Insight on the Formation of Chitosan Nanoparticles through Ionotropic

Jul 30, 2012 - *D.N.B.: Laboratory of Organic Chemical Technology, Department of Chemistry, Aristotle University of Thessaloniki, 541 24 Thessaloniki,...
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Insight on the Formation of Chitosan Nanoparticles through Ionotropic Gelation with Tripolyphosphate Emmanuel N. Koukaras,†,‡ Sofia A. Papadimitriou,‡ Dimitrios N. Bikiaris,*,‡ and George E. Froudakis*,§ †

Laboratory of Molecular Engineering, Department of Physics, University of Patras, Patras GR-26500, Greece Laboratory of Organic Chemical Technology, Department of Chemistry, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece § Department of Chemistry, University of Crete, P.O. Box 1470, Iraklion 714 09, Crete, Greece ‡

S Supporting Information *

ABSTRACT: This work reports details pertaining to the formation of chitosan nanoparticles that we prepare by the ionic gelation method. The molecular interactions of the ionic cross-linking of chitosan with tripolyphosphate have been investigated and elucidated by means of allelectron density functional theory. Solvent effects have been taken into account using implicit models. We have identified primary-interaction ionic cross-linking configurations that we define as H-link, T-link, and M-link, and we have quantified the corresponding interaction energies. H-links, which display high interaction energies and are also spatially broadly accessible, are the most probable cross-linking configurations. At close range, proton transfer has been identified, with maximum interaction energies ranging from 12.3 up to 68.3 kcal/mol depending on the protonation of the tripolyphosphate polyanion and the relative coordination of chitosan with tripolyphosphate. On the basis of our results for the linking types (interaction energies and torsion bias), we propose a simple mechanism for their impact on the chitosan/TPP nanoparticle formation process. We introduce the β ratio, which is derived from the commonly used α ratio but is more fundamental since it additionally takes into account structural details of the oligomers. KEYWORDS: nanoparticles, chitosan, ionotropic gelation, molecular interaction, density functional theory

1. INTRODUCTION Chitosan (CS) is a polysaccharide obtained from deacetylation of chitin. CS is composed of deacetylated β(1−4) 2-amino-2deoxy-β-D-glucan monomers and monomers with N-acetyl groups in place of amino groups that are linked by glycosidic bonds. The percentages of the monomers in the polymer chain determine the degree of deacetylation (DD) of CS. In acidic aqueous solution, chitosan oligomers can self-assemble into nanoparticles through ionic cross-linking with multivalent anions. Chitosan nanoparticles prepared by ionic gelation have been examined in the past decade as delivery media for drugs with low molecular weight.1−12 Compared with alternate promising routes that may be followed for the development of nanoscale drug delivery carriers such as micelles, CS nanoparticles display advantages such as increased stability, mild (on the targeted drug molecules) production methods and conditions, use of water-based solutions and not organic solvents, and simple procedures.13,14 Their stability can be partially attributed to the ionic cross-linking of positively charged chitosan oligomers using polyanions. The CS oligomers themselves are mechanically robust. The advantages presented by this ionic cross-linking inherent to CS nanoparticles prepared by ionic gelation may be transferable to other approaches as well, wherever ionic cross-linking is applicable. Recently, Bronich et al.14 successfully designed and synthesized polymeric micelles modified in such a manner as to include © 2012 American Chemical Society

ionic cross-linked cores, leading to polymer micelles with increased stability. The polyanion most commonly used for the ionic crosslinking is tripolyphosphate (TPP), which is nontoxic. The CS/ TPP molar ratio and the evolved interactions are very crucial for the formation of the nanoparticle mean diameter, since these parameters can affect the drug release properties. However, the mechanism of nanoparticle formation through ionic gelation is not well documented in the literature. It has been suggested that all ionic groups of TPP can participate in interactions with chitosan amine groups.2−4 However, as we show here, an oversimplified model of five interaction sites lacks accuracy, and to the best of our knowledge, this has not been addressed to date. High-accuracy ab initio calculations that additionally take into account solvation effects are needed for a proper and accurate quantitative theoretical description of the interactions that take place both in the process of forming the CS/TPP nanoparticles and between the nanoparticles and encapsulated drugs. In this work, we present results from such calculations and elucidate many aspects of the nanoparticle formation Received: Revised: Accepted: Published: 2856

