RESUMO
In rheumatoid arthritis (RA), macrophages infiltrate joints, while fibroblast-like synovial cells proliferate abnormally, forming a barrier against drug delivery, which hinders effective drug delivery to joint focus. Here we firstly designed a pH-responsive size-adjustable nanoparticle, composed by methotrexate (MTX)-human serum albumin (HSA) complex coating with pH-responsive liposome (Lipo/MTX-HSA) for delivering drugs specifically to inflamed joints in acidic environments. We showed in vitro that the nanoparticles can induce mitochondrial dysfunction, promote apoptosis of fibroblast-like synoviocytes and macrophages, further reduce the secretion of inflammatory factors (TNF-α, IL-1ß, MMP-9), and regulate the inflammatory microenvironment. We also demonstrated similar effects in a rat model of arthritis, in which Lipo/MTX-HSA accumulated in arthritic joints, and at low pH, liposome phospholipid bilayer cleavage released small-sized MTX-HSA, which effectively reduced the number of fibroblast-synoviocytes and macrophages in joints, alleviated joint inflammation, and repaired bone erosion. These findings suggest that microenvironment-responsive size-adjustable nanoparticles show promise as a treatment against rheumatoid arthritis. STATEMENT OF SIGNIFICANCE: Abnormal proliferation of fibroblast synoviocytes poses a physical barrier to effective nanoparticle delivery. We designed size-adjustable nano-delivery systems by preparing liposomes with cholesterol hemisuccinate (CHEM), which were subsequently loaded with small-sized albumin nanoparticles encapsulating the cytotoxic drug MTX (MTX-HSA), termed Lipo/MTX-HSA. Upon tail vein injection, Lipo/MTX-HSA could be aggregated at the site of inflammation via the ELVIS effect in the inflamed joint microenvironment. Specifically, intracellular acidic pH-triggered dissociation of liposomes promoted the release of MTX-HSA, which was further targeted to fibroblasts or across fibroblasts to macrophages to exert anti-inflammatory effects. The results showed that liposomes with adjustable particle size achieved efficient drug delivery, penetration and retention in joint sites; the strategy exerted significant anti-inflammatory effects in the treatment of rheumatoid arthritis by inducing mitochondrial dysfunction to promote apoptosis in fibrosynoviocytes and macrophages.
Assuntos
Apoptose , Artrite Reumatoide , Fibroblastos , Lipossomos , Macrófagos , Metotrexato , Lipossomos/química , Artrite Reumatoide/patologia , Artrite Reumatoide/tratamento farmacológico , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibroblastos/metabolismo , Animais , Concentração de Íons de Hidrogênio , Metotrexato/farmacologia , Metotrexato/química , Apoptose/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Humanos , Ratos , Ratos Sprague-Dawley , Camundongos , Tamanho da Partícula , Masculino , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/patologia , Sinoviócitos/metabolismo , Células RAW 264.7 , Albumina Sérica Humana/química , Albumina Sérica Humana/farmacologia , Nanopartículas/químicaRESUMO
Albumin fusion or conjugation is a well-established technique for tumor delivery and is mainly mediated by albumin-induced caveolae-dependent endocytosis. We report that caveolae-dependent endocytic signaling activated by human serum albumin (HSA) is not sufficiently strong to induce cellular uptake, mainly due to its electrostatic repulsion from the negatively charged cell surface sulfated glycosaminoglycans (GAGs), and fusion of the cell-surface-retained protein with HSA is an effective strategy to activate the HSA-induced endocytic signal, thereby improving its intracellular uptake. In this study, human lactoferrin (hLF), a protein that accumulates on the cell surface along with GAGs, was selected for delivery into human lung adenocarcinoma PC-14 cells. When added exogenously, hLF-fused HSA (hLF-HSA) was successfully endocytosed, whereas the simultaneous addition of HSA and hLF did not result in endocytosis, indicating less efficient activation of endocytic signaling by HSA alone and the importance of its fusion. Importantly, the treatment of cells with chlorate, a known inhibitor of GAG sulfation, dramatically suppressed the endocytosis of hLF-HSA owing to the loss of the hLF-GAG interaction. Therefore, the cell-surface localization of HSA imposed by fusion with the cell-surface-retained protein enhances its binding to the relevant receptor, which improves intracellular delivery as an albumin-fusion platform.
