Fluorescence-Guided Spatial Drug Screening in 3D Colorectal Cancer Spheroids.

The limited recapitulation of critical cancer features in 2D cultures causes poor translatability of preclinical results from in vitro assays to in vivo tumor models. This contributes to slow drug development with a low success rate. 3D cultures better recapitulate the tumor microenvironment, enabling more accurate predictions when screening drug candidates and improving the development of chemotherapeutics. Platinum (Pt) (IV) compounds are promising prodrugs designed to reduce the severe systemic toxicity of widely used Food and Drug Administration (FDA)-approved Pt(II) drugs such as cisplatin. Here, this work presents spatiotemporal evaluations in 3D colorectal cancer (CRC) spheroids of mitochondria-targeting Pt(IV) complexes. CRC spheroids provide a greater pathophysiological recapitulation of in vivo tumors than 2D cultures by a marked upregulation of the ABCG2 chemoresistance marker expression. Furthermore, new 3D-staining protocols are introduced to evaluate the real-time decrease in mitochondria membrane potential (ΔΨ) in CRC spheroids, and a Pt-sensing dye to quantify the Pt mitochondrial accumulation. Finally, this work demonstrates a correlation between in vitro results and the efficacy of the compounds in vivo. Overall, the CRC spheroids represent a fast and cost-effective model to assess the behavior of Pt compounds in vitro and predict their translational potential in CRC treatment.

Design and Characterization of a New Formulation for the Delivery of COVID-19-mRNA Vaccine to the Nasal Mucosa.

Chitosan, a natural polysaccharide derived from chitin, possesses biocompatibility, biodegradability, and mucoadhesive characteristics, making it an attractive material for the delivery of mRNA payloads to the nasal mucosa and promoting their uptake by target cells such as epithelial and immune cells (e.g., dendritic cells and macrophages). In this project, we aimed at developing novel lipid-based nanoformulations for mRNA delivery to counteract the pandemic caused by SARS-CoV-2 virus. The formulations achieved a mRNA encapsulation efficiency of ~80.2% with chitosan-lipid nanoparticles, as measured by the RiboGreen assay. Furthermore, the evaluation of SARS-CoV-2 Spike (S) receptor-binding domain (RBD) expression via ELISA for our vaccine formulations showed transfection levels in human embryonic kidney cells (HEK 293), lung carcinoma cells (A549), and dendritic cells (DC 2.4) equal to 9.9 ± 0.1 ng/mL (174.7 ± 1.1 fold change from untreated cells (UT)), 7.0 ± 0.2 ng/mL (128.1 ± 4.9 fold change from UT), and 0.9 ± 0.0 ng/mL (18.0 ± 0.1 fold change from UT), respectively. Our most promising vaccine formulation was also demonstrated to be amenable to lyophilization with minimal degradation of loaded mRNA, paving the way towards a more accessible and stable vaccine. Preliminary in vivo studies in mice were performed to assess the systemic and local immune responses. Nasal bronchoalveolar lavage fluid (BALF) wash showed that utilizing the optimized formulation resulted in local antibody concentrations and did not trigger any systemic antibody response. However, if further improved and developed, it could potentially contribute to the management of COVID-19 through nasopharyngeal immunization strategies.

Development of an in vitro co-culture model using Caco-2 and J774A.1 cells to mimic intestinal inflammation.

In vitro models that mimic the pathophysiology in vivo are important tools to study mechanisms of disease and assess the pharmacology and toxicity of drugs. In this work, we report the development of a novel model of intestinal inflammation. This model is based on the co-culture of intestinal epithelial Caco-2 cells and murine J774A.1 macrophages. The model is shown to mimic the intestinal barrier in both healthy and inflamed state. In the healthy state, without external stimulation, Caco-2 and J774A.1 cells were co-cultured in one system without affecting the barrier integrity of intestinal epithelial cells and without inducing release of cytokines from macrophages. To mimic the inflamed intestine, Caco-2 cells were primed with an optimised cytokine cocktail (TNF-⍺, IFN-γ and IL-1ß) and J774A.1 cells were pre-exposed to lipopolysaccharide (LPS) and IFN-γ for 24 h before combining the two cell lines into co-culture. In these conditions, a significant disruption of the epithelial barrier and an increase in pro-inflammatory cytokine (TNF-⍺ and IL-6) levels released from macrophages were detected. The data also show that inflammation in the co-culture model was temporary and reversible upon the removal of the inflammatory stimulus. This new in vitro model could be a valuable tool for investigating the safety and efficacy of drugs in the context of intestinal inflammation and provides advantages over other reported co-culture models of intestinal inflammation in terms of cost and simplicity.