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the computations. Solvation effects were taken into account using the polarizable continuum model (PCM)17,18 as implemented in the GAMESS19 package. Tight convergence criteria were enforced on the SCF energy (10−7 au), the oneelectron density (rms of the density matrix up to 10−7), and the norm of the Cartesian gradient (10−4 au). We used spinunrestricted wave functions and ensured that the correct electronic configuration was obtained by performing multiple calculations on different fixed spin states (singlet, triplet, etc.). All of the calculations were performed using the GAMESS19 package. 2.2. Experimental Procedures. CS with a molar mass of 350 000 g/mol and DD > 75% was supplied by Aldrich Chemical. The CS/TPP nanoparticles were prepared according to the ionotropic gelation method.20−24 Blank nanoparticles were obtained upon the addition of an aqueous solution of TPP to a solution of CS in acetic acid (pH 3.5) with final concentrations of 0.5 and 2 mg/mL, respectively. Different concentrations of CS and TPP were used to determine the CS/ TPP ratios providing nanoparticles with optimum size properties (CS/TPP ratio = n/1, where n = 2−8; the final concentration of chitosan was always 2 mg/mL). As previously reported,25 the formation of nanoparticles was a result of the interaction between the negative groups of TPP and the positively charged amino groups of CS. The concentrations of CS and TPP were chosen with respect to the ranges reported21 to produce samples of opalescent suspensions of very small particles and (for one case) an aggregate form. 2.3. Size Measurements on Nanoparticles. The particle size distribution and polydispersity index (PDI) of the CS/TPP nanoparticles was determined by dynamic light scattering (DLS) using a Zetasizer Nano instrument (Nano ZS, ZEN3600, Malvern Instruments, U.K.) operating with a 532 nm laser. A suitable amount of nanoparticles was dispersed in distilled water (pH 7, which was close as possible to body-fluid pH without the use of buffer solutions) to give a total concentration of 1%, and the dispersion was kept at 37 °C (body temperature, as the application of CS/TPP nanoparticles is mainly in the field of drug nanocarriers) under agitation at 100 rpm to achieve a homogeneous state of the sample in order to obtain average values for the size of the nanoparticles. All of the measurements were performed in triplicate, and the results are reported in terms of mean diameter ± SD. 2.4. Morphological Characterization of Nanoparticles. Transmission electron microscopy (TEM) was used to examine the morphology of the nanoparticles prepared in this study. TEM micrographs of nanoparticle samples deposited on copper grids were obtained with a JEOL 120 CX microscope operating at 120 kV.

process, identify interaction sites, calculate interaction energies, and locate energetically favorable relative configurations between oligomers and oligomers−polyanions. We provide information about the role of charge and how positive and negative centers may act as attraction centers for small drug molecules with negative and positive charge, respectively, thus allowing the nanoparticles to deliver both. In Figure 1a, we

Figure 1. Molecular structures of (a) the chitosan oligomer and (b) the tripolyphosphate polyanion and their corresponding simplified schematic representations, shown in (c) and (d) respectively. Depending on the degree of deacetylization and the charge of the amino group, R = H, H2, or COCH3.

show the structure of the CS oligomer in which substitution of R by H, COCH3, and H2 leads to deacetylated, acetylated, and charged monomers, respectively. The structure of the TPP polyanion is shown in Figure 1b. The simplified schematics shown in Figure 1c,d facilitate the representation of more complex configurations. In the convention that we follow for the simplified representations, the polymer chain is implicitly denoted by the dotted terminating lines. For the general case when distinction between acetyl groups and uncharged or charged amino groups is needed, these groups are collectively denoted by dash-terminated solid lines extending from the monomer rings, as shown in the legend of Figure 1. A distinction between amino (either charged or uncharged) and acetyl groups is made by using a simple line or a hooked line accordingly, as also shown in the legend of Figure 1. The distinction between the acetyl groups and the glycosidic bonds is self-evident. Any interactions between CS/TPP nanoparticles and the drugs should be examined as to ensure the lack of reactivity and alteration in the structure of the drugs. However, we did not study interactions of CS/TPP nanoparticles with individual and specific drugs, since they are highly dependent on the structure of the drug and hence beyond the scope of the present work.

