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Neutralizing monoclonal antibodies hold great potential for prevention of human immunodeficiency virus (HIV) acquisition. IgG is the most abundant antibody in human serum, has a long half-life, and potent effector functions, making it a prime candidate for an HIV prevention therapeutic. We combined Positron Emission Tomography imaging and fluorescent microscopy of 64Cu-labeled, photoactivatable-green fluorescent protein HIV (PA-GFP-BaL) and fluorescently labeled HGN194 IgG1 to determine whether intravenously instilled IgG influences viral interaction with mucosal barriers and viral penetration in colorectal tissue 2 h after rectal viral challenge. Our results show that IgG1 did not alter the number of virions found throughout the colon or viral penetration into the epithelium of the rectum or descending colon. A minor increase in virions was observed in the transverse colon of IgG1 treated animals. Overall, the number of viral particles found in the mesenteric lymph nodes was low. However, IgG1 administration resulted in a significant reduction of virions found in mesenteric lymph nodes. Taken together, our results show that HGN194 IgG1 does not prevent virions from penetrating into the colorectal mucosa but may perturb HIV virion access to the lymphatic system.
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Despite the maintenance of YopP/J alleles throughout the human-pathogenic Yersinia lineage, the benefit of YopP/J-induced phagocyte death for Yersinia pathogenesis in animals is not obvious. To determine how the sequence divergence of YopP/J has impacted Yersinia virulence, we examined protein polymorphisms in this type III secreted effector protein across 17 Yersinia species and tested the consequences of polymorphism in a murine model of subacute systemic yersiniosis. Our evolutionary analysis revealed that codon 177 has been subjected to positive selection; the Yersinia enterocolitica residue had been altered from a leucine to a phenylalanine in nearly all Yersinia pseudotuberculosis and Yersinia pestis strains examined. Despite this change being minor, as both leucine and phenylalanine have hydrophobic side chains, reversion of YopJF177 to the ancestral YopJL177 variant yielded a Y. pseudotuberculosis strain with enhanced cytotoxicity toward macrophages, consistent with previous findings. Surprisingly, expression of YopJF177L in the mildly attenuated ksgA- background rendered the strain completely avirulent in mice. Consistent with this hypothesis that YopJ activity relates indirectly to Yersinia pathogenesis in vivo, ksgA- strains lacking functional YopJ failed to kill macrophages but actually regained virulence in animals. Also, treatment with the antiapoptosis drug suramin prevented YopJ-mediated macrophage cytotoxicity and enhanced Y. pseudotuberculosis virulence in vivo. Our results demonstrate that Yersinia-induced cell death is detrimental for bacterial pathogenesis in this animal model of illness and indicate that positive selection has driven YopJ/P and Yersinia evolution toward diminished cytotoxicity and increased virulence, respectively.
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Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Yersiniose/microbiologia , Yersinia/fisiologia , Animais , Proteínas de Bactérias/metabolismo , Suscetibilidade a Doenças , Humanos , Mutação , Virulência/genética , Fatores de Virulência , Yersinia/patogenicidadeRESUMO
Human immunodeficiency virus (HIV) vaccines have not been successful in clinical trials. Dimeric IgA (dIgA) in the form of secretory IgA is the most abundant antibody class in mucosal tissues, making dIgA a prime candidate for potential HIV vaccines. We coupled Positron Emission Tomography (PET) imaging and fluorescent microscopy of 64Cu-labeled, photoactivatable-GFP HIV (PA-GFP-BaL) and fluorescently labeled dIgA to determine how dIgA antibodies influence virus interaction with mucosal barriers and viral penetration in colorectal tissue. Our results show that HIV virions rapidly disseminate throughout the colon two hours after exposure. The presence of dIgA resulted in an increase in virions and penetration depth in the transverse colon. Moreover, virions were found in the mesenteric lymph nodes two hours after viral exposure, and the presence of dIgA led to an increase in virions in mesenteric lymph nodes. Taken together, these technologies enable in vivo and in situ visualization of antibody-virus interactions and detailed investigations of early events in HIV infection.
