RESUMEN

The cells of acute myeloid leukemia are defined by clonal growth and heterogenous immunophenotypes. Chimeric antigen receptors (CARs) commonly recognize molecular targets by single-chain antibody fragments (scFvs) specific to a tumor-associated antigen. However, ScFvs may form aggregates, thus stimulating tonic CAR T-cell activation and reducing CAR T-cell functioning in vivo. Harnessing natural ligands as recognition parts of CARs, specific targeting of membrane receptors can be achieved. Previously, we presented ligand-based Flt3-CAR T-cells targeting the Flt3 receptor. The extracellular part of Flt3-CAR consisted of full-size Flt3Lg. Meanwhile, upon recognition, Flt3-CAR may potentially activate Flt3, triggering proliferative signaling in blast cells. Moreover, the long-lasting presence of Flt3Lg may lead to Flt3 downregulation. In this paper, we present mutated Flt3Lg-based Flt3m-CAR ('m'-for 'mutant') T-cells targeting Flt3. The extracellular part of Flt3m-CAR consists of full-length Flt3Lg-L27P. We have determined that ED50 for recombinant Flt3Lg-L27P produced in CHO cells is at least 10-fold higher than for the wild-type Flt3Lg. We show that the mutation in the recognizing domain of Flt3m-CAR did not affect the specificity of Flt3m-CAR T-cells when compared to Flt3-CAR T-cells. Flt3m-CAR T-cells combine the specificity of ligand-receptor recognition with reduced Flt3Lg-L27P bioactivity, leading to potentially safer immunotherapy.

Asunto(s)

Leucemia Mieloide Aguda , Receptores Quiméricos de Antígenos , Animales , Cricetinae , Humanos , Ligandos , Cricetulus , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/genética , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/genética , Receptores Quiméricos de Antígenos/genética

RESUMEN

Background: Serious side effects caused by paclitaxel formulation, containing toxic solubilizer Cremophor® EL, and its nonspecific accumulation greatly limit clinical paclitaxel application. Aim: To design paclitaxel-loaded copolymer of lactic and glycolic acids nanoparticles decorated with alpha-fetoprotein third domain (rAFP3d-NP) to increase paclitaxel safety profile. Methods: rAFP3d-NP was obtained via carbodiimide technique. Results: The particles were characterized with high paclitaxel loading content of 5% and size of 280 nm. rAFP3d-NP revealed biphasic profile with 67% release of paclitaxel during 220 h. Increased area under the curveinf and mean residence time values after rAFP3d-NP administration confirmed prolonged blood circulation compared with paclitaxel. rAFP3d-NP demonstrated significant tumor growth inhibition at 4T1 and SKOV-3 models. Conclusion: rAFP3d-NP is a promising delivery system for paclitaxel and can be applied similarly for delivery of other hydrophobic drugs.

Asunto(s)

Nanopartículas , Neoplasias , Humanos , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , alfa-Fetoproteínas , Nanopartículas/química , Paclitaxel/química , Polímeros/química , Neoplasias/tratamiento farmacológico , Línea Celular Tumoral , Portadores de Fármacos/química

RESUMEN

Pheophorbide a 17-diethylene glycol ester (XL-8), is a promising high-active derivative of known photosensitizer chlorin e6 used in photodynamic therapy. However, high lipophilicity and poor tumor accumulation limit XL-8 therapeutic application. We developed a novel XL-8 loaded with poly(D,L-lactide-co-glycolide) nanoparticles using the single emulsion-solvent evaporation method. The nanoparticles possessed high XL-8 loading content (4.6%) and encapsulation efficiency (87.7%) and a small size (182 ± 19 nm), and negative surface charge (-22.2 ± 3.8 mV) contributed to a specific intracellular accumulation. Sustained biphasic XL-8 release from nanoparticles enhanced the photosensitizer photostability upon irradiation that could potentially reduce the quantity of the drug applied. Additionally, the encapsulation of XL-8 in the polymer matrix preserved phototoxic activity of the payload. The nanoparticles displayed enhanced cellular internalization. Flow cytometry and confocal laser-scanning microscopy studies revealed rapid XL-8 loaded nanoparticles distribution throughout the cell and initiation of DNA damage, glutathione depletion, and lipid peroxidation via reactive oxygen species formation. The novel nanoformulated XL-8 simultaneously revealed a significant phototoxicity accompanied with enhanced photostability, in contrast with traditional photosensitizers, and demonstrated a great potential for further in vivo studies.

