Carbon-Based Nanomaterials Increase Reactivity of Primary Monocytes towards Various Bacteria and Modulate Their Differentiation into Macrophages.

The evaluation of carbon-based nanomaterials' (C-BNMs') interactions with the immune system, notably their ability to cause inflammation, is a critical step in C-BNM health risk assessment. Particular attention should be given to those C-BNMs that do not cause direct cytotoxicity or inflammation on their own. However, the intracellular presence of these non-biodegradable nanomaterials could dysregulate additional cell functions. This is even more crucial in the case of phagocytes, which are the main mediators of defensive inflammation towards pathogens. Hence, our study was focused on multi-walled carbon nanotubes (MWCNTs) and two different types of graphene platelets (GPs) and whether their intracellular presence modulates a proinflammatory response from human primary monocytes towards common pathogens. Firstly, we confirmed that all tested C-BNMs caused neither direct cytotoxicity nor the release of tumour necrosis factor α (TNF-α), interleukin (IL)-6 or IL-10. However, such pre-exposed monocytes showed increased responsiveness to additional bacterial stimuli. In response to several types of bacteria, monocytes pre-treated with GP1 produced a significantly higher quantity of TNF-α, IL-6 and IL-10. Monocytes pre-treated with MWCNTs produced increased levels of IL-10. All the tested C-BNMs enhanced monocyte phagocytosis and accelerated their differentiation towards macrophages. This study confirms the immunomodulatory potential of C-BNMs.

Overexpression of CD44v8-10 in Colon Polyps-A Possible Key to Early Diagnosis.

Background and aims: The majority of colorectal cancers arise from detectable adenomatous or serrated lesions. Here we demonstrate how deregulated alternative splicing of CD44 gene in diseased colon mucosa results in downregulation of standard isoform of CD44 gene (CD44s) and upregulation of variant isoform CD44v8-10. Our aim is to show that upregulation of CD44v8-10 isoform is a possible marker of precancerous lesion in human colon. Methods: We analysed pairs of fresh biopsy specimen of large intestine in a cohort of 50 patients. We studied and compared alternative splicing profile of CD44 gene in colon polyps and adjoined healthy colon mucosa. We performed end-point and qRT PCR, western blotting, IHC staining and flow cytometry analyses. Results: We detected more than five-fold overexpression of CD44v8-10 isoform and almost twenty-fold downregulation of standard isoform CD44s in colon polyps compared to adjoined healthy tissue with p = 0.018 and p < 0.001 in a cohort of 50 patients. Our results also show that aberrant splicing of CD44 occurs in both biologically distinct subtypes of colorectal adenoma possibly in ESRP-1 specific manner. Conclusion: 92% of the colon polyp positive patients overexpressed CD44v8-10 isoform in their colon polyps while only 36% of them had positive fecal occult blood test which is currently a standard non-invasive screening technique. Impact: We believe that our results are important for further steps leading to application of CD44v8-10 isoform as a biomarker of colorectal precancerosis in non-invasive detection. Early detection of colon precancerosis means successful prevention of colorectal carcinoma.

Myomedin scaffold variants targeted to 10E8 HIV-1 broadly neutralizing antibody mimic gp41 epitope and elicit HIV-1 virus-neutralizing sera in mice.

One of the proposed strategies for the development of a more efficient HIV-1 vaccine is based on the identification of proteins binding to a paratope of chosen broadly neutralizing antibody (bNAb) that will mimic cognate HIV-1 Env (glyco)protein epitope and could be used as potent immunogens for induction of protective virus-neutralizing antibodies in the immunized individuals. To verify this "non-cognate ligand" concept, we developed a highly complex combinatorial library designed on a scaffold of human myomesin-1 protein domain and selected proteins called Myomedins specifically binding to variable regions of HIV-1 broadly neutralizing antibody 10E8. Immunization of mice with these Myomedin variants elicited the production of HIV-1 Env-specific antibodies. Hyperimmune sera bound to Env pseudotyped viruses and weakly/moderately neutralized 54% of tested clade A, B, C, and AE pseudotyped viruses variants in vitro. These results demonstrate that Myomedin variants have the potential to mimic Env epitopes and could be used as potential HIV-1 vaccine components.

Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors.

Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow field-flow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (Rh) measured by online DLS with the Rh values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no self-associations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The Rg/Rh ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot.

Important parameters affecting quality of vitrified donor oocytes.

For group of 281 oocytes obtained from 43 stimulated donors and cryopreserved by vitrification protocol using Cryotop and Kitazato medium we determined important parameters of oocytes collection and vitrification processes which strongly affect the probability that warmed oocytes will produce high-quality embryos for transfer. The probability to obtain high-quality embryos for transfer from vitrified and warmed oocytes was highest when two conditions were fulfilled: 1. oocytes were incubated before vitrification for 7-10 h and 2. stimulated ovaries of donors in one cycle produced a smaller number of oocytes (<7 oocytes from one donor per stimulated cycle). The probable reasons for these observations were: 1. early vitrification (less than 7 h) before final oocyte metaphase II maturation negatively affected the crucial process of post-warm remodelling of spindles and chromosomes, which reduced the fertilization and utilization rates, 2. the evaluated vitrification protocol amplifies negative impact of membrane defects of oocytes of those cohorts containing more than 6 oocytes - freezing places great demands on the integrity and elasticity of the cell membranes. The fact that cryopreservation influences a complex state of oocytes was confirmed by confocal microscopy.

A novel approach to imaging engorged ticks: Micro-CT scanning of Ixodes ricinus fed on blood enriched with gold nanoparticles.

Micro-computed tomography (micro-CT) is an exceptional imaging modality which is limited in visualizing soft biological tissues that need pre-examination contrasting steps, which can cause serious deformation to sizeable specimens like engorged ticks. The aim of this study was to develop a new technique to bypass these limitations and allow the imaging of fed ticks in their natural state. To accomplish this, adult Ixodes ricinus females were allowed to engorge in vitro on blood supplemented with PEGylated gold nanoparticles (PEG-AuNPs). In total, 73/120 females divided into 6 groups engorged on blood enriched with 0.07-2.16 mg PEG-AuNPs per ml of blood. No toxic effect was observed for any of the tested groups compared to the control group, in which 12/20 females engorged on clear blood. The ticks were scanned on a Bruker micro-CT SkyScan 1276. The mean radiodensity of the examined ticks exceeded 0 Hounsfield Units only in the case of the two groups with the highest concentration. The best contrast was observed in ticks engorged on blood with the highest tested concentration of 2.16 mg/mL PEG-AuNPs. In these ticks, the midgut and rectal sac were clearly visible. Also, the midgut lumen volume was computed from segmented image data. The reduction in midgut volume was documented during the egg development process. According to this pilot study, micro-CT of ticks engorged on blood supplemented with contrasting agents in vitro may reveal additional information regarding the engorged ticks' anatomy.

Electrophilic characteristics and aqueous behavior of fatty acid nitroalkenes.

