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The incomplete prediction of prognosis in esophageal squamous cell carcinoma (ESCC) patients is attributed to various therapeutic interventions and complex prognostic factors. Consequently, there is a pressing demand for enhanced predictive biomarkers that can facilitate clinical management and treatment decisions. This study recruited 491 ESCC patients who underwent surgical treatment at Huashan Hospital, Fudan University. We incorporated 14 blood metabolic indicators and identified independent prognostic indicators for overall survival through univariate and multivariate analyses. Subsequently, a metabolism score formula was established based on the biochemical markers. We constructed a nomogram and machine learning models utilizing the metabolism score and clinically significant prognostic features, followed by an evaluation of their predictive accuracy and performance. We identified alkaline phosphatase, free fatty acids, homocysteine, lactate dehydrogenase, and triglycerides as independent prognostic indicators for ESCC. Subsequently, based on these five indicators, we established a metabolism score that serves as an independent prognostic factor in ESCC patients. By utilizing this metabolism score in conjunction with clinical features, a nomogram can precisely predict the prognosis of ESCC patients, achieving an area under the curve (AUC) of 0.89. The random forest (RF) model showed superior predictive ability (AUC = 0.90, accuracy = 86%, Matthews correlation coefficient = 0.55). Finally, we used an RF model with optimal performance to establish an online predictive tool. The metabolism score developed in this study serves as an independent prognostic indicator for ESCC patients.
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As the world's fourth most deadly cancer, colorectal cancer (CRC) still needed the novel therapeutic drugs and target urgently. Although cyclin-dependent kinase 12 (CDK12) has been shown to be implicated in the malignancy of several types of cancer, its functional role and mechanism in CRC remain largely unknown. Here, we found that suppression of CDK12 inhibited tumor growth in CRC by inducing apoptosis. And CDK12 inhibition triggered autophagy by upregulating autophagy related gene 7 (ATG7) expression. Inhibition of autophagy by ATG7 knockdown and chloroquine (CQ) further decreased cell viability induced by CDK12 inhibition. Further mechanism exploration showed that CDK12 interacted with protein kinase B (AKT) regulated autophagy via AKT/forkhead box O3 (AKT/FOXO3) pathway. FOXO3 transcriptionally upregulated ATG7 expression and autophagy when CDK12 inhibition in CRC. Level of CDK12 and p-FOXO3/FOXO3 ratio were correlated with survival in CRC patients. Moreover, CDK12 inhibition improved the efficacy of anti-programmed cell death 1(PD-1) therapy in CRC murine models by enhancing CD8 + T cells infiltration. Thus, our study founded that CDK12 inhibition upregulates ATG7 triggering autophagy via AKT/FOXO3 pathway and enhances anti-PD-1 efficacy in CRC. We revealed the roles of CDK12/FOXO3/ATG7 in regulating CRC progression, suggesting potential biomarkers and therapeutic target for CRC.
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Neoplasias Colorretais , Proteínas Proto-Oncogênicas c-akt , Humanos , Animais , Camundongos , Quinases Ciclina-Dependentes , Apoptose , Autofagia , Neoplasias Colorretais/tratamento farmacológico , Proteína Forkhead Box O3RESUMO
Breast cancer stem cells (BCSCs) play a key role in therapeutic resistance in breast cancer treatments and disease recurrence. This study aimed to develop a combination therapy loaded with pH-sensitive liposomes to kill both BCSCs and the okbulk cancer cells using trastuzumab-sensitive and resistant human epidermal growth factor receptor 2 positive (HER2+) breast cancer cell models. The anti-BCSCs effect and cytotoxicity of all-trans retinoic acid, salinomycin, and bufalin alone or in combination with doxorubicin were compared in HER2+ cell line BT-474 and a validated trastuzumab-resistant cell line, BT-474R. The most potent anti-BCSC agent was selected and loaded into a pH-sensitive liposome system. The effects of the liposomal combination on BCSCs and bulk cancer cells were assessed. Compared with BT-474, the aldehyde dehydrogenase positive BCSC population was elevated in BT-474R (3.9 vs. 23.1%). Bufalin was the most potent agent and suppressed tumorigenesis of BCSCs by â¼50%, and showed strong synergism with doxorubicin in both BT-474 and BT-474R cell lines. The liposomal combination of bufalin and doxorubicin significantly reduced the BCSC population size by 85%, and inhibited both tumorigenesis and self-renewal, although it had little effect on the migration and invasiveness. The cytotoxicity against the bulk cancer cells was also enhanced by the liposomal combination than either formulation alone in both cell lines (p < 0.001). The liposomal bufalin and doxorubicin combination therapy may effectively target both BCSCs and bulk cancer cells for a better outcome in trastuzumab-resistant HER2+ breast cancer.