RESUMEN

The selection of technological parameters for nanoparticle formulation represents a complicated development phase. Therefore, the statistical analysis based on Box-Behnken methodology is widely used to optimize technological processes, including poly(lactic-co-glycolic acid) nanoparticle formulation. In this study, we applied a two-level three-factor design to optimize the preparation of nanoparticles loaded with cobalt (CoTPP), manganese (MnClTPP), and nickel (NiTPP) metalloporphyrins (MeP). The resulting nanoparticles were examined by dynamic light scattering, X-ray diffraction, Fourier transform infrared spectroscopy, MTT test, and hemolytic activity assay. The optimized model of nanoparticle formulation was validated, and the obtained nanoparticles possessed a spherical shape and physicochemical characteristics enabling them to deliver MeP in cancer cells. In vitro hemolysis assay revealed high safety of the formulated MeP-loaded nanoparticles. The MeP release demonstrated a biphasic profile and release mechanism via Fick diffusion, according to release exponent values. Formulated MeP-loaded nanoparticles revealed significant antitumor activity and ability to generate reactive oxygen species. MnClTPP- and CoTPP-nanoparticles specifically accumulated in tissues, preventing wide tissue distribution caused by long-term circulation of the hydrophobic drug. Our results suggest that MnClTPP- and CoTPP-nanoparticles represent the greatest potential for utilization in in anticancer therapy due to their effectiveness and safety.

Asunto(s)

Complejos de Coordinación/farmacocinética , Metaloporfirinas/farmacocinética , Metales/química , Nanopartículas/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Porfirinas/química , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Liberación de Fármacos , Femenino , Células HeLa , Hemólisis/efectos de los fármacos , Humanos , Células MCF-7 , Metaloporfirinas/química , Metaloporfirinas/farmacología , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Nanopartículas/ultraestructura , Ratas Wistar , Espectroscopía Infrarroja por Transformada de Fourier , Distribución Tisular , Difracción de Rayos X

RESUMEN

Relapsed/refractory acute myeloid leukemia (AML) cannot be cured with chemotherapy alone, as the blasts survive the treatment. Chimeric antigen receptor (CAR) approaches for AML are being actively developed. CARs promote immune reactions through recognition of the target molecular epitopes at the surface of cancer cells. The recognition involves the extracellular portion of the CAR protein, which corresponds to either the antibody or the physiological binding partner of the targeted antigen. Here, we design a chimeric receptor with a full-length natural Flt3-ligand recognition module that targets Flt3 tyrosine kinase, known as an adverse marker in AML. We demonstrate specific killing of Flt3-positive THP-1 cells by Flt3-CAR T cells and the lack of cytotoxicity towards Flt3-negative U937 cells. We prove that the inherent cytolytic capacity of T cells is essential for the killing. Finally, we confirm the authenticity of targeting by its competitive dose-dependent inhibition with a soluble Flt3-ligand. The developed system can be viewed as a non-immunogenic functional equivalent of scFv-mediated targeting. The robust in vitro antitumor effects of Flt3-CAR T cells, combined with their low off-target cytotoxicity, hold promise for AML treatment.

RESUMEN

BACKGROUND: Difficult to express peptides are usually produced by co-expression with fusion partners. In this case, a significant mass part of the recombinant product falls on the subsequently removed fusion partner. On the other hand, multimerization of peptides is known to improve its proteolytic stability in E. coli due to the inclusion of body formation, which is sequence specific. Thereby, the peptide itself may serve as a fusion partner and one may produce more than one mole of the desired product per mole of fusion protein. This paper proposes a method for multimeric production of a human alpha-fetoprotein fragment with optimized multimer design and processing. This fragment may further find its application in the cytotoxic drug delivery field or as an inhibitor of endogenous alpha-fetoprotein. RESULTS: Multimerization of the extended alpha-fetoprotein receptor-binding peptide improved its stability in E. coli, and pentamer was found to be the largest stable with the highest expression level. As high as 10 aspartate-proline bonds used to separate peptide repeats were easily hydrolyzed in optimized formic acid-based conditions with 100% multimer conversion. The major product was represented by unaltered functional alpha-fetoprotein fragment while most side-products were its formyl-Pro, formyl-Tyr, and formyl-Lys derivatives. Single-step semi-preparative RP-HPLC was enough to separate unaltered peptide from the hydrolysis mixture. CONCLUSIONS: A recombinant peptide derived from human alpha-fetoprotein can be produced via multimerization with subsequent formic acid hydrolysis and RP-HPLC purification. The reported procedure is characterized by the lower reagent cost in comparison with enzymatic hydrolysis of peptide fusions and solid-phase synthesis. This method may be adopted for different peptide expression, especially with low amino and hydroxy side chain content.