Targeting Non-Coding RNAs for the Development of Novel Hepatocellular Carcinoma Therapeutic Approaches.

Hepatocellular carcinoma (HCC) remains a global health challenge, representing the third leading cause of cancer deaths worldwide. Although therapeutic advances have been made in the few last years, the prognosis remains poor. Thus, there is a dire need to develop novel therapeutic strategies. In this regard, two approaches can be considered: (1) the identification of tumor-targeted delivery systems and (2) the targeting of molecule(s) whose aberrant expression is confined to tumor cells. In this work, we focused on the second approach. Among the different kinds of possible target molecules, we discuss the potential therapeutic value of targeting non-coding RNAs (ncRNAs), which include micro interfering RNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). These molecules represent the most significant RNA transcripts in cells and can regulate many HCC features, including proliferation, apoptosis, invasion and metastasis. In the first part of the review, the main characteristics of HCC and ncRNAs are described. The involvement of ncRNAs in HCC is then presented over five sections: (a) miRNAs, (b) lncRNAs, (c) circRNAs, (d) ncRNAs and drug resistance and (e) ncRNAs and liver fibrosis. Overall, this work provides the reader with the most recent state-of-the-art approaches in this field, highlighting key trends and opportunities for more advanced and efficacious HCC treatments.

Plant-derived extracellular vesicles: Recent advancements and current challenges on their use for biomedical applications.

Extracellular vesicles (EVs) represent a diverse class of lipid bilayer membrane vesicles released by both animal and plant cells. These ubiquitous vesicles are involved in intercellular communication and transport of various biological cargos, including proteins, lipids, and nucleic acids. In recent years, interest in plant-derived EVs has increased tremendously, as they serve as a scalable and sustainable alternative to EVs derived from mammalian sources. In vitro and in vivo findings have demonstrated that these plant-derived vesicles (PDVs) possess intrinsic therapeutic activities that can potentially treat diseases and improve human health. In addition, PDVs can also act as efficient and biocompatible drug carriers. While preclinical studies have shown promising results, there are still several challenges and knowledge gaps that have to be addressed for the successful translation of PDVs into clinical applications, especially in view of the lack of standardised protocols for material handling and PDV isolation from various plant sources. This review provides the readers with a quick overview of the current understanding and research on PDVs, critically analysing the current challenges and highlighting the immense potential of PDVs as a novel class of therapeutics to treat human diseases. It is expected that this work will guide scientists to address the knowledge gaps currently associated with PDVs and promote new advances in plant-based therapeutic solutions.

Investigations on Cellular Uptake Mechanisms and Immunogenicity Profile of Novel Bio-Hybrid Nanovesicles.

In drug delivery, the development of nanovesicles that combine both synthetic and cellular components provides added biocompatibility and targeting specificity in comparison to conventional synthetic carriers such as liposomes. Produced through the fusion of U937 monocytes' membranes and synthetic lipids, our nano-cell vesicle technology systems (nCVTs) showed promising results as targeted cancer treatment. However, no investigation has been conducted yet on the immunogenic profile and the uptake mechanisms of nCVTs. Hence, this study was aimed at exploring the potential cytotoxicity and immune cells' activation by nCVTs, as well as the routes through which cells internalize these biohybrid systems. The endocytic pathways were selectively inhibited to establish if the presence of cellular components in nCVTs affected the internalization route in comparison to both liposomes (made up of synthetic lipids only) and nano-cellular membranes (made up of biological material only). As a result, nCVTs showed an 8-to-40-fold higher cellular internalization than liposomes within the first hour, mainly through receptor-mediated processes (i.e., clathrin- and caveolae-mediated endocytosis), and low immunostimulatory potential (as indicated by the level of IL-1α, IL-6, and TNF-α cytokines) both in vitro and in vivo. These data confirmed that nCVTs preserved surface cues from their parent U937 cells and can be rationally engineered to incorporate ligands that enhance the selective uptake and delivery toward target cells and tissues.

