Genipin-Structured Peptide–Polysaccharide Nanoparticles with

Nov 25, 2014 - Genipin-cross-linked caseinophosphopeptide (CPP)–chitosan (CS) nanoparticles (smaller than 300 nm) showed significantly improved stab...
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Genipin-Structured Peptide−Polysaccharide Nanoparticles with Significantly Improved Resistance to Harsh Gastrointestinal Environments and Their Potential for Oral Delivery of Polyphenols Bing Hu,* Minhao Xie, Chen Zhang, and Xiaoxiong Zeng* College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China S Supporting Information *

ABSTRACT: Genipin-cross-linked caseinophosphopeptide (CPP)−chitosan (CS) nanoparticles (smaller than 300 nm) showed significantly improved stability and adjustable release profile in the gastrointestinal (GI) tract. Optimal purification of the nanoparticles was established by centrifugation to terminate the cross-linking reaction, which was further confirmed and characterized by FT-IR. Results from transmission electron microscopy (TEM), dynamic light scattering (DLS), and electrophoretic mobility (ζ-potential) measurements revealed that genipin cross-linking significantly prevented the bursting of the CPP−CS nanoparticles in simulated stomach acid and their precipitation under neutral intestinal environment. Pepsin showed little impact on the nanoparticle colloid stability; however, trypsin induced their aggregations. Genipin cross-linking slowed the burst release of (−)-epigallocatechin-3-gallate (EGCG) from the nanoparticles. The EGCG-loaded nanoparticles showed strong cytotoxicity against cancer cells; meanwhile, the net nanoparticles demonstrated high biocompatibility. The findings in the present work provide fundamental information for the rational design of biopolymer nanoparticles as an effective delivery systems for polyphenols. KEYWORDS: oral delivery, biopolymer nanoparticles, genipin, structure behavior, centrifugal purification, controlled release, EGCG

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therapeutics to increase their bioavailability.8−10 In our recent study, novel caseinophosphopeptide (CPP)−CS nanoparticles were developed, which were highly biocompatible.11 CPPs, a group of anionic polypeptides, are released from the Nterminus polar region during the tryptic digestion of milk casein proteins. Nevertheless, CS-based nanoparticles have certain disadvantages related to their instability in biological fluids such as limited selectivity in their interaction with mucosal surfaces, which include the highly swelling, even easy dissolution, of the matrix in stomach acid, as well as the precipitation at neutral and alkaline pH in intestinal and physiological environments. Genipin (Scheme 1), a natural iridoid compound, is one of the key bioactive ingredients extracted from the fruit of Gardenia jasminoides E. It is a commonly used herbal medicine or functional food supplement in China for its antidepressant,12 anti-inflammatory,13 antibacterial, antithrombotic,14 hepatoprotective, and choleretic effects.15 Genipin is also a natural watersoluble bifunctional cross-linking reagent, which reacts promptly with amines in a polymer chain spontaneously, thus producing a semi-interpenetrating polymer network within the developed hydrogel system.16 According to previous studies, cross-linking the CS- or protein-based hydrogels, films, or microspheres with genipin can enhance their mechanical stability and prevent their swelling ratio in acidic condition.17−19 Importantly, genipin overcomes the problem of physiological toxicity inherent in the use of some common

INTRODUCTION Over the past decade, polymer nanoparticles have attracted significant attention as oral delivery systems for nutraceuticals because of their ability to interact with mucosal surfaces and facilitate the transport of the associated biomolecules across them.1−3 Despite these potential advantages, polymer nanoparticles face several major barriers including the harsh stomach acid, the sharply varying pH values in the gastrointestinal (GI) tract, digestion enzymes, the mucus layer that lines a majority of the GI tract, and the tight junctions of the epithelium.4 Recently, the endocytosis of polymer nanoparticles by the intestinal epithelium1 and their penetration through the intestinal mucus barriers5 have been paid a lot of attention. However, their stability and structural behaviors in the GI tract, as well as the leakage of the loading drugs, are still challenging, and research in this area is in its infancy. The pH environment in the GI tract is indeed complicated. The normal pH range for stomach acid is between 1.0 and 2.5.6 The pH value in the small intestine is 6.0−7.0, whereas the mean pH in the distal ileum and in the body fluid at intercellular spaces between enterocytes is about 7.4.6 This pH variation makes it difficult to maintain nanoparticle integrity throughout the entirety of the GI tract.4 In addition, polymer nanocarriers could be degraded due to the presence of digestion enzymes. Therefore, they must first withstand the harsh pH and digestive enzyme environment in the GI tract, hold the loading drugs, and reach the drug absorption site in the small intestine; otherwise, nanoparticle-based oral delivery will be unachievable.7 Chitosan (CS) and its derivatives are widely used in the fabrication of promising vehicles for oral delivery of © XXXX American Chemical Society

