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1.
Mol Ther ; 22(2): 397-408, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24145554

RESUMO

Among various proinflammatory cytokines involved in the pathogenesis of rheumatoid arthritis (RA), tumor necrosis factor (TNF)-α plays a pivotal role in the release of other cytokines and induction of chronic inflammation. Even though siRNA has the therapeutic potential, they have a challenge to be delivered into the target cells because of their poor stability in physiological fluids. Herein, we design a nanocomplex of polymerized siRNA (poly-siRNA) targeting TNF-α with thiolated glycol chitosan (tGC) polymers for the treatment of RA. Poly-siRNA is prepared through self-polymerization of thiol groups at the 5' end of sense and antisense strand of siRNA and encapsulated into tGC polymers, resulting in poly-siRNA-tGC nanoparticles (psi-tGC-NPs) with an average diameter of 370 nm. In the macrophage culture system, psi-tGC-NPs exhibit rapid cellular uptake and excellent in vitro TNF-α gene silencing efficacy. Importantly, psi-tGC-NPs show the high accumulation at the arthritic joint sites in collagen-induced arthritis (CIA) mice. Treatment monitoring data obtained by the matrix metalloproteinase 3-specific nanoprobe and microcomputed tomography show that intravenous injection of psi-tGC-NPs significantly inhibits inflammation and bone erosion in CIA mice, comparable to methotrexate (5 mg/kg). Therefore, the availability of psi-tGC-NP therapy that target specific cytokines may herald new era in the treatment of RA.


Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/terapia , Quitosana , Inativação Gênica , Nanopartículas , RNA Interferente Pequeno/genética , Compostos de Sulfidrila , Fator de Necrose Tumoral alfa/genética , Animais , Artrite Experimental , Artrite Reumatoide/patologia , Linhagem Celular , Quitosana/química , Modelos Animais de Doenças , Expressão Gênica , Macrófagos/metabolismo , Masculino , Camundongos , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Compostos de Sulfidrila/química , Resultado do Tratamento , Fator de Necrose Tumoral alfa/sangue
2.
Acc Chem Res ; 44(10): 1018-28, 2011 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-21851104

RESUMO

Therapy and diagnosis are two major categories in the clinical treatment of disease. Recently, the word "theranosis" has been created, combining the words to describe the implementation of these two distinct pursuits simultaneously. For successful theranosis, the efficient delivery of imaging agents and drugs is critical to provide sufficient imaging signal or drug concentration in the targeted disease site. To achieve this purpose, biomedical researchers have developed various nanoparticles composed of organic or inorganic materials. However, the targeted delivery of these nanoparticles in animal models and patients remains a difficult hurdle for many researchers, even if they show useful properties in cell culture condition. In this Account, we review our strategies for developing theranostic nanoparticles to accomplish in vivo targeted delivery of imaging agents and drugs. By applying these rational strategies, we achieved fine multimodal imaging and successful therapy. Our first strategy involves physicochemical optimization of nanoparticles for long circulation and an enhanced permeation and retention (EPR) effect. We accomplished this result by testing various materials in mouse models and optimizing the physical properties of the materials with imaging techniques. Through these experiments, we developed a glycol chitosan nanoparticle (CNP), which is suitable for angiogenic diseases, such as cancers, even without an additional targeting moiety. The in vivo mechanism of this particle was examined through rationally designed experiments. In addition, we evaluated and compared the biodistribution and target-site accumulation of bare and drug-loaded nanoparticles. We then focus on the targeting moieties that bind to cell surface receptors. Small peptides were selected as targeting moieties because of their stability, low cost, size, and activity per unit mass. Through phage display screening, the interleukin-4 receptor binding peptide was discovered, and we combined it with our nanoparticles. This product accumulated efficiently in atherosclerotic regions or tumors during both imaging and therapy. We also developed hyaluronic acid nanoparticles that can bind efficiently to the CD44 antigen receptors abundant in many tumor cells. Their delivery mechanism is based on both physicochemical optimization for the EPR effect and receptor-mediated endocytosis by their hyaluronic acid backbone. Finally, we introduce the stimuli-responsive system related to the chemical and biological changes in the target disease site. Considering the relatively low pH in tumors and ischemic sites, we applied pH-sensitive micelle to optical imaging, magnetic resonance imaging, anticancer drug delivery, and photodynamic therapy. In addition, we successfully evaluated the in vivo imaging of enzyme activity at the target site with an enzyme-specific peptide sequence and CNPs. On the basis of these strategies, we were able to develop self-assembled nanoparticles for in vivo targeted delivery, and successful results were obtained with them in animal models for both imaging and therapy. We anticipate that these rational strategies, as well as our nanoparticles, will be applied in both the diagnosis and therapy of many human diseases. These theranostic nanoparticles are expected to greatly contribute to optimized therapy for individual patients as personalized medicine, in the near future.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/uso terapêutico , Animais , Fenômenos Químicos , Humanos , Injeções Intravenosas , Nanopartículas/administração & dosagem , Nanopartículas/química , Receptores de Superfície Celular/metabolismo
3.
Mol Pharm ; 9(5): 1118-25, 2012 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-22376145

