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1.
Colloids Surf B Biointerfaces ; 181: 989-993, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31382350

RESUMO

Saccharomyces cerevisiae immobilized in electrospun Pluronic F127 dimethacrylate (FDMA) was successfully employed for the production of ethanol in an immobilized cell reactor. Yeast cells were immobilized into fibers formed through the process of electrospinning and cross-linking. The threads had an average diameter of 0.88 µm and were used in continuous-flow immobilized cell reactors. The immobilized cell reactors were able to maintain a high ethanol yield of >90% from day 4 through to the end of the time course at day 14. The reactor was able to achieve a maximum ethanol yield of 94.3%. This study shows that the use of electrospinning is a promising method for continuous ethanol production through immobilized cell-based continuous flow reactors.


Assuntos
Reatores Biológicos , Células Imobilizadas/metabolismo , Etanol/metabolismo , Metacrilatos/metabolismo , Poloxâmero/metabolismo , Saccharomyces cerevisiae/metabolismo , Etanol/química , Fermentação , Metacrilatos/química , Tamanho da Partícula , Poloxâmero/química , Propriedades de Superfície
2.
Eye Contact Lens ; 45(5): 340-345, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31441829

RESUMO

OBJECTIVES: This report aimed to explore whether certain inflammatory mediators were absorbed, extracted, or bound by various contact lens materials. METHODS: Comfilcon A, balafilcon A, omafilcon A, and etafilcon A were soaked in 500 and 100 pg/mL of interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), or interleukin-1 receptor antagonist (IL-1Ra), and also in combined solutions of inflammatory mediators (500 pg/mL or 100 pg/mL) separately. Lenses were then extracted in 1:1 2% trifluoroacetic acid:acetonitrile. The extracted and residual concentrations of inflammatory mediators were determined using enzyme-linked immunosorbent assays. Absorbed (control-residual) and firmly bound (absorbed-extracted) concentrations were calculated for analysis. RESULTS: More MMP-9 was absorbed by omafilcon A (466±9 pg/mL) than balafilcon A (P=0.006; 437±11 pg/mL) or etafilcon A (P=0.001; 428±13 pg/mL) when soaked in 500 pg/mL, but no differences in 100 pg/mL. More MMP-9 remained firmly bound to omafilcon A (P=0.03; 174±3 pg/mL), comfilcon A (P=0.049; 168±34 pg/mL), and balafilcon A (P=0.01; 186±14 pg/mL) than etafilcon A (128±22 pg/mL). There were no differences in IL-8 absorption between lenses; however, more IL-8 remained firmly bound to omafilcon A (P=0.01; 336±25 pg/mL) than etafilcon A (106±133 pg/mL) when soaked in 500 pg/mL. No differences were found in concentrations of absorbed or firmly bound IL-1Ra between materials. When the mediators were combined, IL-8 was absorbed more in etafilcon A (P=0.03) than in other lens materials, but the absorbed IL-8 did not remain firmly bound. CONCLUSIONS: The uptake and extraction of inflammatory mediators from contact lenses was affected by competitive binding between the mediators.


Assuntos
Lentes de Contato Hidrofílicas , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-8/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Ligação Competitiva , Ensaio de Imunoadsorção Enzimática , Hidrogéis/metabolismo , Mediadores da Inflamação , Metacrilatos/metabolismo , Ligação Proteica , Silicones/metabolismo
3.
Protoplasma ; 256(5): 1375-1383, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31079230

RESUMO

In order to gain more insight into the involvement of mitochondrial complex III in the Cd-induced stress, we studied the effect of complex III inhibitors, antimycin A (AA), and myxothiazol (MYXO), on the Cd-induced ROS and NO generation in the barley root tip. Short-term exposure of barley roots to either MYXO or AA provoked a dose-dependent increase in both H2O2 and NO formation. In contrast to H2O2 generation, an enhanced superoxide formation in the transition zone of the root was a characteristic feature of AA-treated roots. MYXO and AA co-treatment had an additive effect on the amount of both H2O2 and NO formed in roots. On the other hand, AA-induced superoxide formation was markedly reversed in roots co-treated with MYXO. Both AA and MYXO exacerbated the Cd-mediated H2O2 or NO generation in the root tip. On the contrary, while AA also exacerbated the Cd-induced superoxide generation, MYXO dose-dependently attenuated it. These data provide strong evidence that ROS generation, a very early symptom of Cd toxicity in roots, is originated in mitochondria. Cd, similarly to AA, generates superoxide by blocking the mitochondrial electron transport chain (ETC) at complex III. In turn, the site of Cd-induced NO generation is not associated with complex III, but ROS formed in mitochondria at this third complex of ETC are probably responsible for enhanced NO generation in barley root under Cd stress.


