Synergistic antitumor effects were investigated for binary mixtures composed of two ionic liquids derived from amino acids (IL–IL mixtures), as well as for mixtures combining a single ionic liquid with methotrexate (MTX), a clinically approved chemotherapeutic agent. These combinations were tested against two human cancer cell lines, A-549 (lung adenocarcinoma) and HT-29 (colon adenocarcinoma). In general, the binary mixtures exhibited enhanced antitumoral activity and selectivity compared to their individual components under both physiological and acidic pH conditions. Notably, these mixtures achieved IC₅₀ values in the nanomolar range, with selectivity indexes in some cases exceeding 100, representing improvements of one to two orders of magnitude over the individual compounds. Supramolecular interaction studies revealed cooperative interactions between long- and short-chained imidazolium derivatives, underscoring the critical role of the amino acid moiety and hydrogen bonding involving amide groups in driving the observed synergistic effects. Furthermore, ¹H NMR and ESI-MS analyses confirmed molecular interactions between the long-chained IL (IL 1) and methotrexate. Confocal fluorescence microscopy, using a fluorescently labelled IL (IL 2), both alone and in combination with IL 1, demonstrated membrane disruption and mitochondrial accumulation, providing further insight into the mechanism of action. In addition, the affinity of individual ILs and IL–IL binary mixtures for salmon sperm DNA was assessed, supporting potential DNA-related mechanisms. Altogether, these findings highlight the potential of these imidazolium-based compounds as promising candidates for combination chemotherapy strategies, and they pave the way for novel applications of ionic liquids in cancer treatment.

The natural products combretastatins A-1 and A-4 are potent antimitotic and vascular-disrupting agents through their binding at the colchicine site in tubulin. However, these compounds suffer from a low water solubility and a tendency to isomerize to the inactive trans stilbenes. In this study, we have prepared a series of 18 cis-restricted triazole analogues of combretastatin A-4 (CA-4), maintaining, in all cases, the 3,4,5-trimethoxy phenyl ring A, with the aim of investigating the substitution pattern on the B-ring in a systematic way. To this end, cytotoxic activities of the cis-restricted analogues of CA-4 prepared were determined in two tumor cell lines, namely, HT-29 and A-549, as well as in the non-tumor cell line HEK-293, to pre-evaluate the selectivity profile of the compounds for the tumor cell lines. The main conclusion was the essential presence of methoxyl or ethoxyl groups at the para position of the B-ring in order to obtain good antitumor activities. Thus, the more active compounds in our study displayed IC50 values in the nanomolar range for the tumor cell lines but not for the normal cells. Consequently, these triazole analogues of CA-4 could serve as promising alternatives to the natural product, although further studies about their biological activity are essential in order to fully determine their viability as therapeutic agents in the treatment of cancer.

A set of fourteen imidazolium ionic liquids, each derived from L-amino acids and featuring distinct hydrophobic and hydrophilic components, were synthesized and physicochemically characterized. Thermal analysis showed transition temperatures remaining below 100 degrees C, being the rigid solid to elastic solid transition temperatures between -27 degrees C to + 57 degrees C. The aggregation studies investigated by fluorescence in water and buffered media showed two critical micellar concentrations (CMC) in aqueous media for compounds 7, 8 and 9, with the formation of well-defined aggregates in the second step. The synthesized ILs were assessed for their effects on two cancer cell lines, namely human lung adenocarcinoma A-549 and colon adenocarcinoma HT-29, under both physiological and acidic pH conditions. Furthermore, the influence of the ILs on embryonic kidney cells (HEK293) was investigated to assess their effect on non-cancerous cells. This study demonstrates that the manipulation of various design vectors allowed for the precise tuning of the structure of ILs, resulting in IC50 values within the micromolar range. ILs featuring a NC12 alkyl side chain in the amide consistently exhibited low IC50 values and a high degree of selectivity against non-tumoral cells under acidic pH conditions. Subsequent investigations were carried out to gain insights into the cellular-level mechanisms of action. These findings provide strong evidence that by meticulously adjusting the molecular structure of these functionalized ILs, it is possible to design potent and selective chemotherapeutic agents.