RESUMEN

The mechanisms of binary catalyst therapy (BCT) and photodynamic therapy (PDT) are based on the formation of reactive oxygen species (ROS). This ROS formation results from specific chemical reactions. In BCT, light exposure does not necessarily initiate ROS formation and BCT application is not limited to regions of tissues that are accessible to illumination like photodynamic therapy (PDT). The principle of BCT is electron transition, resulting in the interaction of a transition metal complex (catalyst) and substrate molecule. MnIII- tetraphenylporphyrin chloride (MnClTPP) in combination with an ascorbic acid (AA) has been proposed as an appropriate candidate for cancer treatment regarding the active agents in BCT. The goal of this study was to determine whether MnClTPP in combination with AA would be a promising agent for BCT. The problem of used MnClTPP's, low solubility in water, was solved by MnClTPP loading into PLGA matrix. H2O2 produced during AA decomposition oxidized MnClTPP to high-reactive oxo-MnV species. MnClTPP in presence AA leads to the production of excessive ROS levels in vitro. ROS are mainly substrates of catalase and superoxide dismutase (H2O2 and O2●-). SOD1 and catalase were identified as the key players of the MnClTPP ROS-induced cell defense system. The cytotoxicity of MnClTPP-loaded nanoparticles (NPs) was greatly increased in the presence of specific catalase inhibitor (3-amino-1,2,4-triazole (3AT)) and superoxide dismutase 1 (SOD1) inhibitor (diethyldithiocarbamate (DDC)). Cell death resulted from the combined activation of caspase-dependent (caspase 3/9 system) and independent pathways, namely the AIF translocation to nuclei. Preliminary acute toxicity and in vivo anticancer studies have been revealed the safe and potent anticancer effect of PLGA-entrapped MnClTPP in combination with AA. The findings indicate that MnClTPP-loaded PLGA NPs are promising agents for BCT.

Asunto(s)

Metaloporfirinas/química , Nanopartículas/administración & dosificación , Neoplasias/tratamiento farmacológico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Especies Reactivas de Oxígeno/metabolismo , Animales , Apoptosis , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Nanopartículas/química , Neoplasias/metabolismo , Neoplasias/patología , Oxidación-Reducción , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Co-encapsulation of abiraterone acetate (AbrA) and docetaxel (Dtx) in polymeric nanoparticles as novel prototypes for prostate cancer treatment combining hormonal and chemotherapy was designed. Nanoparticles (NPs) composed of poly(dl-lactide-co-glycolide) (PLGA) were prepared by single-emulsion solvent evaporation technique and characterized in terms of morphology with atomic force microscopy and transmission electron microscopy. HPLC method for simultaneous determination of AbrA and Dtx encapsulation efficacy was developed. Also differential scanning calorimetry and Fourier-transform infrared spectroscopy were provided. To study the effectiveness of cellular internalization and distribution of NPs with AbrA and Dtx co-encapsulation (NP-AbrA/Dtx), a fluorescence microscopy was utilized. NPs prepared had size 256.3 ±9.4 nm and zeta potential -18.4 ±1.4 mV. Encapsulation efficacy for AbrA was 68.7% and for Dtx was 74.3%. NPs were able to control the AbrA and Dtx release within 24 h. The mathematical model of drug release was performed. The results obtained from confocal microscopy showed the effective accumulation of the NP-AbrA/Dtx in the cytoplasm of cells. Synthesized NPs possessed satisfactory parameters and a biphasic release profile, proceeding by the Fick diffusion mechanism, which provide prolonged release of the drugs and maintenance of their concentration. It was shown that NPs loaded with AbrA and Dtx exhibited a high cytotoxic activity on the LNCaP cell line, similar to the combination of free drugs of AbrA and Dtx, but in contrast to the combination of substances, had a synergistic mechanism of action. Our findings support the potential use of developed NPs in further in vivo studies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1150-1158, 2019.