RESUMO

Immunostimulatory sequences (ISS) are short DNA sequences containing unmethylated CpG dimers that have multiple effects on the host immune system, including the ability to stimulate antigen-specific cytotoxic T lymphocytes (CTLs) and drive Th1-type immune responses. Listeriolysin O (LLO)-containing pH-sensitive liposomes have been shown to efficiently deliver macromolecules to the cytosol of APCs and efficiently stimulate CTLs. We hypothesized that encapsulating ISS-oligodeoxyribonucleotides (ODNs) in this delivery system would enhance the cell-mediated immune response and skew Th1-type responses in protein antigen-based vaccination utilizing LLO-liposomes. In vitro studies indicated that coencapsulation of ISS in LLO-liposomes engendered activation of the NF-κB pathway while maintaining the efficient cytosolic delivery of antigen mediated by the coencapsulated LLO. Antigen-specific CTL responses monitored by using the model antigen ovalbumin (OVA) in mice were enhanced when mice were immunized with OVA and ISS-ODN-containing LLO-liposomes compared with those immunized with OVA-containing LLO-liposomes. The enhanced immune responses were of the Th1-type as monitored by the robust OVA-specific IgG2a induction and the OVA CD8 peptide-stimulated IFN-γ secretion. Our study suggests that including ISS-ODN in LLO-containing pH-sensitive liposomes yields a vaccine delivery system that enhances the cell-mediated immune response and skews this response toward the Th1-type.


Assuntos
Toxinas Bacterianas/química , Proteínas de Choque Térmico/química , Proteínas Hemolisinas/química , Lipossomos/química , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Interferon gama , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/metabolismo
4.
Angew Chem Int Ed Engl ; 51(29): 7203-7, 2012 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-22696263

RESUMO

The condensed version: Thiolated glycol chitosan can form stable nanoparticles with polymerized siRNAs through charge-charge interactions and self-cross-linking (see scheme). This poly-siRNA/glycol chitosan nanoparticles (psi-TGC) provided sufficient in vivo stability for systemic delivery of siRNAs. Knockdown of tumor proteins by psi-TGC resulted in a reduction in tumor size and vascularization.


Assuntos
Quitosana/química , Nanopartículas/química , Neoplasias/terapia , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/uso terapêutico , Animais , Linhagem Celular Tumoral , Terapia Genética , Humanos , Camundongos , Neoplasias/genética , Neoplasias/patologia , RNA Interferente Pequeno/genética , Compostos de Sulfidrila/química , Fator A de Crescimento do Endotélio Vascular/genética
5.
Top Curr Chem (Cham) ; 375(2): 31, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28251564

RESUMO

Nanoparticles based on nanotechnology and biotechnology have emerged as efficient carriers for various biopharmaceutical agents including proteins and genes. In particular, polysaccharides have attracted interest of many researchers in the drug delivery field due to their advantages such as biocompatibility, biodegradability, low toxicity, and ease of modification. A number of polysaccharides including chitosan, hyaluronic acid, and dextran, and their derivatives have been widely used as polymeric backbones for the formation of nanoparticles, which can be provided as valuable gene delivery carriers. In this review, we introduce the chemical and physical natures of different polysaccharides particularly used in biomedical applications, and then discuss recent progress in the development of polysaccharide-based nanoparticles for gene delivery.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética , Nanopartículas/química , Polissacarídeos/química , Tecnologia Biomédica , Vetores Genéticos/química , Humanos
6.
Biomaterials ; 34(37): 9475-85, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24050874

