Mucin coated protein-polyphenol microcarriers for daidzein delivery.

Daidzein, an isoflavone found abundantly in legumes, may benefit from bypassing upper gut absorption to reach the colon where it can be metabolized into the potent estrogen equol by the gut microbiome. To achieve this, we developed mucin coated protein-tannin multilayer microcarriers. Highly porous functionalized calcium carbonate (FCC) microparticles efficiently absorbed daidzein from a dimethyl sulfoxide solution, with a loading capacity of 21.6 ± 1.8 wt% as measured by ultra-high pressure liquid chromatography - mass spectrometry (UPLC-MS). Daidzein-containing FCC microparticles were then coated with a bovine serum albumin (BSA)-tannin n-layer film terminated with mucin ((BSA-TA)n-mucin) by layer-by-layer deposition from corresponding aqueous solutions followed by FCC decomposition with HCl. Raman spectroscopy confirmed mucin-tannin complexation involving both hydrophobic interactions and hydrogen bonding. The resulting multilayer microcarriers contained 54 wt% of nanocrystalline daidzein as confirmed by X-ray diffraction and UPLC-MS. Preliminary screening of several types of mucin coatings using an in vitro INFOGEST digestion model demonstrated that mucin type III from porcine stomach provided the highest protection against upper intestinal digestion. (BSA-TA)8-mucin and (BSA-TA)4-mucin microcarriers retained 71 ± 16.4% and 68 ± 4.6% of daidzein, respectively, at the end of the small intestinal phase. Mucin-free (BSA-TA)8 retained a lower daidzein amount of 46%. Daidzein release and further conversion into equol were observed during in vitro colonic studies with fecal microbiota from a healthy non-equol-producing donor and Slackia equolifaciens. The developed approach has potential for encapsulating other hydrophobic nutraceuticals or therapeutics, enhancing their bioaccessibility in the colon.

Surface-reacted calcium carbonate microparticles as templates for lactoferrin encapsulation.

Microencapsulation helps to improve bioavailability of a functional whey protein, lactoferrin (Lf), in adults. Herein, we report the Lf loading capacity (LC) and retention efficiency (RE) in the microparticles of surface-reacted calcium carbonate (SRCC) of different types and compare them to those of widely used vaterite microparticles. The LCs and REs are analyzed in connection to the total surface area and the volume of intraparticle pores. The best performing SRCC3 demonstrates Lf LC of 11.00 wt% achieved in a single absorption step and 74% RE after two cycles of washing with deionized water. A much larger surface area of SRCC templates and a lower pH required to release Lf do not affect its antitumor activity in MCF-7 assay. Layer-by-Layer assembly of pepsin-tannic acid multilayer shell around Lf-loaded microparticles followed by acidic decomposition of the inorganic core produces microencapsulated Lf with a yield ~36 times higher than from vaterite templates reported earlier, while the scale of encapsulated Lf production is ~12,000 times larger. In vitro digestion tests demonstrate the protection of ~65% of encapsulated Lf from gastric digestion. The developed capsules are prospective candidates for functional foods fortified with Lf.

Arrays of Biocompatible and Mechanically Robust Microchambers Made of Protein-Polyphenol-Clay Multilayer Films.

There is a growing demand for biocompatible and mechanically robust arrays of microcompartments loaded with minute amounts of active substances for sensing or controlled release applications. Here we report on a novel biocompatible composite material, protein-polyphenol-clay (PPC) multilayer film. The material is shown to be strong enough to make robust microchambers retaining the shape and dimensions of truncated square pyramids. We study the mechanical properties and biocompatibility of the PPC microchambers and compare them to those made of synthetic polyelectrolyte multilayer film, poly(styrenesulfonate)-poly(allylammonium) (PSS-PAH). The mechanical properties of the microchambers were characterized under uniaxial compression using nanoindentation with a flat-punch tip. The effective Young's modulus of PPC microchambers, 166 ± 53 MPa, is found to be lower than that of PSS-PAH microchambers, 245 ± 52 MPa. However, the capacity to elastically absorb the energy of the former, 2.4 ± 1.0 MPa, is marginally higher than of the latter, 2.0 ± 1.3 MPa. Arrays of microchambers were sealed onto a polyethylene film, loaded with a model oil-soluble drug, and their biocompatibility was tested using an ex vivo 3D human skin reconstruct model. We found no evidence for toxicity with the PPC microchambers; however, PSS-PAH microchambers stimulated reduced cell density in the epidermis and significantly affected epidermal-dermal attachment. Both materials do not alter skin cell proliferation but affect skin cell differentiation. We interpret that rather than affecting epidermal barrier function, these data suggest the applied plastic films with microchamber arrays affect transpiration, normoxia, and moisture exchange.

