Spotlight on mycobacterial lipid exploitation using nanotechnology for diagnosis, vaccines, and treatments.

Tuberculosis (TB), historically the most significant cause of human morbidity and mortality, has returned as the top infectious disease worldwide, under circumstances worsened by the COVID-19 pandemic's devastating effects on public health. Although Mycobacterium tuberculosis, the causal agent, has been known of for more than a century, the development of tools to control it has been largely neglected. With the advancement of nanotechnology, the possibility of engineering tools at the nanoscale creates unique opportunities to exploit any molecular type. However, little attention has been paid to one of the major attributes of the pathogen, represented by the atypical coat and its abundant lipids. In this review, an overview of the lipids encountered in M. tuberculosis and interest in exploiting them for the development of TB control tools are presented. Then, the amalgamation of nanotechnology with mycobacterial lipids from both reported and future works are discussed.

2D Nanosheets-A New Class of Therapeutic Formulations against Cancer.

Researchers in cancer nanomedicine are exploring a revolutionary multifaceted carrier for treatment and diagnosis, resulting in the proposal of various drug cargos or "magic bullets" in this past decade. Even though different nano-based complexes are registered for clinical trials, very few products enter the final stages each year because of various issues. This prevents the formulations from entering the market and being accessible to patients. In the search for novel materials, the exploitation of 2D nanosheets, including but not limited to the highly acclaimed graphene, has created extensive interest for biomedical applications. A unique set of properties often characterize 2D materials, including semiconductivity, high surface area, and their chemical nature, which allow simple decoration and functionalization procedures, structures with high stability and targeting properties, vectors for controlled and sustained release of drugs, and materials for thermal-based therapies. This review discusses the challenges and opportunities of recently discovered 2D nanosheets for cancer therapeutics, with special attention paid to the most promising design technologies and their potential for clinical translation in the future.

Pathogen associated molecular pattern-decorated mesoporous silica-A colloidal model for studying bacterial-host cell interactions.

Tuberculosis is the top infectious disease worldwide and the development of a vaccine and diagnostic tools to control the disease is a priority that requires a better understanding of the factors involved in the pathogenesis of Mycobacterium tuberculosis, the infectious agent. It is known that bacterial cell surface components are released, interact with immune cell receptors, and may traffic toward host cell structures. Many of these compounds are lipids that have been associated with mycobacterial virulence. However, their hydrophobic nature has frequently hampered their biological study. In this work, silica particles were coated with functional lipids to obtain a colloidal bioinspired system based on nonhydrosoluble glycolipids. Mycobacterium tuberculosis phosphatidylinositol mannosides (PIMs), known to interact with receptors of innate immune cells, were purified from the M. tuberculosis H37Rv type strain, and used to prepare large unilamellar liposomes in combination with zwitterionic phosphatidyl choline. Then, bacillary-like Santa Barbara Amorphous-15 (SBA-15) silica particles were cationized and the vesicle fusion method was used to promote the attachment of anionic PIM-containing lipid bilayers. Thermogravimetric analysis, x-ray diffraction, N2 adsorption-desorption isotherm analysis, Fourier transform infrared spectroscopy, electron microscopy, and zeta potential analyses were used to characterize the materials obtained. The as-prepared PIM-containing colloids, named PIM@SBA-15, showed biocompatibility toward human fibroblasts and were found to colocalize with Toll-like receptor (TLR)2 upon their incubation with THP1-derived macrophages. Furthermore, the particles induced the formation of pseudopods and were internalized into phagocytic cells. In all, these data suggest the usefulness of PIM@SBA-15 particles to better comprehend the interactions between immune cells and PIMs.

Fiber optic interferometric immunosensor based on polydimethilsiloxane (PDMS) and bioactive lipids.

