Dynamic analysis of DNA nanoparticle immobilization to model biomaterial substrates using combinatorial spectroscopic ellipsometry and quartz crystal microbalance with dissipation

Gene expression within cells can be altered through gene delivery approaches, which have tremendous potential for gene therapy, tissue engineering, and diagnostics. Substrate-mediated gene delivery describes the delivery of plasmid DNA or DNA complexed with nonviral vectors to cells from a surface, with the DNA immobilized to a substrate through specific or nonspecific interactions. In this work, DNA-nanoparticle (DNA–NP) adsorption to substrates is evaluated using combinatorial, in situ spectroscopic ellipsometry and quartz crystal microbalance with dissipation (SE/QCM-D), to evaluate the basic dynamic processes involved in the adsorption and immobilization of DNA–NP complexes to substrates. The concentration of DNA–NP solutions influences the adsorbed DNA–NP surface mass, which increases by a factor of approximately 6 (detected by SE) and approximately 4.5-fold (detected by QCM-D), as the DNA concentration increases from 1.5 μg/mL to 15 μg/mL, with an increase in layer porosity. In addition, SE/QCM-D analysis indicates that DNA–NP adsorption rates, surface coverage densities, and volume fractions are dependent on the type of substrate: gold (Au) and silicon dioxide substrates, proteincoated and uncoated substrates, and surfaces modified with alkanethiol self assembled monolayers (SAMs). These studies also demonstrate that the influence of an adsorbed protein layer on resulting DNA–NP immobilization efficiency is substrate dependent. For example, Au surfaces coated with fetal bovine serum (FBS) resulted in two-fold greater mass of adsorbed DNA–NPs, compared to DNA–NP adsorption to FBS-coated SAM substrates. This investigation offers insights into dynamic DNA–NP surface adsorption processes, characteristics of the immobilized DNA–NP layer, and demonstrates substrate-dependent DNA–NP adsorption

Dynamic analysis of DNA nanoparticle immobilization to model biomaterial substrates using combinatorial spectroscopic ellipsometry and quartz crystal microbalance with dissipation

Gene expression within cells can be altered through gene delivery approaches, which have tremendous potential for gene therapy, tissue engineering, and diagnostics. Substrate-mediated gene delivery describes the delivery of plasmid DNA or DNA complexed with nonviral vectors to cells from a surface, with the DNA immobilized to a substrate through specific or nonspecific interactions. In this work, DNA-nanoparticle (DNA–NP) adsorption to substrates is evaluated using combinatorial, in situ spectroscopic ellipsometry and quartz crystal microbalance with dissipation (SE/QCM-D), to evaluate the basic dynamic processes involved in the adsorption and immobilization of DNA–NP complexes to substrates. The concentration of DNA–NP solutions influences the adsorbed DNA–NP surface mass, which increases by a factor of approximately 6 (detected by SE) and approximately 4.5-fold (detected by QCM-D), as the DNA concentration increases from 1.5 μg/mL to 15 μg/mL, with an increase in layer porosity. In addition, SE/QCM-D analysis indicates that DNA–NP adsorption rates, surface coverage densities, and volume fractions are dependent on the type of substrate: gold (Au) and silicon dioxide substrates, proteincoated and uncoated substrates, and surfaces modified with alkanethiol self assembled monolayers (SAMs). These studies also demonstrate that the influence of an adsorbed protein layer on resulting DNA–NP immobilization efficiency is substrate dependent. For example, Au surfaces coated with fetal bovine serum (FBS) resulted in two-fold greater mass of adsorbed DNA–NPs, compared to DNA–NP adsorption to FBS-coated SAM substrates. This investigation offers insights into dynamic DNA–NP surface adsorption processes, characteristics of the immobilized DNA–NP layer, and demonstrates substrate-dependent DNA–NP adsorption

METHOD OFOBTAINING MICROGRAPHS OF TRANSPARENT OR SEMI-TRANSPARENT SPECIMENS USING ANISOTROPIC CONTRAST

Anisotropic contrast methodology in combination with use of sample investigating polarized electromagnetic radiation to provide Jones or Mueller Matrix imaging data corresponding to areas on samples

Combined QCM-D/GE as a tool to characterize stimuli-responsive swelling of and protein adsorption on polymer brushes grafted onto 3D-nanostructures

