Synthesis, characterization and cytocompatibility of lyophilized spherical particles of poly (ɛ-caprolactone) and examination of their impact on the formation of reactive oxygen species

Poli(ε-kaprolakton) (PCL) je biokompatibilan i biodegradabilan poliestar, koji se koristi za različite biomedicinske primene, kao što je kontrolisana dostava lekova, tkivno inženjerstvo, fiksacija koštanih fraktura i druge. Jedan od osnovnih uslova za primenu PCL polimera na polju kontrolisane dostave medikamenata, jesu odgovarajuće morfološke osobine čestica, tj. njihova sferičnost i velika uniformnost. \ud Liofilizacija se koristi u farmaceutskoj industriji prilikom dobijanja najrazličitijih medikamenata, a predstavlja postupak sušenja pri redukovanom pritisku i pri vrlo niskim temperaturama. Iako omogućuje dobijanje čestica dovoljne stabilnosti za distribuciju i skladištenje, ovaj proces generiše izvesne negativne uticaje na strukturu samih formulacija. U cilju prevazilaženja ovakvih nezgoda, formulaciji se dodaju krioprotektanti, supstance koje štite čestice tokom zamrzavanja. Odabir krioprotektanta ni malo nije lak zadatak. Radi obezbeđivanja maksimalne protekcije čestica, potrebno je odabrati pravi tip krioprotektanta i optimizovati njegovu koncentraciju. \ud U radu je opisana sinteza mikrosfera PCL-a fizičkohemijskom rastvarač/nerastvarač metodom i data je komparacija morfoloških karakteristika čestica sušenih u liofilizatoru, uz korišćenje različitih krioprotektanata: poli(L-glutaminske kiseline) (PGA), poli(vinil alkohola) (PVA), saharoze ili glukoze. Uzorci su karakterisani metodama infracrvene spektroskopije sa Fourierovom transformacijom (FT-IR), skenirajuće elektronske mikroskopije (SEM) kao i metodom analize difrakcije laserske svetlosti na česticama (DLS). U analizi svojstava materijala koji je procesiran u cilju potencijalne primene u oblasti biomedicine, veoma je važno ispitati njegovu citokompatibilnost, odnosno meru u kojoj je materijal podesan da koegzistira u prisustvu ćelija, tako da ne utiče na njihov prirodni životni proces, u smislu podsticanja procesa degeneracije i umiranja. Takođe, veoma je važno ispitati i uticaj materijala na prekomerno obrazovanje reaktivnih kiseoničnih vrsta (reactive oxygen species - ROS) i nastanak oksidativnog stresa, koji se dovodi u vezu sa mnogim oboljenjima. Citokompatibilnost uzoraka je ispitana MTT (3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolijum bromid) testom, dok je ROS efekat ispitan DCFH-DA (2′-7′-dihlorodihidrofluorescein diacetat) testom.Poly(ε-caprolactone) (PCL) is a biocompatible and biodegradable polyester, extensively investigated for applications in controlled drug delivery, tissue engineering, bone fracture fixation etc. One of the crucial conditions for the application of polymers in controlled drug delivery are morphological characteristics of the particles, their sphericity and a high uniformity. \ud Freeze drying, also termed lyophilization, is a process of drying in conditions of reduced pressure and very low temperatures. Although this technique was considered as a good method to conserve the integrity of particulate systems, various stresses are generated during the process. To protect nanoparticles from stresses and subsequent aggregation, a cryoprotectant or lyoprotectant is generally used. The selection of a proper cryoprotectant is not straightforward. In some cases, increasing cryoprotectant concentration to a certain level may eventually reach a limit of stabilization or even destabilize nanoparticles. In general, the type of cryoprotectant must be selected and its concentration must be optimized to ensure a maximum stabilization of nanoparticles. \ud The aim of this study was to produce PCL spherical particles using physico-chemical solvent/nonsolvent method, and to evaluate different cryoprotective excipients that were used in order to increase stability of the particles and protect them from freezing stress. Sugars, such as glucose and sucrose, and polymers (PVA and PGA), were added to the formulation to test the effects on the outcome of freeze-dried poly(ε-caprolactone) micro- and nanospheres. Samples were characterized using Fourier Transform Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM) and Dynamic Light Scattering method (DLS). In the analysis of properties of materials processed for applications in biomedicine, it is of crucial importance to test its cytocompatibility, the extent at which the material is able to coexist in the presence of cells without interrupting their living process in terms of promoting degeneration and death. Furthermore, it is very important to investigate the effects on excessive production of reactive oxygen species (ROS) and the oxidative stress, which is responsible for many diseases. In vitro studies used, include MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), testing cytotoxicity as the quality of being toxic to cells, and DCFH-DA assay (2’,7’-dichlordihydrofluorescein-diacetate), testing the possible increase in ROS levels

