RESUMO
For reactive oxygen species (ROS)-sensitive and CD44 receptor-mediated delivery of photosensitizers, chlorin e6 (ce6) tetramer was synthesized using tetra acid (TA) via selenocystamine linkages and then conjugated with hyaluronic acid (HA) (abbreviated as HAseseCe6TA). HAseseCe6TA nanophotosensitizers were fabricated by dialysis procedure. HAseseCe6TA nanophotosensitizers showed spherical morphology with small particle sizes less than 100 nm and monomodal pattern. When H2O2 was added, size distribution was changed to multimodal pattern and morphological observation showed disintegration of nanophotosensitizers, indicating that HAseseCe6TA nanophotosensitizers have ROS sensitivity. Furthermore, H2O2 addition resulted in acceleration of Ce6 release from HAseseCe6TA nanophotosensitizers. In vitro cell culture study, HAseseCe6TA nanophotosensitizers increase Ce6 uptake ratio, ROS production efficiency, and photodynamic therapy efficacy in both B16F10 cells and CT26 cells. Especially, CD44-receptor blocking of cancer cells by pretreatment of HA showed that fluorescence intensity in B16F10 cells was significantly decreased while fluorescence intensity in CT26 cells was not significantly changed, indicating that HAseseCe6TA nanophotosensitizers can be delivered by CD44 receptor-mediated pathway. In vivo animal tumor xenograft study, HAseseCe6TA nanophotosensitizers was selectively delivered to B16F10 tumor rather than CT26 tumor. These results indicated that HAseseCe6TA nanophotosensitizers have ROS sensitivity and have CD44 receptor-recognition properties.
Assuntos
Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/química , Nanopartículas/química , Neoplasias/tratamento farmacológico , Oxirredução/efeitos dos fármacos , Fármacos Fotossensibilizantes/química , Porfirinas/química , Animais , Linhagem Celular Tumoral , Clorofilídeos , Peróxido de Hidrogênio/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Tamanho da Partícula , Fotoquimioterapia/métodos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The main purpose of this study is to synthesize novel types of nanophotosensitizers that are based on hyperbranched chlorin e6 (Ce6) via disulfide linkages. Moreover, hyperbranched Ce6 was conjugated with hyaluronic acid (HA) for CD44-receptor mediated delivery and redox-sensitive photodynamic therapy (PDT) against cancer cells. Hyperbranched Ce6 was considered to make novel types of macromolecular photosensitizer since most of the previous studies regarding nanophotosensizers are concerned with simple conjugation between monomeric units of photosensitizer and polymer materials. Hyperbranched Ce6 was synthesized by conjugation of Ce6 each other while using disulfide linkage. To synthesize Ce6 tetramer, carboxyl groups of Ce6 were conjugated with cystamine and three equivalents of Ce6 were then conjugated again with the end of amine groups of Ce6-cystamine. To synthesize Ce6 decamer as a hyperbranched Ce6, six equivalents of Ce6 was conjugated with the end of Ce6 tetramer via cystamine linkage. Furthermore, HA-cystamine was attached with Ce6 tetramer or Ce6 decamer to synthesize HA-Ce6 tetramer (Ce6tetraHA) or HA-Ce6 decamer (Ce6decaHA) conjugates. Ce6tetraHA and Ce6decaHA nanophotosensitizers showed small diameters of less than 200 nm. The addition of dithiothreitol (DTT) and hyaluronidase (HAse) induced a faster Ce6 release rate in vitro drug release study, which indicated that Ce6tetraHA nanophotosensitizers possess redox-sensitive and HAse-sensitive release properties. Ce6tetraHA nanophotosensitizers showed higher intracellular Ce6 accumulation, higher ROS generation, and higher PDT efficacy than that of Ce6 alone. Ce6tetraHA nanophotosensitizers responded to the CD44 receptor of cancer cell surface, i.e., the pre-treatment of HA blocked CD44 receptor of U87MG or HCT116 cells and then inhibited delivery of nanophotosensitizers in vitro cell culture study. Furthermore, in vivo tumorxenograft study showed that fluorescence intensity in the tumor tissues was stronger than those of other organs, while CD44 receptor blocking by HA pretreatment induced a decrease of fluorescence intensity in tumor tissues when compared to liver. These results indicated that Ce6tetraHA nanophotosensitizers delivered to tumors by redox-sensitive and CD44-sensitive manner.
