Folate-Targeted Liposomal Formulations Improve Effects of Methotrexate in Murine Collagen-Induced Arthritis.

Methotrexate (MTX) is first-line therapy for the treatment of rheumatoid arthritis (RA), however, its use may be limited by side effects notably post-injection malaise. When patients are intolerant or become unresponsive, second-line or antibody therapy may be indicated. A folate-targeted liposomal formulation of MTX (FL-MTX) is tropic to arthritic paws and prevents the onset of collagen-induced arthritis (CIA) in the mouse. We optimized the drug-to-lipid molar ratio to 0.15 and demonstrated the therapeutic efficacy of this form at 2 mg/kg MTX intraperitoneal (i.p.) twice a week. These improved liposomes were present in inflamed joints in proportion to the degree of swelling of the paw and bone remodeling activity. FL-MTX had lower hepatic and renal elimination of MTX than the free substance. FL-MTX provided equivalent results when given i.p. or subcutaneous (s.c.) and FL-MTX 2 mg/kg (drug/lipid 0.15), twice weekly, was similar to or more effective than 35 mg/kg MTX (same route and schedule) in reducing the incidence and swelling in the murine CIA model. These results suggest that FL-MTX is a more potent nanotherapeutic formulation than free MTX treatment. Its potential benefits for patients may include reduced frequency of treatment and lower overall doses for a given response.

Comparing the delivery to the hair bulb of two fluorescent molecules of distinct hydrophilicities by different nanoparticles and a serum formulation.

The follicular route is an important drug penetration pathway in any topical application, either concerning dermatological and cosmetic skin treatments or any transdermal administration regimen. Efficient transport into follicles will depend on drug inherent properties but also on the chosen vehicle. The main study goal was to compare several systems for the delivery to the hair bulb of two fluorescent molecules of different water affinities: the hydrophobic Nile Red and the quite similar but hydrophilic Nile Blue. Three common nanoparticle types were compared in terms of encapsulation efficiency and stability: liposomes, ethosomes and polymeric nanoparticles. A liquid serum-like formulation was also developed, adjusting the final ethanol amount to the type of dye to be solubilized. Then, this formulation and the nanoparticle systems that successfully passed characterization and stability stages were further studied on their ability to reach the bulb. The serum formulation was able to deliver, both drug models, to deeper follicular regions than nanoparticles. Attending to the envisioned zone target of the follicle, the simplest approach proved to be the best choice from all the systems tested in this work. Nonetheless, nanocarriers and the inherent complexity of their manufacturing processes may be justified under very specific requirements.

Design of liposomes as drug delivery system for therapeutic applications.

Liposomes are spherical vesicles consisting of one or more concentric phospholipid bilayers enclosing an aqueous core. Being both nontoxic and biodegradable, liposomes represent a powerful delivery system for several drugs. They have improved the therapeutic efficacy of drugs through stabilizing compounds, overcoming obstacles to cellular and tissue uptake and increasing drug biodistribution to target sites in vivo, while minimizing systemic toxicity. This review offers an overview of liposomes, thought the exploration of their key fundamentals. Initially, the main design aspects to obtain a successful liposomal formulation were addressed, following the techniques for liposome production and drug loading. Before application, liposomes required an extensive characterization to assurance in vitro and in vivo performance. Thus, several properties to characterize liposomes were explored, such as size, polydispersity index, zeta potential, shape, lamellarity, phase behavior, encapsulation efficiency, and in vitro drug release. Topics related with liposomal functionalization and effective targeting strategies were also addressed, as well as stability and some limitations of liposomes. Finally, this review intends to explore the current market liposomes used as a drug delivery system in different therapeutic applications.

Increased Encapsulation Efficiency of Methotrexate in Liposomes for Rheumatoid Arthritis Therapy.

