1. Chitosan/B-lactoglobulin core-shell nanoparticles as nutraceutical carriers Presenter: Jeong Youngjin

2. Tries to develop innovative functional foods that may have physiological benefits or reduce the risks of diseases to improve public health. The incorporation of bioactive compounds—such as peptide, vitamin—into food systems as a potentially simple means of modulating the risks of diseases. However, The effectiveness of such products relies on preserving the bioavailability of the active ingredients. remain poorly available by oral administration due to too short gastric residence time of the dosage form; low permeability and /or solubility within the gut; lack of stability in the environmental conditions encountered in food processes (temperature, oxygen, light) or in the gastro-intestinal tract (pH, enzymes, presence of other nutrients), which limits their activity and potential health benefits. Encapsulation systems can be used to overcome these limitations. The growing interest in the effective and selective delivery of bioactive agents to the site of action has led to the development of new encapsulation materials. Background

3. Chitosan (CS), a copolymer derived from chitin, is a natural nontoxic biopolymer derived from fully or partially deacetylation of chitin, which is structural element in the exoskeleton of crustaceans(crabs, shrimp, etc). CS has biocompatible, biodegradable and antimicrobial properties. CS is allow to soluble at low pH(6.5) aqueous solution and tends to be positively charged, thus can be combined with negatively charged TPP These properties make CS a good candidate for the development of nutraceutical delivery systems for food applications. However, for oral administration, CS matrix are not stable at low pH values, and rapid dissociation and degradation occurred at pH 1.0, which could lead to destruction of sensitive nutraceuticals in the stomach circumstances. To overcome the drawbacks, the CS nanoparticles could be surface coated to enable the protection in the gastric tract. Chitosan

4. B-lactoglobulin, the major wheyprotein in the milk, is a small globular protein widely used as food ingredient because of its nutritional value and its ability to form gels, emulsions, and gelled emulsion. Important functional property of whey proteins -Ability to form cold induced gel matrices by adding cations to a preheated denatured protein suspension, which results in the formation of a network crosslinked via Ca 2+ with carboxylate groups on denatured Blg at ambient temperature. -It is known to be resistant to degradation of the pepsin in the stomach in its native structure or adsorbed at an interface. Hydrolysis of whey proteins by pancreatin enzymes generate bioactive peptides that may exert a number of physiological effects in vivo, e.g. on the gastrointestinal, cardiovascular, endocrine, immune and nervous systems. Due to its attractive techno-functional properties and large availability Blg is an interesting candidate to coat CS nanoparticle to allow a protection when subject to the gastric fluids. B-lactoglobulin (Blg)

5. Preparation of native and denatured blg solutions Formation of CS-Blg nanoparticles 1. Native or denatured Blg solutions at various concentrations and pH values were added to CS solutions in aqueous acetic acid (0.1%) to form CS-Blg complexes with CS concentration of 2.0 mg/ml. 2. Then TPP was dissolved in distilled water at 1.0 mg/ml. Finally, 2 ml of TPP solution was added dropwise to 5ml of CS-Blg complexes, opalescent suspension was formed spontaneously under magnetic stirring at room temperature, and was further examined as nanoparticles. 3. The final pH of the nanoparticle suspension was measured with an Orion 370 pH meter (Orion Research, Inc. MA), and the nanoparticles were characterized immediately. All experiments were performed in triplicates Agitation at room temperature for 1h Hydrating Blg in deionized water Heat at 80 ℃ and 2.4 for 30 min Negligence for 2 h Native Blg Denatured Blg

