Physicochemical Characterization of Ferrocifen Lipid Nanocapsules: Customized Drug Delivery Systems Guided by the Molecular Structure.

Ferrocifens, lipophilic organometallic complexes, comprise a biologically active redox motif [ferrocenyl-ene-p-phenol] which confers very interesting cytotoxic properties to this family. However, because of their highly lipophilic nature, a formulation stage is required before being administered in vivo. In recent decades, ferrocifen lipid nanocapsules (LNCs) have been successfully formulated and have demonstrated anticancer activity on multidrug-resistant cancers in several mice and rat models (glioblastoma, breast cancer, and metastatic melanoma). A recent family of ferrocifens (succinimidoalkyl-ferrociphenols, including P722) appears to be most efficacious on several resistant cancer cell lines, with IC50 values in the nanomolar range together with promising in vivo results on murine ovarian cancer models. As LNCs are composed of an oily core (caprylic/capric triglycerides), modulation of the succinimido-ferrociphenol lipophilicity could be a valuable approach toward improving the drug loading in LNCs. As the drug loading of the diphenol P722 in LNCs was low, it was structurally modified to increase its lipophilicity and thereby the payload in LNCs. Chemical modification led to a series of five succinimido-ferrocifens. Results confirmed that these slight structural modifications led to increased drug loading in LNCs for all ferrocifens, with no reduction of their cytotoxicity on the SKOV3 ovarian cancer cell line. Interestingly, encapsulation of two of the ferrocifens, diester P769 and monophenolic ester (E)-P998, led to the formation of a gel. This was unprecedented behavior, a phenomenon that could be rationalized in terms of the positioning of ferrocifens in LNCs as shown by the decrease of interfacial tension measurements at the water/oil interface. Moreover, these results highlighted the importance of obtaining a gel of this particular motif, in which the acetylated phenolic ring and the succinimidoalkyl moieties are mutually cis relative to the central double bond. Promising perspectives to use these ferrocifen-loaded LNCs to treat glioblastoma could be readily envisaged by local application of the gel in the cavity after tumor resection.

Nanomedicine to target multidrug resistant tumors.

Nanomedicine employs nanotechnologies to develop innovative applications, and more specifically nano-objects in the field of human health, through exploitation of the physical, chemical and biological properties of materials at the nanoscale. The use of nanovehicles capable of transporting and releasing the active therapeutic payload into target cells, particularly in the case of cancer or inflammatory diseases, can also enhance diagnosis. Therefore, nanomedicines improve the benefit/risk ratio of drugs by increasing their bioavailability, selectivity, and efficacy in the target tissue, while reducing the necessary doses and hence diminishing untoward toxicity to healthy tissues. Overcoming multidrug resistance (MDR) to antitumor agents is a central goal of cancer research and therapeutics, making it possible to treat these diseases more accurately and effectively. The adaptability of nanomedicines e.g. modulation of their components, surface functionalization, encapsulation of various active therapeutics as well as the possibility of combining several treatments using a single nanoparticle platform, are characteristics which are perfectly poised to address classical chemoresistance, a major obstacle towards curative cancer therapy. In this review, we discuss an assortment of nanomedicines along with those that should be developed in order to surmount cancer MDR; these include exosomes, natural compounds, lipid nanocapsules, prodrug self-assemblies, and gold nanoparticles.

Multidisciplinary Preclinical Investigations on Three Oxamniquine Analogues as New Drug Candidates for Schistosomiasis*.

Schistosomiasis is a disease of poverty affecting millions of people. Praziquantel (PZQ), with its strengths and weaknesses, is the only treatment available. We previously reported findings on three lead compounds derived from oxamniquine (OXA), an old antischistosomal drug: ferrocene-containing (Fc-CH2 -OXA), ruthenocene-containing (Rc-CH2 -OXA) and benzene-containing (Ph-CH2 -OXA) OXA derivatives. These derivatives showed excellent in vitro activity against both Schistosoma mansoni larvae and adult worms and S. haematobium adult worms, and were also active in vivo against adult S. mansoni. Encouraged by these promising results, we conducted additional in-depth preclinical studies and report in this investigation on metabolic stability studies, in vivo studies on S. haematobium and juvenile S. mansoni, computational simulations, and formulation development. Molecular dynamics simulations supported the in vitro results on the target protein. Though all three compounds were poorly stable within an acidic environment, they were only slightly cleared in the in vitro liver model. This is likely the reason why the promising in vitro activity did not translate into in vivo activity on S. haematobium. This limitation could not be overcome by the formulation of lipid nanocapsules as a way to improve the in vivo activity. Further studies should focus on increasing the compound's bioavailability, to reach an active concentration in the microenvironment of the parasite.