Asunto(s)

Androstenos , Docetaxel , Portadores de Fármacos , Nanopartículas , Neoplasias , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Células A549 , Androstenos/química , Androstenos/farmacocinética , Androstenos/farmacología , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacocinética , Preparaciones de Acción Retardada/farmacología , Docetaxel/química , Docetaxel/farmacocinética , Docetaxel/farmacología , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacología , Humanos , Nanopartículas/química , Nanopartículas/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/farmacocinética , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/farmacología

RESUMEN

PURPOSE: The aim of this study was to compare the physico-chemical and biological properties of polymeric nanoparticles obtained from poly(DL-lactide-co-glycolide) (PLGA) with different ratios of monomers loaded with daunorubicin (DNR). METHODS: DNR-loaded nanoparticles (NPs) were prepared with use of modified simultaneous double-emulsion solvent evaporation/diffusion technique. NPs were characterized using dynamic light scattering, atomic force microscopy, transmission electron microscopy, scanning electron microscopy, and differential scanning calorimetry and Fourier transform infrared spectroscopy. RESULTS: NPs with DNR were differing in size and zeta potential, depending on the type of polymer. The data obtained show that total content of DNR correlates with the values of the binding constant of DNR with polymers. The release of DNR from NPs proceeds predominantly for polymers with lower binding constants. The in vitro study of NPs on the MCF-7 cells showed similar activity of particles and substances while for the anthracycline-resistant MCF-7Adr cells the cytotoxicity of the nanoparticles was 3 to 7 times higher depending on the type of copolymer. CONCLUSIONS: PLGA DNR-loaded nanoparticles can be used to overcome multidrug resistance (MDR) as well as for reducing the frequency of DNR reception due to the prolonged effect, which allows maintaining the concentration of the drug at the required level. The usefulness of binding constant calculations for obtaining nanoparticles with the maximum drug loading was proven. The rate of drug administration and the frequency of administration can be calculated based on the DNR release profiles and release parameters that depend on polymer type.

Asunto(s)

Daunorrubicina/farmacología , Liberación de Fármacos , Ácido Láctico/química , Nanopartículas/química , Ácido Poliglicólico/química , Rastreo Diferencial de Calorimetría , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Cinética , Microscopía de Fuerza Atómica , Nanopartículas/ultraestructura , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Espectroscopía Infrarroja por Transformada de Fourier

RESUMEN

A wide range of methods are known to increase the prokaryotic intracellular recombinant proteins solubility, for instance, growth at low temperature, supplementation of culture media with "chemical chaperones" (proline, glycine-betaine, and trehalose), co-expression with chaperones or highly soluble fusion partners. As an alternative, we have introduced the polyglutamate tag, which, as it has been shown, increased the protein solubility and facilitated folding. In this study we evaluated the minimal quantity of high density negatively charged EEEEVE amino acid repeats (pGlu) necessary to switch the recombinant receptor-binding domain of human alpha-fetoprotein (rbdAFP) expression almost entirely from the inclusion bodies to the soluble cytoplasmic fraction in E. coli. For this purpose, genetic constructs based on pET vectors coding rbdAFP and containing from 1 to 4 additional EEEEVE repeats at the C-terminus have been prepared. It was found that 3 pGlu repeats is the minimal number, that leads to a complete shift of the expression to the soluble cytoplasmic fraction in E. coli SHuffle Express T7 while 4 repeats were required for that in E. coli BL21(DE3). The rbdAFP contained 4 pGlu repeats was purified making use of ion-exchange chromatography and characterized by circular dichroism and ability to bind and accumulate in AFP receptor positive cancer cells in order to check for the structural and specific activity alterations related to the additional polyanionic sequence introduction.

Asunto(s)

Ácido Poliglutámico/metabolismo , Receptores de Péptidos/aislamiento & purificación , Receptores de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Línea Celular Tumoral , Células Cultivadas , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Leucocitos Mononucleares , Ácido Poliglutámico/química , Ácido Poliglutámico/genética , Dominios Proteicos , Pliegue de Proteína , Receptores de Péptidos/química , Receptores de Péptidos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética