RESUMO
Herein, we elucidated the mechanisms and key factors for the tumor-targeting ability of nanoparticles that presented high targeting efficiency for liver tumor. We used several different nanoparticles with sizes of 200-300 nm, including liposome nanoparticles (LNPs), polystyrene nanoparticles (PNPs) and glycol chitosan-5ß-cholanic acid nanoparticles (CNPs). Their sizes are suitable for the enhanced permeation and retention (EPR) effect in literature. Different in vitro characteristics, such as the particle structure, stability, and bioinertness, were carefully analyzed with and without serum proteins. Also, pH-dependent tumor cell uptakes of nanoparticles were studied using fluorescence microscopy. Importantly, CNPs had sufficient stability and bioinertness to maintain their nanoparticle structure in the bloodstream, and they also presented prolonged circulation time in the body (blood circulation half-life T1/2 = about 12.2 h), compared to the control nanoparticles. Finally, employing liver tumor bearing mice, we also observed that CNPs had excellent liver tumor targeting ability in vivo, while LNPs and PNPs demonstrated lower tumor-targeting efficiency due to the nonspecific accumulation in normal liver tissue. Liver tumor models were produced by laparotomy and direct injection of HT29 tumor cells into the left lobe of the liver of athymic nude mice. This study provides valuable information concerning the key factors for the tumor-targeting ability of nanoparticles such as stability, bioinertness, and rapid cellular uptake at targeted tumor tissues.
Assuntos
Quitosana/administração & dosagem , Quitosana/metabolismo , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/metabolismo , Neoplasias Hepáticas/metabolismo , Nanopartículas/química , Animais , Microscopia Crioeletrônica , Eletroforese em Gel de Poliacrilamida , Células HT29 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Eletrônica , Células RAW 264.7 , RatosRESUMO
Imaging techniques including computed tomography, magnetic resonance imaging, and positron emission tomography (PET) offer many potential benefits to diagnosis and treatment of cancers. Each method has its own strong and weak points. Therefore, multimodal imaging techniques have been highlighted as an alternative method for overcoming the limitations of each respective imaging method. In this study, we fabricated PET/optical activatable imaging probe based on glycol chitosan nanoparticles (CNPs) for multimodal imaging. To prepare the dual PET/optical probes based on CNPs, both (64)Cu radiolabeled DOTA complex and activatable matrix metalloproteinase (MMP)-sensitive peptide were chemically conjugated onto azide-functionalized CNPs via bio-orthogonal click chemistry, which was a reaction between azide group and dibenzyl cyclooctyne. The PET/optical activatable imaging probes were visualized by PET and optical imaging system. Biodistribution of probes and activity of MMP were successfully measured in tumor-bearing mice.
Assuntos
Nanopartículas , Nanotecnologia , Neoplasias Experimentais/diagnóstico , Dispositivos Ópticos , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Animais , Linhagem Celular Tumoral , Quitosana/química , Radioisótopos de Cobre , Glicóis/química , Humanos , Células MCF-7 , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Nus , Estrutura Molecular , Nanopartículas/química , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/químicaRESUMO
Active matrix metalloproteinase-3 (MMP-3) is a prognostic marker of rheumatoid arthritis (RA). We recently developed an MMP-3 probe that can specifically detect the active form of MMP-3. The aim of this study was to investigate whether detection and monitoring of active MMP-3 could be useful to predict therapeutic drug responses in a collagen-induced arthritis (CIA) model. During the period of treatment with drugs such as methotrexate (MTX) or infliximab (IFX), MMP-3 mRNA and protein levels were correlated with fluorescence signals in arthritic joint tissues and in the serum of CIA mice. Also, bone volume density and erosion in the knee joints and the paws of CIA mice were measured with microcomputed tomography (micro-CT), X-ray, and histology to confirm drug responses. In joint tissues and serum of CIA mice, strong fluorescence signals induced by the action of active MMP-3 were significantly decreased when drugs were applied. The decrease in RA scores in drug-treated CIA mice led to fluorescence reductions, mainly as a result of down-regulation of MMP-3 mRNA or protein. The micro-CT, X-ray, and histology results clearly showed marked decreases in bone and cartilage destruction, which were consistent with the reduction of fluorescence by down-regulation of active MMP-3 in drug-treated CIA mice. We suggest that the MMP-3 diagnostic kit could be used to detect and monitor the active form of MMP-3 in CIA mice serum during a treatment course and thereby used to predict the drug response or resistance to RA therapies at an earlier stage. We hope that monitoring of active MMP-3 levels in arthritis patients using the MMP-3 diagnostic kit will be a promising tool for drug discovery, drug development, and monitoring of drug responses in RA therapy.