Nanomedicine as a potent strategy in melanoma tumor microenvironment.

Melanoma originated from melanocytes is the most aggressive type of skin cancer. Despite considerable progresses in clinical treatment with the discovery of BRAF or MEK inhibitors and monoclonal antibodies, the durability of response to treatment is often limited to the development of acquired resistance and systemic toxicity. The limited success of conventional treatment highlights the importance of understanding the role of melanoma tumor microenvironment in tumor developement and drug resistance. Nanoparticles represent a promising strategy for the development of new cancer treatments able to improve the bioavailability of drugs and increase their penetration by targeting specifically tumors cells and/or tumor environment. In this review, we will discuss the main influence of tumor microenvironment in melanoma growth and treatment outcome. Furthermore, third generation loaded nanotechnologies represent an exciting tool for detection, treatment, and escape from possible mechanism of resistance mediated by tumor microenvironment, and will be highlighted in this review.

A unique squalenoylated and nonpegylated doxorubicin nanomedicine with systemic long-circulating properties and anticancer activity.

We identified that the chemical linkage of the anticancer drug doxorubicin onto squalene, a natural lipid precursor of the cholesterol's biosynthesis, led to the formation of squalenoyl doxorubicin (SQ-Dox) nanoassemblies of 130-nm mean diameter, with an original "loop-train" structure. This unique nanomedicine demonstrates: (i) high drug payload, (ii) decreased toxicity of the coupled anticancer compound, (iii) improved therapeutic response, (iv) use of biocompatible transporter material, and (v) ease of preparation, all criteria that are not combined in the currently available nanodrugs. Cell culture viability tests and apoptosis assays showed that SQ-Dox nanoassemblies displayed comparable antiproliferative and cytotoxic effects than the native doxorubicin because of the high activity of apoptotic mediators, such as caspase-3 and poly(ADP-ribose) polymerase. In vivo experiments have shown that the SQ-Dox nanomedicine dramatically improved the anticancer efficacy, compared with free doxorubicin. Particularly, the M109 lung tumors that did not respond to doxorubicin treatment were found inhibited by 90% when treated with SQ-Dox nanoassemblies. SQ-Dox nanoassembly-treated MiaPaCa-2 pancreatic tumor xenografts in mice decreased by 95% compared with the tumors in the saline-treated mice, which was significantly higher than the 29% reduction achieved by native doxorubicin. Concerning toxicity, SQ-Dox nanoassemblies showed a fivefold higher maximum-tolerated dose than the free drug, and moreover, the cardiotoxicity study has evidenced that SQ-Dox nanoassemblies did not cause any myocardial lesions, such as those induced by the free doxorubicin treatment. Taken together, these findings demonstrate that SQ-Dox nanoassemblies make tumor cells more sensitive to doxorubicin and reduce the cardiac toxicity, thus providing a remarkable improvement in the drug's therapeutic index.

Design of Polyamine-Grafted Starches for Nucleotide Analogue Delivery: In Vitro Evaluation of the Anticancer Activity.

Nucleotide analogues are a therapeutic class that is very promising and currently used in clinics, notably against viral infectious diseases and cancer. However, their therapeutic potential is often restricted by a poor stability in vivo, the induction of severe side effects, and limited passive intracellular diffusion due to their hydrophilicity. Polysaccharide-based polymers (e.g., starch) have considerable advantages, including a lack of toxicity and the absence of antigenicity. The aim of this study was to develop new cationic starches able to form complexes with nucleotide analogues, thus protecting them and increasing their cell uptake. At the same time, the material should demonstrate good biocompatibility and low cytotoxicity. Different polyamines, (TREN, TEPA, and spermine) were grafted to starch to evaluate the impact of side-chain properties. The resulting cationic starch derivatives were characterized (e.g., degree of modification) and compared in their ability to form polyplexes with ATP as a model nucleotide. Among the tested candidates, the formulation of starch-TEPA and ATP with an N/P ratio of 2 led to nanoparticles with a size of 429 nm, a PdI of 0.054, and a ζ potential of -9 mV. MTT and LDH assays on A549 cell line showed low toxicity for this polymer. Confocal microscopy study proved that the cell internalization was an incubation-time- and energy-dependent process. Most important, starch-TEPA complexed with ddGTP showed significant biological activity on A549 cancer cells compared to that of plain ddGTP at the same concentration.

