Mannosylated Systems for Targeted Delivery of Antibacterial Drugs to Activated Macrophages

Macrophages are a promising target for drug delivery to influence macrophage-associated processes in the body, namely due to the presence of resistant microorganisms in macrophages. In this work, a series of mannosylated carriers based on mannan, polyethylenimine (PEI) and cyclodextrin (CD) was synthesized. The molecular architecture was studied using FTIR and ¹H NMR spectroscopy. The particle size, from small 10–50 nm to large 500 nm, depending on the type of carrier, is potentially applicable for the creation of various medicinal forms: intravenous, oral and inhalation. Non-specific capture by cells with a simultaneous increase in selectivity to CD206+ macrophages was achieved. ConA was used as a model mannose receptor, binding galactosylated (CD206 non-specific) carriers with constants of the order of 10⁴ M⁻¹ and mannosylated conjugates of 10⁶–10⁷ M⁻¹. The results of such primary “ConA-screening” of ligands are in a good agreement in terms of the comparative effectiveness of the interaction of ligands with the CD206+ macrophages: non-specific (up to 10%) absorption of highly charged and small particles; weakly specific uptake of galactosylated polymers (up to 50%); and high affine capture (more than 70–80%) of the ligands with grafted trimannoside was demonstrated using the cytometry method. Double and multi-complexes of antibacterials (moxifloxacin with its adjuvants from the class of terpenoids) were proposed as enhanced forms against resistant pathogens. In vivo pharmacokinetic experiments have shown that polymeric carriers significantly improve the efficiency of the antibiotic: the half-life of moxifloxacin is increased by 2–3 times in conjugate-loaded forms, bio-distribution to the lungs in the first hours after administration of the drug is noticeably greater, and, after 4 h of observation, free moxifloxacin was practically removed from the lungs of rats. Although, in polymer systems, its content is significant—1.2 µg/g. Moreover, the importance of the covalent crosslinking carrier with mannose label was demonstrated. Thus, this paper describes experimental, scientifically based methods of targeted drug delivery to macrophages to create enhanced medicinal forms.     

Validation of a Cleanroom Compliant Sonication-Based Decellularization Technique: A New Concept in Nerve Allograft Production

Defects of the peripheral nervous system are extremely frequent in trauma and surgeries and have high socioeconomic costs. If the direct suture of a lesion is not possible, i.e., nerve gap > 2 cm, it is necessary to use grafts. While the gold standard is the autograft, it has disadvantages related to its harvesting, with an inevitable functional deficit and further morbidity. An alternative to autografting is represented by the acellular nerve allograft (ANA), which avoids disadvantages of autograft harvesting and fresh allograft rejection. In this research, the authors intend to transfer to human nerves a novel technique, previously implemented in animal models, to decellularize nerves. The new method is based on soaking the nerve tissues in decellularizing solutions while associating ultrasounds and freeze–thaw cycles. It is performed without interrupting the sterility chain, so that the new graft may not require post-production γ-ray irradiation, which is suspected to affect the structural and functional quality of tissues. The new method is rapid, safe, and inexpensive if compared with available commercial ANAs. Histology and immunohistochemistry have been adopted to evaluate the new decellularized nerves. The study shows that the new method can be applied to human nerve samples, obtaining similar, and, sometimes better, results compared with the chosen control method, the Hudson technique.     

Medicated Scaffolds Prepared with Hydroxyapatite/Streptomycin Nanoparticles Encapsulated into Polylactide Microfibers