RESUMO
BACKGROUND: Nanoparticles have been extensively investigated for targeted delivery of anticancer drugs. Since the folate receptor is universally over-expressed on the tumor cell membrane, folic acid is often used to modify the fate of nanoparticles in biologicals. METHODS: To fabricate targetable nanoparticles, folic acid was conjugated to a pullulan backbone and poly(DL-lactide-co-glycolide) (PLGA) (abbreviated as FAPuLG) was conjugated. KB cells and NIH3T3-cell-bearing mice were prepared to prove folate receptor targeting of FAPuLG nanoparticles. RESULTS AND DISCUSSION: Nanoparticles of FAPuLG copolymer that self-assembled in water were small with diameters <200 nm. Doxorubicin (DOX) as a model drug was incorporated into the FAPuLG nanoparticles that were used to treat folate receptor over-expressing KB human carcinoma cells. Fluorescence microscopy revealed that DOX-incorporated FAPuLG nanoparticles induced strong red fluorescence in the KB cells in the absence of folic acid. However, fluorescence intensity was decreased by blocking folate receptors. Antitumor activity of FAPuLG nanoparticles against KB cells in vitro was also decreased by blocking folate receptors. In animal study using near-infrared dye-conjugated FAPuLG nanoparticles, fluorescence intensity was significantly higher at KB solid tumor than that of NIH3T3. CONCLUSIONS: The results indicate that FAPuLG nanoparticles can target the folate receptor of tumor cells. FAPuLG nanoparticles are a promising candidate for active targeting of anticancer agents.
RESUMO
This study investigates the use of a new biocompatible block copolymer poly(2-(dimethylamino)ethyl methacrylate-N-(morpholino)ethyl methacrylate (PDMAEMA-b-PMEMA) for the delivery of a particular antisense oligonucleotide targeting E6 gene from human papilloma virus. This antisense oligonucleotide was derivatized with a polyazaaromatic Ru(II) complex which, under visible illumination, is able to produce an irreversible crosslink with the complementary targeted sequence. The purpose of this study is to determine whether by the use of a suitable transfection agent, it is possible to increase the efficiency of the antisense oligonucleotide targeting E6 gene, named Ru-P-4. In a recent study, we showed that Oligofectamine transfected Ru-P-4 antisense oligonucleotide failed to inhibit efficiently the growth of cervical cancer cell line SiHa, contrarily to the Ru-P-6 antisense oligonucleotide, another sequence also targeting the E6 gene. The ability of PDMAEMA-b-PMEMA to form polyplexes with optimal physicochemical characteristics was investigated first. Then the ability of the PDMAEMA-b-PMEMA/Ru-P-4 antisense oligonucleotide polyplexes to transfect two keratinocyte cell lines (SiHa and HaCat) and the capacity of polyplexes to inhibit HPV16+ cervical cancer cell growth was evaluated. PDMAEMA-b-PMEMA base polyplexes at the optimal molar ratio of polymer nitrogen atoms to DNA phosphates (N/P), were able to deliver Ru-P-4 antisense oligonucleotide and to induce a higher growth inhibition in human cervical cancer SiHa cells, compared to other formulations based on Oligofectamine.
Assuntos
Sistemas de Liberação de Medicamentos , Metilmetacrilatos/química , Morfolinos/química , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos/administração & dosagem , Proteínas Oncogênicas Virais/genética , Proteínas Repressoras/genética , Rutênio/química , Células Cultivadas , Feminino , Terapia Genética/instrumentação , Terapia Genética/métodos , Papillomavirus Humano 16/efeitos dos fármacos , Papillomavirus Humano 16/genética , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/virologia , Teste de Materiais , Proteínas Oncogênicas Virais/antagonistas & inibidores , Interferência de RNA , Proteínas Repressoras/antagonistas & inibidores , Transfecção/instrumentação , Transfecção/métodosRESUMO
Skin carcinomas are among the most commonly diagnosed tumors in the world. In this study, we investigated the transfection of immortalized keratinocytes, used as an in vitro model for skin carcinoma, using the antisense technology and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based copolymers. In order to improve the transfection efficiency of the classic PDMAEMA polymers, copolymers were synthesized including a poly(N-morpholino)ethylmethacrylate) (PMEMA) moiety for an improved proton-sponge effect, intended to favour the release of the oligonucleotide from the acidic endosome. These copolymers were synthesized either statistically (with alternating PDMAEMA and PMEMA fragments) or in blocks (one PDMAEMA block followed by one PMEMA block). MTT assays were performed using the PDMAEMA-PMEMA copolymers and revealed no significant cytotoxicity of these polymers at an N/P ratio of 7.3. Using fluorescent oligonucleotides and analyzing transfection efficiency by flow cytometry, we noticed no significant differences between the two kinds of copolymers. However copolymers with a higher DMAEMA content and a higher Mn were also those displaying the highest vectorization efficiency. Confocal microscopy showed that these copolymers induced a fine granular distribution of the transfected antisense oligonucleotides inside the cells. We also assessed the functionality of the transfected antisense oligonucleotide by transfecting immortalized GFP expressing keratinocytes with a GFP antisense oligonucleotide using these copolymers. A significant silencing was achieved with a PDMAEMA-PMEMA in block copolymer (Mn=41,000, 89 % PDMAEMA). Together, these results suggest that PDMAEMA-PMEMA copolymers combining low toxicity, vectorization and proton sponge properties, can be efficiently used to transfect immortalized keratinocytes and so open new perspectives in the therapy of skin carcinomas as well as of other skin diseases of genetic or immunological origin.