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Colo/virologia , Anticorpos Anti-HIV , Infecções por HIV , Imunoglobulina A Secretora , Mucosa/virologia , Animais , Macaca mulatta , Mucosa/imunologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , RetoRESUMO
Antigen accumulation in lymph nodes (LNs) is critical for vaccine efficacy, but understanding of vaccine biodistribution in humans or large animals remains limited. Using the rhesus macaque model, we employed a combination of positron emission tomography (PET) and fluorescence imaging to characterize the whole-animal to tissue-level biodistribution of a subunit vaccine comprised of an HIV envelope trimer protein nanoparticle (trimer-NP) and lipid-conjugated CpG adjuvant (amph-CpG). Following immunization in the thigh, PET imaging revealed vaccine uptake primarily in inguinal and iliac LNs, reaching distances up to 17 cm away from the injection site. Within LNs, trimer-NPs exhibited striking accumulation on the periphery of follicular dendritic cell (FDC) networks in B cell follicles. Comparative imaging of soluble Env trimers (not presented on nanoparticles) in naïve or previously-immunized animals revealed diffuse deposition of trimer antigens in LNs following primary immunization, but concentration on FDCs in pre-immunized animals with high levels of trimer-specific IgG. These data demonstrate the capacity of nanoparticle or "albumin hitchhiking" technologies to concentrate vaccines in genitourinary tract-draining LNs, which may be valuable for promoting mucosal immunity.
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Vacinas contra a AIDS , Vacinas , Adjuvantes Imunológicos , Animais , Macaca mulatta , Tomografia por Emissão de Pósitrons , Distribuição TecidualRESUMO
Providing physicians with new imaging agents to help detect cancer with better sensitivity and specificity has the potential to significantly improve patient outcomes. Development of new imaging agents could offer improved early cancer detection during routine screening or help surgeons identify tumor margins for surgical resection. In this study, we evaluate the optical properties of a colorful class of dyes and pigments that humans routinely encounter. The pigments are often used in tattoo inks and the dyes are FDA approved for the coloring of foods, drugs, and cosmetics. We characterized their absorption, fluorescence and Raman scattering properties in the hopes of identifying a new panel of dyes that offer exceptional imaging contrast. We found that some of these coloring agents, coined as "optical inks", exhibit a multitude of useful optical properties, outperforming some of the clinically approved imaging dyes on the market. The best performing optical inks (Green 8 and Orange 16) were further incorporated into liposomal nanoparticles to assess their tumor targeting and optical imaging potential. Mouse xenograft models of colorectal, cervical and lymphoma tumors were used to evaluate the newly developed nano-based imaging contrast agents. After intravenous injection, fluorescence imaging revealed significant localization of the new "optical ink" liposomal nanoparticles in all three tumor models as opposed to their neighboring healthy tissues (p < 0.05). If further developed, these coloring agents could play important roles in the clinical setting. A more sensitive imaging contrast agent could enable earlier cancer detection or help guide surgical resection of tumors, both of which have been shown to significantly improve patient survival.
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Neoplasias , Tatuagem , Corantes , Meios de Contraste , Humanos , Tinta , Imagem ÓpticaRESUMO
OBJECTIVE: Despite the early promising results of 18F-fluorodeoxyglucose positron emission tomography for assessment of vessel wall inflammation, its accuracy in prospective identification of vulnerable plaques has remained limited. Additionally, previous studies have indicated that 18F-fluorodeoxyglucose uptake alone may not allow for accurate identification of specific macrophage activation states. We aimed to determine whether combined measurement of glucose and glutamine accumulation-the 2 most important bioenergetic substrates for macrophages-improves the distinction of macrophage inflammatory states and can be utilized to image atherosclerosis. APPROACH AND RESULTS: Murine peritoneal macrophages (MΦ) were activated ex vivo into proinflammatory states with either lipopolysaccharide (MΦLPS) or interferon-γ+tumor necrosis factor-α (MΦIFN-γ+TNF-α). An alternative polarization phenotype was induced with interleukin-4 (MΦIL-4). The pronounced increase in 2-deoxyglucose uptake distinguishes MΦLPS from MΦIFN-γ+TNF-α, MΦIL-4, and unstimulated macrophages (MΦ0). Despite having comparable levels of 2-deoxyglucose accumulation, MΦIL-4 can be distinguished from both MΦIFN-γ+TNF-α and MΦ0 based on the enhanced glutamine accumulation, which was associated with increased expression of a glutamine transporter, Slc1a5. Ex vivo autoradiography experiments demonstrated distinct and heterogenous patterns of 18F-fluorodeoxyglucose and 14C-glutamine accumulation in atherosclerotic lesions of low-density lipoprotein receptor-null mice fed a high-fat diet. CONCLUSIONS: Combined assessment of glutamine and 2-deoxyglucose accumulation improves the ex vivo identification of macrophage activation states. Combined ex vivo metabolic imaging demonstrates heterogenous and distinct patterns of substrate accumulation in atherosclerotic lesions. Further studies are required to define the in vivo significance of glutamine uptake in atherosclerosis and its potential application in identification of vulnerable plaques.