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Neoplasias da Mama , Bufanolídeos , Doxorrubicina , Resistencia a Medicamentos Antineoplásicos , Lipossomos , Células-Tronco Neoplásicas , Trastuzumab , Humanos , Doxorrubicina/farmacologia , Doxorrubicina/administração & dosagem , Bufanolídeos/farmacologia , Bufanolídeos/administração & dosagem , Bufanolídeos/química , Células-Tronco Neoplásicas/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Lipossomos/química , Feminino , Trastuzumab/farmacologia , Trastuzumab/administração & dosagem , Linhagem Celular Tumoral , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Receptor ErbB-2/metabolismo , Sobrevivência Celular/efeitos dos fármacosRESUMO
To enhance cytoplasmic delivery efficiency, pH-sensitive liposomes (PSL) have been proposed as a novel strategy. To facilitate clinical translation, this study aims to understand the impact of both size and pH-sensitivity on cellular uptake pathways, intracellular trafficking and pharmacokinetics of liposomes. The large liposomes (130-160 nm) were prepared using thin-film hydration method, while small liposomes (â¼60 nm) were fabricated using microfluidics, for both PSL and non-pH-sensitive liposomes (NPSL). Cellular uptake pathways and intracellular trafficking was investigated through confocal imaging with aid of various endocytosis inhibitors. Intracellular gemcitabine delivery by various liposomal formulations was quantified using HPLC, and the cytotoxicity was assessed via cell viability assays. Pharmacokinetics of gemcitabine loaded in various liposomes was evaluated in rats following intravenous administration. Larger liposomes had a higher loading capacity for hydrophilic gemcitabine (7% vs 4%). Small PSL exhibited superior cellular uptake compared to large PSL or NPSLs. Moreover, the alkalization of endosomes significantly attenuated the cellular uptake of PSL. Large liposomes (PSL and NPSL) predominantly entered cells via clathrin-dependent pathway, whereas small liposomes partially utilized caveolae-dependent pathway. However, the long circulation of the liposomes, as measured by the encapsulated gemcitabine, was compromised by both pH-sensitivity and size reduction (9.5 h vs 5.3 h). Despite this drawback, our results indicate that small PSL holds promise as vectors for the next generation of liposomal nanomedicine, owing to their superior cytoplasmic delivery efficiency.
Large liposomes had higher loading capacity for hydrophilic gemcitabine.Reduction of liposome size enhanced drug release from pH-sensitive liposomes.The internalization efficiency of liposomes was enhanced by pH-sensitivity and size reduction.Larger liposomes (>130 nm) enter cells primarily via clathrin-dependent endocytosis, while smaller liposomes (â¼60 nm) partially through caveolae-mediated pathway, regardless of the pH-sensitivity.The intracellular payload release from pH-sensitive liposomes was decreased by endosome alkalization using chloroquine.Long circulation of the encapsulated gemcitabine was compromised by the pH-sensitivity and size reduction.
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OBJECTIVE: This work aimed to develop a simple HPLC method for the simultaneous quantitative determination of the ultraviolet (UV) filters, hydrophilic benzophenone-4 and lipophilic octocrylene, in the presence of three other commonly used UV filters, avobenzone, octisalate and homosalate. METHODS: Reverse-phased HPLC was performed on a C18 column. A scouting gradient was initially used to determine the approximate mobile phase composition required for efficient analyte elution and separation before further optimization. The assay was validated with regard to specificity, linearity, intra- and inter-day accuracy and precision, limits of detection and limits of quantification. An ultrasound dispersion extraction method for the UV filters from a commercial sunscreen was developed, and the extraction efficiencies from spiked samples were calculated. RESULTS: An acetonitrile-methanol-water mixture (20:67:13, v/v/v), where the water component contained 0.2% trifluoroacetic acid (v/v), was found to be the optimal mobile phase at a flow rate of 1.0 mL/min. The assay was linear between 1.0-100 µg/mL for both benzophenone-4 and octocrylene (both correlation coefficients were above 0.999). There was no interference from the excipients of the sunscreen nor from the three other UV filters. The intra- and inter-day accuracy was between 90.0-104.6% for both analytes. Extraction recoveries from a spiked commercial sunscreen were between 95.4 ± 2.1% to 98.5 ± 2.1% for benzophenone-4, and between 87.3 ± 2.3% and 98.9 ± 3.1% for octocrylene. All validation parameters were within the acceptance criteria set out in the International Council for Harmonization (ICH) guidelines. The HPLC assay showed the extracted quantities of benzophenone-4 and octocrylene from the commercial sunscreen closely matched claimed quantities. CONCLUSION: The developed isocratic HPLC method was suitable for simultaneously determining the hydrophilic benzophenone-4 and lipophilic octocrylene in the presence of other commonly used UV filters. Additionally, the extraction method was simple and effective for accurately quantifying the UV filters in a commercial sunscreen.