A combined setup of quartz crystal microbalance and generalized ellipsometry can be used to comprehensively investigate complex functional coatings comprising stimuli-responsive polymer brushes and 3D nanostructures in a dynamic, noninvasive in situ measurement. While the quartz crystal microbalance detects the overall change in areal mass, for instance, during a swelling or adsorption process, the generalized ellipsometry data can be evaluated in terms of a layered model to distinguish between processes occurring within the intercolumnar space or on top of the anisotropic nanocolumns. Silicon films with anisotropic nanocolumnar morphology were prepared by the glancing angle deposition technique and further functionalized by grafting of poly-(acrylic acid) or poly-(N-isopropylacrylamide) chains. Investigations of the thermoresponsive swelling of the poly-(N-isopropylacrylamide) brush on the Si nanocolumns proved the successful preparation of a stimuli-responsive coating. Furthermore, the potential of these novel coatings in the field of biotechnology was explored by investigation of the adsorption of the model protein bovine serum albumin. Adsorption, retention, and desorption triggered by a change in the pH value is observed using poly-(acrylic acid) functionalized nanostructures, although generalized ellipsometry data revealed that this process occurs only on top of the nanostructures. Poly-(N-isopropylacrylamide) is found to render the nanostructures non-fouling properties. Includes supplemental materials

Combined QCM-D/GE as a tool to characterize stimuli-responsive swelling of and protein adsorption on polymer brushes grafted onto 3D-nanostructures

A combined setup of quartz crystal microbalance and generalized ellipsometry can be used to comprehensively investigate complex functional coatings comprising stimuli-responsive polymer brushes and 3D nanostructures in a dynamic, noninvasive in situ measurement. While the quartz crystal microbalance detects the overall change in areal mass, for instance, during a swelling or adsorption process, the generalized ellipsometry data can be evaluated in terms of a layered model to distinguish between processes occurring within the intercolumnar space or on top of the anisotropic nanocolumns. Silicon films with anisotropic nanocolumnar morphology were prepared by the glancing angle deposition technique and further functionalized by grafting of poly-(acrylic acid) or poly-(N-isopropylacrylamide) chains. Investigations of the thermoresponsive swelling of the poly-(N-isopropylacrylamide) brush on the Si nanocolumns proved the successful preparation of a stimuli-responsive coating. Furthermore, the potential of these novel coatings in the field of biotechnology was explored by investigation of the adsorption of the model protein bovine serum albumin. Adsorption, retention, and desorption triggered by a change in the pH value is observed using poly-(acrylic acid) functionalized nanostructures, although generalized ellipsometry data revealed that this process occurs only on top of the nanostructures. Poly-(N-isopropylacrylamide) is found to render the nanostructures non-fouling properties. Includes supplemental materials

The Role of Surface Chemistry-Induced Cell Characteristics on Nonviral Gene Delivery to Mouse Fibroblasts

Background: Gene delivery approaches serve as a platform to modify gene expression of a cell population with applications including functional genomics, tissue engineering, and gene therapy. The delivery of exogenous genetic material via nonviral vectors has proven to be less toxic and to cause less of an immune response in comparison to viral vectors, but with decreased efficiency of gene transfer. Attempts have been made to improve nonviral gene transfer efficiency by modifying physicochemical properties of gene delivery vectors as well as developing new delivery techniques. In order to further improve and understand nonviral gene delivery, our approach focuses on the cell-material interface, since materials are known to modulate cell behavior, potentially rendering cells more responsive to nonviral gene transfer. In this study, self-assembled monolayers of alkanethiols on gold were employed as model biomaterial interfaces with varying surface chemistries. NIH/3T3 mouse fibroblasts were seeded on the modified surfaces and transfected using either lipid- or polymer- based complexing agents. Results: Transfection was increased in cells on charged hydrophilic surfaces presenting carboxylic acid terminal functional groups, while cells on uncharged hydrophobic surfaces presenting methyl terminations demonstrated reduced transfection for both complexing agents. Surface–induced cellular characteristics that were hypothesized to affect nonviral gene transfer were subsequently investigated. Cells on charged hydrophilic surfaces presented higher cell densities, more cell spreading, more cells with ellipsoid morphologies, and increased quantities of focal adhesions and cytoskeleton features within cells, in contrast to cell on uncharged hydrophobic surfaces, and these cell behaviors were subsequently correlated to transfection characteristics. Conclusions: Extracellular influences on nonviral gene delivery were investigated by evaluating the upregulation and downregulation of transgene expression as a function of the cell behaviors induced by changes in the cells’ microenvronments. This study demonstrates that simple surface modifications can lead to changes in the efficiency of nonviral gene delivery. In addition, statistically significant differences in various surface-induced cell characteristics were statistically correlated to transfection trends in fibroblasts using both lipid and polymer mediated DNA delivery approaches. The correlations between the evaluated complexing agents and cell behaviors (cell density, spreading, shape, cytoskeleton, focal adhesions, and viability) suggest that polymer-mediated transfection is correlated to cell morphological traits while lipid-mediated transfection correlates to proliferative characteristics