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Aterosclerose/diagnóstico por imagem , Desoxiglucose/metabolismo , Fluordesoxiglucose F18 , Glutamina/metabolismo , Macrófagos/metabolismo , Placa Aterosclerótica/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Animais , Aorta/diagnóstico por imagem , Aorta/metabolismo , Aterosclerose/metabolismo , Autorradiografia , Camundongos , Placa Aterosclerótica/metabolismoRESUMO
Purpose To determine the divergence of immunometabolic phenotypes of macrophages stimulated with macrophage colony-stimulating factor (M-CSF) and granulocyte-M-CSF (GM-CSF) and its implications for fluorine 18 (18F) fluorodeoxyglucose (FDG) imaging of atherosclerosis. Materials and Methods This study was approved by the animal care committee. Uptake of 2-deoxyglucose and various indexes of oxidative and glycolytic metabolism were evaluated in nonactivated murine peritoneal macrophages (MΦ0) and macrophages stimulated with M-CSF (MΦM-CSF) or GM-CSF (MΦGM-CSF). Intracellular glucose flux was measured by using stable isotope tracing of glycolytic and tricyclic acid intermediary metabolites. 18F-FDG uptake was evaluated in murine atherosclerotic aortas after stimulation with M-CSF or GM-CSF by using quantitative autoradiography. Results Despite inducing distinct activation states, GM-CSF and M-CSF stimulated progressive but similar levels of increased 2-deoxyglucose uptake in macrophages that reached up to sixfold compared with MΦ0. The expression of glucose transporters, oxidative metabolism, and mitochondrial biogenesis were induced to similar levels in MΦM-CSF and MΦGM-CSF. Unexpectedly, there was a 1.7-fold increase in extracellular acidification rate, a 1.4-fold increase in lactate production, and overexpression of several critical glycolytic enzymes in MΦM-CSF compared with MΦGM-CSF with associated increased glucose flux through glycolytic pathway. Quantitative autoradiography demonstrated a 1.6-fold induction of 18F-FDG uptake in murine atherosclerotic plaques by both M-CSF and GM-CSF. Conclusion The proinflammatory and inflammation-resolving activation states of macrophages induced by GM-CSF and M-CSF in either cell culture or atherosclerotic plaques may not be distinguishable by the assessment of glucose uptake. © RSNA, 2016 Online supplemental material is available for this article.
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Fluordesoxiglucose F18 , Glucose/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Inflamação/diagnóstico por imagem , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/metabolismo , Tomografia por Emissão de Pósitrons , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Inflamação/metabolismo , Camundongos , Compostos RadiofarmacêuticosRESUMO
Liposomes can serve as carriers of radionuclides for diagnostic imaging and therapeutic applications. Herein, procedures are outlined for radiolabeling liposomes with the gamma-emitting radionuclide, technetium-99m (99mTc), for noninvasive detection of disease and for monitoring the pharmacokinetics and biodistribution of liposomal drugs, and/or with therapeutic beta-emitting radionuclides, rhenium-186/188 (186/188Re), for radionuclide therapy. These efficient and practical liposome radiolabeling methods use a post-labeling mechanism to load 99mTc or 186/188Re into preformed liposomes prepared in advance of the labeling procedure. For all liposome radiolabeling methods described, a lipophilic chelator is used to transport 99mTc or 186/188Re across the lipid bilayer of the preformed liposomes. Once within the liposome interior, the pre-encapsulated glutathione or ammonium sulfate (pH) gradient provides for stable entrapment of the 99mTc and 186/188Re within the liposomes. In the first method, 99mTc is transported across the lipid bilayer by the lipophilic chelator, hexamethylpropyleneamine oxime (HMPAO) and 99mTc-HMPAO becomes trapped by interaction with the pre-encapsulated glutathione within the liposomes. In the second method, 99mTc or 186/188Re is transported across the lipid bilayer by the lipophilic chelator, N,N-bis(2-mercaptoethyl)-N',N'-diethylethylenediamine (BMEDA), and 99mTc-BMEDA or 186/188Re-BMEDA becomes trapped by interaction with pre-encapsulated glutathione within the liposomes. In the third method, an ammonium sulfate (pH) gradient loading technique is employed using liposomes with an extraliposomal pH of 7.4 and an interior pH of 5.1. BMEDA, which is lipophilic at pH 7.4, serves as a lipophilic chelator for 99mTc or 186/188Re to transport the radionuclides across the lipid bilayer. Once within the more acidic liposome interior, 99mTc/186/188Re-BMEDA complex becomes protonated and more hydrophilic, which results in stable entrapment of the 99mTc/186/188Re-BMEDA complex within the liposomes. Since many commercially available liposomal drugs use an ammonium sulfate (pH) gradient for drug loading, these liposomal drugs can be directly radiolabeled with 99mTc-BMEDA for noninvasive monitoring of tissue distribution during treatment or with 186/188Re-BMEDA for combination chemo-radionuclide therapy.
