The Role of Inhaled Chitosan-Based Nanoparticles in Lung Cancer Therapy.

Lung cancer is the leading cause of cancer-related mortality worldwide, largely due to the limited efficacy of anticancer drugs, which is primarily attributed to insufficient doses reaching the lungs. Additionally, patients undergoing treatment experience severe systemic adverse effects due to the distribution of anticancer drugs to non-targeted sites. In light of these challenges, there has been a growing interest in pulmonary administration of drugs for the treatment of lung cancer. This route allows drugs to be delivered directly to the lungs, resulting in high local concentrations that can enhance antitumor efficacy while mitigating systemic toxic effects. However, pulmonary administration poses the challenge of overcoming the mechanical, chemical, and immunological defenses of the respiratory tract that prevent the inhaled drug from properly penetrating the lungs. To overcome these drawbacks, the use of nanoparticles in inhaler formulations may be a promising strategy. Nanoparticles can assist in minimizing drug clearance, increasing penetration into the lung epithelium, and enhancing cellular uptake. They can also facilitate increased drug stability, promote controlled drug release, and delivery to target sites, such as the tumor environment. Among them, chitosan-based nanoparticles demonstrate advantages over other polymeric nanocarriers due to their unique biological properties, including antitumor activity and mucoadhesive capacity. These properties have the potential to enhance the efficacy of the drug when administered via the pulmonary route. In view of the above, this paper provides an overview of the research conducted on the delivery of anticancer drug-loaded chitosan-based nanoparticles incorporated into inhaled drug delivery devices for the treatment of lung cancer. Furthermore, the article addresses the use of emerging technologies, such as siRNA (small interfering RNA), in the context of lung cancer therapy. Particularly, recent studies employing chitosan-based nanoparticles for siRNA delivery via the pulmonary route are described.

Susceptibility of Leishmania to novel pentavalent organometallics: Investigating impact on DNA and membrane integrity in antimony(III)-sensitive and -resistant strains.

The aim the present study was to investigate the impact of novel pentavalent organobismuth and organoantimony complexes on membrane integrity and their interaction with DNA, activity against Sb(III)-sensitive and -resistant Leishmania strains and toxicity in mammalian peritoneal macrophages. Ph3M(L)2 type complexes were synthesized, where M = Sb(V) or Bi(V) and L = deprotonated 3-(dimethylamino)benzoic acid or 2-acetylbenzoic acid. Both organobismuth(V) and organoantimony(V) complexes exhibited efficacy at micromolar concentrations against Leishmania amazonensis and L. infantum but only the later ones demonstrated biocompatibility. Ph3Sb(L1)2 and Ph3Bi(L1)2 demonstrated distinct susceptibility profiles compared to inorganic Sb(III)-resistant strains of MRPA-overexpressing L. amazonensis and AQP1-mutated L. guyanensis. These complexes were able to permeate the cell membrane and interact with the Leishmania DNA, suggesting that this effect may contribute to the parasite growth inhibition via apoptosis. Taken altogether, our data substantiate the notion of a distinct mechanism of uptake pathway and action in Leishmania for these organometallic complexes, distinguishing them from the conventional inorganic antimonial drugs.

Development and characterization of liposomal formulations containing sesquiterpene lactones for the treatment of chronic gout.

Gout and hyperuricemia are characterized by high uric acid levels, and their treatment involves medications that have adverse effects. In this study, we evaluated oral liposomal formulations with eremantholide C and goyazensolide as a novel approach to reduce the toxicity associated with these substances while maintaining their anti-hyperuricemic activity. We characterized the formulations and evaluated them based on encapsulation efficiency and stability over 12 months and under simulated physiological environments. We determined the toxicity of the liposomal formulations in Caco-2 cells and the anti-hyperuricemic activity in rats. The formulations exhibited nanometric size, a narrow size distribution, and a negative zeta potential, indicating their stability and uniformity. The efficient encapsulation of the sesquiterpene lactones within the liposomes emphasizes their potential for sustained release and therapeutic efficacy. Stability evaluation revealed a small decrease in the eremantholide C concentration and a remarkable stability in the goyazensolide concentration. In Caco-2 cells, the liposomes did not exert toxicity, but did exhibit an antiproliferative effect. In vivo assays demonstrated that the liposomes reduced serum uric acid levels. Our study represents an advancement in gout and hyperuricemia treatment. The liposomal formulations effectively reduced the toxicity associated with the sesquiterpene lactones while maintaining their therapeutic effects.