Polyelectrolyte-Graphene Oxide Multilayer Composites for Array of Microchambers which are Mechanically Robust and Responsive to NIR Light.

Development of composite polymer/graphene oxide (GO) materials attracts significant attention due to their unique properties. In this work, highly ordered arrays of hollow microchambers made of composite polyelectrolyte/GO multilayers (PEGOMs) are successfully fabricated via layer-by-layer assembly on sacrificial or sustainable templates having imprinted patterns of microwells on their surface. Mechanical and optical properties of PEGOMs are studied by nanoindentation and near-infrared (NIR) absorption spectroscopy. Incorporation of three GO layers in between the polyelectrolyte multilayer stacks increases Young's modulus and critical stress of the microchambers by a factor of 5.6 and 2.6, respectively. Optical density of this PEGOM film is found to decrease gradually from 0.14 at λ = 800 nm to 0.06 at λ = 1500 nm. Remote opening of PEGOM microchambers with NIR laser beam is also demonstrated. One of the possible applications of the developed structures includes micropackaging and delivery systems in biological tissues with remote triggering.

A membrane film sensor with encapsulated fluorescent dyes towards express freshness monitoring of packaged food.

A new Membrane Film Sensor (MFS) has been developed to measure pH of fluids. MFS comprises a polyelectrolyte multilayer film with uniformly distributed compartments (microchambers) where a fluorescent sensing dye is encapsulated. Fabricated film is sealed onto a polyethylene film for a future use. MFS was applied to report changes in golden pomfret fillet upon its storage at 5 °C. MFS pH readings were correlated to bacteriological analysis of fish samples. A hike in pH of fish juices happens after 10 days of storage signaling bacterial spoilage of fish. The design of developed MFS allows easy integration with transparent packaging materials for future development of "SMART" packaging sensing food freshness.

Protein-tannic acid multilayer films: A multifunctional material for microencapsulation of food-derived bioactives.

The benefits of various functional foods are often negated by stomach digestion and poor targeting to the lower gastrointestinal tract. Layer-by-Layer assembled protein-tannic acid (TA) films are suggested as a prospective material for microencapsulation of food-derived bioactive compounds. Bovine serum albumin (BSA)-TA and pepsin-TA films demonstrate linear growth of 2.8±0.1 and 4.2±0.1nm per bi-layer, correspondingly, as shown by ellipsometry. Both multilayer films are stable in simulated gastric fluid but degrade in simulated intestinal fluid. Their corresponding degradation constants are 0.026±0.006 and 0.347±0.005nm-1min-1. Milk proteins possessing enhanced adhesion to human intestinal surface, Immunoglobulin G (IgG) and ß-Lactoglobulin (BLG), are explored to tailor targeting function to BSA-TA multilayer film. BLG does not adsorb onto the multilayer while IgG is successfully incorporated. Microcapsules prepared from the multilayer demonstrate 2.7 and 6.3 times higher adhesion to Caco-2 cells when IgG is introduced as an intermediate and the terminal layer, correspondingly. This developed material has a great potential for oral delivery of numerous active food-derived ingredients.

Formulation for Oral Delivery of Lactoferrin Based on Bovine Serum Albumin and Tannic Acid Multilayer Microcapsules.