Received: September 27, 2014 Revised: November 3, 2014 Accepted: November 25, 2014

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CS NPs, different amounts of genipin were added to the CPP−CS nanoparticle suspension, forming different final genipin concentrations of 0, 0.031, 0.063, 0.125, 0.250, 0.500, 1.000, and 2.000 mg/mL. The reaction mixture was incubated at 37 °C for different times of 3, 6, 12, and 24 h. The cross-linking reaction of the CPP−CS NPs was terminated by separating the nanoparticles from the residual free genipin in the reaction system using a centrifugal purification method that was optimized in following section. The genipin-cross-linked nanoparticles were washed three times using deionized water, precipitated, and collected. Cross-linking of the EGCG-loaded CPP−CS NPs with genipin was operated using the same procedure as described above. Purification of CPP−CS NPs and Genipin-Cross-Linked CPP− CS NPs. The CPP−CS NPs and the genipin-cross-linked CPP−CS NPs were purified from their parental reaction suspension by centrifugation using an Avanti J-25 high-speed centrifuge (Beckman Coulter, Inc., Brea, CA, USA), with different centrifugal forces (25000, 10000, 7500, 5000, and 2500g) and various duration times (15, 30, 45, and 60 min) to optimize the critical centrifuge parameters, under which the nanoparticles could be just precipitated and then recovered to nanoparticle suspension after redispersion. The redispersion property of the nanoparticle precipitate was preliminarily observed by checking whether there were visible particles in the redispersed colloid solution. The nanoparticles were washed three times using deionized water and redispersed to the same volume as that before centrifugation. The size distribution, surface charge, and surface morphology of the purified nanoparticle suspension and their parental ones were characterized by DLS, zeta-potential measurement, and TEM. Nanoparticle Stability and Structure Behavior in Simulated GI pH and Enzyme Environments. Equal amounts of the precipitates of the CPP−CS NPs and the genipin-cross-linked CPP−CS NPs were first redispersed in pH 1.2 (0.1 N HCl), pH 6.2, and pH 7.4 (10 mM PBS) solutions for 2 h, respectively, simulating the pH environments of the GI tract. The size distribution, surface charge, and morphology of the nanoparticles at these distinct pH conditions were characterized by DLS, zeta-potential measurement, and TEM. The nanoparticles that withstood the GI pH environments were further dispersed in 50 mL of 0.1 N HCl (pH 1.2) containing 0.1% pepsin as the simulated gastric fluid (SGF) and 50 mL of 10 mM PBS (pH 7.4) with 1.0% trypsin as the simulated intestinal fluid (SIF) for 2 h, respectively. The samples after gastric and intestinal digestion were isolated from the SGF and SIF, washed, and redispersed in deionized water for the determinations of size distribution, surface charge, and morphology. Cytotoxicity of the EGCG-Loaded CPP−CS NPs Cross-Linked with Genipin against Cancer Cells. Cytotoxicity was measured using the trypan blue dye exclusion test. Human liver HepG2 cells or human gastric BGC823 cells (100,000) were seeded in 6-well plates, in 2.5 mL of medium per well. After 24 h, supernatants were discarded and replaced by fresh EGCG, CPP−CS NPs cross-linked with genipin, or the EGCG-loaded CPP−CS NPs cross-linked with genipin dilutions in cell culture medium in a range of concentrations from 12.5 to 200 μg/mL (0.0273−0.437 mmol/L) for EGCG and from 12.5 to 200 μg/mL (0.0273−0.437 mmol/L) for the nanoparticles. After 24 h of exposure, supernatants were discarded and cells rinsed twice with PBS. Cells exposed to EGCG, CPP−CS NPs cross-linked with genipin, or the EGCG-loaded CPP−CS NPs cross-linked with genipin were harvested with 1× trypsin. Cells were mixed with an aqueous solution of trypan blue 0.4% in PBS (pH 7.0) according to a method previously described.23 Cell counting was performed using Kova counting chambers (Kova Glasstic Slide, Hycor Biomedical) by microscopy. The cell viability was calculated as the ratio between uncolored cells and total cells. All measurements were performed in triplicate. Statistical Analysis. Data were expressed as the mean ± standard deviation (SD) of at least triplicates. The least significant difference (LSD) test and one-way analysis of variance (ANOVA) were used for multiple comparisons by SPSS 16.0. Difference was considered to be statistically significant at p < 0.05.

