A New Strategy Based on Smrho Protein Loaded Chitosan Nanoparticles as a Candidate Oral Vaccine against Schistosomiasis

Background

Schistosomiasis is one of the most important neglected tropical diseases and an effective control is unlikely in the absence of improved sanitation and vaccination. A new approach of oral vaccination with alginate coated chitosan nanoparticles appears interesting because their great stability and the ease of target accessibility, besides of chitosan and alginate immunostimulatory properties. Here we propose a candidate vaccine based on the combination of chitosan-based nanoparticles containing the antigen SmRho and coated with sodium alginate.

Methods and Findings

Our results showed an efficient performance of protein loading of nanoparticles before and after coating with alginate. Characterization of the resulting nanoparticles reported a size around 430 nm and a negative zeta potential. In vitro release studies of protein showed great stability of coated nanoparticles in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Further in vivo studies was performed with different formulations of chitosan nanoparticles and it showed that oral immunization was not able to induce high levels of antibodies, otherwise intramuscular immunization induced high levels of both subtypes IgG1 and IgG2a SmRho specific antibodies. Mice immunized with nanoparticles associated to CpG showed significant modulation of granuloma reaction. Mice from all groups immunized orally with nanoparticles presented significant levels of protection against infection challenge with S. mansoni worms, suggesting an important role of chitosan in inducing a protective immune response. Finally, mice immunized with nanoparticles associated with the antigen SmRho plus CpG had 38% of the granuloma area reduced and also presented 48% of protection against of S. mansoni infection.

Conclusions

Taken together, this results support this new strategy as an efficient delivery system and a potential vaccine against schistosomiasis.     

Antibacterial activity of resin based coatings containing marine microbial extracts

Bioactive agents produced by marine surface‐associated bacteria have been incorporated into experimental antifouling coatings. It was found that coating formulation can affect the action spectra of bacterial extracts involved. Little work has been done on the effect of permeability on the leaching of bioactive compounds from antifouling coatings. The formulations with 1.5% and 1.0% alginate produced significantly larger zones (p < 0.05) than with 0.5% alginate. Test formulations continued to leach active compounds after continuous immersion in seawater for 120 h. The bacterial strains used to produce the active compounds were identified by biochemical tests and/or the analysis of the DNA from a 500 bp section of the gene coding for the 16S rRNA subunit. Strains used were tentatively identified as Planococcus citreus, seven Bacillus strains (including three Bacillus pumilus and three Bacillus lautus strains), Cytophaga fucicola, Cytophaga uliginosa and a Pseudoalteromonas spp.     

Microcultivation of anaerobic bacteria single cells entrapped in alginate microbeads

Alginate microbeads, produced by emulsion/internal gelation, were studied for the entrapment and microcultivation of microbial cells with biotechnological potential. An anaerobic consortium which was selected for its capacity to degrade complex carbohydrates, and a pure culture of cellulose degrading bacteria were used for entrapment studies. Optimization of conditions for the formation of spherical alginate microbeads in sizes between 20 and 80 μm were examined. The best conditions were achieved by combining rapeseed methyl ester as oil phase and stirring at 100 rpm using a rotation impeller. Calcium alginate microbeads produced under these conditions were shown to present morphological stability, with large pores in the internal matrix that favours microcolony development. Finally, single cells were observed inside the beads after the entrapment procedure and microcolony formation was confirmed after cultivation in cellobiose.     

Controlled release of cyclosporin A from liposomes-in-microspheres as an oral delivery system

The aim of this study was to prepare cyclosporin A-loaded liposome (CyA-Lip) as an oral delivery carrier, with their encapsulation into microspheres based on alginate or extracellular polysaccharide (EPS) p-m 10356. The main advantage of liposomes in the microspheres (LIMs) is to improve the restricted drug release property from liposomes and their stability in the stomach environment. Alginate microspheres containing CyA-Lip were prepared with a spray nozzle; CyA-Liploaded EPS microspheres were also prepared using a w/o emulsion method. The shape of the LIMs was spherical and uniform, and the particle size of the alginate-LIMs ranged from 5 to 10 μm, and that of the EPS-LIMs was about 100 μm. In a release test, release rate of CyA in simulated intestinal fluid (SIF) from the LIMs was significantly enhanced compared to that in simulated gastric fluid (SGF). In addition, the CyA release rates were slower from formulations containing the liposomes compared to the microspheres without the liposome. Therefore, alginate-and EPS-LIMs have the potential for the controlled release of CyA and as an oral delivery system.     