We demonstrate a novel and simple means to fabricate optical fiber immunosensors based on Fabry-Perot (F-P) interferometers using polydimethylsiloxane (PDMS) as support for bioactive lipids. The sensors are fabricated following a straightforward dip-coating method producing PDMS end-capped devices. A biosensing platform is realized by subsequent functionalization of the PDMS cap with a previously characterized bioactive lipid antigen cocktail from Mycobacterium fortuitum, used as a surrogate source of antigens for tuberculosis diagnosis. After functionalization of the PDMS, the F-P sensors were immersed in different antibody-containing sera and the registered changes in their spectral features were associated to the interactions between the active lipids and the serum antibodies. Our results show that the proposed PDMS end-capped F-P immunosensors perform well differentiating antibody-containing sera. Furthermore, they offer attractive attributes such as label-free operation, real-time detection capabilities and they are also reusable. The proposed sensors, therefore, serve as an enabling optical immunosensing technique offering excellent potential for developing novel lipidomic analytical tools.

Shock Wave Application Increases the Antineoplastic Effect of Molecular Iodine Supplement in Breast Cancer Xenografts.

We evaluated the effect of oral molecular iodine supplementation and shock wave application under three different conditions on human MDA-MB231 cancer cell xenografts. After tumor volume reached 1 cm3, mice were randomly assigned to groups and treated for 3 weeks. The results revealed that high-dose shock wave treatment (150 shock waves at a pressure of 21.7 MPa, SW150/21.7) generated tissue lesions without decreasing tumor growth, canceled the antineoplastic action of iodine and promoted pro-tumor conditions (increased hypoxia-induced factor [HIF] and vascular endothelial growth factor [VEGF]). In contrast, moderate (SW35/21.7) and low (SW35/9.9) doses of shock waves had significant antineoplastic effects and, in combination with iodine supplement, attenuated the aggressiveness of these cells by decreasing expression of the markers of stem cells (CD44 and Sox2) and invasion (HIF and VEGF). These results allow us to propose the combination of shock waves and iodine as a possible adjuvant in breast cancer therapy.

Mycobacterial glycolipid Di-O-acyl trehalose promotes a tolerogenic profile in dendritic cells.

Due to prolonged coevolution with the human being, Mycobacterium tuberculosis has acquired a sophisticated capacity to evade host immunity and persist in a latent state in the infected individual. As part of this evolutive process, mycobacteria have developed a highly complex cell wall that acts as a protective barrier. Herein we studied the effects of Di-O-acyl trehalose, a cell-wall glycolipid of virulent mycobacteria on murine bone marrow-derived dendritic cells. We have demonstrated that Di-O-Acyl-trehalose promotes a tolerogenic phenotype in bone marrow-derived murine DCs activated with mycobacterial antigens and Toll-like receptor agonists. This phenotype included low expression of antigen presentation and costimulatory molecules and altered cytokine production with downregulation of IL-12 and upregulation of IL-10, an anti-inflammatory cytokine. Additional markers of tolerogenicity were the expression of Indoleamine 2,3-dioxygenase and CD25. Furthermore, Di-O-Acyl-Trehalose promoted the expansion of FoxP3+ regulatory T lymphocytes. A better understanding of mycobacterial cell-wall components involved in the evasion of immunity is a prerequisite to designing better strategies to fight tuberculosis.

Shock wave-induced permeabilization of mammalian cells.

Controlled permeabilization of mammalian cell membranes is fundamental to develop gene and cell therapies based on macromolecular cargo delivery, a process that emerged against an increasing number of health afflictions, including genetic disorders, cancer and infections. Viral vectors have been successfully used for macromolecular delivery; however, they may have unpredictable side effects and have been limited to life-threatening cases. Thus, several chemical and physical methods have been explored to introduce drugs, vaccines, and nucleic acids into cells. One of the most appealing physical methods to deliver genes into cells is shock wave-induced poration. High-speed microjets of fluid, emitted due to the collapse of microbubbles after shock wave passage, represent the most significant mechanism that contributes to cell membrane poration by this technique. Herein, progress in shock wave-induced permeabilization of mammalian cells is presented. After covering the main concepts related to molecular strategies whose applications depend on safer drug delivery methods, the physics behind shock wave phenomena is described. Insights into the use of shock waves for cell membrane permeation are discussed, along with an overview of the two major biomedical applications thereof-i.e., genetic modification and anti-cancer shock wave-assisted chemotherapy. The aim of this review is to summarize 30 years of data showing underwater shock waves as a safe, noninvasive method for macromolecular delivery into mammalian cells, encouraging the development of further research, which is still required before the introduction of this promising tool into clinical practice.