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Diagnóstico por Imagem/métodos , Lipossomos/química , Radioisótopos/química , Compostos Radiofarmacêuticos/química , Rênio/química , Tecnécio/química , Sulfato de Amônio/química , Etilenodiaminas/química , Glutationa/química , Humanos , Concentração de Íons de Hidrogênio , Compostos Organometálicos/química , Compostos de Organotecnécio/química , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios X , Imagem Corporal TotalRESUMO
INTRODUCTION: A major limitation of current liposomal cancer therapies is the inability of liposome therapeutics to penetrate throughout the entire tumor mass. This inhomogeneous distribution of liposome therapeutics within the tumor has been linked to treatment failure and drug resistance. Both liposome particle transport properties and tumor microenvironment characteristics contribute to this challenge in cancer therapy. This limitation is relevant to both intravenously and intratumorally administered liposome therapeutics. AREAS COVERED: Strategies to improve the intratumoral distribution of liposome therapeutics are described. Combination therapies of intravenous liposome therapeutics with pharmacologic agents modulating abnormal tumor vasculature, interstitial fluid pressure, extracellular matrix components, and tumor associated macrophages are discussed. Combination therapies using external stimuli (hyperthermia, radiofrequency ablation, magnetic field, radiation, and ultrasound) with intravenous liposome therapeutics are discussed. Intratumoral convection-enhanced delivery (CED) of liposomal therapeutics is reviewed. EXPERT OPINION: Optimization of the combination therapies and drug delivery protocols are necessary. Further research should be conducted in appropriate cancer types with consideration of physiochemical features of liposomes and their timing sequence. More investigation of the role of tumor associated macrophages in intratumoral distribution is warranted. Intratumoral infusion of liposomes using CED is a promising approach to improve their distribution within the tumor mass.
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Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológico , Animais , Humanos , LipossomosRESUMO
Breast cancer is the leading cause of cancer deaths among women. Paclitaxel (PTX), an important breast cancer medicine, exhibits reduced bioavailability and therapeutic index due to high hydrophobicity and indiscriminate cytotoxicity. PTX encapsulation in one-level active targeting overcomes such barriers, but enhances toxicity to normal tissues with cancer-similar expression profiles. This research attempted to overcome this challenge by increasing selectivity of cancer cell targeting while maintaining an ability to overcome traditional pharmacological barriers. Thus, a multi-core, multi-targeting construct for tumor specific delivery of PTX was fabricated with (i) an inner-core prodrug targeting the cancer-overexpressed cathepsin B through a cathepsin B-cleavable tetrapeptide that conjugates PTX to a poly(amidoamine) dendrimer, and (ii) the encapsulation of this prodrug (PGD) in an outer core of a RES-evading, folate receptor (FR)-targeting liposome. Compared to traditional FR-targeting PTX liposomes, this sequentially active-targeted dendrosome demonstrated better prodrug retention, an increased cytotoxicity to cancer cells (latter being true when FR and cathepsin B activities were both at moderate-to-high levels) and higher tumor reduction. This research may eventually evolve a product platform with reduced systemic toxicity inherent with traditional chemotherapy and localized toxicity inherent to single-target nanoplatforms, thereby allowing for better tolerance of higher therapeutic load in advanced disease states.
