Liposomal Rifabutin-A Promising Antibiotic Repurposing Strategy against Methicillin-Resistant Staphylococcus aureus Infections.

Methicillin-resistant Staphylococcus aureus (M RSA) infections, in particular biofilm-organized bacteria, remain a clinical challenge and a serious health problem. Rifabutin (RFB), an antibiotic of the rifamycins class, has shown in previous work excellent anti-staphylococcal activity. Here, we proposed to load RFB in liposomes aiming to promote the accumulation of RFB at infected sites and consequently enhance the therapeutic potency. Two clinical isolates of MRSA, MRSA-C1 and MRSA-C2, were used to test the developed formulations, as well as the positive control, vancomycin (VCM). RFB in free and liposomal forms displayed high antibacterial activity, with similar potency between tested formulations. In MRSA-C1, minimal inhibitory concentrations (MIC) for Free RFB and liposomal RFB were 0.009 and 0.013 µg/mL, respectively. Minimum biofilm inhibitory concentrations able to inhibit 50% biofilm growth (MBIC50) for Free RFB and liposomal RFB against MRSA-C1 were 0.012 and 0.008 µg/mL, respectively. Confocal microscopy studies demonstrated the rapid internalization of unloaded and RFB-loaded liposomes in the bacterial biofilm matrix. In murine models of systemic MRSA-C1 infection, Balb/c mice were treated with RFB formulations and VCM at 20 and 40 mg/kg of body weight, respectively. The in vivo results demonstrated a significant reduction in bacterial burden and growth index in major organs of mice treated with RFB formulations, as compared to Control and VCM (positive control) groups. Furthermore, the VCM therapeutic dose was two fold higher than the one used for RFB formulations, reinforcing the therapeutic potency of the proposed strategy. In addition, RFB formulations were the only formulations associated with 100% survival. Globally, this study emphasizes the potential of RFB nanoformulations as an effective and safe approach against MRSA infections.

An alternative hybrid lipid nanosystem combining cytotoxic and magnetic properties as a tool to potentiate antitumor effect of 5-fluorouracil.

AIMS: Colorectal cancer is the third most frequent type of cancer and the second leading cause of cancer-related deaths worldwide. The majority of cases are diagnosed at a later stage, leading to the need for more aggressive treatments such as chemotherapy. 5-Fluorouracil (5-FU), known for its high cytotoxic properties has emerged as a chemotherapeutic agent. However, it presents several drawbacks such as lack of specificity and short half-life. To reduce these drawbacks, several strategies have been designed namely chemical modification or association to drug delivery systems. MATERIALS AND METHODS: Current research was focused on the design, physicochemical characterization and in vitro evaluation of a lipid-based system loaded with 5-FU. Furthermore, aiming to maximize preferential targeting and release at tumour sites, a hybrid lipid-based system, combining both therapeutic and magnetic properties was developed and validated. For this purpose, liposomes co-loaded with 5-FU and iron oxide (II, III) nanoparticles were accomplished. KEY FINDINGS: The characterization of the developed nanoformulation was performed in terms of incorporation parameters, mean size and surface charge. In vitro studies assessed in a murine colon cancer cell line confirmed that 5-FU antiproliferative activity was preserved after incorporation in liposomes. In same model, iron oxide (II, III) nanoparticles did not exhibit cytotoxic properties. Additionally, the presence of these nanoparticles was shown to confer magnetic properties to the liposomes, allowing them to respond to external magnetic fields. SIGNIFICANCE: Overall, a lipid nanosystem loading a chemotherapeutic agent displaying magnetic characteristics was successfully designed and physicochemically characterized, for further in vivo applications.

A step forward on the in vitro and in vivo assessment of a novel nanomedicine against melanoma.