Extracellular vesicles in cardiovascular disease.

Cardiovascular disease remains the leading cause of morbidity and mortality globally. Extracellular vesicles (EVs), a group of heterogeneous nanosized cell-derived vesicles, have attracted great interest as liquid biopsy material for biomarker discovery in a variety of diseases including cardiovascular disease. Because EVs inherit bioactive components from parent cells and are able to transfer their contents to recipient cells, EVs hold great promise as potential cell-free therapeutics and drug delivery systems. However, the development of EV-based diagnostics, therapeutics or drug delivery systems has been challenging due to the heterogenicity of EVs in biogenesis, size and cellular origin, the lack of standardized isolation and purification methods as well as the low production yield. In this review, we will provide an overview of the recent advances in EV-based biomarker discovery, highlight the potential usefulness of EVs and EV mimetics for therapeutic treatment and drug delivery in cardiovascular disease. In view of the fast development in this field, we will also discuss the challenges of current methodologies for isolation, purification and fabrication of EVs and potential alternatives.

Dual-Functional Iron Oxide Nanoparticles Coated with Polyvinyl Alcohol/5-Fluorouracil/Zinc-Aluminium-Layered Double Hydroxide for a Simultaneous Drug and Target Delivery System.

Iron oxide nanoparticles are suitable for biomedical applications owing to their ability to anchor to various active agents and drugs, unique magnetic properties, nontoxicity, and biocompatibility. In this work, the physico-chemical and magnetic properties, as well as the cytotoxicity, of Fe3O4 nanoparticles coated with a polymeric carrier and loaded with a 5-fluorouracil (5-FU) anti-cancer drug are discussed. The synthesized Fe3O4 nanoparticles were coated with polyvinyl alcohol and Zn/Al-layered double hydroxide as the drug host. The XRD, DTA/TG, and FTIR analyzes confirmed the presence of the coating layer on the surface of nanoparticles. The results showed a decrease in saturation magnetization of bare Fe3O4 nanoparticles after coating with the PVA/5FU/Zn/Al-LDH layer. In addition, the presence of the coating prevented the agglomeration of nanoparticles. Furthermore, the pseudo-second-order equation governed the kinetics of drug release. Finally, the coated nanoparticles showed stronger activity against liver cancer cells (HepG2) compared to that of the naked 5-FU drug, and displayed no cytotoxicity towards 3T3 fibroblast cell lines. The results of the present study demonstrate the potential of a nano delivery system for cancer treatment.

Synthesis and Characterization of Chitosan-Based Nanodelivery Systems to Enhance the Anticancer Effect of Sorafenib Drug in Hepatocellular Carcinoma and Colorectal Adenocarcinoma Cells.

The formation of two nanodelivery systems, Sorafenib (SF)-loaded chitosan (SF-CS) and their folate-coated (SF-CS-FA) nanoparticles (NPs), were developed to enhance SF drug delivery on human Hepatocellular Carcinoma (HepG2) and Colorectal Adenocarcinoma (HT29) cell lines. The ionic gelation method was adopted to synthesize the NPs. The characterizations were performed by DLS, FESEM, TEM, XRD, TGA, FTIR, and UV-visible spectroscopy. It was found that 83.7 ± 2.4% and 87.9 ± 1.1% of encapsulation efficiency; 18.2 ± 1.3% and 19.9 ± 1.4% of loading content; 76.3 ± 13.7 nm and 81.6 ± 12.9 nm of hydrodynamic size; 60-80 nm and 70-100 nm of TEM; and FESEM sizes of near-spherical shape were observed, respectively, for SF-CS and SF-CS-FA nanoparticles. The SF showed excellent release from the nanoparticles under pH 4.8 PBS solution, indicating a good delivery system for tumor cells. The cytotoxicity study revealed their better anticancer action towards HepG2 and HT29 cell lines compared to the free sorafenib. Moreover, both NPs systems showed negligible toxicity to normal Human Dermal Fibroblast adult cells (HDFa). This is towards an enhanced anticancer drug delivery system with sustained-release properties for better cancer management.

Extracellular Vesicle (EV) biohybrid systems for cancer therapy: Recent advances and future perspectives.

Extracellular vesicles (EVs) are a class of cell-derived lipid-bilayer membrane vesicles secreted by almost all mammalian cells and involved in intercellular communication by shuttling various biological cargoes. Over the last decade, EVs - namely exosomes and microvesicles - have been extensively explored as next-generation nanoscale drug delivery systems (DDSs). This is in large due to their endogenous origin, which enables EVs to circumvent some of the limitations associated with existing cancer therapy approaches (i.e. by preventing recognition by the immune system and improving selectivity towards tumor tissue). However, successful translation of these cell-derived vesicles into clinical applications has been hindered by several factors, among which the loading of exogenous therapeutic molecules still represents a great challenge. In order to address this issue and to further advance these biologically-derived systems as drug carriers, EV-biohybrid nano-DDSs, obtained through the fusion of EVs with conventional synthetic nano-DDSs, have recently been proposed as a valuable alternative as DDSs. Building on the idea of "combining the best of both worlds", a combination of these two unique entities aims to harness the beneficial properties associated with both EVs and conventional nano-DDSs, while overcoming the flaws of the individual components. These biohybrid systems also provide a unique opportunity for exploitation of new synergisms, often leading to improved therapeutic outcomes, thus paving the way for advancements in cancer therapy. This review aims to describe the recent developments of EV-biohybrid nano-DDSs in cancer therapy, to highlight the most promising results and breakthroughs, as well as to provide a glimpse on the possible intrinsic targeting mechanisms of EVs that can be bequeathed to their hybrid systems. Finally, we also provide some insights in the future perspectives of EV-hybrid DDSs.

3D printing of four-in-one oral polypill with multiple release profiles for personalized delivery of caffeine and vitamin B analogues.

Personalized supplementation has found recent momentum with an estimated global market size of USD 1.6 billion in 2019 and an expected CAGR of 8.5% between 2020 and 2028. Alongside this rising trend, a simple, accurate, inexpensive and flexible method to produce personalized dosage forms of a wide variety of supplements would be beneficial to both the industry players and individual consumers. Here, we present a 3D printing method to fabricate a four-in-one oral polypill with multiple release profiles for personalized delivery of caffeine and vitamin B analogues. The 3D printable formulations were fabricated and optimized from existing FDA GRAS excipients based on their viscosity, shear thinning properties, recovery of paste and mechanical strength. In the polypill, vitamin B analogues and caffeine were used as the model dietary ingredients. We performed a standard 2 stage USP in vitro dissolution test of the polypill, and demonstrated that vitamin B1, B3 and B6 could be immediately released within 30 min, while caffeine could be slowly released over a period of 4 h. This demonstrated the ability dietary supplement containing different ingredients with varying release profiles, all within a single polypill. Throughout the formulation and 3D printing process, there were no detectable changes to the dietary ingredients nor any interactions with the excipients. This method serves as an intriguing complement to traditional manufacturing of oral tablets, especially when flexibility in design, dose, volume and release profiles of each dietary ingredient is required, as exemplified in personalized supplementation.

Release of a liver anticancer drug, sorafenib from its PVA/LDH- and PEG/LDH-coated iron oxide nanoparticles for drug delivery applications.

The use of nanocarriers composed of polyethylene glycol- and polyvinyl alcohol-coated vesicles encapsulating active molecules in place of conventional chemotherapy drugs can reduce many of the chemotherapy-associated challenges because of the increased drug concentration at the diseased area in the body. The present study investigated the structure and magnetic properties of iron oxide nanoparticles in the presence of polyvinyl alcohol and polyethylene glycol as the basic surface coating agents. We used superparamagnetic iron oxide nanoparticles (FNPs) as the core and studied their effectiveness when two polymers, namely polyvinyl alcohol (PVA) and polyethylene glycol (PEG), were used as the coating agents together with magnesium-aluminum-layered double hydroxide (MLDH) as the nanocarrier. In addition, the anticancer drug sorafenib (SO), was loaded on MLDH and coated onto the surface of the nanoparticles, to best exploit this nano-drug delivery system for biomedical applications. Samples were prepared by the co-precipitation method, and the resulting formation of the nanoparticles was confirmed by X-ray, FTIR, TEM, SEM, DLS, HPLC, UV-Vis, TGA and VSM. The X-ray diffraction results indicated that all the as-synthesized samples contained highly crystalline and pure Fe3O4. Transmission electron microscopy analysis showed that the shape of FPEGSO-MLDH nanoparticles was generally spherical, with a mean diameter of 17 nm, compared to 19 nm for FPVASO-MLDH. Fourier transform infrared spectroscopy confirmed the presence of nanocarriers with polymer-coating on the surface of iron oxide nanoparticles and the existence of loaded active drug consisting of sorafenib. Thermogravimetric analyses demonstrated the thermal stability of the nanoparticles, which displayed enhanced anticancer effect after coating. Vibrating sample magnetometer (VSM) curves of both produced samples showed superparamagnetic behavior with the high saturation magnetization of 57 emu/g for FPEGSO-MLDH and 49 emu/g for FPVASO-MLDH. The scanning electron microscopy (SEM) images showed a narrow size distribution of both final samples. The SO drug loading and the release behavior from FPEGSO-MLDH and FPVASO-MLDH were assessed by ultraviolet-visible spectroscopy. This evaluation showed around 85% drug release within 72 h, while 74% of sorafenib was released in phosphate buffer solution at pH 4.8. The release profiles of sorafenib from the two designed samples were found to be sustained according to pseudo-second-order kinetics. The cytotoxicity studies confirmed the anti-cancer activity of the coated nanoparticles loaded with SO against liver cancer cells, HepG2. Conversely, the drug delivery system was less toxic than the pure drug towards fibroblast-type 3T3 cells.

Synthesis and Cytotoxicity Study of Magnetite Nanoparticles Coated with Polyethylene Glycol and Sorafenib-Zinc/Aluminium Layered Double Hydroxide.

In the last two decades, the development of novel approaches for cancer treatment has attracted intense attention due to the growing number of patients and the inefficiency of the available current conventional treatments. In this study, superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized by the co-precipitation method in an alkaline medium. Then the nanoparticles were chemically modified by coating them with polyethylene glycol (PEG) and sorafenib (SO)-zinc/aluminum layered double hydroxide (ZLDH) to improve their biocompatibility. The SPIONs and their coated and drug-loaded nanoparticles, M-PEG-SO-ZLDH are of the crystalline phase with the presence of C, O, Al, Fe, Cl, Zn in the latter, indicating the presence of the coating layers on the surface of the SPIONs. The superparamagnetic properties of the bare SPIONs were found to be reduced but retained in its coated drug delivery nanoparticles, M-PEG-SO-ZLDH. The latter has an average particle size of 16 nm and the release of the drug from it was found to be governed by the pseudo-second-order kinetic. The cytotoxicity and biocompatibility evaluation of the drug-loaded magnetic nanoparticles using 3T3 and HepG2 cells using the diphenyltetrazolium bromide (MTT) assays shows that the synthesized nanoparticles were less toxic than the pure drug. This preliminary study indicates that the prepared nanoparticles are suitable to be used for the drug delivery system.

Harnessing the Immunomodulatory Properties of Bacterial Ghosts to Boost the Anti-mycobacterial Protective Immunity.

