RESUMEN
The development of ultra-long circulating nanodrug delivery systems have showed distinct advantage in maintaining the long-lasting tumor retention. Although the relationship between extended tumor retention and ultra-long plasma half-life was apparent, there was still a lack of experimental evidence to reveal the enhancement mechanism. Herein, we proposed a concept of "Sustained Irrigation" effect ("SI" effect) to elucidate that it was through sustained blood irrigation that the ultra-long circulating nanoparticles achieved long-lasting tumor retention. Besides, in order to intuitively verify the "SI" effect, we developed an "ON-OFF-ON" fluorescence switch technology. The ultra-long circulating delivery nanoparticle was constructed by encapsulating the protein with hydrophilic polymer shell. Nanoparticles with ultra-long plasma half-life (t1/2>40 h) fabricated by this method were employed as models for demonstrating the "SI" effect. The recovery of Cy5.5 fluorescence after the laser quenching meant the "fresh" Cy5.5-labeled nanoparticles were entering tumor, which confirmed the ultra-long circulating nanoparticles in blood could sustainedly irrigate to tumor. Our finding revealed the key mechanism by which ultra-long circulating NDDSs enhanced the tumor accumulation and retention, and provided experimental support for the development of ultra-long circulating delivery system in clinic.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/administración & dosificación , Nanopartículas/química , Neoplasias Experimentales/metabolismo , Albúmina Sérica Bovina/administración & dosificación , Animales , Carbocianinas/química , Carbocianinas/farmacocinética , Línea Celular Tumoral , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/farmacocinética , Humanos , Masculino , Ratones , Microscopía Confocal , Microscopía Electrónica de Transmisión , Nanopartículas/ultraestructura , Ratas Sprague-Dawley , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/farmacocinética , Distribución TisularRESUMEN
BACKGROUND: Tumor phototherapy especially photodynamic therapy (PDT) or photothermal therapy (PTT), has been considered as an attractive strategy to elicit significant immunogenic cell death (ICD) at an optimal tumor retention of PDT/PTT agents. Heptamethine cyanine dye (IR-780), a promising PDT/PTT agent, which can be used for near-infrared (NIR) fluorescence/photoacoustic (PA) imaging guided tumor phototherapy, however, the strong hydrophobicity, short circulation time, and potential toxicity in vivo hinder its biomedical applications. To address this challenge, we developed mesoporous polydopamine nanoparticles (MPDA) with excellent biocompatibility, PTT efficacy, and PA imaging ability, facilitating an efficient loading and protection of hydrophobic IR-780. RESULTS: The IR-780 loaded MPDA (IR-780@MPDA) exhibited high loading capacity of IR-780 (49.7 wt%), good physiological solubility and stability, and reduced toxicity. In vivo NIR fluorescence and PA imaging revealed high tumor accumulation of IR-780@MPDA. Furthermore, the combined PDT/PTT of IR-780@MPDA could induce ICD, triggered immunotherapeutic response to breast tumor by the activation of cytotoxic T cells, resulting in significant suppression of tumor growth in vivo. CONCLUSION: This study demonstrated that the as-developed compact and biocompatible platform could induce combined PDT/PTT and accelerate immune activation via excellent tumor accumulation ability, offering multimodal tumor theranostics with negligible systemic toxicity.
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Antineoplásicos , Carbocianinas , Colorantes Fluorescentes , Indoles/química , Nanopartículas/química , Polímeros/química , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Carbocianinas/química , Carbocianinas/farmacocinética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacocinética , Neoplasias Mamarias Animales , Ratones , Fototerapia , Nanomedicina Teranóstica , Distribución TisularRESUMEN
Hypoxia is associated with clinical diseases. Extreme hypoxia leads to multiple organs failure. However, the different effects of hypoxia on brain and visceral organs still need to be clarified, and moreover, characteristics in vulnerable organs suffering from hypoxia remain elusive. In the present study, we first aimed to figure out the hypoxic sensitivity of organs. Adult male mice were exposed to 6% O2 or 8% O2 for 6 h. Control mice were raised under normoxic conditions. In vivo and in vitro imaging of anti-HIF-1α-NMs-cy5.5 nanocomposites showed that the expression level of hypoxia-inducible factor (HIF-1α) was the highest in the liver, followed by kidney and brain. HIF-1α was detected in the hepatocytes of liver, distal convoluted tubules of kidney and neurons of cerebral cortex. The liver, kidney and brain showed distinct metabolic profiles but an identical change in glutamate. Compared with kidney and brain, the liver had more characteristic metabolites and more disturbed metabolic pathways related to glutaminolysis and glycolysis. The level of O-phosphocholine, GTP, NAD and aspartate were upregulated in hypoxic mice brain, which displayed significant positive correlations with the locomotor activity in control mice, but not in hypoxic mice with impaired locomotor activities. Taken together, the liver, kidney and brain are the three main organs of the body that are strongly respond to acute hypoxia, and the liver exhibited the highest hypoxic sensitivity. The metabolic disorders appear to underlie the physiological function changes.