The preparation, characterization, and controlled release of hydroxyapatite (HAp) nanoparticles loaded with streptomycin (STR) was studied. These nanoparticles are highly appropriate for the treatment of bacterial infections and are also promising for the treatment of cancer cells. The analyses involved scanning electron microscopy, dynamic light scattering (DLS) and Z-potential measurements, as well as infrared spectroscopy and X-ray diffraction. Both amorphous (ACP) and crystalline (cHAp) hydroxyapatite nanoparticles were considered since they differ in their release behavior (faster and slower for amorphous and crystalline particles, respectively). The encapsulated nanoparticles were finally incorporated into biodegradable and biocompatible polylactide (PLA) scaffolds. The STR load was carried out following different pathways during the synthesis/precipitation of the nanoparticles (i.e., nucleation steps) and also by simple adsorption once the nanoparticles were formed. The loaded nanoparticles were biocompatible according to the study of the cytotoxicity of extracts using different cell lines. FTIR microspectroscopy was also employed to evaluate the cytotoxic effect on cancer cell lines of nanoparticles internalized by endocytosis. The results were promising when amorphous nanoparticles were employed. The nanoparticles loaded with STR increased their size and changed their superficial negative charge to positive. The nanoparticles’ crystallinity decreased, with the consequence that their crystal sizes reduced, when STR was incorporated into their structure. STR maintained its antibacterial activity, although it was reduced during the adsorption into the nanoparticles formed. The STR release was faster from the amorphous ACP nanoparticles and slower from the crystalline cHAp nanoparticles. However, in both cases, the STR release was slower when incorporated in calcium and phosphate during the synthesis. The biocompatibility of these nanoparticles was assayed by two approximations. When extracts from the nanoparticles were evaluated in cultures of cell lines, no cytotoxic damage was observed at concentrations of less than 10 mg/mL. This demonstrated their biocompatibility. Another experiment using FTIR microspectroscopy evaluated the cytotoxic effect of nanoparticles internalized by endocytosis in cancer cells. The results demonstrated slight damage to the biomacromolecules when the cells were treated with ACP nanoparticles. Both ACP and cHAp nanoparticles were efficiently encapsulated in PLA electrospun matrices, providing functionality and bioactive properties.     

COVID-19 and Lung Mast Cells: The Kallikrein–Kinin Activation Pathway

Mast cells (MCs) have relevant participation in inflammatory and vascular hyperpermeability events, responsible for the action of the kallikrein–kinin system (KKS), that affect patients inflicted by the severe form of COVID-19. Given a higher number of activated MCs present in COVID-19 patients and their association with vascular hyperpermeability events, we investigated the factors that lead to the activation and degranulation of these cells and their harmful effects on the alveolar septum environment provided by the action of its mediators. Therefore, the pyroptotic processes throughout caspase-1 (CASP-1) and alarmin interleukin-33 (IL-33) secretion were investigated, along with the immunoexpression of angiotensin-converting enzyme 2 (ACE2), bradykinin receptor B1 (B1R) and bradykinin receptor B2 (B2R) on post-mortem lung samples from 24 patients affected by COVID-19. The results were compared to 10 patients affected by H1N1pdm09 and 11 control patients. As a result of the inflammatory processes induced by SARS-CoV-2, the activation by immunoglobulin E (IgE) and degranulation of tryptase, as well as Toluidine Blue metachromatic (TB)-stained MCs of the interstitial and perivascular regions of the same groups were also counted. An increased immunoexpression of the tissue biomarkers CASP-1, IL-33, ACE2, B1R and B2R was observed in the alveolar septum of the COVID-19 patients, associated with a higher density of IgE⁺ MCs, tryptase⁺ MCs and TB-stained MCs, in addition to the presence of intra-alveolar edema. These findings suggest the direct correlation of MCs with vascular hyperpermeability, edema and diffuse alveolar damage (DAD) events that affect patients with a severe form of this disease. The role of KKS activation in events involving the exacerbated increase in vascular permeability and its direct link with the conditions that precede intra-alveolar edema, and the consequent DAD, is evidenced. Therapy with drugs that inhibit the activation/degranulation of MCs can prevent the worsening of the prognosis and provide a better outcome for the patient.     

Fetal Undernutrition Modifies Vascular RAS Balance Enhancing Oxidative Damage and Contributing to Remodeling

Fetal stress is known to increase susceptibility to cardiometabolic diseases and hypertension in adult age in a process known as fetal programming. This study investigated the relationship between vascular RAS, oxidative damage and remodeling in fetal programming. Six-month old Sprague-Dawley offspring from mothers that were fed ad libitum (CONTROL) or with 50% intake during the second half of gestation (maternal undernutrition, MUN) were used. qPCR or immunohistochemistry were used to obtain the expression of receptors and enzymes. Plasma levels of carbonyls were measured by spectrophotometry. In mesenteric arteries from MUN rats we detected an upregulation of ACE, ACE2, AT₁ receptors and NADPH oxidase, and lower expression of AT₂, Mas and MrgD receptors compared to CONTROL. Systolic and diastolic blood pressure and plasma levels of carbonyls were higher in MUN than in CONTROL. Vascular morphology evidenced an increased media/lumen ratio and adventitia/lumen ratio, and more connective tissue in MUN compared to CONTROL. In conclusion, fetal undernutrition indices RAS alterations and oxidative damage which may contribute to the remodeling of mesenteric arteries, and increase the risk of adverse cardiovascular events and hypertension.     

