Suchergebnisse

Poloxamer 407 (P407)-based nanoparticles were produced by the high pressure homogenization method for the encapsulation and delivery of methotrexate (MTX), aiming intravenous therapeutic applications. The surface of these nanoparticles was functionalized by conjugation of P407 with folic acid (FA) or with MTX, which served as targeting ligand agents. MTX-P407 conjugate was also developed to increase the final drug cargo. Two hydrophobic derivatives of MTX, MTX di-ethylated ester (MTX-OEt) and the ionic complex MTX-dimethyldioctadecylammonium bromide (MTX-DODAB) were produced and entrapped onto P407-based nanoparticles. All formulations developed revealed a monodisperse character comprising small and narrow nanoparticles (<100 nm). P407 nanoparticles (functionalized with FA) and MTX-P407 nanoparticles, both loaded with MTX-OEt, demonstrated a slow drug release profile. The effect of lipase from Aspergillus oryzae on the hydrolysis of the linkage between the P407 and MTX, and consequent MTX release profile, was also evaluated. We observed a controlled and slow release of MTX (<50% of release after 11 days) in the presence of enzyme. These MTX-P407 nanoparticles loaded with MTX-OEt induced a great effect against Caco-2 cancer cells (40% of cell death after 72 h of incubation), demonstrating higher efficiency than the free MTX at the same concentration. ; This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors also thanks to FCT for funding their scholarship: Jennifer Noro (SFRH/BD/121673/2016) and Carla Silva (SFRH/IF/00186/2015). This work has also received funding from the European Union Horizon 2020 research and innovation program under grant agreement NMP-06-2015-683356 FOLSMART. ...

Polyoxyethanyl--tocopheryl sebacate (PTS) is an amphiphilic compound with self-emulsifying properties known to form micelles. In this work, we report the production of PTS micelles for the encapsulation and delivery of a hydrophobic derivative of methotrexate, MTX di-ethylated (MTX-OEt). We optimized the micelles production by testing two different techniques: auxiliary solvent and sonication. Small and homogeneous micelles ( 40 nm) were obtained through the auxiliary solvent method performed at 30 °C and using 15 mg/mL of PTS. The produced micelles with the most promising physicochemical properties did not induce cytotoxicity when tested in normal human cells (BJ5ta cells), being considered for the encapsulation of MTX-OEt. This prodrug was achieved by Fisher esterification using ethanol, being isolated in good yield (= 68 %). MTX-OEt was efficiently encapsulated onto the produced micelles which preserved their physicochemical properties. The PTS micelles loaded with MTX-OEt, free MTX-OEt and free unmodified MTX revealed similar biological effect against cancer cells (Caco-2 cells). These results demonstrated that the biological activity of MTX is not altered after modification. The developed PTS micelles revealed a promising intracellular delivery performance with potentiality for cancer therapy. ; This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/ BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145- FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. The authors thanks to FCT for funding their scholarship: Jennifer Noro (SFRH/BD/121673/2016) and Carla Silva (SFRH/IF/00186/ 2015). This work has also received funding from the European Union Horizon 2020 research and innovation program under grant agreement NMP-06-2015-683356 FOLSMART. ...

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. ; This work has received funding from the European Union Horizon 2020 research andinnovation programme undergrantagreement NMP06-2015-683356 FOLSMART. This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Diana Guimarães (SFRH/BD/140321/ 2018) and Jennifer Noro (SFRH/BD/121673/2016) holds a scholarship from FCT. SFRH/BD/121673/2016 ...

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. ; This work has received funding from the European Union Horizon 2020 research and innovation programme under grant agreement NMP-06-2015-683356 FOLSMART. This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Diana Guimarães (SFRH/BD/140321/2018) and Jennifer Noro (SFRH/BD/121673/2016) hold a scholarship from FCT. ...

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 nanoparticles disruption inside cancer cells (folate receptor, FR, positive cells). The results present here suggests that interfacial composition of those nanoparticles is of paramount importance for drug trafficking inside cancer cells. ; This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020Programa Operacional Regional do Norte. This work has received funding from the European Union Horizon 2020 research and innovation program under Grant Agreement NMP-06-2015-683356 FOLSMART. Diana Soares da Costa thanks Portuguese Foundation for Science and Technology (FCT) for funding her scholarship SFRH/BPD/85790/2012. ...

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, are 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. ; SFRH/BD/81479/2011 and SFRH/BD/81269/2011 scholarships from Fundação para a Ciência e a Tecnologia (FCT). This work has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This work was supported by FEDER through POFC-COMPETE and by Portuguese funds from FCT through the project ...

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 (FR). 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. ; Eugénia Nogueira (SFRH/BD/81269/2011) and Ana Loureiro (SFRH/BD/81479/2011) hold scholarships from Fundação para a Ciência e a Tecnologia (FCT). This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project "BioHealth − Biotechnology and Bioengineering approaches to improve health quality", Ref. ...

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 5 mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100 nm. 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. ; European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This work was supported by FEDER through POFC – COMPETE and by Portuguese funds from FCT through the project PEst-OE/BIA/UI4050/2014. The authors also thank the FCT Strategic Project of UID/BIO/04469/2013 ...