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BACKGROUND AIMS: Dendritic cell (DC)-based immunotherapy is a promising approach to treat cancer. However, key aspects governing the reproducible manufacturing of high-quality DC remain incompletely defined. Here, we show that the time window between leukapheresis and DC manufacturing is critical. METHODS: Transcriptomic profiling by RNA-seq was used to unbiasedly characterize cellular states during each step of DC manufacturing process, and functional assays were used to determine the anti-tumor activities of DC. RESULTS: During preclinical development of a DC-based cytotherapy platform, CUD-002 (NCT05270720), we found that DC quality varied among different batches, even though commonly used DC maturation markers CD80, CD83 and CD86 were indistinguishable. Multivariate analysis indicated that DC quality was negatively associated with the shipping time from the leukapheresis site to the manufacturing center. To investigate the potential effect of shipping time, we stored leukapheresis materials from three donors for 0, 1, 2 or 3 days before DC manufacturing. For each step, we carried out RNA-seq analysis to unbiasedly characterize cellular states. Integrated bioinformatic analyses indicated that longer storage time reduced the expression of several transcription factors to attenuate interferon pathways. CONCLUSIONS: Consistently, we found that 3-day storage of leukapheresis materials significantly lowered the efficiency to generate DC but also impaired DC responses to inflammatory signals, resulting in inferior antigen-presentation and cytotoxic T-cell activities. Thus, we recommend using leukapheresis materials within 48 h to manufacture therapeutic DCs.
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Leucaférese , Neoplasias , Humanos , Leucaférese/métodos , Neoplasias/metabolismo , Imunoterapia/métodos , Células Dendríticas/fisiologiaRESUMO
BACKGROUND AIMS: Dendritic cell (DC)-based immunotherapy is a promising approach to treat cancer; however, there is no consensus on the manufacturing processes. Cell type heterogeneity in products manufactured by various methods is understudied and may elicit safety concerns from the regulatory perspective. METHODS: We characterized the cell type composition of a recently developed DC vaccine, CUD-002, consisting of DCs loaded with mRNA encoding personalized tumor neoantigens (NCT05270720). RESULTS: Using single-cell transcriptomic analysis as an unbiased approach, we found that >80% cells in the final product were DCs and the rest primarily comprised myelocytes and lymphocytes. Subsequent fluorescence-activated cell sorting analyses confirmed these cellular identities. These results indicate that unintended cells originate from leukapheresis, the first step of the manufacturing process, and thus likely safe. Consistently, no overt toxicity or tumorigenicity was observed in mice inoculated with CUD-002. CONCLUSIONS: Considering that leukapheresis is a widely used procedure for collecting diverse peripheral blood cell types to manufacture various cytotherapies, this study establishes a workflow to analyze and address regulatory considerations on cell type heterogeneity.
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Vacinas Anticâncer , Neoplasias , Vacinas , Animais , Camundongos , Vacinas Anticâncer/genética , Células Dendríticas , Imunoterapia/métodos , Neoplasias/terapia , Análise de Sequência de RNA , Vacinas/metabolismo , Estudos Clínicos como AssuntoRESUMO
In real-world applications, multiple robots need to be dynamically deployed to their appropriate locations as teams while the distance cost between robots and goals is minimized, which is known to be an NP-hard problem. In this paper, a new framework of team-based multi-robot task allocation and path planning is developed for robot exploration missions through a convex optimization-based distance optimal model. A new distance optimal model is proposed to minimize the traveled distance between robots and their goals. The proposed framework fuses task decomposition, allocation, local sub-task allocation, and path planning. To begin, multiple robots are firstly divided and clustered into a variety of teams considering interrelation and dependencies of robots, and task decomposition. Secondly, the teams with various arbitrary shape enclosing intercorrelative robots are approximated and relaxed into circles, which are mathematically formulated to convex optimization problems to minimize the distance between teams, as well as between a robot and their goals. Once the robot teams are deployed into their appropriate locations, the robot locations are further refined by a graph-based Delaunay triangulation method. Thirdly, in the team, a self-organizing map-based neural network (SOMNN) paradigm is developed to complete the dynamical sub-task allocation and path planning, in which the robots are dynamically assigned to their nearby goals locally. Simulation and comparison studies demonstrate the proposed hybrid multi-robot task allocation and path planning framework is effective and efficient.
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Robótica , Algoritmos , Simulação por Computador , Redes Neurais de Computação , Projetos de PesquisaRESUMO
The rate-limiting step for diagnostics development is the discovery and validation of biomarker analytes. We describe a new analyte-agnostic and label-free approach based on colorimetric reactions involving type I polymerization photoinitiators. We demonstrate that a chemically diverse array of hydrogels embedded with cleaved type I photoinitiators could act as microreactors, undergoing colorimetric reactions with bound analytes. The colorimetric signatures produced were visually distinctive and readable with a flatbed document scanner. Signatures of a broad range of sample types were accurately differentiated by unsupervised clustering without knowledge of any analytes bound to the array. The principles described have the potential to enable scalable and cost-effective analysis of complex samples.
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Colorimetria , Língua , Polimerização , HidrogéisRESUMO
Macrophage-based cell therapeutics is an emerging modality to treat cancer and repair tissue damage. A reproducible manufacturing and engineering process is central to fulfilling their therapeutic potential. Here, we establish a robust macrophage-manufacturing platform (Mo-Mac) and demonstrate that macrophage functionality can be enhanced by N1-methylpseudouridine (m1Ψ)-modified mRNA. Using single-cell transcriptomic analysis as an unbiased approach, we found that >90% cells in the final product were macrophages while the rest primarily comprised T cells, B cells, natural killer cells, promyelocytes, promonocytes, and hematopoietic stem cells. This analysis also guided the development of flow-cytometry strategies to assess cell compositions in the manufactured product to meet requirements by the National Medical Products Administration. To modulate macrophage functionality, as an illustrative example we examined whether the engulfment capability of macrophages could be enhanced by mRNA technology. We found that efferocytosis was increased in vitro when macrophages were electroporated with m1Ψ-modified mRNA encoding CD300LF (CD300LF-mRNA-macrophage). Consistently, in a mouse model of acute liver failure, CD300LF-mRNA-macrophages facilitated organ recovery from acetaminophen-induced hepatotoxicity. These results demonstrate a GMP-compliant macrophage-manufacturing process and indicate that macrophages can be engineered by versatile mRNA technology to achieve therapeutic goals.
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Protein phosphorylation plays an important role in the regulation of self-renewal and differentiation of embryonic stem cells. However, the responsible intracellular kinases are not well characterized. Here, we discovered that cyclin K protein was highly expressed in pluripotent embryonic stem cells but low in their differentiated derivatives or tissue-specific stem cells. Upon cell differentiation, the level of cyclin K protein was decreased. Furthermore, knockdown of cyclin K led to cell differentiation, which could be rescued by an expression construct resistant to RNA interference. Surprisingly, cyclin K did not interact with CDK9 protein in cells as thought previously. Instead, it associated with CrkRS (also known as CDK12) and CDC2L5 (also known as CDK13). Similar to cyclin K, both CDK12 and CDK13 proteins were highly expressed in murine embryonic stem cells and were decreased upon cell differentiation. Importantly, knockdown of either kinase resulted in differentiation. Thus, our studies have uncovered two novel protein kinase complexes that maintain self-renewal in embryonic stem cells.
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Diferenciação Celular/fisiologia , Quinases Ciclina-Dependentes/biossíntese , Ciclinas/biossíntese , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/fisiologia , Complexos Multiproteicos/biossíntese , Animais , Linhagem Celular , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Células-Tronco Embrionárias/citologia , Camundongos , Complexos Multiproteicos/genéticaRESUMO
AbstractNear-infrared (NIR) fluorophores are the focus of extensive research for combined molecular imaging and hyperthermia. In this study, we showed that the cyanine dye IR820 has optical and thermal generation properties similar to those of indocyanine green (ICG) but with improved in vitro and in vivo stability. The fluorescent emission of IR820 has a lower quantum yield than ICG but less dependence of the emission peak location on concentration. IR820 demonstrated degradation half-times approximately double those of ICG under all temperature and light conditions in aqueous solution. In hyperthermia applications, IR820 generated lower peak temperatures than ICG (4-9%) after 3-minute laser exposure. However, there was no significant difference in hyperthermia cytotoxicity, with both dyes causing significant cell growth inhibition at concentrations ≥ 5 µM. Fluorescent images of cells with 10 µM IR820 were similar to ICG images. In rats, IR820 resulted in a significantly more intense fluorescence signal and significantly higher organ dye content than for ICG 24 hours after intravenous dye administration (p < .05). Our study shows that IR820 is a feasible agent in experimental models of imaging and hyperthermia and could be an alternative to ICG when greater stability, longer image collection times, or more predictable peak locations are desirable.
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Temperatura Alta , Verde de Indocianina/análogos & derivados , Verde de Indocianina/química , Luz , Abdome , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Hipertermia Induzida , Verde de Indocianina/administração & dosagem , Verde de Indocianina/toxicidade , Injeções Intravenosas , Especificidade de Órgãos/efeitos dos fármacos , Ratos , Ratos Wistar , Soluções , Solventes , Espectrometria de Fluorescência , Espectroscopia de Luz Próxima ao Infravermelho , Fatores de TempoRESUMO
OBJECTIVE: To clone a novel apoptosis-related gene, human PNAS-4, and to get it expressed in E. coli. METHODS: Human PNAS-4 gene was amplified by RT-PCR form A549 human lung adenocarcinoma cells and inserted into pGEX-6P-1 vector. The resulting recombinant plasmid was transformed into E. coli. BL21. Human PNAS-4 protein was expressed with IPTG induction and the purified protein was identified by SDS-PAGE and mass spectrometry. RESULTS: The sequence of the human PNAS-4 gene in the recombinant plasmid was identical with that published in GenBank. The purified fusion protein of human PNAS-4 with relative molecular mass of 50 000 Da was observed in SDS-PAGE analysis, and was identified to be human PNAS-4 by mass spectrometry. CONCLUSION: Human PNAS-4 is expressed and purified successfully, which would ba a foundation for further research on the function of human PNAS-4 gene.
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Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/isolamento & purificação , Vetores Genéticos/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Reguladoras de Apoptose/biossíntese , Carbono-Nitrogênio Liases , Linhagem Celular Tumoral , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Espectrometria de Massas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
With the introduction of autonomy into the precision agriculture process, environmental exploration, disaster response, and other fields, one of the global demands is to navigate autonomous vehicles to completely cover entire unknown environments. In the previous complete coverage path planning (CCPP) research, however, autonomous vehicles need to consider mapping, obstacle avoidance, and route planning simultaneously during operating in the workspace, which results in an extremely complicated and computationally expensive navigation system. In this study, a new framework is developed in light of a hierarchical manner with the obtained environmental information and gradually solving navigation problems layer by layer, consisting of environmental mapping, path generation, CCPP, and dynamic obstacle avoidance. The first layer based on satellite images utilizes a deep learning method to generate the CCPP trajectory through the position of the autonomous vehicle. In the second layer, an obstacle fusion paradigm in the map is developed based on the unmanned aerial vehicle (UAV) onboard sensors. A nature-inspired algorithm is adopted for obstacle avoidance and CCPP re-joint. Equipped with the onboard LIDAR equipment, autonomous vehicles, in the third layer, dynamically avoid moving obstacles. Simulated experiments validate the effectiveness and robustness of the proposed framework.
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The purpose of this study was to compare the cellular uptake and cytotoxicity of targeted and nontargeted doxorubicin (DOX)-loaded poly(d,l-lactide co-glycolide) (PLGA) nanoparticle (NP) drug delivery systems in drug-resistant ovarian (SKOV-3) and uterine (MES-SA/Dx5) cancer cell lines. The cellular uptakes of DOX from nonconjugated DOX-loaded NPs (DNPs) and from HER-2 antibody-conjugated DOX-loaded NPs (ADNPs) in MES-SA/Dx5 cancer cells were higher compared to free DOX. Results also showed higher uptake of DOX from ADNPs in SKOV-3 cells compared with both free DOX and DNPs treatment. Cytotoxicity results at 10 µM extracellular DOX concentration were consistent with the cellular uptake results. Our study concludes that cellular uptake and cytotoxicity of DOX can be improved in MES-SA/Dx5 cells by loading DOX into PLGA NPs. DNPs targeted to membrane receptors may enhance cellular uptake and cytotoxicity in SKOV-3 cells. FROM THE CLINICAL EDITOR: The authors of this study compare the cellular uptake and cytotoxicity of targeted and nontargeted doxorubicin loaded PLGA nanoparticle delivery systems in drug-resistant ovarian and uterine cancer cell lines, concluding that cellular uptake and cytotoxicity of doxorubicin can be improved by the proposed methods.
