| As a consequence of modern drug discovery techniques (i.e., advances in in vitro screening methods, the introduction of combinatorial chemistry), there has been a consistent increase in the number of poor water soluble drug candidate compounds, and currently, more than 40% of new pharmacologically active chemical entities are lipophilic and exhibit poor water solubility. These molecules often suffer from low oral bioavailability, and despite their pharmacological activity, they fail to proceed to advanced stages of research and development. A great challenge facing the pharmaceutical scientist is making there molecules into orally administered medications with sufficient bioavailability. One of the most popular approaches to improve the oral bioavailability of these molecules is the utilization of a lipid based drug delivery system.Lipid and lipophilic excipients can be used to control the digestion, adsorption and efficacy of co-administered lipophilic drugs. In recent years, lipid-based drug delivery systems have gained increasing interest for improving the oral bioavailability of poorly water-soluble drugs, including solutions, suspensions, emulsions, microemulsions, self-emulsifying drug delivery systems, and dried emulsions. The lipid-based drug delivery systems have led over the past years to successful bioavailability enhancement with the immunosuppressive agent cyclosporine A (Neoral(?)), and for the two HIV protease inhibitors ritonavir (Norvir(?)) and saquinavir (Fortovase(?)). Lipid-based drug delivery systems offer a large variety of options, and the success of these delivery systems evolves from the suitable selection of the vehicle composition and rational delivery system design for the drug candidate. The realization that the performance of these delivery systems is affected by lipid digestion and incorporation of lipid digestion products into endogenous micellar species has led to the widespread use of lipid digestion models for in vitro assessment of lipid-based formulations. The other hand is the current development strategies in the area of lipid based drug delivery systems are mostly empirical, demand a large number of animal experiments, and consume time and money. The in vitro digestion models have been developed and widely used, which can provide a very good simulation of the in vivo lipid digestion process. This model is easy-to-handle, repeatable and low-cost. Dynamic lipolysis experiments have enhanced the understanding of the impact of the composition and structure of lipid-based formulations on the digestion and solubilization of lipophilic bioactive components.As a kind of lipid-based drug delivery system, an emulsion generally consists of at least two immiscible liquids (usually oil and water), with one of the liquids being dispersed as small spherical droplets in the other. Emulsion science and technology have been widely used in the food industry to create a considerable number of natural and processed foods, including milk, cream, coffee creamer, soft drinks, fruit beverages, and infant formula. Emulsions have a number of advantages as drug delivery systems since they can easily be prepared from inexpensive food-grade components. There is also a need for edible delivery systems to encapsulate, protect and release bioactive and functional lipophilic constituents within the food and pharmaceutical industries. These delivery systems could be used for a number of purposes:controlling lipid bioavailability; targeting the delivery of bioactive components within the gastrointestinal tract; designing food matrices that delay lipid digestion and therefore induce satiety. Emulsion technology is particularly suited for the design and fabrication of delivery systems for lipids. It has been reported that a number of emulsion-based technologies that can be used as edible delivery systems by the food and other industries, including conventional emulsions, nanoemulsions, multilayer emulsions, solid lipid particles, and filled hydrogel particles. Each of these delivery systems can be produced from food grade (GRAS) ingredients (e.g., lipids, proteins, polysaccharides, surfactants, and minerals) using relatively simple processing operations (e.g., mixing, homogenizing, and thermal processing).This study presented a series of colonic drug delivery systems by combining food and pharmaceutical science. Nowadays, there are many kinds of anti-colon cancer drugs, but unfortunately it is difficult for many potential drugs to effectively express their anti-cancer activity. That’s because some of those drugs are water-insoluble and can easily be digested into small molecules by enzyme within GI tract. Those small molecules will enter the body systematic circulation.Consequently, those drugs can not reach the colon. Our study describes the development and characterization of novel colon-specific delivery systems based on structured emulsions fabricated from natural lipids, food-grade emulsifiers and biopolymers (proteins and polysaccharides). The