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Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Nanoestruturas , Paclitaxel/uso terapêutico , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Linhagem Celular Tumoral , Feminino , Humanos , Lipossomos , Paclitaxel/administração & dosagemRESUMO
One of the major limitations of current cancer therapy is the inability to deliver tumoricidal agents throughout the entire tumor mass using traditional intravenous administration. Nanoparticles carrying beta-emitting therapeutic radionuclides that are delivered using advanced image-guidance have significant potential to improve solid tumor therapy. The use of image-guidance in combination with nanoparticle carriers can improve the delivery of localized radiation to tumors. Nanoparticles labeled with certain beta-emitting radionuclides are intrinsically theranostic agents that can provide information regarding distribution and regional dosimetry within the tumor and the body. Image-guided thermal therapy results in increased uptake of intravenous nanoparticles within tumors, improving therapy. In addition, nanoparticles are ideal carriers for direct intratumoral infusion of beta-emitting radionuclides by convection enhanced delivery, permitting the delivery of localized therapeutic radiation without the requirement of the radionuclide exiting from the nanoparticle. With this approach, very high doses of radiation can be delivered to solid tumors while sparing normal organs. Recent technological developments in image-guidance, convection enhanced delivery and newly developed nanoparticles carrying beta-emitting radionuclides will be reviewed. Examples will be shown describing how this new approach has promise for the treatment of brain, head and neck, and other types of solid tumors.
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Nanopartículas , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , Compostos Radiofarmacêuticos , Radioterapia Guiada por Imagem , Animais , Humanos , Hipertermia Induzida , Nanopartículas/uso terapêutico , Cintilografia , Compostos Radiofarmacêuticos/uso terapêuticoRESUMO
The adjuvant properties of polyglucosamine/squalene-based nanocapsules (PG-nanocapsules) associated with different subunit antigens has been previously reported. Thus, the aim of the present study was to monitor the biodistribution of PG-nanocapsules and their affinity for the draining lymph nodes after subcutaneous (s.c.) injection. The nanocapsules were efficiently radiolabeled with indium-111 ((111)In) (labeling efficiency of 98%). The diameter and zeta potential values of the unlabeled nanocapsules was preserved after the radiolabeling process and only 20% of the (111)In dissociated from the nanocapsules after 48h of incubation in serum. The radiolabeled nanocapsules and the control (111)InCl3 in saline solution (18.5MBq (500µCi) in 100µL) were injected s.c. in New Zealand White rabbits. The γ-scintigraphy imaging analysis revealed a slow clearance of the nanocapsules from the injection site and their progressive accumulation in the popliteal lymph node over time (3.8%±1.2 of the injected dose at 48h). Indeed, the clearance rate of the nanocapsules from the injection site was significantly slower than that of the control (free (111)InCl3), which rapidly drained into systemic circulation and accumulated mainly in excretion organs (i.e. kidneys and liver). In contrast, the biodistribution of nanocapsules was preferably limited to the lymphatic circulation. These results suggest that the immune potentiating effect previously observed for PG-nanocapsules is mainly due to the formation of a depot at the injection site, which was followed by a slow drainage into the lymphatic system and a prolonged retention in the lymph nodes.
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Linfonodos/diagnóstico por imagem , Nanocápsulas , Polissacarídeos/farmacocinética , Adjuvantes Imunológicos/farmacocinética , Animais , Glucosamina/farmacocinética , Linfocintigrafia , Masculino , Coelhos , Distribuição TecidualRESUMO
World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible (1, 2, 3,4,5). These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) (6). In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed. To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom (7). The Appendix in Lee et al. demonstrates the procedures of phantom fabrication (7). In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. (8).
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Imageamento por Ressonância Magnética/instrumentação , Neoplasias/diagnóstico , Imagens de Fantasmas , Tomografia Computadorizada por Raios X/instrumentação , Ultrassonografia/instrumentação , Humanos , Neoplasias/diagnóstico por imagem , Neoplasias/patologiaRESUMO
The prevalence of hepatocellular carcinoma (HCC) was diminished from 60% to 18% at 15 months of age in C3HeB/FeJ male transgenic mice expressing hMGMT in our previous studies. To directly test if the methyltransferase activity is required for diminished tumor prevalence, two separate lines of transgenic mice bearing an enzymatically inactive form of hMGMT were used. In these lines, cysteine 145 was substituted with alanine (C145A). Expression of the hMGMT C145A transgene in liver was demonstrated by Northern blots and Western blots. Immunohistochemistry revealed predominantly nuclear localization of the hMGMT C145A protein. hMGMT C145A transgenic mice were crossed with lacI transgenic mice to assess mutant frequencies in the presence of the mutant protein. Mutant frequencies were similar among livers of lacI × hMGMT C145A bi-transgenic mice and lacI × wild-type (WT) mice. DNA sequence analysis of recovered lacI mutants revealed similar mutation spectra for hMGMT C145A and WT mice. The prevalence of HCC was also similar for the two tested lines of hMGMT C145A mice, 45% and 48% prevalence with median tumor sizes of 11 and 8 mm, and WT mice, 40% prevalence and median tumor size of 10 mm. These results provide evidence that residue C145 in hMGMT is required to reduce the prevalence of HCC in C3HeB/FeJ mice transgenic for hMGMT.