Ionizable Lipid Nanoparticle-Mediated TRAIL mRNA Delivery in the Tumor Microenvironment to Inhibit Colon Cancer Progression.

Introduction: Immunotherapy has revolutionized cancer treatment by harnessing the immune system to enhance antitumor responses while minimizing off-target effects. Among the promising cancer-specific therapies, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted significant attention. Methods: Here, we developed an ionizable lipid nanoparticle (LNP) platform to deliver TRAIL mRNA (LNP-TRAIL) directly to the tumor microenvironment (TME) to induce tumor cell death. Our LNP-TRAIL was formulated via microfluidic mixing and the induction of tumor cell death was assessed in vitro. Next, we investigated the ability of LNP-TRAIL to inhibit colon cancer progression in vivo in combination with a TME normalization approach using Losartan (Los) or angiotensin 1-7 (Ang(1-7)) to reduce vascular compression and deposition of extracellular matrix in mice. Results: Our results demonstrated that LNP-TRAIL induced tumor cell death in vitro and effectively inhibited colon cancer progression in vivo, particularly when combined with TME normalization induced by treatment Los or Ang(1-7). In addition, potent tumor cell death as well as enhanced apoptosis and necrosis was found in the tumor tissue of a group treated with LNP-TRAIL combined with TME normalization. Discussion: Together, our data demonstrate the potential of the LNP to deliver TRAIL mRNA to the TME and to induce tumor cell death, especially when combined with TME normalization. Therefore, these findings provide important insights for the development of novel therapeutic strategies for the immunotherapy of solid tumors.

Nanoparticle-based DNA vaccine protects against SARS-CoV-2 variants in female preclinical models.

A safe and effective vaccine with long-term protection against SARS-CoV-2 variants of concern (VOCs) is a global health priority. Here, we develop lipid nanoparticles (LNPs) to provide safe and effective delivery of plasmid DNA (pDNA) and show protection against VOCs in female small animal models. Using a library of LNPs encapsulating unique barcoded DNA (b-DNA), we screen for b-DNA delivery after intramuscular administration. The top-performing LNPs are further tested for their capacity of pDNA uptake in antigen-presenting cells in vitro. The lead LNP is used to encapsulate pDNA encoding the HexaPro version of SARS-CoV-2 spike (LNP-HPS) and immunogenicity and protection is tested in vivo. LNP-HPS elicit a robust protective effect against SARS-CoV-2 Gamma (P.1), correlating with reduced lethality, decreased viral load in the lungs and reduced lung damage. LNP-HPS induce potent humoral and T cell responses against P.1, and generate high levels of neutralizing antibodies against P.1 and Omicron (B.1.1.529). Our findings indicate that the protective efficacy and immunogenicity elicited by LNP-HPS are comparable to those achieved by the approved COVID-19 vaccine from Biontech/Pfizer in animal models. Together, these findings suggest that LNP-HPS hold great promise as a vaccine candidate against VOCs.

Distearoyl phosphatidylglycerol and dioleoyl phosphatidylglycerol increase the retention and reduce the toxicity of amphotericin B-loaded in nanoemulsions.

Aim: To develop nanoemulsions (NEs) loading amphotericin B (AmB) and to evaluate the influence of different excipients on the stability and the supramolecular organization, retention and toxicity of AmB. Materials & methods: The NEs were developed from different oils, surfactants, external media and anionic lipids (disteaoryl phosphatidylglycerol [DSPG] and dioleoyl phosphatidylglycerol [DOPG]). Their impact on the size, pH, zeta potential, AmB encapsulation efficiency, AmB retention and hemolytic potential of the NEs was evaluated. Results & conclusion: The use of soybean oil (lipid matrix), Span 80 (surfactant), phosphate buffer (external phase) and DSPG or DOPG (hydrophobic ion pair) provided better NE stability, higher AmB retention within the NEs and a safer formulation profile in hemolysis tests.