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Portadores de Fármacos/toxicidade , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Anticorpos/imunologia , Transporte Biológico/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Química Farmacêutica , Doxorrubicina/farmacologia , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Cinética , Luz , Microscopia Confocal , Nanopartículas/química , Tamanho da Partícula , Espalhamento de Radiação , Eletricidade Estática , Frações Subcelulares/metabolismoRESUMO
PURPOSE: To test the effectiveness of a dual-agent-loaded PLGA nanoparticulate drug delivery system containing doxorubicin (DOX) and indocyanine green (ICG) in a DOX-sensitive cell line and two resistant cell lines that have different resistance mechanisms. METHODS: The DOX-sensitive MES-SA uterine sarcoma cell line was used as a negative control. The two resistant cell lines were uterine sarcoma MES-SA/Dx5, which overexpresses the multidrug resistance exporter P-glycoprotein, and ovarian carcinoma SKOV-3, which is less sensitive to doxorubicin due to a p53 gene mutation. The cellular uptake, subcellular localization and cytotoxicity of the two agents when delivered via nanoparticles (NPs) were compared to their free-form administration. RESULTS: The cellular uptake and cytotoxicity of DOX delivered by NPs were comparable to the free form in MES-SA and SKOV-3, but much higher in MES-SA/Dx5, indicating the capability of the NPs to overcome P-glycoprotein resistance mechanisms. NP-encapsulated ICG showed slightly different subcellular localization, but similar fluorescence intensity when compared to free ICG, and retained the ability to generate heat for hyperthermia delivery. CONCLUSION: The dual-agent-loaded system allowed for the simultaneous delivery of hyperthermia and chemotherapy, and this combinational treatment greatly improved cytotoxicity in MES-SA/Dx5 cells and to a lesser extent in SKOV-3 cells.
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Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Portadores de Fármacos/química , Ácido Láctico/química , Nanopartículas/química , Compostos Orgânicos/administração & dosagem , Ácido Poliglicólico/química , Antineoplásicos/farmacocinética , Transporte Biológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Hipertermia Induzida/métodos , Microscopia Eletrônica de Varredura , Fenômenos Ópticos , Compostos Orgânicos/farmacocinética , Compostos Orgânicos/farmacologia , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Solubilidade , Propriedades de SuperfícieRESUMO
Embryonic stem cells (ESCs) are self-renewing pluripotent cells, capable of differentiating into all somatic cell types. The molecular control of self-renewal is relatively well-characterized, whereas how ESCs exit pluripotent state to differentiate is poorly understood. Here we identify two genes are required for differentiation and dozens of intergenic regions that potentially regulate ESC differentiation. We used PiggyBac (PB) transposon-based approach to randomly mutate the genome of ESCs, and generated hundreds of clones that resisted differentiation signals. Each clone was sequenced to determine genomic regions mutated by PB insertion. Intriguingly, many mutations were localized among intergenic regions and we identified two genes are required for differentiation. This study should facilitate further exploration of novel molecular determinants of embryonic stem cell differentiation.