Assuntos
Albuminas , Neoplasias , Humanos , Endocitose , Transporte Biológico , Albumina Sérica Humana/farmacologia , Transdução de SinaisRESUMO
BACKGROUND: Liver transplantation is a life-saving treatment in end-stage liver failure. Hemorheological features as blood fluidity and red blood cell aggregation may alter effective tissue perfusion, graft function and hemodynamic variables. OBJECTIVE: The aim of the study is to investigate effect of albumin infusion on red blood cell deformability and aggregation, blood viscosity and hemodynamics in liver transplant patients. METHODS: Seventeen live or cadaveric donors were included in this prospective study. Hemorheological and hemodynamic measurements were performed in order to evaluate the effects of albumin infusion in perioperative period. RESULTS: Erythrocyte aggregation was significantly reduced 90 minutes after albumin infusion (pâ<â0.01). Mean blood viscosity revealed significant decrease at 20ârpm and 50ârpm after 90 minutes of albumin infusion (pâ<â0.05). Plasma viscosity decreased significantly compared to the value before albumin infusion at 20ârpm (pâ<â0.05). Albumin replacement improved hemodynamic variables in patients with low blood pressure and cardiac index measurements (pâ>â0.05). CONCLUSIONS: Human albumin infusion led to decrease in whole blood and plasma viscosities, red blood cell aggregation and induced blood pressure and cardiac index elevation in perioperative liver transplant patients. Determination of hemodynamic and hemorheological effects of human albumin replacement in various patient populations may serve beneficial clinical data.
Assuntos
Hemorreologia , Transplante de Fígado , Humanos , Estudos Prospectivos , Deformação Eritrocítica , Agregação Eritrocítica , Viscosidade Sanguínea , Albumina Sérica Humana/farmacologiaRESUMO
Platelet aggregation contributes to the pathogenesis of cardiovascular diseases. After activation it leads to dense granule secretion and 5-HT release. The question arises; how platelet aggregation is endogenously controlled during blood circulation. In preliminary studies, we observed that human platelets aggregate more rapidly when suspended in buffer as compared to those suspended in plasma (PRP). These observations point to the presence of an endogenous substance that may inhibit arachidonic acid- induced platelet aggregation. An analysis of plasma Cohn fractions demonstrated that most of the plasma inhibitory activity was associated with albumin-rich and α-globulin rich protein fractions. The identity of plasma endogenous inhibitors of platelet aggregation (EIPA) was established by affinity chromatography on Cibacron Blue F3G-A for specific removal of albumin. The association of α-globulins to EIPA activity was recognized as due to haptoglobin by affinity chromatography on a column of hemoglobin-sepharose. In addition, we also found that the distribution of EIPA activity varies according to sex and physiological state. These findings reveal that EIPA may act by modulation of arachidonic acid metabolism or sequestering the fatty acid substrate.
Assuntos
Agregação Plaquetária , Serotonina , Humanos , Serotonina/metabolismo , Serotonina/farmacologia , Haptoglobinas/metabolismo , Haptoglobinas/farmacologia , Albumina Sérica , Ácido Araquidônico/farmacologia , Ácido Araquidônico/metabolismo , Albumina Sérica Humana/metabolismo , Albumina Sérica Humana/farmacologia , Plaquetas/metabolismoRESUMO
BACKGROUND: We recently demonstrated that oxygen-glucose deprivation (OGD) and unconjugated bilirubin (UCB) can damage mature and immature organotypic hippocampal slices and induce an oxidative stress similar to what occurs in jaundiced term and preterm infants with hypoxic-ischemic encephalopathy (HIE). OBJECTIVES: To assess the effects of OGD and UCB on the expression of heme-oxygenase 1 (HO-1) and oxidative stress-related enzymes in an in vitro model of HIE. METHODS: Mature and immature organotypic hippocampal slices were exposed to 30-min OGD and to 24 h UCB or UCB plus human serum albumin (HSA). The expression of HO-1, superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase (GPX), and nuclear factor erythroid-related factor 2 were analyzed by real-time PCR. RESULTS: In mature slices, OGD did not affect the expression of HO-1 and oxidative stress-induced enzymes. The addition of UCB was associated with the upregulation of HO-1 and Nrf2 that is abolished by the presence of equimolar amount of HSA. In immature slices, OGD induced the downregulation of CAT, GPX, and Nrf2 expression and the addition of UCB further decreased GPX. The addition of UCB and HSA reverted the effects of OGD and UCB on gene expression. CONCLUSIONS: In an in vitro model of HIE in term infants, we did not observe neuroprotective changes of the expression of HO-1 and genes involved in antioxidant defenses. Conversely, in an in vitro model of HIE in preterm infants, we observed a harmful decrease of the expression of genes encoding for antioxidant enzymes.