Oral formulation of Wnt inhibitor complex reduces inflammation and fibrosis in intraperitoneal implants in vivo.

The use of implantable biomaterials to replace physiological and anatomical functions has been widely investigated in the clinic. However, the selection of biomaterials is crucial for long-term function, and the implantation of certain biomaterials can cause inflammatory and fibrotic processes, triggering a foreign body reaction that leads to loss of function and consequent need for removal. Specifically, the Wnt signaling pathway controls the healing process of the human body, and its dysregulation can result in inflammation and fibrosis, such as in peritoneal fibrosis. Here, we assessed the effects of daily oral administration of a Wnt pathway inhibitor complex (CD:LGK974) to reduce the inflammatory, fibrotic, and angiogenic processes caused by intraperitoneal implants. CD:LGK974 significantly reduced the infiltration of immune cells and release of inflammatory cytokines in the implant region compared to the control groups. Furthermore, CD:LGK974 inhibited collagen deposition and reduced the expression of pro-fibrotic α-SMA and TGF-ß1, confirming fibrosis reduction. Finally, the CD:LGK974 complex decreased VEGF levels and both the number and area of blood vessels formed, suggesting decreased angiogenesis. This work introduces a potential new application of the Wnt inhibitor complex to reduce peritoneal fibrosis and the rejection of implants at the intraperitoneal site, possibly allowing for longer-term functionality of existing clinical biomaterials.

Cholesterol improves stability of amphotericin B nanoemulsion: promising use in the treatment of cutaneous leishmaniasis.

Aim: Amphotericin B (AmB) is an antileishmanial drug with high toxicity; however, this drawback might overcome by decreasing the AmB self-aggregation state. This work aimed at evaluating the influence of cholesterol on the aggregation state of AmB loaded in a nanoemulsion (NE-AmB) for the treatment of cutaneous leishmaniasis. NE-AmB (1, 4 and 8 mg/kg/day) was administered intravenously to animals infected by Leishmania major every 2 days for a total of five injections. Results: Ultraviolet-visible spectroscopy and circular dichroism studies demonstrated that cholesterol reduced AmB aggregation state in NE. NE-AmB was stable after 180 days, and its hemolytic toxicity was lower than that observed for the conventional AmB. NE-AmB administered intravenously into animals infected by Leishmania major at 8 mg/kg was capable of stabilizing the lesion size and reducing the parasitic load. Conclusion: These findings support the NE potential as a stable nanocarrier for AmB in the treatment of cutaneous leishmaniasis.

Ionizable Lipid Nanoparticle-Mediated Delivery of Plasmid DNA in Cardiomyocytes.

Introduction: Gene therapy is a promising approach to be applied in cardiac regeneration after myocardial infarction and gene correction for inherited cardiomyopathies. However, cardiomyocytes are crucial cell types that are considered hard-to-transfect. The entrapment of nucleic acids in non-viral vectors, such as lipid nanoparticles (LNPs), is an attractive approach for safe and effective delivery. Methods: Here, a mini-library of engineered LNPs was developed for pDNA delivery in cardiomyocytes. LNPs were characterized and screened for pDNA delivery in cardiomyocytes and identified a lead LNP formulation with enhanced transfection efficiency. Results: By varying lipid molar ratios, the LNP formulation was optimized to deliver pDNA in cardiomyocytes with enhanced gene expression in vitro and in vivo, with negligible toxicity. In vitro, our lead LNP was able to reach a gene expression greater than 80%. The in vivo treatment with lead LNPs induced a twofold increase in GFP expression in heart tissue compared to control. In addition, levels of circulating myeloid cells and inflammatory cytokines remained without significant changes in the heart after LNP treatment. It was also demonstrated that cardiac cell function was not affected after LNP treatment. Conclusion: Collectively, our results highlight the potential of LNPs as an efficient delivery vector for pDNA to cardiomyocytes. This study suggests that LNPs hold promise to improve gene therapy for treatment of cardiovascular disease.

Formulation of Amphotericin B in PEGylated Liposomes for Improved Treatment of Cutaneous Leishmaniasis by Parenteral and Oral Routes.