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Encéfalo/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Animales , Conducta Animal , Western Blotting , Carbocianinas/química , Carbocianinas/farmacocinética , Hipoxia/fisiopatología , Espectroscopía de Resonancia Magnética , Masculino , Ratones Endogámicos BALB C , Imagen Molecular , Nanocompuestos/químicaRESUMEN
There has been considerable interest in the clinical use of exosomes as delivery vehicles for treatments as well as for promising diagnostic biomarkers, but the physiological distribution of exosomes must be further elucidated to validate their efficacy and safety. Here, we aimed to develop novel methods to monitor exosome biodistribution in vivo using positron emission tomography (PET) and optical imaging. Exosomes were isolated from cultured mouse breast cancer cells and labeled for PET and optical imaging. In mice, radiolabeled and fluorescently labeled exosomes were injected both via lymphatic and hematogenous metastatic routes. PET and fluorescence images were obtained and quantified. Radioactivity and fluorescence intensity of ex vivo organs were measured. PET signals from exosomes in the lymphatic metastatic route were observed in the draining sentinel lymph nodes. Immunohistochemistry revealed greater exosome uptake in brachial and axillary versus inguinal lymph nodes. Following administration through the hematogenous metastasis pathway, accumulation of exosomes was clearly observed in the lungs, liver, and spleen. Exosomes from tumor cells were successfully labeled with 64Cu (or 68Ga) and fluorescence and were visualized via PET and optical imaging, suggesting that this simultaneous and rapid labeling method could provide valuable information for further exosome translational research and clinical applications.
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Exosomas , Colorantes Fluorescentes/farmacocinética , Imagen Multimodal/métodos , Animales , Carbocianinas/química , Carbocianinas/farmacocinética , Radioisótopos de Cobre , Vías de Administración de Medicamentos , Exosomas/química , Femenino , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/química , Radioisótopos de Galio , Compuestos Heterocíclicos con 1 Anillo/química , Inyecciones Intravenosas , Marcaje Isotópico/métodos , Ratones Endogámicos BALB C , Tomografía de Emisión de Positrones/métodos , Distribución TisularRESUMEN
Brain endothelial cells (BECs) hinder macromolecules from reaching brain parenchyma, necessitating the evaluation and engineering of therapeutic immunoglobulin γ (IgG) for improved brain delivery. Emerging fluorescent-based approaches to assess IgG brain exposure can expedite and complement current methods; however, alterations in IgG pharmacokinetics following fluorophore conjugation, which remain unexplained, indicate that conjugation may confound analysis of native IgG processing. Here, changes in transcytosis and intracellular processing of IgG conjugates (with sulfonated cyanine 5) were examined using human induced pluripotent stem cell-derived BECs (iBECs). Above a critical degree of labeling, transcytosis rates increased significantly but could be attenuated by nonspecific protein competition. Concurrent increases in intracellular accumulation, which was not attributable to disrupted binding by the neonatal Fc receptor (FcRn), are indicative of indirect reduction of FcRn-mediated recycling that agrees with reported aberrations in the pharmacokinetics of certain unconjugated IgGs. Overall, these findings support the notion that certain fluorophore-IgG conjugates can engage in adsorptive interactions with cell surface moieties, reminiscent of phenomena exhibited by cationized IgG, and provide in vitro criteria to identify changes in IgG processing following fluorophore conjugation.
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Barrera Hematoencefálica/metabolismo , Carbocianinas/farmacocinética , Células Endoteliales/metabolismo , Colorantes Fluorescentes/farmacocinética , Inmunoconjugados/farmacocinética , Inmunoglobulina G/metabolismo , Transcitosis , Adsorción , Barrera Hematoencefálica/efectos de los fármacos , Línea Celular , Células Endoteliales/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Receptores Fc/metabolismoRESUMEN
Photodynamic therapy (PDT), as one of the most powerful photo-therapeutic strategies for cancer treatment with minimum invasiveness, can effectively damage local tumor cells and significantly induce systemic antitumor immunity. However, current nanotechnology-assisted PDT-immunomodulators have either poor penetration for deep tumors or low singlet oxygen generation. Herein, we construct a novel theranostic nanocarrier (HA-PEG-CyI, HPC) by inducing the self-assembly of PEGylated CyI and attaching the ligand HA to its surface. The prepared HPC can be used as an ideal PDT-immunomodulator for synergistic cancer therapy. CyI is an iodinated-cyanine dye with enhanced singlet oxygen generation ability as well as excellent photo-to-photothermal and near-infrared fluorescence imaging properties. Under 808 nm laser irradiation, the prepared HPC can generate both reactive oxygen species (ROS) and elevate temperature which can subsequently result in apoptosis and necrosis at tumor sites. Moreover, the HPC-induced cell death can generate a series of acute inflammatory reactions, leading to systemic immunity induction and secondary death of tumor cells, which further results in reducing tumor recurrence. In vitro and in vivo results show that HPC can enhance the tumor targeting efficacy, generate ROS efficiently and exhibit a high temperature response under NIR irradiation, which working together can activate immune responses for synergistic phototherapy on tumor cells. Accordingly, the proposed multi-functional HPC nanocarriers represent an important advance in PDT and can be used as a superior cancer treatment strategy with great promise for clinical applications.