Fatty acid nitroalkenes (NO2-FA) are endogenously-generated products of the reaction of metabolic and inflammatory-derived nitrogen dioxide (.NO2) with unsaturated fatty acids. These species mediate signaling actions and induce adaptive responses in preclinical models of inflammatory and metabolic diseases. The nitroalkene substituent possesses an electrophilic nature, resulting in rapid and reversible reactions with biological nucleophiles such as cysteine, thus supporting post-translational modifications (PTM) of proteins having susceptible nucleophilic centers. These reactions contribute to enzyme regulation, modulation of inflammation and cell proliferation and the regulation of gene expression responses. Herein, focus is placed on the reduction-oxidation (redox) characteristics and stability of specific NO2-FA regioisomers having biological and clinical relevance; nitro-oleic acid (NO2-OA), bis-allylic nitro-linoleic acid (NO2-LA) and the conjugated diene-containing nitro-conjugated linoleic acid (NO2-cLA). Cyclic and alternating-current voltammetry and chronopotentiometry were used to the study of reduction potentials of these NO2-FA. R-NO2 reduction was observed around -0.8 V (vs. Ag/AgCl/3 M KCl) and is related to relative NO2-FA electrophilicity. This reduction process could be utilized for the evaluation of NO2-FA stability in aqueous milieu, shown herein to be pH dependent. In addition, electron paramagnetic resonance (EPR) spectroscopy was used to define the stability of the nitroalkene moiety under aqueous conditions, specifically under conditions where nitric oxide (.NO) release could be detected. The experimental data were supported by density functional theory calculations using 6-311++G (d,p) basis set and B3LYP functional. Based on experimental and computational approaches, the relative electrophilicities of these NO2-FA are NO2-cLA >> NO2-LA > NO2-OA. Micellarization and vesiculation largely define these biophysical characteristics in aqueous, nucleophile-free conditions. At concentrations below the critical micellar concentration (CMC), monomeric NO2-FA predominate, while at greater concentrations a micellar phase consisting of self-assembled lipid structures predominates. The CMC, determined by dynamic light scattering in 0.1 M phosphate buffer (pH 7.4) at 25 °C, was 6.9 (NO2-LA) 10.6 (NO2-OA) and 42.3 µM (NO2-cLA), respectively. In aggregate, this study provides new insight into the biophysical properties of NO2-FA that are important for better understanding the cell signaling and pharmacological potential of this class of mediators.

Thrombus Imaging Using 3D Printed Middle Cerebral Artery Model and Preclinical Imaging Techniques: Application to Thrombus Targeting and Thrombolytic Studies.

Diseases with the highest burden for society such as stroke, myocardial infarction, pulmonary embolism, and others are due to blood clots. Preclinical and clinical techniques to study blood clots are important tools for translational research of new diagnostic and therapeutic modalities that target blood clots. In this study, we employed a three-dimensional (3D) printed middle cerebral artery model to image clots under flow conditions using preclinical imaging techniques including fluorescent whole-body imaging, magnetic resonance imaging (MRI), and computed X-ray microtomography (microCT). Both liposome-based, fibrin-targeted, and non-targeted contrast agents were proven to provide a sufficient signal for clot imaging within the model under flow conditions. The application of the model for clot targeting studies and thrombolytic studies using preclinical imaging techniques is shown here. For the first time, a novel method of thrombus labeling utilizing barium sulphate (Micropaque®) is presented here as an example of successfully employed contrast agents for in vitro experiments evaluating the time-course of thrombolysis and thus the efficacy of a thrombolytic drug, recombinant tissue plasminogen activator (rtPA). Finally, the proof-of-concept of in vivo clot imaging in a middle cerebral artery occlusion (MCAO) rat model using barium sulphate-labelled clots is presented, confirming the great potential of such an approach to make experiments comparable between in vitro and in vivo models, finally leading to a reduction in animals needed.

Hypromellose - A traditional pharmaceutical excipient with modern applications in oral and oromucosal drug delivery.

Hydroxypropylmethylcellulose (HPMC), also known as Hypromellose, is a traditional pharmaceutical excipient widely exploited in oral sustained drug release matrix systems. The choice of numerous viscosity grades and molecular weights available from different manufacturers provides a great variability in its physical-chemical properties and is a basis for its broad successful application in pharmaceutical research, development, and manufacturing. The excellent mucoadhesive properties of HPMC predetermine its use in oromucosal delivery systems including mucoadhesive tablets and films. HPMC also possesses desirable properties for formulating amorphous solid dispersions increasing the oral bioavailability of poorly soluble drugs. Printability and electrospinnability of HPMC are promising features for its application in 3D printed drug products and nanofiber-based drug delivery systems. Nanoparticle-based formulations are extensively explored as antigen and protein carriers for the formulation of oral vaccines, and oral delivery of biologicals including insulin, respectively. HPMC, being a traditional pharmaceutical excipient, has an irreplaceable role in the development of new pharmaceutical technologies, and new drug products leading to continuous manufacturing processes, and personalized medicine. This review firstly provides information on the physical-chemical properties of HPMC and a comprehensive overview of its application in traditional oral drug formulations. Secondly, this review focuses on the application of HPMC in modern pharmaceutical technologies including spray drying, hot-melt extrusion, 3D printing, nanoprecipitation and electrospinning leading to the formulation of printlets, nanoparticle-, microparticle-, and nanofiber-based delivery systems for oral and oromucosal application. Hypromellose is an excellent excipient for formulation of classical dosage forms and advanced drug delivery systems. New methods of hypromellose processing include spray draying, hot-melt extrusion, 3D printing, and electrospinning.

