Ionizable Lipid Nanoparticle-Mediated TRAIL mRNA Delivery in the Tumor Microenvironment to Inhibit Colon Cancer Progression.

Introduction: Immunotherapy has revolutionized cancer treatment by harnessing the immune system to enhance antitumor responses while minimizing off-target effects. Among the promising cancer-specific therapies, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted significant attention. Methods: Here, we developed an ionizable lipid nanoparticle (LNP) platform to deliver TRAIL mRNA (LNP-TRAIL) directly to the tumor microenvironment (TME) to induce tumor cell death. Our LNP-TRAIL was formulated via microfluidic mixing and the induction of tumor cell death was assessed in vitro. Next, we investigated the ability of LNP-TRAIL to inhibit colon cancer progression in vivo in combination with a TME normalization approach using Losartan (Los) or angiotensin 1-7 (Ang(1-7)) to reduce vascular compression and deposition of extracellular matrix in mice. Results: Our results demonstrated that LNP-TRAIL induced tumor cell death in vitro and effectively inhibited colon cancer progression in vivo, particularly when combined with TME normalization induced by treatment Los or Ang(1-7). In addition, potent tumor cell death as well as enhanced apoptosis and necrosis was found in the tumor tissue of a group treated with LNP-TRAIL combined with TME normalization. Discussion: Together, our data demonstrate the potential of the LNP to deliver TRAIL mRNA to the TME and to induce tumor cell death, especially when combined with TME normalization. Therefore, these findings provide important insights for the development of novel therapeutic strategies for the immunotherapy of solid tumors.

Delivery of Plasmid DNA by Ionizable Lipid Nanoparticles to Induce CAR Expression in T Cells.

Introduction: Chimeric antigen receptor (CAR) cell therapy represents a hallmark in cancer immunotherapy, with significant clinical results in the treatment of hematological tumors. However, current approved methods to engineer T cells to express CAR use viral vectors, which are integrative and have been associated with severe adverse effects due to constitutive expression of CAR. In this context, non-viral vectors such as ionizable lipid nanoparticles (LNPs) arise as an alternative to engineer CAR T cells with transient expression of CAR. Methods: Here, we formulated a mini-library of LNPs to deliver pDNA to T cells by varying the molar ratios of excipient lipids in each formulation. LNPs were characterized and screened in vitro using a T cell line (Jurkat). The optimized formulation was used ex vivo to engineer T cells derived from human peripheral blood mononuclear cells (PBMCs) for the expression of an anti-CD19 CAR (CAR-CD19BBz). The effectiveness of these CAR T cells was assessed in vitro against Raji (CD19+) cells. Results: LNPs formulated with different molar ratios of excipient lipids efficiently delivered pDNA to Jurkat cells with low cytotoxicity compared to conventional transfection methods, such as electroporation and lipofectamine. We show that CAR-CD19BBz expression in T cells was transient after transfection with LNPs. Jurkat cells transfected with our top-performing LNPs underwent activation when exposed to CD19+ target cells. Using our top-performing LNP-9-CAR, we were able to engineer human primary T cells to express CAR-CD19BBz, which elicited significant specific killing of CD19+ target cells in vitro. Conclusion: Collectively, our results show that LNP-mediated delivery of pDNA is a suitable method to engineer human T cells to express CAR, which holds promise for improving the production methods and broader application of this therapy in the future.

High-Density-Immune-Complex Regulatory Macrophages Promote Recovery of Experimental Colitis in Mice.

Macrophages not only play a fundamental role in the pathogenesis of inflammatory bowel disease (IBD), but they also play a major role in preserving intestinal homeostasis. In this work, we evaluated the role of macrophages in IBD and investigated whether the functional reprogramming of macrophages to a very specific phenotype could decrease disease pathogenesis. Thus, macrophages were stimulated in the presence of high-density immune complexes which strongly upregulate their production of IL-10 and downregulate pro-inflammatory cytokines. The transfer of these high-density-immune-complex regulatory macrophages into mice with colitis was examined as a potential therapy proposal to control the disease. Animals subjected to colitis induction received these high-density-immune-complex regulatory macrophages, and then the Disease Activity Index (DAI), and macroscopic and microscopic lesions were measured. The treated group showed a dramatic improvement in all parameters analyzed, with no difference with the control group. The colon was macroscopically normal in appearance and size, and microscopically colon architecture was preserved. The immunofluorescence migration assay showed that these cells migrated to the inflamed intestine, being able to locally produce the cytokine IL-10, which could explain the dramatic improvement in the clinical and pathological condition of the animals. Thus, our results demonstrate that the polarization of macrophages to a high IL-10 producer profile after stimulation with high-density immune complexes was decisive in controlling experimental colitis, and that macrophages are a potential therapeutic target to be explored in the control of colitis.

Nanostructured lipid carriers as a novel tool to deliver sclareol: physicochemical characterisation and evaluation in human cancer cell lines

Sclareol (SC) is arousing great interest due to its cytostatic and cytotoxic activities in several cancer cell lines. However, its hydrophobicity is a limiting factor for its in vivo administration. One way to solve this problem is through nanoencapsulation. Therefore, solid lipid nanoparticles (SLN-SC) and nanostructured lipid carriers (NLC-SC) loaded with SC were produced and compared regarding their physicochemical properties. NLC-SC showed better SC encapsulation than SLN-SC and was chosen to be compared with free SC in human cancer cell lines (MDA-MB-231 and HCT-116). Free SC had slightly higher cytotoxicity than NLC-SC and produced subdiploid DNA content in both cell lines. On the other hand, NLC-SC led to subdiploid content in MDA-MB-231 cells and G2/M checkpoint arrest in HCT-116 cells. These findings suggest that SC encapsulation in NLC is a way to allow the in vivo administration of SC and might alter its biological properties

Photobiomodulation drives pericyte mobilization towards skin regeneration.