RESUMO

The safe and effective systemic delivery of siRNA is a prerequisite for the successful development of siRNA-based cancer therapeutics. For the enhanced delivery of siRNA, cationic lipids and polymers have been widely used as siRNA carriers to form electrolyte complexes with anionic siRNA. However, the considerable toxicity of strong cationic-charged molecules hampers their clinical use. In this study, we utilized human serum albumin (HSA), which is the most abundant of the plasma proteins, as a siRNA carrier for systemic tumor-targeted siRNA delivery. Both HSA and siRNA molecules were thiol-introduced to improve the binding affinity for each other. The resulting thiolated HSA (tHSA) and polymerized siRNA (psi) formed stable nanosized complexes (psi-tHSAs) by chemical crosslinking and self-crosslinking. After internalization, the psi-tHSAs showed target gene silencing activity in vitro comparable to conventional Lipofectamine™-siRNA complexes, without remarkable cytotoxicity. After intravenous injection in tumor-bearing mice, psi-tHSAs accumulated specifically at the tumor sites, leading to efficient gene silencing in the tumors in a sequential manner. The therapeutic VEGF siRNA was loaded into psi-tHSAs, which significantly inhibited tumor-related angiogenesis in PC-3 tumor xenografts and resulted in retarding the growth of PC-3 tumors. The results showed that self-crosslinked psi-tHSA nanocarriers might provide a promising approach for the systemic siRNA therapy of various human cancers.


Assuntos
Portadores de Fármacos/química , Neoplasias/terapia , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Albumina Sérica/química , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Neoplasias/irrigação sanguínea , Neoplasias/genética , Neovascularização Patológica/genética , Neovascularização Patológica/terapia , Polimerização , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Compostos de Sulfidrila/química , Fator A de Crescimento do Endotélio Vascular/genética
7.
Theranostics ; 2(2): 198-206, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22375158

RESUMO

PURPOSE: A fluorogenic peptide probe-immobilized diagnostic kit was used to analyze MMP activity in the synovial fluids (SFs) from patients with osteoarthritis (OA) and acute inflammatory conditions of the knee joint. METHODS: The MMP diagnostic kit containing a polymer-conjugated MMP probe immobilized on a 96-well plate was utilized for high-throughput screening of MMP activity in SFs from OA patients (n = 33) and patients with acute inflammatory conditions of the knee joint (n = 5). RESULTS: Compared to SF from OA patients, SF from patients with acute inflammatory conditions of the knee joint presented stronger NIR fluorescent signals. In gelatin zymography, most samples from patients with acute inflammatory conditions of the knee joint also displayed 92 kDa (pro-form) MMP-9 and faint 84 kDa (active form) MMP-9, while SF from OA patients did not display detectable MMP-9 activity . CONCLUSION: The presence of a strong fluorescence signal from the MMP diagnostic kit corresponded well with patients with acute inflammatory conditions of the knee joint. The results suggest that our MMP diagnostic kit can be useful in differentiation between early stages of OA and acute inflammatory conditions of the knee joint.

8.
Biomaterials ; 33(12): 3485-93, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22322197

RESUMO

The purpose of this study is to determine the correlation between the distribution of nanoparticles in the vitreous and retina and their surface properties after intravitreal injection. For this purpose, we synthesized seven kinds of nanoparticles through self-assembly of amphiphilic polymer conjugates in aqueous condition. They showed similar size but different surface properties. They were labeled with fluorescent dyes for efficient tracking. After intravitreal injection of these nanoparticles into a rodent eye, their time-dependent distribution in the vitreous and retina was determined in stacking tissue images by confocal microscopy. The results demonstrated that the surface property of nanoparticles is a key factor in determining their distribution in the vitreous and retina after intravitreal injection. In addition, immunohistochemistry and TEM images of retina tissues suggested the important mechanism related with Mülller cells for intravitreally administered nanoparticles to overcome the physical barrier of inner limiting membrane and to penetrate into the deeper retinal structures. Therefore, we expect that this study can provide valuable information for biomedical researchers to develop optimized nanoparticles as drug or gene carriers for retinal and optic nerve disorders such as glaucoma, age-related macular degeneration, and diabetic retinopathy.