Methotrexate (MTX) is a common drug used to treat rheumatoid arthritis. Due to the excessive side effects, encapsulation of MTX in liposomes is considered an effective delivery system, reducing drug toxicity, while maintaining its efficacy. The ethanol injection method is an interesting technique for liposome production, due to its simplicity, fast implementation, and reproducibility. However, this method occasionally requires the extrusion process, to obtain suitable size distribution, and achieve a low level of MTX encapsulation. Here, we develop a novel pre-concentration method, based on the principles of the ethanol injection, using an initial aqueous volume of 20% and 1:1 ratio of organic:aqueous phase (v/v). The liposomes obtained present small values of size and polydispersity index, without the extrusion process, and a higher MTX encapsulation (efficiency higher than 30%), suitable characteristics for in vivo application. The great potential of MTX to interact at the surface of the lipid bilayer was shown by nuclear magnetic resonance (NMR) studies, revealing mutual interactions between the drug and the main phospholipid via hydrogen bonding. In vivo experiments reveal that liposomes encapsulating MTX significantly increase the biological benefit in arthritic mice. This approach shows a significant advance in MTX therapeutic applications.

Quantification of drugs encapsulated in liposomes by 1H NMR.

Liposomes are one of the most important and extensively studied drug delivery system due to their ability to encapsulate different kinds of drugs. Exploiting the advantages of 1H Nuclear Magnetic Resonance (NMR) spectrometry, we established a rapid and easy method for quantification of drugs encapsulated in liposomes. An internal standard, pyridine, was used for quantitative determination of drug concentration. Two different drugs were involved in this work, one hydrophilic, methotrexate disodium salt, and another hydrophobic, tamoxifen. The specificity and selectivity of the suggested method were evaluated by the absence of overlapping of at least one signal of each drug with pyridine in the NMR spectrum. The accuracy and precision of the method were assessed by adding a known amount of each drug to unloaded liposomes. Results obtained by quantitative NMR (qNMR) were validated and confirmed by comparing with two other traditional techniques, Ultraviolet-Visible (UV-vis) spectrophotometry and High-Performance Liquid Chromatography (HPLC). It was found that the results were consistent with the ones obtained from our proposed qNMR method. Considering all the experiments conducted in this study, we deliberate that qNMR can be a suitable tool for the determination of drugs encapsulated in liposomes.

Protective Effect of Saccharides on Freeze-Dried Liposomes Encapsulating Drugs.

The production of freeze-dried liposomes encapsulating drugs is considered a key challenge since the drugs are prone to leakage. The aim of this work was to study the effect of different saccharides on preserving the stability and drug retention capacity of a previously developed liposomal formulation, when subjected to a freeze-drying process. The protective role of trehalose, lactose, glucose, mannitol and sucrose, known for their cryo/lyoprotective effect, was tested by addition of different concentrations to liposomes. Sucrose, in a concentration dependent manner (8:1 sugar:lipids mass ratio) proved to be a suitable cryo/lyoprotectant of these liposomes. Effectively, this saccharide prevents the fusion or/and aggregation of the liposomal formulation, protecting the integrity of the freeze-dried empty liposomes. The liposomal formulation containing sucrose was studied in terms of morphology, concentration, and anticancer drugs retention ability. The study involved two drugs encapsulated in the aqueous core, methotrexate (MTX) and doxorubicin (DOX), and one drug located in the lipid bilayer, tamoxifen (TAM). After the freeze-drying process, liposomes with sucrose encapsulating drugs revealed high physical stability, maintaining their narrow and monodisperse character, however high leakage of the drugs encapsulated in the aqueous core was observed. Otherwise, no significant drug leakage was detected on liposomes containing the TAM, which maintained its biological activity after the freeze-drying process. These findings reveal that sucrose is a good candidate for the cryo/lyoprotection of liposomes with drugs located in the lipid bilayer.

Internalization of Methotrexate Conjugates by Folate Receptor-α.

The folate antagonist methotrexate is a cytotoxic drug used in the treatment of several cancer types. The entry of methotrexate into the cell is mediated by two main transport systems: the reduced folate carrier and membrane-associated folate receptors. These transporters differ considerably in their mechanism of (anti)folate uptake, substrate specificity, and tissue specificity. Although the mechanism of action of the reduced folate carrier is fairly well-established, that of the folate receptor has remained unknown. The development of specific folate receptor-targeted antifolates would be accelerated if additional mechanistic data were to become available. In this work, we used two fluorescently labeled conjugates of methotrexate, differently linked at the terminal groups, to clarify the uptake mechanism by folate receptor-α. The results demonstrate the importance of methotrexate amino groups in the interaction with folate receptor-α.

Absence of Albumin Improves in Vitro Cellular Uptake and Disruption of Poloxamer 407-Based Nanoparticles inside Cancer Cells.