Assuntos
Hipóxia-Isquemia Encefálica , Fator 2 Relacionado a NF-E2 , Antioxidantes/farmacologia , Bilirrubina , Catalase/genética , Catalase/metabolismo , Catalase/farmacologia , Expressão Gênica , Glucose , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Peroxidase/farmacologia , Heme/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Hipóxia-Isquemia Encefálica/metabolismo , Recém-Nascido , Recém-Nascido Prematuro , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/farmacologia , Estresse Oxidativo , Oxigênio/metabolismo , Albumina Sérica Humana/farmacologia , Superóxido Dismutase-1/genéticaRESUMO
Flucloxacillin (FLX) induces adverse liver reactions, which has been reported to be related to human leukocyte antigen (HLA)-B*57:01. In a previous study, abacavir-induced hypersensitivity was induced in HLA-B*57:01-transgenic mice (B*57:01-Tg), originally constructed by our group (Susukida et al., 2021). In this study, B*57:01-Tg mice were used to reproduce FLX-induced liver injury. However, treatment of B*57:01-Tg mice with FLX alone did not increase serum ALT levels. Immune-deficient B*57:01-Tg/PD-1-/-mice were produced by mating B*57:01-Tg with PD-1-/- mice. The immune response of B*57:01-Tg/PD-1-/- mice was further modulated by co-administration of CpG-oligodeoxynucleotides and anti-CD4 mAb. Nevertheless, immune regulation in B*57:01-Tg mice did not contribute to the onset of FLX-induced liver injury or immune activation. Moreover, we generated an FLX-human serum albumin (HSA) conjugate and showed that FLX covalently bound to HSA in a time-dependent manner. The FLX-HSA conjugate was administered to the B*57:01-Tg mice. The immune response was investigated using flow cytometry, revealing the phenotype of CD44highCD62Llow in CD8+ T cells (TEM cells). Administration of the FLX-HSA conjugate resulted in an HLA-B*57:01 restricted immune response as shown by the stimulation of TEM cells in the draining lymph nodes. In conclusion, administration of FLX alone to B*57:01-Tg mice did not induce liver injury or immune activation. Immune system sensitivity does not play a decisive role in this process. The conjugation of FLX and HSA results in specific TEM cell stimulation, which suggests that HLA-B*57:01 drives a stronger interaction with CD8+ T cells. These results suggest that patients carrying HLA-B*57:01 could be more susceptible to a conjugate of FLX and albumin and drive CD8+ T cell activation, which may be a vital risk factor for FLX-induced liver injury. In addition, the application of the FLX-HSA adduct may be an effective method for the construction of FLX-induced idiosyncratic liver injury in mice.
Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Floxacilina , Animais , Linfócitos T CD8-Positivos , Floxacilina/farmacologia , Antígenos HLA-B/genética , Humanos , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Oligodesoxirribonucleotídeos/farmacologia , Receptor de Morte Celular Programada 1 , Albumina Sérica Humana/farmacologiaRESUMO
Human interleukin-15 (hIL-15) has attracted a considerable attention as a promising cancer immunotherapeutic due to its function to directly stimulate the proliferation and cytotoxic activity of NK and T cells. Nevertheless, a relatively short half-life of hIL-15 requires repeated administration and higher doses, causing serious side effects. Here, we demonstrate an enhanced blood half-life and biological activity of hIL-15 through genetic fusion of a human serum albumin-specific protein binder (rHSA). The fusion construct (rHSA-IL15) was observed to maintain respective binding activities for both hIL-15 receptor α and human serum albumin. The rHSA-IL15 led to a significant increase in the secretion of Granzyme B and INF-γ by immune cells compare to free hIL-15, expanding the population of activated T cell subset such as CD4 + T and CD8+ T cells. The terminal half-life of the rHSA-IL15 was prolonged by around a 40-fold in transgenic mice expressing human serum albumin, compared to free hIL-15. The rHSA-IL15 resulted in distinct anti-tumor activities in xenograft SCC (squamous cell carcinoma) mouse and allograft melanoma mouse models through activation of NK and CD8+ T cells. The rHSA-IL15 is expected to be used in cancer immunotherapy, assisting in the development of other cytokines as immunotherapeutic agents with greater efficacy.