OBJECTIF: Ces travaux visaient à développer une méthode de chromatographie en phase liquide à haute performance simple pour la détermination quantitative simultanée de la benzophénone-4 hydrophile et de l'octocrylène lipophile, des filtres ultraviolets (UV), en présence de trois autres filtres UV couramment utilisés, l'avobenzone, l'octisalate et l'homosalate. MÉTHODES: Une chromatographie en phase liquide à haute performance en phase inverse a été réalisée sur une colonne C18. Un gradient de référence a été initialement utilisé pour déterminer la composition approximative de la phase mobile requise pour une élution et une séparation efficace de l'analyte avant une optimisation plus poussée. Le dosage a été validé en termes de spécificité, de linéarité, d'exactitude et de précision intra- et inter-journalières, de limites de détection et de limites de quantification. Une méthode d'extraction par dispersion ultrasonique des filtres UV d'une crème solaire commerciale a été mise au point, et les efficacités d'extraction des échantillons artificiellement traités ont été calculées. RÉSULTATS: Un mélange acétonitrile-méthanol-eau (20:67:13, v/v/v), où la composante eau contenait 0,2 % d'acide trifluoroacétique (v/v), s'est avéré être la phase mobile optimale à un débit de 1,0 ml/min. Le dosage était linéaire entre 1,0 et 100 µg/ml pour la benzophénone-4 et l'octocrylène (les deux coefficients de corrélation étaient supérieurs à 0,999). Aucune interférence n'a été observée entre les excipients de l'écran solaire et les trois autres filtres UV. La précision intra et inter-journalière était comprise entre 90,0 et 104,6 % pour les deux analytes. Les récupérations par extraction à partir d'une crème solaire commerciale artificiellement traitée étaient comprises entre 95,4 % ± 2,1 % et 98,5 % ± 2,1 % pour la benzophénone-4, et entre 87,3 % ± 2,3 % et 98,9 % ± 3,1 % pour l'octocrylène. Tous les paramètres de validation étaient conformes aux critères d'acceptation définis dans les lignes directrices du Conseil international d'harmonisation (ICH). Le dosage par chromatographie en phase liquide à haute performance a montré que les quantités extraites de benzophénone-4 et d'octocrylène de la crème solaire commerciale correspondaient étroitement aux quantités revendiquées. CONCLUSION: La méthode de chromatographie en phase liquide à haute performance isocratique mise au point a permis de déterminer simultanément la benzophénone-4 hydrophile et l'octocrylène lipophile en présence d'autres filtres UV couramment utilisés. En outre, la méthode d'extraction était simple et efficace pour quantifier avec précision les filtres UV dans une crème solaire commerciale.
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Protetores Solares , Raios Ultravioleta , Protetores Solares/química , Cromatografia Líquida de Alta Pressão/métodos , ÁguaRESUMO
The anti-HER2 antibody trastuzumab has been proven to be an effective targeting ligand for drug delivery. This study investigates the structural integrity of trastuzumab under different stress factors in formulation development and its long-term stability. A validated size exclusion high performance liquid chromatographic (SEC-HPLC) method was first developed. The stability of trastuzumab (0.21-21 mg/ml) under stress conditions (mechanical, freeze-and-thaw, pH and temperature) and long-term storage in the presence of formulation excipients were monitored for up to 12 months, using both the SEC-HPLC method and sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The anti-proliferation activity of the reconstituted antibody stored at 4 °C against HER2+ BT-474 breast cells was also monitored over 12 months. The developed SEC-HPLC method was sensitive and accurate. Solutions of trastuzumab were resistant to mechanical stress and repeated freeze-and-thaw; but were unstable under acidic (pH 2.0 and 4.0) and alkaline (pH 10.0 and 12.0) environments. The samples degraded over 5 days at 60 °C, and within 24 h at 75 °C. Low temperature (-80 °C or 4 °C) and low concentration (0.21 mg/ml) favoured the long-term stability. The anti-proliferation activity was conserved at 4 °C for at least 12 months. This study provided valuable stability information in developing trastuzumab involved nano-formulation as well as in clinical settings.
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Nanomedicina , Trastuzumab/farmacologia , Cromatografia Líquida de Alta Pressão/métodos , Estabilidade de Medicamentos , Eletroforese em Gel de PoliacrilamidaRESUMO
While delivery of chemotherapeutics to cancer cells by nanomedicines can improve therapeutic outcomes, many fail due to the low drug loading (DL), poor cellular uptake and endosomal entrapment. This study investigated the potential to overcome these limitations using pH-sensitive liposomes (PSL) empowered by the use of calcium acetate. An acidic dinitrobenzamide mustard prodrug SN25860 was used as a model drug, with non pH-sensitive liposomes (NPSL) as a reference. Calcium acetate as a remote loading agent allowed to engineer PSL- and NPSL-SN25860 with DL of > 31.1% (w/w). The IC50 of PSL-SN25860 was 21- and 141-fold lower than NPSL and free drug, respectively. At 48 h following injection of PSL-SN25860, NPSL-SN25860 and the free drug, drug concentrations in EMT6-nfsB murine breast tumors were 56.3 µg/g, 6.76 µg/g and undetectable (< 0.015 µg/g), respectively (n = 3). Meanwhile, the ex vivo tumor clonogenic assay showed 9.1%, 19.4% and 42.7% cell survival in the respective tumors. Live-cell imaging and co-localization analysis suggested endosomal escape was accomplished by destabilization of PSL followed by release of Ca2+ in endosomes allowing induction of a proton sponge effect. Subsequent endosomal rupture was observed approximately 30 min following endocytosis of PSL containing Ca2+. Additionally, calcium in liposomes promoted internalization of both PSL and NPSL. Taken together, this study demonstrated multifaceted functions of calcium acetate in promoting drug loading into liposomes, cellular uptake, and endosomal escape of PSL for efficient cytoplasmic drug delivery. The results shed light on designing nano-platforms for cytoplasmic delivery of various therapeutics.