Characterization of calcium alginate and chitosan-treated calcium alginate gel beads entrapping allyl isothiocyanate

Calcium alginate (CA), chitosan-coated calcium alginate (CCA-I), and chitosan–calcium alginate complex (CCA-II) gel beads, in which an oil-in-water emulsion containing allyl isothiocyanate (AITC) was entrapped, were prepared and characterized for efficient oral delivery of AITC. The AITC entrapment efficiency was 81% for CA gel beads, whereas about 30% lower values were determined for the chitosan-treated gel beads. Swelling studies showed that all the gel beads suddenly shrunk in simulated gastric fluid (pH 1.2). In simulated intestinal fluid (pH 7.4), CA and CCA-I gel beads rapidly disintegrated, whereas CCA-II gel beads highly swelled without degradation probably due to the strong chitosan–alginate complexation. Release studies revealed that most entrapped AITC was released during the shrinkage, degradation, or swelling of the gel beads, and the chitosan treatments, especially the chitosan–alginate complexation, were effective in suppressing the release. CCA-II gel beads showed the highest bead stability and AITC retention under simulated gastrointestinal pH conditions.     

Assessment of Phosphinothricin Acetyltransferase (PAT) Degradation From Transgenic Zoysiagrass Digested with Simulated Gastric Fluid (SGF)

The in vitro pepsin digestion assay is the international standard for assessing the safety or risk of novel proteins newly produced in transgenic crops. However, this protocol, based on the degradation of protein purified from Escherichia coli, has recently been criticized for problems such as its objective detection limit. Here, we estimated the digestion stability of the phosphinothricin acetyltransferase (PAT) protein in soluble proteins as well as from leaf tissue powder in simulated gastric fluid (SGF). Our line of genetically modified zoysiagrass carried a single copy of the bar gene, which entered a chromosomal region not encoding protein. We designated it as Jeju Green 21 (JG21). From total soluble proteins extracted from JG21 leaves, digestibility of the PAT protein in SGF was examined by enzymatic assays, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western gel blots, and an immunodetection strip kit. The degradability of pure PAT protein obtained from E. coli was clearly apparent within at least 30 s. However, PAT degradation in leaf tissue powder was significantly delayed, indicating that some matrices in that powder might have influenced its digestion stability by SGF. Nevertheless, degradation of the powder (real-life) sample was complete within at least 5 min, suggesting that this protein produced in JG21 zoysiagrass can be digested harmlessly in the stomachs of humans or livestock.     

Polysaccharide Encrusted Multilayered Nano-Colloidal System of Andrographolide for Improved Hepatoprotection

Andrographolide (AP), a phytoconstituent of Andrographis paniculata is reported as a potent hepatoprotective agent. However, utility of this molecule is restricted due to its low aqueous solubility, gastric instability and hence low bioavailability. It was aimed to formulate and characterize AP-loaded, natural biopolymer stabilized, multilayered nano-hydrocolloid delivery system. Nanoemulsion (NE) was formulated using layer-by-layer (LbL) technology via electrostatic deposition of chitosan over alginate encrusted o/w NE by ultra-sonication. Improved transparency and stability of NE were observed with increasing sonication time. Best stability was obtained after 20 min sonication and particle size of the multilayered NE was measured in the range of 90.8–167.8 nm. Transmission electron microscopy confirmed the progressive layering of nanosized NE. Higher magnitude of zeta potential (i.e., 22.9 to 31.01 mV) confirmed higher stability and coating of alginate layer over NE surface for the period of 3 months. NE showed strategic release pattern when assessed in vitro in various simulated biological fluids of GIT in timed pattern. Multilayered NE showed significant modulation in liver function test (ALT, ALP, AST, TBIL, DBIL, and liver glycogen) and serum cytokines (TNF-α, IL-6, IL-10, and IL-β) when assessed in vivo in galactosamine-lipopolysaccharide intoxicated mice. In conclusion, the andrographolide engrained multi-layered NE enhanced the solubility, stability and henceforth assured the increased availability in simulated biological fluids. The in vivo study exhibited the significantly improved hepatoprotection by andrographolide when delivered in stable multi-layered NE carrier systems.     