Liposomal amphotericin B (AmB) or AmBisome® is the most effective and safe therapeutic agent for visceral leishmaniasis (VL), but its clinical efficacy is limited in cutaneous leishmaniasis (CL) and HIV/VL co-infection. The aim of this work was to develop a formulation of AmB in PEGylated liposomes and compare its efficacy to AmBisome® in a murine model of CL. Formulations of AmB in conventional and PEGylated liposomes were characterized for particle size and morphology, drug encapsulation efficiency and aggregation state. Those were compared to AmBisome® in Leishmania amazonensis-infected BALB/c mice for their effects on the lesion size growth and parasite load. The conventional and PEGylated formulations showed vesicles with 100-130 nm diameter and low polydispersity, incorporating more than 95% of AmB under the non-aggregated form. Following parenteral administration in the murine model of CL, the PEGylated formulation of AmB significantly reduced the lesion size growth and parasite load, in comparison to control groups, in contrast to conventional liposomal AmB. The PEGylated formulation of AmB was also effective when given by oral route on a 2-day regimen. This work reports for the first time that PEGylated liposomal AmB can improve the treatment of experimental cutaneous leishmaniasis by both parenteral and oral routes.

Comparative evaluation of meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes and immunotherapy using an anti-canine IL-10 receptor-blocking monoclonal antibody on canine visceral leishmaniasis.

This study compared the therapeutic potential of the chemotherapy using meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes (Nano Sbv) and immunotherapy with anti-canine IL-10 receptor-blocking monoclonal antibody (Anti IL-10R) on canine visceral leishmaniasis (CVL). Twenty mongrel dogs naturally infected by L. infantum, displaying clinical signs of visceral leishmaniasis were randomly divided in two groups. In the first one, nine dogs received six intravenous doses of a mixture of conventional and PEGylated liposomes containing meglumine antimoniate at 6.5 mg Sb/kg/dose. In the second one, eleven dogs received two intramuscular doses of 4 mg of anti-canine IL-10 receptor-blocking monoclonal antibody. The animals were evaluated before (T0) and 30, 90, and 180 days after treatments. Our major results demonstrated that both treatments were able to maintain hematological and biochemical parameters, increase circulating T lymphocytes subpopulations, increase the IFN-γ producing T-CD4 lymphocytes, restore the lymphoproliferative capacity and improve the clinical status. However, although these improvements were observed in the initial post-treatment times, they did not maintain until the end of the experimental follow-up. We believe that the use of booster doses or the association of chemotherapy and immunotherapy (immunochemotherapy) is promising to improve the effectiveness of treating CVL for improving the clinical signs and possibly reducing the parasite burden in dogs infected with Leishmania infantum.

Liposomal Amphotericin B for Treatment of Leishmaniasis: From the Identification of Critical Physicochemical Attributes to the Design of Effective Topical and Oral Formulations.

The liposomal amphotericin B (AmB) formulation, AmBisome®, still represents the best therapeutic option for cutaneous and visceral leishmaniasis. However, its clinical efficacy depends on the patient's immunological status, the clinical manifestation and the endemic region. Moreover, the need for parenteral administration, its side effects and high cost significantly limit its use in developing countries. This review reports the progress achieved thus far toward the understanding of the mechanism responsible for the reduced toxicity of liposomal AmB formulations and the factors that influence their efficacy against leishmaniasis. It also presents the recent advances in the development of more effective liposomal AmB formulations, including topical and oral liposome formulations. The critical role of the AmB aggregation state and release rate in the reduction of drug toxicity and in the drug efficacy by non-invasive routes is emphasized. This paper is expected to guide future research and development of innovative liposomal formulations of AmB.

Use of liposomal nanoformulations in antileishmania therapy: challenges and perspectives.

Leishmaniasis is a parasitic disease treatable and curable, however, the chemotherapeutic agents for their treatment are limited. In South American countries, pentavalent antimonials are still the first line of treatment for cutaneous leishmaniasis with an efficacy of about 75%, but the toxicity of the drug causes serious side effects and remains as the main obstacle for treatment. New knowledge aimed to improve drug delivery into the intracellular environment is essential, especially for drugs currently used in the clinic, to develop new anti-Leishmania formulations. In the present study, we analysed the scientific literature to highlight the progress achieved in the last decade regarding the use of nanotechnology for improving the current leishmaniasis treatments. Results allowed us to conclude that the encapsulated Glucantime liposomal formulation can be improved by means of nanoparticle functionalization processes, resulting in new drug delivery systems that can be potentially proposed as alternative therapies for leishmaniasis treatment.

