Monodisperse Chitosan Nanoparticles for Mucosal Drug Delivery

Biomacromolecules 2004, 5, 2461-2468

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Monodisperse Chitosan Nanoparticles for Mucosal Drug Delivery Hong Zhang,† Megan Oh,† Christine Allen,*,‡ and Eugenia Kumacheva*,† Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada, and Department of Pharmaceutical Sciences, University of Toronto, 19 Russell Street, Toronto, Ontario M5S 2S2, Canada Received June 30, 2004; Revised Manuscript Received August 6, 2004

Chitosan nanoparticles (CS NPs) of a controlled size (below 100 nm) and narrow size distribution were obtained through the process of ionic gelation between CS and sodium tripolyphosphate (TPP). A high degree of CS deacetylation and narrow polymer molecular weight distribution were demonstrated to be critical for the controlling particle size distribution. Properties of the CS NPs were examined at different temperatures, values of pH, and ratios of CS to TPP. The model protein, bovine serum albumin, was encapsulated into the NPs, and the in vitro release profiles were examined in physiologically relevant media at 37 °C. Introduction Developments in genomics and proteomics continue to give rise to the identification of peptides and proteins with therapeutic potential.1 Traditionally, administration of peptide and protein drugs has relied on the parenteral route.1,2 However, major efforts have been aimed at developing effective formulation technologies suitable for mucosal delivery via oral, nasal, or pulmonary administration of these macromolecules.1-3 To date, mucosal delivery of a wide range of macromolecules has been explored including group B streptococcus,4 tetanus toxoid,5 insulin,6 and β-lactoglobulin.7 Mucosal delivery of macromolecules is reliant on adsorption at mucosal sites or transport across epithelial barriers.8 Many different strategies have been attempted as a means to improve the mucosal delivery of macromolecules.1-3,9 One interesting approach is the encapsulation of peptides and proteins in lipid- or polymer-based carriers.9-11 The delivery systems that have been studied include polymeric nanoparticles (NPs), microspheres, micelles, and liposomes.9-11 In vitro and in vivo evaluation of these technologies has enabled identification of some of the favorable properties for protein and peptide delivery systems. For example, it has been found that delivery systems with mean diameters in the range of hundreds of nanometers have a greater ability to penetrate the epithelia when compared to particles in the micrometer size range.12 The use of polymer materials such as poly(lactic acid) and poly(2-hydroxy acid) was limited by their instability in gastrointestinal fluids13 and poor transport across mucosal barriers.14 By contrast, polymer materials such as chitosan (CS) acted as absorption enhanc* To whom correspondence should be addressed. E-mail: ekumache@ chem.utoronto.ca (E.K.); [email protected] (C.A.). † Department of Chemistry, University of Toronto. ‡ Department of Pharmaceutical Sciences, University of Toronto.

ers, as they functioned to increase the transport of molecules across mucosal barriers.11,15 CS, has been reported to be a biocompatible, biodegradable, and nontoxic polysaccharide.16 In recent years, CS has also been investigated extensively as a carrier for mucosal drug delivery due to its mucoadhesive property, as well as its established ability to act as an absorption enhancer.17-19 To date, there have been a variety of reports on the preparation of CS particles. For example, Ohya et al.20 prepared CS particles with mean dimensions from 250 to 300 nm using a water-in-oil emulsion method. This method involved ultrasonication of a solution of CS in acetic acid mixed with toluene followed by chemical cross-linking of the CS particles with glutaraldehyde. Ultrasonication and emulsification techniques have also been employed to prepare CS-alginate particulate systems.21,22 The mean diameter of the particles varied from 450 nm21 to 8 µm.22 Alonso et al.23 reported the use of an ionic gelation method to prepare CS NPs. In this method, an anionic cross-linking agent, sodium tripolyphosphate (TPP), was added to an aqueous solution of CS in acetic acid. Positively charged CS NPs were formed through the inter- and intra-cross-linking of the amino groups of CS with the negatively charged phosphate groups of TPP. The authors demonstrated the encapsulation of bovine serum albumin (BSA) by the CS NPs with the maximum loading capacity (LC) of 51 wt %. Several advantages of this method included the use of aqueous solutions, the preparation of particles with a small size, the manipulation of particle size by the variation in pH values, and the possibility of encapsulation of proteins or DNA during particle formation. The potential drawbacks of this method were the relatively weak interactions between CS and TPP under low and high pH conditions; thus, NP stability could be challenged in the low pH environment within the stomach. The size of particles influences the drug release rates and the suitability of particles for a particular route of administra-