Lactoferrin (Lf) has considerable potential as a functional ingredient in food, cosmetic and pharmaceutical applications. However, the bioavailability of Lf is limited as it is susceptible to digestive enzymes in gastrointestinal tract. The shells comprising alternate layers of bovine serum albumin (BSA) and tannic acid (TA) were tested as Lf encapsulation system for oral administration. Lf absorption by freshly prepared porous 3 µm CaCO3 particles followed by Layer-by-Layer assembly of the BSA-TA shells and dissolution of the CaCO3 cores was suggested as the most efficient and harmless Lf loading method. The microcapsules showed high stability in gastric conditions and effectively protected encapsulated proteins from digestion. Protective efficiency was found to be 76 ± 6% and 85 ± 2%, for (BSA-TA)4 and (BSA-TA)8 shells, respectively. The transit of Lf along the gastrointestinal tract (GIT) of mice was followed in vivo and ex vivo using NIR luminescence. We have demonstrated that microcapsules released Lf in small intestine allowing 6.5 times higher concentration than in control group dosed with the same amount of free Lf. Significant amounts of Lf released from microcapsules were then absorbed into bloodstream and accumulated in liver. Suggested encapsulation system has a great potential for functional foods providing lactoferrin.

Patterned Microstructure Fabrication: Polyelectrolyte Complexes vs Polyelectrolyte Multilayers.

Polyelectrolyte complexes (PEC) are formed by mixing the solutions of oppositely charged polyelectrolytes, which were hitherto deemed "impossible" to process, since they are infusible and brittle when dry. Here, we describe the process of fabricating free-standing micro-patterned PEC films containing array of hollow or filled microchambers by one-step casting with small applied pressure and a PDMS mould. These structures are compared with polyelectrolyte multilayers (PEM) thin films having array of hollow microchambers produced from a layer-by-layer self-assembly of the same polyelectrolytes on the same PDMS moulds. PEM microchambers "cap" and "wall" thickness depend on the number of PEM bilayers, while the "cap" and "wall" of the PEC microchambers can be tuned by varying the applied pressure and the type of patterned mould. The proposed PEC production process omits layering approaches currently employed for PEMs, reducing the production time from ~2 days down to 2 hours. The error-free structured PEC area was found to be significantly larger compared to the currently-employed microcontact printing for PEMs. The sensitivity of PEC chambers towards aqueous environments was found to be higher compared to those composed of PEM.

Multilayer Capsules of Bovine Serum Albumin and Tannic Acid for Controlled Release by Enzymatic Degradation.

With the purpose to replace expensive and significantly cytotoxic positively charged polypeptides in biodegradable capsules formed via Layer-by-Layer (LbL) assembly, multilayers of bovine serum albumin (BSA) and tannic acid (TA) are obtained and employed for encapsulation and release of model drugs with different solubility in water: hydrophilic-tetramethylrhodamine-isothiocyanate-labeled BSA (TRITC-BSA) and hydrophobic 3,4,9,10-tetra-(hectoxy-carbonyl)-perylene (THCP). Hydrogen bonding is proposed to be predominant within thus formed BSA/TA films. The TRITC-BSA-loaded capsules comprising 6 bilayers of the protein and polyphenol are benchmarked against the shells composed of dextran sulfate (DS) and poly-l-arginine (PARG) on degradability by two proteolytic enzymes with different cleavage site specificity (i.e., α-chymotrypsin and trypsin) and toxicity for murine RAW264.7 macrophage cells. Capsules of both types possess low cytotoxicity taken at concentrations equal or below 50 capsules per cell, and evident susceptibility to α-chymotrypsin resulted in release of TRITC-BSA. While the BSA/TA-based capsules clearly display resistance to treatment with trypsin, the assemblies of DS/PARG extensively degrade. Successful encapsulation of THCP in the TRITC-BSA/TA/BSA multilayer is confirmed, and the release of the model drug is observed in response to treatment with α-chymotrypsin. The thickness, surface morphology, and enzyme-catalyzed degradation process of the BSA/TA-based films are investigated on a planar multilayer comprising 40 bilayers of the protein and polyphenol deposited on a silicon wafer. The developed BSA/TA-based capsules with a protease-specific degradation mechanism are proposed to find applications in personal care, pharmacology, and the development of drug delivery systems including those intravenous injectable and having site-specific release capability.