2. METHODS 2.1. Theoretical Methods. The theoretical study was performed using all-electron calculations within the framework of density functional theory (DFT) and the generalized gradient approximation. The interaction energies and maximum-interaction-energy configurations of chitosan oligomers with TPP and of ionically cross-linked chitosan oligomers were computed by performing potential energy surface (PES) scans with respect to various distances and orientations. All of the geometry optimizations, PES scans, and interaction energy computations were performed using Grimme’s B97-D functional,15 which includes long-range dispersion corrections, with the def-TZVP16 basis set, which is of triple-ζ quality. The performance of the B97-D functional for noncovalently bound systems and pure van der Waals complexes is exceptionally good.15 Symmetry constraints were not imposed at any point in

3. RESULTS AND DISCUSSION 3.1. Nanoparticle Preparation and Characterization. CS/TPP nanoparticles can easily be prepared upon addition of a TPP solution to a CS solution under magnetic stirring, since the creation of nanoparticles depends mainly on the evolved ionic interaction of CS with TPP that eventually leads to the reduction of the aqueous solubility of CS. The CS/TPP ratio is critical and controls the size and the size distribution of the nanoparticles. The size characteristics have been found to affect the biological performance of CS/TPP nanoparticles.26 For this reason, we studied the effect of the CS/TPP ratio on the size characteristics of the nanoparticles to find the optimum ratio that results in nanoparticles with small size and a narrow size 2857

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reported by Zhang et al.4 who found an optimum ratio of 5/1. We attribute this small discrepancy mainly to the difference in the DD of the CS (they reported DD = 91.4%). The same behavior was reported by Fan et al.27 in their recent work on low-molecular-weight chitosan. 3.2. Potential Energy Hypersurfaces and Interaction Energies. We identified the sites of maximum interaction between a TPP polyanion and a CS pentamer by performing geometry optimizations for a multitude of relative CS−TPP initial configurations, examining both perpendicular and parallel coordination. We focused on the oxygen and phosphorus atoms of TPP and the amino and hydroxyl groups of CS. In an attempt to perform a nearly exhaustive search, we also included initial configurations that were unlikely to lead to strong interactions. In Figure 3a we show the configuration exhibiting the strongest interaction. The fully unprotonated TPP (P3O105−)

distribution. In doing so, we also compared and identified possible agreement of these results with those obtained using the theoretical approach to identify which CS/TPP ratio and what kind of ionic interactions give the optimum size for the nanoparticles. In Figure 2, the particle size distributions

Figure 2. (top) Particle size distribution obtained by DLS. (bottom) Extracted particle sizes at various CS−TPP weight ratios.

obtained by DLS for a range of CS/TPP ratios are presented. From these curves, the nanoparticle diameters, particle size distributions, and PDIs were calculated, and the results are presented in Table 1. The characterization as narrow or broad

Figure 3. (a) Configuration giving the strongest interaction between an initially unprotonated TPP polyanion (P3O105−) and a CS pentamer. (b) Single-site TPP−CS interaction configuration (local minimum) that gives the strongest interaction for the N-approach. Configuration (b) is the main configuration encountered in ionic cross-linking.

Table 1. Particle Size Distributions and Polydispersity Indexes Determined by DLS CS/TPP w/w ratio

nanoparticle diameter (nm)a

particle size distribution

2/1

531 ± 15

0.098

3/1

396 ± 10

0.067

4/1

342 ± 8

0.054

5/1

458 ± 4

0.078

6/1

498 ± 7

0.086

7/1

531 ± 12

0.093

8/1

615 ± 9

0.079

a

PDI broad unimodal narrow unimodal narrow unimodal broad unimodal broad unimodal broad unimodal broad unimodal

physical appearance and opacity aggregate

was examined in this case. The interaction energy was computed by a PES scan with respect to the average OTPP···NCS distance over the two participating NH3+ groups, and we denote this as the parallel approach, or P-approach. The dimensions (especially the length) of TPP are very suitable for this configuration, which consists of two individual interactions, each localized at a CS amino group. Since other type of polyanions have been and are being considered as ionic cross-linking agents, this should be taken into account during the selection process. The single-site TPP− CS interaction energy was calculated by a PES scan with respect to the OTPP···NCS distance with the TPP nearly perpendicular to CS. The structure giving the maximum interaction from this PES scan is shown in Figure 3b, and we denote it as the Napproach. At each point of the scan, for the selected constant distance, a partial geometry optimization was performed, allowing the structures of CS and TPP to relax. The OTPP···NCS distance is from the edge oxygen atom of TPP to the nitrogen atom of CS closest to the approaching TPP, as shown in Figure 3b. The resulting interaction energies as functions of the OTPP···NCS distance (RO···N) are shown in Figure 4. The upper range of the PES scan was 16 Å, but in Figure 4 we have displayed the range up to only 12 Å to improve the clarity of

opalescent suspension opalescent suspension opalescent suspension opalescent suspension opalescent suspension opalescent suspension