Novel nanoparticles based on Poloxamer 407 and vegetable oil were produced by high pressure homogenization. Functionalization of those nanoparticles was made by incorporation of folic acid (FA)-Poloxamer 407 conjugate. These nanoparticles showed suitable characteristics for intravenous therapeutic applications similarly to PEGylated albumin-based nanoparticles, previously described by our research group. Here, we found that the absence of albumin at the interface of Poloxamer 407-based nanoparticles improves the overall process of in vitro cellular uptake and nanoparticle disruption inside cancer cells (folate receptor, FR, positive cells). The results presented here suggest that interfacial composition of those nanoparticles is of paramount importance for drug trafficking inside cancer cells.

OBP fused with cell-penetrating peptides promotes liposomal transduction.

Cell-penetrating peptides (CPPs) have been applied as novel transport systems with the ability to facilitate the delivery of peptides, proteins, and oligonucleotides into cells. Herein, we designed different fusion proteins composed by pig odorant binding protein (OBP-I) and three CPPs, namely Tat, pVEC and Pep-1. A new methodology using liposomes as reservoirs and OBP:CPPs as carriers was developed as an advanced system to capture odorant molecules. 1-aminoanthracene (1-AMA) was used as a model molecule to evaluate the transduction ability of OBP:CPPs into the reservoirs. The transduction efficiency was dependent on the initial capacity of OBP:CPPs to bind 1-AMA and on the penetration of liposomes promoted by the CPPs. An encapsulation efficiency of 42% was obtained with OBP:Tat fusion protein. The presence of Tat peptide increased the 1-AMA transduction of 1.3 and 2.5 fold compared with Pep-1 and pVEC, respectively. This work expands the application of OBPs and CPPs on the design of promising capture and delivery systems for textile and cosmetic applications.

Fab antibody fragment-functionalized liposomes for specific targeting of antigen-positive cells.

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers.

Neutral PEGylated liposomal formulation for efficient folate-mediated delivery of MCL1 siRNA to activated macrophages.

Cationic liposomes are efficient vectors for systemic delivery of therapeutic small interfering RNA (siRNA), taking advantage of RNA interference (RNAi), a naturally occurring gene-silencing mechanism in mammalian cells. However, toxicity at high concentrations, short circulating half-lives and lack of specificity restrict their successful application in a wider scale. The purpose of this study was to evaluate the efficiency of neutral liposomes containing polyethylene glycol (PEG) to encapsulate siRNA in their aqueous core. This formulation will reduce drastically the toxicity associated to cationic liposomes by bringing surface charge to almost zero, increasing stealth degree and therefore circulation time. In this study, we evaluate the efficiency of folate-targeted liposomes for specific delivery of siRNA to activated macrophages, key effector cells in rheumatoid arthritis (RA) pathology which specifically express folate receptor ß (FRß). Myeloid cell leukaemia-1 (Mcl-1) is a protein essential for synovial macrophage survival, since Mcl-1 suppression results in the induction of apoptosis. The effect of MCL1 siRNA incorporated in liposomal formulation was assessed in primary human macrophages and successful inhibition of Mcl-1 expression was achieved. Here we show that the neutral liposomal derived from DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) formulation developed is efficient to encapsulate MCL1 siRNA and silencing gene expression in activated human macrophages.

Update on Therapeutic Approaches for Rheumatoid Arthritis.

Rheumatoid arthritis is a common chronic inflammatory and destructive arthropathy that consumes considerable personal, social and economic costs. It consists of a syndrome of pain, stiffness and symmetrical inflammation of the synovial membrane (synovitis) of freely moveable joints such as the knee (diarthrodial joints). Although the etiology of rheumatoid arthritis is unclear, the disease is characterized by inflammation of the synovial lining of diarthrodial joints, high synovial proliferation and an influx of inflammatory cells, macrophages and lymphocytes through angiogenic blood vessels. Diseasemodifying antirheumatic drugs slow disease progression and can induce disease remission in some patients. Methotrexate is the first line therapy, but if patients become intolerant to this drug, biologic agents should be used. The development of biological substances for the treatment of rheumatic conditions has been accompanied by ongoing health economic discussions regarding the implementation of these highly effective, but accordingly, highly priced drugs are the standard treatment guidelines of rheumatic diseases. In this way, more efficient strategies have to be identified. Despite numerous reviews in rheumatoid arthritis in the last years, this area is in constant development and updates are an urgent need to incorporate new advances in rheumatoid arthritis research. This review highlights the immunopathogenesis rationale for the current therapeutic strategies in rheumatoid arthritis.

