Development and characterization of a temozolomide-loaded nanoemulsion and the effect of ferrocene pre and co-treatments in glioblastoma cell models.

BACKGROUND: Glioblastoma is a severe brain tumor that requires aggressive treatment involving surgery, radiotherapy, and chemotherapy, offering a survival rate of only 15 months. Fortunately, recent nanotechnology progress has enabled novel approaches and, alongside ferrocenes' unique properties of cytotoxicity, sensitization, and interaction with reactive oxygen species, have brought new possibilities to complement chemotherapy in nanocarrier systems, enhancing treatment results. METHODS: In this work, we developed and characterized a temozolomide-loaded nanoemulsion and evaluated its cytotoxic potential in combination with ferrocene in the temozolomide-resistant T98G and temozolomide-sensitive U87 cell lines. The effects of the treatments were assessed through acute assays of cell viability, cell death, mitochondrial alterations, and a treatment protocol simulation based on different two-cycle regimens. RESULTS: Temozolomide nanoemulsion showed a z-average diameter of 173.37 ± 0.86 nm and a zeta potential of - 6.53 ± 1.13 mV. Physicochemical characterization revealed that temozolomide is probably associated with nanoemulsion droplets instead of being entrapped within the nanostructure, allowing a rapid drug release. In combination with ferrocene, temozolomide nanoemulsion reduced glioblastoma cell viability in both acute and two-cycle regimen assays. The combined treatment approach also reversed T98G's temozolomide-resistant profile by altering the mitochondrial membrane potential of the cells, thus increasing reactive oxygen species generation, and ultimately inducing cell death. CONCLUSIONS: Altogether, our results indicate that using nanoemulsion containing temozolomide in combination with ferrocene is an effective approach to improve glioblastoma therapy outcomes.

pH/GSH dual-responsive supramolecular nanomedicine for hypoxia-activated combination therapy.

Moderate oxygen (O2) supply and uneven distribution of oxygen at the tumor site usually hinder the therapeutic efficacy of hypoxia-activated prodrugs. In this report, we designed a ferrocene-containing supramolecular nanomedicine (PFC/GOD-TPZ) with the PEG corona and disulfide-bond cross-linked core to co-encapsulate 4-di-N-oxide tirapazamine (TPZ) and glucose oxidase (GOD). The PEG corona of PFC/GOD-TPZ could be weakly acidic tumor pH-responsively detached for an enhanced cellular internalization, while the disulfide-bond cross-linked core could be cleavaged by intracellular glutathione (GSH) to present a GSH-triggered drug-release behavior. Subsequently, the cascade reactions, including catalytic reactions among the released GOD, glucose, and O2 to generate H2O2 and the subsequent Fenton reaction between ferrocene and H2O2, occurred. With the depletion of O2, the non-toxic TPZ was activated and converted into the cytotoxic therapeutic agent benzotriazinyl (BTZ) radical under the exacerbated hypoxic microenvironment. Collectively, the PFC/GOD-TPZ provides a promising strategy for effective combination therapy of GOD-mediated starvation therapy, chemodynamic therapy (CDT), and hypoxia-activated chemotherapy (CT).

A self-assembly nano-prodrug for triple-negative breast cancer combined treatment by ferroptosis therapy and chemotherapy.

Chemotherapeutics have been recommended as the standard protocol for inoperable patients with triple-negative breast cancer (TNBC) at advanced stage, yet limited success has been achieved in prolonging survival rates by this monotherapy. A major reason for this failure is the chemo-resistance from traditional apoptotic pathways resulting in poor therapeutic effect. Ferroptosis has become a powerful modality of no-apoptotic cell death, which can effectively evade chemo-resistance in apoptotic pathways. Herein, we propose an active-targeting small-molecular self-assembly nano-prodrug for co-delivery of chemotherapeutics (CPT), Ferrocene (Fc) and GPX4 inhibitor (RSL3) to overcome the chemo-resistance from traditional apoptotic pathways. In this nano-prodrug, the disulfide linkage not only serves as a GSH-responsive trigger, but also exhibits a stable self-assembly behavior that forms nanoparticle. Interestingly, the RSL3 can be loaded during this self-assembly process that forms a three-components nano-prodrug. In tumor environment, the high GSH level can disassemble the nano-prodrug to trigger the release of the parent drug, which can improve the therapeutic effect by synergistic effects of ferroptosis and apoptosis. In different TNBC mice models, the nano-prodrug is encapsulated into RGD-modified phospholipid micelles (DSPE-PEG2000-RGD) and exhibits high anti-tumor and anti-metastasis efficacy, especially in orthotopic models. The application of ferroptosis to assist the enhancement of chemotherapeutics may serve as a promising strategy for TNBC treatment. STATEMENT OF SIGNIFICANCE: Chemotherapeutics have been recommended as the standard of care for palliative and adjuvant treatment in patients with triple-negative breast cancer (TNBC), yet limited success has been achieved in prolonging the overall survival of patients by this monotherapy. A major reason for this failure is the chemo-resistance from traditional apoptotic pathways resulting in poor therapeutic effect. Thus, the co-delivery of the apoptosis and ferroptosis drug may overcome or evade the resistance in chemotherapy-induced apoptotic pathways and provide a promising strategy to combat TNBC. In this work, we developed a small-molecular self-assembly nano-prodrug for co-delivery of chemotherapeutics (CPT), Ferrocene (Fc) and ferroptosis resistance inhibitor (RSL3), which could overcome the chemo-resistance and improve the therapeutic effect by synergistic effects of ferroptosis and apoptosis.