Antileishmanial activity of fullerol and its liposomal formulation in experimental models of visceral leishmaniasis.

Visceral leishmaniasis (VL) is a systemic parasitic disease that leads to high rates of morbidity and mortality in humans worldwide. There is a great need to develop new drugs and novel strategies to make chemotherapy for this disease more efficacious and well tolerated. Recent reports on the immunomodulatory effects and the low toxicity of the spherical carbon nanostructure fullerol led us to investigate in vitro and in vivo antileishmanial activity in free and encapsulated forms in liposomes. When assayed against intramacrophagic Leishmania amastigotes, fullerol showed a dose-dependent reduction of the infection index with IC50 of 0.042 mg/mL. When given daily by i.p. route for 20 days (0.05 mg/kg/d) in a murine model of acute VL, fullerol promoted significant reduction in the liver parasite load. To improve the delivery of fullerol to the infection sites, liposomal formulations were prepared by the dehydration-rehydration method. When evaluated in the acute VL model, liposomal fullerol (Lip-Ful) formulations given i.p. at 0.05 and 0.2 mg/kg with 4-days intervals were more effective than the free form, with significant parasite reductions in both liver and spleen. Lip-Ful at 0.2 mg/kg promoted complete parasite elimination in the liver. The antileishmanial activity of Lip-Ful was further confirmed in a chronic model of VL. Lip-Ful was also found to induce secretion of pro-inflammatory TNF-α, IFN-γ and IL-1ß cytokines. In conclusion, this work reports for the first time the antileishmanial activity of fullerol and introduces an innovative approach for treatment of VL based on the association of this nanostructure with liposomes.

Preclinical Gold Complexes as Oral Drug Candidates to Treat Leishmaniasis Are Potent Trypanothione Reductase Inhibitors.

The drugs currently used to treat leishmaniases have limitations concerning cost, efficacy, and safety, making the search for new therapeutic approaches urgent. We found that the gold(I)-derived complexes were active against L. infantum and L. braziliensis intracellular amastigotes with IC50 values ranging from 0.5 to 5.5 µM. All gold(I) complexes were potent inhibitors of trypanothione reductase (TR), with enzyme IC50 values ranging from 1 to 7.8 µM. Triethylphosphine-derived complexes enhanced reactive oxygen species (ROS) production and decreased mitochondrial respiration after 2 h of exposure, indicating that gold(I) complexes cause oxidative stress by direct ROS production, by causing mitochondrial damage or by impairing TR activity and thus accumulating ROS. There was no cross-resistance to antimony; in fact, SbR (antimony-resistant mutants) strains were hypersensitive to some of the complexes. BALB/c mice infected with luciferase-expressing L. braziliensis or L. amazonensis and treated orally with 12.5 mg/kg/day of AdT Et (3) or AdO Et (4) presented reduced lesion size and parasite burden, as revealed by bioimaging. The combination of (3) and miltefosine allowed for a 50% reduction in miltefosine treatment time. Complexes 3 and 4 presented favorable pharmacokinetic and toxicity profiles that encourage further drug development studies. Gold(I) complexes are promising antileishmanial agents, with a potential for therapeutic use, including in leishmaniasis caused by antimony-resistant parasites.

Therapeutic Efficacy of a Mixed Formulation of Conventional and PEGylated Liposomes Containing Meglumine Antimoniate, Combined with Allopurinol, in Dogs Naturally Infected with Leishmania infantum.