Assuntos
Antirreumáticos/uso terapêutico , Colágeno/toxicidade , Metaloproteinase 3 da Matriz/metabolismo , Sondas Moleculares/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Experimental/enzimologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/enzimologia , Modelos Animais de Doenças , Infliximab , Metotrexato/uso terapêutico , CamundongosRESUMO
PURPOSE: The application of gold nanoparticles (AuNPs) in biomedical field was limited due to the low stability in the biological condition. Herein, to enhance stability and tumor targeting ability of AuNPs, their surface was modified with biocompatible glycol chitosan (GC) and the in vivo biodistribution of GC coated AuNPs (GC-AuNPs) were studied through computed tomography (CT). METHODS: Polymer-coated gold nanoparticles were produced using GC as a reducing agent and a stabilizer. Their feasibility in biomedical application was explored through CT in tumor-bearing mice. RESULTS: Stability of gold nanoparticles increased in the physiological condition due to the GC coating layer on the surface. Tomographic images of tumor were successfully obtained in the tumor-xenografted animal model when the GC-AuNPs were used as a CT contrast agent. The tumor targeting property of the gold nanoparticles was due to the properties of GC because GC-AuNPs were accumulated in the tumor, while most of heparin-coated nanoparticles were found in the liver and spleen. CONCLUSIONS: The polymer properties on the surface played an important role in the behavior of gold nanoparticles in the biological condition and the enhanced stability and tumor targeting property of nanoparticles were inherited from GC on the surface.
Assuntos
Materiais Biocompatíveis , Quitosana/química , Meios de Contraste/química , Ouro/química , Nanopartículas Metálicas/química , Neoplasias/diagnóstico , Animais , Sobrevivência Celular/efeitos dos fármacos , Quitosana/toxicidade , Meios de Contraste/farmacocinética , Humanos , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/secundário , Camundongos , Neoplasias/patologia , Radiografia , Distribuição TecidualRESUMO
The activity of rheumatoid arthritis (RA) correlates with the expression of proteases. Among several proteases, matrix metalloproteinase-3 (MMP-3) is one of the biological markers used to diagnose RA. The active form of MMP-3 is a key enzyme involved in RA-associated destruction of cartilage and bone. Thus, detection of active MMP-3 in serum or in vivo is very important for early diagnosis of RA. In this study, a soluble MMP-3 probe was prepared to monitor RA progression by detecting expression of active MMP-3 in collagen-induced arthritis (CIA) mice in vivo in both serum and fibroblast-like synoviocytes (FLSs). The MMP-3 probe exhibited strong sensitivity to MMP-3 and moderate sensitivity to MMP-7 at nanomolecular concentrations, but was not sensitive to other MMPs such as MMP-2, MMP-9, and MMP-13. In an optical imaging study, the MMP-3 probe produced early and strong NIR fluorescence signals prior to observation of erythema and swelling in CIA mice. The MMP-3 probe was able to rapidly and selectively detect and monitor active MMP-3 in diluted serum from CIA mice. Furthermore, histological data demonstrated that activated FLSs in arthritic knee joints expressed active MMP-3. Together, our results demonstrated that the MMP-3 probe may be useful for detecting active MMP-3 for diagnosis of RA. More importantly, the MMP-3 probe was able to detect active MMP-3 in diluted serum with high sensitivity. Therefore, the MMP-3 probe developed in this study may be a very promising probe, useful as a biomarker for early detection and diagnosis of RA.