10.1021/bm0496211 CCC: $27.50 © 2004 American Chemical Society Published on Web 09/25/2004

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tion.24 Furthermore, a narrow distribution in particle dimensions ensures that drug delivered by all particles has the same biological fate. Several groups have explored the effect of preparative conditions on the dimensions of CS particles obtained by ionic gelation.23,25-27 Typically, the mean diameters of the particles varied from 200 to 745 nm. It has been found that the concentrations of CS and TPP and the CS/TPP ratio in aqueous solution have a large influence on the size of the NPs obtained:23 increase in the amount of TPP led to a higher degree of CS cross-linking and a decrease in NP dimensions. Xu and Du28 prepared CS NPs with diameters in the range of 20-200 nm. The smaller particles were prepared by decreasing the concentration ratio of acetic acid/CS from 1.75 (reported by Calvo and Alonso)23 to 0.5. However, transmission electron microscopy (TEM) analysis of the NPs demonstrated that these particles have a broad size distribution. It has also been found that an increase in CS molecular weight leads to a moderate increase in NP size. The mean diameter of CS NPs increased from 220 to 250 nm when the polymer molecular weight increased from 11 to 70 kDa.25 This effect was explained by the change in viscosity of CS solution and the increased ability of lower molecular weight CS to form smaller structures. It was also found that insulin encapsulated within the smaller NPs formed from low molecular weight CS was released more rapidly than insulin entrapped in the NPs formed from a higher molecular weight CS. The faster release rate was presumably a result of the insufficiently strong interactions between the low molecular weight CS and the encapsulated agent. A wide variety of drugs, such as insulin,25 tetanus toxoid,19 and plasmid DNA,29 have been encapsulated into CS NPs. Ma and co-workers27 investigated the effect of the acidity of the medium on the size of the insulin-loaded CS NPs. The size of CS NPs increased with the increasing pH as a result of the weaker interactions present between CS and TPP. The same group reported the effect of pH on the LC and association efficiency (AE) of CS NPs with insulin. To our knowledge, however, there have been no reports on the preparation of monodisperse CS NPs and the effect of CS treatment, preparation temperature, and ratio of CS to TPP on NP size distribution. Herein, we used ionic gelation of CS and TPP to prepare monodisperse CS NPs with a predetermined size. Our approach was based on the following considerations. First, we assumed that fractionation of commercially available CS would narrow the molecular weight distribution of the polymer and would allow us to achieve better control over NP size. Second, we increased the degree of deacetylation (DD) of commercial CS assuming that a higher degree of cross-linking of amino groups would reasult in a more compact NP structure. We examined the effect of each modification step on the variation of NP size and size distribution and demonstrated that a combination of these steps enabled us to produce CS NPs in the size range from 90 to 200 nm with a narrow size distribution. In the next stage, we examined the variation in NP size as a function of pH, CS/TPP ratio, and temperature. Furthermore, we loaded the model protein BSA into the CS particles and