structure, preparation, and utilization of each type of delivery system for controlling lipid digestion are discussed. This knowledge can be used to select the most appropriate emulsion-based delivery system for specific applications, such as encapsulation, controlled digestion, and targeted release. Dynamic and static light scattering, confocal microscopy, and an in vitro digestion model (pH-stat) were used to characterize the structure, stability and digestibility of the structured emulsions.Main study contents and conclusions:1. Emulsion-Based Delivery Systems for tributyrin, a potential colon Cancer preventative agentTributyrin, a short-chain triglyceride oil used as a food additive, has been reported to be a potential preventive agent against colon cancer. The purpose of this study was to develop tributyrin delivery systems based on food-grade oil-in-water emulsions that could potentially be incorporated into foods. Emulsions containing only tributyrin as the lipid phase were highly unstable to droplet growth due to Ostwald ripening (OR) because of the relatively high water solubility of this low molecular weight triacylglycerol. The stability of the emulsions to OR could be greatly improved by incorporating≥15-25% corn oil (a food-grade oil with a low water solubility) into the lipid phase. In addition, the tendency for droplet sedimentation to occur was reduced because the density contrast between the lipid and water phases was reduced in the mixed tributyrin/corn oil systems. The potential anticarcinogenic ability of the tributyrin emulsions was demonstrated using a cell culture model. We studied both emulsifier type and oil type impact cell viability in colon cancer cell culture models. The efficacy of the emulsifiers at inhibiting cell viability decreased in the following order:Tween 20 (a non-ionic surfactant)>β-lactoglobulin and caseinate (two milk proteins)> lecithin (a phospholipid). The possible reduction of cell viability by emulsifiers therefore needs to be taken into account when interpreting the results of cell culture models. Treatments with emulsions containing tributyrin significantly inhibited the viability of HT29 colon carcinoma cells. Tributyrin was found to appreciably inhibit cell viability (> 50% reduction) at levels exceeding about 0.02 wt%(i.e.,> 0.7 mM). These results have important implications for the development and testing of nutraceuticals encapsulated in food-grade delivery systems as anticancer agents.2. Standardize and modulate in vitro digestion model by pH-stat and mathematical methodsThe control of lipid digestibility within the human gastrointestinal tract is important for the development of many functional food and pharmaceutical products. The influence of product composition and microstructure on lipid digestibility is typically studied using in vitro digestion methods. The pH-stat method is widely used to characterize the in vitro digestibility of lipids under simulated small intestine conditions. This method measures the fraction of free fatty acids (FFA) released from triacylglycerols over time by monitoring the alkali concentration required to maintain the pH at a constant pre-set value (e.g., pH 7.0). The purpose of this series of experiments was to optimize the in vitro digestion model so that it could be used to rationalize the design of structured emulsion-based delivery systems designed for controlling lipid digestion.The impact of various experimental factors on lipid digestion in oil-in-water emulsions using a pH-stat method was studied. The rate and extent of lipid digestion was found to increase with:increasing lipase (from 0 to 5 mg/ml); decreasing bile extract (from 20 to 0 mg/ml); increasing CaCl2 (from 0 to 20 mM); decreasing lipid (from 2.5 to 0.1 wt%); decreasing droplet diameter (from d= 800 to 200 nm); and decreasing fatty acid molecular weight (medium chain triglycerides versus corn oil). These affects are interpreted in terms of the surface area of lipid exposed to the aqueous phase, and factors affecting the accumulation of reaction products (fatty acids) at the oil-water interface.3. Control the functional performance and lipid digestibility of emulsion-based delivery systems by Biopolymer multilayer nanolaminated coatingsMulti-component biopolymer coatings were formed around lipid droplets by using an interfacial electrostatic deposition approach. The results showed the digestion and release of bioactive lipophilic components encapsulated within emulsion-based delivery systems could be controlled by coating the lipid droplets with multilayer biopolymer coatings. The emulsions were stabilized by a protein emulsifiers (β-lactoglobulin or sodium caseinate), and then coated by different polysaccharides at a certain pH value. Lipid droplets surrounded byβ-lactoglobulin-chitosan-alginate/pectin,β-lactoglobulin-alginate-chitosan, caseinate-chitosan-pectin and caseinate-pectin-chitosan-pectin coatings were prepared. The results illuminated that the polysaccharide coatings could