Gadolinium labelled nanoliposomes as the platform for MRI theranostics: in vitro safety study in liver cells and macrophages.

Gadolinium (Gd)-based contrast agents are extensively used for magnetic resonance imaging (MRI). Liposomes are potential nanocarrier-based biocompatible platforms for development of new generations of MRI diagnostics. Liposomes with Gd-complexes (Gd-lip) co-encapsulated with thrombolytic agents can serve both for imaging and treatment of various pathological states including stroke. In this study, we evaluated nanosafety of Gd-lip containing PE-DTPA chelating Gd+3 prepared by lipid film hydration method. We detected no cytotoxicity of Gd-lip in human liver cells including cancer HepG2, progenitor (non-differentiated) HepaRG, and differentiated HepaRG cells. Furthermore, no potential side effects of Gd-lip were found using a complex system including general biomarkers of toxicity, such as induction of early response genes, oxidative, heat shock and endoplasmic reticulum stress, DNA damage responses, induction of xenobiotic metabolizing enzymes, and changes in sphingolipid metabolism in differentiated HepaRG. Moreover, Gd-lip did not show pro-inflammatory effects, as assessed in an assay based on activation of inflammasome NLRP3 in a model of human macrophages, and release of eicosanoids from HepaRG cells. In conclusion, this in vitro study indicates potential in vivo safety of Gd-lip with respect to hepatotoxicity and immunopathology caused by inflammation.

Proinflammatory Effect of Carbon-Based Nanomaterials: In Vitro Study on Stimulation of Inflammasome NLRP3 via Destabilisation of Lysosomes.

Carbon-based nanomaterials (C-BNM) have recently attracted an increased attention as the materials with potential applications in industry and medicine. Bioresistance and proinflammatory potential of C-BNM is the main obstacle for their medicinal application which was documented in vivo and in vitro. However, there are still limited data especially on graphene derivatives such as graphene platelets (GP). In this work, we compared multi-walled carbon nanotubes (MWCNT) and two different types of pristine GP in their potential to activate inflammasome NLRP3 (The nod-like receptor family pyrin domain containing 3) in vitro. Our study is focused on exposure of THP-1/THP1-null cells and peripheral blood monocytes to C-BNM as representative models of canonical and alternative pathways, respectively. Although all nanomaterials were extensively accumulated in the cytoplasm, increasing doses of all C-BNM did not lead to cell death. We observed direct activation of NLRP3 via destabilization of lysosomes and release of cathepsin B into cytoplasm only in the case of MWCNTs. Direct activation of NLRP3 by both GP was statistically insignificant but could be induced by synergic action with muramyl dipeptide (MDP), as a representative molecule of the family of pathogen-associated molecular patterns (PAMPs). This study demonstrates a possible proinflammatory potential of GP and MWCNT acting through NLRP3 activation.

Preparation of nanoliposomes by microfluidic mixing in herring-bone channel and the role of membrane fluidity in liposomes formation.

Introduction of microfluidic mixing technique opens a new door for preparation of the liposomes and lipid-based nanoparticles by on-chip technologies that are applicable in a laboratory and industrial scale. This study demonstrates the role of phospholipid bilayer fragment as the key intermediate in the mechanism of liposome formation by microfluidic mixing in the channel with "herring-bone" geometry used with the instrument NanoAssemblr. The fluidity of the lipid bilayer expressed as fluorescence anisotropy of the probe N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) was found to be the basic parameter affecting the final size of formed liposomes prepared by microfluidic mixing of an ethanol solution of lipids and water phase. Both saturated and unsaturated lipids together with various content of cholesterol were used for liposome preparation and it was demonstrated, that an increase in fluidity results in a decrease of liposome size as analyzed by DLS. Gadolinium chelating lipids were used to visualize the fine structure of liposomes and bilayer fragments by CryoTEM. Experimental data and theoretical calculations are in good accordance with the theory of lipid disc micelle vesiculation.