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Carbocianinas , Portadores de Fármacos , Hidrocarburos Yodados , Factores Inmunológicos , Nanoestructuras , Neoplasias Experimentales , Fotoquimioterapia , Animales , Apoptosis/efectos de los fármacos , Carbocianinas/química , Carbocianinas/farmacocinética , Carbocianinas/farmacología , Línea Celular Tumoral , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacología , Femenino , Humanos , Hidrocarburos Yodados/química , Hidrocarburos Yodados/farmacocinética , Hidrocarburos Yodados/farmacología , Factores Inmunológicos/química , Factores Inmunológicos/farmacocinética , Factores Inmunológicos/farmacología , Ratones , Ratones Endogámicos BALB C , Nanoestructuras/química , Nanoestructuras/uso terapéutico , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Células RAW 264.7RESUMEN
Prostate cancer surgery is currently being revolutionized by the use of prostate-specific membrane antigen (PSMA)-targeted radiotracers, for example, 99mTc-labeled PSMA tracer analogs for radioguided surgery. The purpose of this study was to develop a second-generation 99mTc-labeled PSMA-targeted tracer incorporating a fluorescent dye. Methods: Several PSMA-targeted hybrid tracers were synthesized: glutamic acid-urea-lysine (EuK)-Cy5-mas3, EuK-(SO3)Cy5-mas3, EuK-Cy5(SO3)-mas3, EuK-(Ar)Cy5-mas3, and EuK-Cy5(Ar)-mas3; the Cy5 dye acts as a functional backbone between the EuK targeting vector and the 2-mercaptoacetyl-seryl-seryl-seryl (mas3) chelate to study the dye's interaction with PSMA's amphipathic entrance funnel. The compounds were evaluated for their photophysical and chemical properties and PSMA affinity. After radiolabeling with 99mTc, we performed in vivo SPECT imaging, biodistribution, and fluorescence imaging on BALB/c nude mice with orthotopically transplanted PC346C tumors. Results: The dye composition influenced the photophysical properties (brightness range 0.3-1.5 × 104 M-1 × cm-1), plasma protein interactions (range 85.0% ± 2.3%-90.7% ± 1.3% bound to serum, range 76% ± 0%-89% ± 6% stability in serum), PSMA affinity (half-maximal inhibitory concentration [IC50] range 19.2 ± 5.8-175.3 ± 61.1 nM) and in vivo characteristics (tumor-to-prostate and tumor-to-muscle ratios range 0.02 ± 0.00-154.73 ± 28.48 and 0.46 ± 0.28-5,157.50 ± 949.17, respectively; renal, splenic, and salivary retention). Even though all tracer analogs allowed tumor identification with SPECT and fluorescence imaging, 99mTc-EuK-(SO3)Cy5-mas3 had the most promising properties (e.g., half-maximal inhibitory concentration, 19.2 ± 5.8, tumor-to-muscle ratio, 5,157.50 ± 949.17). Conclusion: Our findings demonstrate the intrinsic integration of a fluorophore in the pharmacophore in PSMA-targeted small-molecule tracers. In this design, having 1 sulfonate on the indole moiety adjacent to EuK (99mTc-EuK-(SO3)Cy5-mas3) yielded the most promising tracer candidate for imaging of PSMA.
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Antígenos de Superficie/química , Antígenos de Superficie/metabolismo , Carbocianinas/química , Carbocianinas/farmacocinética , Colorantes/química , Colorantes/farmacocinética , Glutamato Carboxipeptidasa II/química , Glutamato Carboxipeptidasa II/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Humanos , Ratones , Trazadores Radiactivos , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
The renal primary cilium is a small microtubule-based appendage thought to have mechano/chemosensory roles detecting changes in the fluid passing through the nephron. Mutations affecting cilium structure or function of ciliary-localized proteins result in a spectrum of diseases termed ciliopathies, with prevalent phenotypes such as the formation of renal cysts and fibrosis. While many studies have been conducted using fixed kidney sections or live imaging of cells in culture to investigate the cilium, examination in the context of a living murine kidney remains to be conducted. Previously, our lab generated the SSTR3GFP mouse to study cilium dynamics in vivo and found novel cilium behaviors that occurred following alteration of heart rate, blood pressure, and tubule flow. In this manuscript, we utilize multiple transgenic mouse models and an abdominal window imaging approach to observe primary cilia and tubule flow dynamics, immune cell movement, and renal Ca2+ signaling as it occurs in real time within a live mouse kidney. We present this window method as an approach that can be used in combination with various fluorescently labeled transgenic mice to investigate renal physiology, pathology, and function in vivo in longitudinal studies for as long as 5weeks.