Assuntos
Materiais Biocompatíveis/química , Queratinócitos/efeitos dos fármacos , Metacrilatos/química , Nylons/química , Polímeros/química , Materiais Biocompatíveis/farmacologia , Células Cultivadas , Endossomos/efeitos dos fármacos , Endossomos/genética , Endossomos/metabolismo , Técnicas de Transferência de Genes , Humanos , Queratinócitos/metabolismo , Metacrilatos/farmacologia , Nylons/farmacologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Transfecção/métodosRESUMO
Sorafenib-incoporated nanoparticles were prepared using a block copolymer that is composed of dextran and poly(DL-lactide-co-glycolide) [DexbLG] for antitumor drug delivery. Sorafenib-incorporated nanoparticles were prepared by a nanoprecipitation-dialysis method. Sorafenib-incorporated DexbLG nanoparticles were uniformly distributed in an aqueous solution regardless of the content of sorafenib. Transmission electron microscopy of the sorafenib-incorporated DexbLG nanoparticles revealed a spherical shape with a diameter < 300 nm. Sorafenib-incorporated DexbLG nanoparticles at a polymer/drug weight ratio of 40:5 showed a relatively uniform size and morphology. Higher initial drug feeding was associated with increased drug content in nanoparticles and in nanoparticle size. A drug release study revealed a decreased drug release rate with increasing drug content. In an in vitro anti-proliferation assay using human cholangiocarcinoma cells, sorafenib-incorporated DexbLG nanoparticles showed a similar antitumor activity as sorafenib. Sorafenib-incorporated DexbLG nanoparticles are promising candidates as vehicles for antitumor drug targeting.
RESUMO
Diblock copolymers composed of poly(epsilon-caprolactone) (PCL) and poly(N,N-dimethylamino-2-ethyl methacrylate) (PDMAEMA), or methoxy polyethylene glycol(PEG), were synthesized via a combination of ring-opening polymerization and atom-transfer radical polymerization in order to prepare polymeric nanoparticles as an antifungal drug carrier. Amphotericin B (AmB), a natural antibiotic, was incorporated into the polymeric nanoparticles. The physical properties of AmB-incorporated polymeric nanoparticles with PCL-b-PDMAEMA and PCL-b-PEG were studied in relation to morphology and particle size. In the aggregation state study, AmB-incorporated PCL-b- PDMAEMA nanoparticles exhibited a monomeric state pattern of free AmB, whereas AmB-incorporated PCL-b- PEG nanoparticles displayed an aggregated pattern. In in vitro hemolysis tests with human red blood cells, AmBincorporated PCL-b-PDMAEMA nanoparticles were seen to be 10 times less cytotoxic than free AmB (5 microgram/ml). In addition, an improved antifungal activity of AmBincorporated polymeric nanoparticles was observed through antifungal activity tests using Candida albicans, whereas polymeric nanoparticles themselves were seen not to affect activity. Finally, in vitro AmB release studies were conducted, proving the potential of AmB-incorporated PCL-b-PDMAEMA nanoparticles as a new formulation candidate for AmB.
Assuntos
Anfotericina B/química , Antifúngicos/química , Portadores de Fármacos/síntese química , Metacrilatos/síntese química , Nanopartículas/química , Nylons/síntese química , Poliésteres/síntese química , Polímeros/síntese química , Anfotericina B/farmacologia , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Portadores de Fármacos/química , Hemólise/efeitos dos fármacos , Humanos , Metacrilatos/química , Nylons/química , Poliésteres/química , Polimerização , Polímeros/químicaRESUMO
Two different and well-defined methacrylate-based (co)polymers were employed as a polymeric siRNA delivery system. siRNA, poly(2-(dimethylamino) ethyl methacrylate) homopolymers (PDMAEMA) and poly(2-(dimethylamino) ethyl methacrylate)-b-poly (ethyleneglycol) alpha-methoxy, omega-methacrylate (PDMAEMA-b-PMAPEG) palm-tree-like copolymer ternary complexes were prepared using a rapid and simple two-step mixing protocol involving noncovalent post-PEGylation, and physicochemical properties including hydrodynamic diameter, zeta-potential and siRNA condensation efficiency were characterized. Transfection efficiency, intracellular uptake, and cytotoxicity of ternary complexes were also evaluated. Ternary complexes provide efficient condensation and compaction of siRNA within the cationic core of complexes. Noncovalent post-PEGylation provides the ternary complexes with enzymatic and serum stability without harming complex formation and condensation of siRNA. Thereby, under an optimal N/P ratio, ternary complexes exhibited brilliant gene silencing efficiency with low cytotoxicity in media containing 10% serum. Confocal microscopy clearly showed efficient and even intracellular uptake of complexes by cells via endocytosis. This study highlights the excellent properties of noncovalent post-PEGylated ternary complexes produced by rapid and simple mixing. Accordingly, these findings suggest that the formation of ternary complexes could be utilized as a safe and effective polymeric siRNA delivery strategy.
