Artificial Extracellular Vesicles Generated from T Cells Using Different Induction Techniques.

Cell therapy is at the forefront of biomedicine in oncology and regenerative medicine. However, there are still significant challenges to their wider clinical application such as limited efficacy, side effects, and logistical difficulties. One of the potential approaches that could overcome these problems is based on extracellular vesicles (EVs) as a cell-free therapy modality. One of the major obstacles in the translation of EVs into practice is their low yield of production, which is insufficient to achieve therapeutic amounts. Here, we evaluated two primary approaches of artificial vesicle induction in primary T cells and the SupT1 cell line-cytochalasin B as a chemical inducer and ultrasonication as a physical inducer. We found that both methods are capable of producing artificial vesicles, but cytochalasin B induction leads to vesicle yield compared to natural secretion, while ultrasonication leads to a three-fold increase in particle yield. Cytochalasin B induces the formation of vesicles full of cytoplasmic compartments without nuclear fraction, while ultrasonication induces the formation of particles rich in membranes and membrane-related components such as CD3 or HLAII proteins. The most effective approach for T-cell induction in terms of the number of vesicles seems to be the combination of anti-CD3/CD28 antibody activation with ultrasonication, which leads to a seven-fold yield increase in particles with a high content of functionally important proteins (CD3, granzyme B, and HLA II).

Pillar[5]arene/albumin biosupramolecular systems for simultaneous native protein preservation and encapsulation of a water-soluble substrate.

The growing resistance of pathogens, bacteria, viruses, and fungi to a number of drugs has encouraged researchers to use natural and synthetic biomimetic systems to overcome this challenge. Multicomponent systems are an attractive approach for drug design and multitarget therapy. In this study, we report the assembly of a three-component (pillar[5]arene, bovine serum albumin, and methyl orange) biosupramolecular system as a potential drug delivery system. We estimated the cytotoxic activity and transfection ability of pillar[5]arene derivatives and investigated the effect of the nature of macrocycle functions (L-phenylalanine, glycine, L-alanine) on the native conformation of serum albumin in a three-component system. NMR, UV-vis, fluorescence, CD spectroscopy, DLS, and molecular docking studies were performed in order to confirm the structure and possible pillar[5]arene/bovine serum albumin/methyl orange interactions occurring during the association process. Results indicate that pillar[5]arene with L-phenylalanine fragments retains the native form of BSA to the maximum extent and forms more stable associates.

Immunotherapy Strategy for Systemic Autoimmune Diseases: Betting on CAR-T Cells and Antibodies.

Systemic autoimmune diseases (SAIDs), such as systemic lupus erythematosus (SLE), systemic sclerosis (SSc) and rheumatoid arthritis (RA), are fully related to the unregulated innate and adaptive immune systems involved in their pathogenesis. They have similar pathogenic characteristics, including the interferon signature, loss of tolerance to self-nuclear antigens, and enhanced tissue damage like necrosis and fibrosis. Glucocorticoids and immunosuppressants, which have limited specificity and are prone to tolerance, are used as the first-line therapy. A plethora of novel immunotherapies have been developed, including monoclonal and bispecific antibodies, and other biological agents to target cellular and soluble factors involved in disease pathogenesis, such as B cells, co-stimulatory molecules, cytokines or their receptors, and signaling molecules. Many of these have shown encouraging results in clinical trials. CAR-T cell therapy is considered the most promising technique for curing autoimmune diseases, with recent successes in the treatment of SLE and SSc. Here, we overview novel therapeutic approaches based on CAR-T cells and antibodies for targeting systemic autoimmune diseases.

Decasubstituted Pillar[5]arene Derivatives Containing L-Tryptophan and L-Phenylalanine Residues: Non-Covalent Binding and Release of Fluorescein from Nanoparticles.

