The Assembly Mechanism Of Chitosan Based Nanoparticles As Delivery System For Tea Catechins For Enhancing Its Bioavailability

Chemoprevention, especially through the use of dietary phytochemicals capable of interfering with one or more stages of carcinogenesis process, is a promising approach for cancer management. Among the phytochemicals, tea catechins are a well-studied group of chemopreventive agents and have shown remarkable chemopreventive potential in a wide range of cell culture and preclinical studies, which are ideal candidates for preparing nanochemoprevention. Recently, encapsulation of phytochemicals using nanoparticle-mediated delivery systems has been approved as an effective strategy to enhance the outcome of chemoprevention, which is denominated as nanochemoprevention. However, most of these synthetic nanoparticle materials are much more suitable for parenteral injections rather than oral consumption. In fact, oral consumption is the most desirable and acceptable form of delivery of bioactive dietary phytochemicals. In this manner of consumption, the low bioavailability of dietary phytochemicals, such as EGCG is primarily caused by their poor small intestinal absorption. Therefore, design of nanoparticle materials that are suitable for oral consumption is very important for further development of nanochemoprevention. In the purpose of oral consumption and minimizing carrier-induced undesirable cytotoxicity, we believe that no better are food-grade polymers suitable in developing such delivery system.In present study, a new HPLC method was developed for simultaneous determination of major active components in green tea including the methylated derivatives of tea catechins, which provided a rapid, sensitive, repeatable and practical analysis method for further research assays of determining the encapsulation efficiency, in vitro release profile as well as penetration and absorption of nano-encapsulated EGCG. Food grade CS with the property of enhancing intestinal absorption was employed as the major material to prepare nanoparticles for encapsulation of tea catechins. Firstly, tea catechins were encapsulated in chitosan-tripolyphosphate (CS-TPP) nanoparticles which was used to be carriers for macromolecular protein drugs. Then, in order to overcome the shortcomings of CS-TPP nanoparticles in encapsulation of tea catechins such as low encapsulation efficiency and quick burst release of tea catechins, we introduced bioactive caseinophosphopeptides (CPP) rather than TPP in preparation of CS nanoparticles. Thermodynamic properties associated with complex formation including surface charge, particle size, complex morphology and binding constant were measured. An interaction model was proposed to explain the physical chemistry insights of the structure of the novel nanocomplexes formed. CPP were applied to crosslink CS forming mono-dispersed nanoparticles and bind with EGCG for trapping it inside the nanoparticles. Cytotoxicity of nanoparticles, cellular uptake fate, bioactivity and intestinal absorption of nano-encapsulated EGCG were determined by cell assays.The primary research results include:1. A new HPLC method was developed for simultaneous determination of fourteen components in tea including gallic acid, four major tea catechins, four of their epimers, two methylated catechins, and three purine alkaloids within 15 min total analysis time. This method is sensitive, reproducible, and represents a 2.5-7 fold reduction (15 min, as opposed to 40-105 min) in HPLC analysis time from existing analytical methods for analysis of purine alkaloids, GA, tea catechins including their epimers and O-methylated derivatives.2. CS-TPP nanoparticles with a mean diameter of 128.5 nm in a narrow size distribution (PDI 0.185) were obtained. The zeta potential of the CS-TPP nanoparticles was about+42.5 mV. Based on TEM images, the CS-TPP nanoparticles were regular spherical in shape. The particle size and surface charge of CS-TPP nanoparticles could be controlled by changing the fabrication conditions. The encapsulation efficiency of tea catechins in the CS-TPP nanoparticles were in the range of 25.84-47.37%. The controlled release of tea catechins using CS-TPP nanoparticles was achievable, however, with a quite quick burst release ratio in the range of 50-60% at the initial 12h.3. The water soluble negatively charged peptides from casein were identified as caseinophosphopeptides (CPPs) with different amount of clusters of phosphorylated seryl residues by HPLC-MS-MS results. At low CS/CPPs mass ratio, negatively charged CPPs easily bound to and cross-linked the positively charged CS molecules partially neutralized the CS chains by forming negatively charged intrapolymer nanocomplex saturated with CPPs (CPPNPs). Subsequently, the sharp increase in the particle size with increasing CS/CPPs mass ratio might be contributed to the bridging of the nearly-neutralized CPPNPs and the formation of associative interpolymer complexes. Further increase of CS concentration caused the formation of positively charged CS-CPPs nanocomplexes, and the electrostatic repulsion resulted in the breakdown of the bridges within the associative complexes and the formation of isolated, positively charged spherical CS/CPPs nanocomplexes. Furthermore, the electrostatic repulsion between the positively charged CS/CPPs nanocomplexes and excess amount of free CS allowed the adsorption of free CS to the surface of nanocomplexes and the desorption of CS from the nanocomplexes to reach equilibrium, causing the particle sizes (115nm) and surface charges (+29.4mV) of nanocomplexes to maintain at steady values. The interactions between the peptides and CS were mainly driven by electrostatic interactions with the binding constant Kcs=4.6×104M-1. Hydrophobic driven also occurred during the interaction between CS and CPPs. The phosphorylated groups, Asp and Glu in the CPPs might be the dominant sites for interaction with-NH3+ on the CS molecular chains. The effect of the peptideCS interaction on the peptide conformation is an important question. Our preliminary circular dichroism (CD) results indicate that the content of P-sheet increased from 17% to 40% upon binding to chitosan. This suggests that peptide unfolding might occur during the chitosanpeptide interaction process.4.The particle size of the CS-CPPs nanoparticles loaded with EGCG was 150±4.3 nm (n= 3) with surface charge of 32.2±3.3 mV (n= 3). The PDIs of the suspensions ranged from 0.05 to 0.14, indicating a homogeneous dispersion of nanoparticles. QCMD results indicated the rigid binding among the CS, EGCG and CPPs molecules. And the binding amount of CPPs increased with EGCG concentration. The binding between CPPs and EGCG provided direct encapsulation and entrapping drive for keeping EGCG inside the nanoparticles. The encapsulation efficiency of EGCG with CS-CPP nanoparticles (70.5-81.7%) was about one hundred percent higher than that with CS-TPP nanoparticle (25.8-47.4%). Compared with the release profile of EGCG in previous CS-TPP nanoparticles (around 45-60% at 12 h), the burst release of EGCG in CS-CPP nanoparticles (around 23-40% at 12 h) was slowed down in a much more controllable manner.5. Introduction of CPPs decreased the cytotoxicity of CS nanoparticles significantly (p < 0.05). Nano-encapsulated EGCG entered HepG2 cancer cells to exert significantly (p< 0.05) stronger antioxidant activity. The process of nanoparticle uptake was dose and time dependent in the studied time and concentration range. The intestinal permeability of EGCG was enhanced significantly as delivered by the nanoparticles from 3.50×10-7 cm/s to 1.34 ×10-6 cm/s. Controlled intestinal absorption of EGCG at high rates was successively achieved after encapsulated by the nanoparticles. Therefore, CS-CPPs nanoparticles are efficient carriers for enhancing the bioavailability of EGCG.