Scheme 1. Genipin Reacted with Amino Groups in CS and Peptide To Yield Two Main Cross-Linking Reactions (Taking CS, for Example)

synthetic chemicals as cross-linking agents, such as glutaraldehyde, ethylene glycol, diglycidyl ether, and diisocyanate.20 Herein, the CPP−CS (NPs) were cross-linked with genipin, and the critical centrifuge parameters for purification of the biopolymer nanoparticles were optimized. The morphology, particle size, and surface charge of the nanoparticles before and after the centrifugal purification were characterized using TEM, DLS, and electrophoretic mobility (ζ-potential) measurements. The cross-linking reaction was confirmed and characterized by FT-IR. The structure behaviors of the nanoparticles, including morphology, particle size, and surface charge, as well as the controlled release profile of EGCG, from the nanoparticles under different digestion pH and enzyme conditions were investigated systematically. EGCG is the most abundant and bioactive polyphenol in green tea. However, the bioavailability of EGCG is very low.21 The anticancer activities of the EGCGloaded CPP−CS nanoparticles cross-linked with genipin were investigated by in vitro cell assays.

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MATERIALS AND METHODS

Materials. CS of molecular weight 100 kDa with a 90% deacetylation degree, derived from crab shell, was obtained from Golden-Shell Biochemical Co. Ltd. (Hangzhou, China). CPPs were prepared and identified with HPLC-MS-MS as described in our previous study.22 EGCG (purity > 98%) and trypan blue were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Genipin (purity > 98%) was purchased from Linchuan Zhixin Biotechnology Co., Ltd. (Jiangxi, China). High-purity grade pepsin with activity of 3000−3.5 NF U/mg and trypsin with activity of >250 NF U/mg were purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Dimethyl sulfoxide (DMSO) and acetic acid were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Human gastric BGC823 and liver HepG2 cancer cells were obtained from the Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Wisent Biotechnology (Wisent Inc., Quebec, Canada). All other chemicals were of analytical grade. General Procedure for Preparation of CPP−CS NPs and EGCG-Loaded CPP−CS NPs. CPP−CS NPs were prepared according to our previously reported procedure.22 Briefly, CS was dissolved in 1% (w/v) acetic acid solution with sonication until the solution was transparent. The aqueous solution of CPP was obtained at a suitable concentration. Both CS and CPP solutions were adjusted to pH 6.2 with 1.0 N HCl or NaOH solution. Subsequently, CS solution was added to CPP solution under stirring at room temperature. For the preparation of EGCG-loaded CPP−CS NPs, an aqueous solution of EGCG was added to a CPP solution before the addition of CS solution. The formation of CPP−CS NPs started spontaneously via the CPP-initiated ionic gelation mechanism. Cross-Linking of CPP−CS NPs and EGCG-Loaded CPP−CS NPs with Genipin. For the preparation of genipin-cross-linked CPP− B

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Figure 1. (A) Particle size and surface charge of the genipin cross-linked CPP−CS nanoparticles (NPs) with different concentrations of genipin; (B) schematic plot of the intra-cross-linkage of the CPP−CS NPs.

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RESULTS AND DISCUSSION Cross-Linking the CPP−CS NPs with Genipin. Covalent cross-linking of the CPP−CS NPs with genipin took place spontaneously when genipin was added to the parental suspension. The appearance of blue coloration in the reaction mixture was traditionally considered as rapid evidence that the cross-linking reaction was taking place.17 The cross-linking reaction occurred by means of a complex mechanism.24 When the CPP−CS NPs were submitted to cross-linking with genipin, a nucleophilic attack by the amino groups of CS (peptide) on the olefinic carbon atom at C-3 in genipin occurred, followed by opening the dihydropyran ring and attack by the secondary amino group on the newly formed aldehydo group (Scheme 1). Genipin could form multiple cross-linkings among CS and CPP. In the product, short chains of condensed genipin acted as cross-linking bridges. The cross-linking reaction of the CPP− CS NPs with genipin could be further characterized by FT-IR, which will be presented and discussed later. Theoretically, genipin might cause intra- and inter-crosslinkage of the CPP−CS NPs, leading to changes in particle size and surface charge. Therefore, the effect of different genipin concentrations on the physicochemical properties of the CPP− CS NPs was first investigated. Figure 1A shows the particle size and surface charge of the CPP−CS NPs and the covalently cross-linked CPP−CS NPs with different genipin concentrations. The particle size of CPP−CS NPs before cross-linking was 236.5 ± 26.3 nm (n = 3) with a polydispersity index (PDI) of 0.152 ± 0.032 (n = 3), which indicated a homogeneous dispersion of nanoparticles. Zeta-potential analysis revealed a surface charge of 26.6 ± 1.4 mV (n = 3). After cross-linking with different genipin concentrations at 37 °C for 24 h, no significant change in particle size and surface charge of the nanoparticles could be observed. It could be found that the particle size decreased slightly with the increase of genipin concentrations, which might be related to the tighter crosslinking of the nanoparticles at higher concentration of genipin. The PDIs of the genipin cross-linked CPP−CS NPs were in the range of 0.108−0.204, which also indicated a homogeneous dispersion of nanoparticles. Therefore, it could be concluded that genipin mainly produced intra-cross-linkage of the CPP− CS NPs (Figure 1B), leading to little influence on their physicochemical properties. Purification of Nanoparticles. CS-based polyelectrolyte nanoparticles are some of the most widely utilized engineered nanocarriers in bioimaging and biomedical therapeutics.10,25

