Biofilm contamination on an implanted medical device represents a particularity resilient reservoir of infection that inevitably leads to device failure. In this study, we demonstrate that an ...atmospheric pressure air plasma treatment can simultaneously eradicate biofilm contamination while beneficially functionalizing the underlying surface, creating long-lasting characteristics that inhibit microbial recolonization and promote fibroblast proliferation. By comparing two contrasting plasma treatments the interplay between plasma generated reactive species, biofilm contamination and the underlying surface was uncovered. The composition, wettability and topography of titanium surfaces were characterized using X-ray photoelectron spectroscopy, water contact angle measurements and atomic force microscopy. Exposure to plasma generated chemical species created nanoscale surface features and the introduction of oxygen and nitrogen containing functional groups, resulting in changes to the surface wettability. Using a polymicrobial biofilm model comprising of E. coli and S. epidermidis, it was shown that plasma can effectively eliminate biofilm contamination from the surface, while simultaneously functionalizing the surface to inhibit recolonization. To assess the biocompatibility of treated surfaces the adhesion and proliferation of murine fibroblasts was assessed using fluorescent microscopy, cell viability assays and flow cytometry. It was shown that plasma exposure led to surface characteristics that promote fibroblast adhesion and proliferation.
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•Low temperature plasma simultaneously decontaminates and functionalizes titanium.•Plasma exposure creates nanoscale features and superhydrophilic characteristics.•Plasma exposure is highly effective in eliminating polymicrobial biofilms.•Plasma treated surface inhibits microbial recolonization and biofilm formation.•Biocompatibility improved through enhanced cell adhesion and proliferation.
Wound healing is a complex biological response to injury characterized by a sequence of interdependent and overlapping physiological actions. To study wound healing and cutaneous regeneration ...processes, the complexity of wound healing requires the use of animal models. In this chapter, we describe the protocol to generate skin wounds in a mouse model. In the mouse splinted excisional wound model, two full-thickness wounds are firstly created on the mouse dorsum, which is followed by application of silicone splint around wounded area. A splinting ring tightly adheres to the skin around full-thickness wound, preventing wound contraction and replicating human processes of re-epithelialization and new tissue formation. The wound is easily accessible for treatment as well as for daily monitoring and quantifying the wound closure.This technique represents valuable approach for the study of wound healing mechanisms and for evaluation of new therapeutic modalities. In this protocol, we describe how to utilize the model to study the effect of gene electrotransfer of plasmid DNA coding for antiangiogenic molecules. Additionally, we also present how to precisely regulate electrical parameters and modify electrode composition to reach optimal therapeutic effectiveness of gene electrotransfer into skin around wounded area.
The antibacterial and cell-proliferative character of atmospheric pressure plasma jets (APPJs) helps in the healing process of chronic wounds. However, control of the plasma-biological target ...interface remains an open issue. High vacuum ultraviolet/ultraviolet (VUV/UV) radiation and RONS flux from plasma may cause damage of a treated tissue; therefore, controlled interaction is essential. VUV/UV emission from argon APPJs and radiation control with aerosol injection in plasma effluent is the focus of this research. The aerosol effect on radiation is studied by a fluorescent target capable of resolving the plasma oxidation footprint. In addition, DNA damage is evaluated by plasmid DNA radiation assay and cell proliferation assay to assess safety aspects of the plasma jet, the effect of VUV/UV radiation, and its control with aerosol injection. Inevitable emission of VUV/UV radiation from plasmas during treatment is demonstrated in this work. Plasma has no antiproliferative effect on fibroblasts in short treatments (t < 60 s), while long exposure has a cytotoxic effect, resulting in decreased cell survival. Radiation has no effect on cell survival in the medium due to absorption. However, a strong cytotoxic effect on the attached fibroblasts without the medium is apparent. VUV/UV radiation contributes 70% of the integral plasma effect in induction of single- and double-strand DNA breaks and cytotoxicity of the attached cells without the medium. Survival of the attached cells increases by 10% when aerosol is introduced between plasma and the cells. Injection of aerosol in the plasma effluent can help to control the plasma–cell/tissue interaction. Aerosol droplets in the effluent partially absorb UV emission from the plasma, limiting photon flux in the direction of the biological target. Herein, cold and safe plasma-aerosol treatment and a safe operational mode of treatment are demonstrated in a murine model.