Assuntos
Interleucina-15 , Neoplasias , Animais , Linfócitos T CD8-Positivos , Proliferação de Células , Meia-Vida , Humanos , Interleucina-15/genética , Interleucina-15/metabolismo , Interleucina-2 , Camundongos , Neoplasias/tratamento farmacológico , Albumina Sérica , Albumina Sérica Humana/farmacologiaRESUMO
KRAS mutation and NF-κB both play crucial role in pancreatic cancer; in addition, defensin, the peptide mediator in innate immunity, can inhibit NF-κB. Assuming a strategy that targets both NF-κB and concomitantly the mutated KRAS indirectly via intensive macropinocytosis, we designed and generated a recombinant protein DF2-HSA which consists of two molecules of human beta-defensin 2 (HBD2) and a moiety of human serum albumin (HSA). As shown, the recombinant protein DF2-HSA markedly down-regulated NF-κB in both KRAS mutant MIA PaCa-2 cells and wild type BxPC-3 cells. Determined by confocal microscopy, the uptake of DF2-HSA in MIA PaCa-2 cells was more intense than that in BxPC-3 cells. The uptake was blocked by the specific inhibitor EIPA, indicating that DF2-HSA internalized via macropinocytosis. DF2-HSA displayed more potent cytotoxicity to cancer cells than HBD2. DF2-HSA induced apoptosis in cancer cells. Notably, DF2-HSA inhibited tumor cell spheroid formation, an effect comparable to that of salinomycin. DF2-HSA inhibited tumor cell migration and invasion. As detected with scanning electron microscopy, DF2-HSA strongly depleted filopodia on cell surface; and salinomycin induced similar changes. By in vivo imaging, DF2-HSA displayed intense tumor-site accumulation and lasting retention for over 14 days; however, HBD2 showed much less tumor-site accumulation and a shorter retention time for only 24 h. DF2-HSA suppressed the growth of pancreatic cancer MIA PaCa-2 xenograft in athymic mice; and its combination with gemcitabine achieved higher antitumor efficacy. In summary, the recombinant defensin/HSA fusion protein that inhibits NF-κb associated with intensive macropinocytosis is highly effective against pancreatic cancer.
Assuntos
NF-kappa B , Neoplasias Pancreáticas , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Camundongos , NF-kappa B/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Albumina Sérica Humana/metabolismo , Albumina Sérica Humana/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias PancreáticasRESUMO
Stem cells are an important therapeutic source for recovery and regeneration, as their ability of self-renewal and differentiation offers an unlimited supply of highly specialized cells for therapeutic transplantation. Growth factors and serum are essential for maintaining the characteristics of stem cells in culture and for inducing differentiation. Because growth factors are produced mainly in bacterial (Escherichia coli) or animal cells, the use of such growth factors raises safety concerns that need to be resolved for the commercialization of stem cell therapeutics. To overcome this problem, studies on proteins produced in plants have been conducted. Here, we describe the functions of plant-derived fibroblast growth factor 2 (FGF2) and human serum albumin in the maintenance and differentiation of human-induced pluripotent stem cells (hiPSCs). Plant-derived FGF2 and human epidermal growth factor EGF were able to differentiate hiPSCs into neural stem cells (NSCs). These NSCs could differentiate into neuronal and glial cells. Our results imply that culturing stem cells in animal-free culture medium, which is composed of plant-derived proteins, would facilitate stem cell application research, for example, for cell therapy, by reducing contamination risk.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Albumina Sérica Humana/farmacologia , Animais , Linhagem Celular , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/farmacologia , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neurais/metabolismo , Oryza/genética , Oryza/metabolismo , Fenótipo , Proteínas de Plantas/farmacologia , Proteínas Recombinantes/farmacologia , Albumina Sérica Humana/genética , Albumina Sérica Humana/metabolismoRESUMO
Glioblastoma is one of the most difficult to treat cancers with poor prognosis and survival of around one year from diagnosis. Effective treatments are desperately needed. This work aims to prepare temozolomide acid (TMZA) loaded albumin nanoparticles, for the first time, to target glioblastoma (GL261) and brain cancer stem cells (BL6). TMZA was loaded into human serum albumin nanoparticles (HSA NPs) using the desolvation method. A response surface 3-level factorial design was used to study the effect of different formulation parameters on the drug loading and particle size of NPs. The optimum conditions were found to be: 4 mg TMZA with 0.05% sodium cholate. This yielded NPs with particle size and drug loading of 111.7 nm and 5.5% respectively. The selected formula was found to have good shelf life and serum stability but with a relatively fast drug release pattern. The optimized NPs showed excellent cellular uptake with â¼ 50 and 100% of cells were positive for NP uptake after 24 h incubation with both GL261 and BL6 glioblastoma cell lines, respectively. The selected formula showed high cytotoxicity with Ì´ 20% cell viability at 1 mM TMZA after 72 h incubation time. Finally, the fluorescently labelled NPs showed co-localization with the bioluminescent syngeneic BL6 intra-cranial tumour mouse model after intravenous administration.