Freeze-drying method to produce a range of PCL particles with tailored morphological properties

Poly (ε-caprolactone) (PCL) is a widely investigated bioresorbable polymer and it has been extensively used in numerous biomaterials applications especially in tissue engineering and drug delivery systems. Freeze-dried particles of poly (ε-caprolactone), with different morphological characteristics (spherical or cube in shape), were prepared by physicochemical method with solvent/non-solvent systems and by using the different types of cryoprotectants. Natural polymer poly (L-glutamic acid) (PGA) as well as disaccharide, saccharose, were used as cryoprotectant i.e. substance that is used to protect particles from freezing damage (damage due to ice formation). PGA has dual role in the synthesis; besides as cryoprotectant, it acts as stabilizer of the particles i.e. to prevent their agglomeration. The samples were characterized by Fourier transform infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM). The biocompatibility of the samples was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The formation of intracellular reactive oxygen species was measured spectrophotometrically using a fluorescent probe

Effects of different cryoprotectants on morphology of lyophilized poly(ε-caprolactone) micro and nanospheres

A common limitation of using polymeric micro and nanoparticles in long-term conservation is due to their poor physical and chemical stability. Freeze-drying is one of the most convenient methods that enable further reconstitution of micro and nanoparticles for therapeutical use. Nevertheless, this process generates various stresses during freezing and desiccation steps. The aim of this study was to evaluate different cryoprotectants (protective excipients that are usually added to increase stability upon storage and protect the particles from freezing stress): sugars (glucose and sucrose) and polymers (PVA and PGA), on the outcome of freeze-dried poly(ε-caprolactone) micro and nanospheres. The best freeze-drying results in terms of morphological characteristics, analyzed with SEM, were achieved with glucose at concentration of 1%. The FTIR analysis confirmed that the molecular structure of PCL particles remained the same after the addition cryoprotectants

Fixation and Permeabilization Approaches for Scanning Electrochemical Microscopy of Living Cells

Scanning electrochemical microscopy (SECM) has been widely used for the electrochemical imaging of dynamic topographical and metabolic changes in alive adherent mammalian cells. However, extracting intracellular information by SECM is challenging, since it requires redox species to travel in and out the lipid cell membrane. Herein, we present cell fixation and permeabilization approaches as an alternative tool for visualizing cell properties by SECM. With this aim, adherent cells were analyzed in the SECM feedback mode in three different conditions: i) alive; ii) fixed and iii) fixed and permeabilized. The fixation was carried out with formaldehyde and does not damage lipid membranes. Therefore, this strategy can be used for the SECM investigation of cell topography or the passive transport of the redox mediator into the cells. Additional permeabilization of the cell membrane after fixation enables the analysis of the intracellular content through the coupling of SECM with immunoassay strategies for the detection of specific biomarkers. The latter was successfully applied as an easy and fast screening approach to detect the expression of the melanoma associated marker tyrosinase in adherent melanoma cell lines corresponding to different cancer progression stages using the SECM substrate generation – tip collection mode. The present approach is simple, fast and reliable and can open new ways to analyze cell cultures with electrochemically based scanning probe techniques

Locus of control, negative live events and psychopathological symptoms in collectivist adolescents