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Técnicas Biosensibles , Señalización del Calcio/fisiología , Cilios/ultraestructura , Microscopía Intravital/métodos , Túbulos Renales/ultraestructura , Microscopía Confocal/métodos , Animales , Presión Sanguínea/fisiología , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/metabolismo , Carbocianinas/química , Carbocianinas/farmacocinética , Cilios/fisiología , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacocinética , Expresión Génica , Genes Reporteros , Glicoconjugados/química , Glicoconjugados/farmacocinética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Frecuencia Cardíaca/fisiología , Microscopía Intravital/instrumentación , Túbulos Renales/fisiología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Transgénicos , Microscopía Confocal/instrumentación , Receptores CCR2/genética , Receptores CCR2/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reología , Proteína Fluorescente RojaRESUMEN
A detailed understanding of the cellular uptake and trafficking of nanomaterials is essential for the design of "smart" intracellular drug delivery vehicles. Typically, cellular interactions can be tailored by endowing materials with specific properties, for example, through the introduction of charges or targeting groups. In this study, water-soluble carboxylated N-acylated poly(amino ester)-based comb polymers of different degree of polymerization and side-chain modification were synthesized via a combination of spontaneous zwitterionic copolymerization and redox-initiated reversible addition-fragmentation chain-transfer polymerization and fully characterized by 1H NMR spectroscopy and size exclusion chromatography. The comb polymers showed no cell toxicity against NIH/3T3 and N27 cell lines nor hemolysis. Detailed cellular association and uptake studies by flow cytometry and confocal laser scanning microscopy (CLSM) revealed that the carboxylated polymers were capable of passively diffusing cell membranes and targeting mitochondria. The interplay of pendant carboxylic acids of the comb polymers and the Cy5-label was identified as major driving force for this behavior, which was demonstrated to be applicable in NIH/3T3 and N27 cell lines. Blocking of the carboxylic acids through modification with 2-methoxyethylamine and poly(2-ethyl-2-oxazoline) or replacement of the dye label with a different dye (e.g., fluorescein) resulted in an alteration of the cellular uptake mechanism toward endocytosis as demonstrated by CLSM. In contrast, partial modification of the carboxylic acid groups allowed to retain the cellular interaction, hence, rendering these comb polymers a highly functional mitochondria targeted carrier platform for future drug delivery applications and imaging purposes.
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Carbocianinas , Membrana Celular/metabolismo , Portadores de Fármacos , Mitocondrias/metabolismo , Polímeros , Animales , Carbocianinas/química , Carbocianinas/farmacocinética , Carbocianinas/farmacología , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacología , Endocitosis , Citometría de Flujo , Ratones , Microscopía Confocal , Células 3T3 NIH , Polímeros/química , Polímeros/farmacocinética , Polímeros/farmacología , RatasRESUMEN
A major restriction on optical imaging techniques is the range of available fluorophores that are compatible with aqueous media without aggregation, absorb light above 750 nm with high extinction coefficients, fluoresce with relatively high quantum yields, and resist photodecomposition. Indocyanine green (ICG or A in this paper) is an important example of a fluorophore that fits this description. Other dyes that are becoming increasingly prevalent are select heptamethine cyanine dyes (Cy7) which feature a cyclohexyl framework to rigidify the conjugated alkenes, and meso-chlorine substitution; MHI-148 (B) is one example. Methods: Research described here was initiated to uncover the consequences of a simple isoelectronic substitution to MHI-148 that replaces a cyclohexyl methylene with a dialkyl ammonium fragment. Solubility experiments were carried out in aqueous and cell culture media, photophysical properties including fluorescence quantum yields, brightness and stability were measured. Moreover, in vivo pharmacokinetics, distribution and tumor seeking properties were also explored. Results: Modification to incorporate dialkyl ammonium fragment leads to a brighter, more photostable fluorophore, with a decreased tendency to aggregation, complementary solubility characteristics, and a lower cytotoxicity. Conclusion: All the above-mentioned parameters are favorable for many anticipated applications of the new dye we now call quaternary cyanine-7 or QuatCy.