6. Influence of the pH value on the nanoparticle properties Measurements of particle size and zeta potential of the nanoparticles were performed with photon correlation spectroscopy and laser Doppler Anemometry, using a Mastersizer 2000 and Zetasizer 2000, respectively (Malvern Instruments, South borough, MA). The size measurements were performed at 25 ℃ and at a 90 ℃ scattering angle. It was recorded for 180 s for each measurement. The mean hydrodynamic diameter was generated by cumulative analysis. The zeta potential measurements were performed using an aqueous dip cell in the automatic mode. At low pH value (pH<3), majority of the amino groups (over 90%) are protonated to form an extended molecular chain in the acid solution due to strong repulsion existing among positively charged amino groups. Therefore a more extended spherical shape is formed upon addition of the TPP solution. While with increase of the pH value (pH 3–9), positive charges would be neutralized with gradual deprotonation of the amino groups, resulting in a less extended molecular chain of CS to form uniform nanoparticles with small size. 이온과 입자가 안정하게 존재하는 이론적인 경계. 제타포텐셜의 크기는 coloidal 시스템의 포텐셜적인 안정성을 나타냄. Suspension 에 있는 모든 입자가 큰 음전하 혹은 양전하의 제타전위를 가지고 있을 때 서로 많이 반발하는 경향을 가지게 되고 이들은 서로 결합 하려 하지 않는다. 그러나 낮은 제타전위를 가지 게 될 경우 입자들이 서로 반발하는 힘이 줄어 들게 되고 결국 응집이 일어나게 된다. 일반적으로 안정하거나 불안정한 suspension 을 나 누는 기준은 +30 or – 30 mV 이다.

7. CS-Blg nanoparticles TEM photographs (Fig. 1(a)) show Surface and interior morphology of CS-Blg nanoparticles prepared with native Blg at pH 6.1 with C Blg of 2.0 mg/ml. Fig. 1(b) show the particle size about 100 nm, indicating that nanoparticles are formed—they appear spherical in shape with smooth surfaces. The interior structure of CS-Blg nanoparticles demonstrates a circular shape consisting of a dark core and a light shell. Fig. 1. Surface and interior morphologyof CS-blg nanoparticles prepared with native blg at pH 6.1 with Cblg of 2.0 mg/ml.

8. To determine the association efficiency(AE) and loading efficiency(LE) of Blg on the nanoparticles, triplicate batches of nanoparticles were centrifuged at 30,000g (RC5C Sovall Instruments Dupont, Newton, Conn.), 20 ℃ for 30 min, and the Blg content in the supernatant was determined by UV spectrophotometry (HP 8453 UV–Visible, spectrophotometer, Palo alto, CA) at 280 nm. The pellet was vacum dried and weighted. The AE and LE values were calculated by the following formulae: Blg coating properties

9. The effects of pH values on AE and LE of Blg on CS-TPP core are shown in Fig. 2. Three regions with two transition points at pH 4.3 and 5.9 in the pH range used were observed for both native and denatured Blg. -pH value < 4.3 A small amount of blg were coated on CS-TPP core. Both CS and Blg are positively charged, strong repulsion prevents association of Blg on CS-TPP core. -4.3 < pH value < 5.9 pH value was higher than 4.3, AE values for both native and denatured blg began to increase steadily The pI of Blg exists, intraionic attractions between COO- and NH3 + result in seldom residual ionic groups on Blg. In this pH range, hydrophobic interactions and hydrogen bondings between Blg and CS are supposed to dominate to explain the steadily increase of the AE value -pH value > 5.9 AE value increased sharply to a maximum of 42.8% for native blg at pH 6.1 and 63.4% for denatured blg at pH 7.1. CS is positivelyc harged, while Blg becomes negatively charged, the driving force for Blg association thus changed from hydrophobic interactions gradually to electrostatic attractions Influence of the pH value on the nanoparticle properties 42.8% at pH 6.1 ↓ 63.4% at pH 7.1 ↓ 4.3 5.3 5.9