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Elementos de DNA Transponíveis , Células-Tronco Embrionárias Murinas/citologia , Mutagênese Insercional/métodos , Animais , Diferenciação Celular , Proliferação de Células , Sistema de Sinalização das MAP Quinases , Camundongos , Mutação , Análise de Sequência de DNARESUMO
The assembly of prereplicative complex (pre-RC) during G1 phase must be tightly controlled to sustain cell proliferation and maintain genomic stability. Mechanisms to prevent pre-RC formation in G2/M and S phases are well appreciated, whereas how cells ensure efficient pre-RC assembly during G1 is less clear. Here we report that cyclin K regulates pre-RC formation. We find that cyclin K expression positively correlates with cell proliferation, and knockdown of cyclin K or its cognate kinase CDK12 prevents the assembly of pre-RC in G1 phase. Mechanistically we uncover that cyclin K promotes pre-RC assembly by restricting cyclin E1 activity in G1. We identify a cyclin K-dependent, novel phosphorylation site in cyclin E1 that disrupts its interaction with CDK2. Importantly, this antagonistic relationship is largely recapitulated in cyclin E1-overexpressing tumors. We discuss the implications of our findings in light of recent reports linking cyclin K and CDK12 to human tumorigenesis.
Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Proliferação de Células , Ciclina E/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/antagonistas & inibidores , Ciclinas/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Instabilidade Genômica , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/genéticaRESUMO
Anthracyclines cause severe irreversible cardiac toxicity. The study of changes in cardiac permeability with chemotherapy could enhance the understanding of mechanisms behind cardiac damage, and provide useful information to evaluate anthracycline cardiotoxicity. Thirty-six rats (12 Sprague-Dawley, 12 Wistar, 12 Fischer-344) were randomly assigned to control (n = 21) or doxorubicin (n = 15), and injected i.p. with a cumulative dose of 18 mg/kg doxorubicin in saline (vehicle) or vehicle alone over 12 days. Echocardiography was performed at baseline and on day 11. An isolated heart experiment was done on day 12 to obtain perfused heart pressure values, and to measure cardiac capillary permeability using a Texas Red/sodium fluorescein multiple indicator dilution method. Control animals had significantly lower average permeability-surface-area-products (0.035 ± 0.013 cm(3)/s) than doxorubicin animals (0.066 ± 0.023 cm(3)/s), PSP ± SD, p < 0.001. These permeability changes correlated with significant functional changes. There was a significant decline in cardiac function with a deleterious effect of chemotherapy on fractional shortening (p < 0.001), left ventricular developed pressure (p < 0.001), contractility (p < 0.001), and relaxation (p = 0.02). Based on our results, cardiac capillary permeability changes can be detected after in vivo chemotherapy treatment using our fluorescent multiple indicator dilution technique, and may provide valuable information in evaluating cardiotoxicity of novel drugs.
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Antibióticos Antineoplásicos , Permeabilidade Capilar/efeitos dos fármacos , Cardiotoxinas , Doxorrubicina , Coração/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/efeitos adversos , Antibióticos Antineoplásicos/farmacocinética , Cardiotoxicidade/metabolismo , Cardiotoxicidade/fisiopatologia , Cardiotoxinas/efeitos adversos , Cardiotoxinas/farmacocinética , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacocinética , Ecocardiografia/efeitos dos fármacos , Fluorescência , Coração/fisiologia , Miocárdio/metabolismo , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Ratos Wistar , Pressão Ventricular/efeitos dos fármacosRESUMO
Near-infrared dyes can be used as theranostic agents in cancer management, based on their optical imaging and localized hyperthermia capabilities. However, their clinical translatability is limited by issues such as photobleaching, short circulation times, and nonspecific biodistribution. Nanoconjugate formulations of cyanine dyes, such as IR820, may be able to overcome some of these limitations. We covalently conjugated IR820 with 6 kDa polyethylene glycol (PEG)-diamine to create a nanoconjugate (IRPDcov) with potential for in vivo applications. The conjugation process resulted in nearly spherical, uniformly distributed nanoparticles of approximately 150 nm diameter and zeta potential -0.4±0.3 mV. The IRPDcov formulation retained the ability to fluoresce and to cause hyperthermia-mediated cell-growth inhibition, with enhanced internalization and significantly enhanced cytotoxic hyperthermia effects in cancer cells compared with free dye. Additionally, IRPDcov demonstrated a significantly longer (P<0.05) plasma half-life, elimination half-life, and area under the curve (AUC) value compared with IR820, indicating larger overall exposure to the theranostic agent in mice. The IRPDcov conjugate had different organ localization than did free IR820, with potential reduced accumulation in the kidneys and significantly lower (P<0.05) accumulation in the lungs. Some potential advantages of IR820-PEG-diamine nanoconjugates may include passive targeting of tumor tissue through the enhanced permeability and retention effect, prolonged circulation times resulting in increased windows for combined diagnosis and therapy, and further opportunities for functionalization, targeting, and customization. The conjugation of PEG-diamine with a near-infrared dye provides a multifunctional delivery vector whose localization can be monitored with noninvasive techniques and that may also serve for guided hyperthermia cancer treatments.