Targeting Human Thrombus by Liposomes Modified with Anti-Fibrin Protein Binders.

Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bß chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.

Proteins mimicking epitope of HIV-1 virus neutralizing antibody induce virus-neutralizing sera in mice.

BACKGROUND: The development of an effective vaccine preventing HIV-1 infection is hindered by the enormous antigenic variability and unique biochemical and immunological properties of HIV-1 Env glycoprotein, the most promising target for HIV-1 neutralizing antibody. Functional studies of rare elite neutralizers led to the discovery of broadly neutralizing antibodies. METHODS: We employed a highly complex combinatorial protein library derived from a 5 kDa albumin-binding domain scaffold, fused with support protein of total 38 kDa, to screen for binders of broadly neutralizing antibody VRC01 paratope. The most specific binders were used for immunization of experimental mice to elicit Env-specific antibodies and to test their neutralization activity using a panel of HIV-1 clade C and B pseudoviruses. FINDINGS: Three most specific binders designated as VRA017, VRA019, and VRA177 exhibited high specificity to VRC01 antibody. Immunized mice produced Env-binding antibodies which neutralize eight of twelve HIV-1 Tier 2 pseudoviruses. Molecular modelling revealed a shape complementarity between VRA proteins and a part of VRC01 gp120 interacting surface. INTERPRETATION: This strategy based on the identification of protein replicas of broadly neutralizing antibody paratope represents a novel approach in HIV-1 vaccine development. This approach is not affected by low immunogenicity of neutralization-sensitive epitopes, variability, and unique biochemical properties of HIV-1 Env used as a crucial antigen in the majority of contemporary tested vaccines. FUND: Czech Health Research Council 15-32198A, Ministry of Health, Czech Republic.

Application of Advanced Microscopic Methods to Study the Interaction of Carboxylated Fluorescent Nanodiamonds with Membrane Structures in THP-1 Cells: Activation of Inflammasome NLRP3 as the Result of Lysosome Destabilization.

Nanodiamonds (ND), especially fluorescent NDs, represent potentially applicable drug and probe carriers for in vitro/in vivo applications. The main purpose of this study was to relate physical-chemical properties of carboxylated NDs to their intracellular distribution and impact on membranes and cell immunity-activation of inflammasome in the in vitro THP-1 cell line model. Dynamic light scattering, nanoparticle tracking analysis, and microscopic methods were used to characterize ND particles and their intracellular distribution. Fluorescent NDs penetrated the cell membranes by both macropinocytosis and mechanical cutting through cell membranes. We proved accumulation of fluorescent NDs in lysosomes. In this case, lysosomes were destabilized and cathepsin B was released into the cytoplasm and triggered pathways leading to activation of inflammasome NLRP3, as detected in THP-1 cells. Activation of inflammasome by NDs represents an important event that could underlie the described toxicological effects in vivo induced by NDs. According to our knowledge, this is the first in vitro study demonstrating direct activation of inflammasome by NDs. These findings are important for understanding the mechanism(s) of action of ND complexes and explain the ambiguity of the existing toxicological data.

Bioimmunological activities of Candida glabrata cellular mannan.

Candida glabrata is a second most common human opportunistic pathogen which causes superficial but also life-threatening systemic candidosis. According to the localisation of mannans and mannoproteins in the outermost layer of the cell wall, mannan detection could be one of the first steps in the cell recognition of Candida cells by the host innate immune system. Mannans from the cell wall provide important immunomodulatory activities, comprising stimulation of cytokine production, induction of dendritic cells (DCs) maturation and T-cell immunity. The model of DCs represents a promising tool to study immunomodulatory interventions throughout the vaccine development. Activated DCs induce, activate and polarise T-cell responses by expression of distinct maturation markers and cytokines regulating the adaptive immune responses. In addition, they are uniquely adept at decoding the fungus-associated information and translate it in qualitatively different T helper responses. We find out, that C. glabrata mannan is able to induce proliferation of splenocytes and to increase the production of TNF-α and IL-4. Next, increased the expression of co-stimulatory molecules CD80 and CD86 and the proportion of CD4+CD25+ and CD4+CD28+ T cells during in vitro stimulation of splenocytes. Reported results provide C. glabrata mannan capability to modulate cytokine production, DCs activation and antigen presentation activity, influencing T-cell phenotype in response to stimulation.