Assuntos
Resinas Acrílicas/química , Portadores de Fármacos/química , Metacrilatos/química , Nylons/química , Polietilenoglicóis/química , RNA Interferente Pequeno/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Estabilidade de Medicamentos , Inativação Gênica/efeitos dos fármacos , Humanos , Masculino , Microscopia Confocal , Modelos Moleculares , Tamanho da Partícula , RNA Interferente Pequeno/genética , Transfecção , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Hederacolchiside A1 was used to progressively permeabilize the membrane of human melanoma MEL-5 cells. Holes formation was followed by Scanning Electron Microscopy and interaction of the saponin with cholesterol and phospholipids by TOF-SIMS. 2D-LC-MS/MS and 2D-SDS-PAGE show that the release of soluble proteins into serum-free culture media increases with time. This can lead to a new rapid and efficient strategy to analyze the cytosolic subproteome and it opens the door to get information from the cytosolic compartment for clinical proteomic studies.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Proteínas/análise , Proteômica/métodos , Saponinas/farmacologia , Animais , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/ultraestrutura , Forma Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Colesterol/análise , Cromatografia Líquida de Alta Pressão/métodos , Citosol/metabolismo , Eletroforese em Gel Bidimensional , Espaço Extracelular/química , Hemólise/efeitos dos fármacos , Humanos , Hidroliases/análise , Micelas , Microscopia Eletrônica de Varredura , Microscopia de Contraste de Fase , Estrutura Molecular , Fosfolipídeos/análise , Saponinas/química , Ovinos , Espectrometria de Massa de Íon Secundário , Espectrometria de Massas em TandemRESUMO
Non-covalently molecularly imprinted polymers (MIPs) for beta2-amino acids were prepared for the first time. N-(2-chlorobenzyloxycarbonyl)-(R)-beta2-homophenylalanine (N-2-ClZ-(R)-beta2-HPhe) was imprinted with methacrylic acid (MAA) and/or 4-vinylpyridine (4-VPy) as the functional monomers, with ethylene glycol dimethacrylate (EDMA) as the cross-linker. The MIPs made with different ratios of MAA:4-VPy were studied in HPLC mode. The results show that MIPs made with 4-VPy yielded the best chiral separation factor (alpha= 1.86) for the template molecule. The importance for an efficient separation of pi-stacking interactions between the MIPs and the template molecule is demonstrated. Racemates of Z-alpha-amino acids and beta-amino acid analogues of the template were either not or poorly resolved by the MIPs, thus demonstrating the close three-dimensional complementarity of the MIPs' recognition sites with the template.
Assuntos
Aminoácidos/isolamento & purificação , Animais , Cromatografia Líquida de Alta Pressão/métodos , Polímeros , EstereoisomerismoRESUMO
The binding affinity for a 12-bp dsDNA of Antennapedia helix 3 analogues, major groove binders, has been measured by displacement of prebound ethidium bromide, a fluorescent displacement assay proposed for minor groove binders by Boger et al.(J. Am. Chem. Soc., 2000, 122, 6382-6394). Relative binding affinities determined by this method were compared to those obtained by gel mobility shift and footprinting assays for the 12-bp dsDNA and a 178-bp DNA fragment. The present work demonstrates that the fluorescence displacement assay is suitable for rapid screening of major groove binders, even though about 60 to 70% of the prebound ethidium bromide is displaced by these peptides. Total (100%) displacement of ethidium bromide was serendipitously achieved by addition in the peptide sequence, at the N-terminus, of a S-3-nitro-2-pyridinesulfenyl-N-acetyl-cysteine residue. S-3-nitro-2-pyridinesulfenylcysteine was shown to (i) bind to dsDNA with a micromolar affinity and (ii) direct within DNA grooves a peptide with no affinity for dsDNA.