Economic analysis of logging residues production

Magistro darbe tiriamas miško kirtimo liekanų ruošos ekonominė analizė. Darbo objektas: miško kirtimo liekanų ruošos ekonominė analizė VĮ Valstybinių miškų urėdijos Tauragės regioniniame padalinyje. Darbo tikslas: atlikti miško kirtimo liekanų ruošos ekonominę analizę. Darbo metodai: miško kirtimo liekanų ruošos ekonominė analizė 2017–2017 metais. Darbo rezultatai: Įvertinus VĮ Valstybinių miškų urėdijos Tauragės regioniniame padalinyje surinktus duomenis apie biokuro gamybą ir prekybą 2015–2017 metais, matomas ženklus pagaminamų ir parduodamų kiekių svyravimas. Susumavus visų metų pagamintą ir parduotą biokurą pastebima, kad per pastaruosius metus Tauragės regioninis padalinys pagamindavo ir parduodavo nuo 2,3 iki 4,5 tūkst. m³, vidutiniškai 3,4 tūkst. m³ kirtimo liekanų kurui, tokiu būdu vykdydamas VĮ Valstybinių miškų urėdijos nustatytas kirtimo liekanų ruošos užduotis. Kasmet buvo parduodamos ne visos paruoštos kirtimo liekanos. 2016 metais buvo parduota tik 2,3 tūkst. m³ kirtimo liekanų, nes dalies liekanų pardavimas vėlavo. Skiedrų Tauragės padalinys pagamino vidutiniškai 11,2 tūkst. m³, arba 37,2 tūkst. m³ per trejus metus. Tauragės regioniniame padalinyje 2015–2017 metais skiedra buvo gaminama iš įvairių sortimentų - malkinės medienos (ML-1; ML-2; ML-3) plokščių medienos (PM-SP; PM-LP) ir kirtimo liekanų (KA, KAD). Skiedrų kiekis buvo matuojamas po sumalimo pakrovus į transportą bei taikant glaudumo koeficientus. Nagrinėjamu laikotarpiu skiedrų gamybos apimtys išaugo nuo 5,3 tūkst. m³ 2015 metais iki 15–17 tūkst. m³ 2016–2017 metais. VĮ Valstybinių miškų urėdija Tauragės regioniniame padalinyje kirtimo liekanų gamybos vidutinė savikaina siekia 7,0 Eur/m³, skiedros gamybos savikaina 52,36 Eur/m³. Esant kirtimo liekanų vidutinei kainai 12,2 Eur/m³, o skiedros kainai 61,34 Eur/m³, abiejų produkcijos rūšių gamyba yra pelninga. Skiedrų transportavimas į biokuro katilines padidina išlaidas 20 %. Atlikta apklausa (anketa 1 priedas) regioniniuose padaliniuose atskleidė, kad kirtimo liekanų surenkama nuo 5 tūkst. m³ iki 17 tūkst.m³. Surenkamus kiekinius įtakoja padalinių vadovai (38 %), centrinė vadovybė (47 %) ir dalinai gamtos sąlygos, augavietė bei gruntas (15 %). Kirtimo liekanos yra naudingos (atsakė 90 %). Iš plynų kirtimo biržių išvežami kirtimo liekanų kiekiai tinkami (64 %), bet ne visada visos KA yra parduodamos. Skiedras gamina trys padaliniai: žaliavas daugiausiai sudaro 60 % malkinė mediena, 40 % kirtimo liekanos ir iš jų bendrai pagamina skiedros vidutiniškai 14–18 tūkst. m³. Į technologijų investavimą (antrinės produkcijos biokuro) gaminimui, regioninių padalinių 46 % neinvestuoja, 46 % noriai investuoja, o 8 % planuoja investuoti. Skiedros realizuojamos visos kiek pagaminama.Economic analysis of logging residues production in VĮ Valstybinių miškų urėdija Tauragės regioninis padalinys. Subjects of research – VIVMU Tauragės regioninis padalinys. Aim of the research – to carry out economic analysis of logging residues. Methodology – comparative analysis, scientific literature analysis, accounting data processing methods, quantitative analysis. Reluts. Production of biofuel in Tauragė regional division of State Forest Enterprise is being investigated in the article as well as economic analysis of the process is being carried out. The amount of forest biofuel production and sales in 2015–2017 is being examined and analyzed. Calculations of the costs and profitability of collection and hauling of forest biofuel are presented. The results of research showed that Tauragė regional division of State Forest Enterprise during 2015–2017 produced and sold 11,2 thous. m³ of logging residues, 37,2 thous. m³ of wood chips and 386,4 thous. m³ of commercial roundwood. In order to find out the quantities of biofuel production produced, biofuel piles and chips in trailers were measured. Knowing the quantities of the products after grinding into the chips, the use of material labor and monetary resources, which showed profitability of forest logging residues production, were calculated.Miškų ir ekologijos fakultetasMiškotvarkos ir medienotyros instituta