Assuntos
Glioma , Nanopartículas/uso terapêutico , Osteonectina/metabolismo , Albumina Sérica Humana/farmacologia , Temozolomida , Animais , Antineoplásicos Alquilantes/administração & dosagem , Antineoplásicos Alquilantes/farmacocinética , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Composição de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Glioma/tratamento farmacológico , Glioma/metabolismo , Glioma/patologia , Camundongos , Tamanho da Partícula , Temozolomida/administração & dosagem , Temozolomida/farmacocinética , Distribuição TecidualRESUMO
Recently, we designed an inventive paradigm in nanomedicine-drug-free macromolecular therapeutics (DFMT). The ability of DFMT to induce apoptosis is based on biorecognition at cell surface, and crosslinking of receptors without the participation of low molecular weight drugs. The system is composed of two nanoconjugates: a bispecific engager, antibody or Fab' fragment-morpholino oligonucleotide (MORF1) conjugate; the second nanoconjugate is a multivalent effector, human serum albumin (HSA) decorated with multiple copies of complementary MORF2. Here, we intend to demonstrate that DFMT is a platform that will be effective on other receptors than previously validated CD20. We appraised the impact of daratumumab (DARA)- and isatuximab (ISA)-based DFMT to crosslink CD38 receptors on CD38+ lymphoma (Raji, Daudi) and multiple myeloma cells (RPMI 8226, ANBL-6). The biological properties of DFMTs were determined by flow cytometry, confocal fluorescence microscopy, reactive oxygen species determination, lysosomal enlargement, homotypic cell adhesion, and the hybridization of nanoconjugates. The data revealed that the level of apoptosis induction correlated with CD38 expression, the nanoconjugates meet at the cell surface, mitochondrial signaling pathway is strongly involved, insertion of a flexible spacer in the structure of the macromolecular effector enhances apoptosis, and simultaneous crosslinking of CD38 and CD20 receptors increases apoptosis.
Assuntos
ADP-Ribosil Ciclase 1/genética , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Morfolinos/química , Morfolinos/genética , Morfolinos/farmacologia , Mieloma Múltiplo/patologia , Nanoconjugados/química , Albumina Sérica Humana/química , Albumina Sérica Humana/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
The short half-life of coagulation factor IX (FIX) for haemophilia B (HB) therapy has been prolonged through fusion with human serum albumin (HSA), which drives the neonatal Fc receptor (FcRn)-mediated recycling of the chimera. However, patients would greatly benefit from further FIX-HSA half-life extension. In the present study, we designed a FIX-HSA variant through the engineering of both fusion partners. First, we developed a novel cleavable linker combining the two FIX activation sites, which resulted in improved HSA release. Second, insertion of the FIX R338L (Padua) substitution conferred hyperactive features (sevenfold higher specific activity) as for FIX Padua alone. Furthermore, we exploited an engineered HSA (QMP), which conferred enhanced human (h)FcRn binding [dissociation constant (KD ) 0·5 nM] over wild-type FIX-HSA (KD 164·4 nM). In hFcRn transgenic mice, Padua-QMP displayed a significantly prolonged half-life (2·7 days, P < 0·0001) versus FIX-HSA (1 day). Overall, we developed a novel FIX-HSA protein with improved activity and extended half-life. These combined properties may result in a prolonged functional profile above the therapeutic threshold, and thus in a potentially widened therapeutic window able to improve HB therapy. This rational engineering of both partners may pave the way for new fusion strategies for the design of engineered biotherapeutics.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Fator IX/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Albumina Sérica Humana/farmacologia , Animais , Fator IX/genética , Feminino , Meia-Vida , Hemofilia B/sangue , Hemofilia B/tratamento farmacológico , Humanos , Masculino , Camundongos Transgênicos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Albumina Sérica Humana/genéticaRESUMO
PURPOSE: Different commercial human embryo culture mediums can alter embryo quality and change birthweight. One component that could be contributing to variations but is not widely investigated is human serum albumin (HSA). HSA plays a multitude of roles during embryo culture and is a carrier for molecules including lipids. It remains unclear if lipid composition of HSA varies among commercial products and its effects on embryo quality, implantation, and fetal outcomes are relatively unknown. METHODS: Utilizing a mouse model of embryo culture, we cultured zygotes until the blastocyst stage (72-h culture) in G1/G2 containing either Vitrolife HSA, Sage HSA, or Recombinant HSA at 10%. Blastocyst quality (development, total cell number, superoxide generation), blastocyst lipid content (neutral lipids, non-esterified fatty acids, phospholipids, and triglycerides), implantation, and fetal lengths and weights were assessed. Fatty acid quantification of HSA source was assessed by standard thin-layer chromatography. RESULTS: Sage HSA had the greatest fatty acid composition, with an eightfold increase in saturated fatty acids. This coincided with reduced blastocyst development, increased superoxide generation, neutral lipids and triglycerides levels of blastocysts, and decreased implantation rates (p < 0.05). Unexpectedly, while Recombinant HSA had the lowest overall lipids it had 70-fold increase in palmitoleic acid and the lowest fetal weights (p < 0.05). CONCLUSION: Indicates the importance of a balance between different types/amount of lipids, and an "optimal ratio" required for embryo and fetal development. Therefore, the lipid content of HSA should be considered when choosing a suitable HSA source for use in clinical IVF.