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Antineoplásicos/química , Diaminas/química , Verde de Indocianina/análogos & derivados , Nanoconjugados/química , Imagem Óptica/métodos , Polietilenoglicóis/química , Algoritmos , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Diaminas/toxicidade , Humanos , Hipertermia Induzida , Verde de Indocianina/química , Verde de Indocianina/farmacocinética , Verde de Indocianina/toxicidade , Camundongos , Nanoconjugados/toxicidade , Nanotecnologia , Polietilenoglicóis/toxicidade , Cirurgia Assistida por Computador , Distribuição TecidualRESUMO
BACKGROUND: In the past decade, researchers have focused on developing new biomaterials for cancer therapy that combine imaging and therapeutic agents. In our study, we use a new biocompatible and biodegradable polymer, termed poly(glycerol malate co-dodecanedioate) (PGMD), for the synthesis of nanoparticles (NPs) and loading of near-infrared (NIR) dyes. IR820 was chosen for the purpose of imaging and hyperthermia (HT). HT is currently used in clinical trials for cancer therapy in combination with radiotherapy and chemotherapy. One of the potential problems of HT is that it can up-regulate hypoxia-inducible factor-1 (HIF-1) expression and enhance vascular endothelial growth factor (VEGF) secretion. RESULTS: We explored cellular response after rapid, short-term and low thermal dose laser-IR820-PGMD NPs (laser/NPs) induced-heating, and compared it to slow, long-term and high thermal dose heating by a cell incubator. The expression levels of the reactive oxygen species (ROS), HIF-1 and VEGF following the two different modes of heating. The cytotoxicity of NPs after laser/NP HT resulted in higher cell killing compared to incubator HT. The ROS level was highly elevated under incubator HT, but remained at the baseline level under the laser/NP HT. Our results show that elevated ROS expression inside the cells could result in the promotion of HIF-1 expression after incubator induced-HT. The VEGF secretion was also significantly enhanced compared to laser/NP HT, possibly due to the promotion of HIF-1. In vitro cell imaging and in vivo healthy mice imaging showed that IR820-PGMD NPs can be used for optical imaging. CONCLUSION: IR820-PGMD NPs were developed and used for both imaging and therapy purposes. Rapid and short-term laser/NP HT, with a low thermal dose, does not up-regulate HIF-1 and VEGF expression, whereas slow and long term incubator HT, with a high thermal dose, enhances the expression of both transcription factors.
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In this study, we prepared novel poly(Glycerol malate co-dodecanedioate) (PGMD) NPs containing an imaging/hyperthermia agent (IR820) and a chemotherapeutic agent (doxorubicin, DOX). The PGMD polymer was prepared by thermal condensation. IR820 and DOX loaded PGMD NPs were prepared using the single oil emulsion technique. The size of the NPs measured was around 150 nm. Drug loading efficiency of DOX and IR820 was around 4% and 8%, respectively. An acidic environment (pH=5.0) induced higher DOX release as compared to pH=7.4. DOX release was also enhanced by exposure to laser, which increased the temperature to 42°C. Cytotoxicity of the drug loaded NPs was comparable in MES-SA but was higher in Dx5 cells compared to free drug (p<0.05). The combination of hyperthermia and chemotherapy improved cytotoxicity in both cell lines. The NP formulation significantly improved the plasma half-life of IR820 in mice after tail vein injection.