Squalenoylation of Chitosan: A Platform for Drug Delivery?

The present study describes the synthesis of chitosan-squalene (chitosan-SQ), a unique amphiphilic chitosan derivative, which enables the efficient formation of nanoparticles in acetate buffer by self-assembly. The influence of different parameters on the nanoparticle size such as percentage of substitution, pH of the acetate buffer, concentration in chitosan-SQ, and time of stirring was studied. It could be demonstrated that this new polymer was nontoxic to cells, biodegradable, and preserved the anti-infective properties of the initial chitosan.1 Moreover, chitosan-SQ showed good carrier properties by allowing the encapsulation of both hydrophilic and hydrophobic model drug compounds.

Self-assembly of polyisoprenoyl gemcitabine conjugates: influence of supramolecular organization on their biological activity.

An amphiphilic prodrug of gemcitabine, a cytidine analogue used clinically against various tumors, had been previously synthesized by covalent coupling to squalene, a natural isoprenoid chain. The resulting bioconjugate self-assembled spontaneously in water as nanoparticles, displaying an impressive activity both in vitro and in vivo. The aim of the present study was to determine the influence of the length of the isoprene moiety on the structure of the nanoparticles, in an attempt to establish a relationship between the chemical structure of the prodrug, its supramolecular organization, and its pharmacological activity. Remarkably, gemcitabine-squalene and gemcitabine-5-isoprenes, which differ only in the position of two methyl groups on the hydrophobic chain, displayed different supramolecular organizations and different anticancer activities on some cell lines. This difference in activity was related to the ability of nanoparticles to be internalized by cells.

Nanomedicines via the pulmonary route: a promising strategy to reach the target?

Over the past decades, research on nanomedicines as innovative tools in combating complex pathologies has increased tenfold, spanning fields from infectiology and ophthalmology to oncology. This process has further accelerated since the introduction of SARS-CoV-2 vaccines. When it comes to human health, nano-objects are designed to protect, transport, and improve the solubility of compounds to allow the delivery of active ingredients on their targets. Nanomedicines can be administered by different routes, such as intravenous, oral, intramuscular, or pulmonary routes. In the latter route, nanomedicines can be aerosolized or nebulized to reach the deep lung. This review summarizes existing nanomedicines proposed for inhalation administration, from their synthesis to their potential clinical use. It also outlines the respiratory organs, their structure, and particularities, with a specific emphasis on how these factors impact the administration of nanomedicines. Furthermore, the review addresses the organs accessible through pulmonary administration, along with various pathologies such as infections, genetic diseases, or cancer that can be addressed through inhaled nanotherapeutics. Finally, it examines the existing devices suitable for the aerosolization of nanomedicines and the range of nanomedicines in clinical development.

Diversely substituted poly(N-vinyl amide) derivatives towards non-toxic, stealth and pH-responsive lipid nanocapsules.

Surface modification of lipid nanocapsules (LNC) is necessary to impart stealth properties to these drug carriers and enhance their accumulation into the tumor microenvironment. While pegylation is commonly used to prolong the circulation time of LNC, the increased presence of anti-PEG antibodies in the human population and the internalization issues associated to the PEG shell are strong incentives to search alternatives. This work describes the development of amphiphilic poly(N-vinyl amide)-based (co)polymers, including pH-responsive ones, and their use as LNC modifiers towards improved drug delivery systems. RAFT polymerization gave access to a series of LNC modifiers composed of poly(N-methyl-N-vinyl acetamide), poly(N-vinyl pyrrolidone) or pH-responsive vinylimidazole-based sequence bearing a variety of lipophilic end-groups, namely octadecyl, dioctadecyl or phospholipid groups, for anchoring to the LNC. Decoration of the LNC with these families of poly(N-vinyl amide) derivatives was achieved via both post-insertion and per-formulation methods. This offered valuable and non-toxic LNC protection from opsonization by complement activation, emphasized the benefit of dioctadecyl in the per-formulation approach and highlighted the great potential of poly(N-methyl-N-vinyl acetamide) as PEG alternative. Moreover, incorporation of imidazole moieties in the shell of the carrier imparted pH-responsiveness to the LNC likely to increase the cellular uptake in the acidic tumor microenvironment, opening up new possibilities in the field of active targeting.