Assuntos
Artrite Experimental/enzimologia , Artrite Reumatoide/enzimologia , Metaloproteinase 3 da Matriz/metabolismo , Membrana Sinovial/enzimologia , Animais , Artrite Experimental/sangue , Artrite Experimental/metabolismo , Artrite Reumatoide/sangue , Artrite Reumatoide/metabolismo , Western Blotting , Modelos Animais de Doenças , Ativação Enzimática , Masculino , Metaloproteinase 3 da Matriz/sangue , Camundongos , Camundongos Endogâmicos DBA , Imagem Molecular , Membrana Sinovial/citologia , Membrana Sinovial/metabolismoRESUMO
An efficient and straightforward method for radiolabeling nanoparticles is urgently needed to understand the in vivo biodistribution of nanoparticles. Herein, we investigated a facile and highly efficient strategy to prepare radiolabeled glycol chitosan nanoparticles with (64)Cu via a strain-promoted azide-alkyne cycloaddition strategy, which is often referred to as click chemistry. First, the azide (N3) group, which allows for the preparation of radiolabeled nanoparticles by copper-free click chemistry, was incorporated to glycol chitosan nanoparticles (CNPs). Second, the strained cyclooctyne derivative, dibenzyl cyclooctyne (DBCO) conjugated with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator, was synthesized for preparing the preradiolabeled alkyne complex with (64)Cu radionuclide. Following incubation with the (64)Cu-radiolabeled DBCO complex (DBCO-PEG4-Lys-DOTA-(64)Cu with high specific activity, 18.5 GBq/µmol), the azide-functionalized CNPs were radiolabeled successfully with (64)Cu, with a high radiolabeling efficiency and a high radiolabeling yield (>98%). Importantly, the radiolabeling of CNPs by copper-free click chemistry was accomplished within 30 min, with great efficiency in aqueous conditions. In addition, we found that the (64)Cu-radiolabeled CNPs ((64)Cu-CNPs) did not show any significant effect on the physicochemical properties, such as size, zeta potential, or spherical morphology. After (64)Cu-CNPs were intravenously administered to tumor-bearing mice, the real-time, in vivo biodistribution and tumor-targeting ability of (64)Cu-CNPs were quantitatively evaluated by microPET images of tumor-bearing mice. These results demonstrate the benefit of copper-free click chemistry as a facile, preradiolabeling approach to conveniently radiolabel nanoparticles for evaluating the real-time in vivo biodistribution of nanoparticles.
Assuntos
Quitosana/química , Química Click/métodos , Radioisótopos de Cobre/química , Nanopartículas/química , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos NusRESUMO
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/metabolismoRESUMO
OBJECTIVE: Early treatment based on an early diagnosis of rheumatoid arthritis (RA) could halt progression of the disease, but early diagnosis is often difficult. Matrix metalloproteinase 3 (MMP-3) is thought to be particularly important in the pathogenesis of RA. The aim of this study was to investigate whether an MMP-3-specific polymeric probe could be used for early diagnosis and for visualizing the progression of arthritis, using a near-infrared fluorescence (NIRF) imaging system. METHODS: The MMP-3-specific polymeric probe was developed by conjugating NIRF dye, MMP substrate peptide, and dark quencher to self-assembled chitosan nanoparticles. One hour after intravenous administration of the probe, fluorescent images of mice with collagen-induced arthritis at different stages of disease development were obtained. The correlation between the fluorescence recovered in in vivo imaging when using an MMP-3-specific polymeric probe and up-regulated MMP-3 activity in the joint tissues was evaluated by Western blotting and immunohistochemical staining. Histologic analysis and micro-computed tomography (micro-CT) were also used to assess arthritis progression. RESULTS: A significantly higher NIRF signal was recovered from arthritic joints compared with normal joints at 14 days after the first immunization, before any erythema or swelling could be observed with the naked eye or any erosion was detected by histologic analysis or micro-CT. The results of immunohistochemical analysis and Western blotting confirmed that the fluorescence recovered in the in vivo imaging was related to up-regulated MMP-3 activity in the joint tissues. CONCLUSION: An MMP-3-specific polymeric probe provided clear early diagnosis of arthritis and visualization of arthritis progression using an NIRF imaging system. This approach could be used for early diagnosis and for monitoring drug and surgical therapies in individual cases.
Assuntos
Artrite Experimental/diagnóstico , Artrite Experimental/metabolismo , Diagnóstico por Imagem/métodos , Diagnóstico Precoce , Corantes Fluorescentes/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Animais , Artrite Experimental/patologia , Artrografia , Biomarcadores/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Nanopartículas , Tomografia Computadorizada por Raios XRESUMO
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 inâ 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/genéticaRESUMO
We report a new apoptosis nanoprobe (Apo-NP) designed on the basis of a polymer nanoparticle platform. This simple one-step technique is capable of boosting fluorescence signals upon apoptosis in living cells, enabling real-time imaging of apoptosis in single cells and in vivo. The Apo-NP efficiently delivers chemically labeled, dual-quenched caspase-3-sensitive fluorogenic peptides into cells, allowing caspase-3-dependent strong fluorescence amplification to be imaged in apoptotic cells in real-time and at high resolution. The design platform of the Apo-NP is flexible and can be fine-tuned for a wide array of applications such as identification of caspase-related apoptosis in pathologies and for monitoring therapeutic efficacy of apoptotic drugs in cancer treatment.
Assuntos
Apoptose , Corantes Fluorescentes/química , Imagem Molecular , Nanopartículas/química , Polímeros/química , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/química , Caspase 3/metabolismo , Doxorrubicina/farmacologia , Corantes Fluorescentes/administração & dosagem , Humanos , Medições Luminescentes , Camundongos , Estrutura Molecular , Nanopartículas/administração & dosagem , Neoplasias Experimentais , Tamanho da Partícula , Polímeros/administração & dosagem , Propriedades de Superfície , Células Tumorais CultivadasRESUMO
Among the classical collagenases, matrix metalloproteinase-13 (called MMP-13, collagenase-3) is one of the most important components for cartilage destruction of osteoarthritis (OA) developments. Despite many efforts, the detection methods of MMP-13 activity have been met with limited success in vivo, in part, due to the low sensitivity and low selectivity by homology of MMP family. Previously, we demonstrated the use of strongly dark-quenched fluorogenic probe allowed for the visual detection of MMP-13 in vitro and in OA-induced rat models. In this study, we described the optimization of MMP-13 fluorogenic probe for OA detection in vivo. Three candidate probes demonstrated recovered fluorescent intensity proportional with MMP-13 concentrations, respectively; however, Probe 2 exhibited both high signal amplification and selective recognition for MMP-13, not MMP-2 and MMP-9 in vitro. When Probe 2 was applied to OA-induced rat models, clear visualization of MMP-13 activity in OA-induced cartilage was obtained. Optimized MMP-13 fluorogenic probe can be applied to detect and image OA and have potential for evaluating the in vivo efficacy of MMP-13 inhibitors which are being tested for therapeutic treatment of OA.
Assuntos
Carbocianinas/metabolismo , Corantes Fluorescentes/metabolismo , Metaloproteinase 13 da Matriz/análise , Metaloproteinase 13 da Matriz/metabolismo , Imagem Molecular/métodos , Sondas Moleculares/metabolismo , Osteoartrite/enzimologia , Animais , Carbocianinas/química , Modelos Animais de Doenças , Corantes Fluorescentes/química , Humanos , Cinética , Sondas Moleculares/química , Osteoartrite/diagnóstico , Peptídeos/química , Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Sensibilidade e Especificidade , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
We developed a new apoptosis imaging probe with gold nanoparticles (AuNPs). A near-infrared fluorescence dye was attached to AuNP surface through the bridge of peptide substrate (DEVD). The fluorescence was quenched in physiological conditions due to the quenching effect of AuNP, and the quenched fluorescence was recovered after the DEVD had been cleaved by caspase-3, the enzyme involved in apoptotic process. The adhesion of DEVD substrates on AuNP surface was accomplished by conjugation of the 3,4-dihydroxy phenylalanine (DOPA) groups which are adhesive to inorganic surface and rich in mussels. This surface modification with DEVD substrates by DOPA groups resulted in increased stability of AuNP in cytosol condition for hours. Moreover, the cleavage of substrate and the dequenching process are very fast, and the cells did not need to be fixed for imaging. Therefore, the real-time monitoring of caspase activity could be achieved in live cells, which enabled early detection of apoptosis compared to a conventional apoptosis kit such as Annexin V-FITC. Therefore, our apoptosis imaging has great potential as a simple, inexpensive, and efficient apoptosis imaging probe for biomedical applications.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Ouro/farmacologia , Nanopartículas Metálicas/química , Imagem Molecular/métodos , Caspase 3/química , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/química , Ouro/química , Células HeLa , Humanos , Levodopa/química , Óptica e Fotônica , Tamanho da Partícula , Peptídeos/química , Propriedades de SuperfícieRESUMO
Herein, we developed matrix metalloprotease (MMP) sensitive gold nanorods (MMP-AuNR) for cancer imaging and therapy. It was feasible to absorb NIR laser and convert into heat as well as visualize MMP activity. We showed the possibility of gold nanorods as a hyperthermal therapeutic agent and MMP sensitive imaging agent both in vitro and in vivo condition. The results suggested potential application of MMP-AuNR for simultaneous cancer diagnosis and therapy.