Folate-targeted nanoparticles for rheumatoid arthritis therapy.

Rheumatoid arthritis (RA) is the most common inflammatory rheumatic disease, affecting almost 1% of the world population. Although the cause of RA remains unknown, the complex interaction between immune mediators (cytokines and effector cells) is responsible for the joint damage that begins at the synovial membrane. Activated macrophages are critical in the pathogenesis of RA and showed specifically express a receptor for the vitamin folic acid (FA), folate receptor ß (FRß). This particular receptor allows internalization of FA-coupled cargo. In this review we will address the potential of nanoparticles as an effective drug delivery system for therapies that will directly target activated macrophages. Special attention will be given to stealth degree of the nanoparticles as a strategy to avoid clearance by macrophages of the mononuclear phagocytic system (MPS). This review summarizes the application of FA-target nanoparticles as drug delivery systems for RA and proposes prospective future directions. FROM THE CLINICAL EDITOR: Rheumatoid arthritis is a debilitating autoimmune disease of the joints which affects many people worldwide. Up till now, there is a lack of optimal therapy against this disease. In this review article, the authors outlined in depth the current mechanism of disease for rheumatoid arthritis and described the latest research in using folic acid-targeted nanoparticles to target synovial macrophages in the fight against rheumatoid arthritis.

Assessment of liposome disruption to quantify drug delivery in vitro.

Efficient liposome disruption inside the cells is a key for success with any type of drug delivery system. The efficacy of drug delivery is currently evaluated by direct visualization of labeled liposomes internalized by cells, not addressing objectively the release and distribution of the drug. Here, we propose a novel method to easily assess liposome disruption and drug release into the cytoplasm. We propose the encapsulation of the cationic dye Hoechst 34580 to detect an increase in blue fluorescence due to its specific binding to negatively charged DNA. For that, the dye needs to be released inside the cell and translocated to the nucleus. The present approach correlates the intensity of detected fluorescent dye with liposome disruption and consequently assesses drug delivery within the cells.

Design of liposomal formulations for cell targeting.

Liposomes have gained extensive attention as carriers for a wide range of drugs due to being both nontoxic and biodegradable as they are composed of substances naturally occurring in biological membranes. Active targeting for cells has explored specific modification of the liposome surface by functionalizing it with specific targeting ligands in order to increase accumulation and intracellular uptake into target cells. None of the Food and Drug Administration-licensed liposomes or lipid nanoparticles are coated with ligands or target moieties to delivery for homing drugs to target tissues, cells or subcellular organelles. Targeted therapies (with or without controlled drug release) are an emerging and relevant research area. Despite of the numerous liposomes reviews published in the last decades, this area is in constant development. Updates urgently needed to integrate new advances in targeted liposomes research. This review highlights the evolution of liposomes from passive to active targeting and challenges in the development of targeted liposomes for specific therapies.

Enhancing Methotrexate Tolerance with Folate Tagged Liposomes in Arthritic Mice.

Methotrexate is the first line of treatment of rheumatoid arthritis. Since many patients become unresponsive to methotrexate treatment, only very expensive biological therapies are effective and increased methotrexate tolerance strategies need to be identified. Here we propose the encapsulation of methotrexate in a new liposomal formulation using a hydrophobic fragment of surfactant protein conjugated to a linker and folate to enhance their tolerance and efficacy. In this study we aim to evaluate the efficiency of this system to treat rheumatoid arthritis, by targeting folate receptor ß present at the surface of activated macrophages, key effector cells in this pathology. The specificity of our liposomal formulation to target folate receptor ß was investigated both in vitro as in vivo using a mouse model of arthritis (collagen-induced arthritis in DBA/1J mice strain). In both systems, the liposomal constructs were shown to be highly specific and efficient in targeting folate receptor ß. These liposomal formulations also significantly increase the clinical benefit of the encapsulated methotrexate in vivo in arthritic mice, together with reduced expression of CD39 and CD73 ectonucleotidases by joint-infiltrating macrophages. Thus, our formulation might be a promising cost effective way to treat rheumatoid arthritis and delay or reduce methotrexate intolerance.