However, scarce data on purification of the CS-based nanoparticles from the reaction systems have been reported. Excess free CS, the corresponding negative polyelectrolytes, and the unloaded free cargoes are expected to remain in the product solution and should be removed before use, which otherwise could cause misleading results, such as in fluorescence labeling experiments. Furthermore, purification of the polymer nanoparticles is essential for further modification of nanoparticle surface properties, such as conjugating with functional groups, layer by layer coating with multilayers, and tests of the polymer nanoparticles’ effects on biological tissues. In the present study, separation of the covalently cross-linked CPP−CS NPs from the free genipin, CS, and CPP remaining in the reaction system was of primary importance to terminate the spontaneous cross-linking reaction and to characterize the physicochemical and biological properties of the nanoparticles. Centrifugation is most commonly used to separate nanoparticles, probably because of the easy availability of the instrument.26 This purification method also offers more flexibility to vary the final volume of the treated nanoparticle suspension.27 However, during centrifugation, the polymer nanoparticles are prone to cluster and mesh, leading to precipitation, even a polymer film that can never be redispersed as nanoparticle suspension. Therefore, a critical centrifuge parameter has to be optimized, under which the nanoparticles could just be precipitated and could also be recovered to nanoparticle suspension with similar physicochemical properties as the original ones. We summarized the centrifuge speed and duration employed in various studies to purify the CSbased nanoparticles (Supporting Information, Table S1) and found that the use of different parameters (e.g., rpm vs g-force) in the papers challenged attempts to cross-compare the studies. Even if a similar unit expression was adopted, the centrifuge forces and durations were seemingly selected arbitrarily (Supporting Information, Table S1). To address the above-mentioned issues, in the present study, the critical centrifuge parameters for purification of the CPP− CS NPs and the genipin-cross-linked CPP−CS NPs were optimized with different centrifuge forces (25000, 10000, 7500, 5000, and 2500g) and various centrifugal times (15, 30, 45, and 60 min). The g-force (g) can be widely used and crosscompared, independent of different types of centrifuge equipment. As different genipin concentrations had little impact on the physicochemical properties of the nanoparticles, C

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we first took the cross-linked CPP−CS NPs with a genipin concentration of 0.125 mg/mL as the test sample. The redispersion properties of the nanoparticle precipitate were preliminarily checked whether there were visible particles in the redispersed colloid solution. Eventually, it was found that, under a centrifuge force of 2500g for 45 min, the nanoparticles could be just fully precipitated (Figure 2A) and then redispersed without visible particles (Figure 2B). The purified nanoparticles were washed and centrifuged three times before further characterization.