Melanoma is the most aggressive form of skin cancer, with increasing incidence and mortality rates. To overcome current treatment limitations, a hybrid molecule (HM) combining a triazene and a sulfur L-tyrosine analogue, was recently synthesized, incorporated in long blood circulating liposomes (LIP HM) and validated in an immunocompetent melanoma model. The present work constitutes a step forward in the therapeutic assessment of HM formulations. Here, human melanoma cells, A375 and MNT-1, were used and dacarbazine (DTIC), a triazene drug clinically available as first-line treatment for melanoma, constituted the positive control. In cell cycle analysis, A375 cells, after 24-h incubation with HM (60 µM) and DTIC (70 µM), resulted in a 1.2 fold increase (related to control) in the percentage of cells in G0/G1 phase. The therapeutic activity was evaluated in a human murine melanoma model (subcutaneously injected with A375 cells) to most closely resemble the human pathology. Animals treated with LIP HM exhibited the highest antimelanoma effect resulting in a 6-, 5- and 4-fold reduction on tumor volume compared to negative control, Free HM and DTIC groups, respectively. No toxic side effects were detected. Overall, these results constitute another step forward in the validation of the antimelanoma activity of LIP HM, using a murine model that more accurately simulates the pathology that occurs in human patients.

Advancing Medicine with Lipid-Based Nanosystems-The Successful Case of Liposomes.

Nanomedicine, a promising area of medicine, employs nanosized tools for the diagnosis, prevention, and treatment of disease. Particularly, liposomes, lipid-based nanovesicles, are currently one of the most successful nanosystems, with extensive applications in the clinic and an increasing pipeline of products in preclinical and clinical development. These versatile nanotechnological tools are biocompatible and biodegradable, and can load a variety of molecules and, ultimately, improve the therapeutic performance of drugs while minimizing undesired side effects. In this review, we provide a brief description on liposomes' composition and classification and mainly focus on their clinical use in various areas, including disease management (e.g., cancer, fungal and bacterial infections, ocular pathologies), analgesia, vaccination, diagnostics, and immunosuppression in organ transplantation. Herein are described examples of current liposomal products already in the clinic, as well as the most recent clinical trials involving liposomes as effective and safe nanomedicine tools.

Liposomal Delivery of Saquinavir to Macrophages Overcomes Cathepsin Blockade by Mycobacterium tuberculosis and Helps Control the Phagosomal Replicative Niches.

Mycobacterium tuberculosis is able to establish a chronic colonization of lung macrophages in a controlled replication manner, giving rise to a so-called latent infection. Conversely, when intracellular bacteria undergo actively uncontrolled replication rates, they provide the switch for the active infection called tuberculosis to occur. Our group found that the pathogen is able to manipulate the activity of endolysosomal enzymes, cathepsins, directly at the level of gene expression or indirectly by regulating their natural inhibitors, cystatins. To provide evidence for the crucial role of cathepsin manipulation for the success of tuberculosis bacilli in their intracellular survival, we used liposomal delivery of saquinavir. This protease inhibitor was previously found to be able to increase cathepsin proteolytic activity, overcoming the pathogen induced blockade. In this study, we demonstrate that incorporation in liposomes was able to increase the efficiency of saquinavir internalization in macrophages, reducing cytotoxicity at higher concentrations. Consequently, our results show a significant impact on the intracellular killing not only to reference and clinical strains susceptible to current antibiotic therapy but also to multidrug- and extensively drug-resistant (XDR) Mtb strains. Altogether, this indicates the manipulation of cathepsins as a fine-tuning strategy used by the pathogen to survive and replicate in host cells.

Preclinical validation of a new hybrid molecule loaded in liposomes for melanoma management.

The aggressiveness of melanoma and lack of effective therapies incite the discovery of novel strategies. Recently, a new dual acting hybrid molecule (HM), combining a triazene and a ʟ-tyrosine analogue, was synthesized. HM was designed to specifically be activated by tyrosinase, the enzyme involved in melanin biosynthesis and overexpressed in melanoma. HM displayed remarkable superior antiproliferative activity towards various cancer cell lines compared with temozolomide (TMZ), a triazene drug in clinical use, that acts through DNA alkylation. In B16-F10 cells, HM induced a cell cycle arrest at phase G0/G1 with a 2.8-fold decrease in cell proliferation index. Also, compared to control cells, HM led to a concentration-dependent reduction in tyrosinase activity and increase in caspase 3/7 activity. To maximize the therapeutic performance of HM in vivo, its incorporation in long blood circulating liposomes, containing poly(ethylene glycol) (PEG) at their surface, was performed for passively targeting tumour sites. HM liposomes (LIP HM) exhibited high stability in biological fluids. Preclinical studies demonstrated its safety for systemic administration and in a subcutaneous murine melanoma model, significantly reduced tumour progression. In a metastatic murine melanoma model, a superior antitumour effect was also observed for mice receiving LIP HM, with markedly reduction of lung metastases compared to positive control group (TMZ). Biodistribution studies using 111In-labelled LIP HM demonstrated its ability for passively targeting tumour sites, thus correlating with the high therapeutic effect observed in the two experimental murine melanoma models. Overall, our proposed nanotherapeutic strategy was validated as an effective and safe alternative against melanoma.