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Lipossomos , Neoplasias , Animais , Cálcio , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Endossomos , Concentração de Íons de Hidrogênio , Lipossomos/farmacologia , Camundongos , PrótonsRESUMO
Poisoning is a significant cause of injury-related death worldwide. Dialysis is usually ineffective in removing the toxin once it has been absorbed because of drug protein binding and high volumes of distribution. In this work, we explore whether the addition of liposomes to peritoneal dialysate could extract protein bound amitriptyline. Liposomes were prepared using the thin film hydration method. In the in vitro experiment, 3 mL of 20% albumin with a concentration of 6000 nmol/L amitriptyline in a proprietary dialysis cartridge was dialysed against 125 mL of phosphate-buffered saline with and without 80 mg 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) liposomes. In the in vivo arm, peritoneal dialysis was undertaken in 6 rats with pH gradient liposome augmented dialysate after intravenous amitriptyline injection. Peritoneal blood flow was estimated by CO2 extraction. Total amitriptyline extracted was compared to freely dissolved (non-protein bound) and total amitriptyline perfusing the membrane during the peritoneal dwell. Mean liposome size for DOPG and acidic centre pH gradient liposomes was 119 nm and 430 nm, respectively. In the in vitro experiment, more amitriptyline was extracted into the liposome containing dialysate than the control dialysate (40 +/- 2 nmol/L vs. 27 +/- 1 nmol/L). In the in vivo experiment, the total amitriptyline in dialysate was 5240 +/- 2750 nmol. Mean total free amitriptyline perfusing the peritoneal membrane was 93 +/- 46 nmol. Mean total blood amitriptyline perfusing the peritoneal membrane was 23,920 +/- 6920 nmol. Two of the six animals were excluded due to overestimation of peritoneal blood flow. This exploratory work suggests the addition of liposome nanoparticles to peritoneal dialysate extracted protein bound amitriptyline from blood.
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Amitriptilina , Diálise Peritoneal , Albuminas , Animais , Dióxido de Carbono , Soluções para Diálise , Lipossomos/uso terapêutico , Diálise Peritoneal/métodos , Fosfatos , Proteínas , Força Próton-Motriz , Ratos , Diálise RenalRESUMO
Up to now, insufficient drug accumulation in tumor remains a major challenge for nanochemotherapy. However, the spherical nanocarriers with large diameter, which are beneficial for blood circulation and tumor extravasation, cannot travel deep in a tumor. Additionally, high tumor interstitial fluid pressure (IFP) in the tumor microenvironment may promote the efflux of the penetrable nanodrugs. Therefore, the size and shape of nanocarriers as well as the tumoral IFP can be regulated synchronously for improved tumor penetration and combined chemotherapy. Herein, a novel dual-functional polymer-polypeptide (Biotin-PEG2000-GKGPRQITITK) for both verified tumor targeting and responsiveness was synthesized to construct the "peel" of nanopomegranate-like nanovectors (DI-MPL), in which docetaxel-loaded micelles was encapsulated as "seeds". Interestingly, DI-MPL was endowed multi-abilities of tunable size/shape switch and controlled release of IFP alleviator imatinib (IM), which were developed with one and the same strategy-alteration of membrane fluidity under the cleavage of polymer-polypeptide and PEGylation. As a result, the peel of DI-MPL could turn into small pieces with the seed scattered out in response to matrix metalloproteinase-9 (MMP-9), making nanopomegranate (180 nm) switch into spheres/disks (40 nm), during which IM is released to reduce IFP synchronously. With prominent tumor penetration ability in both multicellular tumor spheroids (MCTS) and tumor tissue, DI-MPL exhibited optimal inhibition of MCTS growth and the enhanced chemotherapy in comparison to other preparations. Meanwhile, the improved penetrability of DI-MPL in tumor tissue was found to be related to the reduced IFP, which is achieved via inhibiting expression of phosphorylated platelet-derived growth factor receptor-ß (p-PDGFR-ß) by IM. Altogether, the bilateral adjusting strategies from nanocarrier size/shape and tumoral IFP with a single enzyme-responsive material could provide a potential combined chemotherapy to improve tumor penetration.