MicroRNA-targeting nanomedicines for the treatment of intervertebral disc degeneration.

Low back pain stands as a pervasive global health concern, afflicting almost 80% of adults at some point in their lives with nearly 40% attributable to intervertebral disc degeneration (IVDD). As only symptomatic relief can be offered to patients there is a dire need for innovative treatments.Given the accumulating evidence that multiple microRNAs (miRs) are dysregulated during IVDD, they could have a huge potential against this debilitating condition. The way miRs can profoundly modulate signaling pathways and influence several cellular processes at once is particularly exciting to tackle this multifaceted disorder. However, miR delivery encounters extracellular and intracellular biological barriers. A promising technology to address this challenge is the vectorization of miRs within nanoparticles, providing both protection and enhancing their uptake within the scarce target cells of the degenerated IVD. This comprehensive review presents the diverse spectrum of miRs' connection with IVDD and demonstrates their therapeutic potential when vectorized in nanomedicines.

Self-assembly of squalene-based nucleolipids: relating the chemical structure of the bioconjugates to the architecture of the nanoparticles.

Squalene-based nucleolipids, including anticancer or antiviral prodrugs, gave rise to nanoparticles displaying a diversity of structures upon nanoprecipitation in water. Synchrotron small-angle X-ray scattering and cryo-TEM imaging revealed that both the nature of the nucleoside and the position of the squalene moiety relative to the nucleobase determined the self-assembly of the corresponding bioconjugates. It was found that small chemical differences resulted in major differences in the self-organization of nucleolipids when squalene was grafted onto the nucleobase whereas only lamellar phases were observed when squalene was linked to the sugar moiety. The key role of hydrogen bonds between nucleobases in the formation of the lamellar phases was suggested, in agreement with molecular simulations. These findings provide a way to fine tune the supramolecular organization of squalene-based prodrugs, with the aim of improving their pharmacological activity.

Thioredoxin Reductase and Organometallic Complexes: A Pivotal System to Tackle Multidrug Resistant Tumors?

Cancers classified as multidrug-resistant (MDR) are a family of diseases with poor prognosis despite access to increasingly sophisticated treatments. Several mechanisms explain these resistances involving both tumor cells and their microenvironment. It is now recognized that a multi-targeting approach offers a promising strategy to treat these MDR tumors. Inhibition of thioredoxin reductase (TrxR), a key enzyme in maintaining redox balance in cells, is a well-identified target for this approach. Auranofin was the first inorganic gold complex to be described as a powerful inhibitor of TrxR. In this review, we will first recall the main results obtained with this metallodrug. Then, we will focus on organometallic complexes reported as TrxR inhibitors. These include gold(I), gold(III) complexes and metallocifens, i.e., organometallic complexes of Fe and Os derived from tamoxifen. In these families of complexes, similarities and differences in the molecular mechanisms of TrxR inhibition will be highlighted. Finally, the possible relationship between TrxR inhibition and cytotoxicity will be discussed and put into perspective with their mode of action.

Ferrocifen stealth LNCs and conventional chemotherapy: A promising combination against multidrug-resistant ovarian adenocarcinoma.