Assuntos
Antimicina A/metabolismo , Cádmio/metabolismo , Hordeum/química , Peróxido de Hidrogênio/metabolismo , Óxido Nítrico/metabolismo , Raízes de Plantas/química , Superóxidos/metabolismo , Metacrilatos/metabolismo , Tiazóis/metabolismo
4.
Mycotoxin Res ; 35(3): 293-307, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30949955

RESUMO

This study attempts to evaluate the potential aflatoxin binder activity of a molecularly imprinted polymer (TMU95) synthesized to target the aflatoxin B1 (AFB1) analog molecule in comparison to a commercial toxin binder (CTB). Adsorption experiments were carried out to assess the ability to bind to AFB1 at various pH values. The strength of binding was investigated by the chemisorption index. The isothermal analysis was used to determine the maximum adsorption capacity values. The ability of TMU95 and CTB to adsorb essential minerals was evaluated and the obtained data suggested that CTB would significantly reduce availability of them compared to TMU95. The in vivo efficacy of TMU95 as an aflatoxin (AF) binder in duckling exposed to aflatoxin-contaminated feed from 4 to 18 days of age in comparison to the CTB was also assessed. TMU95 and CTB were effective in reducing the adverse effects caused by AFs on feed conversion ratio of duckling (p ≤ 0.01), and also showed a minor reduction of injuries caused by AFs on visceral organs enlargement (p ≤ 0.01). It was concluded that TMU95 could absorb AFB1 in vitro efficiently and had beneficial health effects that could alleviate some of the toxic effects of AFs on growing duckling performance similar to CTB.


Assuntos
Aflatoxina B1/metabolismo , Ração Animal/análise , Metacrilatos/metabolismo , Polímeros/química , Adsorção , Aflatoxina B1/toxicidade , Ração Animal/toxicidade , Animais , Patos , Contaminação de Alimentos , Concentração de Íons de Hidrogênio , Cinética , Trato Gastrointestinal Inferior/efeitos dos fármacos , Trato Gastrointestinal Inferior/patologia , Baço/efeitos dos fármacos , Baço/patologia
5.
Acta Pharmacol Sin ; 40(8): 1106-1118, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30792487

RESUMO

Cinnamic acid and its analogues (pyragrel and ozagrel) undergo chain-shortened (ß-oxidative) and reductive metabolism on acyl side chain. In this study, we characterized the ß-oxidative and reductive metabolism on acyl side chain of cinnamic acid and its analogues using primary rat hepatocytes, hepatic mitochondrial, and microsomal systems. A compartmental model including parent compounds and metabolites was developed to characterize in vivo ß-oxidative and reductive metabolism following an intravenous dose of parent compounds to rats. The fitted total in vivo clearance values were further compared with the in vitro values predicted by the well-stirred model. We showed that hepatic microsomal CYP450s did not catalyze ß-oxidative or reductive metabolism of the three compounds. Similar to ß-oxidation of fatty acids, ß-oxidative metabolism on their acyl side chain occurred mainly in mitochondria, which was highly dependent on ATP, CoA and NAD+. Fatty acids and NADH inhibited the ß-oxidative metabolism. Reductive metabolism occurred in both mitochondria and microsomes. Reduction in mitochondria was ATP-, CoA-, and NAD(P)H-dependent and reversible, which was suppressed by enoyl reductase inhibitor triclosan. Reduction in microsomes was ATP-, CoA-, and NADPH-dependent but little affected by triclosan. Both plasma concentrations of ß-oxidative metabolites and reductive metabolites were successfully fitted using the compartmental model. The estimated total in vivo clearance values were consistent with those predicted from hepatocytes and organelles, implicating significance of in vitro kinetics. These findings demonstrate the roles of hepatic mitochondria and microsomes in ß-oxidative and reductive metabolism on acyl side chain of cinnamic acid and its analogues along with their metabolic characteristics.