Polymeric nano-system for macrophage reprogramming and intracellular MRSA eradication.

Intracellular Methicillin-Resistant Staphylococcus aureus (MRSA) remains a major factor of refractory and recurrent infections, which cannot be well addressed by antibiotic therapy. Here, we design a cellular infectious microenvironment-activatable polymeric nano-system to mediate targeted intracellular drug delivery for macrophage reprogramming and intracellular MRSA eradication. The polymeric nano-system is composed of a ferrocene-decorated polymeric nanovesicle formulated from poly(ferrocenemethyl methacrylate)-block-poly(2-methacryloyloxyethyl phosphorylcholine) (PFMMA-b-PMPC) copolymer with co-encapsulation of clofazimine (CFZ) and interferon-γ (IFN-γ). The cellular-targeting PMPC motifs render specific internalization by macrophages and allow efficient intracellular accumulation. Following the internalization, the ferrocene-derived polymer backbone sequentially undergoes hydrophobic-to-hydrophilic transition, charge reversal and Fe release in response to intracellular hydrogen peroxide over-produced upon infection, eventually triggering endosomal escape and on-site cytosolic drug delivery. The released IFN-γ reverses the immunosuppressive status of infected macrophages by reprogramming anti-inflammatory M2 to pro-inflammatory M1 phenotype. Meanwhile, intracellular Fe2+-mediated Fenton reaction together with antibiotic CFZ contributes to increased intracellular hydroxyl radical (•OH) generation. Ultimately, the nano-system achieves robust potency in ablating intracellular MRSA and antibiotic-tolerant persisters by synchronous immune modulation and efficient •OH killing, providing an innovative train of thought for intracellular MRSA control.

Induction of immunogenic cell death in murine colon cancer cells by ferrocene-containing redox phospholipid polymers.

Immunogenic cell death (ICD), activated by damage-associated molecular patterns (DAMPs), is an apoptotic cell death process that elicits antitumor immunity. Although anticancer drugs that can induce ICD are promising for cancer treatment, the design strategy for ICD inducers remains unclear. In this study, we demonstrated the cell-penetrating redox phospholipid polymer poly(2-methacryloyloxyethyl phosphorylcholine-co-vinyl ferrocene) (pMFc) inducing ICD in murine colon cancer CT26 cells. pMFc produced oxidative stress by extracting electrons from CT26 cells and induced the release of DAMPs, such as calreticulin, adenosine triphosphate, and high-mobility group box 1. Moreover, the injection of pMFc-treated CT26 cells inhibited tumor formation in subsequently challenged CT26 cells, indicating that pMFc elicited antitumor immunity through ICD. Using in vivo therapy, intratumoral injections of pMFc induced complete tumor regression in 20% (1/5) of mice. These results suggested that the redox phospholipid polymer provides a new option for ICD-inducing anticancer polymers.

Ferrocene-based multifunctional nanoparticles for combined chemo/chemodynamic/photothermal therapy.

Ferrocene and its derivatives have great potential for biomedical applications, but few related studies have been reported. In this study, copper ions and ferrocene derivatives were used for the first time to construct the ferrocene-based nanoparticles (Cu-Fc) with a hydrated particle size of approximately 220 nm. Their good photothermal conversion properties were verified in vitro and in vivo for the first time, indicating that they could be used as a novel photothermal agent for tumor treatment. In addition, the nanoparticles exhibited efficient Fenton effect under weakly acidic conditions, indicating that they can generate hydroxyl radicals (OH) to kill tumors in the weakly acidic environment of the tumor-specific microenvironment. More importantly, the nanoparticles can deplete glutathione (GSH), thus further enhancing Fenton effect-mediated chemodynamic therapy (CDT). Multifunctional ferrocene-based nanoparticles (DOX@Cu-Fc) were obtained after loading the chemotherapeutic drug doxorubicin hydrochloride (DOX). The results of in vitro and in vivo experiments showed that DOX@Cu-Fc could enhance tumor treatment by the combination of chemo/CDT/photothermal therapy (PTT).