Shock Wave-Induced Damage and Poration in Eukaryotic Cell Membranes.

Shock waves are known to permeabilize eukaryotic cell membranes, which may be a powerful tool for a variety of drug delivery applications. However, the mechanisms involved in shock wave-mediated membrane permeabilization are still poorly understood. In this study, the effects on both the permeability and the ultrastructural features of two human cell lineages were investigated after the application of underwater shock waves in vitro. Scanning Electron Microscopy of cells derived from a human embryo kidney (HEK)-293 and Michigan Cancer Foundation (MCF)-7 cells, an immortalized culture derived from human breast adenocarcinoma, showed a small amount of microvilli (as compared to control cells), the presence of hole-like structures, and a decrease in cell size after shock wave exposure. Interestingly, these effects were accompanied by the permeabilization of acid and macromolecular dyes and gene transfection. Trypan blue exclusion assays indicated that cell membranes were porated during shock wave treatment but resealed after a few seconds. Deformations of the cell membrane lasted for at least 5 min, allowing their observation in fixed cells. For each cell line, different shock wave parameters were needed to achieve cell membrane poration. This difference was correlated to successful gene transfection by shock waves. Our results demonstrate, for the first time, that shock waves induce transient micro- and submicrosized deformations at the cell membrane, leading to cell transfection and cell survival. They also indicate that ultrastructural analyses of cell surfaces may constitute a useful way to match the use of shock waves to different cells and settings.

Shock waves and DNA-cationic lipid assemblies: a synergistic approach to express exogenous genes in human cells.

Cationic lipid/DNA complexes (lipoplexes) represent a powerful tool for cell transfection; however, their use is still limited by important concerns, including toxicity and poor internalization into deep tissues. In this work, we investigated the use of shock wave-induced acoustic cavitation in vitro for the transfection of lipoplexes in human embryo kidney 293 cells. We selected shock waves with the ability to internalize 10-kDa fluorescein isothiocyanate-dextran into cells while maintaining survival rates above 50%. Cell transfection was tested using the green fluorescent protein-encoding plasmid pCX::GFPGPI2. Confocal microscopy and fluorescence-assisted cell sorting analyses revealed successful transfection after treatments ranging from 1 to 3 min using 60 to 180 shock waves at peak amplitudes of 12.3 ± 1.5 MPa. Interestingly, the combination of shock waves and lipoplexes induced a 3.1- and 3.8-fold increase in the expression of the reporter gene compared with the use of lipoplexes or shock waves alone, respectively. These results indicate that cationic DNA assembly and shock waves act in a synergistic manner to promote transfection of human cells, revealing a potential approach for non-invasive site-specific gene therapy.

Growth hormone (GH) and GH-releasing hormone (GHRH): Co-localization and action in the chicken testis.

Growth hormone (GH) gene expression is not confined to the pituitary gland and occurs in many extrapituitary tissues, including the chicken testis. The regulation and function of GH in extrapituitary tissues is, however, largely unknown. The possibility that chicken testicular GH might be regulated by GH-releasing hormone (GHRH), as in the avian pituitary gland, was investigated in the present study. GHRH co-localized with GH in the germinal epithelium and in interstitial zones within the chicken testes, particularly in the spermatogonia and spermatocytes. In testicular cell cultures, exogenous human GHRH1-44 induced (at 1, 10 and 100nM) a dose-related increase in GH release. Western blot analysis showed a heterogeneous pattern in the GH moieties released during GHRH stimulation. 26kDa monomer GH was the most abundant moiety under basal conditions, but 15 and 17kDa isoforms were more abundant after GHRH stimulation. GHRH treatment also increased the abundance of PCNA (proliferating cell nuclear antigen) immunoreactivity in the testes. This may have been GH-mediated, since exogenous GH similarly increased the incorporation of ((3)H)-thymidine into cultured testicular cells and increased their metabolic activity, as determined by increased MTT reduction. Furthermore, GH and GHRH immunoneutralization blocked GHRH-stimulated proliferative activity. In summary, these results indicate that GHRH stimulates testicular GH secretion in an autocrine or paracrine manner. Data also demonstrate proliferative actions of GHRH on testicular cell number and suggest that this action is mediated by local GH production.