Sensitive systems with controlled release of drugs or diagnostic markers are attractive for solving the problems of biomedicine and antitumor therapy. In this study, new decasubstituted pillar[5]arene derivatives containing L-Tryptophan and L-Phenylalanine residues have been synthesized as pH-responsive drug nanocarriers. Fluorescein dye (Fluo) was loaded into the pillar[5]arene associates and used as a spectroscopic probe to evaluate the release in buffered solutions with pH 4.5, 7.4, and 9.2. The nature of the substituents in the pillar[5]arene structure has a huge influence on the rate of delivering. When the dye was loaded into the associates based on pillar[5]arene derivatives containing L-Tryptophan, the Fluo release occurs in the neutral (pH = 7.4) and alkaline (pH = 9.2) buffered solutions. When the dye was loaded into the associates based on pillar[5]arene with L-Phenylalanine fragments, the absence of release was observed in every pH evaluated. This happens as the result of different packing of the dye in the structure of the associate. This fact was confirmed by different fluorescence mechanisms (aggregation-caused quenching and aggregation-induced emission) and association constants. It was shown that the macrocycle with L-Phenylalanine fragments binds the dye more efficiently (lgKa = 3.92). The experimental results indicate that the pillar[5]arene derivatives with amino acids fragments have a high potential to be used as a pH-responsive drug delivery devices, especially for promoting the intracellular delivering, due to its nanometric size.

Aggregation, Cytotoxicity and DNA Binding in a Series of Calix[4]arene Amphiphile Containing Aminotriazole Groups.

The present work focuses on the study of the aggregation and complexing properties of calixarenes as potential DNA condensation agents for gene delivery. In the current study, 1,4-triazole derivatives of calix[4]arenes 7 and 8 containing monoammonium fragments were synthesized. The synthesized compound's structure was characterized by using various spectroscopic techniques (FTIR, HRESI MS, ¹H NMR and ¹³C NMR). The interactions between a series of calix[4]arene-containing aminotriazole groups (triazole-containing macrocycles with diethylenetriammonium fragments (3 and 4) and triazole-containing macrocycles with monoammonium fragments (7 and 8)) and calf thymus DNA were carried out via UV absorption, fluorescence spectroscopy, dynamic light scattering and zeta potential measurements. The role of the binding forces of calixarene-DNA complexes was analyzed. Photophysical and morphological studies revealed the interaction of the calixarenes 3, 4 and 8 with ct-DNA, which transformed the fibrous structure of ct-DNA to completely condensed compact structures that are 50 nm in diameter. The cytotoxic properties of calixarenes 3, 4, 7 and 8 against cancerous cells (MCF7, PC-3) as well as a healthy cell line (HSF) were investigated. Compound 4 was found to have the highest toxic effect on MCF7 breast adenocarcinoma (IC50 3.3 µM).

Anticancer therapeutic strategies for targeting mutant p53-Y220C.

The tumor suppressor p53 is a transcription factor with a powerful antitumor activity that is controlled by its negative regulator murine double minute 2 (MDM2, also termed HDM2 in humans) through a feedback mechanism. At the same time, TP53 is the most frequently mutated gene in human cancers. Mutant p53 proteins lose wild-type p53 tumor suppression functions but acquire new oncogenic properties, among which are deregulating cell proliferation, increasing chemoresistance, disrupting tissue architecture, and promoting migration, invasion and metastasis as well as several other pro-oncogenic activities. The oncogenic p53 mutation Y220C creates an extended surface crevice in the DNA-binding domain destabilizing p53 and causing its denaturation and aggregation. This cavity accommodates stabilizing small molecules that have therapeutic values. The development of suitable small-molecule stabilizers is one of the therapeutic strategies for reactivating the Y220C mutant protein. In this review, we summarize approaches that target p53-Y220C, including reactivating this mutation with small molecules that bind Y220C to the hydrophobic pocket and developing immunotherapies as the goal for the near future, which target tumor cells that express the p53-Y220C neoantigen.