Reported as mean ± standard deviation.

is in accordance with that used in the experimental work of Fan et al.27 Furthermore, TEM micrographs of the prepared chitosan nanoparticles (provided in the Supporting Information) indicate that CS/TPP nanoparticles were roughly spherical in shape. It is clear that there is an optimum CS/ TPP ratio where nanoparticles with smaller sizes are produced. It appears that the optimum CS/TPP w/w ratio among these samples is near 4/1, which gave nanoparticles with sizes of 340 nm, while for other CS/TPP ratios, the size of the nanoparticles tended to increase. This behavior is in agreement with that 2858

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(RO···N ≈ 2.79 Å), after a double proton transfer, this energy also decreases, in this case by 33.6 kcal/mol to the new value of 34.7 kcal/mol. Each of these two proton transfers takes place between the TPP polyanion and one of the two interacting amino groups (i.e., one proton transfer per amino group). Both the drop in energy due to the proton transfer and the resulting interaction energy are significantly higher than the corresponding values in the case of the perpendicular configuration, but neither one is twice as large, as might be expected. This can be collectively explained by considering that (i) the electrostatic component of the interaction decreases nonuniformly upon successive proton transfers, by an approximate factor of [25 − (5 − n)2] for the nth proton transfer, and (ii) during the process we allowed for structural relaxation, so the process corresponds to an adiabatic proton transfer. We emphasize here that although we refer to successive proton transfers, this is only a simplification to facilitate the description of the effects. In both cases, the final maximum interaction energy is significantly higher than what would be expected for a single O···HN hydrogen bond.31,32 In addition to the electrostatic components that we have already mentioned, these high values can be partially attributed to long-range, weaker secondary interactions that take place between an alternate oxygen atom of TPP and a nearby hydroxyl group of CS. The dependence on the pH of the solution is implicitly taken into account by additionally examining initially protonated TPP anions. In mildly acidic solutions, the amino groups of CS are protonated, thus attributing a positive charge to these groups. Depending on the pH of the solutions, TPP polyanions will be partially protonated as well.5,33 In this respect, in addition to the PES scan for the fully unprotonated TPP, P3O105−, we also performed an N-approach PES scan for the initially protonated TPP, H4P3O10−. In the bottom panel of Figure 4, we show the corresponding interaction energy for the N-approach. As opposed to the previous case with the initially unprotonated TPP, H4P3O10− does not induce a proton transfer from the protonated amino groups at close range. Furthermore, the maximum interaction energy was calculated to be 12.3 kcal/ mol, which is significantly lower than in the previous case but is still higher than what would be expected for a single O···HN hydrogen bond.31,32 Having identified the maximum-interaction configurations and the corresponding energies for the two limiting cases, P3O105− and H4P3O10−, we continued by examining two CS oligomers cross-linked with an initially unprotonated TPP. After setting up an initial configuration, we performed fully unrestricted geometry optimizations. We additionally examined any effects that may depend on the length of the CS oligomers by performing the same procedure using CS trimers, tetramers, and pentamers. Interestingly, even though the CS oligomers were initially oriented parallel to each other, in every case their final orientation was such that the planes defined by their chain axes and the TPP primary axis formed a dihedral angle of 120− 135°. This tendency induces a bias in the orientation of the cross-linked CS oligomers that is expected to affect the CS/ TPP nanoparticle formation mechanism, especially at low TPP concentrations. In Figure 5 we show the final configuration for the case of the CS trimers. The two planes correspond to a CS chain and the TPP as described above, and the dihedral angle that they form is 124.8°. This effect became irregular when we increased the chain length to pentamers, since in that case the length was sufficient to permit weak direct oligomer−oligomer interactions.