Assuntos
Blastocisto/citologia , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Fertilização in vitro/métodos , Desenvolvimento Fetal/efeitos dos fármacos , Lipídeos/análise , Albumina Sérica Humana/farmacologia , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Implantação do Embrião , Transferência Embrionária , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Masculino , Camundongos , ZigotoRESUMO
Nanoparticulate albumin bound paclitaxel (nab-paclitaxel, nab-PTX) is among the most widely prescribed nanomedicines in clinical use, yet it remains unclear how nanoformulation affects nab-PTX behaviour in the tumour microenvironment. Here, we quantified the biodistribution of the albumin carrier and its chemotherapeutic payload in optically cleared tumours of genetically engineered mouse models, and compared the behaviour of nab-PTX with other clinically relevant nanoparticles. We found that nab-PTX uptake is profoundly and distinctly affected by cancer-cell autonomous RAS signalling, and RAS/RAF/MEK/ERK inhibition blocked its selective delivery and efficacy. In contrast, a targeted screen revealed that IGF1R kinase inhibitors enhance uptake and efficacy of nab-PTX by mimicking glucose deprivation and promoting macropinocytosis via AMPK, a nutrient sensor in cells. This study thus shows how nanoparticulate albumin bound drug efficacy can be therapeutically improved by reprogramming nutrient signalling and enhancing macropinocytosis in cancer cells.
Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutação , Nanopartículas , Neoplasias Experimentais/tratamento farmacológico , Paclitaxel , Proteínas Proto-Oncogênicas p21(ras)/genética , Albumina Sérica Humana , Animais , Linhagem Celular Tumoral , Glucose/deficiência , Glucose/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Nanopartículas/química , Nanopartículas/uso terapêutico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Paclitaxel/farmacocinética , Paclitaxel/farmacologia , Pinocitose , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células RAW 264.7 , Albumina Sérica Humana/química , Albumina Sérica Humana/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genéticaRESUMO
Fluid resuscitation following severe inflammation-induced hypoperfusion is critical for the restoration of hemodynamics and the prevention of multiorgan dysfunction syndrome during septic shock. Fluid resuscitation with commercially available crystalloid and colloid solutions only provides transient benefits, followed by fluid extravasation and tissue edema through the inflamed endothelium. The increased molecular weight (M.W.) of polymerized human serum albumin (PolyHSA) can limit fluid extravasation, leading to restoration of hemodynamics. In this prospective study, we evaluated how fluid resuscitation with PolyHSA impacts the hemodynamic and immune response in a lipopolysaccharide (LPS) induced endotoxemia mouse model. Additionally, we evaluated fluid resuscitation with PolyHSA in a model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Resuscitation with PolyHSA attenuated the immune response and improved the maintenance of systemic hemodynamics and restoration of microcirculatory hemodynamics. This decrease in inflammatory immune response and maintenance of vascular wall shear stress likely contributes to the maintenance of vascular integrity following fluid resuscitation with PolyHSA. The sustained restoration of perfusion, decrease in pro-inflammatory immune response, and improved vascular integrity that results from the high M.W. of PolyHSA indicates that a PolyHSA based solution is a potential resuscitation fluid for endotoxic and septic shock.
Assuntos
Endotoxemia/tratamento farmacológico , Hidratação/métodos , Microcirculação/efeitos dos fármacos , Albumina Sérica Humana/administração & dosagem , Choque Séptico/tratamento farmacológico , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Lipopolissacarídeos/efeitos adversos , Masculino , Mesocricetus , Camundongos , Estudos Prospectivos , Albumina Sérica Humana/farmacologia , Choque Séptico/etiologia , Choque Séptico/imunologiaRESUMO
Uveal melanoma (UM) is an intraocular tumor which is almost lethal at the metastatic stage due to the lack of effective treatments. In this regard, we have developed an albumin-based nanostructure (ABN) containing AZD8055 (ABN-AZD), which is a potent mTOR kinase inhibitor, for its efficient delivery to the tumors. The drug has been conjugated to ABN using tailored linkers that have a disulfide moiety, allowing its release selectively and effectively in the presence of an elevated concentration of glutathione, such as inside the tumoral cells. Our therapeutic approach induced significant cellular toxicity in uveal melanoma cells, but not in non-tumoral keratinocytes, highlighting the excellent selectivity of the system. In addition, these nanostructures showed excellent activity in vivo, decreasing the tumor surface compared to the free AZD8055 in mice models. Remarkably, the results obtained were achieved employing a dose 23 times lower than those used in previous reports.
