Labelling of polyelectrolytes with fluorescent dyes and monitoring of electrostatic coating of bacterial cells with microscopy and calorimetry

Žive bakterijske celice so danes nepogrešljive na mnogih področjih biotehnologije in biomedicine. Za te namene pogosto spreminjamo površinske lastnosti celic. Ena od možnosti za modifikacijo celične površine je oblaganje celic z metodo nanoplastenja, pri kateri se plasti polielektrolitov nalagajo na nabito površino oblaganca na podlagi elektrostatskih interakcij. V magistrski nalogi smo spremljali tvorbo polikationske plasti na negativno nabiti površini bakterijskih celic. To je prva naloga, ki preučuje nanoplastenje celic s fluorescenčno mikroskopijo z vzbujenim praznjenjem emisije (STED). Namen je bil optimizacija postopka označevanja polikationov s fluorescentnim barvilom in izbor barvila, ki na uporabljenem STED mikroskopu izkazuje boljšo ločljivost metode kot na konfokalnem mikroskopu. Sistematično smo nanoplastenje s polielektroliti preučili na dveh vrstah celic (E. coli in P. stutzeri) z meritvijo zeta potenciala (ZP) in STED mikroskopijo. Nanooblaganje smo spremljali še z izotermno titracijsko kalorimetrijo (ITC). Rezultati kažejo izboljšanje ločljivosti na STED v primerjavi s konfokalnim mikroskopom pri barvilu STAR RED, ne pa pri ATTO 594 in rodamin-B-izotiocianatu. S STAR RED smo označili polikatione (visoko- in nizkomolekularna polietilenimin - PEI in polialilamin klorid - PAH) in z njimi obložili celice E. coli in P. stutzeri. ZP celic se je spremenil iz negativnega pred nanooblaganjem v pozitivnega po nanooblaganju. STED slike kažejo nastanek nanoobloge na celični površini. Ugotovili smo, da polikationi prehajajo v notranjost P. stutzeri, ne pa v E. coli, nismo pa dokazali razlik v prehajanju polikationov glede na molekulsko maso. Pri ITC so krivulje odvisnosti toplotnega toka od količine dodanega polielektrolita pri E. coli dosegale plato, pri katerem predpostavimo zasedenost celotne celične površine s polikationom. Pri P. stutzeri je bilo prisotno sipanje podatkov okoli pričakovane (sigmoidne) oblike krivulje zaradi prehajanja polielektrolita v notranjost. Nanooblaganje preučevanih bakterijskih celic s PEI in PAH je bilo uspešno, saj se je površinski naboj spremenil iz negativnega v pozitivnega, tvorba nanoobloge pa je vidna tudi s STED. Permeabilnost celične stene za polikation je manj odvisna od njegove molekulske mase in bolj od vrste celic, kar smo dokazali z opazovanjem lokalizacije polikationov po nanooblaganju s STED mikroskopijo. Z ITC potrdimo doseganje nasičenja celične površine s polikationom.Live bacterial cells are used in many biotechnological and biomedical applications that often require modifications of the cellular surface. A possible method for cell surface modification is nanocoating via layer-by-layer method, which is based on electrostatically driven deposition of polyelectrolytes onto a charged cell surface. We observed the formation of the polycation layer on a negatively charged bacterial cell surface. This is the first master thesis studying nanocoating of cells with stimulated emission depletion microscopy (STED). The aim of this work was to optimize a protocol for fluorescent labelling of polycations with a fluorescent dye and determine the appropriate dye, manifesting an improvement in resolution on the used STED microscope when compared to confocal microscope. A systematic study of nanocoating of two bacterial species (E. coli and P. stutzeri) was done by measuring zeta potential (ZP) and observing the nanocoated cells with STED. We also followed the nanocoating process with isothermal titration calorimetry (ITC). STAR RED showed an improved resolution in STED when compared to confocal microscopy, whereas ATTO 594 and rhodamine-B-isothiocyanate did not. We labelled polycations (high and low molecular weight polyethyleneimines - PEI and polyallylamine hidrochlorides - PAH) with STAR RED and used them to coat E. coli and P. stutzeri. STED microscopy showed the formation of a polyelectrolyte layer on the cell surface. Polycations permeated into P. stutzeri and not into E. coli. We did not observe a difference in cell wall penetration between high and low molecular weight polymers. ZP changed from negative before to positive after nanocoating. ITC termograms, showing the dependance of heat rate on the amount of added polymer, reach a plateau in the case of E. coli, suggesting achievement of cell surface saturation with polycation. In case of P. stutzeri the curve is not the expected sigmoidal shape as polycations permeate into the cell. We conclude the nanocoating of studied bacterial cells with PEI and PAH was successful, as surface charge changed from negative to positive. Permeation of polycations through the cell wall depends less on molecular weight and more on species, which was proven by observing the localization of fluorescently labelled polycations after nanocoating with STED. ITC confirmed the saturation of the cellular surface with the polycation

Drug delivery strategies for curcumin and other natural Nrf2 modulators of oxidative stress-related diseases