Ovarian cancer is one of the deadliest epithelial malignancies in women, owing to the multidrug resistance that restricts the success of conventional chemotherapy, carboplatin and paclitaxel. High grade serous ovarian carcinoma can be classified into two subtypes, the chemosensitive High OXPHOS and the Low OXPHOS tumour, less sensitive to chemotherapy. This difference of treatment efficacy could be explained by the redox status of these tumours, High OXPHOS exhibiting a chronic oxidative stress and an accumulation of reactive oxygen species. Ferrocifens, bio-organometallic compounds, are believed to be ROS producers with a good cytotoxicity on ovarian cancer cell lines. The aim of this study was to evaluate the in vivo efficacy of ferrocifen stealth lipid nanocapsules on High and Low OXPHOS ovarian Patient-Derived Xenograft models, alone or in combination to standard chemotherapy. Accordingly, two ferrocifens, P53 and P722, were encapsulated in stealth LNCs. The treatment by stealth P722-LNCs in combination with standard chemotherapy induced, with a concentration eight time lower than in stealth P53-LNCs, similar tumour reduction on a Low OXPHOS model, allowing us to conclude that P722 could be a leading ferrocifen to treat ovarian cancer. This combination of treatments may represent a promising synergistic approach to treat resistant ovarian adenocarcinoma.

Lipid nanocapsules for intracellular delivery of microRNA: A first step towards intervertebral disc degeneration therapy.

Approximately 40% of cases of lower back pain are caused by disc degeneration disease (DDD). It is well established that microRNA (miR) dysregulation is a key player in various diseases, and its impact on DDD has recently been highlighted. RNAi (miR in particular) is increasingly being considered as a novel therapeutic tool. However, free miR is degraded rapidly in vivo, and its protection is thus a prerequisite. Nanoparticular platforms, such as lipid nanocapsules (LNC), could be specifically adapted for miR delivery, allowing the transfer and release of miR in the cell cytoplasm. The objective of the current study was to formulate and characterize miR-loaded LNC to establish their in vitro potential (cell internalization, bioactivity) as well as to determine the safety and feasibility of in situ intervertebral disc (IVD) injection of miR LNC in a healthy sheep model. Using a miR library, miR-155 was clearly identified as being involved in the DDD process and was selected for further assessment. miR-155-loaded LNC (miR-155 LNC) were successfully formulated using a phase inversion process, with the addition of lipoplexes in the cooling step. Following purification, miR-155 LNC were fully characterized, and the optimized formulation had an average diameter of 75 nm, a polydispersity index below 0.1, and a positive zeta potential. By fluorescence spectroscopy, an encapsulation efficiency (EE) of 75.6% and a drug loading (DL) of 0.6% were obtained, corresponding to a sufficient amount of miR per mL of LNC to potentially have a biological effect. The sustained release of miR-155 from LNC was demonstrated compared with free miR-155: only 22% was released after 2 h and 58% after 24 h. miR-155 protection against endonuclease degradation by LNC was confirmed by gel electrophoresis, a sine qua non condition for it to be administered in vivo. Cell viability assays were performed on human adipose stromal cells (hASCs) and ovine Nucleus pulposus cells (oNP), and a cytotoxicity of <30% was obtained at the considered concentrations. Additionally, miR-155 LNC cell internalization was demonstrated by flow cytometry and confocal imaging. Moreover, downregulation of total ERK1/2 in hASCs and oNP cells, after miR-155 LNC treatment, was demonstrated by Western blot and quantitative reverse-transcription PCR (qRT-PCR), thus confirming maintenance of its bioactivity after formulation and internalization. Finally, the feasibility and safety of miR-155 LNC in situ injection (compared to control groups: blank LNC and sham condition) was demonstrated in healthy sheep by imaging (MRI and T2wsi measurement) and histology (Boos' scoring) analysis. T2wsi was measured, and no significant difference was observed three months after the injection between the different conditions. No histological impact was observed, with no significant difference in Boos' scoring between the different conditions. All these results suggest LNC may be a potent strategy for the encapsulation and delivery of miR (particularly miR-155) and can be considered as a first step towards IVD regenerative medicine.

Impact of anti-PDGFRα antibody surface functionalization on LNC uptake by oligodendrocyte progenitor cells.