Assuntos
Cinamatos/metabolismo , Metacrilatos/metabolismo , Pirazinas/metabolismo , Animais , Cinamatos/química , Cinamatos/farmacocinética , Ácidos Graxos/metabolismo , Hepatócitos/metabolismo , Masculino , Metacrilatos/química , Metacrilatos/farmacocinética , Microssomos Hepáticos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Estrutura Molecular , NAD/metabolismo , Oxirredução/efeitos dos fármacos , Pirazinas/química , Pirazinas/farmacocinética , Ratos Sprague-Dawley , Triclosan/farmacologia
6.
Physiol Res ; 67(Suppl 2): S293-S303, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30379551

RESUMO

In this review we summarize several synthetic approaches to the advanced synthesis of star-like polymer-based drug carriers. Moreover, their application as nanomedicines for therapy or the diagnosis of neoplastic diseases and their biodistribution are reviewed in detail. From a broad spectrum of star-like systems, we focus only on fully water-soluble systems, mainly based on poly(ethylene glycol) or N-(2-hydroxypropyl)methacrylamide polymer and copolymer arms and polyamidoamine dendrimers serving as the core of the star-like systems.


Assuntos
Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Polímeros/química , Polímeros/metabolismo , Animais , Portadores de Fármacos/administração & dosagem , Humanos , Metacrilatos/administração & dosagem , Metacrilatos/química , Metacrilatos/metabolismo , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/metabolismo , Polímeros/administração & dosagem , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologia
7.
Physiol Res ; 67(Suppl 2): S357-S365, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30379556

RESUMO

Novel star polymers based on the water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and cyclodextrin were synthesized and the physico-chemical behavior of these precursors was studied. Semitelechelic HPMA copolymers were grafted onto the cyclodextrin core, thus forming star-like structure. Both prepared systems were designed as possible polymer carriers for the controlled release of cytostatic drugs, which after the drug release and degradation will be eliminated from the organism. Two synthesis approaches were used to obtain similar polymer carriers with different degradation rates. All the polymers were prepared by reversible addition-fragmentation chain-transfer polymerization, which guarantees low dispersity of the prepared systems.


Assuntos
Química Farmacêutica/métodos , Ciclodextrinas/síntese química , Polímeros/síntese química , Água/química , Ciclodextrinas/metabolismo , Metacrilatos/síntese química , Metacrilatos/metabolismo , Polímeros/metabolismo , Solubilidade , Água/metabolismo
8.
Bioconjug Chem ; 29(7): 2181-2194, 2018 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-29712427

RESUMO

Methacrylate-based polymers represent promising nonviral gene delivery vectors, since they offer a large variety of polymer architectures and functionalities, which are beneficial for specific demands in gene delivery. In combination with controlled radical polymerization techniques, such as the reversible addition-fragmentation chain transfer polymerization, the synthesis of well-defined polymers is possible. In this study we prepared a library of defined linear polymers based on (2-aminoethyl)-methacrylate (AEMA), N-methyl-(2-aminoethyl)-methacrylate (MAEMA), and N,N-dimethyl-(2-aminoethyl)-methacrylate (DMAEMA) monomers, bearing pendant primary, secondary, and tertiary amino groups, and investigated the influence of the substitution pattern on their gene delivery capability. The polymers and the corresponding plasmid DNA complexes were investigated regarding their physicochemical characteristics, cytocompatibility, and transfection performance. The nonviral transfection by methacrylate-based polyplexes differs significantly from poly(ethylene imine)-based polyplexes, as a successful transfection is not affected by the buffer capacity. We observed that polyplexes containing a high content of primary amino groups (AEMA) offered the highest transfection efficiency, whereas polyplexes bearing tertiary amino groups (DMAEMA) exhibited the lowest transfection efficiency. Further insights into the uptake and release mechanisms could be identified by fluorescence and transmission electron microscopy, emphasizing the theory of membrane-pore formation for the time-efficient endosomal release of methacrylate-based vectors.


Assuntos
Técnicas de Transferência de Genes , Metacrilatos/metabolismo , Polímeros/metabolismo , Aminas/química , Membrana Celular/metabolismo , Endossomos/metabolismo , Humanos , Polimerização , Porosidade , Relação Estrutura-Atividade
9.
Chem Commun (Camb) ; 54(49): 6252-6255, 2018 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-29736504

RESUMO

Fluorescent unimolecular micelles (FUMs) with multicolor emission acting as fluorescent nanoagents for optical fluorescence imaging have, for the first time, been reported. The FUMs show good water-solubility, ultra-small size, and enhanced biocompatibility, which endow the FUMs with versatile applications including organelle labeling, multicolor markers and high tumor accumulation, revealing that our design can serve as a rational strategy for the development of UM-based fluorescent nanoagents for bioprocess monitoring.