Histamine H4 Receptor Agonism Induces Antitumor Effects in Human T-Cell Lymphoma

The discovery of the human histamine H4 receptor (H4R) has contributed to our understanding of the role of histamine in numerous physiological and pathological conditions, including tumor development and progression. The lymph nodes of patients with malignant lymphomas have shown to contain high levels of histamine, however, less is known regarding the expression and function of the H4R in T-cell lymphoma (TCL). In this work we demonstrate the expression of H4R isoforms (mRNA and protein) in three human aggressive TCL (OCI-Ly12, Karpas 299, and HuT78). Histamine and specific H4R agonists (VUF8430 and JNJ28610244) significantly reduced cell viability in a dose-dependent manner (p < 0.05). The combined treatment with the H4R antagonist (JNJ7777120, 10 µM) reversed the effects of the H4R ligands. Importantly, we screened a drug repurposing library of 433 FDA-approved compounds (1 μM) in combination with histamine (10 μM) in Hut78 cells. Histamine produced a favorable antitumor effect with 18 of these compounds, including the histone deacetylase inhibitor panobinostat. Apoptosis, proliferation, and oxidative stress studies confirmed the antitumoral effects of the combination. We conclude that the H4R is expressed in TCL, and it is involved in histamine-mediated responses.     

Cold Atmospheric Plasma Apoptotic and Oxidative Effects on MCF7 and HCC1806 Human Breast Cancer Cells

Breast cancer (BC) is a malignant neoplasia with the highest incidence and mortality rates in women worldwide. Currently, therapies include surgery, radiotherapy, and chemotherapy, including targeted therapies in some cases. However, treatments are often associated with serious adverse effects. Looking for new options in BC treatment, we evaluated the therapeutic potential of cold atmospheric plasma (CAP) in two cell lines (MCF7 and HCC1806) with distinct histological features. Apoptosis seemed to be the most prevalent type of death, as corroborated by several biochemical features, including phosphatidylserine exposure, the disruption of mitochondrial membrane potential, an increase in BAX/BCL2 ratio and procaspase 3 loss. Moreover, the accumulation of cells in the G2/M phase of the cell cycle points to the loss of replication ability and decreased survival. Despite reported toxic concentrations of peroxides in culture media exposed to plasma, intracellular peroxide concentration was overall decreased accompanying a reduction in GSH levels shortly after plasma exposure in both cell lines. In HCC1806, elevated nitric oxide (NO) concentration accompanied by reduced superoxide levels suggests that these cells are capable of converting plasma-derived nitrites into NO that competes with superoxide dismutase (SOD) for superoxide to form peroxinitrite. The concomitant inhibition of the antioxidative activity of cells during CAP treatment, particularly the inhibition of cytochrome c oxidase with sodium azide, synergistically increased plasma toxicity. Thus, this in vitro research enlightens the therapeutic potential of CAP in the treatment of breast cancer, elucidating its possible mechanisms of action.     

Upregulation of Cathepsin X in Glioblastoma: Interplay with γ-Enolase and the Effects of Selective Cathepsin X Inhibitors

Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Understanding GBM pathobiology and discovering novel therapeutic targets are critical to finding efficient treatments. Upregulation of the lysosomal cysteine carboxypeptidase cathepsin X has been linked to immune dysfunction and neurodegenerative diseases, but its role in cancer and particularly in GBM progression in patients is unknown. In this study, cathepsin X expression and activity were found to be upregulated in human GBM tissues compared to low-grade gliomas and nontumor brain tissues. Cathepsin X was localized in GBM cells as well as in tumor-associated macrophages and microglia. Subsequently, potent irreversible (AMS36) and reversible (Z7) selective cathepsin X inhibitors were tested in vitro. Selective cathepsin X inhibitors decreased the viability of patient-derived GBM cells as well as macrophages and microglia that were cultured in conditioned media of GBM cells. We next examined the expression pattern of neuron-specific enzyme γ-enolase, which is the target of cathepsin X. We found that there was a correlation between high proteolytic activity of cathepsin X and C-terminal cleavage of γ-enolase and that cathepsin X and γ-enolase were colocalized in GBM tissues, preferentially in GBM-associated macrophages and microglia. Taken together, our results on patient-derived material suggest that cathepsin X is involved in GBM progression and is a potential target for therapeutic approaches against GBM.