Assuntos
Nanopartículas/administração & dosagem , Nanopartículas/análise , Retina/metabolismo , Corpo Vítreo/metabolismo , Animais , Sistemas de Liberação de Medicamentos , Injeções Intravítreas , Nanopartículas/ultraestrutura , Ratos , Ratos Long-Evans , Retina/ultraestrutura , Propriedades de Superfície , Corpo Vítreo/ultraestrutura
9.
J Control Release ; 152(1): 21-9, 2011 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-21457740

RESUMO

This study reports that tumor-targeting glycol chitosan nanoparticles with physically loaded and chemically conjugated photosensitizers can be used in photodynamic therapy (PDT). First, the hydrophobic photosensitizer, chlorin e6 (Ce6), was physically loaded onto the hydrophobically-modified glycol chitosan nanoparticles (HGC), which were prepared by self-assembling amphiphilic glycol chitosan-5ß-cholanic acid conjugates under aqueous conditions. Second, the Ce6s were chemically conjugated to the glycol chitosan polymers, resulting in amphiphilic glycol chitosan-Ce6 conjugates that formed self-assembled nanoparticles in aqueous condition. Both Ce6-loaded glycol chitosan nanoparticles (HGC-Ce6) and Ce6-conjugated chitosan nanoparticles (GC-Ce6) had similar average diameters of 300 to 350 nm, a similar in vitro singlet oxygen generation efficacy under buffer conditions, and a rapid cellular uptake profile in the cell culture system. However, compared to GC-Ce6, HGC-Ce6 showed a burst of drug release in vitro, whereby 65% of physically loaded drugs were rapidly released from the particles within 6.5h in the buffer condition. When injected through the tail vein into tumor bearing mice, HGC-Ce6 did not accumulate efficiently in tumor tissue, reflecting the burst in the release of the physically loaded drug, while GC-Ce6 showed a prolonged circulation profile and a more efficient tumor accumulation, which resulted in high therapeutic efficacy. These comparative studies with drug-loaded and drug-conjugated nanoparticles showed that the photosensitizer-conjugated glycol chitosan nanoparticles with excellent tumor targeting properties have potential for PDT in cancer treatment.


Assuntos
Quitosana/administração & dosagem , Nanopartículas/administração & dosagem , Neoplasias Experimentais/tratamento farmacológico , Fármacos Fotossensibilizantes/administração & dosagem , Porfirinas/administração & dosagem , Animais , Quitosana/química , Quitosana/farmacocinética , Clorofilídeos , Células HT29 , Humanos , Camundongos , Fotoquimioterapia , Oxigênio Singlete/metabolismo , Distribuição Tecidual
10.
J Control Release ; 141(3): 339-46, 2010 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-19836427

RESUMO

Small interfering RNA (siRNA) is a promising biological strategy for treatment of diverse diseases, but the therapeutic application of siRNA has been limited by its instability and poor cellular uptake efficiency. Although the development of various gene delivery systems has increased the siRNA delivery efficiency, many problems still remain to be resolved before the clinical application of siRNA. In this study, we suggest reducible polymerized siRNA a possible solution for low delivery efficiency of siRNA. Dithiol-modified red fluorescent protein (RFP) siRNAs at the 5'-ends of both sense and anti-sense strands were disulfide-polymerized. Polymerized siRNA (poly-siRNA) was composed of 30% oligomeric siRNA (50 approximately 300 bps) and 66% polymeric siRNA (above approximately 300 bps) as fractions, and was reducible in reducing solution through disulfide bond cleavage. Poly-siRNA formed more condensed and nano-sized complexes with low molecular weight polyethylenimine (PEI) by strong electrostatic interaction based on the higher charge density of poly-siRNA, compared with siRNA (mono-siRNA). The compact poly-siRNA/PEI complexes prevented the loss and degradation of siRNA from a polyanion competitor and RNases in serum. Furthermore, poly-siRNA/PEI complexes exhibited superior intracellular uptake by murine melanoma cells (B16F10), and was accompanied with RFP gene silencing efficiency of about 80%, compared to untreated cells. These results sufficiently support that strong polyanionic and reducible poly-siRNA can be utilized as a novel powerful therapeutic strategy for human diseases.


Assuntos
Inativação Gênica , Polietilenoimina/metabolismo , Polímeros/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Sequência de Bases , Dissulfetos/metabolismo , Corantes Fluorescentes/metabolismo , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Peso Molecular , Polietilenoimina/química , Polímeros/química , RNA Interferente Pequeno/química , Eletricidade Estática , Proteína Vermelha Fluorescente
11.
J Control Release ; 144(2): 134-43, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20184928