Introduction: Electroporation allows efficient delivery of DNA into cells and tissues, thereby improving the expression of therapeutic or immunogenic proteins that are encoded by plasmid DNA. This ...simple and versatile method holds a great potential and could address unmet medical needs such as the prevention or treatment of many cancers or infectious diseases.
Areas covered: This review explores the electroporation mechanism and the parameters affecting its efficacy. An analysis of past and current clinical trials focused on DNA electroporation is presented. The pathologies addressed, the protocol used, the treatment outcome and the tolerability are highlighted. In addition, several of the possible optimization strategies for improving patient compliance and therapeutic efficacy are discussed such as plasmid design, use of genetic adjuvants for DNA vaccines, choice of appropriate delivery site and electrodes as well as pulse parameters.
Expert opinion: The growing number of clinical trials and the results already available underline the strong potential of DNA electroporation which combines both safety and efficiency. Nevertheless, it remains critical to further increase fundamental knowledge to refine future strategies, to develop concerted and common DNA electroporation protocols and to continue exploring new electroporation-based therapeutic options.
DNA vaccination against cancer has become a promising strategy for inducing a specific and long-lasting antitumor immunity. However, DNA vaccines fail to generate potent immune responses when used as ...a single therapy. To enhance their activity into the tumor, a DNA vaccine against murine P815 mastocytoma was combined with antibodies directed against the immune checkpoints CTLA4 and PD1. The combination of these two strategies delayed tumor growth and enhanced specific antitumor immune cell infiltration in comparison to the corresponding single therapies. The combination also promoted IFNg, IL12 and granzyme B production in the tumor microenvironment and decreased the formation of liver metastasis in a very early phase of tumor development, enabling 90% survival. These results underline the complementarity of DNA vaccination and immune checkpoint blockers in inducing a potent immune response, by exploiting the generation of antigen-specific T cells by the vaccine and the ability of immune checkpoint blockers to enhance T cell activity and infiltration in the tumor. These findings suggest how and why a rational combination therapy can overcome the limits of DNA vaccination but could also allow responses to immune checkpoint blockers in a larger proportion of subjects.
Biomedical applications of plasma require its efficacy for specific purposes and equally importantly its safety. Herein the safety aspects of cold plasma created with simple atmospheric pressure ...plasma jet produced with helium gas and electrode discharge are evaluated in skin damage on mouse, at different duration of exposure and gas flow rates. The extent of skin damage and treatments are systematically evaluated using stereomicroscope, labelling with fluorescent dyes, histology, infrared imaging and optical emission spectroscopy. The analyses reveal early and late skin damages as a consequence of plasma treatment, and are attributed to direct and indirect effects of plasma. The results indicate that direct skin damage progresses with longer treatment time and increasing gas flow rates which reflect changes in plasma properties. With increasing flow rates, the temperature on treated skin grows and the RONS formation rises. The direct effects were plasma treatment dependent, whereas the disclosed late-secondary effects were more independent on discharge parameters and related to diffusion of RONS species. Thermal effects and skin heating are related to plasma-coupling properties and are separated from the effects of other RONS. It is demonstrated that cumulative topical treatment with helium plasma jet could lead to skin damage. How these damages can be mitigated is discussed in order to provide guidance, when using atmospheric pressure plasma jets for skin treatments.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The exact mechanisms of nucleic acid (NA) delivery with gene electrotransfer (GET) are still unknown, which represents a limitation for its broader use. Further, not knowing the effects that ...different experimental electrical and biological parameters have on GET additionally hinders GET optimization, resulting in the majority of research being performed using a trial-and-error approach. To explore the current state of knowledge, we conducted a systematic literature review of GET papers in in vitro conditions and performed meta-analyses of the reported GET efficiency. For now, there is no universal GET strategy that would be appropriate for all experimental aims. Apart from the availability of the required electroporation device and electrodes, the choice of an optimal GET approach depends on parameters such as the electroporation medium; type and origin of cells; and the size, concentration, promoter, and type of the NA to be transfected. Equally important are appropriate controls and the measurement or evaluation of the output pulses to allow a fair and unbiased evaluation of the experimental results. Since many experimental electrical and biological parameters can affect GET, it is important that all used parameters are adequately reported to enable the comparison of results, as well as potentially faster and more efficient experiment planning and optimization.