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Docetaxel/administração & dosagem , Portadores de Fármacos/química , Mesilato de Imatinib/administração & dosagem , Fluidez de Membrana , Neoplasias/tratamento farmacológico , Animais , Biotina/química , Linhagem Celular Tumoral , Modelos Animais de Doenças , Docetaxel/farmacocinética , Composição de Medicamentos/métodos , Líquido Extracelular , Feminino , Humanos , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Nanosferas/química , Neoplasias/patologia , Peptídeos/química , Peptídeos/metabolismo , Polietilenoglicóis/química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Distribuição Tecidual , Microambiente Tumoral/efeitos dos fármacosRESUMO
The COVID-19 pandemic has left scientists and clinicians no choice but a race to find solutions to save lives while controlling the rapid spreading. Messenger RNA (mRNA)-based vaccines have become the front-runners because of their safety profiles, precise and reproducible immune response with more cost-effective and faster production than other types of vaccines. However, the physicochemical properties of naked mRNA necessitate innovative delivery technologies to ferry these 'messengers' to ribosomes inside cells by crossing various barriers and subsequently induce an immune response. Intracellular delivery followed by endosomal escape represents the key strategies for cytoplasmic delivery of mRNA vaccines to the target. This Perspective provides insights into how state-of-the-art nanotechnology helps break the delivery barriers and advance the development of mRNA vaccines. The challenges remaining and future perspectives are outlined.
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Vacinas contra COVID-19/uso terapêutico , COVID-19/prevenção & controle , Citoplasma/metabolismo , Portadores de Fármacos , Lipídeos/química , Nanopartículas , Ribossomos/metabolismo , Vacinas Sintéticas/uso terapêutico , Vacina de mRNA-1273 contra 2019-nCoV , Animais , Vacina BNT162 , COVID-19/imunologia , COVID-19/virologia , Vacinas contra COVID-19/química , Vacinas contra COVID-19/farmacocinética , Composição de Medicamentos , Humanos , Nanomedicina , Vacinas Sintéticas/química , Vacinas de mRNARESUMO
PURPOSE: PEGylated pH-sensitive liposomes (PSL) dual-loaded with gemcitabine and curcumin were investigated for the potential application in gemcitabine-resistant pancreatic ductal adenocarcinoma (PDAC) treatment. Curcumin was employed as an inhibitor of the efflux transporter, multidrug resistance protein 5 (MRP5) in PDAC cells. METHODS: Liposomes were prepared with gemcitabine in the core and curcumin in the bilayers. The effects of curcumin on pH-sensitivity and 'endosome escape' of PSL with different PEGylation were investigated using a calcein self-quench assay. The effects of curcumin on intracellular gemcitabine concentrations, and cytotoxicity to a MIA PaCa-2 PDAC cell line was evaluated. The pharmacokinetics were investigated in rats following intravenous injection. RESULTS: The addition of curcumin to the PSL bilayers (0.2-1 mol%)slightly decreased the pH-sensitivity of PSL, but to a less extent than PEGylation (0-5 mol%). Co-treatment with curcumin increased gemcitabine cellular accumulation in a concentration-dependent manner, and resulted in synergistic cytotoxicity towards MIA PaCa-2cells.Both these effects were augmented by the use of PSL, particularly when the two drugs were co-loaded in PSL. In rats, the dual-drug loaded PSL produced significantly reduced (p < 0.05) plasma clearance (CL) and volume of distribution (Vd) for both drugs, alongside 3 to 4-fold increases in the area-under-the-concentration-time curves compared to the free drugs. Additionally, curcumin slightly increase the plasma concentrations of gemcitabine possibly also via the MRP5 inhibition effect. CONCLUSION: Co-delivery of curcumin with gemcitabine using PSL not only increased the intracellular gemcitabine concentration thus cytotoxicity to MIA PaCa-2 cells but also significantly improved the pharmacokinetic profiles for both drugs. Graphical Abstract.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Curcumina/farmacologia , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Área Sob a Curva , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Curcumina/uso terapêutico , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Liberação Controlada de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Concentração de Íons de Hidrogênio , Lipossomos , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Neoplasias Pancreáticas/patologia , Polietilenoglicóis/química , Ratos , GencitabinaRESUMO
Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer membrane-enclosed vesicles and act like 'messages in a bottle' in cell-cell communication by transporting their cargoes to recipient cells. Small EVs (sEVs, < 200 nm) are highly researched recently and have been harnessed as novel delivery systems for the treatment of various diseases, including neurodegenerative disorders, cardiovascular diseases, and most importantly cancer primarily because of their non-immunogenicity, tissue penetration and cell-tropism. This review will first provide a comprehensive overview of sEVs regarding the current understanding on their properties, biogenesis, new classification by the ISEV, composition, as well as their roles in cancer development (thereby called "oncosomes"). The primary focus will be given to the current state of sEVs as natural nanocarriers for cancer drug delivery, the technologies and challenges involved in sEV isolation and characterization, therapeutic cargo loading, and surface modification to enhance tumor-targeting. We will also provide examples of sEV products under clinical trials. Furthermore, the current challenges as well as the advance in "sEV mimetics" to address some of the sEVs limitations is briefly discussed. We seek to advance our understanding of sEVs to unlock their full potential as superior drug delivery vehicles in cancer therapy.