10. The LE values which measure the amount of Blg loaded on unit weight of nanoparticles are also strongly pH dependent and showed similar changing tendency of AE values with two transition points as a function of pH, as demonstrated in Fig. 2(b), Confirming change of interactions between CS and Blg at different pH values. However, the maximum LE values were obtained at pH 6.1 for native Blg and pH 6.5 for denatured blg, and with further increase of the pH, LE values for both native and denatured Blg decreased. A reasonable explanation for this decrease could be conformation change of the absorbed Blg. When denatured by heating, Blg molecular chains tend to be random coils. Near pI, while with the remaining hydrophobic portion of the molecule lying close to the surface. Thus steric hindrance between proteins prevents further adsorption of the denatured Blg. Therefore, one unit weight of CS-nano allows more adsorption of the native Blg, which leads to a relatively high LE value The LE value of native Blg loaded on unit weight of CS-TPP core are much higher than those of denatured Blg in all pH values investigated in this study, as obviously shown in Fig 2(b). Influence of the pH value on the nanoparticle properties 4.3 5.3 5.9 pH 6.1 ↓ pH 6.5 ↓

11. Table 2 depicts the effects of initial Blg concentrations (C Blg ) on nanoparticle properties at various pH values. No obvious differences in the mean diameter, polydispersity and zeta potential among CS-Blg nanoparticles prepared with different C Blg at any specified pH value were observed, indicating that Cblg does not play an important role in regulating particle size, size distribution and surface charges of the CS-blg nanoparticles. Influence of Blg initial concentration on the nanoparticle properties Native Blg Denatured Blg

12. In order to investigate the feasibility of CS-Blg nanoparticles as carriers for nutraceuticals. In vitro release properties of BB into simulated gastric-intestinal tract were evaluated for CS Blg nanoparticles formed with a C Blg of 2.0 mg/ml at pH 6.1 for native Blg and pH 6.5 for denatured Blg (Labeled as nanonative and nanodenatured). Nanoparticles prepared with denatured Blg was also crosslinked with CaCl 2 (10 mmol/l) over night to form a network with formation between carboxylate groups of denatured Blg and Ca 2 + (Labeled as nanocrosslinked). This work was intended to obtain a optimal shell structure of the nanoparticles. Brilliant blue (BB) was used as model molecules. Since BB is a small molecule with high anionic charge densities, strong electrostatic interactions between BB and amino groups of CS played a very important role in BB encapsulation. The molecules encapsulated nanoparticles were prepared by incorporating BB into the CS-Blg complexes to a final concentration of 0.2 mg/ml, prior to the formation of the nanoparticles. For the determination of the drug encapsulation capacity, the BB encapsulated CS-Blg nanoparticles were separated from the aqueous suspension medium by ultracentrifugation with 30,000g at 20 ℃ for 30 min. The amount of free BB in the clear supernatant was determined by measuring the UV–vis absorption at 570nm (HP 8453 UV–Visible, spectrophotometer, Palo alto, CA). BB encapsulation capacity(EC) was calculated with the following equation: Brilliant blue release

13. Release test to target organ Without enzyme at pH 1.2 pH 1.2 ↓ pH 7.5 ↓ With pepsin and pancreatin With pepsin

14. Conclusion This work has elaborated CS-Blg core–shell nanoparticles by cold ionic gelation of chitosan (CS) and Blg mixtures with TPP. The coating properties of native and denatured Blg on CS- TPP core were highly sensitive to formulation pH and C Blg. Optimal nanoparticles with size of about 100nm were obtained at pH 6.1 for native Blg and pH 6.5 for denatured Blg at C Blg of 2.0 mg/ml, where the native Blg exhibits an end- on conformation and denatured Blg forms a tail projected from the positively charged surface with both of their hydrophobic portions of the molecule lying close to the CS-TPP core to allow a maximum Blg loading. The BB release experiments indicate that Blg in its native formation is an interesting candidate to coat CS nanoparticle to allow a protection of nutraceutical coumpounds when subjected to the gastric fluids due to favorable properties to resist acid and pepsin degradation in the gastric fluids. When transferred to the intestinal fluids, the outside shells of these nanoparticles were degraded by pancreat.