Figure 4. (top) Interaction energies of an initially unprotonated TPP polyanion (P3O105−) for the P-approach (green triangles, dashed lines) and N-approach (black circles, solid lines). RO···N is the distance between the edge oxygen atom of TPP and the nearest nitrogen atom of CS. (bottom) Interaction energy of an initially protonated TPP anion (H4P3O10−) for the N-approach. For clarity, we have not included distances above 12 Å.

the steep region. For distances above 12 Å the curve continues with the same limiting trend. Both the N-approach and Papproach interaction energies are important for the implementation of an accurate mesoscopic model of CS/TPP nanoparticles. The interaction energy Eint at each point of the PES scan was calculated using an equation of the general form E int = ECS + TPP − (ECS + E TPP)

(1)

where ECS+TPP, ECS, and ETPP are the energies of the combined system, the CS oligomer, and the TPP polyanion, respectively. The Eint values include corrections for basis-set superposition error (BSSE), which were calculated using the counterpoise (CP) method. However, when a counterpoise correction is applied in combination with a solvent reaction field, any basis functions of the ghost atoms lying outside the reaction-field cavity may artificially increase the outlying charge and thus produce erroneous results. Therefore, we computed the interaction energy within the solvation model, performed the CP method in the gas phase, and corrected the interaction energies with the calculated BSSE corrections. The overall charge of the CS pentamers here was set to 5e, that is, we considered fully protonated CS oligomers. The pKa of chitosan is DD-dependent, with values ranging from 6.4 to 7.1.28 For DD > 75%, the pKa tends to 6.5.29 At pH values well below the pKa, such as the values that we used, most of the amino groups (95%) are protonated, making chitosan water-soluble.30 The maximum interaction energy at the perpendicularconfiguration local minimum in Figure 3b is 49.4 kcal/mol. The interaction energy curve becomes very steep at close range, for RO···N < 3.50 Å. At the shorter distance of RO···N = 2.78 Å, a proton transfer from a CS amino group to TPP reduces the interaction energy by 21.2 kcal/mol to a new value of 28.2 kcal/ mol. The configuration with the global maximum interaction is shown in Figure 3a, which corresponds to a computed maximum interaction energy of 68.3 kcal/mol. At close range 2859

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as well as the increased efficiency of the nanoparticle formation processes at a CS/TPP w/w ratio (α ratio) of 5/1. However, even on the sole basis of spatial arguments, the C2/c group symmetry with a unit cell containing four Na5P3O10 molecules cannot be reproduced using a CS oligomer−P3O10 combination. Also, the optimum α ratio of 5/1 is rather fortuitous, since this is a w/w ratio and not a monomer/TPP ratio. The α ratio, which is commonly used in the literature, does not provide direct insight into the proportions between individual molecular units on an atomistic level. Depending on the DD, a CS oligomer chain is composed of specific content of deacetylated (D-glucosamine) and acetylated (Nacetyl-D-glucosamine) monomers. These two monomers have different molecular weights, which for any given α ratio directly affect the correspondence between CS monomers and TPP polyanions. We introduce the β ratio, which quantifies the correspondence of the CS monomers to the TPP polyanions, as well as the active βi ratio, which takes into account only the deacetylated monomers (i.e., the monomers that participate in the ionic cross-linking), through the following equations:

Figure 5. Cross-linked CS trimers with initially unprotonated TPP. The CS oligomers form dihedral angles of ∼125° about the TPP linking axis.

We denote the general cross-linking configuration shown in Figure 5, either with or without the dihedral bias effect, as an Hlink. This is one of the three primary cross-linking configurations that we define. In view of the high value of the interaction energy as well as the extensive area (on either side of TPP) accessible for the formation of H-links, this is the most probable cross-linking type. In Figure 6a, we provide a simplified schematic representation of an H-link based on the correspondences given in Figure 1. In Figure 6b, we show a schematic of the second primary configuration, which we denote as a T-link. In a T-link, the nonbridging oxygen atoms bonded to the central phosphorus atom of TPP interact with an amino group of CS. The CS−TPP interaction in a T-link configuration is significantly more localized compared with an H-link, making it less significant with a reduced probability to occur. The probability is further reduced since the prerequisite for a T-link is the support of TPP through an H-link or an Mlink. Nevertheless, the corresponding maximum interaction energy is significant, and we computed it to be 48.3 kcal/mol for the case of P3O105− and 13.5 kcal/mol for H4P3O10−. The third and final primary cross-linking configuration, which we denote as an M-link and show in Figure 3a, corresponds to one TPP mounted on the CS oligomer and interacting with two amino groups. Schematically, an M-link can be represented similarly to a T-link with a relative shift by one monomer between TPP and CS. The primary cross-linking configurations that we have introduced do not favor the viewpoint that TPP forms five ionic cross-linking points with CS. This viewpoint may have its origins from the stability of the phase-II crystalline structure of sodium tripolyphosphate, Na5P3O10, at ordinary temperatures