Therapeutic proteins and nanotechnology: immune response and stealth bioengineered constructs.

With unique potentials for organ drug delivery and targeting, intravenous administration of drugs has represented a key tool in biomedicine. A major concern of this route is the rapid capture and destruction of foreign substances by circulating immune cells. Knowledge about the inter-relationships between drugs and blood cells is essential for a better control in drug stability and bioavailability. In this review, both classical pathways and novel insights into the immune mechanisms leading to drug clearance after systemic delivery are described. Drug surface chemistry and size have been identified as critical factors for the activation of host immune responses, and their modification has been extensively explored in order to evade immune surveillance. Common strategies to camouflage drug surfaces through polymer-grafting are presented, with special emphasis on Poly(Ethylene Glycol) (PEG) linkages, one of the most diverse strategies for modifying biomolecular surfaces. Finally, the use of "smart shields", such as PEG attachments shed at particular intracellular conditions, is briefly overviewed as an interesting approach for balancing circulation half lives VS bioavailability in polymer-grafted formulations.

Nonprotein structures from mycobacteria: emerging actors for tuberculosis control.

Immune response to Mycobacterium tuberculosis, the causal agent of tuberculosis, is critical for protection. For many decades, consistent to classical biochemistry, most studies regarding immunity to the tubercle bacilli focused mainly on protein structures. But the atypical, highly impermeable and waxy coat of mycobacteria captured the interest of structural biologists very early, allowing the description of amazing molecules, such as previously unknown carbohydrates or fatty acids of astonishing lengths. From their discovery, cell wall components were identified as important structural pillars, but also as molecular motifs able to alter the human immune response. Recently, as new developments have emerged, classical conceptions of mycobacterial immune modulators have been giving place to unexpected discoveries that, at the turn of the last century, completely changed our perception of immunity vis-à-vis fat compounds. In this paper, current knowledge about chemical and ultrastructural features of mycobacterial cell-wall is overviewed, with an emphasis on the relationships between cell-wall nonpeptide molecules and immune response. Remarks regarding the potential of these molecules for the development of new tools against tuberculosis are finally discussed.

Mycobacterial di-O-acyl trehalose inhibits Th-1 cytokine gene expression in murine cells by down-modulation of MAPK signaling.

Protection against tuberculosis (TB) is based on cell-mediated immune responses. TB is often characterized by immunological dysfunction of peripheral blood mononuclear cells, especially at chronic stages. Lipids from the Mycobacterium tuberculosis cell wall have been shown to produce various suppressive effects on cell-mediated immunity. The cell-surface lipid di-O-acyl-trehalose (DAT) is able to inhibit T-cell proliferation and cytokine secretion in cells from naïve mice. In the present study, we addressed the mechanisms involved in the suppressive effect caused by DAT. We found that DAT decreased the proliferation of spleen cells induced with PMA-ionomycin, suggesting that the suppressive mechanisms target intracellular functions just after phospholipase C-gamma activation. Addressing this possibility, the effect of DAT was found to involve down-modulation of the di-acyl glycerol-dependent activation of the MAPK-ERK1/2 pathway, one of the crucial signaling pathways leading to adaptive cell immune response against TB. Moreover, the inhibitory effect of DAT on proliferation was reproduced in antigen-stimulated T cells from M. tuberculosis-infected mice, involving the lowering of Th1-type cytokine transcription levels. The present findings thus reveal a new kind of bioactivity for a long-known M. tuberculosis cell wall lipid, DAT.

The use of fatty acid methyl esters as biomarkers to determine aerobic, facultatively aerobic and anaerobic communities in wastewater treatment systems.