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Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Fígado/patologia , O(6)-Metilguanina-DNA Metiltransferase/genética , Substituição de Aminoácidos , Animais , Carcinoma Hepatocelular/enzimologia , Ativação Enzimática , Humanos , Fígado/enzimologia , Fígado/metabolismo , Neoplasias Hepáticas/enzimologia , Masculino , Camundongos , Camundongos Transgênicos , O(6)-Metilguanina-DNA Metiltransferase/análise , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , TransgenesRESUMO
The major hurdle in the formulation of liposome-encapsulated haemoglobin (LEH) is the oxidation of haemoglobin (Hb) into methaemoglobin during storage and after administration. In order to reduce this oxidative degradation, we tested various reducing conditions in the presence of catalase. We found that at 37°C more than 50% of Hb oxidized to methaemoglobin within 24 h, whereas in presence of catalase, the oxidation was significantly reduced. The effect of catalase was further enhanced by a reduction mixture containing ß-NAD, d-glucose, adenine, inosine, MgCl2, KCl, KH2PO4 and Na2HPO4; only 14% methaemoglobin was generated in the presence of catalase and reduction mixture. Contrary to the expectation, glutathione, deferoxamine and homocysteine enhanced Hb oxidation. The presence of CRM inside liposomes (250 nm) significantly decreased Hb oxidation. The results suggest that catalase and a well-defined mixture of co-factors may help control Hb oxidation for improvement in the functional life of LEH.
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Substitutos Sanguíneos/administração & dosagem , Substitutos Sanguíneos/metabolismo , Hemoglobinas/administração & dosagem , Hemoglobinas/metabolismo , Lipossomos/química , Animais , Substitutos Sanguíneos/química , Catalase/metabolismo , Bovinos , Hemoglobinas/química , Humanos , Metemoglobina/química , Metemoglobina/metabolismo , Estabilidade ProteicaRESUMO
OBJECTIVES: To investigate the relationship between particle size and gastric emptying in rodents using radiolabeled insoluble polymethyl methacrylate (PMMA) microcapsules/beads. METHODS: PMMA microcapsules (50-500 µm) and beads (0.5-3 mm) loaded with technetium-99 m diethylenetriamine pentaacetic acid ((99m) Tc-DTPA) were administered to ICR mice or Sprague Dawley (SD) rats by oral gavage. Gamma scintiscans were acquired initially following administration and then at hourly intervals to 4 hours. RESULTS: Scintiscans revealed that the smallest PMMA microcapsules (50-100 µm) or beads (0.5-1 mm) were impeded in the stomach and emptied slower than large particles in both rodent species. In mice, no significant difference in gastric emptying was found with microcapsules between 100 and 300 µm in diameter (p = 0.25) and particles more than 300 µm could not be administered. In rats, capsules containing 0.5-3 mm beads were stuck to the esophagus (up to 1 hour), this was a limitation of dosing beads of this size because they cannot be suspended in a liquid media for oral gavage purposes. Beads with diameters of 2-3 mm stayed in the stomach for up to 4 hours. CONCLUSIONS: The cut-off emptying size in ICR mice could not be determined, due to the limitation of current available dosing methods. The cut-off emptying size in SD rats was between 1.5 and 2 mm. Therefore, particles with a diameter greater than 2 mm should not be used for gastric emptying studies of intact particles in SD rats, as their emptying is retarded in the stomach.