This document is the authors’ version of the final accepted manuscript, published online 21.10.2019 by Personality and Individual Differences. - Correspondence concerning this article should be addressed to Prof. Paulo Moreira, Instituto de Psicologia e de Ciências da Educação, Universidade Lusíada, Rua de Moçambique 21 e 71, Porto 4100-348, Portugal. Email: [email protected] current research trend is the identification of psychosocial variables that moderate and/or mediate the association between stressors and psychopathological symptoms. Research has shown Locus of Control (LoC) is a key cognitive component of this psychological process in adolescents from individualist cultures. It is unclear whether this finding can be generalized to collectivist adolescents given that LoC is argued to be a culturally relative construct. The study examined the moderating and mediating effects of LoC on the relationship between negative events and psychopathological symptoms (anxiety and depressive symptoms) in adolescents from collectivist countries (n = 2800). Consistent with prior research, negative life events and external LoC were associated with more psychopathological symptoms. Unlike past studies with samples from individualist countries, the study did not produce clear evidence that LoC moderated or mediated this relationship. Results are discussed in terms of cultural differences in the (un)desirability of external control

Atomic Force Microscopy of Biological Systems: Quantitative Imaging and Nanomotion Detection

The core of this thesis is the application of different modalities of atomic force microscopy to study living systems. After a brief introduction, quantitative imaging state of the art is presented and the applications for mammalian, plant, bacterial, and yeast cells are reviewed. The same chapter contains research effort about exploring nanomechanical properties of bone cells exposed to conditions that simulate the ones in outer space, in order to investigate the changes in architectural rearrangements of cells during space flight. The next section takes the focus on probing bacterial adhesion sites with an antibody-modified tip, a research important for the understanding of how bacterial cells adhere to the host and establish an infection. Plant cells and their nanomechanical profiles are also covered in the chapter. A large portion of the thesis consists in development of a new nanomechanical sensing technique, and the exploration of its potential applications. The technique is based on the AFM detection method and focuses on transducing small fluctuations that define living systems into a measurable mechanical change in the cantilever. The fact that there is a disproportionate rise in antimicrobial-resistant bacteria and that our lives depend on the new drugs and diagnostic tools to fight them has fueled the first and largest application of the technique â rapid antimicrobial susceptibility resting. Chapter 3 starts with the introduction of the technique, basic principles and guidelines for its use, and continues on potential applications. Proof of principle of the technique is presented, showing experimental results using blind clinical samples of bloodstream infectious agents. A separate section is devoted to investigations concerning slow-growing bacteria and the application of the technique to rapidly determine which antibiotic would best work against them. It then expands on the applications towards detecting metabolic activity of mitochondria and their response to substrates and inhibition. Furthermore, the method has been described as a rapid anti-cancer profiling tool that might pave the way towards a diagnostic platform for personalized medical treatment. The final section of the third chapter contains the current knowledge of the origins of oscillatory movement that governs the nanomotion technique. It presents different hypothesis and results towards exploring them. The thesis ends with a summary of the main ideas and conclusions about the presented work and future perspectives

Mitochondrial activity detected by cantilever based sensor

Our team recently demonstrated that cantilever based devices can detect signature of life in a chemistry independent manner. In this technique, the organism of interest is attached onto a classical AFM cantilever. If alive, it induces nanometre scale oscillations of the cantilever that disappear once the organism is killed. The technique was successfully used on bacteria, yeast, vegetal and mammalian cells. In this work we demonstrate that the method can also be applied to sub-cellular organelles, such as mitochondria. Mitochondria are involved in cellular energy production and are present in most eukaryotic cells. Nowadays, it is believed that mitochondria were originally prokaryotes that colonized eukaryotic cells and that live in an endosymbiotic way ever since. Here we present that mitochondria are also animated by nanometre scale oscillations that depend on their metabolic state and that stop once they are inhibited. This observation opens novel avenues to investigate the numerous mitochondria-related diseases in humans

Detecting nanoscale vibrations as signature of life

The existence of life in extreme conditions, in particular in extraterrestrial environments, is certainly one of the most intriguing scientific questions of our time. In this report, we demonstrate the use of an innovative nanoscale motion sensor in life-searching experiments in Earth-bound and interplanetary missions. This technique exploits the sensitivity of nanomechanical oscillators to transduce the small fluctuations that characterize living systems. The intensity of such movements is an indication of the viability of living specimens and conveys information related to their metabolic activity. Here, we show that the nanomotion detector can assess the viability of a vast range of biological specimens and that it could be the perfect complement to conventional chemical life-detection assays. Indeed, by combining chemical and dynamical measurements, we could achieve an unprecedented depth in the characterization of life in extreme and extraterrestrial environments.status: publishe