Decapsulation of Dextran by Destruction of Polyelectrolyte Microcapsule Nanoscale Shell by Bacillus subtilis Bacteria

Gulyaev Y.V., Cherepenin V.A., Vdovin V.A., Taranov I.V., Sukhorukov G.B., Gorin D.A., Khomutov G.B.

A technology of remote activation of specially created polyelectrolyte nanocomposite microcapsules (PNMCs) by pulse microwave action is developed. The sensitivity of the synthesized PNMCs to the external microwave action is caused by the presence of layers of ferric oxide nanoparticles in their shells. A setup for remote pulse microwave action on the PNMC is constructed. The effect of decapsulation of polyelectrolyte nanocomposite microcapsules under the remote action of microwave pulse is detected. Destruction of the PNMC shell is recorded by the methods of the transmission electron microscopy.

Bukreeva T.V., Orlova O.A., Sulyanov S.N., Grigoriev Y.V., Dorovatovskiy P.V.

A new method for modifying polyelectrolyte capsule shells by magnetic nanoparticles is proposed: the in situ synthesis of magnetite by chemical condensation. The capsule cores were spherical calcium carbonate microparticles, while polymer shells were prepared using the layer-by-layer electrostatic adsorption of polyallylamine and polystyrene sulfonate. After in situ synthesis, nanoparticles of different shapes are formed on the capsule shell; the main crystalline phase of nanoparticles is magnetite. The thus obtained nanocomposite capsules are highly sensitive to external magnetic fields.

Based on the method of the layer-by-layer (LbL) adsorption of oppositely charged polyelectrolytes, sodium alginate (Alg) and poly-L-lysine (PLL), novel biodegradable microcapsules have been prepared for delivery of biological active substances (BAS). Porous spherical CaCO3 microparticles were used as templates. The template cores were coated with several layers of oppositely charged polyelectrolytes forming shell on the core surface. The core-shell microparticles were converted into hollow microcapsules by means of core dissolution with EDTA. Mild conditions for microcapsules preparation allow to perform incorporation of various biomolecules maintaining their bioactivity. Biocompatibility and biodegradability of the polyelectrolytes give a possibility to use the microcapsules as the target delivery systems. Chymotrypsin entrapped into the microcapsules was used as a model enzyme. The immobilized enzyme retained about 86% of the activity compared to a native chymotrypsin. The resultant microcapsules were stable in acidic medium and could be easily decomposed by trypsin treatment in slightly alkaline medium. Chymotrypsin was shown to be active after its release from the microcapsules decomposed by the trypsin treatment. Thus, the microcapsules prepared by the LbL technique can be used for the development of new type of BAS delivery systems in humans and animals.

Marchenko I.V., Plotnikov G.S., Baranov A.N., Saletskii A.M., Bukreeva T.V.

Polyelectrolyte capsules with Rhodamine 6G molecules included into the shell are obtained in this work. The inclusion of dye molecules into the shell can provide the destruction of capsules by photosensitization. Measurements of the dye fluorescence intensity in the shell show that the energy of photoexcited molecules is effectively dissipated by the surrounding organic matrix. The capsule suspension is laser irradiated in the absorption band of Rhodamine 6G molecules. Measurements of the size distribution of capsules before and after laser irradiation reveal that the capsules are destroyed by laser illumination.

Balkundi S.S., Veerabadran N.G., Eby D.M., Johnson G.R., Lvov Y.M.

Layer-by-layer assembly uses alternating charged layers of polyionic polymers to coat materials sequentially in a sheath of functionalized nanofilms. Bacterial spores were encapsulated in organized ultrathin shells using layer-by-layer assembly in order to assess the biomaterial as a suitable core and determine the physiological effects of the coating. The shells were constructed on Bacillus subtilis spores using biocompatible polymers polyglutamic acid, polylysine, albumin, lysozyme, gelatin A, protamine sulfate, and chondroitin sulfate. The assembly process was monitored by measuring the electrical surface potential (zeta-potential) of the particles at each stage of assembly. Fluorescent laser confocal microscopy and scanning electron microscopy confirmed the formation of uniform coatings on the spores. The coating surface charge and thickness (20-100 nm) could be selectively tuned by using appropriate polymers and the number of bilayers assembled. The effect of each coating type on germination was assessed and compared to native spores. The coated spores were viable, but the kinetics and extent of germination were changed from control spores in all instances. The results and insight gained from the experiments may be used to design various bioinspired systems. The spores can be made dormant for a desired amount of time using the LbL encapsulation technique and can be made active when appropriate.

Muñoz Javier A., Kreft O., Semmling M., Kempter S., Skirtach A.G., Bruns O.T., del Pino P., Bedard M.F., Rädler J., Käs J., Plank C., Sukhorukov G.B., Parak W.J.