Assuntos
Carcinoma de Células Escamosas , Diagnóstico por Imagem/métodos , Ouro , Terapia a Laser/métodos , Metaloproteinases da Matriz/metabolismo , Fototerapia/métodos , Neoplasias Cutâneas , Animais , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/terapia , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Fluorescência , Ouro/química , Ouro/uso terapêutico , Temperatura Alta , Luz , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/química , Camundongos , Camundongos Nus , Microscopia Eletrônica de Transmissão , Nanotubos/química , Transplante de Neoplasias , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/terapia , Espectrometria de Fluorescência , Espectroscopia de Luz Próxima ao Infravermelho , TemperaturaRESUMO
Matrix metalloproteinases (MMPs) have been shown to be abundant in pathological conditions such as cancer, osteoarthritis (OA), and rheumatoid arthritis (RA). The extent of MMPs detected in biological samples provides important clinical information for diagnosis, prognosis, and therapeutic monitoring of various diseases relating with MMPs. Herein, we developed a new high-throughput MMP diagnostic kit (MMP-D-KIT) based on a 96-well plate by immobilizing MMP-13 specific fluorogenic peptide probes (MMP peptide probe), which is a pair consisting of a near-infrared (NIR) fluorophore (Cy5.5) and a quencher (BHQ-3), onto the biocompatible glycol chitosan (GC) polymer anchored 96-well plate. When MMP enzymes were simply added and incubated in a MMP-D-KIT, the fluorescence of each well was recovered and the fluorescence intensity showed distinct difference within minutes through NIR fluorescence imaging system. The fluorescence was recovered not only by MMP-13 activity, but also by other MMPs activity. Furthermore, recovery of NIR fluorescent signals in MMP-D-KIT was proportional to concentrations of immobilized MMP peptide probe-GC conjugates and, importantly, MMP concentration. The MMP-D-KIT is most specific for target MMP, compared with other enzymes including caspase-3 and 20s proteasome. Additionally, the MMP-D-KIT was used to detect MMP activity in biological samples such as synovial fluid from 12 OA patients (grades 1-4 based on the Kellgren-Lawrence grading scale). It was found that the fluorescence intensity measured using MMP-D-KIT decidedly correlates with the progression of OA. The MMP-D-KIT could be applicable in detecting MMP activities in various biological samples and evaluating the effects of MMP inhibitors in a rapid and easy fashion.
Assuntos
Corantes Fluorescentes/química , Proteínas Imobilizadas/metabolismo , Metaloproteinases da Matriz/metabolismo , Sondas Moleculares/química , Peptídeos/química , Kit de Reagentes para Diagnóstico , Fluorescência , Corantes Fluorescentes/análise , Humanos , Proteínas Imobilizadas/química , Metaloproteinases da Matriz/análise , Metaloproteinases da Matriz/química , Sondas Moleculares/análise , Osteoartrite do Joelho/enzimologia , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
We report tumor targeting nanoparticles for optical/MR dual imaging based on self-assembled glycol chitosan to be a potential multimodal imaging probe. To develop an optical/MR dual imaging probe, biocompatible and water-soluble glycol chitosan (M(w) = 50 kDa) were chemically modified with 5beta-cholanic acid (CA), resulting in amphiphilic glycol chitosan-5beta-cholanic acid conjugates (GC-CA). For optical imaging near-infrared fluorescence (NIRF) dye, Cy5.5, was conjugated to GC-CA resulting in Cy5-labeled GC-CA conjugates (Cy5.5-GC-CA). Moreover, in order to chelate gadolinium (Gd(III)) in the Cy5.5-GC-CA conjugates, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was directly conjugated in Cy5.5-GC-CA. Finally, the excess GdCl(3) was added to DOTA modified Cy5.5-GC-CA conjugates in distilled water (pH 5.5). The freshly prepared Gd(III) encapsulated Cy5.5-GC-CA conjugates were spontaneously self-assembled into stable Cy5.5 labeled and Gd(III) encapsulated chitosan nanoparticles (Cy5.5-CNP-Gd(III)). The Cy5.5-CNP-Gd(III) was spherical in shape and approximately 350 nm in size. From the cellular experiment, it was demonstrated that Cy5.5-CNP-Gd(III) were efficiently taken up and distributed in cytoplasm (NIRF filter; red). When the Cy5.5-GC-Gd(III) were systemically administrated into the tail vein of tumor-bearing mice, large amounts of nanoparticles were successfully localized within the tumor, which was confirmed by noninvasive near-infrared fluorescence and MR imaging system simultaneously. These results revealed that the dual-modal imaging probe of Cy5.5-CNP-Gd(III) has the potential to be used as an optical/MR dual imaging agent for cancer treatment.