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Antineoplásicos/administração & dosagem , Portadores de Fármacos/química , Vesículas Extracelulares/química , Nanopartículas/química , Neoplasias/tratamento farmacológico , Ensaios Clínicos como Assunto , Humanos , Tamanho da Partícula , Resultado do TratamentoRESUMO
Conventional non-pH-sensitive liposomes for cytoplasmic delivery of protein suffer from poor efficiency. Here we investigated mannosylated pH-sensitive liposomes (MAN-PSL) for cytoplasmic delivery of protein to macrophages RAW 264.7 using PSL and non-pH-sensitive liposomes for comparison. We characterised the pH-dependent fluorescence of green fluorescent protein (GFP) and encapsulated it in liposomes as an intracellular trafficking tracer. GFP showed a reversed 'S'-shaped pH-fluorescence curve with a dramatic signal loss at acidic pH. GFP stored at 4 °C with light protection showed a half-life of 10 days (pH 5-8). The entrapment efficiency of GFP was dominated by the volume ratio of intraliposomal core to external medium for thin-film hydration. Mannosylation did not affect the pH-responsiveness of PSL. Confocal microscopy elucidated that mannosylation promoted the cellular uptake of PSL. For both these liposomes, the strongest, homogeneously distributed GFP fluorescence in the cytoplasm was found at 3 h, confirming efficient endosomal escape of GFP. Conversely, internalisation of non-pH-sensitive liposomes was slow (peaked at 12 h) and both Nile Red and GFP signals remained weak and punctuated in the cytosol. In conclusion, GFP performed as a probe for endosome escape of liposomal cargo. Mannosylation facilitated the internalisation of PSL without compromising their endosomal escape ability.
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Citoplasma/metabolismo , Endossomos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Macrófagos/metabolismo , Manose/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Citoplasma/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Proteínas de Fluorescência Verde/administração & dosagem , Proteínas de Fluorescência Verde/síntese química , Concentração de Íons de Hidrogênio , Lipossomos , Substâncias Luminescentes/administração & dosagem , Substâncias Luminescentes/síntese química , Substâncias Luminescentes/metabolismo , Macrófagos/efeitos dos fármacos , Manose/administração & dosagem , Manose/síntese química , Camundongos , Microscopia Confocal/métodos , Células RAW 264.7RESUMO
Hepatocellular carcinoma (HCC), one of the most lethal malignancies worldwide, has limited efficient therapeutic options. Here, we first demonstrated that simultaneously targeting poly (ADP-ribose) polymerase (PARP) and autophagy could evoke striking synergistic lethality in HCC cells. Specifically, we found that the PARP inhibitor Niraparib induced cytotoxicity accompanied by significant autophagy formation and autophagic flux in HCC cells. Further experiments showed that Niraparib induced suppression of the Akt/mTOR pathway and activation of the Erk1/2 cascade, two typical signaling pathways related to autophagy. In addition, the accumulation of reactive oxygen species was triggered, which was involved in Niraparib-induced autophagy. Blocking autophagy by chloroquine (CQ) in combination with Niraparib further enhanced cytotoxicity, induced apoptosis and inhibited colony formation in HCC cells. Synergistic inhibition was also observed in Huh7 xenografts in vivo. Mechanistically, we showed that autophagy inhibition abrogated Niraparib-induced cell-cycle arrest and checkpoint activation. Cotreatment with CQ and Niraparib promoted the formation of γ-H2AX foci while inhibiting the recruitment of the homologous recombination repair protein RAD51 to double-strand break sites. Thus, the present study developed a novel promising strategy for the management of HCC in the clinic and highlighted a potential approach to expand the application of PARP inhibitors.
Assuntos
Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Cloroquina/farmacologia , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Histonas/genética , Humanos , Indazóis/farmacologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Proteína Oncogênica v-akt/genética , Piperidinas/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
In this work, we report on an ATP-responsive low-molecular-weight polyethylenimine (LMW-PEI)-based supramolecular assembly. It formed via host-guest interaction between PEI (MW = 1.8 kDa)-α-cyclodextrin (α-CD) conjugates and PEI1.8k-phenylboronic acid (PBA) conjugates. The host-guest interaction between PEI1.8k-α-CD and PEI1.8k-PBA was confirmed by the 2D-NOESY chromatogram experiment and competition test. The ATP-responsive property of the supramolecular assembly was evaluated by a series of ATP-triggered degradation and siRNA release studies in terms of fluorescence resonance energy transfer, agarose gel electrophoresis assay, and the time course monitoring of the particle size and morphology. Confocal laser scanning microscopy confirmed the intracellular disassembly of the supramolecular polymer and the release of siRNA. The supramolecular assembly showed high buffering capability and was capable of protecting siRNA from RNase degradation. It had high cytocompatibility according to in vitro cytotoxicity and hemolysis assays. LMW-PEI-based supramolecular assembly facilitated cellular entry of siRNA via energy-dependent endocytosis. Moreover, the assembly/SR-A siRNA polyplexes at N/P ratio of 30 was most effective in knocking down SR-A mRNA and inhibiting uptake of modified LDL. Taken together, this work shows that ATP-responsive LMW-PEI-based supramolecular assembly is a promising gene vector and has potential application in treating atherosclerosis.