with free polymers. According to our previous study, it was mainly the excess free CS that was around the nanoparticles, allowing adsorption of free CS onto the surface of the nanoparticles and desorption of CS from the nanoparticles to reach equilibrium.22 In addition, CS aggregates including certain crystal structures (marked with red arrows) could be observed in the background of the TEM images of the nanoparticle sample before centrifugation (Figure 3A,C). Comparatively, after centrifugal purification, in the TEM images of both the CPP−CS NPs (Figure 3B) and the genipin-cross-linked CPP−CS NPs (Figure 3D), the free CS surrounding the nanoparticles and the CS aggregates in the background disappeared, and the background of the images became clear. This phenomenon indicated that the CPP−CS NPs and genipin-cross-linked CPP−CS NPs had been successfully separated from the reaction system by the centrifugal purification method. The purified nanoparticles were regular spheres as well and dispersed homogeneously without aggregations (Figure 3B,D). No significant variations in the nanoparticle structure and morphology can be found by comparing the CPP−CS NPs with the genipin-cross-linked CPP−CS NPs themselves. The particle size and surface charge of the CPP−CS nanoparticle and genipin-cross-linked CPP-CS nanoparticle samples before and after centrifugal purification were further determined by DLS and zeta-potential measurement, which are shown in Figure 4. The mean particle size of the parental CPP−CS NPs and genipin-cross-linked CPP−CS NPs before centrifugation were 269.2 ± 19.6 nm (Figure 4A) and 246.2 ± 17.0 nm (Figure 4C), with PDIs of 0.165 and 0.142, respectively, indicating that the nanoparticles dispersed homogeneously. After centrifugation and redispersion, the particles size of the purified CPP−CS NPs (279.4 ± 21.1 nm) and genipin-cross-linked nanoparticles (245.3 ± 18.3 nm) were almost the same as that of their parental nanoparticles before centrifugation. This result was in accordance with the TEM result (Figure 3). After centrifugation, the surface charge of both the CPP−CS NPs and the genipin-cross-linked CPP−CS NPs increased from 20.3 ± 1.5 to 25.9 ± 3.8 mV and from 24.3 ± 3.5 to 32.4 ± 6.1 mV, respectively. This phenomenon might be related to the removal of the shielding effect caused by the excess free CS around the nanoparticles (Figure 3). Therefore, operation at 2500g for 45 min was the critical centrifuge parameter for purification of the CPP−CS NPs and the genipin-cross-linked CPP−CS NPs for terminating the cross-linking reaction and for further separating the nanoparticles from the simulated GI fluids. The nanoparticles themselves maintained their structure and physicochemical properties after centrifugal purification. The CPP−CS NPs and the genipin-cross-linked CPP−CS NPs with different genipin concentrations (0.063, 0.125, and 0.250 mg/mL) and various reaction times (3, 6, 12, and 24 h) were purified by the centrifuge method and lyophilized for further FT-IR analysis. FT-IR Analysis. To characterize the cross-linking reaction, we acquired the FT-IR spectra of the purified CPP−CS NPs and genipin-cross-linked nanoparticles with different genipin concentrations and reaction times (Figure 5). In Figure 5A, the covalently cross-linked CPP−CS NPs were obtained after cross-linking reaction for 3 h with different genipin concentrations of 0.063, 0.125, and 0.250 mg/mL. In Figure 5B, the genipin cross-linked nanoparticles with a fixed genipin concentration of 0.125 mg/mL were purified at different reaction times of 3, 6, 12, and 24 h. The main characteristic

Figure 2. Centrifugation and redispersion of the genipin cross-linked CPP−CS NPs operating at a centrifuge force of 2500g and duration of 45 min (genipin concentration = 0.125 mg/mL).

Figure 3 shows the structure and morphology of the CPP− CS nanoparticle and the genipin-cross-linked CPP−CS nano-

Figure 3. TEM images of CPP−CS NPs (A, B) and genipin-crosslinked CPP−CS NPs (C, D) before (A, C) and after (B, D) purification with centrifugation at a centrifuge force of 2500g and duration of 45 min (genipin concentration = 0.125 mg/mL).

particle samples before and after centrifugal purification at 2500g for 45 min, which were characterized using TEM. It can be seen that the parental CPP−CS NPs (Figure 3A) and genipin cross-linked CPP−CS NPs (Figure 3C) were regularly spherical in shape, dispersed homogeneously, and surrounded D

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Figure 4. Particle size distribution of the CPP−CS NPs (A, B) and the genipin cross-linked CPP−CS NPs (C, D) before and after centrifugal purification at a centrifuge force of 2500g and duration of 45 min (genipin concentration = 0.125 mg/mL).

absorption bands of the CPP−CS NPs appeared at 1625, 1518, and 1150−1040 cm−1, which corresponded to amide I and amide II groups and glycosidic linkage (COC), respectively.28 The amide I bands originated from CO stretching vibrations coupled to NH bending vibration. The amide II bands arise from the NH bending vibrations coupled to CN stretching vibrations. After cross-linking with genipin, the characteristic absorption band of amide I at 1625.15 cm−1 shifted to 1636.80 cm−1 with the increase of genipin concentration from 0.063 to 0.250 mg/mL, indicating the formation of secondary amides as a result of the reaction between the ester groups on genipin and the amino groups on CS.29 A similar phenomenon also appeared in Figure 5B; the absorption band of amide I at 1624.72 cm−1 shifted to 1634.92 cm−1 with the increase of reaction time from 3 to 24 h. Furthermore, in both panels A and B of Figure 5, two new very weak absorptions at 1719 and 1700 cm−1 appeared after crosslinking with genipin, and the intensity of these two absorptions increased with the elevation of the genipin concentration and prolonged reaction time. The appearance and increased absorption of the peak at 1719 cm−1 corresponded to the formation of aldehydo that was the reaction product between the carboxymethyl group of genipin and the amino groups on the surface of CPP−CS NPs30 (insets of Figure 5). The absorption at lower wavenumber (1700 cm−1) represented the aldehydo that formed intermolecular hydrogen bonding with other groups. After cross-linking with genipin, there were still many amino groups available on the surface of the CPP−CS NPs, which could be used as reaction sites for further surface modification. Stability of Nanoparticles under Simulated GI pH Conditions. To study the stability of nanoparticles under simulated GI pH conditions, equal amounts of the purified CPP−CS nanoparticle and genipin-cross-linked CPP−CS nanoparticle precipitates were redispersed in pH 1.2 (0.1 N HCl), pH 6.2, and pH 7.4 buffers (PBS), respectively, for 2 h. Then, the samples in different pH conditions were submitted to characterization of particle size and morphology using DLS and TEM. The particle size and PDI of the CPP−CS NPs and the genipin-cross-linked CPP−CS NPs in different pH conditions