Metal Coordination and Biological Screening of a Schiff Base Derived from 8-Hydroxyquinoline and Benzothiazole.

Designing new metallodrugs for anticancer therapy is a driving force in the scientific community. Aiming to contribute to this field, we hereby report the development of a Schiff base (H2L) derived from the condensation of 2-carbaldehyde-8-hydroxyquinoline with 2-hydrazinobenzothiazole and its complexation with transition metal ions. All compounds were characterised by analytical and spectroscopic techniques, which disclosed their structure: [Cu(HL)Cl], [Cu(HL)2], [Ni(HL)(acetate)], [Ni(HL)2], [Ru(HL)Cl(DMSO)], [VO(HL)2] and [Fe(HL)2Cl(H2O)]. Different binding modes were proposed, showing the ligand's coordination versatility. The ligand proton dissociation constants were determined, and the tested compounds showed high lipophilicity and light sensitivity. The stability of all complexes in aqueous media and their ability to bind to albumin were screened. Based on an antiproliferative in vitro screening, [Ni(HL)(acetate)] and [Ru(HL)Cl(DMSO)] were selected for further studies aiming to investigate their mechanisms of action and therapeutic potential towards colon cancer. The complexes displayed IC50 < 21 µM towards murine (CT-26) and human (HCT-116) colon cancer cell lines. Importantly, both complexes exhibited superior antiproliferative properties compared to the clinically approved 5-fluorouracil. [Ni(HL)(acetate)] induced cell cycle arrest in S phase in CT-26 cells. For [Ru(HL)Cl(DMSO)] this effect was observed in both colon cancer cell lines. Additionally, both compounds significantly inhibited cell migration particularly in the human colon cancer cell line, HCT-116. Overall, the therapeutic potential of both metal complexes was demonstrated.

Nanomedicines in the treatment of colon cancer: a focus on metallodrugs.

Worldwide, colon cancer (CC) represents the fourth most common type of cancer and the fifth major cause of cancer-associated deaths. Surgical resection is considered the standard therapeutic choice for CC in early stages. However, in latter stages of the disease, adjuvant chemotherapy is essential for an appropriate management of this pathology. Metal-based complexes displaying cytotoxic properties towards tumor cells emerge as potential chemotherapeutic options. One metallodrug, oxaliplatin, was already approved for clinical use, playing an important role in the treatment of CC patients. Unfortunately, most of the newly designed metal-based complexes exhibit lack of selectivity against cancer cells, low solubility and permeability, high dose-limiting toxicity, and emergence of resistances. Nanodelivery systems enable the incorporation of metallodrugs at adequate payloads, solving the above-referred drawbacks. Moreover, drug delivery systems, depending on their physicochemical properties, are able to release the incorporated material preferentially at affected tissues/organs, enhancing the therapeutic activity in vivo, with concomitant fewer side effects. In this review, the general features and therapeutic management of CC will be addressed, with a special focus on preclinical or clinical studies using metal-based compounds. Furthermore, the use of different nanodelivery systems will also be described as tools to potentiate the therapeutic index of metallodrugs for the management of CC.

The Challenging Melanoma Landscape: From Early Drug Discovery to Clinical Approval.