Peptide Anchor for Folate-Targeted Liposomal Delivery.

Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.

Size controlled protein nanoemulsions for active targeting of folate receptor positive cells.

Bovine serum albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri-block copolymer (Poloxamer 407), which presents a central hydrophobic chain of polyoxypropylene (PPO) and two identical lateral hydrophilic chains of polyethylene glycol (PEG). We observed a linear correlation between tri-block copolymer concentration and size - the use of 5mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100nm. Molecular dynamics and fluorescent tagging of the tri-block copolymer highlight their mechanistic role on the size of emulsions. This novel method enables the fabrication of highly stable albumin emulsions in the nano-size range, highly desirable for controlled drug delivery. Folic Acid (FA)-tagged protein nanoemulsions were shown to promote specific folate receptor (FR)-mediated targeting in FR positive cells. The novel strategy presented here enables the construction of size controlled, functionalized protein-based nanoemulsions with excellent characteristics for active targeting in cancer therapy.

Folic acid-tagged protein nanoemulsions loaded with CORM-2 enhance the survival of mice bearing subcutaneous A20 lymphoma tumors.

Folic Acid (FA)-tagged protein nanoemulsions were found to be preferentially internalized on B-cell lymphoma cell line (A20 cell line), which, for the first time, is reported to express folate receptor (FR)-alpha. Carbon monoxide releasing molecule-2 (CORM-2) was incorporated in the oil phase of the initial formulation. FA-functionalized nanoemulsions loaded with CORM-2 exhibited a considerable antitumor effect and an increased survival of BALB/c mice bearing subcutaneous A20 lymphoma tumors. The developed nanoemulsions also demonstrated to be well tolerated by these immunocompetent mice. Thus, the results obtained in this study demonstrate that FA-tagged protein nanoemulsions can be successfully used in cancer therapy, with the important ability to delivery drugs intracellularly. FROM THE CLINICAL EDITOR: In this research, the authors developed folic acid tagged nanoemulsions containing a carbon monoxide releasing protein molecule for targeted cancer cell treatment. In-vitro and in-vivo experiments showed efficacy against B-cell lymphoma cells. The same nanocarrier platform could be applied to other tumor cells expressing folate receptors on the cell surface.

Participation of Candida albicans transcription factor RLM1 in cell wall biogenesis and virulence.

Candida albicans cell wall is important for growth and interaction with the environment. RLM1 is one of the putative transcription factors involved in the cell wall integrity pathway, which plays an important role in the maintenance of the cell wall integrity. In this work we investigated the involvement of RLM1 in the cell wall biogenesis and in virulence. Newly constructed C. albicans Δ/Δrlm1 mutants showed typical cell wall weakening phenotypes, such as hypersensitivity to Congo Red, Calcofluor White, and caspofungin (phenotype reverted in the presence of sorbitol), confirming the involvement of RLM1 in the cell wall integrity. Additionally, the cell wall of C. albicans Δ/Δrlm1 showed a significant increase in chitin (213%) and reduction in mannans (60%), in comparison with the wild-type, results that are consistent with cell wall remodelling. Microarray analysis in the absence of any stress showed that deletion of RLM1 in C. albicans significantly down-regulated genes involved in carbohydrate catabolism such as DAK2, GLK4, NHT1 and TPS1, up-regulated genes involved in the utilization of alternative carbon sources, like AGP2, SOU1, SAP6, CIT1 or GAL4, and genes involved in cell adhesion like ECE1, ALS1, ALS3, HWP1 or RBT1. In agreement with the microarray results adhesion assays showed an increased amount of adhering cells and total biomass in the mutant strain, in comparison with the wild-type. C. albicans mutant Δ/Δrlm1 strain was also found to be less virulent than the wild-type and complemented strains in the murine model of disseminated candidiasis. Overall, we showed that in the absence of RLM1 the modifications in the cell wall composition alter yeast interaction with the environment, with consequences in adhesion ability and virulence. The gene expression findings suggest that this gene participates in the cell wall biogenesis, with the mutant rearranging its metabolic pathways to allow the use of alternative carbon sources.