are summarized in Table 1. It can be seen that, under pH 6.2, the CPP−CS NPs and the genipin-cross-linked CPP−CS NPs (reaction for 3 or 24 h) had similar particle sizes with PDI ranging from 0.095 to 0.189. After dispersion in 0.1 N HCl for 2 h, stimulating the pH environment in the stomach, the average count rate (ACR) of the CPP−CS nanoparticle suspension under laser decreased sharply from 342.8 kcps (pH 6.2) to 3.6 kcps (pH 1.2), indicating the disappearance of the nanoparticles under pH 1.2. ACR is a parameter that is influenced by the number and size of the particles in a suspension. In the experimental condition used in the present study, the detected particle size values were legal and reasonable only when the ACR value was in the range of 100−500 kcps. Therefore, under an ACR of 3.6 kcps, the detected particle size was illegal, which was recorded as “nd” in Table 1. Dispersed in pH 7.4 PBS buffer for 2 h, the particle size of the CPP−CS NPs was 310.6 ± 24.9 nm, which was larger than that under pH 6.2. Comparatively, after crosslinking with genipin (reaction for both 3 and 24 h), the CPP− CS NPs became very stable with just slight changes in particles size and PDI values under different simulating GI fluid pH values (pH 1.2, 6.2, and 7.4). The morphology and structure of the samples under different pH conditions were further investigated by TEM. Figure 6 shows the structure and morphology of the CPP− CS NPs and the corresponding genipin cross-linked CPP−CS NPs (reaction for 3 h) after they were dispersed in pH 1.2, 6.2, and 7.4 buffers for 2 h, respectivley. Both the CPP−CS NPs (Figure 6B) and the genipin-cross-linked nanoparticles (Figure 6B′) were regular spheres and dispersed well under pH 6.2. Under pH 1.2 (Figure 6A), it can be seen that the CPP−CS NPs were dissoved with cloudy morphology and the release of the inner composition peptides (black dots marked with arrows), which was consistent with the DLS result of the sharp decrease of ACR value. This phenomenon was caused by the strong repusion among the highly protonated amino groups in CS under strong acidic condition, leading to the burst of the CPP−CS NPs. On the other hand, under pH 7.4, the CS capping on the surface of the nanoparticles seemed to precipitate in the insoluble state, which could be seen clearly E

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Figure 5. FT-IR spectra of the purified genipin cross-linked nanoparticles with different genipin concentrations (A) and reaction times (B).

Table 1. Effects of pH on Particle Size and Polydispersity Index (PDI) Values of the CPP−CS NPs and the Genipin-CrossLinked CPP−CS NPsa G-CPP−CS NPs for 3 hb

CPP−CS NPs

G-CPP−CS NPs for 24 hc

pH value

particle size (nm)

PDI

particle size (nm)

PDI

particle size (nm)

PDI

1.2 6.2 7.4

nd 269.4 ± 15.2 310.6 ± 24.9

nd 0.095 0.174

289.4 ± 18.2 266.4 ± 13.3 269.4 ± 16.6

0.099 0.189 0.143

276.1 ± 8.5 268.1 ± 18.9 266.9 ± 16.9

0.141 0.143 0.129

Genipin concentration was 0.125 mg/mL. Values are expressed as the mean ± standard deviation (n = 3). bThe parental CPP−CS nanoparticle suspension was incubated with genipin for 3 h. Then, the cross-linked NPs were purified by the centrifugal purification method. cThe parental CPP− CS nanoparticle suspension was incubated with genipin for 24 h. Then, the cross-linked NPs were purified by the centrifugal purification method. a

changes on structure and morphology of the CPP−CS NPs could be observed after they were dispersed in pH 1.2, 6.2, and 7.4 buffers for 2 h, respectively (Figure 6A′,B′,C′), which was consistent with the DLS results in Table 2. This meant that cross-linking with genipin, a simple and green method, could significantly enhance the stability of the CPP−CS NPs and successfully extend their effective scope in GI fluid, which was

in the inset image of Figure 6C. A similar result was also reported by Tang et al.31 As the pKa value of the amine groups on CS is approximately pH 6.5, CS precipitates at neutral pH due to deprotonation of amine groups. This property of CS suggests that the CS NPs can be effective as a mucoadhesive agent and an absorption enhancer only in a limited area of the intestinal lumen where the pH values are below or close to its pKa. In contrast, after cross-linking with genipin, no significant F

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Figure 6. TEM images of the CPP−CS NPs (A, B, C) and genipin cross-linked CPP−CS NPs (A′, B′, C′) under different simulated GI pH environments.

digestion enzymes on the physicochemical properties of the polymer nanoparticles, equal amounts of the purified genipincross-linked CPP−CS NPs were incubated in SGF and SIF for 2 h, respectively. After incubation for 30 and 120 min, the nanoparticle samples were isolated from the SGF and SIF according to the centrifugal purification method as mentioned above. Their structure were characterized by TEM, DLS, and zeta-potential measurement. After incubation with SGF for either 30 or 120 min, no significant changes could be observed in the particle size, PDI value, and zeta potential of the genipincross-linked CPP−CS NPs compared with those of the original ones (Table 2). It can be seen from Figure 7A,B that the nanoparticles remained stable and dispersed well after incubation with the SGF for either 30 and 120 min, which was consistent with the DLS results. In fact, the stability of nanocarriers in gastic fluid has attracted a lot of attention in the rational design of oral delivery nanodevices. It was found that the negatively charged nanodevices, such as poly(lactic acid) (PLA) nanoparticle and 32 β-lactoglobulin (β-lg)-stabilized emulsions,33,34 underwent aggregation or flocculation in gastric conditions, which were mainly caused by neutralization of their surface charge. Comparatively, in the present study, the genipin-cross-linked CPP−CS NPs were stable and well dispersed in SGF, which

Table 2. Effects of Digestion Enzymes on Particle Size, Polydispersity index (PDI) Value, and Surface Charge (Zeta Potential) of the Genipin Cross-Linked CPP−CS NPs (GCPP−CS NPs)a sample G-CPP−CS NPs G-CPP−CS NPs incubated with SGF for 30 min G-CPP−CS NPs incubated with SGF for 120 min G-CPP−CS NPs incubated with SIF for 30 min G-CPP−CS NPs incubated with SIF for 120 min

mean particle size (nm)

PDI

zeta potential (mV)

245.3 ± 18.3 262.7 ± 25.1

0.125 0.097

32.4 ± 6.1 29.6 ± 11.5

251.8 ± 31.3

0.176

32.6 ± 7.5

523.8 ± 62.9

0.104

34.4 ± 5.6

1388.9 ± 152.1

0.285

32.1 ± 5.3

a

Genipin concentration was 0.125 mg/mL. Values are expressed as mean ± standard deviation (n = 3).

the prerequirement for intestinal mucosal delivery of therapeutics. Structure of Nanoparticles under Simulated GI Enzyme Conditions. Despite the genipin-cross-linked CPP−CS NPs were able to maintain their stability under the GI pH environments, the presence of digestion enzymes was another big challege for them. To investigate the effects of

Figure 7. TEM images of the genipin-cross-linked CPP−CS NPs under simulated gastric fluid for 30 min (A) and 120 min (B) and under simulated intestinal fluid for 30 min (C) and 120 min (D). G

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should be mainly attributed to their highly positive surface charge. It could be concluded that pepsin in the stomach had little impact on the colloid structure and physicochemical properties of the genipin-cross-linked CPP−CS NPs. After incubation with the SIF for 30 min, the particle size increased from 245.3 ± 18.3 to 523.8 ± 62.9 nm and further sharply to 1388.9 ± 152.1 nm when the incubation time was prolonged to 120 min (Table 2). Meanwhile, the PDI of the nanoparticle suspension increased to 0.285 (Table 2) with the incubation time of 120 min, which indicated the increased heterogeneity of the nanoparticle suspension. Observed with TEM (Figure 7C), it could be seen that aggregations of the genipin-cross-linked CPP−CS NPs appeared after the nanoparticles were incubated with SIF for 30 min. The degree of aggregation increased with prolonged incubation time. When the nanoparticles were incubated with SIF for 120 min, much bigger clusters of the nanoparticles could be seen (Figure 7D), compared with those with an incubation time of 30 min (Figure 7C). The nanoparticles inside the aggregations/clusters were still integral and compact. Therefore, it could be concluded that the increase of particle size was mainly caused by the aggregation of the nanopaticles, rather than the swelling of the nanoparticles. Similar results were also reported by Tobı ́o et al., that PLA nanoparticles interacted with the digestive enzymes, thus leading to their agglomeration in the incubation medium.35 In Vitro Release Profile of EGCG from the CPP−CS NPs and the Genipin-Cross-Linked CPP−CS NPs in Simulated GI Environments. The encapsulation efficiencies of EGCG in the CPP−CS NPs and genipin-cross-linked CPP−CS NPs were around 71% in the present stduy, which was consistent with our previous result.21 The in vitro release of EGCG from the CPP− CS NPs and the genipin-cross-linked CPP−CS NPs in simulated gastric and intestinal environments is shown in Figure 8. Compared with the non-cross-linked nanoparticles, cross-linking the CPP−CS NPs with genipin significantly (p < 0.05) reduced the burst release or leakage of EGCG from the CPP−CS NPs either in simulated gastric or in intestinal environments. It should be related to the significantly enhanced structure stability of the nanoparticles in simulated gastric and intestinal environments after cross-linking with genipin (Figure 6). Furthermore, for the genipin-cross-linked CPP−CS NPs, the release of EGCG from the nanoparticles slowed significantly (p < 0.05) to a much more controllable manner with the increase of the cross-linking degree. This meant that the controlled release profile of the EGCG from the nanoparticles could be modulated effectively through changing the genipin cross-linking degree. For the genipin-cross-linked nanoparticles, the release rate of EGCG in the simulated intestinal environment was higher than that in the gastric environment, which should be caused by the instability and aggregation of the nanoparticles under intestinal conditions (Figure 7). This release profile was especially desirable for oral delivery systems. In Figure 8B, the decrease of the measured release rate after 120 min was mainly caused by the instability and degradation of the EGCG under neutral and alkaline environments,36,37 which also indicated it was necessary to encapsulate EGCG in polymer nanoparticles. Preventing the burst release of loading drugs, especially small molecular and hydrophilic drugs, from the delivery systems in the GI tract is important. The results in the present study indicated that the release profile of the payloads from the genipin-cross-linked CPP−CS NPs could be adjusted through changing the genipin

Figure 8. Release profile of EGCG from the CPP−CS NPs and the genipin-cross-linked CPP−CS NPs in simulated gastric (A) and intestinal (B) environments.

cross-linking degree, which would favor the controlled release of the payloads at the sites of their action. Anticancer Activities of the EGCG-Loaded CPP−CS NPs Cross-Linked with Genipin. The cytotoxicity of the EGCG-loaded CPP−CS NPs cross-linked with genipin against gastrointestinal cancer cells (human gastric BGC823 cells) and human liver HepG2 cells was investigated. In our previous studies, it had been found that the CPP−CS NPs could significantly enhance the intestinal absorption of EGCG in an in vitro cell assay, and the uptake of the nanoparticles played an important role in this process.12 Generally, the oral drugs and delivery systems would first transport to the liver. To evaluate the anticancer potential of the EGCG-loaded CPP−CS NPs cross-linked with genipin, we conducted a trypan blue assay to determine gastric cell and live cell density. The viable cells could exclude the trypan blue dye, which could not be stained. The trypan blue assay showed that the EGCG-loaded CPP−CS NPs cross-linked with genipin exhibited cell growth inhibition similar to that of EGCG (Figure 9). The naked CPP−CS NPs cross-linked with genipin did not show cytotoxicity against the cells. In BGC823 cancer cells, the IC50 values of the EGCGloaded CPP−CS NPs cross-linked with genipin and free EGCG were 0.107 and 0.123 mmol/L (calculated in terms of EGCG concentration), respectively, which in HepG2 cells were 0.208 H

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the biopolymer nanoparticles under simulated GI conditions were thoroughly characterized, providing basic information for the rational design of oral delivery nanoparticles. In addition, the established centrifugal purification method will provide technical reference for further purification of other polymer nanoparticles. The EGCG-loaded CPP−CS NPs cross-linked with genipin demonstrated significant anticancer activities, similar to that of free EGCG. Therefore, the novel polymer nanoparticles showed promise as an efficient oral delivery vehicle of polyphenols.

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ASSOCIATED CONTENT

* Supporting Information S

Methods for characterization of nanoparticles and determination of encapsulation efficiency and in vitro release profile of EGCG.; summary of the centrifuge parameters used to prepare chitosan-based nanoparticles in previous studies. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(B.H.) E-mail: [email protected]. Phone: (+86)13915983201. *(X.Z.) E-mail: [email protected]. Phone: (+86)-2584396791. Fax: (+86)-25-84396791. Funding

This work was supported by The Natural Science Foundation of Jiangsu Province, China (BK2012367), Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Specialized Research Fund for the Doctoral Program of Higher Education of China (20120097120001), and an Undergraduate Student Research Trainning (SRT) program from Nanjing Agricultural University (1318A05). Figure 9. Cell viability of the human gastric BGC24 (A) and liver HepG2 (B) cancer cells incubated with the CPP−CS NPs cross-linked with genipin (G-CPP−CS NPs), EGCG, and EGCG-loaded CPP−CS NPs cross-linked with genipin (EGCG loading G-CPP−CS NPs).

Notes

The authors declare no competing financial interest.

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REFERENCES

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