Ferrocene and glucose oxidase-installed multifunctional hydrogel reactors for local cancer therapy.

A hyaluronic acid (HA)-based one-pot hydrogel reactor with single syringe injection and immediate gelation was developed for starvation therapy (ST), chemodynamic therapy (CDT), ferroptosis, and photothermal therapy (PTT) against breast cancer. A rheologically tuned hydrogel network, composed of HA-phenylboronic acid (HP) and HA-dopamine (HD), was designed by introducing a boronate ester linkage (phenylboronic acid-dopamine interaction) and polydopamine bond (pH control). Ferrocene (Fc)-conjugated HP (Fc-HP) was synthesized to achieve ferroptosis, Fenton reaction-involved toxic hydroxyl radical (•OH) generation, and photothermal ablation in cancer therapy. Glucose oxidase (GOx) was entrapped in the pH-modulated Fc-HP (Fc-HP°)/HD hydrogel network for converting intracellular glucose to H2O2 to enable its own supply. The GOx/Fc combination-installed hydrogel reactor system can provide sustained ST/CDT/PTT functions along with ferroptosis. Injection of Fc-HP°/HD/GOx hydrogel with single-syringe injectability, shear-thinning feature, and self-healing capability offered a slow biodegradation rate and high safety profiles. Peritumorally injected Fc-HP°/HD/GOx hydrogel also efficiently suppressed the growth of breast cancer based on multifunctional therapeutic approaches with reduced dosing frequency. Hyperthermia induced by near-infrared (NIR) laser absorption may amplify the therapeutic effects of free radicals. It is expected that this Fc-HP°/HD/GOx hydrogel system can be applied to local cancer therapy with high efficacy and safety profiles.

pH-responsive Mannose-modified ferrocene Metal-Organic frameworks with rare earth for Tumor-targeted synchronous Chemo/Chemodynamic therapy.

The combination of chemodynamic therapy (CDT) and chemotherapy is a promising strategy to achieve enhanced anticancer effects. Metal-organic frameworks (MOFs), as multifunctional drug delivery vehicles, have received extensive attention in the biomedical field. Carbohydrate has excellent biocompatibility and targeting ability, which can be used as a targeting ligand due to a specific recognition with glycoprotein receptors that overexpress on cancer cell membranes. Herein, the pH-responsive mannose-modified ferrocene MOFs with rare earth metal were synthesized via coordination-driven self-assembly of 1,1'-Ferrocenedicarboxylic acid and ytterbium chloride. Subsequently, DOX@Fc-MOFs-Mann nanoparticles (NPs) were obtained by loading doxorubicin (DOX) and modifying mannose (Mann), where DOX@Fc-MOFs-Mann NPs were able to precisely target HepG2 cells via mannose receptor and slowly decompose in the acidic environment of tumor to release ferrocene, DOX, and Yb3+. Fe2+ in ferrocene effectively activated Fenton reaction to produce high levels of reactive oxygen species (ROS) for irreversible induction of cell apoptosis or necroptosis. Combined with the chemotherapy (CT) ability of DOX, Yb3+ further induced cell death through its own toxicity to successfully achieved the rare earth metal synergistic CDT and CT combination therapy. This synergistic CDT and CT strategy not only opens up new horizons for rare earth metals in biomedical applications but also provides new inspiration into the construction of glycosyl-modified MOFs.

Supramolecular Polymerization-Induced Nanoassemblies for Self-Augmented Cascade Chemotherapy and Chemodynamic Therapy of Tumor.

The clinical application of chemodynamic therapy is impeded by the insufficient intracellular H2 O2 level in tumor tissues. Herein, we developed a supramolecular nanoparticle via a simple one-step supramolecular polymerization-induced self-assembly process using platinum (IV) complex-modified ß-cyclodextrin-ferrocene conjugates as supramolecular monomers. The supramolecular nanoparticles could dissociate rapidly upon exposure to endogenous H2 O2 in the tumor and release hydroxyl radicals as well as platinum (IV) prodrugs in situ, which is reduced into cisplatin to significantly promote the generation of H2 O2 in the tumor tissue. Thus, the supramolecular nanomedicine overcomes the limitation of conventional chemodynamic therapy via the self-augmented cascade radical generation and drug release. In addition, dissociated supramolecular nanoparticles could be readily excreted from the body via renal clearance to effectively avoid systemic toxicity and ensure long term biocompatibility of the nanomedicine. This work may provide new insights on the design and development of novel supramolecular nanoassemblies for cascade chemo/chemodynamic therapy.