Influence of encapsulation on the survival of probiotics in food matrix under simulated stress conditions

The main objective of the present study was to evaluate the influence of encapsulation by extrusion technique using two hydrogels, namely; sodium alginate (Na-ALG) and whey protein isolate (WPI) on Bifidobacterium bifidium viability and stability of yoghurt under simulated gastrointestinal conditions. Probiotic bacteria (free or encapsulated) were added to yogurt for four weeks to test their viability and stability. Physicochemical and sensory analysis of yoghurt were conducted. Viability of B. bifidium in the simulated gastrointestinal conditions pH 2 and pH 7.5 was determined. Also, the efficiency of encapsulated final yield of the microcapsules was determined. With storage time, the pH of yoghurt containing encapsulated bacteria increased more than that of yoghurt containing free probiotic bacteria, resulting in a decrease in acidity. When compared to yoghurt containing encapsulated bacteria, the lactose level of yoghurt containing free probiotic bacteria decreased over time. The viscosity of yoghurt containing encapsulated WPI remained stable over the storage period, with syneresis remaining stable. The sensory properties of yoghurt containing free probiotics deteriorated over time. Cell viability was significantly reduced in yoghurt-containing free probiotics compared to other treated yoghurts. Cell viability in free probiotics yoghurt was lower than in encapsulated ones when exposed to simulated gastric and intestinal juice. In conclusion, WPI- encapsulated probiotics showed better stability over 28 days of storage in both yoghurt and gastrointestinal conditions, followed by sodium alginate.     

In vitro evaluation of the activity of microencapsulated carvacrol against Escherichia coli with K88 pili

Aims: The aim of the current study is to develop encapsulation of essential oils for oral delivery to the small intestine of pigs in order to retain their antimicrobial activity. Methods and Results: Carvacrol was used as a model essential oil and successfully encapsulated in microcapsules made from Ca‐alginate hydrogel using an emulsion–extrusion technology with high encapsulation efficiency. This encapsulation method did not compromise the antimicrobial activity when tested against Escherichia coli K88 in a culture medium, as well as in a simulated gastrointestinal model. In the simulated gastrointestinal model, <20% of encapsulated carvacrol was released in the simulated gastric fluid; the rest was nearly completely released in the intestinal fluid after 6 h of incubation. Conclusions: Encapsulation in Ca‐alginate microcapsules could effectively reduce the early absorption of carvacrol in the upper gastrointestinal tract after oral administration, therefore, retains its potential antibacterial activity for the small intestine. Significance and Impact of the Study: The developed encapsulation method is expected to be suitable for encapsulation of other essential oils. The results from this study would increase the likelihood of success in the application of essential oils as antimicrobial agents for controlling enteric diseases in pigs.     

Development of chondrocyte-laden alginate hydrogels with modulated microstructure and properties for cartilage regeneration

Alginate hydrogel is an attractive biomaterial for cell microencapsulation. The microarchitecture of hydrogels can regulate cellular functions. This study aims to investigate the applicability of sodium citrate buffer (SCB) as a culture medium supplement for modulating the microstructure of alginate microbeads to provide a favorable microenvironment for chondrogenic induction. The chondrocyte-laden microbeads, with and without TGF-β3 incorporation, were produced through an encapsulator. The obtained small-sized microbeads (~300 μm) were exposed to a treatment medium containing SCB, composed of varied concentrations of sodium citrate (1.10–1.57 mM), sodium chloride (3.00–4.29 mM), and ethylenediaminetetraacetic acid (0.60–0.86 mM) to partially degrade their crosslinked structure for 3 days, followed by culture in a normal medium until day 21. Scanning electron microscope micrographs demonstrated a loose hydrogel network with an enhanced pore size in the SCB-treated microbeads. Increasing the concentration of SCB in the treatment medium reduced the calcium content of the microbeads via a Na⁺/Ca²⁺ exchange process and improved the water absorption of the microbeads, resulting in a higher swelling ratio. All the tested SCB concentrations were non-cytotoxic. Increases in aggrecan and type II collagen gene expression and their corresponding extracellular matrix accumulation, glycosaminoglycans, and type II collagen were vividly detected in the TGF-β3-containing microbeads with increasing SCB concentrations in the treatment medium. Our findings highlighted that the combination of SCB treatment and TGF-β3 incorporation in the chondrocyte-laden microbeads is a promising strategy for enhancing cartilage regeneration, which may contribute to a versatile application in cell delivery and tissue engineering.     