Assuntos
Trifosfato de Adenosina/química , Técnicas de Transferência de Genes , Nanoconjugados/química , Polietilenoimina/química , RNA Interferente Pequeno/química , Animais , Ácidos Borônicos/química , Células Cultivadas , Ciclodextrinas/química , Endocitose , Hemólise/efeitos dos fármacos , Camundongos , Nanoconjugados/toxicidade , Células RAW 264.7 , CoelhosRESUMO
Doxorubicin (DOX) is a representative antibiotic of terpenoids and clinically used in the treatment of various malignant tumors. However, its application is limited by the cardiotoxocity. Curdione, an extract from Rhizoma Curcumae, has many promising pharmacological effects including protecting acute liver injury and cerebral ischemia. It is still unknown whether curdione has a protective function for DOX-induced cardiotoxicity. In our study, we investigated the protective effects of curdione against DOX-induced cardiotoxicity. Our results showed that curdione attenuated DOX-induced growth inhibition and release of lactic dehydrogenase in a concentration-dependent manner. And curdione ameliorated the histopathological damage, reduced the elevation of serum creatine kinase-MB isoenzyme (CK-MB) and lactic dehydrogenase by DOX. Furthermore, curdione inhibited DOX-induced cell apoptosis and modulated the expression of Bcl-2 and Bax proteins, as well as abrogated DOX-induced reactive oxygen species accumulation and prevented mitochondria dysfunction. Further study indicated that curdione decreased DOX-induced phosphorylation of extracellular signal-regulated kinase1/2 (Erk1/2) and c-Jun-N-terminal kinase and activated nuclear factor-erythroid 2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signal pathway. Our results suggested that curdione maybe is a new and feasible strategy to prevent DOX-induced cardiotoxicity through monitoring multiple targets.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Doxorrubicina/toxicidade , Cardiopatias/prevenção & controle , Heme Oxigenase (Desciclizante)/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Sesquiterpenos de Germacrano/farmacologia , Animais , Cardiotoxicidade , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Cardiopatias/induzido quimicamente , Cardiopatias/enzimologia , Cardiopatias/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Fosforilação , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Liposome supported peritoneal dialysis is a recently described technique which may eventually be applicable in the clinical scenario of the intoxicated patient. We evaluated the hypothesis that intravenous injection of lipid emulsion (ILE) would augment acidic pH gradient liposome supported peritoneal dialysis (LSPD). Orogastrically amitriptyline dosed rats were treated with either Sodium bicarbonate (NaHCO3) intravenously and standard intraperitoneal dialysate (Group A); NaHCO3 intravenously and LSPD (Group B); or ILE and LSPD (Group C). The primary endpoint was dialysate amitriptyline concentration after a 60 min dwell. Secondary analysis included an estimate of extraction ratio for peritoneal blood flow (ERs). There were significantly higher intraperitoneal concentrations of amitriptyline and ERs in the two groups treated with LSPD (Group B, p = 0.02, Group C, p < 0.01 vs. Group A). There was no observed effect for ILE on intraperitoneal amitriptyline concentration or ERs (p > 0.20). LSPD increased the amitriptyline concentration in peritoneal dialysate. No further increase was demonstrated with ILE. This may be either because such an effect is absent, or type II error. Exploratory analysis suggests LSPD may be driven by total rather than free drug concentrations.