Assuntos
Melanoma/tratamento farmacológico , Morfolinas , Nanoestruturas , Albumina Sérica Humana , Neoplasias Uveais/tratamento farmacológico , Animais , Células Alimentadoras , Humanos , Melanoma/enzimologia , Camundongos , Camundongos Nus , Morfolinas/química , Morfolinas/farmacologia , Nanoestruturas/química , Nanoestruturas/uso terapêutico , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Albumina Sérica Humana/química , Albumina Sérica Humana/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Neoplasias Uveais/enzimologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Albumin has been regarded as a potent antioxidant with free radical scavenging activities. Oxidative stress and neuronal apoptosis are responsible for its highly damaging effects on brain injury after intracerebral hemorrhage (ICH). Here, the present study investigated the neuroprotective effect of albumin against early brain injury after ICH and the potential underlying mechanisms. METHODS: Adult male Sprague-Dawley rats were subjected to intrastriatal injection of autologous blood to induce ICH. Human serum albumin was given by intravenous injection 1 h after ICH. U0126, an inhibitor of extracellular signal-regulated kinase (ERK1/2), and ML385, an inhibitor of nuclear factor-E2-related factor 2 (Nrf2), were intraperitoneally administered 1 h before ICH induction. Short- and long-term neurobehavioral tests, western blotting, immunofluorescence staining, oxidative stress evaluations, and apoptosis measurements were performed. RESULTS: Endogenous expression of albumin (peaked at 5 days) and heme oxygenase 1 (HO-1, peaked at 24 h) was increased after ICH compared with the sham group. Albumin and HO-1 were colocalized with neurons. Compared with vehicle, albumin treatment significantly improved short- and long-term neurobehavioral deficits and reduced oxidative stress and neuronal death at 72 h after ICH. Moreover, albumin treatment significantly promoted the phosphorylation of ERK1/2; increased the expression of Nrf2, HO-1, and Bcl-2; and downregulated the expression of Romo1 and Bax. U0126 and ML385 abolished the treatment effects of albumin on behavior and protein levels after ICH. CONCLUSIONS: Albumin attenuated oxidative stress-related neuronal death may in part via the ERK/Nrf2/HO-1 signaling pathway after ICH in rats. Our study suggests that albumin may be a novel therapeutic method to ameliorate brain injury after ICH.
Assuntos
Apoptose/efeitos dos fármacos , Hemorragia Cerebral/patologia , Heme Oxigenase-1/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Albumina Sérica Humana/farmacologia , Animais , Hemorragia Cerebral/metabolismo , Humanos , Masculino , Memória/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Nitrosylation of sulfhydryl (SH) groups of cysteine (Cys) moieties is an important post-translational modification (PTM), often on a par with phosphorylation. S-Nitrosoalbumin (ALB-Cys34SNO; SNALB) in plasma and S-nitrosohemoglobin (Hb-Cysß93SNO; HbSNO) in red blood cells are considered the most abundant high-molecular-mass pools of nitric oxide (NO) bioactivity in the human circulation. SNALB per se is not an NO donor. Yet, it acts as a vasodilator and an inhibitor of platelet aggregation. SNALB can be formed by nitrosation of the sole reduced Cys group of albumin (Cys34) by nitrosating species such as nitrous acid (HONO) and nitrous anhydride (N2O3), two unstable intermediates of NO autoxidation. SNALB can also be formed by the transfer (S-transnitrosylation) of the nitrosyl group (NO+) of a low-molecular-mass (LMM) S-nitrosothiol (RSNO) to ALB-Cys34SH. In the present study, the effects of LMM thiols on the inhibitory potential of ALB-Cys34SNO on human washed platelets were investigated. ALB-Cys34SNO was prepared by reacting n-butylnitrite with albumin after selective extraction from plasma of a healthy donor on HiTrapBlue Sepharose cartridges. ALB-Cys34SNO was used in platelet aggregation measurements after extended purification on HiTrapBlue Sepharose and enrichment by ultrafiltration (cutoff, 20 kDa). All tested LMM cysteinyl thiols (R-CysSH) including L-cysteine and L-homocysteine (at 10 µM) were found to mediate the collagen-induced (1 µg/mL) aggregation of human washed platelets by SNALB (range, 0-10 µM) by cGMP-dependent and cGMP-independent mechanisms. The LMM thiols themselves did not affect platelet aggregation. It is assumed that the underlying mechanism involves S-transnitrosylation of SH groups of the platelet surface by LMM RSNO formed through the reaction of SNALB with the thiols: ALB-Cys34SNO + R-CysSH â ALB-Cys34SH + R-CysSNO. Such S-transnitrosylation reactions may be accompanied by release of NO finally resulting in cGMP-dependent and cGMP-independent mechanisms.