Assuntos
Corantes Fluorescentes/metabolismo , Metacrilatos/metabolismo , Micelas , Neoplasias/diagnóstico por imagem , Polietilenoglicóis/metabolismo , beta-Ciclodextrinas/metabolismo , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Carbocianinas/síntese química , Carbocianinas/química , Carbocianinas/metabolismo , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Feminino , Fluoresceínas/síntese química , Fluoresceínas/química , Fluoresceínas/metabolismo , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Lisossomos/metabolismo , Metacrilatos/síntese química , Metacrilatos/química , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Tamanho da Partícula , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Rodaminas/síntese química , Rodaminas/química , Rodaminas/metabolismo , Solubilidade , beta-Ciclodextrinas/síntese química , beta-Ciclodextrinas/química
10.
J Control Release ; 282: 140-147, 2018 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-29518467

RESUMO

The clinical success of chimeric antigen receptor (CAR) T cell immunotherapy in treating multiple blood cancers has created a need for efficient methods of ex vivo gene delivery to primary human T cells for cell engineering. Here, we synthesize and evaluate a panel of cationic polymers for gene delivery to both cultured and primary human T cells. We show that a subset of comb- and sunflower-shaped pHEMA-g-pDMAEMA polymers can mediate transfection with efficiencies up to 50% in the Jurkat human T cell line with minimal concomitant toxicity (>90% viability). We then optimize primary human T cell transfection conditions including activation time, cell density, DNA dose, culture media, and cytokine treatment. We demonstrate transfection of both CD4+ and CD8+ primary human T cells with messenger RNA and plasmid DNA at efficiencies up to 25 and 18%, respectively, with similarly high viability.


Assuntos
DNA/administração & dosagem , Portadores de Fármacos/química , Metacrilatos/química , Nylons/química , Poli-Hidroxietil Metacrilato/química , RNA Mensageiro/administração & dosagem , Linfócitos T/metabolismo , Transfecção/métodos , Sobrevivência Celular/efeitos dos fármacos , DNA/genética , Portadores de Fármacos/metabolismo , Portadores de Fármacos/toxicidade , Humanos , Células Jurkat , Metacrilatos/metabolismo , Metacrilatos/toxicidade , Nylons/metabolismo , Nylons/toxicidade , Plasmídeos/administração & dosagem , Plasmídeos/genética , Poli-Hidroxietil Metacrilato/metabolismo , Poli-Hidroxietil Metacrilato/toxicidade , RNA Mensageiro/genética , Linfócitos T/efeitos dos fármacos
11.
Sci Rep ; 8(1): 2427, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29402945

RESUMO

Yeast Ndi1 is a monotopic alternative NADH dehydrogenase. Its crystal structure in complex with the electron acceptor, ubiquinone, has been determined. However, there has been controversy regarding the ubiquinone binding site. To address these points, we identified the first competitive inhibitor of Ndi1, stigmatellin, along with new mixed-type inhibitors, AC0-12 and myxothiazol, and thereby determined the crystal structures of Ndi1 in complexes with the inhibitors. Two separate binding sites of stigmatellin, STG-1 and STG-2, were observed. The electron density at STG-1, located at the vicinity of the FAD cofactor, further demonstrated two binding modes: STG-1a and STG-1b. AC0-12 and myxothiazol are also located at the vicinity of FAD. The comparison of the binding modes among stigmatellin at STG-1, AC0-12, and myxothiazol revealed a unique position for the aliphatic tail of stigmatellin at STG-1a. Mutations of amino acid residues that interact with this aliphatic tail at STG-1a reduced the affinity of Ndi1 for ubiquinone. In conclusion, the position of the aliphatic tail of stigmatellin at STG-1a provides a structural basis for its competitive inhibition of Ndi1. The inherent binding site of ubiquinone is suggested to overlap with STG-1a that is distinct from the binding site for NADH.


Assuntos
Coenzimas/química , Complexo I de Transporte de Elétrons/química , Flavina-Adenina Dinucleotídeo/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Ubiquinona/química , Sequência de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Coenzimas/metabolismo , Cristalografia por Raios X , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Expressão Gênica , Cinética , Metacrilatos/química , Metacrilatos/metabolismo , Modelos Moleculares , Mutação , Polienos/química , Polienos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termodinâmica , Tiazóis/química , Tiazóis/metabolismo , Ubiquinona/metabolismo
12.
Nucl Med Biol ; 58: 59-66, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29413458