RESUMO

Here, we designed a new nano-sized siRNA carrier system composed of biocompatible/biodegradable glycol chitosan polymer (GC) and strongly positively charged polyethylenimine (PEI) polymers. In order to make a stable and tumor-homing nano-sized carrier, each polymer was modified with hydrophobic 5beta-cholanic acid, and they were simply mixed to form self-assembled GC-PEI nanoparticles (GC-PEI NPs), due to the strong hydrophobic interactions of 5beta-cholanic acids in the polymers. The freshly prepared GC-PEI NPs showed a stable nanoparticle structure (350nm) and they presented a strongly positive-charged surface (zeta potential=23.8) that is enough to complex tightly with negatively charged RFP-siRNAs, designed for inhibiting red fluorescent protein (RFP) expression. The siRNA encapsulated nanoparticles (siRNA-GC-PEI NPs) formed more compact and stable nanoparticle structures (250nm) at 1: 5 weight ratio of siRNA to GC-PEI nanoparticles. In vitro RFP expressing B16F10 tumor cell (RFP/B16F10) culture system, the siRNA-GC-PEI NPs presented a rapid time-dependent cellular uptake profile within 1h. Moreover, the internalized siRNA-GC-PEI NPs lead to specific mRNA breaks down. Furthermore, our new formulation of siRNA-GC-PEI NPs presented a significant inhibition of RFP gene expression of RFP/B16F10-bearing mice, due to their higher tumor-targeting ability. These results revealed the promising potential of GC-PEI NPs as a stable and effective nano-sized siRNA delivery system for cancer treatment.


Assuntos
Nanopartículas/química , Polímeros/química , Polímeros/metabolismo , RNA Interferente Pequeno/genética , Animais , Quitosana , Ácidos Cólicos , Interações Hidrofóbicas e Hidrofílicas , Proteínas Luminescentes , Masculino , Camundongos , Camundongos Nus , Neoplasias/genética , Neoplasias/metabolismo , Polietilenoimina/química , Polietilenoimina/metabolismo , RNA Interferente Pequeno/metabolismo , Proteína Vermelha Fluorescente
12.
Infect Immun ; 70(9): 4841-50, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12183528

RESUMO

Human macrophage chemoattractant protein 1 (MCP-1) is a potent mediator of macrophage migration and therefore plays an essential role in early events of inflammation. In endothelial cells, at least three independent pathways regulate MCP-1 expression by NF-kappaB and AP-1. Orientia tsutsugamushi causes vasculitis in humans by replicating inside macrophages and endothelial cells. In the present study, we investigated the cis-acting and trans-acting elements involved in O. tsutsugamushi-induced MCP-1 gene expression in human umbilical vein endothelial cells (HUVEC). Although NF-kappaB activation was observed in HUVEC infected with O. tsutsugamushi, inhibition of NF-kappaB activation did not affect the MCP-1 expression. However, treatment of HUVEC with extracellular signal-regulated kinase (ERK) kinase inhibitor or p38 mitogen-activated protein kinase (MAPK) inhibitor suppressed expression of MCP-1 mRNA concomitant with downregulation of activator protein 1 (AP-1) activation. Deletion of triphorbol acetate response elements (TRE) at position -69 to -63 of MCP-1 gene abolished inducible promoter activity. Deletion of TRE at position -69 to -63-96 to -90 or deletion of NF-kappaB-binding site at position -69 to -63-88 to -79 did not affect the inducibility of promoter. Site-directed mutagenesis of the NF-kappaB binding sites at positions -2640 to -2632, -2612 to -2603 in the enhancer region, or the AP-1 biding site at position -2276 to -2270 decreased the inducible activity of the promoter. Taken together, AP-1 activation by both the ERK pathway and the p38 MAPK pathway as well as their binding to TRE at position -69 to -63 in proximal promoter and TRE at position -2276 to -2270 in enhancer region is altogether essential in induction of MCP-1 mRNA in HUVEC infected with O. tsutsugamushi. Although NF-kappaB activation is not essential per se, the kappaB site in the enhancer region is important in MCP-1 induction of HUVEC. This discrepancy in the involvement of the NF-kappaB may be due to the function of chromatin structures and other transcription cofactors in the regulation of MCP-1 gene expression in response to O. tsutsugamushi infectioin.


Assuntos
Quimiocina CCL2/genética , Endotélio Vascular/metabolismo , Endotélio Vascular/microbiologia , Orientia tsutsugamushi/patogenicidade , Prolina/análogos & derivados , Fator de Transcrição AP-1/metabolismo , Antioxidantes/farmacologia , Sequência de Bases , Células Cultivadas , DNA/genética , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Luciferases/genética , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , NF-kappa B/metabolismo , Prolina/farmacologia , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tifo por Ácaros/etiologia , Tiocarbamatos/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Transfecção , Vasculite/etiologia
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