Oxidative stress is associated with a wide range of diseases characterised by oxidant-mediated disturbances of various signalling pathways and cellular damage. The only effective strategy for the prevention of cellular damage is to limit the production of oxidants and support their efficient removal. The implication of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in the cellular redox status has spurred new interest in the use of its natural modulators (e.g., curcumin, resveratrol). Unfortunately, most natural Nrf2 modulators are poorly soluble and show extensive pre-systemic metabolism, low oral bioavailability, and rapid elimination, which necessitates formulation strategies to circumvent these limitations. This paper provides a brief introduction on the cellular and molecular mechanisms involved in Nrf2 modulation and an overview of commonly studied formulations for the improvement of oral bioavailability and in vivo pharmacokinetics of Nrf2 modulators. Some formulations that have also been studied in vivo are discussed, including solid dispersions, self-microemulsifying drug delivery systems, and nanotechnology approaches, such as polymeric and solid lipid nanoparticles, nanocrystals, and micelles. Lastly, brief considerations of nano drug delivery systems for the delivery of Nrf2 modulators to the brain, are provided. The literature reviewed shows that the formulations discussed can provide various improvements to the bioavailability and pharmacokinetics of natural Nrf2 modulators. This has been demonstrated in animal models and clinical studies, thereby increasing the potential for the translation of natural Nrf2 modulators into clinical practice

Incorporation of Ethylcellulose Microparticles Containing a Model Drug with a Bitter Taste into Nanofibrous Mats by the Electrospinning Technique—Preliminary Studies

Electrospinning is considered a simple and comprehensive technique to formulate ultrafine fibres by using an electric field. Polymeric nanofibers constitute promising materials in biomedical applications as drug delivery systems. For their preparation, both natural and synthetic polymers are utilised. Owing to the potential use of electrospun nanofibers as an orodispersible drug dosage form, ethylcellulose microparticles containing the antihistamine drug rupatadine fumarate, prepared by the spray drying technique to conceal the drug’s bitter taste, were incorporated into nanofibers. The obtained nanofibrous mats were evaluated for morphology, mechanical strength, disintegration time, the drug solid state and acceptability in terms of taste masking efficiency. Preliminary studies showed that hypromellose used as a single polymer was not a suitable substance for the manufacturing of nanofibers. Therefore, in order to facilitate the obtention of homogeneous nonwovens, different grades of polyethylene oxide (2,000,000–2M-Da and 4,000,000–4M-Da) were added, which improved the quality of the prepared mats. Nanofibers of the most satisfactory quality were obtained from hypromellose (6.5% w/v) and PEO (2M, 0.5% w/v). SEM image analysis has shown that the nanofibers were homogeneous and smooth and possessed a fast disintegration time (below 30 s) and an adequate drug content with a simultaneous taste-masking effect (as indicated by the in vivo and in vitro methods). However, further studies are necessary to refine their mechanical characteristics

Incorporation of ethylcellulose microparticles containing a model drug with a bitter taste into nanofibrous mats by the electrospinning technique - preliminary studies

Electrospinning is considered a simple and comprehensive technique to formulate ultrafine fibres by using an electric field. Polymeric nanofibers constitute promising materials in biomedical applications as drug delivery systems. For their preparation, both natural and synthetic polymers are utilised. Owing to the potential use of electrospun nanofibers as an orodispersible drug dosage form, ethylcellulose microparticles containing the antihistamine drug rupatadine fumarate, prepared by the spray drying technique to conceal the drug’s bitter taste, were incorporated into nanofibers. The obtained nanofibrous mats were evaluated for morphology, mechanical strength, disintegration time, the drug solid state and acceptability in terms of taste masking efficiency. Preliminary studies showed that hypromellose used as a single polymer was not a suitable substance for the manufacturing of nanofibers. Therefore, in order to facilitate the obtention of homogeneous nonwovens, different grades of polyethylene oxide (2,000,000–2M-Da and 4,000,000–4M-Da) were added, which improved the quality of the prepared mats. Nanofibers of the most satisfactory quality were obtained from hypromellose (6.5% w/v) and PEO (2M, 0.5% w/v). SEM image analysis has shown that the nanofibers were homogeneous and smooth and possessed a fast disintegration time (below 30 s) and an adequate drug content with a simultaneous taste-masking effect (as indicated by the in vivo and in vitro methods). However, further studies are necessary to refine their mechanical characteristics

Nanofibers with genotyped Bacillus strains exhibiting antibacterial and immunomodulatory activity

Biofilm-associated diseases such as periodontitis are widespread and challenging to treat which calls for new strategies for their effective management. Probiotics represent a promising approach for targeted treatment of dysbiosis in biofilm and modulation of host immune response. In this interdisciplinary study, nanofibers with two autochthonous Bacillus strains 27.3.Z and 25.2.M were developed. The strains were isolated from the oral microbiota of healthy individuals, and their genomes were sequenced and screened for genes associated with antimicrobial and immunomodulatory activities, virulence factors, and transferability of resistance to antibiotics. Spores of two Bacillus strains were incorporated individually or in combination into hydrophilic poly(ethylene oxide) (PEO) and composite PEO/alginate nanofibers. The nanofiber mats were characterised by a high loading of viable spores (> 7 log CFU/mg) and they maintained viability during electrospinning and 6 months of storage at room temperature. Spores were rapidly released from PEO nanofibers, while presence of alginate in the nanofibers prolonged their release. All formulations exhibited swelling, followed by transformation of the nanofiber mat into a hydrogel and polymer erosion mediating spore release kinetics. The investigated Bacillus strains released metabolites, which were not cytotoxic to peripheral blood mononuclear cells (PBMCs) in vitro. Moreover, their metabolites exhibited antibacterial activity against two periodontopathogens, an antiproliferative effect on PBMCs, and inhibition of PBMC expression of proinflammatory cytokines. In summary, the developed nanofiber-based delivery system represents a promising therapeutic approach to combat biofilm-associated disease on two fronts, namely via modulation of the local microbiota with probiotic bacteria and host immune response with their metabolites