RESUMO

BACKGROUND: For the evaluation of macromolecular drug delivery systems suitable pre-clinical monitoring of potential nanocarrier systems is needed. In this regard, both short-term as well as long-term in vivo tracking is crucial to understand structure-property relationships of polymer carrier systems and their resulting pharmacokinetic profile. Based on former studies revealing favorable in vivo characteristics for 18F-labeled random (ran) copolymers consisting of N-(2-hydroxypropyl)methacrylamide (HPMA) and lauryl methacrylate (LMA) - including prolonged plasma half-life as well as enhanced tumor accumulation - the presented work focuses on their long-term investigation in the living organism. METHODS: In this respect, four different HPMA-based polymers (homopolymers as well as random copolymers with LMA as hydrophobic segment) were synthesized and subsequent radioactive labeling was accomplished via the longer-lived radioisotope 131I. In vivo results, concentrating on the pharmacokinetics of a high molecular weight HPMA-ran-LMA copolymer, were obtained by means of biodistribution and metabolism studies in the Walker 256 mammary carcinoma model over a time-span of up to three days. Besides, a direct comparison with the 18F-radiolabeled polymer was drawn. To consider physico-chemical differences between the differently labeled polymer (18F or 131I) on the critical micelle concentration (CMC) and the size of the polymeric micelles, those properties were determined using the 19F- or 127I-functionalized polymer. Special emphasis was laid on the time-dependent correlation between blood circulation properties and corresponding tumor accumulation, particularly regarding the enhanced permeability and retention (EPR) effect. RESULTS: Studies revealed, at first, differences in the short time (2h) body distribution, despite the very similar properties (molecular structure, CMC and size of the micellar aggregates) of the non-radioactive 19F- and 127I-functionalized polymers. Long-term investigations with the 131I-labeled polymer demonstrated that, despite a polymer clearance from the blood within 72h, there was still an increase in tumor uptake observed over time. Regarding the stability of the 131I-label, ex vivo biodistribution experiments, considering the uptake in the thyroid, indicated low metabolism rates. CONCLUSION: The observed in vivo characteristics strongly underline the EPR effect. The findings illustrate the need to combine information of different labeling approaches and in vivo evaluation techniques to generate an overall pharmacokinetic picture of potential nanocarriers in the pre-clinical setting. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENTS: The in vivo behavior of the investigated HPMA-ran-LMA copolymer demonstrates great potential in terms of an effective accumulation in the tumor.


Assuntos
Radioisótopos do Iodo , Ácidos Láuricos/química , Ácidos Láuricos/farmacocinética , Metacrilatos/química , Metacrilatos/farmacocinética , Polímeros/química , Polímeros/farmacocinética , Animais , Linhagem Celular Tumoral , Marcação por Isótopo , Ácidos Láuricos/metabolismo , Metacrilatos/metabolismo , Polímeros/metabolismo , Ratos , Distribuição Tecidual
13.
Biochemistry ; 57(22): 3126-3129, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29293329

RESUMO

Incubation of (±)-2-methyl-3-ketobutyryl-SNAC (3) and (±)-2-methyl-3-ketopentanoyl-SNAC (4) with BonKR2 or OxaKR5, ketoreductase domains from the bongkrekic acid (1) and oxazolomycin (2) polyketide synthases, in the presence of NADPH gave in each case the corresponding (2 R,3 S)-2-methyl-3-hydroxybutyryl-SNAC (5) or (2 R,3 S)-2-methyl-3-hydroxypentanoyl-SNAC (6) products, as established by chiral gas chromatography-mass spectrometry analysis of the derived methyl esters. Identical results were obtained by BonKR2- and OxaKR5-catalyzed reduction of chemoenzymatically prepared (2 R)-2-methyl-3-ketopentanoyl-EryACP6, (2 R)-2-methyl-3-ketobutyryl-BonACP2 (12), and (2 R)-2-methyl-3-ketopentanoyl-BonACP2 (13). The paired dehydratase domains, BonDH2 and OxaDH5, were then shown to catalyze the reversible syn dehydration of (2 R,3 S)-2-methyl-3-hydroxybutyryl-BonACP2 (14) to give the corresponding trisubstituted ( Z)-2-methylbutenoyl-BonACP2 (16).


Assuntos
Hidroliases/fisiologia , Policetídeo Sintases/química , Proteínas de Bactérias/química , Biocatálise , Hidroliases/química , Metacrilatos/metabolismo , NADP/metabolismo , Policetídeo Sintases/fisiologia , Estereoisomerismo , Especificidade por Substrato/fisiologia
14.
Mol Cancer Ther ; 16(12): 2701-2710, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28830983