Photobiomodulation is being widely applied for improving dermal or mucosal wound healing. However, the underlying cellular and molecular processes that directly contribute to its effects remain poorly understood. Pericytes are relevant cells involved in the wound microenvironment and could be one of the main targets of photobiomodulation due to their plasticity and perivascular localization. Herein, we investigate tissue repair under the photobiomodulation stimulus using a pericyte labeled (or reporter) transgenic mice. Using a model of two contralateral back wounds, one the control and the other photoactivated daily (660 nm, 20 mW, 0.71 W/cm2, 5 J/cm2, 7 s, 0.14 J), we showed an overall influx of immune and undifferentiated cells and higher mobilization of a potent pericyte subpopulation (Type-2 pericytes) in the photoactivated wounds in comparison to the controls. Doppler analysis showed a significant increase in the blood flow in the photoactivated wounds, while marked vascular supply was observed histologically. Histochemical analysis has indicated more advanced stages of tissue repair after photoactivation. These data suggest that photobiomodulation significantly accelerates tissue repair through its vascular effects with direct recruitment of pericytes to the injury site.

The Tumor Microenvironment: Focus on Extracellular Matrix.

The extracellular matrix (ECM) regulates the development and maintains tissue homeostasis. The ECM is composed of a complex network of molecules presenting distinct biochemical properties to regulate cell growth, survival, motility, and differentiation. Among their components, proteoglycans (PGs) are considered one of the main components of ECM. Its composition, biomechanics, and anisotropy are exquisitely tuned to reflect the physiological state of the tissue. The loss of ECM's homeostasis is seen as one of the hallmarks of cancer and, typically, defines transitional events in tumor progression and metastasis. In this chapter, we discuss the types of proteoglycans and their roles in cancer. It has been observed that the amount of some ECM components is increased, while others are decreased, depending on the type of tumor. However, both conditions corroborate with tumor progression and malignancy. Therefore, ECM components have an increasingly important role in carcinogenesis and this leads us to believe that their understanding may be a key in the discovery of new anti-tumor therapies. In this book, the main ECM components will be discussed in more detail in each chapter.

Sensory Ganglia-Specific TNF Expression Is Associated With Persistent Nociception After Resolution of Inflammation.

Joint pain is a distressing symptom of arthritis, and it is frequently persistent even after treatments which reduce local inflammation. Continuous production of algogenic factors activate/sensitize nociceptors in the joint structures and contribute to persistent pain, a challenging and difficult condition to treat. TNF is a crucial cytokine for the pathogenesis of several rheumatic diseases, and its inhibition is a mainstay of treatment to control joint symptoms, including pain. Here, we sought to investigate the inflammatory changes and the role of TNF in dorsal root ganglia (DRG) during persistent hypernociception after the resolution of acute joint inflammation. Using a model of antigen-induced arthritis, the peak of joint inflammation occurred 12-24 h after local antigen injection and was characterized by an intense influx of neutrophils, pro-inflammatory cytokine production, and joint damage. We found that inflammatory parameters in the joint returned to basal levels between 6 and 8 days after antigen-challenge, characterizing the resolving phase of joint inflammation. Mechanical hyperalgesia was persistent up to 14 days after joint insult. The persistent nociception was associated with the inflammatory status of DRG after cessation of acute joint inflammation. The late state of neuroinflammation in the ipsilateral side was evidenced by gene expression of TNF, TNFR2, IL-6, IL-1ß, CXCL2, COX2, and iNOS in lumbar DRG (L3-L5) and leukocyte adhesion in the lumbar intumescent vessels between days 6 and 8. Moreover, there were signs of resident macrophage activation in DRG, as evidenced by an increase in Iba1-positive cells. Intrathecal or systemic injection of etanercept, an agent clinically utilized for TNF neutralization, at day 7 post arthritis induction, alleviated the persistent joint hyperalgesia by specific action in DRG. Our data suggest that neuroinflammation in DRG after the resolution of acute joint inflammation drives continuous neural sensitization resulting in persistent joint nociception in a TNF-dependent mechanism.

Novel nitroaromatic compound activates autophagy and apoptosis pathways in HL60 cells.

N-(2-butanoyloxyethyl)-4-(chloromethyl)-3-nitrobenzamide (NBCN) is a nitroaromatic bioreducible compound with cytotoxic effects in cancer cell lines. The aim of this work was to investigate the molecular mechanisms involved in cell death promoted by NBCN in HL60 cells. We observed that NBCN treatment increased intracellular ROS and reduced mitochondria membrane potential (ΔΨm). NBCN treatment also induced morphological changes, phosphatidylserine exposure, cell cycle arrest in G2/M-phase, DNA condensation and fragmentation, but it did not show cytotoxic effects on normal human peripheral blood mononuclear cells (PBMCs). NBCN-induced caspase 3- and 9-dependent DNA fragmentation, which was blocked by pretreatment with the broad-spectrum caspase inhibitor, z-VAD-fmk. Flow cytometry analysis demonstrated that NBCN also increased of the number of autophagic vesicles in HL60 cells, which was not observed when cells were pre-treated with bafilomycin A1. Taken together, these results indicate that NBCN triggered the mitochondrial apoptotic pathway and led to the onset of autophagic cell death, which contributed to its cytotoxic effects.