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investigated the release behavior of the BSA-loaded CS NPs in buffer solutions. Experimental Section Materials. CS with molecular weight from 50 to 190 kDa and TPP were purchased from Aldrich, Canada. The minimum DD of CS was 75% (the data provided by the company). BSA crystals were provided by Aldrich, Canada. Micro BCA protein assay kit was purchased from Pierce, U.S.A. Acetic acid was supplied by Fisher Scientific, Canada. The deionized water was obtained from the Millipore Milli-Q water purification system. Deacetylation of CS. Post-deacetylation of CS was carried out according to the previously reported procedures.30 CS was dissolved in an aqueous 2 wt % solution of acetic acid to the concentration of 1.75 ( 0.25 wt %. The solution was then poured in a small stream into an aqueous 47 wt % NaOH solution. A sediment of CS immediately formed. The mixture of CS precipitate and alkaline solution was then refluxed for 1 h at 100 °C under nitrogen. Then the CS sediment was washed in water at 80 °C for neutralization. The above procedures were repeated once to achieve a higher DD of CS. The DD of CS was characterized in Fourier transform infrared (FT-IR) experiments using a Paragon 500 spectrometer (Perkin-Elmer) following the previously reported procedures.31 In brief, CS films were cast on a ZnSe IR crystal window (25 mm in diameter and 2 mm in thickness, Aldrich, U.S.A.) from a 1 wt % solution of CS in aqueous 1 vol % acetic acid. After incubation at 105 ( 5 °C for 12 h, the films were immersed for 6 h into a mixture of methanol and aqueous ammonia (volume ratio of 7/3) to neutralize the amino groups. The films were then washed with distilled water, rinsed with methanol, dried for 12 h in a vacuum oven at 70 °C, and stored in a desiccator. The DD of commercial and post-deacetylated CSs was determined using the methods reported by Baxter et al.32 and Miya et al.33 For DD < 90%, the DD was calculated as DD ) [1 - (A1655/A3450)/1.33] × 100

(1)

where A1655 is the absorbance of the amide I band at 1655 cm-1 and A3450 is the absorbance of the OH vibration band at 3450 cm-1 as an internal reference. For DD > 90%, DD was determined using the dependence between the ratio A1655/ A2867 and the DD reported by Miya et al.,33 where A2867 is the absorbance of the CH stretching band at 2867 cm-1 as an internal reference. The DD of CS samples obtained from FT-IR experiments was confirmed by elemental analysis (model 2400 elemental analyzer, Perkin-Elmer) in the CHN mode. The DD was calculated according to DD ) [1 - (C/N - 5.145)/(6.861 - 5.145)] × 100 (2) where C/N is the ratio of carbon to nitrogen.34 Fractionation of CS. Fractionation of CS was conducted by adding dropwise an aqueous 10 wt % solution of NaOH into a 1.75 wt % CS solution in acetic acid. CS precipitate

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was collected at different values of pH. The obtained precipitate was dried in a vacuum oven for 24 h at 50 °C. Intrinsic viscosity [η] of CS solutions (0.5 g of CS in the solution of 0.2 M HAc/0.1 M NaAc) was measured using an Ubbelohde capillary viscometer with a capillary diameter of 1.52 ( 0.03 mm (model C359, International Research Glassware, U.S.A.). The temperature was equilibrated at 30.0 ( 0.1 °C. The viscosity-average molecular weight of CS was calculated using the Mark-Houwink equation [η] ) KMR where K ) 1.64 × 10-30 × DD14

(3)

R ) 1.82 - 1.02 × 10-2 × DD

(4)

where DD is the DD of CS.35 Preparation of CS NPs. CS was dissolved in an aqueous solution of acetic acid at concentrations of 0.05, 0.10, 0.15, and 0.20 wt %. The concentration of acetic acid was 1.75 times as high as that of CS. (For example, 0.10 wt % CS solution was prepared in 0.175 wt % acetic acid solution.) Then, 1.0 mL of TPP solution was added dropwise to 5.0 mL of CS solution in a 20-mL glass vial under magnetic stirring at 600 ( 20 rpm measured by a tachometer (StewartWarner, model 757-W) using an octagonal stirring bar (1/2 in. L × 5/6 in. D, Fisher Scientific, Canada). The mixture was then stirred for additional 10 min. The weight ratios of CS/TPP were 4:1, 4.5:1, 5:1, 5.5:1, and 6:1. At least five batches of CS NPs were obtained for each recipe. Characterization of CS NPs. The size, size distribution, and electrokinetic potential (ζ potential) of CS NPs were measured using photocorrelation spectroscopy (PCS; Zetasizer 3000HS, Malvern Instruments, U.K.). Experiments examining the variation in CS particle size at different ratios of CS/TPP, temperature, and pH were carried out on the DynaPro PCS spectrometer (Protein Solution, Ltd., U.K.). Each batch was analyzed in triplicate. TEM experiments were carried out on a Hitachi model 600 electron microscope at a 75-kV accelerating voltage. A droplet of CS dispersion was placed onto a copper grid covered with carbon (Electron Microscope Sciences, Inc., U.S.A.) and allowed to dry. In Vitro Evaluation of BSA Release from CS NPs. BSA was added to 5 mL of a 0.10 wt % CS acetic acid solution prior to the addition of 1 mL of 0.10 wt % TPP. The weight ratio of BSA to CS was 0.3:1, 0.5:1, or 0.8:1. The AE and LC were evaluated by isolating the particles by ultracentrifugation and measuring the concentration of BSA remaining in the supernatant. The values of AE and LC were calculated using eqs 5 and 6, respectively. AE ) (A - B)/A × 100

(5)

LC ) (A - B)/C × 100

(6)

where A is the total amount of BSA added during preparation, B is the amount of BSA remaining in the supernatant, and C is the weight of the CS NPs. The in vitro release of BSA from the CS NPs was evaluated over a 10-day period in both simulated gastric fluid without enzymes at pH 1.2 and simulated intestinal fluid without enzymes at pH 7.5. Formulations including CS NPs

Figure 1. FT-IR spectra of (a) commercial and (b) post-deacetylated CSs.

with BSA/CS weight ratios of 15.9, 31.2, and 40.6% were analyzed using the following procedures. A total of 5 mL of release buffer was added to each of a series of 5 mg of protein-loaded CS NPs (in 20-mL glass vials). The vials were incubated at 37 °C without stirring. At specific time points, the CS NPs were isolated from buffer by ultracentrifugation at 24 000g for 30 min at 10 °C. The concentration of BSA in the supernatant was measured using the Micro BCA assay with UV measurements (model 5000 UV-vis-NIR spectrophotometer, Varian, U.S.A.) at a wavelength of 562 nm. Each batch was analyzed in triplicate. Results and Discussion Deacetylation. Several attempts have been made to obtain CS with a DD above 90%.36,37 The process of refluxing CS alkaline solution for 1 h at about 140 °C, however, led to the degradation of CS molecules. In contrast, N-deacetylation of chitin in an alkaline solution intermitted by washing of the intermediate product produced highly deacetylated CS.30 In the present report, a CS solution in acetic acid was slowly added into the alkaline solution. The precipitated biopolymer was washed with water and redissolved in acetic acid to repeat deacetylation. The DD of commercial and post-deacetylated CSs characterized by FT-IR spectroscopy is shown in Figure 1. Two types of baselines are drawn for commercial (DD < 90%, Figure 1a) and post-deacetylated (DD > 90%, Figure 1b) CSs following the approaches of Baxter et al.32 and Miya et al.,33 respectively. While for commercial CS absorbance the of amide Ι bond at 1655 cm-1 was strong, for the postdeacetylated sample the intensity band was apparently reduced, indicating that the DD for post-treated CS was increased. Using the absorbance of the internal reference at 3450 cm-1 for commercial CS and 2867 cm-1 for post-

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Table 1. DD of Commercial and Post-Deacetylated CSs measurement method

IR spectroscopy

elemental analysis

commercial CS post-deacetylated CS

80.2% 94.2%

78.1% 91.4%

Table 2. Physicochemical Properties of CS NPs CS nanosparticles fraction- deacetyl- deacetylated and commercial ated ated fractionated diametera (nm)

153 ( 2 499 ( 2

170 ( 4 602 ( 4

polydispersityb [η] Mv (kDa)

0.68 91.0 131

0.60 73.9 107

20 ( 2 50 ( 2 151 ( 2 0.24 158 123

92 ( 1

0.17 131 99.4

a Particle size was characterized in PBS buffer at pH ) 3. b Polydispersity was given by the PCS technique.

deacetylated CS, we determined the DDs of commercial and post-deacetylated CSs to be 80.2 and 94.2%, respectively. In principle, an error could arise from using the 3450 cm-1 absorption band for the low DD of CS as the internal reference because of the effect of adsorbed water on the intensity of this band. Domszy and Roberts,31 however, showed that the DD obtained using the baseline approach was not dependent on the presence of moisture: increase in the adsorbed water content caused an increase of absorption at 1640 cm-1 as well as at 3450 cm-1. Thus, absorption of the amide Ι band at 1655 cm-1 was altered and compensated for any increase in the reference band at 3450 cm-1. In the highly deacetylated CS, the effect of moisture on CH vibration could be considered to be weaker than that at 3450 cm-1.33 According to the results of elemental analysis of the CS samples prior to and following post-deacetylation, the fractions of C, N, and H elements in commercial CS were 45.38, 8.22, and 6.72%, respectively. The calculated DD was 78.1%, in agreement with the DD ) 75% provided by Aldrich, Canada, and close to DD ) 80.2% obtained by FTIR. The fractions of C, N, and H in a post-deacetylated sample were 45.01, 8.52, and 6.79%, respectively. The calculated DD was 91.4%. Thus, post-deacetylation increased DD by 13.3% in comparison with commercial samples. The DDs of CS samples obtained by both infrared spectroscopic measurement and elemental analysis are summarized in Table 1. Fractionation. Upon addition of NaOH solution to the CS solution in acetic acid, CS precipitation occurred at pH ) 5.8. Collection of CS precipitates was carried out in the range of pH values from 5.8 to 7.4. For preparation of CS NPs, we only used the fraction obtained in the range 6.8 < pH< 7.2. The intrinsic viscosity and the viscosity-average molecular weight (Mv) of CS samples calculated using the MarkHouwink equation35 are given in Table 2 for commercial, post-deacetylated, and fractionated CSs and for CS after a combined treatment. We note that viscometric characterization of the molecular weight of CS, a cationic polymer, may not provide an absolute value of polymer molecular weight.

Nevertheless, Mv of commercial CS is 131 kDa, in reasonable agreement with the molecular weight provided by Aldrich, Canada. We also stress that after post-deacetylation the value of Mv of CS underwent only a slight change, which confirmed that polymer N-deacetylation in NaOH solution intermitted by washing of the intermediate CS fractions did not result in macromolecular chain degradation. Finally, we conclude that polymer fractionation as well as combined fractionation and post-deacetylation treatment allowed us to obtain a sample with a lower molecular weight and lower polydispersity. Size and Size Distribution of CS NPs. To explore the effect of post-deacetylation and fractionation of CS on the formation of polymer NPs, we examined the variation in their size, size distribution, ζ potential, and morphology following different steps in modification of commercial CS. For all batches and a particular recipe the variation in particle size and ζ potential was within 6%. Figure 2a-d shows the size distribution of CS NPs prepared from CS used as received and, following fractionation, post-deacetylation and combined treatments, respectively. Figure 2a features a bimodal particle size distribution with average particle sizes of 153 and 500 nm, respectively. The volume fraction of the bigger particles was 81.5% according to the PCS measurements. Figure 2b shows the size distribution of CS NPs prepared from fractionated CS. There still exists a bimodal size distribution with mean particle sizes of 170 and 600 nm. The dimensions of particles are close to those in Figure 2a, but the fraction of larger beads decreased from 81.5 to 36.8%. This result indicated that polymer fractionation to a certain extent controlled particle size and size distribution. For NPs obtained from the post-deacetylated polymer, Figure 2c shows a major peak for 150-nm-sized particles; that is, NP dimensions were significantly smaller than those in Figure 2a,b. The size distribution of the CS beads is also significantly narrower, although two small peaks exist for 20- and 50-nm-sized NPs. Small CS particles were presumably obtained by gelation of low molecular weight CS fragments which had been generated during the process of postdeacetylation. CS modification by polymer fractionation or post-deacetylation to a certain extent helped decrease the particle size and narrow the particle size distribution. However, small and monodisperse NPs were obtained only following both postdeacetylation and fractionation. After a combined CS treatment, we obtained 92-nm-sized CS particles with a narrow size distribution and polydispersity of 0.17 (Figure 2d). Table 2 gives a summary of these results. We conclude that particle size and size distribution were influenced by a higher DD and polymer molecular weight distribution. Additional N-deacetylation of commercial CS led to a higher degree of cross-linking of CS with TPP, producing denser and smaller CS particles. The effect of polymer fractionation was not straightforward. It could be anticipated, in accord with the results of Janes and Alonso,25 that low molecular weight CS species would form smaller particles: shorter polymer chains easily penetrated into the partially formed CS-TPP associates to produce smaller and denser beads. In our case, however, we