RESUMO

Polymeric nanocarriers such as N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers deliver drugs to solid tumors and avoid the systemic toxicity of conventional chemotherapy. Because HPMA copolymers can target sites of inflammation and accumulate within innate immune cells, we hypothesized that HPMA copolymers could target tumor-associated macrophages (TAM) in both primary and metastatic tumor microenvironments. We verified this hypothesis, first in preliminary experiments with isolated bone marrow macrophage cultures in vitro and subsequently in a spontaneously metastatic murine breast cancer model generated from a well-established, cytogenetically characterized 4T1 breast cancer cell line. Using our standardized experimental conditions, we detected primary orthotopic tumor growth at 7 days and metastatic tumors at 28 days after orthotopic transplantation of 4T1 cells into the mammary fat pad. We investigated the uptake of HPMA copolymer conjugated with Alexa Fluor 647 and folic acid (P-Alexa647-FA) and HPMA copolymer conjugated with IRDye 800CW (P-IRDye), following their retroorbital injection into the primary and metastatic tumor-bearing mice. A significant uptake of P-IRDye was observed at all primary and metastatic tumor sites in these mice, and the P-Alexa647-FA signal was found specifically within CD11b+ TAMs costained with pan-macrophage marker CD68. These findings demonstrate, for the first time, a novel capacity of a P-Alexa647-FA conjugate to colocalize to CD11b+CD68+ TAMs in both primary and metastatic breast tumors. This underscores the potential of this HPMA nanocarrier to deliver functional therapeutics that specifically target tumor-promoting macrophage activation and/or polarization during tumor development. Mol Cancer Ther; 16(12); 2701-10. ©2017 AACR.


Assuntos
Neoplasias da Mama/genética , Macrófagos/metabolismo , Metacrilatos/metabolismo , Animais , Neoplasias da Mama/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Metástase Neoplásica , Polímeros
15.
J Drug Target ; 25(9-10): 818-828, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28728446

RESUMO

'Polymer Enzyme Liposome Therapy' (PELT) is a two-step anticancer approach in which a liposomal drug and polymer-phospholipase conjugate are administered sequentially to target the tumour interstitium by the enhanced permeability and retention effect, and trigger rapid, local, drug release. To date, however, the concept has only been described theoretically. We synthesised two polymer conjugates of phospholipase C (PLC) and A2 (PLA2) and evaluated their ability to trigger anthracycline release from the clinically used liposomes, Caelyx® and DaunoXome®. N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-PLC and a dextrin-PLA2 were synthesised and their enzymatic activity characterised. Doxorubicin release from polyethyleneglycol-coated (PEGylated) Caelyx® was relatively slow (<20%, 60 min), whereas daunomycin was rapidly released from non-PEGylated DaunoXome® (∼87%) by both enzymes. Incubation with dextrin-PLA2 triggered significantly less daunomycin release than HPMA copolymer-PLC, but when dextrin-PLA2 was pre-incubated with α-amylase, the rate of daunomycin release increased. DaunoXome®'s diameter increased in the presence of PLA2, while Caelyx®'s diameter was unaffected by free or conjugated PLA2. Dextrin-PLA2 potentiated the cytotoxicity of DaunoXome® to MCF-7 cells to a greater extent than free PLA2, while combining dextrin-PLA2 with Caelyx® resulted in antagonism, even in the presence of α-amylase, presumably due to steric hindrance by PEG. Our findings suggest that in vivo studies to evaluate PELT combinations should be further evaluated.


Assuntos
Dextrinas/metabolismo , Doxorrubicina/análogos & derivados , Metacrilatos/metabolismo , Fosfolipases A2/metabolismo , Polímeros/metabolismo , Fosfolipases Tipo C/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Dextrinas/administração & dosagem , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Doxorrubicina/metabolismo , Humanos , Lipossomos , Células MCF-7 , Metacrilatos/administração & dosagem , Fosfolipases A2/administração & dosagem , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/metabolismo , Polímeros/administração & dosagem , Fosfolipases Tipo C/administração & dosagem
16.
Colloids Surf B Biointerfaces ; 156: 38-43, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28500977

RESUMO

Liposomes are generally used as delivery systems, as they are capable of encapsulating a wide variety of molecules (i.e. plasmids, recombinant proteins, therapeutic drugs). However, liposomal drug delivery have to fulfill different requirements, such as the effective internalization by the target cells and avoidance of the degradative activity of the intracellular compartments. The use of polymer lipid complexes (PLCs), by including different polymers in the liposome formulation, could improve internalization and intracellular release of drugs. The aim of the present work is to study the mechanisms of cellular uptaking and the intracellular trafficking of PLCs formed with cholesterol-poly(2-(dimethylamino)ethyl methacrylate) CHO-PDMAEMA and lecithin (LC CHO-PD). Calcein-loaded liposomes were used to determine cellular uptake and intracellular localization by flow cytometry and confocal microscopy. Incorporation of CHO-PDMAEMA to lecithin liposomes enhanced the internalization capacity of PLCs. Internalization of PLCs by human epithelial-like cells (HEK-293) diminished at 4°C, suggesting uptake by endocytosis. PLCs showed no co-localization with acidic compartments after internalization. Experiments with endocytosis inhibitors and co-localization of liposomes and albumin, suggested the caveolae endocytic pathway as the most probable route for intracellular trafficking of PLCs. In this work, we demonstrated an efficient uptake of LC CHO-PDs by human epithelial-like cells (HEK-293) through the non-degradative caveolae endocytic pathway. The mode of internalization and the intracellular fate of liposomes under study, suggest a promising use of LC CHO-PDs as drug delivery systems.