The treatment of dogs naturally infected with Leishmania infantum using meglumine antimoniate (MA) encapsulated in conventional liposomes (LC) in association with allopurinol has been previously reported to promote a marked reduction in the parasite burden in the main infection sites. Here, a new assay in naturally infected dogs was performed using a novel liposome formulation of MA consisting of a mixture of conventional and long-circulating (PEGylated) liposomes (LCP), with expected broader distribution among affected tissues of the mononuclear phagocyte system. Experimental groups of naturally infected dogs were as follows: LCP plus Allop, receiving LCP intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg of body weight/dose) at 4-day intervals plus allopurinol at 30 mg/kg/12 h per os (p.o.) during 130 days (LCP+Allop); LC plus Allop, receiving LC intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg/dose) plus allopurinol during 130 days (LC+Allop); Allop, treated with allopurinol only; and a nontreated control. Parasite loads were evaluated by quantitative PCR in liver, spleen, and bone marrow tissue and by immunohistochemistry in the ear skin, before treatment, just after treatment, and 4 months later. The LCP+Allop and LC+Allop groups, but not the Allop group, showed significant suppression of the parasites in the liver, spleen, and bone marrow 4 months after treatment compared to the pretreatment period or the control group. Only LCP+Allop group showed significantly lower parasite burden in the skin in comparison to the control group. On the basis of clinical staging and parasitological evaluations, the LCP formulation exhibited a more favorable therapeutic profile than the LC one, being therefore promising for the treatment of canine visceral leishmaniasis.

Nanostructured Systems Improve the Antimicrobial Potential of the Essential Oil from Cymbopogon densiflorus Leaves.

The physicochemical characteristics of nanostructured suspensions are important prerequisites for the success of new drug development. This work aimed to develop nanometric systems containing Cymbopogon densiflorus leaf essential oil and to evaluate their antimicrobial activity. The essential oil was isolated by hydrodistillation from leaves and analyzed by GC-MS. The main constituents were found to be trans-p-mentha-2,8-dien-1-ol, cis-p-mentha-2,8-dien-1-ol, trans-p-mentha-1(7),8-dien-2-ol, cis-piperitol, and cis-p-mentha-1(7),8-dien-2-ol. In silico prediction analysis suggested that this oil possesses antimicrobial potential and the main mechanism of action might be the peptidoglycan glycosyltransferase inhibition. Nanoemulsions were prepared by the phase inversion method, and liposomes were made by the film hydration method. Qualitative evaluation of the antimicrobial activity was performed by the diffusion disk assay with 24 microorganisms; all of them were found to be sensitive to the essential oil. Subsequently, this property was quantified by the serial microdilution technique, where the nanoformulations demonstrated improved activity in comparison with the free oil. Bactericidal action was tested by the propidium iodide method, which revealed that free essential oil and nanoemulsion increased cytoplasmic membrane permeability, while no difference was observed between negative control and liposome. These results were confirmed by images obtained using transmission electron microscopy. This study has shown an optimization in the antimicrobial activity of C. densiflorus essential oil by a nanoemulsion and a liposomal formulation of the active substances.

Corrigendum: Characterization of Aspergillus fumigatus Extracellular Vesicles and Their Effects on Macrophages and Neutrophils Functions.

[This corrects the article DOI: 10.3389/fmicb.2019.02008.].

Characterization of Aspergillus fumigatus Extracellular Vesicles and Their Effects on Macrophages and Neutrophils Functions.

Extracellular vesicles (EVs) has been considered an alternative process for intercellular communication. EVs release by filamentous fungi and the role of vesicular secretion during fungus-host cells interaction remain unknown. Here, we identified the secretion of EVs from the pathogenic filamentous fungus, Aspergillus fumigatus. Analysis of the structure of EVs demonstrated that A. fumigatus produces round shaped bilayer structures ranging from 100 to 200 nm size, containing ergosterol and a myriad of proteins involved in REDOX, cell wall remodeling and metabolic functions of the fungus. We demonstrated that macrophages can phagocytose A. fumigatus EVs. Phagocytic cells, stimulated with EVs, increased fungal clearance after A. fumigatus conidia challenge. EVs were also able to induce the production of TNF-α and CCL2 by macrophages and a synergistic effect was observed in the production of these mediators when the cells were challenged with the conidia. In bone marrow-derived neutrophils (BMDN) treated with EVs, there was enhancement of the production of TNF-α and IL-1ß in response to conidia. Together, our results demonstrate, for the first time, that A. fumigatus produces EVs containing a diverse set of proteins involved in fungal physiology and virulence. Moreover, EVs are biologically active and stimulate production of inflammatory mediators and fungal clearance.