Melanoma is recognized as the most dangerous type of skin cancer, with high mortality and resistance to currently used treatments. To overcome the limitations of the available therapeutic options, the discovery and development of new, more effective, and safer therapies is required. In this review, the different research steps involved in the process of antimelanoma drug evaluation and selection are explored, including information regarding in silico, in vitro, and in vivo experiments, as well as clinical trial phases. Details are given about the most used cell lines and assays to perform both two- and three-dimensional in vitro screening of drug candidates towards melanoma. For in vivo studies, murine models are, undoubtedly, the most widely used for assessing the therapeutic potential of new compounds and to study the underlying mechanisms of action. Here, the main melanoma murine models are described as well as other animal species. A section is dedicated to ongoing clinical studies, demonstrating the wide interest and successful efforts devoted to melanoma therapy, in particular at advanced stages of the disease, and a final section includes some considerations regarding approval for marketing by regulatory agencies. Overall, considerable commitment is being directed to the continuous development of optimized experimental models, important for the understanding of melanoma biology and for the evaluation and validation of novel therapeutic strategies.

Sulfur Analogues of Tyrosine in the Development of Triazene Hybrid Compounds: A New Strategy against Melanoma.

Malignant melanoma is the major cause of death from skin cancer. Treatment of metastatic melanoma remains an enormous challenge. In this study we developed hybrid compounds and studied their potential use in malignant melanoma chemotherapy. They were designed to act by a double mechanism of action, being composed of two pharmacophores: the tyrosine sulfur analogue 4-S-cysteaminylphenol (4-S-CAP, 10), with immunomodulatory properties and specific melanocytotoxic activity, and triazene 4, with DNA alkylating properties. The design of these compounds aims to achieve selective activation by the enzyme tyrosinase overexpressed in melanoma cells. Compounds 11a-e, 13a, and 13b were found to be excellent tyrosinase substrates (0.5 min ≤ t 1/2 ≤ 3.7 min). Furthermore, derivatives 11 and 13 were evaluated for their molecular properties, hepatotoxicity, in vivo toxicity profile, and assessment of cytotoxic activity in melanoma and non-melanoma cell lines. The results were compared with those obtained for temozolomide, a triazene used in melanoma therapy. It was discovered that the hybrids are selective and effective drugs, representing a valuable model for the development of new multitarget melanoma therapy. In particular, compound 10 may be an important component for these strategies that use a metabolic pathway of melanin synthesis. Molecular hybridization of 10 with triazenes 4 renders the hybrids (11 and 13) unexpectedly devoid of hepatotoxicity while maintaining cytotoxic activity in malignant cells.

Therapeutic potential of a copper complex loaded in pH-sensitive long circulating liposomes for colon cancer management.

Colorectal carcinoma is a complex malignancy and current therapies are hampered by systemic toxicity and tumor resistance to treatment. In the field of cancer therapy, copper (Cu) compounds hold great promise, with some reaching clinical trials. However, the anticancer potential of Cu complexes has not yet been fully disclosed due to speciation in biological systems, leading to inactivation and/or potential side effects. This is the case of the widely studied Cu(II) complexes featuring phenanthroline ligands, with potent antiproliferative effects in vitro, but often failing in vivo. Aiming to overcome these limitations and maximize its anticancer effects in vivo, the Cu(II) complex (Cu(1,10-phenanthroline)Cl2) (Cuphen), displaying IC50 values <6 µM against different tumor cell lines, was loaded in long circulating liposomes with pH-sensitive properties (F1, DMPC:CHEMS:DSPE-PEG; F2, DOPE:CHEMS:DMPC:DSPE-PEG). This enabled a pH-dependent Cuphen release, with F1 and F2 releasing 36/78% and 47/94% of Cuphen at pH 6/4.5, respectively. The so formed nanoformulations preserved Cuphen effects towards cancer cell lines, with F2 presenting IC50 of 2.7 µM and 4.9 µM towards colon cancer CT-26 and HCT-116 cells, respectively. Additional in vitro studies confirmed that Cuphen antiproliferative activity towards colon cancer cells does not rely on cell cycle effect. Furthermore, in these cells, Cuphen reduced glycerol permeation and impaired cell migration. At 24 h incubation, wound closure was reduced by Cuphen, with migration values of 29% vs 54% (control) and 45% (1,10-phenanthroline) in CT-26 cells, and 33% vs ~44% (control and 1,10-phenanthroline) in HCT-116 cells. These effects were probably due to inhibition of aquaglyceroporins, membrane water and glycerol channels that are often abnormally expressed in tumors. In a syngeneic murine colon cancer model, F2 significantly reduced tumor progression, compared to the control group and to mice treated with free Cuphen or with the ligand, 1,10-phenanthroline, without eliciting toxic side effects. F2 led to a tumor volume reduction of ca. 50%. This was confirmed by RTV analysis, where F2 reached a value of 1.3 vs 4.4 (Control), 5.8 (Phen) and 3.8 (free Cuphen). These results clearly demonstrated the important role of the Cu(II) for the observed biological activity that was maximized following the association to a lipid-based nanosystem. Overall, this study represents a step forward in the development of pH-sensitive nanotherapeutic strategies of metallodrugs for colon cancer management.