Twenty-one styryl carbamates have been designed, synthetized and characterized by their physical properties. Afterwards, their potential activity as tumor microenvironment disruptors has been evaluated. First, antiproliferative studies on tumor cell lines HT-29, A-549 and MCF-7 and on non-tumor cell line HEK-293 were carried out. Then, the antiproliferative activity of some selected compounds was evaluated on co-cultures of A-549 and immune cells, Jurkat T or monocytes THP-1. To determine their potential as oncoinflammation regulators, the secretion levels of IL-6, TNF-α and SAA-1 were, also, determined from these co-culture assays. Then, the effect on the expression levels of anticancer targets PD-L1 in both A-549 and monocytes THP-1 were established and, finally, the expression of CD80 and CD11b in THP-1. Compounds 9–13 bearing halophenyl carbamate units exhibited promising results as TME disruptors and inflammation regulators.

Pulsed laser fragmentation in liquid (PLFL) is one of the synthesis routes for producing high-purity carbon quantum dots (CQDs) with less toxic by-products in a straightforward system. The limited literature regarding the application of PLFL-synthesized CQDs motivated this study to exploit them as fluorescent probes in glucose sensing. The design of a fluorescent-based non-enzymatic glucose sensor was achieved by implementing CQDs and 3-aminophenylboronic acid (APBA) as fluorescent probes and glucose receptors, respectively. The CQDs were fabricated by PLFL through an Nd:YAG nanosecond laser, starting from the carbon powder dispersion in ethanol. Then, a laser-assisted post-modification process with oxidizing acid was implemented to produce waterdispersible CQDs for easy functionalization. Functionalizing these CQDs with APBA (CQDs-APBA) resulted in a blue-shifted fluorescence emission with a 35% quantum yield and high photostability. The CQDs-APBA exhibited a turn-off response at increasing glucose concentrations. This method offers good sensitivity with a linear detection range of glucose with a concentration of 0.165 to 8 mM and a detection limit of 165 mu M. The applicability of this sensor to real analytes such as saliva obtained a satisfactory result with good reproducibility. This work proves that CQDs synthesized from PLFL can also be an alternative fluorescent probe for fluorescent-based sensing applications.

Thirteen benzylethoxyaryl ureas have been synthesized and biologically evaluated as multitarget inhibitors of VEGFR-2 and PD-L1 proteins to overcome resistance phenomena offered by cancer. The antiproliferative activity of these molecules on several tumor cell lines (HT-29 and A549), on the endothelial cell line HMEC-1, on immune cells (Jurkat T) and on the non-tumor cell line HEK-293 has been determined. Selective indexes (SI) have been also determined and compounds bearing p-substituted phenyl urea unit together with a diaryl carbamate exhibited high SI values. Further studies on these selected compounds to determine their potential as small molecule immune potentiators (SMIPs) and as antitumor agents have been performed. From these studies, we have concluded that the designed ureas have good tumor antiangiogenic properties, exhibit good inhibition of CD11b expression, and regulate pathways involved in CD8 T-cell activity. These properties suggest that these compounds could be potentially useful in the development of new cancer immune treatments.

The combination of polymers and low molecular weight (LMW) compounds is a powerful approach to prepare new supramolecular materials. Here we prepare two-component hydrogels made by a well-known and biologically active polymer, hyaluronic acid (HA), and a dipeptide-based supramolecular gelator. We undertake a detailed study of materials with different compositions including macroscopic (hydrogel formation, rheology) and micro/nanoscopic characterization (electron microscopy, X-ray powder diffraction). We observe that the two components mutually benefit in the new materials: a minimum amount of HA helps to reduce the polymorphism of the LMW network leading to reproducible hydrogels with improved mechanical properties; the LMW component network holds HA without the need for an irreversible covalent crosslinking. These materials have a great potential for biomedical application as, for instance, extracellular matrix mimetics for cell growth. As a proof of concept, we have observed that this material is effective for cell growth in suspension and avoids cell sedimentation even in the presence of competing cell-adhesive surfaces. This may be of interest to advanced cell delivery techniques.

Twenty-seven compounds bearing a tetrazole ring as a central unit have been designed, synthetized and biologically evaluated. Studies have been performed in order to compare the effect of tetrazole derivatives bearing amine electron-donor or nitro electron-acceptor groups. The antiproliferative activity has been determined in monoculture studies on tumor cell lines HT-29, A-549, MCF-7 and on non-tumor cell line HEK-293 as well as in co-culture studies (HT-29/THP-1). All the compounds have been studied as PD-L1 (Programmed Death Ligand 1), VEGFR-2 (Vascular Endothelial Growth Factor 2), CD-47 (Cluster of Differentiation 47) and c-Myc inhibitors. The effect on TNF-α secretion has also been determined. Bromoderivatives 23, 24 and chloroderivatives 26, 27 have demonstrated an apoptotic effect on HT-29 cancer cells. Compounds bearing an amine group have shown very promising effects as TME immunomodulatory agents.