improve the environmental stability and modify the interfacial properties of protein-stabilized emulsions. The droplets in primary emulsions, which had an outer protein coating, changed from positive to negative charged when the pH was increased from 3 to 7. As a result these emulsions were unstable to droplet aggregation at intermediate pH values because of the low net charge on the droplets near the isoelectric point of the adsorbed proteins. After coated by polysaccharides, the stability of the emulsions to pH changes (3 to 7) depended strongly on the order of biopolymers within the nanolaminated coatings and on the nature of the outer coating. When chitosan was the outer layer, the emulsions were stable to droplet aggregation from pH 3 to 6, but highly unstable at higher pH (>6.5)values because of the low net charge on the droplets at neutral pH. When alginate or pectin was the outer layer, there was a little aggregation at pH 3 because of the low net negative charge on the droplets, but could be improved if higher anionic polysaccharide levels were used. Further, up to five bilayers were prepared by deposition of alginate and chitosan onto the surface of lipid droplets. The mean diameter and electronic charge after adding another layer were measured. The mean diameter increased at the beginning layers and there was no obvious change. The droplet charge converted to positive by adding chitosan and negative by adding alginate. The |ζ| for each layer was kept at-30 mV.The influence of the structural organization of the nanolaminated biopolymer coatings surrounding the lipid droplets on their in vitro digestibility by pancreatic lipase was also studied. An in vitro lipid digestion model (pH stat) indicated that polysaccharide coatings could only delay the rate of lipid digestibility, but failed to completely prevent it. The digestion rate was also depended on some factors, like compositions of outer layers, Ca2+ concentration. At low Ca2+ concentration, the biopolymer nanolaminate could not greatly affect the digestion rate, but at high Ca2+ concentration, the biopolymer nanolaminate decreased and delayed the lipid digestion rate.90% lipids was digested from several minutes to more than 2 h. This study has important implications for the design of delivery systems to control the digestion and release of lipophilic components in the human GI.For layer-by-layer coatings with alginate and chitosan, the preliminary pH-stat resulted showed that more than two bilayers could significantly decrease and prevent the rate and extent of lipid digestion. The extent of lipid digestion after 2 h was decreased from about 90% to 50-60%. With more layers, the extent of lipid digestion could be decrease to 5-10%. The decreasing trend was also depended on the lipid types. With increasing the content of shorter chain triglyceride, more layers would be needed to inhibit the lipid digestion. When the coating layer was three bilayer,12% lipids would be digested after 2 h for 50% TB, while 34% for 80% TB.4. Controlling Lipid Digestibility by ionically cross-linked polysaccharides and polyelectrolyte complexation coatingSodium tripolyphosphate (TPP) was used to cross-link chitosan-coated lipid droplets. Relatively, high TPP levels (～0.004 wt%) promoted droplet aggregation and gravitational separation, which was attributed to charge neutralization and interdroplet cross-linking. Cross-linked chitosan-coated lipid droplets were formed at lower TPP levels that were relatively small (d≈450 nm), cationic (ζ≈+60 mV), and stable to particle aggregation and gravitational separation (pH 3,21 days). However, these droplets were highly unstable at pH 7 because of a reduction in net particle charge and weakened electrostatic repulsion. An in vitro lipid digestion model (pH stat) was used to study the impact of the chitosan coating on the digestibility of lipid droplets by pancreatic lipase (pH 7, bile salts, pancreatic lipase, and 5.0 mM CaCl2). The rate of lipid digestion decreased when the lipid droplets were coated with chitosan and decreased further when the chitosan coating was cross-linked with TPP. Indeed, both cross-linked and non-cross-linked chitosan coatings were able to prevent lipid digestion under conditions simulating the small intestine. This study has important implications for the design of structured emulsions with controlled lipid digestibility and for the targeted delivery of lipophilic functional components to specific regions within the gastrointestinal tract.Alginate/chitosan complex coaceravates encapsulating lipid were prepared by two methods:the direct method and the indirect method. In the direct method, a mixture of protein-coated lipid droplets and sodium alginate was dropped into a solution containing calcium chloride and chitosan. In the indirect method, a mixture of protein-coated lipid droplets and sodium alginate was dropped into a solution containing calcium chloride, and then the resulting mixture was mixed with a chitosan solution. For both