N-Oxy lipid-based click chemistry for orthogonal coupling of mannan onto nanoliposomes prepared by microfluidic mixing: Synthesis of lipids, characterisation of mannan-coated nanoliposomes and in vitro stimulation of dendritic cells.

New synthetic aminooxy lipid was designed and synthesized as a building block for the formulation of functionalised nanoliposomes (presenting onto the outer surface of aminooxy groups) by microfluidic mixing. Orthogonal binding of cellular mannan (Candida glabrata (CCY 26-20-1) onto the outer surface of functionalised nanoliposomes was modified by orthogonal binding of reducing termini of mannans to oxime lipids via a click chemistry reaction based on aminooxy coupling (oxime ligation). The aminooxy lipid was proved as a suitable active component for preparation of functionalised nanoliposomes by the microfluidic mixing method performed with the instrument NanoAssemblr™. This "on-chip technology" can be easily scaled-up. The structure of mannan-liposomes was visualized by transmission and scanning electron microscopy, including immunogold staining of recombinant mannan receptor bound onto mannosylated-liposomes. The observed structures are in a good correlation with data obtained by DLS, NTA, and TPRS methods. In vitro experiments on human and mouse dendritic cells demonstrate selective internalisation of fluorochrome-labelled mannan-liposomes and their ability to stimulate DC comparable to lipopolysaccharide. We describe a potentially new drug delivery platform for mannan receptor-targeted antimicrobial drugs as well as for immunotherapeutics. Furthermore, the platform based on mannans bound orthogonally onto the surface of nanoliposomes represents a self-adjuvanted carrier for construction of liposome-based recombinant vaccines for both systemic and mucosal routes of administration.

Hyaluronic Acid Surface Modified Liposomes Prepared via Orthogonal Aminoxy Coupling: Synthesis of Nontoxic Aminoxylipids Based on Symmetrically α-Branched Fatty Acids, Preparation of Liposomes by Microfluidic Mixing, and Targeting to Cancer Cells Expressing CD44.

New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling ( N-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.

Lasioglossins LLIII affect the morphogenesis of Candida albicans and reduces the duration of experimental vaginal candidiasis in mice.

Lasioglossins are a group of peptides with identified antimicrobial activity. The inhibitory effects of two synthetic lasioglossin derivatives, LLIII and D-isomeric variant LLIII-D, on morphological changes in Candida albicans in vitro and the effect of local administration of LLIII during experimental murine candidiasis were investigated. C. albicans blastoconidia were grown in the presence of lasioglossin LLIII or LLIII-D at concentrations of 11.5 µM and 21 µM, respectively, for 1, 2 and 3 days and their viability determined by flow cytometry using eosin Y staining. Morphological changes were examined by light and fluorescent microscopy. The Candida-inhibitory effect of daily intravaginal administration of 0.7 or 1.4 µg of LLIII was assessed in mice with experimentally-induced vaginal candidiasis. LLIII and LLIII-D lasioglossins exhibited candidacidal activity in vitro (>76% after 24 hr and >84% after 48 hr of incubation). After 72 hr incubation of Candida with low concentration of lasioglossins, an increase in viability was detected, probably due to a Candida antimicrobial peptides evasion strategy. Furthermore, lasioglossins inhibited temperature-induced morphotype changes toward hyphae and pseudohyphae with sporadic occurrence of atypical cells with two or enlarged nuclei, suggesting interference with mitosis or cytokinesis. Local application of LLIII reduced the duration of experimental candidiasis with no evidence of adverse effects. Lasioglossin LLIII is a promising candidate for development as an antimicrobial drug for treating the vaginal candidiasis.