A Novel Hybrid Nanosystem Integrating Cytotoxic and Magnetic Properties as a Tool to Potentiate Melanoma Therapy.

Cancer is a major health concern and the prognosis is often poor. Significant advances in nanotechnology are now driving a revolution in cancer detection and treatment. The goal of this study was to develop a novel hybrid nanosystem for melanoma treatment, integrating therapeutic and magnetic targeting modalities. Hence, we designed long circulating and pH-sensitive liposomes loading both dichloro(1,10-phenanthroline) copper (II) (Cuphen), a cytotoxic metallodrug, and iron oxide nanoparticles (IONPs). The synthetized IONPs were characterized by transmission electron microscopy and dynamic light scattering. Lipid-based nanoformulations were prepared by the dehydration rehydration method, followed by an extrusion step for reducing and homogenizing the mean size. Liposomes were characterized in terms of incorporation parameters and mean size. High Cuphen loadings were obtained and the presence of IONPs slightly reduced Cuphen incorporation parameters. Cuphen antiproliferative properties were preserved after association to liposomes and IONPs (at 2 mg/mL) did not interfere on cellular proliferation of murine and human melanoma cell lines. Moreover, the developed nanoformulations displayed magnetic properties. The absence of hemolytic activity for formulations under study demonstrated their safety for parenteral administration. In conclusion, a lipid-based nanosystem loading the cytotoxic metallodrug, Cuphen, and displaying magnetic properties was successfully designed.

The Aquaporin-3-Inhibiting Potential of Polyoxotungstates.

Polyoxometalates (POMs) are of increasing interest due to their proven anticancer activities. Aquaporins (AQPs) were found to be overexpressed in tumors bringing particular attention to their inhibitors as anticancer drugs. Herein, we report for the first time the ability of polyoxotungstates (POTs), such as of Wells-Dawson P2W18, P2W12, and P2W15, and Preyssler P5W30 structures, to affect aquaporin-3 (AQP3) activity and impair melanoma cell migration. The tested POTs were revealed to inhibit AQP3 function with different effects, with P2W18, P2W12, and P5W30 being the most potent (50% inhibitory concentration (IC50) = 0.8, 2.8, and 3.2 µM), and P2W15 being the weakest (IC50 > 100 µM). The selectivity of P2W18 toward AQP3 was confirmed in yeast cells transformed with human aquaglyceroporins. The effect of P2W12 and P2W18 on melanoma cells that highly express AQP3 revealed an impairment of cell migration between 55% and 65% after 24 h, indicating that the anticancer properties of these compounds may in part be due to the blockage of AQP3-mediated permeability. Altogether, our data revealed that P2W18 strongly affects AQP3 activity and cancer cell growth, unveiling its potential as an anticancer drug against tumors where AQP3 is highly expressed.

Inhalable locust bean gum microparticles co-associating isoniazid and rifabutin: Therapeutic assessment in a murine model of tuberculosis infection.