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Esvaziamento Gástrico , Polimetil Metacrilato/química , Compostos Radiofarmacêuticos/farmacocinética , Pentetato de Tecnécio Tc 99m/farmacocinética , Animais , Trato Gastrointestinal/diagnóstico por imagem , Trato Gastrointestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Tamanho da Partícula , Polimetil Metacrilato/administração & dosagem , Cintilografia , Compostos Radiofarmacêuticos/administração & dosagem , Ratos , Ratos Sprague-Dawley , Pentetato de Tecnécio Tc 99m/administração & dosagemRESUMO
Camptothecin (CPT) represents a potent anticancer drug. However, its therapeutic use is impaired by both drug solubility, hydrolysis, and protein interactions in vivo. Use of liposomes as a drug-formulation approach could overcome some of these challenges. The aim of this study was to perform a mechanistic study of the incorporation and retention of the lipophilic parent CPT compound in different liposome formulations using radiolabeled CPT and thus to be able to identify promising CPT delivery systems. In this context, we also wanted to establish an appropriate mouse tumor model, in vivo scintigraphic imaging, and biodistribution methodology for testing the most promising formulation. CPT retention in various liposome formulations after incubation in buffer and serum was determined. The HT-29 mouse tumor model, (111)In-labeled liposomes, as well as (3)H-labeled CPT were used to investigate the biodistribution of liposomes and drug. The ability of different liposome formulations to retain CPT in buffer was influenced by lipid concentration and drug/lipid ratio, rather than lipid composition. The tested formulations were cleared from the blood in the following order: CPT solution > CPT liposomes > (111)In-labeled liposomes, and liposomes mainly accumulated in the liver. Lipid composition did not influence CPT retention to the same extent as earlier observed from incorporation studies. The set-up for the biodistribution study works well and is suited for future in vivo studies on CPT liposomes. The biodistribution study showed that liposomes circulated longer than free drug, but premature release of drug from liposomes occurred. Further studies to develop formulations with higher retention potential and prolonged circulation are desired.
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Camptotecina/administração & dosagem , Sistemas de Liberação de Medicamentos , Lipossomos/administração & dosagem , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Camptotecina/química , Humanos , Lipídeos , Lipossomos/química , Camundongos , Neoplasias/patologia , Solubilidade , Distribuição TecidualRESUMO
Most diagnosed early stage breast cancer cases are treated by lumpectomy and adjuvant radiation therapy, which significantly decreases the locoregional recurrence but causes inevitable toxicity to normal tissue. By using a technique of preparing liposomes carrying technetium-99m ((99m)Tc), rhenium-186 ((186)Re), or rhenium-188 ((188)Re) radionuclides, as well as chemotherapeutic agents, or their combination, for cancer therapy with real time image-monitoring of pharmacokinetics and prediction of therapy effect, this study investigated the potential of a novel targeted focal radiotherapy with low systemic toxicity using radioactive immunoliposomes to treat both the surgical cavity and draining lymph nodes in a rat breast cancer xenograft positive surgical margin model. Immunoliposomes modified with either panitumumab (anti-EGFR) or bevacizumab (anti-VEGF) were remote loaded with (99m)Tc diagnostic radionuclide, and injected into the surgical cavity of female nude rats with positive margins postlumpectomy. Locoregional retention and systemic distribution of (99m)Tc-immunoliposomes were investigated by nuclear imaging, stereofluorescent microscopic imaging, and gamma counting. Histopathological examination of excised draining lymph nodes was performed. The locoregional retention of (99m)Tc-immunoliposomes in each animal was influenced by the physiological characteristics of the surgical site of individual animals. Panitumumab- and bevacizumab-liposome groups had higher intracavitary retention compared with the control liposome groups. Draining lymph node uptake was influenced by both the intracavitary radioactivity retention level and metastasis status. The panitumumab-liposome group had higher accumulation on the residual tumor surface and in the metastatic lymph nodes. Radioactive liposomes that were cleared from the cavity were metabolized quickly and accumulated at low levels in vital organs. Therapeutic radionuclide-carrying specifically targeted panitumumab- and bevacizumab-liposomes have increased potential compared to non-antibody targeted liposomes for postlumpectomy focal therapy to eradicate remaining breast cancer cells inside the cavity and draining lymph nodes with low systemic toxicity.