4-Aminoquinoline-ferrocene Hybrids as Potential Antimalarials.

BACKGROUND: Malaria is a deadly disease. It is mostly treated using 4- aminoquinoline derivatives such as chloroquine etc. because it is well-tolerated, displays low toxicity, and after administration, it is rapidly absorbed. The combination of 4-aminoquinoline with other classes of antimalarial drugs has been reported to be an effective approach for the treatment of malaria. Furthermore, some patents reported hybrids 4-aminoquinolines containing ferrocene moiety with potent antimalarial activity. OBJECTIVE: The objective of the current study is to prepare 4-aminoquinoline-ferrocene hybrids via esterification and amidation reactions. The compounds were characterized via FTIR, LC-MS and NMR spectroscopy. In vitro screening against chloroquine-sensitive P. falciparum parasite (NF54) at concentrations (1 µM and 5 µM) and an inhibitory concentration (full dose-response) was studied. METHODS: The compounds were prepared via known reactions and monitored by Thin Layer Chromatography. The compounds were purified by column chromatography and characterized using FTIR, NMR and MS. In vitro antiplasmodial evaluation was performed against asexual parasite and chloroquine was used as a reference drug. RESULTS: The percentage inhibition effects of the hybrid compounds were in a range of 97.9-102% at 5 µM and 36-96% at 1 µM. Furthermore, the IC50 values of the compounds were in the range of 0.7-1.6 µM when compared to the parent drug, 4-ferrocenylketobutanoic acid. CONCLUSION: The hybrid compounds displayed significant antimalarial activity when compared to the parent drug. However, they were not as effective as chloroquine on the drug-sensitive parasite. The findings revealed that 4-aminoquinolines and ferrocene are potential scaffolds for developing potent antimalarials.

Lipid Compositions in Infant Formulas Affect the Solubilization of Antimalarial Drugs Artefenomel (OZ439) and Ferroquine during Digestion.

Recent studies have shown that the solubilization of two antimalarial drug candidates, artefenomel (OZ439) and ferroquine (FQ), designed to provide a single-dose combination therapy for uncomplicated malaria can be enhanced using milk as a lipid-based formulation. However, milk as an excipient faces significant quality and regulatory hurdles. We therefore have investigated infant formula as a potential alternative formulation approach. The significance of the lipid species present in a formula with different lipid compositions upon the solubilization of OZ439 and FQ during digestion has been investigated. Synchrotron small-angle X-ray scattering was used to measure the diffraction from a dispersed drug during digestion and thereby determine the extent of drug solubilization. High-performance liquid chromatography was used to quantify the amount of drug partitioned into the digested lipid phases. Our results show that both the lipid species and the amount of lipids administered were key determinants for the solubilization of OZ439, while the solubilization of FQ was independent of the lipid composition. Infant formulas could therefore be designed and used as milk substitutes to tailor the desired level of drug solubilization while circumventing the variability of components in naturally derived milk. The enhanced solubilization of OZ439 was achieved during the digestion of medium-chain triacylglycerols (MCT), indicating the potential applicability of MCT-fortified infant formula powder as a lipid-based formulation for the oral delivery of OZ439 and FQ.

Ferrocene Functionalized Upconversion Nanoparticle Nanosystem with Efficient Near-Infrared-Light-Promoted Fenton-Like Reaction for Tumor Growth Suppression.

By taking advantage of the efficient Förster resonance energy transfer (FRET) between near-infrared (NIR)-responsive lanthanide-doped upconversion nanoparticles (UCNPs) and Fenton reagent ferrocenyl compounds (Fc), a series of Fc-UCNPs was designed by functionalizing NaYF4:Yb,Tm nanoparticles with Fc1-Fc5 via surface-coordination chemistry. Fc-UCNP-Lipo nanosystems were then constructed by encapsulating Fc-UCNP inside liposomes for efficient delivery. Fc-UCNP can effectively release ·OH via a NIR-promoted Fenton-like reaction. In vitro and in vivo studies of Fc1-UCNP-Lipo confirmed the preferential accumulation in a tumor site followed by an enhanced uptake of cancer cells. After cellular internalization, the released Fc1-UCNP can effectively promote ·OH generation for tumor growth suppression. Such a Fc1-UCNP-Lipo nanosystem exhibits advantages such as easy fabrication, low drug dosage, and no ferrous ion release.