Figure 2. Size distribution of NPs produced from (a) commercial, (b) fractionated, and (c) post-deacetylated CSs and (d) both post-deacetylated and fractionated CS.

Figure 3. ζ-potential profiles of NPs prepared with (a) commercial and (b) deacetylated CSs.

measured a largely bimodal particle size distribution for the commercial and fractionated CS. ζ Potential of CS NPs. Figure 3 shows the variation in the ζ potential of the NPs prepared from commercial CS and CS after combined post-deacetylation and fractionation treatment. Two fractions of NPs were formed from commercial CS (Figure 3a): positively charged particles with ζ ) +49.5 mV and an average dimension of 153 nm as well as neutral submicrobeads with ζ ) 0 and an average size of 500 nm. Overall, the charge distribution for CS NPs was broad, ranging from -30 to +75 mV. In contrast, the NPs prepared from post-deacetylated and fractionated CS featured a single peak at ζ ) +47.2 mV (Figure 3b). These results shed light on the mechanism of reduction in the NP size and enhancement of the NP size distribution following polymer fractionation and especially post-deacetylation. Two types of NPs (small positively charged particles and large neutral beads) were obtained because of the relatively low DD and broad molecular weight distribution of commercial CS. CS with a high density of amino groups uniformly distributed along the chain formed smaller NPs

as a result of enhanced ionic interactions between amino groups and the TPP ions. The presence of the higher positive charge on this particles indicated that free (non-cross-linked) amino groups remained on the particle surface. In principle, these surface groups could be later used for bioconjugation with peptides for the targeted drug delivery. The CS molecules with a low amino group density produced larger particles because of the formation of a smaller number of ionic linkages. A low electrokinetic potential of these particles indicated that a positive charge of amino groups was almost neutralized by TPP. Morphology of CS NPs. Figure 4 shows typical TEM images of CS NPs prepared from the commercial and modified biopolymers. In Figure 4a the particles prepared from commercial CS are polydisperse with mean particle size in the range from 150 to 340 nm, comparable to the PCS results shown in Figure 2a. The morphology of CS NPs prepared from fractionated CS was very similar to that for commercial CS (not included in this figure). By contrary, the TEM image of particles prepared from post-deacetylated CS featured reduced polydispersity and dimensions from 145

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Figure 4. TEM images of NPs prepared with (a) commercial and (b) post-deacetylated CSs and (c) both post-deacetylated and fractionated CS. Table 3. Variation in CS Particle Size at Different CS/TPP Ratios and Temperatures temperaturea (°C)

CS/TPP (weight ratio)

25

32

37

ζ potentialb (mV)

4.0 4.5 5.0 5.5 6.0

127 ( 2 115 ( 2 109 ( 4 129 ( 3 135 ( 4

132 ( 3 127 ( 2 112 ( 4 121 ( 4 139 ( 3

131 ( 3 127 ( 4 117 ( 4 135 ( 4 141 ( 3

44.7 ( 2.1 45.7 ( 1.8 46.0 ( 0.7 48.3 ( 1.0 49.4 ( 0.7

a Particle size at different temperatures was characterized by protein solutions. b ζ potential was given by the PCS technique at 25 °C.