Thirteen benzylethylenearyl ureas and one carbamate have been designed, synthesized and biologically evaluated as regards their antiproliferative activity on tumor cell lines HEK-293 and A-549 and on the immune and endothelial Jurkat T cells and HMEC-1, respectively. A relationship between the structure of the synthetic compounds and their antiproliferative activity has been established. Compounds C.1, C.3, C.12 and C.14 were chosen for further biological studies in order to determine their potential as oncoimmunomodulating agents. Some compounds exhibited significant inhibitory effects on both total PD-L1 and VEGFR-2 in HT-29 cell line, showing urea C.12 active against both targets. Some compounds could reduce more than 50% of cancer living cells, compared to non-treated ones, when assessed in co-cultures using HT-29 and THP-1 cells. In addition, these compounds showed no effect on the immune cell viability and significantly reduce CD11b expression, which is a promising target for immune modulation in anti-cancer therapies.

The rise in antibiotic-resistant bacteria caused by the excessive use of antibiotics has led to the urgent exploration of alternative antimicrobial solutions. Among these alternatives, antimicrobial proteins, and peptides (Apps) have garnered attention due to their wide-ranging antimicrobial effects. This study focuses on evaluating the antimicrobial properties of Solanum lycopersicum heme-binding protein 2 (SlHBP2), an apoplastic protein extracted from tomato plants treated with 1-Methyl tryptophan (1-MT), against Pseudomonas syringae pv. tomato DC3000 ( Pst ). Computational studies indicate that SlHBP2 is annotated as a SOUL heme-binding family protein. Remarkably, recombinant SlHBP2 demonstrated significant efficacy in inhibiting the growth of Pst within a concentration range of 3–25 μg/mL. Moreover, SlHBP2 exhibited potent antimicrobial effects against other microorganisms, including Xanthomonas vesicatoria ( Xv ), Clavibacter michiganensis subsp . michiganensis ( Cmm ), and Botrytis cinerea . To understand the mechanism of action employed by SlHBP2 against Pst , various techniques such as microscopy and fluorescence assays were employed. The results revealed that SlHBP2 disrupts the bacterial cell wall and causes leakage of intracellular contents. To summarize, the findings suggest that SlHBP2 has significant antimicrobial properties, making it a potential antimicrobial agent against a wide range of pathogens. Although further studies are warranted to explore the full potential of SlHBP2 and its suitability in various applications.

This work focuses on the development of thirteen benzylethylenearyl ureas and one carbamate. After the synthesis and purification of the compounds, we studied their antiproliferative action on cell lines, such as HEK-293, and cancer ones, such as HT-29, MCF-7 or A-549, on the immune Jurkat T-cells and endothelial cells HMEC-1. Compounds C.1, C.3, C.12 and C.14 were selected for further biological studies to establish their potential as immunomodulating agents. Some of the derivatives exhibited significant inhibitory effects on both targets: PD-L1 and VEGFR-2 in the HT-29 cell line, showing that urea C.12 is active against both targets. Some compounds could inhibit more than 50% of cancer cell proliferation compared to non-treated ones when assessed in co-cultures using HT-29 and THP-1 cells. In addition, they significantly reduced CD11b expression, which is a promising target for immune modulation in anticancer immunotherapies.

Fourteen triazole-scaffold derivatives were synthetized and biologically evaluated as potential oncoimmunomodultator agents by targeting both PD-L1 and c-Myc. First, the antiproliferative activity of these molecules on the monocultures of several tumor cell lines (HT-29, A-549, and MCF-7) and on the non-tumor cell line HEK-293 was studied. Then, the effects on the mentioned biological targets were also evaluated. Finally, the effect on cancer cell viability when the molecules were co-cultured with immune cells (Jurkat T cells or THP-1) was also determined. Compounds bearing a bromoophenyl group were selected because of their excellent results, and their effect on IL-6 secretion was also studied. In conclusion, we found compounds that are capable of downregulating c-Myc, as well as influencing and altering the distribution of PD-L1 in tumor cells; the compounds are thus capable of influencing the behavior of defensive cells towards cancer cells. p-Bromophenyltriazol 3 is the most active of these as a PD-L1 and c-Myc downregulator and as a potential immunomodulator agent. Moreover, it exhibits an interesting action on inflammation-related cytokine IL-6.

Thirty-nine aryl azoles, thirteen triazoles and twenty-seven tetrazoles, have been synthetized and biologically evaluated to determine their activity as tumor microenvironment disruptors. Antiproliferative studies have been performed on tumor cell lines HT-29, A-549 and MCF-7 and on non-tumor cell line HEK-293. It has been studied in HT-29 the expression levels of biological targets which are involved in tumor microenvironment processes, such as PD-L1, CD-47, c-Myc and VEGFR-2. In addition, antiproliferative activity was evaluated when HT-29 were co-cultured with THP-1 monocytes and the secretion levels of IL-6 were also determined in these co-cultures. The angiogenesis effect of some selected compounds on HMEC-1 was also evaluated as well as their action against vasculogenic mimicry on HEK-293. Compounds bearing an amino group in the phenyl ring and a halogen atom in the benzyl ring showed promising results as tumor microenvironment disrupting agents. The most outstanding compound decrease dramatically the population of HT-29 cells when co-cultured with THP-1 monocytes and the levels of IL-6 secreted, as well as it showed moderate effects over PD-L1, CD-47 and c-Myc.

Twenty-six triazole-based derivatives were designed for targeting both PD-L1 (programmed death receptor ligand 1) and VEGFR-2 (vascular endothelial growth factor receptor 2). These compounds were synthetized and biologically evaluated as multitarget inhibitors of VEGFR-2, PD-L1 and c-Myc proteins. The antiproliferative activity of these molecules on several tumor cell lines (HT-29, A-549, and MCF-7) and on the non-tumor cell line HEK-293 was determined. The effects on the abovementioned biological targets were evaluated for some selected compounds. Compound 23, bearing a p-chlorophenyl group, showed better results than sorafenib in regard to the downregulation of VEGFR-2 and a similar effect to BMS-8 on both PD-L1 and c-Myc proteins. The antiangiogenic and antivascular activities of chloro derivatives were also established by endothelial microtube formation assay on Matrigel®.

The innate immunity toll-like receptor 4 (TLR4) system is a receptor of paramount importance as a therapeutic target. Virtual screening following a "computer-aided drug repurposing" approach was applied to the discovery of novel TLR4 modulators with a non-lipopolysaccharide-like structure. We screened almost 29,000 approved drugs and drug-like molecules from commercial, public, and in-house academia chemical libraries and, after biological assays, identified several compounds with TLR4 antagonist activity. Our computational protocol showed to be a robust approach for the identification of hits with drug-like scaffolds as possible inhibitors of the TLR4 innate immune pathways. Our collaborative work broadens the chemical diversity for inspiration of new classes of TLR4 modulators.

The antitumor activity of imidazolium salts is highly dependent upon their lipophilicity that can be tuned by the introduction of different hydrophobic substituents on the nitrogen atoms of the imidazolium ring of the molecule. Taking this into consideration, we have synthesized and characterized a series of tripodal imidazolium salts derived from L-valine and L-phenylalanine containing different hydrophobic groups and tested them against four cancer cell lines at physiological and acidic pH. At acidic pH (6.2) the anticancer activity of some of the tripodal compounds changes dramatically, and this parameter is crucial to control their cytotoxicity and selectivity. Moreover, several of these compounds displayed selectivity against the control healthy cell line higher than four. The transmembrane anion transport studies revealed moderate transport abilities suggesting that the observed biological activity is likely not the result of just their transport activity. The observed trends in biological activity at acidic pH agree well with the results for the CF leakage assay. These results strongly suggest that this class of compounds can serve as potential chemotherapeutic agents.

The use of nanocarriers for intracellular transport of actives has been extensively studied in recent years and represents a central area of nanomedicine. The main novelty of this paper lies on the use of nanogels formed by a low-molecular-weight gelator (1). Here, non-polymeric, molecular nanogels are successfully used for intracellular transport of two photodynamic therapy (PDT) agents, Rose Bengal (RB) and hypericin (HYP). The two photosensitizers (PSs) exhibit different drawbacks for their use in clinical applications. HYP is poorly water-soluble, while the cellular uptake of RB is hindered due to its dianionic character at physiological pH values. Additionally, both PSs tend to aggregate precluding an effective PDT. Despite the different nature of these PSs, nanogels from gelator 1 provide, in both cases, an efficient intracellular transport into human colon adenocarcinoma cells (HT-29) and a notably improved PDT efficiency, as assessed by confocal laser scanning microscopy and flow cytometry. Furthermore, no significant dark toxicity of the nanogels is observed, supporting the biocompatibility of the delivery system. The developed nanogels are highly reproducible due to their non-polymeric nature, and their synthesis is easily scaled up. The results presented here thus confirm the potential of molecular nanogels as valuable nanocarriers, capable of entrapping both hydrophobic and hydrophilic actives, for PDT of cancer.

Twenty-one styryl and phenethyl aryl ureas have been synthetized and biologically evaluated as multitarget inhibitors of Vascular endothelial growth factor receptor-2 VEGFR-2 and programmed death-ligand-1 (PD-L1) proteins in order to overcome resistance phenomena offered by cancer. The antiproliferative activity of these molecules on several tumor cell lines (HT-29, MCF-7, HeLa and A549), on the endothelial cell line human microvascular endothelial cells (HMEC)-1 and on the non-tumor cell line human embryonic kidney cells (HEK)-293 has been determined. Some derivatives were evaluated for their antiangiogenic properties such as their ability to inhibit microvessel formation using HMEC-1 or their effect on VEGFR-2 in both cancer and endothelial cell lines. In addition, the immunomodulator action of a number of selected compounds was also studied on PD-L1 and c-Myc proteins. Compounds 16 and 23 (Z) and (E)-styryl p-bromophenyl urea, respectively, showed better results than sorafenib in down-regulation of VEGFR-2 and also improved the effect of the anti-PD-L1 compound BMS-8 on both targets, PD-L1 and c-Myc proteins.

Background: Six N-acyl derivatives of aminocombretastatin A-4 have been synthesized and evaluated according to their interaction with tubulin and as c-Myc downregulators. Aims: In search of new promising anti-cancer agents. Objective: This study is focused on the synthesis and the biological evaluation of N-acyl derivatives of aminocombretastatin A-4 (CA-4). Docking studies were carried out to find out whether the synthetic derivatives could bind to tubulin at the colchicine site in a conformation similar to that of CA4. The synthetic derivatives' effect on the proliferation of several cancer cells and non-cancer cells has been measured. Their effect on tubulin polymerization, cell cycle distribution, the microtubule network and c-Myc expression has also been evaluated. Method: A set of six N-acyl derivatives was achieved by means of a peptide-type coupling of aminocombretastatin A-4 and the corresponding carboxylic acid. The synthetic compounds' ability to inhibit cell proliferation was measured by MTT assay against three human carcinoma cell lines (colorectal HT-29, lung A549, and breast adenocarcinoma MCF-7) and one non-tumor cell line (HEK293). Turbidimetry time-course measurements evaluated the inhibition of tubulin polymerization. The action of the synthetic derivatives on cell cycle distribution was measured by flow cytometry and their effects on the microtubule network were determined by immunofluorescence microscopy. Finally, the downregulation of the synthetic derivatives on c-Myc protein was quantified by ELISA assay, while the effect on c-Myc gene was measured by RT-qPCR analysis. Results: Derivatives bearing pentanoyl (compound 2), hexanoyl (compound 3), and heptanoyl (compound 4) side chains show anti-proliferative activities on the HT-29 line in the low nanomolar range, with values similar to that exhibited by AmCA-4 but far exceeding those of CA-4. Compounds 1 (butanoyl side chain) and 2-3 inhibit tubulin polymerization in vitro in a manner similar to that of CA-4 and AmCA-4 whereas compounds 4, 5 (octanoyl side chain) and 6 (dodecanoyl side chain) may be considered as partial inhibitors of tubulin polymerization. While all derivatives are able to accumulate cells in G2/M phase, compounds with the longest acyl chains (5 and 6) are the least active ones in this particular action. Moreover, compounds 2-3 were the most active ones as c-Myc downregulators. Conclusion: Our studies show that the most active compounds in the disruption of the microtubule network are also the most potent ones in the downregulation of c-Myc expression. Other: Compounds 2 and 3 are good candidates for in vivo studies as they combine the best antimitotic and c-Myc downregulation activities at low doses.