Assuntos
Amitriptilina/administração & dosagem , Antidepressivos Tricíclicos/administração & dosagem , Emulsões Gordurosas Intravenosas/administração & dosagem , Lipossomos , Diálise Peritoneal/métodos , Administração Intravenosa , Animais , Feminino , Concentração de Íons de Hidrogênio , Peritônio/irrigação sanguínea , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional , Bicarbonato de Sódio/administração & dosagemRESUMO
PURPOSE: To fabricate an acid-cleavable PEG polymer for the development of PEG-cleavable pH-sensitive liposomes (CL-pPSL), and to investigate their ability for endosomal escape and long circulation. METHODS: PEG-benzaldehyde-hydrazone-cholesteryl hemisuccinate (PEGB-Hz-CHEMS) containing hydrazone and ester bonds was synthesised and used to fabricate a dual pH-sensitive CL-pPSL. Non-cleavable PEGylated pH-sensitive liposome (pPSL) was used as a reference and gemcitabine as a model drug. The cell uptake and endosomal escape were investigated in pancreatic cancer Mia PaCa-2 cells and pharmacokinetics were studied in rats. RESULTS: The CL-pPSL showed accelerated drug release at endosomal pH 5.0 compared to pPSL. Compared to pPSL, CL-pPSL released their fluorescent payload to cytosol more efficiently and showed a 1.4-fold increase in intracellular gemcitabine concentration and higher cytotoxicity. In rats, injection of gemcitabine loaded CL-pPSL resulted in a slightly smaller Vd (149 ± 27 ml/kg; 170 ± 30 ml/kg) and shorter terminal T1/2 (5.4 ± 0.3 h; 5.8 ± 0.6 h) (both p > 0.05) but a significantly lower AUC (p < 0.01), than pPSL, due to the lower PEGylation degree (1.7 mol%) which means a 'mushroom' configuration of PEG. A five-time increase in the dose with CL-pPSL resulted in a 11-fold increase in AUC and a longer T1/2 (8.2 ± 0.5 h). CONCLUSION: The PEG-detachment from the CL-pPSL enhanced endosome escape efficiency compared with pPSL, without significantly compromising their stealth abilities.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Benzaldeídos/metabolismo , Preparações de Ação Retardada/metabolismo , Desoxicitidina/análogos & derivados , Hidrazonas/metabolismo , Lipossomos/metabolismo , Polietilenoglicóis/metabolismo , Animais , Antimetabólitos Antineoplásicos/farmacocinética , Benzaldeídos/química , Linhagem Celular Tumoral , Ésteres do Colesterol/química , Ésteres do Colesterol/metabolismo , Preparações de Ação Retardada/química , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacocinética , Endossomos/metabolismo , Humanos , Hidrazonas/química , Concentração de Íons de Hidrogênio , Lipossomos/química , Polietilenoglicóis/química , Ratos , Ratos Sprague-Dawley , GencitabinaRESUMO
PURPOSE: To enhance therapeutic efficacy and prevent phlebitis caused by Asulacrine (ASL) precipitation post intravenous injection, ASL-loaded hybrid micelles with size below 40 nm were developed to improve drug retention and tumor penetration. METHODS: ASL-micelles were prepared using different weight ratios of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethyleneglycol-2000 (DSPE-PEG2000) and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) polymers. Stability of micelles was optimized in terms of critical micelle concentration (CMC) and drug release properties. The encapsulation efficiency (EE) and drug loading were determined using an established dialysis-mathematic fitting method. Multicellular spheroids (MCTS) penetration and cytotoxicity were investigated on MCF-7 cell line. Pharmacokinetics of ASL-micelles was evaluated in rats with ASL-solution as control. RESULTS: The ASL-micelles prepared with DSPE-PEG2000 and TPGS (1:1, w/w) exhibited small size (~18.5 nm), higher EE (~94.12%), better sustained in vitro drug release with lower CMC which may be ascribed to the interaction between drug and carriers. Compared to free ASL, ASL-micelles showed better MCTS penetration capacity and more potent cytotoxicity. Pharmacokinetic studies demonstrated that the half-life and AUC values of ASL-micelles were approximately 1.37-fold and 3.49-fold greater than that of free ASL. CONCLUSIONS: The optimized DSPE-PEG2000/TPGS micelles could serve as a promising vehicle to improve drug retention and penetration in tumor.
Assuntos
Amsacrina/análogos & derivados , Antineoplásicos/farmacocinética , Micelas , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Amsacrina/química , Amsacrina/farmacocinética , Amsacrina/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Técnicas de Cultura de Células , Sobrevivência Celular , Preparações de Ação Retardada , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Meia-Vida , Humanos , Células MCF-7 , Masculino , Nanopartículas/química , Tamanho da Partícula , Permeabilidade , Ratos , Ratos Sprague-Dawley , Propriedades de Superfície , Vitamina E/químicaRESUMO
Acetaminophen (APAP) overdose is currently the leading cause of acute liver disease, but therapeutic treatment strategies are commonly limited. Although dihydroquercetin (DHQ) is an attractive botanical antioxidant, its protective potential for liver disease remains elusive. The present study investigated the protective effects of DHQ against APAP-induced hepatic cytotoxicity. Primary mouse hepatocytes were treated with different concentrations of DHQ followed by APAP administration. Our data showed that DHQ relieved APAP-induced growth inhibition and lactate dehydrogenase (LDH) release in a dose-dependent manner, as well as inhibited APAP-induced necrosis and extracellular signal regulated kinase-c-Jun-N-terminal kinase (ERK-JNK) stress. In addition, reactive oxygen species (ROS) accumulation and mitochondria dysfunction were also reversed by DHQ treatment. Further study revealed that DHQ induced phosphorylation of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) cascade and thus modulated expression of anti-apoptotic Bcl-2 family proteins. Moreover, DHQ induced autophagy which mediated its protective effects in hepatocytes. The protection was abrogated through pharmacological blockage of autophagy by chloroquine (CQ). These studies demonstrated, for the first time, that DHQ possessed hepatocellular protective effects in the context of APAP-induced cytotoxicity and subsequently revealed that the mechanisms comprised activation of JAK2/STAT3 signaling pathway and autophagy. These altogether highlighted the significant therapeutic potential of this agent during acute liver failure and other types of liver diseases.