to 158 nm. These CS particles had a much more regular close-to-spherical shape. Figure 4c shows NPs prepared from post-deacetylated and fractionated CS. The individual particles had an average size of about 98 nm. The image analysis of the TEM image (Figure 4c) revealed polydispersity (defined as the standard deviation divided by particle mean size) of 8.3%. Effect of the CS/TPP Ratio on Particle Size. In further experiments we characterized the properties of NPs obtained from fractionated and post-deacetylated polymers. We studied the effects of pH, temperature, and CS/TPP ratio on the variation in dimensions of CS NPs, as well as encapsulation and in vitro release of BSA from the NPs. Table 3 shows the change in dimensions of NPs prepared at different weight ratios of CS to TPP (denoted as R) in the temperature range from 25 to 37 °C. It indicates that the value of R is critical for the formation of CS NPs. The particle size increases when R is either greater or lower than 5. We note that CS NPs could only be produced in a specific concentration range of CS and TPP, beyond or below which either aggregation occurred or no particles were formed. In the present work, the typical concentrations were from 2.5 to 4 mg/mL for CS and from 0.15 to 0.75 mg/mL for TPP. These results agreed with earlier findings.23 A nonmonotonic variation in NP size at different pH values compared well with the previous results of Fernańdez-Urrusuno et al.26 who reported the preparation of 320-nm-sized CS NPs at R ) 5, whereas at R ) 4 and R ) 6 larger beads were obtained. The decrease in particle size at R ) 5 can be explained as follows. A polyfunctional cross-linking agent TPP can make five ionic cross-linking points with amino groups of CS. Addition of TPP to CS in a 5:1 weight ratio presumably led to the most efficient cross-linking of amino groups producing the most compact particle structure, while at R ) 6 (a larger

Figure 5. Change in CS particle size as a function of pH at various temperatures: (() 25, (9) 32, (2) 37, (×) 42, (× with line) 47, and (b) 55 °C at a CS/TPP weight ratio of 5:1.

CS fraction) cross-linking was less efficient, causing an increase in NP size. For R ) 4, partial bridging of CS particles with TPP resulted in an increase of average size. Table 3 shows the values of the ζ potential of NPs obtained at different CS/TPP ratios. The value of ζ potential of NPs increased with an increase of CS/TPP ratio from 4 to 6, indicating an increase in the number of free amino groups on the surface of CS NPs. Effect of pH and Temperature on Particle Size. The variation in size of CS NPs in phosphate-buffered saline (PBS) solution at the various values of pH is shown in Figure 5. In the temperature range from 25 to 37 °C, the NP size slightly increased with an increase of pH from 3 to 5 at various temperatures, while at pH ) 2 there was a strong temperature-dependent increase in NP dimensions. The dependence of NP size on the variation in pH originated from the nature of ionic gelation used for the production of CS NPs. The degree of deprotonation of amino groups of CS increased when the pH increased from 2 to 5. Ultimately, deprotonation of amino groups (pKa ) 6.4) led to weaker ionic interactions between the amino groups and TPP anions. A sharp increase in particle size (exceeding 1 µm) was observed for pH ) 6 (not included in Figure 5). On the other hand, pKa3 and pKa4 of TPP are 2.3 and 6.3, respectively;38 thus, in the range 3 < pH < 6.3 no additional anions were generated for cross-linking of CS molecules and the NP size did not significantly change in the temperature range studied. At pH ) 2, however, an increase in temper-


Figure 6. AE (light gray) and LC (dark gray) of CS-BSA NPs as a function of BSA loading concentration (mean (SD, n ) 3).