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Carbocianinas/síntesis química , Colorantes Fluorescentes/síntesis química , Neoplasias/diagnóstico por imagen , Imagen Óptica/métodos , Animales , Carbocianinas/administración & dosificación , Carbocianinas/efectos adversos , Carbocianinas/farmacocinética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/efectos adversos , Colorantes Fluorescentes/farmacocinética , Ratones , Estructura Molecular , SolubilidadRESUMEN
Phototheranostics, which combines deep tissue imaging and phototherapy [photodynamic therapy (PDT) and/or photothermal therapy (PTT)] via light irradiation, is a promising strategy to treat tumors. Near-infrared (NIR) cyanine dyes are researched as potential phototheranostics reagents for their excellent photophysical properties. However, the low singlet oxygen generation efficiency of cyanine dyes often leads to inadequate therapeutic efficacy for tumors. Herein, we modified an indocyanine green derivative Cy7 with heavy atom iodine to form a novel NIR dye CyI to improve the reactive oxygen species (ROS) production and heat generation while, at the same time, maintain their fluorescence characteristics for in vivo noninvasive imaging. More importantly, in vitro and in vivo therapeutic results illustrated that CyI could quickly and simultaneously generate enhanced ROS and heat to induce more cancer cell apoptosis and higher inhibition rates in deep HepG2 tumors than other noniodinated NIR dyes upon NIR irradiation. Besides, low toxicity of the resulted iodinated NIR dyes was confirmed by in vivo biodistribution and acute toxicity. Results indicate that this low toxic NIR dye could be an ideal phototheranostics agent for deep tumor treatments. Our study presents a novel approach to achieve the fast-synergistic PDT/PTT treatment in deep tissues.
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Carbocianinas , Hidrocarburos Yodados , Hipertermia Inducida , Neoplasias Experimentales , Fototerapia , Animales , Carbocianinas/química , Carbocianinas/farmacocinética , Carbocianinas/farmacología , Células Hep G2 , Humanos , Hidrocarburos Yodados/química , Hidrocarburos Yodados/farmacocinética , Hidrocarburos Yodados/farmacología , Ratones , Ratones Desnudos , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Especies Reactivas de Oxígeno/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
It is highly desirable to realize real-time monitoring of the drug delivery/release process in cancer treatment. Herein, a monitorable mitochondria-specific DNAtrain (MitoDNAtrs) was developed for image-guided drug delivery and synergistic cancer therapy. In this system, mitochondria-targeting Cy5.5 dye served as the "locomotive" to guide the DNA "vehicle" selectively accumulating in the cancer cells in a detectable manner. More importantly, Cy5.5 showed reactive oxygen species (ROS) generation ability, which made it a promising adjuvant chemotherapy amplifier for cancer theranostics.
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Antineoplásicos/administración & dosificación , Carbocianinas/farmacocinética , Sistemas de Liberación de Medicamentos/métodos , Mitocondrias/efectos de los fármacos , Carbocianinas/química , ADN/química , ADN/farmacocinética , Doxorrubicina/administración & dosificación , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Portadores de Fármacos/farmacología , Sinergismo Farmacológico , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacocinética , Humanos , Células MCF-7 , Mitocondrias/metabolismo , Sulfobromoftaleína/farmacología , Nanomedicina Teranóstica/métodosRESUMEN
Colorectal cancer is a common malignant tumour with a low 5-year survival rate. A combination therapy with high selectivity and easy controllability is a pressing need for the effective treatment of such cancer. In this study, an indocyanine green derivative (Cy7)-conjugated lipid with a terminal carboxyl group was synthesized, which could self-assemble with a cerasome-forming lipid (CFL) into nanoparticles (NPs) by encapsulating doxorubicin (DOX) to achieve combined photothermal chemotherapy. The resulting Gly@Cy7-Si-DOX NPs with a surface covalent silicate framework showed excellent morphological stability and colloidal stability. Specifically, the conjugated Cy7 was covalently conjugated in the liposomal bilayer, resulting in high drug loading content, high photostability, and high photothermal conversion efficiency, which enabled the resulting nanoparticles to be an effective platform for photothermal therapy. Meanwhile, the encapsulated DOX leaked only slightly under physiological conditions due to the silicate surface of Gly@Cy7-Si-DOX NPs and exhibited controlled release in a weakly acidic environment or under near-infrared (NIR) light irradiation for chemotherapy. Gly@Cy7-Si-DOX NPs were efficiently taken up by tumour cells. Upon light irradiation, the released DOX entered the nuclei of tumour cells, as observed by confocal microscopy and flow cytometry. In vitro cell experiments indicated that both healthy cells and tumour cells were viable under treatment with only Gly@Cy7-Si-DOX NPs, indicating the encapsulated DOX was stably confined to the NPs, and cells were significantly killed when treated with both Gly@Cy7-Si-DOX NPs and NIR laser irradiation. After i.v. administration, Gly@Cy7-Si-DOX NPs accumulated at the tumour site, as monitored by near-infrared fluorescence imaging. A significant tumour inhibition rate (95.8%) was also achieved in a HT-29 colorectal cancer model when treated with Gly@Cy7-Si-DOX NPs plus irradiation. Therefore, the Gly@Cy7-Si-DOX NPs hold great promise for controllable combined colorectal cancer photothermal chemotherapy.