β=α

M TPP MCS

(2)

βi = dβ

(3)

with MCS = dMDG + (1 − d)MNADG

(4)

where d is the DD of CS and MTPP, MDG, and MNADG are the molecular weights of TPP, the deacetylated CS monomer, and the acetylated CS monomer, respectively. In Figure 6c, we show a simplified schematic of possible combinations of H-link and T-link ionic cross-linking configurations. In all three of the linking types shown in Figure 6, there is a high degree of correspondence between CS monomers and TPP polyanions, and thus, these correspond to low β (and α) ratios. The β ratio, which can be adjusted by altering the TPP content, has an impact on the CS/TPP nanoparticle formation process. Our results suggest that at low β ratios, the high concentration of TPP permits the formation of dense H-links, which are less affected by the dihedral bias (shown in Figure 6), with parallel CS chains. Such parallel Hlinked CS chains should exhibit longer lengths as a result of random (parallel) displacements in the linking between chains.

Figure 6. Simplified schematics of the primary ionic cross-linking configurations (a) H-link and (b) T-link. Combinations of primary linking types lead to (c) secondary linking types. In configuration (c), the dotted monomer structures are off-plane and form T-links with the TPP units below them. 2860

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Greece; tel, (+302810) 545055; fax, (+302810) 545001; e-mail, [email protected].

We propose that further cross-linking between such H-linked CS chains leads to the formation of compact H-linked CS nanofibers of longer lengths relative to the average length of the individual CS chains. These long (compared to the average CS chain length) nanofibers construct larger and stiffer nanoparticle cores (which act as the nanoparticle seeds) relative to what would be formed by individual CS chains, with internal areas of localized increased density. At high β (and α) ratios, the dihedral bias deters the formation of parallel CS chains and impels the formation of more irregular nanoparticle cores, which as a result are less stiff and of smaller size. At even higher β ratios, the very low concentration of TPP results in low nanoparticle core densities because of the increased distance between successive H-links, ultimately leading to an increased nanoparticle size.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to acknowledge co-funding of this research by European Union-European Regional Development Fund and Greek Ministry of Εducation/EYDE-ETAK through program ESPA 2007-2013/EPAN II/Action “SYNERGASIA” (project 09SYN-33-484).