Rigid particles as well as soft capsules can be ingested by cells and stored in acidic compartments around the nucleus. TEM and fluorescence images show that the rigid particles retain their original spherical shape whereas the hollow and thus more flexible capsules are deformed and squeezed upon the incorporation process. Though soft capsules are deformed upon uptake, the cargo loaded into the capsules is not released into the cytosol.

Sukhorukov B.I., Tikhonenko S.A., Saburova E.A., Dubrovskii A.V., Dybovskaya Y.N., Shabarchina L.I.

Encapsulation of enzymes (lactate dehydrogenase and urease) in polyelectrolyte shells was assessed with a view to designing enzymic microdiagnostics for low-molecular compounds in native biological fluids. Polyelectrolyte microcapsules were prepared with two polyanions [poly(styrenesulfonate) PSS and dextran sulfate DS] and two polycations [poly(allylamine) PAA and poly(diallyldimethylammonium) PDADMA]; calcium carbonate microspherulites with embedded enzymes served as “cores.” It was demonstrated that the main problem in making such a biosensor is to select a pair of oppositely charged polyelectrolytes that would be optimal for enzyme functioning. The best pairs were PAA/DS and PAA/PSS for lactate dehydrogenase, and PSS/PAA and PSS/PDADMA for urease. We designed and prepared enzyme-containing microcapsules differing in polyelectrolyte composition and number of layers, and investigated their properties.

Tam N.K., Uyen N.Q., Hong H.A., Duc L.H., Hoa T.T., Serra C.R., Henriques A.O., Cutting S.M.

ABSTRACT Bacillus subtilis is considered a soil organism for which endospore formation provides a means to ensure long-term survival in the environment. We have addressed here the question of what happens to a spore when ingested. Spores displaying on their surface a heterologous antigen, tetanus toxin fragment C (TTFC), were shown to generate anti-TTFC responses not to the antigen contained in the primary oral inoculum but to those displayed on spores that had germinated and then resporulated. We then used reverse transcription-PCR to determine expression of vegetative genes and sporulation-specific genes in the mouse gut following oral dosing with spores. Significant levels of germination and sporulation were documented. Using natural isolates of B. subtilis that could form biofilms, we showed that these strains could persist in the mouse gut for significantly longer than the laboratory strain. Moreover, these isolates could grow and sporulate anaerobically and exhibited a novel phenomenon of being able to form spores in almost half the time required for the laboratory isolate. This suggests that spores are not transient passengers of the gastrointestinal tract but have adapted to carry out their entire life cycle within this environment. This is the first report showing an intestinal life cycle of B. subtilis and suggests that other Bacillus species could also be members of the gut microflora.

De Geest B.G., Déjugnat C., Sukhorukov G.B., Braeckmans K., De Smedt S.C., Demeester J.

Skirtach A.G., Antipov A.A., Shchukin D.G., Sukhorukov G.B.

We present a novel method for remote release of an encapsulated material from polyelectrolyte capsules based on laser light illumination. Two different components were introduced in the polyelectrolyte shells of PAH/PSS capsules-either Ag nanoparticles or IR dye-to induce absorption of light. Under laser illumination the capsules containing Ag nanoparticles or IR dye were deformed or cut, thus providing a venue for remote release of encapsulated materials. The experiments were conducted with a low-power near-infrared continuous-wave laser diode.

Atrih A., Foster S.J.

Sukhorukov G.B., Antipov A.A., Voigt A., Donath E., Möhwald H.

pH-Controlled encapsulation in and release of macromolecules from polyelectrolyte capsules of a few microns in diameter is demonstrated. Capsules were prepared via alternating adsorption of the oppositely charged polymers poly(allylamine hydrochloride) and poly(styrene sulfonate) onto decomposable melamin formaldehyde cores. The capsules were open for macromolecules at pH values below 6 and closed at pH > 8.

Riesenman P.J., Nicholson W.L.

ABSTRACT Spores of Bacillus subtilis possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of B. subtilis (containing the gerE36 and/or cotE::cat mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H 2 O 2 ) and various artificial and solar UV treatments. Spores of strains carrying mutations in gerE and/or cotE were very sensitive to lysozyme and to 5% H 2 O 2 , as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing the gerE36 mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing the cotE::cat mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the gerE36 and cotE::cat mutations behaved like gerE36 mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.

Dubas S.T., Schlenoff J.B.

The dependence of polyelectrolyte multilayer thickness on salt concentration, salt type, solvent quality, deposition time, and polymer concentration is evaluated. Polymers are deposited on spinning silicon wafers. For the strong polycation/polyanion pair studied, film thickness is approximately proportional to the number of layers and the salt concentration. The irreversibility of overall molecule adsorption is indicated by the lack of exchange of surface (radiolabeled) for solution polymer. The hydrophobic nature of the driving force for polymer sorption is illustrated by the choice of salt counterion or solvent. Analyzed within the framework of ion exchange, the net energy of ion pair formation is not high, at most a few kT. Salt, competing with polymer segments for the surface, permits localized rearrangements. In the mechanism proposed, excess polymer is accommodated within several layers, rather than in one layer of loops and tails. Steric barriers coupled with slow conformational changes are respons...