Assuntos
Colesterol/metabolismo , Endocitose , Lipossomos , Metacrilatos/metabolismo , Nylons/metabolismo , Células HEK293 , Humanos
17.
J Pharm Pharmacol ; 69(8): 978-990, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28480594

RESUMO

OBJECTIVE: Uncontrolled rapid release of drugs can reduce their therapeutic efficacy and cause undesirable toxicity; however, controlled release from reservoir materials helps overcome this issue. The aims of this study were to determine the release profiles of ketoprofen and spironolactone from a pH-responsive self-assembling DPA-MPC-DPA triblock copolymer gel and elucidate underlying physiochemical properties. METHODS: Drug release profiles from DPA50 -MPC250 -DPA50 gel (pH 7.5), over 32 h (37 °C), were determined using UV-Vis spectroscopy. Nanoparticle size was measured by dynamic light scattering (DLS) and critical micelle concentration (CMC) by pyrene fluorescence. Polymer gel viscosity was examined via rheology, nanoparticle morphology investigated using scanning transmission electron microscopy (STEM) and the gel matrix observed using cryo-scanning electron microscopy (Cryo-SEM). KEY FINDINGS: DPA50 -MPC250 -DPA50 copolymer (15% w/v) formed a free-standing gel (pH 7.5) that controlled drug release relative to free drugs. The copolymer possessed a low CMC, nanoparticle size increased with copolymer concentration, and DLS data were consistent with STEM. The gel displayed thermostable viscosity at physiological temperatures, and the gel matrix was a nanostructured aggregation of smaller nanoparticles. CONCLUSIONS: The DPA50 -MPC250 -DPA50 copolymer gel could be used as a drug delivery system to provide the controlled drug release of ketoprofen and spironolactone.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Cetoprofeno/química , Metacrilatos/química , Nanoestruturas/química , Fosforilcolina/análogos & derivados , Ácidos Polimetacrílicos/química , Espironolactona/química , Preparações de Ação Retardada/química , Preparações de Ação Retardada/metabolismo , Liberação Controlada de Fármacos , Géis , Cetoprofeno/metabolismo , Metacrilatos/metabolismo , Fosforilcolina/química , Fosforilcolina/metabolismo , Ácidos Polimetacrílicos/metabolismo , Espironolactona/metabolismo
18.
Mol Pharm ; 14(5): 1405-1417, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28263073

RESUMO

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers have been studied as an efficient carrier for drug delivery and tumor imaging. However, as with many macromolecular platforms, the substantial accumulation of HPMA copolymer by the mononuclear phagocyte system (MPS)-associated tissues, such as the blood, liver, and spleen, has inhibited its clinical translation. Our laboratory is pursuing approaches to improve the diagnostic and radiotherapeutic effectiveness of HPMA copolymers by reducing the nontarget accumulation. Specifically, we have been investigating the use of a cathepsin S (Cat S)-cleavable peptidic linkers to degrade multiblock HPMA copolymers to increase MPS-associated tissue clearance. In this study, we further our investigation into this area by exploring the impact of copolymer block size on the biological performance of Cat S-degradable HPMA copolymers. Using a variety of in vitro and in vivo techniques, including dual labeling of the copolymer and peptide components, we investigated the constructs using HPAC pancreatic ductal adenocarcinoma models. The smaller copolymer block size (S-CMP) demonstrated significantly faster Cat S cleavage kinetics relative to the larger system (L-CMP). Confocal microscopy demonstrated that both constructs could be much more efficiently internalized by human monocyte-differentiated macrophage (hMDM) compared to HPAC cells. In the biodistribution studies, the multiblock copolymers with a smaller block size exhibited faster clearance and lower nontarget retention while still achieving good tumor targeting and retention. Based on the radioisotopic ratios, fragmentation and clearance of the copolymer constructs were higher in the liver compared to the spleen and tumor. Overall, these results indicate that block size plays an important role in the biological performance of Cat S-degradable polymeric constructs.