Chitosan coated alginate beads for the survival of microencapsulated Lactobacillus plantarum in pomegranate juice

This work studied the effect of multi-layer coating of alginate beads on the survival of encapsulated Lactobacillus plantarum in simulated gastric solution and during storage in pomegranate juice at 4°C. Uncoated, single and double chitosan coated beads were examined. The survival of the cells in simulated gastric solution (pH 1.5) was improved in the case of the chitosan coated beads by 0.5-2 logs compared to the uncoated beads. The cell concentration in pomegranate juice after six weeks of storage was higher than 5.5logCFU/mL for single and double coated beads, whereas for free cells and uncoated beads the cells died after 4 weeks of storage. In simulated gastric solution, the size of the beads decreased and their hardness increased with time; however, the opposite trend was observed for pomegranate juice, indicating that there is no correlation between cell survival and the hardness of the beads.     

Preparation and Evaluation of Differently Sulfonated Styrene–Divinylbenzene Cross-linked Copolymer Cationic Exchange Resins as Novel Carriers for Drug Delivery

The differently sulfonated styrene–divinylbenzene cross-linked copolymer cationic exchange resins were prepared by oil-in-water polymerization and varied degrees of sulfonation. Several characteristics of the obtained resins were evaluated, i.e., Fourier transform infrared spectra, the ion-exchange capacity, microscopic morphology, size, and swelling. The resin characteristics were altered in relation to the degree of sulfonation, proving that differently sulfonated resins could be prepared. The behavior of chlorpheniramine (CPM) loading and in vitro release in the USP simulated gastric (SGF) and intestinal fluids (SIF) of the obtained resins were also evaluated. The CPM loaded in the resinates (drug-loaded resins) increased with the increasing degree of sulfonic group and hence the drug binding site in the employed resins. The CPM release was lower from the resins with the higher degree of sulfonic group due to the increase in the diffusive path depth. The CPM release was obviously lower in SGF than SIF because CPM, a weak base drug, ionized to a greater extent in SGF and then preferred binding with rather than releasing from the resins. In conclusion, the differently sulfonated resins could be utilized as novel carriers for drug delivery.     

Microencapsulation of Bacteriophage Felix O1 into Chitosan-Alginate Microspheres for Oral Delivery

This paper reports the development of microencapsulated bacteriophage Felix O1 for oral delivery using a chitosan-alginate-CaCl₂ system. In vitro studies were used to determine the effects of simulated gastric fluid (SGF) and bile salts on the viability of free and encapsulated phage. Free phage Felix O1 was found to be extremely sensitive to acidic environments and was not detectable after a 5-min exposure to pHs below 3.7. In contrast, the number of microencapsulated phage decreased by 0.67 log units only, even at pH 2.4, for the same period of incubation. The viable count of microencapsulated phage decreased only 2.58 log units during a 1-h exposure to SGF with pepsin at pH 2.4. After 3 h of incubation in 1 and 2% bile solutions, the free phage count decreased by 1.29 and 1.67 log units, respectively, while the viability of encapsulated phage was fully maintained. Encapsulated phage was completely released from the microspheres upon exposure to simulated intestinal fluid (pH 6.8) within 6 h. The encapsulated phage in wet microspheres retained full viability when stored at 4°C for the duration of the testing period (6 weeks). With the use of trehalose as a stabilizing agent, the microencapsulated phage in dried form had a 12.6% survival rate after storage for 6 weeks. The current encapsulation technique enables a large proportion of bacteriophage Felix O1 to remain bioactive in a simulated gastrointestinal tract environment, which indicates that these microspheres may facilitate delivery of therapeutic phage to the gut.     

Antifouling Coatings Influence both Abundance and Community Structure of Colonizing Biofilms: a Case Study in the Northwestern Mediterranean Sea

When immersed in seawater, substrates are rapidly colonized by both micro- and macroorganisms. This process is responsible for important economic and ecological prejudices, particularly when related to ship hulls or aquaculture nets. Commercial antifouling coatings are supposed to reduce biofouling, i.e., micro- and macrofoulers. In this study, biofilms that primarily settled on seven different coatings (polyvinyl chloride [PVC], a fouling release coating [FRC], and five self-polishing copolymer coatings [SPC], including four commercial ones) were quantitatively studied, after 1 month of immersion in summer in the Toulon Bay (Northwestern Mediterranean Sea, France), by using flow cytometry (FCM), microscopy, and denaturing gradient gel electrophoresis. FCM was used after a pretreatment to separate cells from the biofilm matrix, in order to determine densities of heterotrophic bacteria, picocyanobacteria, and pico- and nanoeukaryotes on these coatings. Among diatoms, the only microphytobenthic class identified by microscopy, Licmophora, Navicula, and Nitzschia were determined to be the dominant taxa. Overall, biocide-free coatings showed higher densities than all other coatings, except for one biocidal coating, whatever the group of microorganisms. Heterotrophic bacteria always showed the highest densities, and diatoms showed the lowest, but the relative abundances of these groups varied depending on the coating. In particular, the copper-free SPC failed to prevent diatom settlement, whereas the pyrithione-free SPC exhibited high picocyanobacterial density. These results highlight the interest in FCM for antifouling coating assessment as well as specific selection among microbial communities by antifouling coatings.     

Development of a Gastric Absorptive,Immediate Responsive,Oral Protein-Loaded Versatile Polymeric Delivery System

A multifunctional platform to deliver three diverse proteins of insulin, interferon beta (INF-β) and erythropoietin (EPO), using a novel copolymeric microparticulate system of TMC-PEGDMA-MAA, was synthesised as an intelligent pH-responsive 2-fold gastric and intestinal absorptive system. Physiochemical and physicomechanical studies proved the degree of crystallinity that supported the controlled protein delivery of the microparticulate system. The copolymer was tableted before undertaking in vitro and in vivo analysis. After 2.5 h in simulated gastric fluid (SGF), insulin showed a fractional release of 3.2% in comparison to simulated intestinal fluid (SIF), in which a maximum of 83% of insulin was released. Similarly, INF-β and EPO released 3 and 9.7% in SGF and a maximum of 74 and 81.3% in SIF, respectively. In vivo studies demonstrated a significant decrease in blood glucose by 54.19% within 4 h post-dosing, and the comparator formulation provided 74.6% decrease in blood glucose within the same time period. INF-β peak bioavailable dose in serum was calculated to be 1.3% in comparison to an SC formulation having a peak concentration of 0.9%, demonstrating steady-state release for 24 h. EPO-loaded copolymeric microparticles had a 1.6% peak bioavailable concentration, in comparison to the 6.34% peak concentration after 8 h from the SC comparator formulation.     

Effect of Polyglycerol Esters of Fatty Acids on the Physicochemical Properties and Stability of β-Carotene Emulsions during Digestion in Simulated Gastric Fluid

The effects of six different polyglycerol esters of fatty acids (PGEs) and two different particle sizes produced using various processing parameters on the physicochemical properties and stability of the β-carotene emulsions during digestion in simulated gastric fluid (SGF) were investigated. β-Carotene emulsions were prepared by high-pressure homogenization using β-carotene (0.1% w/w) in soybean oil as the oil phase and 1% (w/w) PGE in Milli-Q water as the water phase. The particle size of β-carotene emulsions was measured by a laser diffraction technique, and the stability of emulsions was interpreted in terms of the increase in particle size and span value of emulsion droplets and the retention of β-carotene during digestion in SGF. The average particle size ranges of emulsions were 0.17 to 0.27 μm for fine emulsions and 1.16 to 1.59 μm for coarse emulsions. In the prepared β-carotene emulsions, the particle size decreased with increasing polymerization of the glycerol in PGEs, and the higher polymerization of the glycerol also increased the stability of emulsions during digestion in SGF. Although the β-carotene content in the emulsions significantly decreased with increasing digestion period, loss of β-carotene was more severe in unstable emulsions than in stable emulsions, suggesting that the particles incorporated into droplets could provide some protective barrier for decreasing the β-carotene degradation. Therefore, β-carotene emulsions stabilized by PGEs with high polymerization of the glycerol may be useful for further applications in food and drug formulations. Decaglycerol monooleate (MO750) was demonstrated to be the most effective emulsifier in stabilizing β-carotene emulsions in this study.     

