be related to stress or irritation.3 In contrast, the response elicited by insulin-loaded CS-TPP nanoparticles was significantly enhanced (maximum glucose decrease of ∼35% at 45 min after dosing). A similar maximum hypoglycaemic pick was observed following administration of CS-TPP-ALG18 or CS-TPP-ALG32 insulin-loaded nanoparticles. Interestingly, the hypoglycaemic effect corre-

sponding to the CS-TPP-ALG18 formulation, was significantly prolonged by up to 5 h with respect to the control. In agreement with previous studies using the same animal model and experimental protocol,2,28 these results confirm that CS-based nanoparticles enhance the systemic absorption of insulin after nasal administration. Moreover, these results indicate that the presence of ALG in the nanoparticles leads to a prolonged hypoglycaemic response for up to 5 h. Chitosan is known to induce transient widening of tight junctions between cells49,50 that results in an increased cell permeability.39 However, a series of studies have led us to accept that the mechanism of action whereby nanoparticles enhance the nasal absorption of macromolecules might differ from that of CS solutions.2,28 In fact, the results of the present study again confirm that CS nanoparticles are able to reduce glucose plasma levels to a greater extent than the same insulin dose administered as a solution. As previously reported,28 the ability to increase the nasal absorption of insulin, could be explained in terms of two mechanisms. First, the bioadhesion of nanoparticles leads to an important accumulation of CS in the epithelium, thus enabling the CS to open the tight junctions concomitant with the delivery of the associated insulin. This hypothesis has been supported by the observed ability of CS-based nanoparticles to reversibly decrease the transepithelial resistance (TEER) in epithelial cell cultures. Second, there is also the possibility that a fraction of the nanoparticles could enter the epithelial cells and deliver the associated insulin intracellularly. This hypothesis comes from the observed presence of CS-TPP-cyclodextrin nanoparticles inside the nasal epithelium after intranasal administration to conscious rats.28 With regard to the bioadhesive behavior of the nanoparticles, it is noteworthy that the presence of ALG could further contribute to this mechanism due to its high affinity for Ca2+.51 We could also speculate that this affinity could be responsible for the prolonged insulin absorption observed with the ALG18 formulation. However, we cannot discard the possibility that


the insulin is delivered more slowly in vivo from this formulation due to a stronger interaction with the ALG-containing nanocarrier.

Conclusions A new type of hybrid CS-TPP-ALG nanoparticle has been generated by the concomitant ionic cross-linking of CS to TPP and the formation of an electrostatic complex between oppositely charged groups in CS and ALG. The average colloidal size of these nanoparticles can be modulated in three distinct ways: (1) by the overall concentration of the added electrolyte; (2) by controlling the mass ratio of ALG and CS; and (3) by varying the molecular weight of the ALG incorporated. This system was able to associate insulin, our model peptide, with efficiencies up to 50.7% and to release it in vitro in the monomeric bioactive form as determined by CD spectroscopy. This last result is consistent with the capacity of insulin-loaded nanoparticles to enhance the systemic absorption of insulin after nasal administration to conscious rabbits. Additionally, it was observed that the duration of the hypoglycaemic response was affected by the Mw of the ALG incorporated into the nanoparticles. Acknowledgment. Financial support of the European Union from the NANOBIOSACCHARIDES project (Ref. No. 013882 of call FP6-2003-NMP-TI-3-Main) is gratefully acknowledged. We thank Mr. Rafael Romero of USC for his help and advice during in vivo assays and Francesca Ungaro of University of Napoli during CD experiments, as well as Anna Coggiola, Geoffroy Henriet, Melany De Prijck, and Paula Tavares, students under ERASMUS training at USC, for their help during experimental work. Supporting Information Available. Experimental layout utilized for the optimization of the conditions for the preparation of nanoparticles; FTIR spectroscopy results used to probe the interactions and secondary structure of insulin in CS-TPP and CS-TPP-ALG insulin-loaded nanoparticles. This information is available free of charge via the Internet at http://pubs.acs.org.

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BM9001377

Chitosan-Alginate Blended Nanoparticles as Carriers for the

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