Assuntos
Catepsinas/química , Metacrilatos/química , Polímeros/química , Animais , Sistemas de Liberação de Medicamentos , Humanos , Metacrilatos/metabolismo , Camundongos , Microscopia Confocal , Neoplasias Pancreáticas/metabolismo , Polímeros/síntese química , Polímeros/metabolismo
19.
Curr Pharm Des ; 23(18): 2685-2694, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28201971

RESUMO

BACKGROUND: Light delivery in photodynamic therapy is a challenging issue in deep cancer treatment. To solve this problem, photosensitizers are conjugated to X-ray luminescent nanoparticles. When the complexes are stimulated by X-rays during radiotherapy, the nanoparticles generate light and activate the photosensitizers. METHOD: Core-shell molecularly imprinted polymers (MIPs) were prepared against mitoxantrone (MX) in which TiO2 nanoparticles were applied as a core, diacrylated polycaprolctone as a biodegradable cross-linker and methacrylic acid (MAA) or 4-vinylpyridin (4-VP) as the functional monomer. TiO2 was selected as a scintillator, MX as a photosensitizer and MIP as a drug delivery system in order to evaluate the possibility of using photodynamic therapy (PDT) during radiotherapy in the next studies. Binding properties of polymers and drug release profile were studied and the optimized MIP was characterized by SEM, TEM, EDS, FT-IR and XRD. Also, cytotoxicity and free radical production were also studied in vitro. RESULTS: Data indicated that MAA-based MIP had superior binding properties compared to its non-imprinted polymer (NIP) and higher imprinting factor value than MIP-4VP. Drug release experiments indicated higher MX released amount from MAA-based MIP than the other polymers. MAA-based MIP was selected as an optimized carrier for MX delivery system. According to the results, the size of MX-MIP@TiO2 was reported to be less than 75 nm. The free radical production and cytotoxicity of nanoparticles were also evaluated in vitro. CONCLUSION: The results of the present work proposed the possibility of applying MIP layer as a drug delivery system around TiO2 nanoparticles.


Assuntos
Sistemas de Liberação de Medicamentos , Metacrilatos/administração & dosagem , Mitoxantrona/administração & dosagem , Nanopartículas/administração & dosagem , Poliésteres/administração & dosagem , Titânio/administração & dosagem , Acrilatos/administração & dosagem , Acrilatos/química , Acrilatos/metabolismo , Linhagem Celular Tumoral , Radicais Livres/antagonistas & inibidores , Radicais Livres/metabolismo , Humanos , Metacrilatos/química , Metacrilatos/metabolismo , Mitoxantrona/química , Mitoxantrona/metabolismo , Nanopartículas/química , Nanopartículas/metabolismo , Poliésteres/química , Poliésteres/metabolismo , Titânio/química , Titânio/metabolismo , Difração de Raios X
20.
Regul Toxicol Pharmacol ; 84: 77-93, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28087335

RESUMO

All of the lower alkyl methacrylates are high production chemicals with potential for human exposure. The genotoxicity of seven mono-functional alkyl esters of methacrylic acid, i.e. methyl methacrylate, ethyl methacrylate, hydroxyethyl methacrylate, n-, i- and t-butyl methacrylate and 2 ethyl hexyl methacrylate, as well as methacrylic acid itself, the acyl component common to all, is reviewed and compared with the lack of carcinogenicity of methyl methacrylate, the representative member of the series so evaluated. Also reviewed are the similarity of structure, chemical and biological reactivity, metabolism and common metabolic products of this group of compounds which allows a category approach for assessing genotoxicity. As a class, the lower alkyl methacrylates are universally negative for gene mutations in prokaryotes but do exhibit high dose clastogenicity in mammalian cells in vitro. There is no convincing evidence that these compounds induce genotoxic effects in vivo in either sub-mammalian or mammalian species. This dichotomy of effects can be explained by the potential genotoxic intermediates generated in vitro. This genotoxic profile of the lower alkyl methacrylates is consistent with the lack of carcinogenicity of methyl methacrylate.


Assuntos
Dano ao DNA , Metacrilatos/toxicidade , Testes de Mutagenicidade/métodos , Animais , Biotransformação , Testes de Carcinogenicidade , Linhagem Celular , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/genética , Relação Dose-Resposta a Droga , Humanos , Metacrilatos/química , Metacrilatos/metabolismo , Estrutura Molecular , Mutagênese , Medição de Risco , Relação Estrutura-Atividade
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