Tuberculosis (TB) pathogenesis is characterized by inadequate immune cell activation and delayed T cell response in the host. Recent immunotherapeutic efforts have been directed at stimulating innate immunity and enhancing interactions between antigen presenting cells and T cells subsets to improve the protective immunity against TB. In this study, we investigated the immunostimulatory properties of bacterial ghosts (BG) as a novel approach to potentiate the host immunity against mycobacterial infection. BG are intact cytoplasm-free Escherichia coli envelopes and have been developed as bacterial vaccines and adjuvant/delivery system in cancer immunotherapy. However, BG have yet to be exploited as immunopotentiators in the context of infectious diseases. Here, we showed that BG are potent inducers of dendritic cells (DC), which led to enhanced T cell proliferation and differentiation into effector cells. BG also induced macrophage activation, which was associated with enhanced nitric oxide production, a key anti-mycobacterial weapon. We further demonstrated that the immunostimulatory capability of BG far exceeds that of LPS and involves both TLR4-dependent and independent pathways. Consistently, BG treatment, but not LPS treatment, reduced the bacterial burden in infected mice, which correlated with increased influx of innate and adaptive effector immune cells and increased production of key cytokines in the lungs. Finally and importantly, enhanced bacilli killing was seen in mice co-administered with BG and second-line TB drugs bedaquiline and delamanid. Overall, this work paves the way for BG as potent immunostimulators that may be harnessed to improve mycobacteria killing at the site of infection.

Design, Synthesis and Evaluation of New Indolylpyrimidylpiperazines for Gastrointestinal Cancer Therapy.

Human A3 adenosine receptor hA3AR has been implicated in gastrointestinal cancer, where its cellular expression has been found increased, thus suggesting its potential as a molecular target for novel anticancer compounds. Observation made in our previous work indicated the importance of the carbonyl group of amide in the indolylpyrimidylpiperazine (IPP) for its human A2A adenosine receptor (hA2AAR) subtype binding selectivity over the other AR subtypes. Taking this observation into account, we structurally modified an indolylpyrimidylpiperazine (IPP) scaffold, 1 (a non-selective adenosine receptors' ligand) into a modified IPP (mIPP) scaffold by switching the position of the carbonyl group, resulting in the formation of both ketone and tertiary amine groups in the new scaffold. Results showed that such modification diminished the A2A activity and instead conferred hA3AR agonistic activity. Among the new mIPP derivatives (3-6), compound 4 showed potential as a hA3AR partial agonist, with an Emax of 30% and EC50 of 2.89 ± 0.55 µM. In the cytotoxicity assays, compound 4 also exhibited higher cytotoxicity against both colorectal and liver cancer cells as compared to normal cells. Overall, this new series of compounds provide a promising starting point for further development of potent and selective hA3AR partial agonists for the treatment of gastrointestinal cancers.

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines to develop functionalized ligands to target adenosine receptors: fluorescent ligands as an example.

A series of adenosine receptor antagonists bearing a reactive linker was developed. Functionalization of these derivatives is useful to easily obtain multi-target ligands, receptor probes, drug delivery systems, and diagnostic or theranostic systems. The pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine scaffold was chosen as a pharmacophore for the adenosine receptors. It was substituted at the 5 position with reactive linkers of different lengths. Then, these compounds were used to synthesise probes for the adenosine receptors by functionalization with a fluorescent moiety. Both series of compounds were evaluated for their binding at the four adenosine receptor subtypes. Different affinity and selectivity profiles were observed towards hA1, hA2A and hA3 adenosine receptors. In particular, fluorescent compounds behave as dual hA2A/hA3 ligands. Computational studies suggested different binding modes for developed compounds at the three receptors. Both molecular docking and supervised molecular dynamics (SuMD) simulations confirmed that the preferred binding mode at the single receptor was driven by the substitution present at the 5 position. Obtained results rationalized the compounds' binding profile at the adenosine receptors and pave the way for the development of more potent conjugable and conjugated ligands targeting these membrane receptors.

Carbon nanotube bottles for incorporation, release and enhanced cytotoxic effect of cisplatin.