The use of fatty acid methyl esters (FAME) as biomarkers to identify groups of microorganisms was studied. A database was constructed using previously published results that identify FAME biomarkers for aerobic, anaerobic and facultatively aerobic bacteria. FAME profiles obtained from pure cultures were utilized to confirm the predicted presence of biomarkers. Principal component analysis demonstrated that the FAME profiles can be used to determine the incidence of these bacterial groups. The presence of aerobic, anaerobic and facultatively aerobic bacteria in the communities, in four bioreactors being used to treat different wastewaters, was investigated by applying FAME biomarkers.

Mycobacterial trehalose-containing glycolipid with immunomodulatory activity on human CD4+ and CD8+ T-cells.

Protection against Mycobacterium tuberculosis is based on cell-mediated immunity, most importantly involving CD4+ and CD8+ T-cell subsets. One of the key features of the tubercle bacillus is its cell envelope, characterized by extremely abundant and specific lipids. The cell-surface glycolipid 2,3-di-O-acyl-trehalose (DAT) has been consistently found in M. tuberculosis strains. In this study, analysis of proliferation, activation markers and cytokine release was performed in human peripheral blood mononuclear cells (PBMC) activated in the presence and absence of DAT. We present evidence that mycobacterial DAT is able to reduce antigen-induced proliferation of human CD4+ and CD8+ T-cell subsets. We show that the effect is associated with a decrease of cells expressing the T-cell surface activation markers CD25 and CD69, and down-modulation of IL-2, IL-12, TNF-alpha and IL-10 cytokines. Data indicating that fine acyl chain structural variations in the trehalose-containing lipid may be involved in the degree of immune modulation are also presented.

Peptide mimotopes of Mycobacterium tuberculosis carbohydrate immunodeterminants.

Cell-surface saccharides of Mycobacterium tuberculosis appear to be crucial factors in tuberculosis pathogenicity and could be useful antigens in tuberculosis immunodiagnosis. In the present study, we report the successful antigenic and immunogenic mimicry of mannose-containing cell-wall compounds of M. tuberculosis by dodecamer peptides identified by phage-display technology. Using a rabbit antiserum raised against M. tuberculosis cell-surface saccharides as a target for biopanning, peptides with three different consensus sequences were identified. Phage-displayed and chemically synthesized peptides bound to the anticarbohydrate antiserum. Rabbit antibodies elicited against the peptide QEPLMGTVPIRAGGGS recognize the mannosylated M. tuberculosis cell-wall antigens arabinomannan and lipoarabinomannan, and the glycosylated recombinant protein alanine/proline-rich antigen. Furthermore, antibodies were also able to react with mannan from Saccharomyces cerevisiae, but not with phosphatidylinositol dimannosides or arabinogalactan from mycobacteria. These results suggest that the immunogenic peptide mimics oligomannosidic epitopes. Interestingly, this report provides evidence that, in contrast with previously known carbohydrate mimotopes, no aromatic residues are necessary in a peptide sequence for mimicking unusual glycoconjugates synthesized by mycobacteria. The possible usefulness of the identified peptide mimotopes as surrogate reagents for immunodiagnosis and for the study of functional roles of the native non-peptide epitopes is discussed.

6,6'-Dimycoloyl trehalose from a rapidly growing Mycobacterium: an alternative antigen for tuberculosis serodiagnosis.

Mycobacterial O-acyltrehaloses have been described as highly specific and sensitive reagents for tuberculosis immunodiagnosis. An O-acyltrehalose-containing lipid fraction from the rapidly growing Mycobacterium fortuitum was found to include additional antigens, which presented high cross-reactivity with sera from tuberculosis-infected patients. Based on a combination of selective chemical degradations, thin-layer-chromatography analyses and (1)H-nuclear magnetic resonance spectroscopy, the antigenic by-product was identified as 6,6'-dimycoloyl trehalose, the so-called cord factor. The lipid was purified and tested in ELISA for pulmonary tuberculosis serodiagnosis. Sensitivity and specificity of the test were found to be 66.6-74.1% and 95.2-99.0%, respectively, showing a slightly higher efficiency as compared to the ELISA performed using 6,6'-dimycoloyl trehalose from Mycobacterium tuberculosis. No cross-reactivity was found with sera from Nocardia-infected individuals.