Naturally inspired polyelectrolyte multilayer composite films synthesised through layer-by-layer assembly and chemically infiltrated with CaCO3.

Naturally occurring composite structures like antler bone and nacre have a highly ordered structural design at the nanoscale. Nature's successful architecture has attracted widespread interest in mimicking such systems artificially, the goal being to design tough composite materials with adaptable mechanical properties. Here we report results on synthesis pathways towards fabricating such materials, including a chemical infiltration route where calcium carbonate particles nucleate and grow inside polyelectrolyte multilayers assembled via a layer-by-layer route. SEM analysis demonstrates a considerable change in the morphology of thin films upon chemical infiltration. The depth of mineralisation within the multilayer is confirmed by TOF-SIMS studies of both mineralised and non-mineralised thin films. TGA was used to calculate the overall content of CaCO3 within multilayer films. Infiltrated multilayers have shown up to 60% w/w of calcium carbonate which is comparable to structures like bones. X-ray diffraction to characterise the crystallographic structure and micromechanical testing involving nano-indentation have also been conducted. The Young's modulus of mineralised multilayer thin films significantly increased up to 10 GPa after infiltration in comparison to the non-mineralised multilayers with a modulus of only 3.8 GPa, while the increase in hardness is almost 50-fold. Thus, the synthetic composites can be compared with natural biomineralised tissues like nacre, ultimately replicating the natural strength of biomimetic materials on the nanoscale.

Active drug release systems: current status, applications and perspectives.

Active drug release systems offer an important privilege to manage the dosage, time and sometimes site of drug release after the implantation procedure has been performed. Once developed, they could cover such applications as hormone therapy, implantation surgery, and delivery of immunization boosters. A number of existing approaches towards such systems include arrays of microreservoirs equipped with stimuli-responsive actuators or valves. The very first developed system has reached the stage of in-human trials recently. A breakthrough could happen if microreservoirs themselves are made of responsive material susceptible towards remote triggers. A promising candidate is a material made of Layer-by-Layer assembled films which currently are widely exploited only as passive implantable drug release systems.

Layer-by-layer assembled multilayer shells for encapsulation and release of fragrance.

Layer-by-layer assembled shells are prospective candidates for encapsulation, stabilization, storage, and release of fragrances. A shell comprising four alternative layers of a protein and a polyphenol is employed to encapsulate the dispersed phase of a fragrance-containing oil-in-water emulsion. The model fragrance used in this work consists of 10 ingredients, covering a range of typically employed aroma molecules, all premixed in equal mass and with sunflower oil acting as the base. The encapsulated emulsion is stable after 2 months of storage at 4 °C as revealed by static light scattering and confocal laser scanning microscopy. Gas chromatography/mass spectrometry data show that the encapsulation efficiency of 8 out of 10 fragrance ingredients depends on the water solubility: the less water-soluble an ingredient, the more of it is encapsulated. The amount of these fragrance ingredients remaining encapsulated decreases linearly upon emulsion incubation at 40 °C and the multilayer shell does not hinder their release. The other two fragrance ingredients having the lowest saturation vapor pressure demonstrate sustained release over 5 days of incubation at 40 °C. The composition of released fragrance remains almost constant over 3 days of incubation, upon further incubation it becomes enriched with these two ingredients when others start to be depleted.

Individually addressable patterned multilayer microchambers for site-specific release-on-demand.

Patterned arrays of light-responsive microchambers are suggested as candidates for site-specific release of chemicals in small and precisely defined quantities on demand. A composite film is made of poly(allylammonium)-poly(styrene sulfonate) multilayers and gold nanoparticles incorporated between subsequent stacks of polyelectrolytes. The film shaped as microchambers is loaded with colloid particles or oil-soluble molecules. The microchambers are sealed onto a glass slide precoated with an adhesive poly(diallyldimethylammonium)-poly(styrene sulfonate) multilayer film. A focused laser beam is used for remote addressing the individual microchambers and site-specific release of the loaded cargo.