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Carbocianinas/química , Neoplasias Colorrectales/diagnóstico por imagen , Neoplasias Colorrectales/terapia , Colorantes/química , Liposomas/química , Imagen Óptica , Fototerapia/métodos , Animales , Carbocianinas/farmacocinética , Neoplasias Colorrectales/patología , Doxorrubicina/química , Doxorrubicina/farmacología , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Femenino , Células HT29 , Humanos , Rayos Infrarrojos , Ratones , Nanopartículas/química , Distribución TisularRESUMEN
We performed in vivo/ex vivo/polyacrylamide gel electrophoresis (PAGE) fluorescence imaging of near-infrared fluorescence (NIRF)-labeled siRNA (Cy5.5-siGL3) in mice to investigate the validity of each fluorescence imaging result as the biodistribution/biostability assessment of siRNA. Statistically significant correlations could be obtained between the in vivo and ex vivo fluorescence intensities of Cy5.5 in the relevant regions/tissues, except the lung region/tissue after intravenous administration. On PAGE fluorescence images with the naked formulation, there was no band corresponding to intact Cy5.5-siGL3 from all the tissues evaluated after intravenous administration, indicating that the fluorescence detected by in vivo and ex vivo fluorescence imaging was derived from degraded Cy5.5-siGL3 or free Cy5.5 cleaved from Cy5.5-siGL3. However, the band was detected from the lungs after intratracheal administration of the naked formulation, confirming higher stability of siRNA on the respiratory epithelium than in the blood. Regarding the polyethyleneimine formulation, the band was detected from all the tissues evaluated after intravenous administration and from the lungs after intratracheal administration, verifying the enhanced stability of siRNA in the body. These results clearly indicated the necessity of comprehensive analysis from in vivo/ex vivo/PAGE fluorescence imaging to precisely assess the distribution and stability of NIRF-labeled oligonucleotides including siRNA in the body.
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Carbocianinas/administración & dosificación , Polietileneimina/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Administración por Inhalación , Administración Intravenosa , Animales , Carbocianinas/farmacocinética , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Luciferasas de Luciérnaga/genética , Pulmón/metabolismo , Ratones Endogámicos ICR , Imagen Óptica , Polietileneimina/farmacocinética , ARN Interferente Pequeño/farmacocinética , Distribución TisularRESUMEN
Near-infrared (NIR) fluorescence imaging is a promising new medical imaging modality. Associated with a targeting molecule, NIR fluorophores can accumulate selectively in tissues of interest and become valuable tools for the diagnosis and therapy of various pathologies. To facilitate the design of targeted NIR imaging agents, it is important to identify simple and affordable fluorescent probes, allowing rapid labelling of biovectors such as proteins, ideally in a site-specific manner. Here, we demonstrate that heptamethine cyanine based fluorophores, such as IR-783, that contain a chloro-cyclohexyl moiety within their polymethine chain can react selectively, at neutral pH, with cysteine residues in proteins to give stable, site-specifically labelled conjugates, that emit in the NIR spectral window. This reaction is exemplified with the labelling of peptides and two protein models: albumin and a Fab' antibody fragment. The resulting fluorescent proteins are stable and suitable for in vivo NIR imaging applications, as shown on a mice model. This straightforward one-step procedure, that does not require the prior derivatisation of the fluorophore with a bioconjugatable handle, should facilitate the production and use of near-infrared labelled proteins in life sciences.
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Carbocianinas/química , Cisteína/química , Colorantes Fluorescentes/química , Rayos Infrarrojos , Proteínas/química , Secuencia de Aminoácidos , Animales , Carbocianinas/farmacocinética , Línea Celular Tumoral , Colorantes Fluorescentes/farmacocinética , Halogenación , Ratones , Imagen Óptica , Péptidos/química , Coloración y Etiquetado , Distribución TisularRESUMEN
Molecular entities that localize in tumor tissue are clinically important for targeted delivery of diagnostic, imaging, and therapeutic reagents. Often these targeting entities are designed for specific receptors (e.g., EGFR or integrin receptors). However, there is a subset of cyanine-7 dyes that apparently localize in every type of solid tumor tissue (at least, no exceptions have been reported so far), and they persist there for several days. Consequently, these dyes can be used for near-IR optical imaging of tumors in animal studies, they can be conjugated with cytotoxic species to give experimental theranostics, and there is potential for expanding their use into the development of clinically useful derivatives. Data presented in the literature and in this work indicate that the half-lives of these compounds in serum at 37 °C is on the order of minutes to a few hours, so what accounts for the persistent fluorescence of these dyes in tumor tissue over periods of several days? Literature, solely based on tissue culture experiments featuring a particular receptor blocker, indicates that uptake of these dyes is mediated by the organic anion transporter proteins (OATPs). Data presented in this paper agrees with that conclusion for short-term uptake, but significantly expands understanding of the likely reasons for long-term uptake and persistent tumor localization in vivo.