methods, filled hydrogel particles consisting of lipid droplets dispersed within alginate/chitosan complex coacervates were obtained. The size of the hydrogel particles could be varied by altering the sodium alginate (0.2-0.5%, w/v), chitosan (0.2-0.5%, w/v), and calcium chloride (0.02-0.05%, w/v) concentrations used. The lipid digestibility of these hydrogel particles was evaluated using the in vitro digestion method (pH stat). The results showed that particle structure and lipid digestibility were mainly determined by chitosan and calcium chloride concentrations, as well as the preparation method used. The in vitro digestion results showed that the hydrogel particles had little effect on the rate and extent of digestion at low chitosan/chloride concentrations (～100% digestion within 30 min), but greatly delayed digestion at high concentration by an amount depending on the preparation method (～100% digestion from several minutes to more than 1 h). The results obtained showed that lipid digestibility could be controlled by rational selection of the system composition and preparation procedure.5. Control of Lipase Digestibility of Emulsified Lipids by Encapsulation within Calcium Alginate BeadsThis study focused on the development of filled hydrogel particles, consisting of lipid droplets trapped within calcium alginate beads, designed to control the digestion and release of encapsulated lipids. These particles remained intact when the pH was varied from 1 to 7, but they exhibited distinct shrinkage at pH 1 and 2. The free fatty acids released from the filled hydrogel particles after addition of lipase were monitored using a pH-stat in vitro digestion model. Encapsulation of lipid droplets within calcium alginate beads (d= 2.4 mm) reduced the free fatty acids released from around 100% to less than 12% after 120 minutes. The effects of beads size, cross-linking degree, lipid types were investigated. The rate and extent of lipid digestion increased with decreasing bead size (from 3.4 to 0.8 mm), decreasing degree of cross-linking (i.e., lower calcium or alginate concentrations), and decreasing triglyceride molecular weight (i.e., tributyrin> MCT≈corn oil). Calcium alginate beads with mean diameters of 0.82±0.15,2.37±0.16, and 3.37±0.21 mm were prepared and are referred to as "small", "medium" and "large" beads, respectively. The rate and extent of lipid digestibility increased with decreasing bead diameter e.g., 11.3,11.8 and 20.5% FFAs were released after 2 hours digestion for large, medium and small beads. When alginate concentration was 0.25%,0.5% 1.0%,2.0%, after 2 hours of digestion the percentage of free fatty acids released were 84.1±9.2%, 60.6±2.2%,30.6±1.1%,17.5±1.8%, respectively. When Ca2+ concentration was 0.5%, 1.0%,2.0%5.0%,10.0%, after 2 hours of digestion the percentage of free fatty acids released were 50.0±0.8%,45.4±2.0%,26.4±8.4%,19.1±3.5%,16.9±1.2. The rate and extent of lipid digestion was relatively slow for beads loaded with either CO or MCT droplets, e.g.,< 10% FFAs were released after nearly 2 hours of digestion. But when the lipids was 50% CO/50%TB, over 35% lipids were released after nearly 2 hours digestion. These results have important implications for the design of delivery systems to protect and release bioactive components in the human body.Overall, this work has developed a number of food-grade delivery systems to control the digestibility and release of lipophilic bioactive components.6. Preparation and protein release of blending carboxylmethyl chitosan/poly(vinyl alcohol) hydrogelBlending hydrogels were widely applied in medical fields. They can provide many advantages, such as biocompatibility, biodegradability. Many materials and methods were used to obtain blending hydrogels. In this work, the carboxymethyl chitosan (CMCS) and poly(vinyl alcohol) (PVA) blending hydrogels were prepared by the freezing and thawed technique. The properties of the prepared hydrogels, such as gel fraction, swelling and pH-responsive behaviors were investigated. The gel fraction increased with the increasing time of freezing and thawing through the gravimetric analysis. It was also found that the equilibrium degree of swelling properties improved obviously due to the addition of CMCS compared to pure PVA hydrogel. The blending hydrogel with composition CMCS/PVA (80/20) possessed the highest swelling ratio. The results of the influence of pH value on the swelling behavior showed the minimum swelling ratios of hydrogels happened near the isoelectric point of CMCS. The protein release studies were performed in various pH conditions; the release was much slower in acid condition than in basic condition. The release burst in the first 15 h and then steadily increased. The addition of CMCS could improve the physical property of pure PVA hydrogels and provide pH sensitivity. It is concluded that PVA hydrogels based-CMCS could be potentially applied as an oral delivery system for protein drug. |
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