Adhesion of microorganisms to bovine submaxillary mucin coatings: effect of coating deposition conditions

The adhesion of Staphylococcus epidermidis, Escherichia coli, and Candida albicans on mucin coatings was evaluated to explore the feasibility of using the coating to increase the infection resistance of biomaterials. Coatings of bovine submaxillary mucin (BSM) were deposited on a base layer consisting of a poly(acrylic acid-b-methyl methacrylate) (PAA-b-PMMA) diblock copolymer. This bi-layer system exploits the mucoadhesive interactions of the PAA block to aid the adhesion of mucin to the substratum, whereas the PMMA block prevents dissolution of the coating in aqueous environments. The thickness of the mucin coating was adjusted by varying the pH of the solution from which it was deposited. Thin mucin coatings decreased the numbers of bacteria but increased the numbers of C. albicans adhering to the copolymer and control surfaces. Increasing the mucin film thickness resulted in a further lowering of the density of adhering S. epidermidis cells, but it did not affect the density of E. coli. In contrast, the density of C. albicans increased with an increase in mucin thickness.     

Alginate from the macroalgae <Emphasis Type="Bold">Sargassum sinicola</Emphasis> as a novel source for microbial immobilization material in wastewater treatment and plant growth promotion

Alginate extracted from the macroalgae Sargassum sinicola was used as the raw material for co-immobilization of the microalgae Chlorella sorokiniana and growth-promoting bacterium Azospirillum brasilense for wastewater treatment and as an inoculant carrier of A. brasilense for plant growth promotion. The composition, structure, viscosity, color, and phenolic compound content of the alginate were analyzed and compared with commercially available alginate produced from the macroalgae Macrocystis pyrifera. From ¹H NMR analysis of alginate, S. sinicola was found to have more guluronic acid (F _G=0.64) than it had mannuronic acid (F _M=0.38) and had a viscosity of 13.5 m Pa s compared to 50 m Pa s for M. pyrifera. The S. sinicola alginate had dark brown color, reducing light penetration, with more phenolic compounds than M. pyrifera alginate. Nonetheless, growth of C. sorokiniana and A. brasilense in S. sinicola alginate was not significantly different than the growth in M. pyrifera alginate beads. Nutrient removal from wastewater by the co-immobilized microorganisms was similar for both types of alginate beads, and so was the growth enhancement of tomato plants inoculated with microbeads containing A. brasilense. This study shows the potential use of S. sinicola alginate as a raw material for cell immobilization for wastewater treatment and plant growth promotion.     

Survival, root colonisation and biocontrol capacities of Pseudomonas fluorescens F113 LacZY in dry alginate microbeads

Cells of Pseudomonas fluorescens F113 LacZY were encapsulated in alginate and their survival and ability to colonise sugar beet were evaluated. To assess survival, the formulation, composed of dry alginate microbeads of 300- to 700-μm diameter, was stored 1 year at 28±2 and 4±2°C and then tested against pathogenic fungi Pythium ultimum and Rhizoctonia solani in in vitro inhibition experiments. The same material was also used as inoculant for protection of sugar beet against Py. ultimum in microcosm experiments. The results obtained indicated that, although drying alginate beads resulted in a significant reduction of bacterial viability, the use of microbeads enabled a satisfactory level of root colonisation and protection, at least under microcosm conditions. The capability of the encapsulated cells to produce the antifungal metabolite 2,4-diacetylphloroglucinol (Phl) was not significantly affected by 12 months storage. Journal of Industrial Microbiology & Biotechnology (2001) 27, 337–342. Received 07 September 2000/ Accepted in revised form 08 May 2001     

Impact of Layer Structure on Physical Stability and Lipase Digestibility of Lipid Droplets Coated by Biopolymer Nanolaminated Coatings

The objective of this study was to investigate the influence of the structure of nanolaminated biopolymer coatings surrounding lipid droplets on their physical stability and in vitro digestibility by pancreatic lipase. Caseinate (Ca) was used as an amphoteric emulsifier, pectin (P) was used as an anionic polyelectrolyte, and chitosan (C) was used as a cationic polyelectrolyte. The electrostatic layer-by-layer deposition approach was used to prepare multilayer emulsions containing lipid droplets coated by: (1) the same coating composition but different layer order (Ca–P–C and Ca–C–P); (2) the same outer layer but different coating compositions (e.g., Ca–P, Ca–P–C–P, and Ca–C–P). 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. The lipid droplets in all of the multilayer emulsions were largely digested by lipase within 30 min when monitored using an in vitro digestion model (pH-Stat). This information could be useful for the rational design of delivery systems for lipophilic bioactive compounds that need to be encapsulated within foods but released in the human body.