Assuntos
Quitosana , Imageamento por Ressonância Magnética , Nanopartículas , Neoplasias/diagnóstico , Quitosana/síntese química , Quitosana/química , Ácidos Cólicos/química , Fluorescência , Corantes Fluorescentes/química , Gadolínio/química , Nanopartículas/química , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
Herein, we prepared tumor-targeting peptide (AP peptide; CRKRLDRN) conjugated pH-responsive polymeric micelles (pH-PMs) in cancer therapy by active and pH-responsive tumor targeting delivery systems, simultaneously. The active tumor targeting and tumoral pH-responsive polymeric micelles were prepared by mixing AP peptide conjugated PEG-poly(d,l-lactic acid) block copolymer (AP-PEG-PLA) into the pH-responsive micelles of methyl ether poly(ethylene glycol) (MPEG)-poly(beta-amino ester) (PAE) block copolymer (MPEG-PAE). These mixed amphiphilic block copolymers were self-assembled to form stable AP peptide-conjugated and pH-responsive AP-PEG-PLA/MPEG-PAE micelles (AP-pH-PMs) with an average size of 150 nm. The AP-pH-PMs containing 10 wt % of AP-PEG-PLA showed a sharp pH-dependent micellization/demicellization transition at the tumoral acid pH. Also, they presented the pH-dependent drug release profile at the acidic pH of 6.4. The fluorescence dye, TRITC, encapsulated AP-pH-PMs (TRITC-AP-pH-PMs) presented the higher tumor-specific targeting ability in vitro cancer cell culture system and in vivo tumor-bearing mice, compared to control pH-responsive micelles of MPEG-PAE. For the cancer therapy, the anticancer drug, doxorubicin (DOX), was efficiently encapsulated into the AP-pH-PMs (DOX-AP-pH-PMs) with a higher loading efficiency. DOX-AP-pH-PMs efficiently deliver anticancer drugs in MDA-MB231 human breast tumor-bearing mice, resulted in excellent anticancer therapeutic efficacy, compared to free DOX and DOX encapsulated MEG-PAE micelles, indicating the excellent tumor targeting ability of AP-pH-PMs. Therefore, these tumor-targeting peptide-conjugated and pH-responsive polymeric micelles have great potential application in cancer therapy.
Assuntos
Portadores de Fármacos/química , Micelas , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Oligopeptídeos/química , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Doxorrubicina/química , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Portadores de Fármacos/metabolismo , Portadores de Fármacos/farmacocinética , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Oligopeptídeos/metabolismo , Polietilenoglicóis/química , Polímeros/química , Fatores de Tempo , Distribuição TecidualRESUMO
When car crash experiments are performed using cadavers or dummies, the active muscles' reaction on crash situations cannot be observed. The aim of this study is to estimate muscles' response of the major muscle groups using three-dimensional musculoskeletal model by dynamic simulations of low-speed sled-impact. The three-dimensional musculoskeletal models of eight subjects were developed, including 241 degrees of freedom and 86 muscles. The muscle parameters considering limb lengths and the force-generating properties of the muscles were redefined by optimization to fit for each subject. Kinematic data and external forces measured by motion tracking system and dynamometer were then input as boundary conditions. Through a least-squares optimization algorithm, active muscles' responses were calculated during inverse dynamic analysis tracking the motion of each subject. Electromyography for major muscles at elbow, knee, and ankle joints was measured to validate each model. For low-speed sled-impact crash, experiment and simulation with optimized and unoptimized muscle parameters were performed at 9.4 m/h and 10 m/h and muscle activities were compared among them. The muscle activities with optimized parameters were closer to experimental measurements than the results without optimization. In addition, the extensor muscle activities at knee, ankle, and elbow joint were found considerably at impact time, unlike previous studies using cadaver or dummies. This study demonstrated the need to optimize the muscle parameters to predict impact situation correctly in computational studies using musculoskeletal models. And to improve accuracy of analysis for car crash injury using humanlike dummies, muscle reflex function, major extensor muscles' response at elbow, knee, and ankle joints, should be considered.