Impairment of oligodendrocyte progenitor cell (OPC) differentiation into oligodendrocytes and chronic inflammation are key determinants of poor remyelination observed in diseases such as multiple sclerosis. For many pro-myelinating molecules, the therapeutic potential is hindered by poor solubility or limited access to the targeted cells. A promising approach to improve the delivery of those molecules to OPC is to encapsulate them in functionalized Lipid Nanocapsules (LNC). We aimed to develop the first OPC-targeting LNC, by grafting an anti-PDGFRα antibody on the surface of the LNC using several strategies and evaluating the interaction with PDGFRα via ELISA. We found that only site-selective click-chemistry grafting maintained anti-PDGFRα/PDGFRα association, which was confirmed in vitro on primary rat OPC. In conclusion, we demonstrated that it was possible to produce anti-PDGFRα functionalized LNC, we confirmed the antibody's ability to recognize its receptor after grafting and we optimized techniques to characterize antibody functionalized LNC.

Microbiota and nanoparticles: Description and interactions.

The healthy human body is inhabited with a large number of bacteria, forming natural flora. It is even estimated that for a human body, its amount of DNA is less important that its bacterial genetic material. This flora plays major roles in the sickness and health of the human body and any change in its composition may lead to different diseases. Nanoparticles are widely used in numerous fields: cosmetics, food, industry, and as drug delivery carrier in the medical field. Being included in these various applications, nanoparticles may interact with the human body at various levels and with different mechanisms. These interactions differ depending on the nanoparticle nature, its structure, its concentration and manifest in different ways on the microbiota, leading to its destabilization, its restoring or showing no toxic effect. Nanoparticles may also be used as a vehicle to regulate the microbiota or to treat some of its diseases.

Ferrocifen Loaded Lipid Nanocapsules: A Promising Anticancer Medication against Multidrug Resistant Tumors.

Resistance of cancer cells to current chemotherapeutic drugs has obliged the scientific community to seek innovative compounds. Ferrocifens, lipophilic organometallic compounds composed of a tamoxifen scaffold covalently bound to a ferrocene moiety, have shown very interesting antiproliferative, cytotoxic and immunologic effects. The formation of ferrocenyl quinone methide plays a crucial role in the multifaceted activity of ferrocifens. Lipid nanocapsules (LNCs), meanwhile, are nanoparticles obtained by a free organic solvent process. LNCs consist of an oily core surrounded by amphiphilic surfactants and are perfectly adapted to encapsulate these hydrophobic compounds. The different in vitro and in vivo experiments performed with this ferrocifen-loaded nanocarrier have revealed promising results in several multidrug-resistant cancer cell lines such as glioblastoma, breast cancer and metastatic melanoma, alone or in combination with other therapies. This review provides an exhaustive summary of the use of ferrocifen-loaded LNCs as a promising nanomedicine, outlining the ferrocifen mechanisms of action on cancer cells, the nanocarrier formulation process and the in vivo results obtained over the last two decades.

The evolution of nucleosidic analogues: self-assembly of prodrugs into nanoparticles for cancer drug delivery.

Nucleoside and nucleotide analogs are essential tools in our limited arsenal in the fight against cancer. However, these structures face severe drawbacks such as rapid plasma degradation or hydrophilicity, limiting their clinical application. Here, different aspects of nucleoside and nucleotide analogs have been exposed, while providing their shortcomings. Aiming to improve their fate in the body and combating their drawbacks, two different approaches have been discussed, the prodrug and nanocarrier technologies. Finally, a novel approach called "PUFAylation" based on both the prodrug and nanocarrier technologies has been introduced, promising to be the supreme method to create a novel nucleoside or nucleotide analog based formulation, with enhanced efficacy and highly reduced toxicity.

Microbiota and cancer: In vitro and in vivo models to evaluate nanomedicines.

Nanomedicine implication in cancer treatment and diagnosis studies witness huge attention, especially with the promising results obtained in preclinical studies. Despite this, only few nanomedicines succeeded to pass clinical phase. The human microbiota plays obvious roles in cancer development. Nanoparticles have been successfully used to modulate human microbiota and notably tumor associated microbiota. Taking the microbiota involvement under consideration when testing nanomedicines for cancer treatment might be a way to improve the poor translation from preclinical to clinical trials. Co-culture models of bacteria and cancer cells, as well as animal cancer-microbiota models offer a better representation for the tumor microenvironment and so potentially better platforms to test nanomedicine efficacy in cancer treatment. These models would allow closer representation of human cancer and might smoothen the passage from preclinical to clinical cancer studies for nanomedicine efficacy.