One Stone Two Birds: Zr-Fc Metal-Organic Framework Nanosheet for Synergistic Photothermal and Chemodynamic Cancer Therapy.

Metal-organic frameworks (MOFs) have been identified as promising materials for the delivery of therapeutics to cure cancer owing to their intrinsic porous structure. However, in a majority of cases, MOFs act as only a delivery cargo for anticancer drugs while little attention has been focused on the utilization of their intriguing physical and chemical properties for potential anticancer purposes. Herein for the first time, an ultrathin (16.4 nm thick) ferrocene-based MOF (Zr-Fc MOF) nanosheet has been synthesized for synergistic photothermal therapy (PTT) and Fenton reaction-based chemodynamic (CDT) therapy to cure cancer without additional drugs. The Zr-Fc MOF nanosheet acts not only as an excellent photothermal agent with a prominent photothermal conversion efficiency of 53% at 808 nm but also as an efficient Fenton catalyst to promote the conversion of H2O2 into hydroxyl radical (•OH). As a consequence, an excellent therapeutic performance has been achieved in vitro as well as in vivo through this combinational effect. This work aims to construct an "all-in-one" MOF nanoplatform for PTT and CDT treatments without incorporating any additional therapeutics, which may launch a new era in the investigation of MOF-based synergistic therapy platforms for cancer therapy.

Ferrocenes as new anticancer drug candidates: Determination of the mechanism of action.

Chemotherapy plays an essential role in the management of cancer worldwide. However, it is a non-specific treatment limited by major drawbacks, thus identification and testing of new promising molecular structures representing potential drug candidates are urgently needed. In this work, ferrocene complexes as potential antitumor drugs that display cytotoxicity in low micromolar concentrations against ovarian cancer cells A2780 and SK-OV-3 were investigated to identify their mode of action. Their mechanism of cellular accumulation was studied using differential pulse voltammetry and inductively coupled plasma - mass spectrometry. Their mode of cell death induction was determined by changes in the mitochondrial membrane potential, production of reactive oxygen species and by Annexin V staining. Transferrin receptors were identified as key mediators of intracellular accumulation of ferrocenes and the extent of cellular uptake reflected the anticancer activity of individual compounds. Functional analysis revealed activation of intrinsic apoptosis as a dominant mechanism leading to regulated cell death induced in ovarian cancer cells by ferrocenes. Ferrocenes represent a group of promising sandwich organometallic complexes exerting cytotoxic activity. We suggest their application not only as standalone chemotherapeutics but also as modifying substituents of known drugs to improve their antitumor effects.

Chemical Tools for Targeted Amplification of Reactive Oxygen Species in Neutrophils.

A number of chemical compounds are known, which amplify the availability of reactive oxygen species (ROS) in neutrophils both in vitro and in vivo. They can be roughly classified into NADPH oxidase 2 (NOX2)-dependent and NOX2-independent reagents. NOX2 activation is triggered by protein kinase C agonists (e.g., phorbol esters, transition metal ions), redox mediators (e.g., paraquat) or formyl peptide receptor (FPR) agonists (e.g., aromatic hydrazine derivatives). NOX2-independent mechanisms are realized by reagents affecting glutathione homeostasis (e.g., l-buthionine sulfoximine), modulators of the mitochondrial respiratory chain (e.g., ionophores, inositol mimics, and agonists of peroxisome proliferator-activated receptor γ) and chemical ROS amplifiers [e.g., aminoferrocene-based prodrugs (ABPs)]. Since a number of inflammatory and autoimmune diseases, as well as cancer and bacterial infections, are triggered or enhanced by aberrant ROS production in neutrophils, it is tempting to use ROS amplifiers as drugs for the treatment of these diseases. However, since the known reagents are not cell specific, their application for treatment likely causes systemic enhancement of oxidative stress, leading to severe side effects. Cell-targeted ROS enhancement can be achieved either by using conjugates of ROS amplifiers with ligands binding to receptors expressed on neutrophils (e.g., the GPI-anchored myeloid differentiation marker Ly6G or FPR) or by designing reagents activated by neutrophil function [e.g., phagocytic activity or enzymatic activity of neutrophil elastase (NE)]. Since binding of an artificial ligand to a receptor may trigger or inhibit priming of neutrophils the latter approach has a smaller potential for severe side effects and is probably better suitable for therapy. Here, we review current approaches for the use of ROS amplifiers and discuss their applicability for treatment. As an example, we suggest a possible design of neutrophil-specific ROS amplifiers, which are based on NE-activated ABPs.