Assuntos
Acidentes de Trânsito , Modelos Biológicos , Fenômenos Fisiológicos Musculoesqueléticos , Adulto , Algoritmos , Fenômenos Biomecânicos , Engenharia Biomédica , Simulação por Computador , Eletromiografia , Humanos , Imageamento Tridimensional , Articulações/fisiologia , Análise dos Mínimos Quadrados , Masculino , Torque , Adulto JovemRESUMO
We report here a new protease activatable strategy based on a polymer nanoparticle platform. This nanosensor delivers chemically labeled matrix metalloproteinase (MMP)-activatable fluorogenic peptides to the specific MMPs of interest in vivo. Intravenous administration of the nanosensor in an MMP-positive SCC-7 xenograft tumor and a colon cancer mouse model verified the enzyme specificity of the nanosensor in vivo. The design platform of the nanosensor is flexible and can be fine-tuned for a wide array of applications such as the detection of biomarkers, early diagnosis of disease, and monitoring therapeutic efficacy.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias do Colo/enzimologia , Corantes Fluorescentes , Metaloproteinases da Matriz/análise , Nanotecnologia/instrumentação , Polímeros/química , Espectroscopia de Luz Próxima ao Infravermelho/instrumentação , Animais , Linhagem Celular Tumoral , Portadores de Fármacos/química , Desenho de Equipamento , Análise de Falha de Equipamento , Camundongos , TransdutoresRESUMO
The early detection of osteoarthritis (OA) is currently a key challenge in the field of rheumatology. Biochemical studies of OA have indicated that matrix metalloproteinase-13 (MMP-13) plays a central role in cartilage degradation. In this study, we describe the potential use of a dark-quenched fluorogenic MMP-13 probe to image MMP-13 in both in vitro and rat models. The imaging technique involved using a MMP-13 peptide substrate, near-infrared (NIR) dye, and a NIR dark quencher. The results from this study demonstrate that the use of a dark-quenched fluorogenic probe allows for the visual detection of MMP-13 in vitro and in OA-induced rat models. In particular, by targeting this OA biomarker, the symptoms of the early and late stages of OA can be readily monitored, imaged, and analyzed in a rapid and efficient fashion. We anticipate that this simple and highly efficient fluorogenic probe will assist in the clinical management of patients with OA, not only for early diagnosis but also to assess individual patient responses to new drug treatments.
Assuntos
Cartilagem Articular/patologia , Diagnóstico por Imagem/métodos , Corantes Fluorescentes , Metaloproteinase 13 da Matriz/metabolismo , Osteoartrite/patologia , Espectrometria de Fluorescência/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Osteoartrite/enzimologia , RatosRESUMO
Antiangiogenic peptide drugs have received much attention in the fields of tumor therapy and tumor imaging because they show promise in the targeting of integrins such as alpha(v)beta(3) on angiogenic endothelial cells. However, systemic antiangiogenic peptide drugs have short half-lives in vivo, resulting in fast serum clearance via the kidney, and thus the therapeutic effects of such drugs remain modest. In this study, we prepared self-assembled glycol chitosan nanoparticles and explored whether this construct might function as a prolonged and sustained drug delivery system for RGD peptide, used as an antiangiogenic model drug in cancer therapy. Glycol chitosan hydrophobically modified with 5beta-cholanic acid (HGC) formed nanoparticles with a diameter of 230 nm, and RGD peptide was easily encapsulated into HGC nanoparticles (yielding RGD-HGC nanoparticles) with a high loading efficiency (>85%). In vitro work demonstrated that RGD-HGC showed prolonged and sustained release of RGD, lasting for 1 week. RGD-HGC also inhibited HUVEC adhesion to a beta ig-h3 protein-coated surface, indicating an antiangiogenic effect of the RGD peptide in the HGC nanoparticles. In an in vivo study, the antiangiogenic peptide drug formulation of RGD-HGC markedly inhibited bFGF-induced angiogenesis and decreased hemoglobin content in Matrigel plugs. Intratumoral administration of RGD-HGC significantly decreased tumor growth and microvessel density compared to native RGD peptide injected either intravenously or intratumorally, because the RGD-HGC formulation strongly enhanced the antiangiogenic and antitumoral efficacy of RGD peptide by affording prolonged and sustained RGD peptide delivery locally and regionally in solid tumors.