ature from 25 to 37 °C significantly affected the NP size. This effect can be explained on the basis of the van’t Hoff equation: for exothermic dissociation the value of the dissociation constant decreases at elevated temperatures. By making an analogy with the dissociation between phosphoric acid and TPP, we note that the first dissociation of H3PO4 (pKa1 ) 2.15) has an enthalpy of -7.95 kJ/mol.39 Therefore, a smaller number of TPP anions were generated at a higher temperature, which resulted in a lower degree of ionic gelation and an increase in NP size. Encapsulation and in Vitro Release of BSA from CS NPs. The model protein, BSA, was encapsulated into the CS NPs to evaluate their suitability as a delivery system for proteins and peptides. NP formulations were prepared by the addition of different amounts of BSA to dispersions of CS NPs with BSA/CS weight ratios of 0.3:1, 0.5:1, or 0.8:1. The AE of the NPs for BSA was found to be 23.1, 47.7, and 62.3% for BSA/CS weight ratios of 0.3:1, 0.5:1, and 0.8:1, respectively, while the LC was 15.9, 31.2, and 40.6%, respectively (Figure 6). The LC of the CS NPs for BSA is relatively high and comparable to that obtained for other hydrogel particle systems.23,40 The in vitro release of BSA from the NPs was measured over a 10-day period in both simulated gastric fluid (pH ) 1.2) and simulated intestinal fluid (pH ) 7.5) at 37 °C. As shown in Figure 7, the release of BSA from CS NPs incubated in simulated gastric fluid is much faster than that for NPs incubated in simulated intestinal fluid. In fact, 7590% of the total BSA loaded in the NPs was released within the initial 24 h following incubation in gastric fluid. The accelerated release of the protein from CS NPs incubated in the low pH media is likely owed to the reduced interactions present within the NPs at this pH. As mentioned previously, the size of CS NPs increased when the particles are incubated in solutions of pH < 2 owing to the reduction in TPP ions present, which in turn reduces the extent of interaction within the particles. By contrast, the release of BSA at pH ) 7.5 is slower than that at pH ) 1.2. The decreased rate for release at this high pH is likely due to the fact that the NPs are partly collapsed under these conditions with BSA entrapped inside and interacting with CS via hydrogen bonding and ionic interactions.


Figure 7. In vitro release of BSA from CS NPs in simulated gastric fluid (pH ) 1.2, dotted lines) and simulated intestinal fluid (pH ) 7.5, solid lines) at various LCs: (() 15.9, (9) 31.2, and (2) 40.6% (mean (SD, n ) 3).

The relatively fast release of BSA obtained under low pH conditions indicates that it may be challenging to use the TPP cross-linked CS NPs for oral delivery of peptides and proteins. The release rate of the encapsulated macromolecules may be reduced by addition of biocompatible polymers [such as poly(ethylene glycol) or alginate] to the formulation used. The CS NPs described in the present work can be suitable, however, for mucosal delivery of drugs via nasal or pulmonary routes of administration. Conclusions We report the preparation of CS NPs in the size range of 90 to 200 nm achieved by combined post-deacetylation and fractionation of commercially available CS. We examined the effect of each step of CS modification and demonstrated that post-deacetylation is critical in producing small monodisperse CS NPs. The dimensions of the CS NPs were dependent on the pH of the solution and the weight ratio of CS to TPP, yet independent of temperature in the pH range from 3 to 5. It was found that the model protein, BSA, could be loaded into the CS NPs at protein-to-material weight ratios of up to 40%. In addition, the CS NPs were found to provide sustained release of this protein in simulated intestinal fluid (pH ) 7.5) over a 6-day period. These small, uniformly sized particles are suitable as a delivery system for the mucosal delivery of vaccines, peptides, and proteins. Acknowledgment. The authors appreciate Jiguang Zhang for assistance in TEM measurements. H.Z. is grateful to the Ontario Graduate Scholarship Program. E.K. is grateful to the Canada Research Chair funding. References and Notes (1) Lee, H. J. Arch. Pharm. Res. 2002, 25, 572-584. (2) Cleland, J. L.; Daugherty, A.; Mrsny, R. Curr. Opin. Biotechnol. 2001, 12, 212-219. (3) Carino, G. P.; Mathiowitz, E. AdV. Drug DeliVery ReV. 1999, 35, 249-257. (4) Hunter, S. K.; Andracki, M. E.; Kreig, A. M. Am. J. Obstet. Gynecol. 2001, 185, 1174-1179. (5) Jung, T.; Kamm, W.; Breitenbach, A.; Klebe, G.; Kissel, T. Pharm. Res. 2002, 19, 1105-1113.

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