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(1) Janes, K. A.; Alonso, M. J. Depolymerized chitosan nanoparticles for protein delivery: Preparation and characterization. J. Appl. Polym. Sci. 2003, 88, 2769−2776. (2) Sun, P.; Li, P.; Li, Y.-M.; Wei, Q.; Tian, L.-H. A pH-sensitive chitosan−tripolyphosphate hydrogel beads for controlled glipizide delivery. J. Biomed. Mater. Res., Part B 2011, 97, 175−183. (3) Vyas, A.; Saraf, S.; Saraf, S. Encapsulation of cyclodextrin complexed simvastatin in chitosan nanocarriers: A novel technique for oral delivery. J. Inclusion Phenom. Macrocyclic Chem. 2010, 66, 251− 259. (4) Zhang, H.; Oh, M.; Allen, C.; Kumacheva, E. Monodisperse chitosan nanoparticles for mucosal drug delivery. Biomacromolecules 2004, 5, 2461−2468. (5) Pati, F.; Adhikari, B.; Dhara, S. Development of chitosan− tripolyphosphate fibers through pH dependent ionotropic gelation. Carbohydr. Res. 2011, 346, 2582−2588. (6) Morris, G. A.; Castile, J.; Smith, A.; Adams, G. G.; Harding, S. E. The effect of prolonged storage at different temperatures on the particle size distribution of tripolyphosphate (TPP)−chitosan nanoparticles. Carbohydr. Polym. 2011, 84, 1430−1434. (7) Yang, H.-C.; Hon, M.-H. The effect of the molecular weight of chitosan nanoparticles and its application on drug delivery. Microchem. J. 2009, 92, 87−91. (8) Papadimitriou, S.; Bikiaris, D.; Avgoustakis, K.; Karavas, E.; Georgarakis, M. Chitosan nanoparticles loaded with dorzolamide and pramipexole. Carbohydr. Polym. 2008, 73, 44−54. (9) Agnihotri, S. A.; Mallikarjuna, N. N.; Aminabhavi, T. M. Recent advances on chitosan-based micro- and nanoparticles in drug delivery. J. Controlled Release 2004, 100, 5−28. (10) Zhang, Y.; Yang, Y.; Tang, K.; Hu, X.; Zou, G. Physicochemical characterization and antioxidant activity of quercetin-loaded chitosan nanoparticles. J. Appl. Polym. Sci. 2008, 107, 891−897. (11) Lu, B.; Xiong, S.-B.; Yang, H.; Yin, X.-D.; Zhao, R.-B. Mitoxantrone-loaded BSA nanospheres and chitosan nanospheres for local injection against breast cancer and its lymph node metastases: I: Formulation and in vitro characterization. Int. J. Pharmaceut. 2006, 307, 168−174. (12) Jafarinejad, S.; Gilani, K.; Moazeni, E.; Ghazi-Khansari, M.; Najafabadi, A. R.; Mohajel, N. Development of chitosan-based nanoparticles for pulmonary delivery of itraconazole as dry powder formulation. Powder Technol. 2012, 222, 65−70. (13) Liu, Z.; Jiao, Y.; Wang, Y.; Zhou, C.; Zhang, Z. Polysaccharidesbased nanoparticles as drug delivery systems. Adv. Drug Delivery Rev. 2008, 60, 1650−1662. (14) Bronich, T. K.; Keifer, P. A.; Shlyakhtenko, L. S.; Kabanov, A. V. Polymer micelle with cross-linked ionic core. J. Am. Chem. Soc. 2005, 127, 8236−8237. (15) Grimme, S. Semiempirical GGA-type density functional constructed with a long-range dispersion correction. J. Comput. Chem. 2006, 27, 1787−1799. (16) Schäfer, A.; Huber, C.; Ahlrichs, R. Fully optimized contracted Gaussian basis sets of triple zeta valence quality for atoms Li to Kr. J. Chem. Phys. 1994, 100, 5829−5836.

4. CONCLUSIONS We have prepared chitosan/TPP nanoparticles with a range of sizes from 340 to 615 nm using the ionic gelation method. We found the smallest nanoparticle sizes at a CS/TPP w/w ratio near 4/1, in agreement with similar work by other groups. Accurate DFT computations with long-range corrections were employed to elucidate and quantify systematically the intermolecular interactions responsible for the ionic crosslinking. We have identified the maximum-interaction relative configurations of TPP and CS oligomers, which we define as the primary ionic cross-linking types H-links, M-links and Tlinks. The corresponding interaction energies were quantified by performing potential energy hypersurface scans, and maximum values of 68.3 and 49.4 kcal/mol were found for M-links and H-links, respectively, using initially unprotonated TPP, P3O105−. Following an adiabatic proton transfer, these values decreased to 28.2 and 34.7 kcal/mol, respectively. For initially protonated TPP, H4P3O10−, we found a maximum interaction energy of 12.3 kcal/mol for H-links. For the T-links, we found interaction energies of 48.3 and 13.5 kcal/mol for P3O105− and H4P3O10−, respectively. On the basis of the high interaction energies and the extensive area accessible for their formation, H-links are the most probable cross-linking types. An examination of two chitosan chains cross-linked with TPP revealed the existence of a torsion bias. We have proposed that this bias affects the initial stages of the nanoparticle formation process and have made a connection to the TPP content. In doing so, we have defined the β ratio and the active (interacting) βi ratio, which take into account structural details of the chitosan chains and as a result are more fundamental quantities than the α ratio commonly used in the literature.

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ASSOCIATED CONTENT

S Supporting Information *

Cartesian coordinates of (constrained or free) maximuminteraction M-link, H-link, and cross-linked configurations and a TEM micrograph of the prepared CS nanoparticles. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*D.N.B.: Laboratory of Organic Chemical Technology, Department of Chemistry, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece; tel/fax, 0030-2310-997812; email, [email protected]. G.E.F.: Department of Chemistry, University of Crete, P.O. Box 1470, Iraklion 714 09, Crete, 2861

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dx.doi.org/10.1021/mp300162j | Mol. Pharmaceutics 2012, 9, 2856−2862