Tuberculosis is a leading cause of death worldwide. Although the development of new antimycobacterial drugs is an obvious and necessary strategy to address the disease, improving the therapeutic performance of drugs already approved constitutes a valuable alternative approach. As the lung is the most affected organ, where M. tuberculosis is able to survive and proliferate, the direct pulmonary delivery of antitubercular drugs comprises a highly promising therapeutic strategy. In this work, spray-dried locust bean gum (LBG) microparticles were used to deliver a combination of two first line antitubercular drugs, isoniazid (INH) and rifabutin (RFB), to the alveolar zone, where macrophages hosting the bacteria reside. LBG is expected to mediate favoured macrophage uptake of microparticles, leading to enhanced therapeutic effect. The therapeutic effect of LBG/INH/RFB microparticles was evaluated in a murine model infected with M. tuberculosis, strain H37Rv and compared with oral co-therapy of INH and RFB in the free form. The pulmonary administration of LBG/INH/RFB microparticles 5 times per week was the only treatment schedule that provided negative growth index values in lung (-0.22), spleen (-0.14) and liver (-0.26) even using a lower therapeutic dose for both antibiotics. For the control group, the respective values were +1.95, +0.75 and +0.96.

Emergent Nanotechnological Strategies for Systemic Chemotherapy against Melanoma.

Melanoma is an aggressive form of skin cancer, being one of the deadliest cancers in the world. The current treatment options involve surgery, radiotherapy, targeted therapy, immunotherapy and the use of chemotherapeutic agents. Although the last approach is the most used, the high toxicity and the lack of efficacy in advanced stages of the disease have demanded the search for novel bioactive molecules and/or efficient drug delivery systems. The current review aims to discuss the most recent advances on the elucidation of potential targets for melanoma treatment, such as aquaporin-3 and tyrosinase. In addition, the role of nanotechnology as a valuable strategy to effectively deliver selective drugs is emphasized, either incorporating/encapsulating synthetic molecules or natural-derived compounds in lipid-based nanosystems such as liposomes. Nanoformulated compounds have been explored for their improved anticancer activity against melanoma and promising results have been obtained. Indeed, they displayed improved physicochemical properties and higher accumulation in tumoral tissues, which potentiated the efficacy of the compounds in pre-clinical experiments. Overall, these experiments opened new doors for the discovery and development of more effective drug formulations for melanoma treatment.

Copper complex nanoformulations featuring highly promising therapeutic potential in murine melanoma models.

AIM: Preclinical evaluation of a cytotoxic copper (II) complex formulated in long circulating nanoliposomes for melanoma treatment. MATERIALS & METHODS: Liposomal nanoformulations of the copper complex were characterized in terms of thermodynamic behavior (differential scanning calorimeter), pH-sensitivity (spectrophotometry) and antiproliferative effects against murine melanoma B16F10 cells in vitro. Preclinical studies were performed in a C57BL/6 syngeneic melanoma model. RESULTS: Nanoformulations were thermodynamically stable, and CHEMS-containing nanoliposomes were pH-sensitive and preserved the antiproliferative properties of the copper compound. These nanoformulations significantly impaired tumor progression in vivo, devoid of toxic side effects, compared with control mice or mice treated with the free metallodrug. CONCLUSION: Copper complex-containing nanoliposomes demonstrate high anticancer efficacy and safety, constituting a step forward to the development of more effective therapeutic strategies against melanoma.

Current Trends in Cancer Nanotheranostics: Metallic, Polymeric, and Lipid-Based Systems.

Theranostics has emerged in recent years to provide an efficient and safer alternative in cancer management. This review presents an updated description of nanotheranostic formulations under development for skin cancer (including melanoma), head and neck, thyroid, breast, gynecologic, prostate, and colon cancers, brain-related cancer, and hepatocellular carcinoma. With this focus, we appraised the clinical advantages and drawbacks of metallic, polymeric, and lipid-based nanosystems, such as low invasiveness, low toxicity to the surrounding healthy tissues, high precision, deeper tissue penetration, and dosage adjustment in a real-time setting. Particularly recognizing the increased complexity and multimodality in this area, multifunctional hybrid nanoparticles, comprising different nanomaterials and functionalized with targeting moieties and/or anticancer drugs, present the best characteristics for theranostics. Several examples, focusing on their design, composition, imaging and treatment modalities, and in vitro and in vivo characterization, are detailed herein. Briefly, all studies followed a common trend in the design of these theranostics modalities, such as the use of materials and/or drugs that share both inherent imaging (e.g., contrast agents) and therapeutic properties (e.g., heating or production reactive oxygen species). This rationale allows one to apparently overcome the heterogeneity, complexity, and harsh conditions of tumor microenvironments, leading to the development of successful targeted therapies.