Adhesion of polyelectrolyte multilayers: sealing and transfer of microchamber arrays.

Polyelectrolyte multilayer (PEM) films with array of responsive microchambers are promising candidates for site-specific release of chemicals in small and precisely defined quantities on demand. It requires effective sealing of the microchambers toward a support to prevent leakage of a cargo. In this paper, we study the pressure-induced adhesion of poly(allylammonium)-poly(4-styrenesulfonate) (PAH-PSS) multilayers assembled on different templates toward the poly(4-styrenesulfonate)-poly(diallyldimethylammonium) multilayer. The tensile bond strength increases from 0.4 to 3.5 MPa upon the increase of PAH-PSS bilayers from 10 to 40, if assembled on a silicon template. Weaker tensile bond strength of 0.35 MPa between the PAH-PSS multilayer and a poly(methylmethacrylate) (PMMA) template results in adhesive break at this interface and allows mechanical removal of the template. The successful PEM transfer is demonstrated for templates of various geometrical patterns, while the tensile break of a multilayer film happens for the others.

Peculiarities of polyelectrolyte multilayer assembly on patterned surfaces.

The layer-by-layer assembly of poly(diallyldimethylammonium chloride) and poly(sodium 4-styrenesulfonate) is studied on templates with imprinted arrays of microwells ranging from 2 to 25 µm and different aspect ratios. The thickness and microstructure of polyelectrolyte multilayers (PEMs) are measured using scanning electron microscopy. At 0.2 M ionic strength, the PEM film evenly coats the template both inside and outside the microwells. If the film is thinner than the critical value of about 400 nm, PEM microstructures collapse upon dissolving the template. Euler's model of critical stress is used to describe the collapse. At 2 M ionic strength, a substantially thinner PEM film is assembled inside the 25 µm wells than outside. If the well diameter is reduced to 7 and 2 µm, a much thicker PEM film is formed inside the microwells. These observations have been attributed to the changing of polyelectrolyte conformation in the solutions.

Kinetic stability of water-dispersed oil droplets encapsulated in a polyelectrolyte multilayer shell.

The original theoretical model of polyelectrolyte adsorption onto water-dispersed colloid particles is extended to the system of polydisperse droplets of sunflower oil. Polycation (poly(allylamine hydrochloride)) and polyanion (poly(sodium 4-styrenesulfonate)) are taken in the theoretically projected concentrations to perform Layer-by-Layer assembly of a multilayer shell on the surface of oil droplets preliminary stabilized with a protein emulsifier (bovine serum albumin). The velocity of gravitational separation in suspension of encapsulated oil droplets is theoretically predicted and experimentally measured depending on the coating shell's thickness, aiming to clarify the mechanism to control over the separation process. Combining the theory and experimental data, the mass density of a polyelectrolyte multilayer shell assembled in a Layer-by-Layer fashion is obtained. Polyelectrolyte multilayer coated oil droplets are characterized by means of ζ-potential, and particle size measurements, and visualized by scanning electron microscopy.

Patterned microcontainers as novel functional elements for microTAS and LOC.

Using nanoimprint lithography, arrays of highly ordered patterns of polyelectrolyte multilayer microcapsules consisting of alternating layers of poly(allylamine hydrochloride) and poly(sodium 4-styrene sulfonate) have been achieved. Anchoring the capsules on a pre-patterned substrate facilitates the utilization of their various capabilities in lab-on-a chip devices. In this paper we have demonstrated a very effective method to entrap soft capsules into surface cavities. Supported microcapsules were applied as the depots for loading and storage of macromolecular cargo (glucose oxidase and peroxidase) and as preserved microvessels for the cascade of enzymatic reactions. The loading of capsules was achieved under a pre-determined pH environment. This development is potentially useful for the realization of novel multianalytical systems for catalytic, bio-affinity and pH detection with protected sensing molecules.