Carbon nanotubes (CNTs) have emerged as promising drug delivery systems particularly for cancer therapy, due to their abilities to overcome some of the challenges faced by cancer treatment, namely non-specificity, poor permeability into tumour tissues, and poor stability of anticancer drugs. Encapsulation of anticancer agents inside CNTs provides protection from external deactivating agents. However, the open ends of the CNTs leave the encapsulated drugs exposed to the environment and eventually their uncontrolled release before reaching the desired target. In this study, we report the successful encapsulation of cisplatin, a FDA-approved chemotherapeutic drug, into multi-walled carbon nanotubes and the capping at the ends with functionalised gold nanoparticles to achieve a "carbon nanotube bottle" structure. In this proof-of-concept study, these caps did not prevent the encapsulation of drug in the inner space of CNTs; on the contrary, we achieved higher drug loading inside the nanotubes in comparison with data reported in literature. In addition, we demonstrated that encapsulated cisplatin could be delivered in living cells under physiological conditions to exert its pharmacological action.

Dual-Targeting Dual-Action Platinum(IV) Platform for Enhanced Anticancer Activity and Reduced Nephrotoxicity.

A novel and highly efficient dual-targeting platform was designed to ensure targeted in vivo delivery of dual-action PtIV prodrugs. The dual targeting was established by liposomal encapsulation of PtIV complexes, thereby utilizing the enhanced permeability and retention (EPR) effect as the first stage of targeting to attain a high accumulation of the drug-loaded liposomes in the tumor. After the release of the PtIV prodrug inside cancer cells, a second stage of targeting directed a portion of the PtIV prodrugs to the mitochondria. Upon intracellular reduction, these PtIV prodrugs released two bioactive molecules, acting both on the mitochondrial and on the nuclear DNA. Our PtIV system showed excellent activity in vitro and in vivo, characterized by a cytotoxicity in a low micromolar range and complete tumor remission, respectively. Notably, marked in vivo activity was accompanied by reduced kidney toxicity, highlighting the unique therapeutic potential of our novel dual-targeting dual-action platform.

New Water-Soluble Copper(II) Complexes with Morpholine-Thiosemicarbazone Hybrids: Insights into the Anticancer and Antibacterial Mode of Action.

Six morpholine-(iso)thiosemicarbazone hybrids HL1-HL6 and their Cu(II) complexes with good-to-moderate solubility and stability in water were synthesized and characterized. Cu(II) complexes [Cu(L1-6)Cl] (1-6) formed weak dimeric associates in the solid state, which did not remain intact in solution as evidenced by ESI-MS. The lead proligands and Cu(II) complexes displayed higher antiproliferative activity in cancer cells than triapine. In addition, complexes 2-5 were found to specifically inhibit the growth of Gram-positive bacteria Staphylococcus aureus with MIC50 values at 2-5 µg/mL. Insights into the processes controlling intracellular accumulation and mechanism of action were investigated for 2 and 5, including the role of ribonucleotide reductase (RNR) inhibition, endoplasmic reticulum stress induction, and regulation of other cancer signaling pathways. Their ability to moderately inhibit R2 RNR protein in the presence of dithiothreitol is likely related to Fe chelating properties of the proligands liberated upon reduction.

[1,2,4]Triazolo[1,5-c]pyrimidines as adenosine receptor antagonists: Modifications at the 8 position to reach selectivity towards A3 adenosine receptor subtype.

[1,2,4]Triazolo[1,5-c]pyrimidine is a promising platform to develop adenosine receptor antagonists. Here, we tried to investigate the effect of the substituent at the 8 position of [1,2,4]triazolo[1,5-c]pyrimidine derivatives on affinity and selectivity at the human A3 adenosine receptor subtype. In particular, we have introduced both esters and amides, principally with a benzylic nature. In addition, a small series of 5-substituted [1,2,4]triazolo[1,5-c]pyrimidines was designed in order to complete the structure-activity relationship analysis. Several of these new compounds showed affinity towards human A3 adenosine receptor in the low nanomolar range, with the most potent derivative of the series bringing a 4-ethylbenzylester at the 8 position (compound 18, hA3AR Ki = 1.21 nM). Docking studies performed on the synthesized compounds inside models of human A1, A2A and A3 adenosine receptors showed similar binding modes, comparable with the typical crystallographic binding mode of the inverse agonist ZM-241,385.