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The purpose of this study was to prepare targeted Poly lactide-co-glycolide (PLGA) nanoparticles with simultaneous entrapment of indocyanine green (ICG) and doxorubicin (DOX) by surface decorating them with tumor specific monoclonal antibodies in order to achieve simultaneous therapy and imaging. ICG was chosen as an imaging and hyperthermia agent and DOX was used as a chemotherapeutic agent. ICG and DOX were incorporated into PLGA nanoparticles using the oil-in-water emulsion solvent evaporation technique. These nanoparticles were further surface decorated with antibodies against Human Epithelial Receptor-2 (HER-2) using carbodiimide chemistry. The uptake of antibody conjugated ICG-DOX-PLGA nanoparticles (AIDNP) was enhanced in SKOV-3 (HER-2 overexpressing cell lines) compared to their non-conjugated counterparts (ICG-DOX-PLGA nanoparticles (IDNP)). The uptake of antibody conjugated ICG-DOX-PLGA nanoparticles, however, was similar in MES-SA and MES-SA/Dx5 cancer cells (HER-2 negative cell lines), which were used as negative controls. The cytotoxicity results after laser treatment (808 nm, 6.7 W/cm(2)) showed an enhanced toxicity in treatment of SKOV-3. The negative controls exhibited comparable cytotoxicity with or without exposure to the laser. Thus, this study showed that these antibody conjugated ICG-DOX-PLGA nanoparticles have potential for combinatorial chemotherapy and hyperthermia.
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Antibióticos Antineoplásicos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Doxorrubicina/administração & dosagem , Verde de Indocianina/administração & dosagem , Terapia a Laser , Nanopartículas/administração & dosagem , Antibióticos Antineoplásicos/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Humanos , Verde de Indocianina/química , Ácido Láctico/química , Nanopartículas/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Receptor ErbB-2/imunologiaRESUMO
Primordial dwarfism (PD) is characterized by global growth failure, both during embryogenesis and postnatally. Loss-of-function germline mutations in La ribonucleoprotein domain family, member 7 (LAPR7) have recently been linked to PD. Paradoxically, LARP7 deficiency was previously assumed to be associated with increased cell growth and proliferation via activation of positive transcription elongation factor b (P-TEFb). Here, we show that Larp7 deficiency likely does not significantly increase P-TEFb activity. We further discover that Larp7 knockdown does not affect pluripotency but instead primes embryonic stem cells (ESCs) for differentiation via downregulation of Lin28, a positive regulator of organismal growth. Mechanistically, we show that Larp7 interacts with a poly(A) polymerase Star-PAP to maintain Lin28 mRNA stability. We propose that proper regulation of Lin28 and PTEFb is essential for embryonic cells to achieve a sufficient number of cell divisions prior to differentiation and ultimately to maintain proper organismal size.
Assuntos
Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Nanismo/genética , Nanismo/metabolismo , Nanismo/patologia , Elonguina , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Polinucleotídeo Adenililtransferase/antagonistas & inibidores , Polinucleotídeo Adenililtransferase/genética , Polinucleotídeo Adenililtransferase/metabolismo , Ligação Proteica , Interferência de RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/antagonistas & inibidores , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Germ cell and embryonic stem cells are inextricably linked in many aspects. Remarkably both can generate all somatic cell types in organisms. Yet the molecular regulation accounting for these similarities is not fully understood. Cyclin K was previously thought to associate with CDK9 to regulate gene expression. However, we and others have recently shown that its cognate interacting partners are CDK12 and CDK13 in mammalian cells. We further demonstrated that cyclin K is essential for embryonic stem cell maintenance. In this study, we examined the expression of cyclin K in various murine and human tissues. We found that cyclin K is highly expressed in mammalian testes in a developmentally regulated manner. During neonatal spermatogenesis, cyclin K is highly expressed in gonocytes and spermatogonial stem cells. In adult testes, cyclin K can be detected in spermatogonial stem cells but is absent in differentiating spermatogonia, spermatids and spermatozoa. Interestingly, the strongest expression of cyclin K is detected in primary spermatocytes. In addition, we found that cyclin K is highly expressed in human testicular cancers. Knockdown of cyclin K in a testicular cancer cell line markedly reduces cell proliferation. Collectively, we suggest that cyclin K may be a novel molecular link between germ cell development, cancer development and embryonic stem cell maintenance.