Assuntos
Imunotoxinas/administração & dosagem , Lipossomos/administração & dosagem , Linfonodos/diagnóstico por imagem , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/radioterapia , Radioisótopos/administração & dosagem , Compostos Radiofarmacêuticos/administração & dosagem , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/química , Bevacizumab , Feminino , Imunotoxinas/química , Lipossomos/química , Neoplasias Mamárias Experimentais/prevenção & controle , Neoplasias Mamárias Experimentais/cirurgia , Mastectomia Segmentar/métodos , Panitumumabe , Tamanho da Partícula , Radioisótopos/química , Cintilografia , Compostos Radiofarmacêuticos/química , Ratos , Ratos Nus , Rênio/administração & dosagem , Rênio/química , Tecnécio/administração & dosagem , Tecnécio/químicaRESUMO
BACKGROUND: Gold nanoshells are excellent agents for photothermal ablation cancer therapy and are currently under clinical trial for solid tumors. Previous studies showed that passive delivery of gold nanoshells through intravenous administration resulted in limited tumor accumulation, which represents a major challenge for this therapy. In this report, the impact of direct intratumoral administration on the pharmacokinetics and biodistribution of the nanoshells was systematically investigated. METHODS: The gold nanoshells were labeled with the radionuclide, copper-64 ((64)Cu). Intratumoral infusion of (64)Cu-nanoshells and two controls, ie, (64)Cu-DOTA (1,4,7,10-tetraazaciclododecane- 1,4,7,10-tetraacetic acid) and (64)Cu-DOTA-PEG (polyethylene glycol), as well as intravenous injection of (64)Cu-nanoshells were performed in nude rats, each with a head and neck squamous cell carcinoma xenograft. The pharmacokinetics was determined by radioactive counting of serial blood samples collected from the rats at different time points post-injection. Using positron emission tomography/computed tomography imaging, the in vivo distribution of (64)Cu-nanoshells and the controls was monitored at various time points after injection. Organ biodistribution in the rats at 46 hours was analyzed by radioactive counting and compared between the different groups. RESULTS: The resulting pharmacokinetic curves indicated a similar trend between the intratumorally injected agents, but a significant difference with the intravenously injected (64)Cu-nanoshells. Positron emission tomography images and organ biodistribution results on rats after intratumoral administration showed higher retention of (64)Cu-nanoshells in tumors and less concentration in other healthy organs, with a significant difference from the controls. It was also found that, compared with intravenous injection, tumor concentrations of (64)Cu-nanoshells improved substantially and were stable at 44 hours post-injection. CONCLUSION: There was a higher intratumoral retention of (64)Cu-nanoshells and a lower concentration in other healthy tissues, suggesting that intratumoral administration is a potentially better approach for nanoshell-based photothermal therapy.
Assuntos
Radioisótopos de Cobre/administração & dosagem , Nanoconchas/administração & dosagem , Animais , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/radioterapia , Linhagem Celular Tumoral , Radioisótopos de Cobre/farmacocinética , Estudos de Viabilidade , Ouro , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Injeções Intralesionais , Masculino , Imagem Multimodal , Nanomedicina , Tomografia por Emissão de Pósitrons , Ratos , Ratos Nus , Distribuição Tecidual , Tomografia Computadorizada por Raios X , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Liposomes are effective lipid nanoparticle drug delivery systems, which can also be functionalized with noninvasive multimodality imaging agents with each modality providing distinct information and having synergistic advantages in diagnosis, monitoring of disease treatment, and evaluation of liposomal drug pharmacokinetics. We designed and constructed a multifunctional theranostic liposomal drug delivery system, which integrated multimodality magnetic resonance (MR), near-infrared (NIR) fluorescent and nuclear imaging of liposomal drug delivery, and therapy monitoring and prediction. The premanufactured liposomes were composed of DSPC/cholesterol/Gd-DOTA-DSPE/DOTA-DSPE with the molar ratio of 39:35:25:1 and having ammonium sulfate/pH gradient. A lipidized NIR fluorescent tracer, IRDye-DSPE, was effectively postinserted into the premanufactured liposomes. Doxorubicin could be effectively postloaded into the multifunctional liposomes. The multifunctional doxorubicin-liposomes could also be stably radiolabeled with (99m)Tc or (64)Cu for single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging, respectively. MR images displayed the high-resolution micro-intratumoral distribution of the liposomes in squamous cell carcinoma of head and neck (SCCHN) tumor xenografts in nude rats after intratumoral injection. NIR fluorescent, SPECT, and PET images also clearly showed either the high intratumoral retention or distribution of the multifunctional liposomes. This multifunctional drug carrying liposome system is promising for disease theranostics allowing noninvasive multimodality NIR fluorescent, MR, SPECT, and PET imaging of their in vivo behavior and capitalizing on the inherent advantages of each modality.