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Benzotiazoles/metabolismo , Carbocianinas/metabolismo , Colorantes Fluorescentes/metabolismo , Neoplasias/metabolismo , Benzotiazoles/química , Benzotiazoles/farmacocinética , Carbocianinas/química , Carbocianinas/farmacocinética , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacocinética , Humanos , Modelos Moleculares , Neoplasias/diagnóstico por imagen , Imagen Óptica/métodos , Transportadores de Anión Orgánico/metabolismo , Albúmina Sérica Humana/metabolismoRESUMEN
The endothelial cells that form the blood-brain barrier (BBB) are coated with glycocalyx, on the luminal side, and with the basement membrane and astrocyte endfeet, on the abluminal side. However, it is unclear how exactly the glycocalyx and extravascular structures contribute to BBB properties. We used two-photon microscopy in anesthetized mice to record passive transport of four different-sized molecules-sodium fluorescein (376 Da), Alexa Fluor (643 Da), 40-kDa dextran, and 150-kDa dextran-from blood to brain, at the level of single cortical capillaries. Both fluorescein and Alexa penetrated nearly the entire glycocalyx volume, but the dextrans penetrated less than 60% of the volume. This suggested that the glycocalyx was a barrier for large but not small molecules. The estimated permeability of the endothelium was the same for fluorescein and Alexa but several-fold lower for the larger dextrans. In the extravascular compartment, co-localized with astrocyte endfeet, diffusion coefficients of the dyes were an order of magnitude lower than in the brain parenchyma. This suggested that the astrocyte endfeet and basement membrane also contributed to BBB properties. In conclusion, the passive transport of small and large hydrophilic molecules through the BBB was determined by three separate barriers: the glycocalyx, the endothelium, and the extravascular compartment. All three barriers must be taken into account in drug delivery studies and when considering BBB dysfunction in disease states.
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Barrera Hematoencefálica/metabolismo , Endotelio Vascular/metabolismo , Glicocálix/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/fisiología , Carbocianinas/farmacocinética , Carbocianinas/farmacología , Fluoresceína/farmacocinética , Fluoresceína/farmacología , Masculino , Ratones , Microscopía de Fluorescencia por Excitación MultifotónicaRESUMEN
Rationale: Successful treatment of pancreatic cancer remains a challenge due to desmoplasia and prevalence of KRAS mutation. While hedgehog (Hh) ligand levels are upregulated in pancreatic cancer cells and contribute to desmoplasia, there is significant downregulation of tumor suppressor let-7b, which targets mutant KRAS, C-MYC and several other genes involved in pancreatic cancer progression, invasion, and metastasis. We recently explored combination therapy of GDC-0449 (Hh inhibitor) and let-7b mimic using poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (PEG-b-PCC-g-DC-g-TEPA) micelles in pancreatic tumor mouse model. Here, our objective was to determine the biodistribution (BD), pharmacokinetics (PK), therapeutic efficacy and toxicity of this micellar formulation. Methods: We determined the PK of micelles encapsulating Cy5.5-let-7b and GDC-0449 following intravenous injection in orthotopic pancreatic tumor-bearing NSG mice at doses of 2 mg/kg and 10 mg/kg, respectively. Mice were scanned for fluorescence by IVIS to determine the biodistribution of Cy5.5-let-7b at the whole-body level, and its concentration in plasma and major organs was determined by measuring fluorescence using a fluorimeter and by real-time RT-PCR. GDC-0449 concentration was determined by LC/MS/MS. Therapeutic efficacy and toxicity of the micellar formulation of let-7b and GDC-0449 was also determined after two weeks of treatment. Results: The use of a micellar formulation markedly prolonged the elimination half-life (t1/2, e) of Cy5.5-let-7b in plasma from 0.49 ± 0.19 h to 2.65 ± 0.46 h and increased the area-under-the-curve (AUC 0-∞ ) by 7-fold, while t1/2,e and AUC 0-∞ of GDC-0449 were increased by 1.78-fold and 3.2-fold, respectively. The micelles significantly decreased the clearance of both encapsulated let-7b mimic and GDC-0449 compared to the emulsion formulation. Compared to the emulsion counterpart, the micellar formulation elevated the delivery of Cy5.5-let-7b and GDC-0449 to the orthotopic pancreatic tumor tissue by 7.8- and 4.2-fold, respectively. Furthermore, there was a significant reduction in tumor volume and negligible systemic toxicity as evident by hematological parameters and histological evaluation. Conclusion: PEG-b-PCC-g-DC-g-TEPA micelles carrying GDC-0449 and let-7b mimic have great potential to improve drug delivery for pancreatic cancer treatment.