We aimed to explore whether the combination of intradermal DNA vaccination, to boost immune response against melanoma antigens, and immune checkpoint blockade, to alleviate immunosuppression, ...improves antitumor effectiveness in a murine B16F10 melanoma tumor model. Compared to single treatments, a combination of intradermal DNA vaccination (ovalbumin or gp100 plasmid adjuvanted with IL12 plasmid) and immune checkpoint CTLA-4/PD-1 blockade resulted in a significant delay in tumor growth and prolonged survival of treated mice. Strong activation of the immune response induced by combined treatment resulted in a significant antigen-specific immune response, with elevated production of antigen-specific IgG antibodies and increased intratumoral CD8+ infiltration. These results indicate a potential application of the combined DNA vaccination and immune checkpoint blockade, specifically, to enhance the efficacy of DNA vaccines and to overcome the resistance to immune checkpoint inhibitors in certain cancer types.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
DNA vakcine predstavljajo obetaven pristop imunoterapije raka, predvsem zaradi njihove enostavnosti, stabilnosti in varnosti. DNA vakcine temeljijo na vnosu plazmidne DNA z zapisom za enega ali več ...tumorskih antigenov, udeleženih v nastanku, napredovanju ali zasevanju rakavih celic. Z vnosom DNA vakcine spodbudimo imunski odziv usmerjen proti tumorskemu antigenu, kar potencialno vodi do uničenja rakavih celic. Kljub številnim prednostim DNA vakcin pred klasičnimi vakcinami njihovo uporabo v humani kliniki omejuje prešibak antigen-specifični imunski odziv. Za izboljšanje imunogenosti DNA vakcin se razvoj osredotoča na optimizacijo sestave plazmidne DNA, raziskovanje novih tumorskih antigenov, razvoj novih dostavnih sistemov in sočasna uporaba adjuvantnih in imunomodulatornih molekul. Tovrstni pristopi so del številnih kliničnih raziskav na področju imunoterapije raka in predstavljajo korak bližje k izboljšanju imunogenosti DNA vakcin, učinkovitejšemu uničenju rakavih celic in lažjemu prenosu DNA vakcinacije v humano klinično prakso.
The accuracy of techniques such as microarrays, reverse transcription polymerase chain reaction, and whole transcriptome shotgun sequencing is critically dependent on RNA quality. We have repeatedly ...observed extensive RNA degradation following trypsinization, a routine procedure used to dissociate adherent tissue culture cells prior to RNA extraction. This study investigated the cause of this degradation and identifies an alternative procedure that enables extraction of intact high-quality RNA. Trypsinization and several alternative procedures were used to dissociate a range of different cell lines prior to RNA extraction. The contribution of exogenous ribonucleases or induction of endogenous ribonucleases by trypsin reagent proteases to RNA degradation was examined. Trypsinization resulted in a complete degradation of RNA regardless of cell line type, differentiation stage, or passage number. This occurred when intact RNA was incubated directly with trypsin and was not suppressed by inhibiting trypsin’s protease activity. Prevention of degradation by sodium hypochlorite treatment of trypsin reagent identified the presence of ribonucleases in trypsin derived from animal pancreas. Consistent extraction of high-quality RNA requires the use of direct cell lysis with a phenol guanidine-based reagent or an animal origin-free protease-based dissociation agent if enzymatic detachment prior to RNA extraction cannot be avoided.
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