Donath E., Sukhorukov G.B., Caruso F., Davis S.A., Möhwald H.

Exact control of the film thickness of polyelectrolyte shells (a transmission electron microscopy image is shown) is achieved by colloid-templated consecutive adsorption of polyanions and polycations followed by decomposition of the templating core. Possible areas of application for these shells range from the pharmaceutical, food, cosmetic, and paint industries to catalysis and microcrystallization.

Kim A.L., Musin E.V., Chebykin Y.S., Tikhonenko S.A.

Polyelectrolyte microcapsules (PMC) based on polyallylamine and polystyrene sulfonate are utilized in various fields of human activity, including medicine, textiles, and the food industry, among others. However, characteristics such as microcapsule size, shell thickness, and pore size are not sufficiently studied and systematized, even though they determine the possibility of using microcapsules in applied tasks. The aim of this review is to identify general patterns and gaps in the study of the morphology of polyelectrolyte microcapsules obtained by the alternate adsorption of polystyrene sulfonate and polyallylamine on different solid cores. First and foremost, it was found that the morphological change in polyelectrolyte microcapsules formed on different cores exhibits a significant difference in response to varying stimuli. Factors such as ionic strength, the acidity of the medium, and temperature have different effects on the size of the microcapsules, the thickness of their shells, and the number and size of their pores. At present, the morphology of the microcapsules formed on the melamine formaldehyde core has been most studied, while the morphology of microcapsules formed on other types of cores is scarcely studied. In addition, modern methods of nanoscale system analysis will allow for an objective assessment of PMC characteristics and provide a fresh perspective on the subject of research.

Dubrovskii A.V., Kim A.L., Musin E.V., Tikhonenko S.A.

AbstractPolyelectrolyte microcapsules can be applied as microcontainers for the delivery of a wide range of substances, and it is important to search for new methods for capsule destruction and releasing substances from them. In this work, we studied the possibility of using sodium dodecyl sulfonate (SDS) for the release of fluorescein isothiocyanate—dextran from six-layer microcapsules composed of PAH and PSS. It was shown that the presence of SDS in the medium, at a concentration of 3000 μg/ml, leads to the destruction of polyelectrolyte microcapsules and the release of the substance from them (54% of the amount of the encapsulated substance), while the main part of the FITC-dextran released during the first hours of incubation. At an SDS concentration of 100 μg/ml, the substance released is uniform and is 44% in 24 h. At SDS concentrations from 50 to 100 μg/ml, the process of destruction of microcapsules proceeds more slowly. At SDS concentrations from 10 to 50 μg/ml, microcapsules are not degraded.

Musin E.V., Kim A.L., Tikhonenko S.A.

Controlled release of substance from polyelectrolyte microcapsules is a triggered degradation of the microcapsule membrane that is extensive enough to release the contained substances out into the environment. Membrane degradation can be a result of enzymatic digestion, ultrasound or light exposure, heating, application of a magnetic field, pH or ionic strength changes in the solution or bacteria-mediated processes. This technology can be used for the targeted release of drugs, and for the development of self-healing materials and new generation pesticides.

Musin E.V., Kim A.L., Dubrovskii A.V., Ariskina E.V., Kudryashova E.B., Tikhonenko S.A.

Antimicrobial resistance is a global public health threat. One of the possible ways to solve this problem is phage therapy, but the instability of bacteriophages hinders the development of this approach. A bacteriophage delivery system that stabilizes the phage is one of the possible solutions to this problem. This study is dedicated to exploring methods to create encapsulated forms of bacteriophages for delivery. We studied the effect of proteolytic enzymes on the destruction of the polyelectrolyte microcapsule shell and revealed that protease from Streptomyces griseus was able to destroy the membrane of the microcapsule (dextran sulfate/polyarginine)3 ((DS/PArg)3). In addition, the protease decreased the activity of the bacteriophage in the second hour of incubation, and the phage lost activity after 16 h. It was found that a medium with pH 9.02 did not affect the survival of the bacteriophage or E. coli. The bacteriophages were encapsulated into polyelectrolyte microcapsules (DS/PArg)3. It was established that it is impossible to use microcapsules as a means of delivering bacteriophages since the bacteriophages are inactivated. When bacteriophages were included inside a CaCO3 core, it was demonstrated that the phage retained activity before and after the dissolution of the CaCO3 particle. From the results of this study, we recommend using CaCO3 microparticles as a container for bacteriophage delivery through the acidic stomach barrier.