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Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Portadores de Fármacos/administración & dosificación , Micelas , MicroARNs/farmacología , MicroARNs/farmacocinética , Neoplasias Pancreáticas/tratamiento farmacológico , Anilidas/farmacocinética , Anilidas/farmacología , Estructuras Animales/química , Animales , Carbocianinas/farmacocinética , Cromatografía Liquida , Modelos Animales de Enfermedad , Fluorometría , Ratones , Imagen Óptica , Neoplasias Pancreáticas/patología , Plasma/química , Piridinas/farmacocinética , Piridinas/farmacología , Espectrometría de Masas en Tándem , Resultado del Tratamiento , Imagen de Cuerpo EnteroRESUMEN
Photodynamic therapy (PDT) of cancer is dependent on three primary components: photosensitizer (PS), light and oxygen. Because these components are interdependent and vary during the dynamic process of PDT, assessing PDT efficacy may not be trivial. Therefore, it has become necessary to develop pre-treatment planning, on-line monitoring and dosimetry strategies during PDT, which become more critical for two or more chromophore systems, for example, PS-CD (Photosensitizer-Cyanine dye) conjugates developed in our laboratory for fluorescence-imaging and PDT of cancer. In this study, we observed a significant impact of variable light dosimetry; (i) high light fluence and fluence rate (light dose: 135 J/cm², fluence rate: 75 mW/cm²) and (ii) low light fluence and fluence rate (128 J/cm² and 14 mW/cm² and 128 J/cm² and 7 mW/cm²) in photobleaching of the individual chromophores of PS-CD conjugates and their long-term tumor response. The fluorescence at the near-infrared (NIR) region of the PS-NIR fluorophore conjugate was assessed intermittently via fluorescence imaging. The loss of fluorescence, photobleaching, caused by singlet oxygen from the PS was mapped continuously during PDT. The tumor responses (BALB/c mice bearing Colon26 tumors) were assessed after PDT by measuring tumor sizes daily. Our results showed distinctive photobleaching kinetics rates between the PS and CD. Interestingly, compared to higher light fluence, the tumors exposed at low light fluence showed reduced photobleaching and enhanced long-term PDT efficacy. The presence of NIR fluorophore in PS-CD conjugates provides an opportunity of fluorescence imaging and monitoring the photobleaching rate of the CD moiety for large and deeply seated tumors and assessing PDT tumor response in real-time.
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Clorofila/análogos & derivados , Neoplasias del Colon/diagnóstico por imagen , Neoplasias del Colon/tratamiento farmacológico , Glicoconjugados/farmacología , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Radiometría/métodos , Animales , Carbocianinas/química , Carbocianinas/farmacocinética , Clorofila/síntesis química , Clorofila/farmacología , Neoplasias del Colon/patología , Relación Dosis-Respuesta en la Radiación , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacocinética , Glicoconjugados/síntesis química , Indoles/química , Indoles/farmacocinética , Rayos Infrarrojos , Ratones , Ratones Endogámicos BALB C , Imagen Óptica/métodos , Fotoblanqueo , Fotoquimioterapia/instrumentación , Fármacos Fotosensibilizantes/síntesis química , Propionatos/química , Propionatos/farmacocinética , Oxígeno Singlete/química , Oxígeno Singlete/metabolismo , Espectrometría de Fluorescencia/métodos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fluorescence-based whole-body imaging is widely used in the evaluation of nanoparticles (NPs) in small animals, often combined with quantitative analysis to indicate their spatiotemporal distribution following systemic administration. An underlying assumption is that the fluorescence label represents NPs and the intensity increases with the amount of NPs and/or the labeling dyes accumulated in the region of interest. We prepare DiR-loaded poly(lactic- co-glycolic acid) (PLGA) NPs with different surface layers (polyethylene glycol with and without folate terminus) and compare the distribution of fluorescence signals in a mouse model of folate-receptor-expressing tumors by near-infrared fluorescence whole-body imaging. Unexpectedly, we observe that fluorescence distribution patterns differ far more dramatically with DiR loading than with the surface ligand, reaching opposite conclusions with the same type of NPs (tumor-specific delivery vs predominant liver accumulation). Analysis of DiR-loaded PLGA NPs reveals that fluorescence quenching, dequenching, and signal saturation, which occur with the increasing dye content and local NP concentration, are responsible for the conflicting interpretations. This study highlights the critical need for validating fluorescence labeling of NPs in the quantitative analysis of whole-body imaging. In light of our observation, we make suggestions for future whole-body fluorescence imaging in the in vivo evaluation of NP behaviors.