Assuntos
Plaquetas/efeitos dos fármacos , Compostos Nitrosos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Albumina Sérica Humana/farmacologia , Compostos de Sulfidrila/química , Plaquetas/metabolismo , Humanos , Óxido Nítrico/metabolismo , Compostos Nitrosos/química , Processamento de Proteína Pós-Traducional , S-Nitrosotióis/química , S-Nitrosotióis/farmacologia , Albumina Sérica Humana/química , Compostos de Sulfidrila/farmacologiaRESUMO
Half-life extension strategies to reduce the intravitreal dosing frequency of biomolecules for the treatment of retinal neovascular diseases are attracting increasing interest. This study investigated ocular and systemic pharmacokinetics of the trivalent nanobody BI-X (with affinity to VEGF, Ang-2 and human albumin) in cynomolgus monkeys after intravitreal injection. BI-X concentrations were measured in serial samples of plasma, vitreous humor, aqueous humor and retina. Ocular pharmacokinetics of BI-X exhibited two phases. Initially up to 2-4 weeks after dosing, BI-X concentrations in vitreal, aqueous humor and retina declined with half-lives of around 3 days, which is comparable to macromolecules with a similar molecular weight. Thereafter, only vitreal concentrations were measurable, with a terminal half-life of 13.2 days, which is considerably longer than expected based on the BI-X molecular weight or hydrodynamic radius. It is hypothesized that binding of BI-X to low levels of intraocular albumin resulted in this half-life extension. BI-X was detectable in plasma up to 10 weeks post-dosing. Plasma pharmacokinetics of BI-X exhibited a similar biphasic disposition profile to the vitreous body, with a terminal half-life of 11.8 days, thus reflecting input kinetics from the eye. In conclusion, an important half-life extension principle based on vitreal albumin binding could be confirmed in a primate model, and the data obtained can potentially be translated to humans taking into account the differing vitreal albumin concentrations.
Assuntos
Inibidores da Angiogênese/farmacocinética , Angiopoietina-2/metabolismo , Albumina Sérica Humana/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Corpo Vítreo/metabolismo , Animais , Área Sob a Curva , Sinergismo Farmacológico , Feminino , Meia-Vida , Injeções Intravítreas , Macaca fascicularis , MasculinoRESUMO
BACKGROUND: Dysregulation of transcription and cytokine expression has been implicated in the pathogenesis of a variety inflammatory diseases. The resulting imbalance between inflammatory and resolving transcriptional programs can cause an overabundance of pro-inflammatory, classically activated macrophage type 1 (M1) and/or helper T cell type 1 (Th1) products, such as IFNγ, TNFα, IL1-ß, and IL12, that prevent immune switching to resolution and healing. The low molecular weight fraction of human serum albumin (LMWF5A) is a novel biologic drug that is currently under clinical investigation for the treatment of osteoarthritis and the hyper-inflammatory response associated with COVID-19. This study aims to elucidate transcriptional mechanisms of action involved with the ability of LMWF5A to reduce pro-inflammatory cytokine release. METHODS: ELISA arrays were used to identify cytokines and chemokines influenced by LMWF5A treatment of LPS-stimulated peripheral blood mononuclear cells (PBMC). The resulting profiles were analyzed by gene enrichment to gain mechanistic insight into the biologic processes and transcription factors (TFs) underlying the identified differentially expressed cytokines. DNA-binding ELISAs, luciferase reporter assays, and TNFα or IL-1ß relative potency were then employed to confirm the involvement of enriched pathways and TFs. RESULTS: LMWF5A was found to significantly inhibit a distinct set of pro-inflammatory cytokines (TNFα, IL-1ß, IL-12, CXCL9, CXCL10, and CXCL11) associated with pro-inflammatory M1/Th1 immune profiles. Gene enrichment analysis also suggests these cytokines are, in part, regulated by NF-κB and STAT transcription factors. Data from DNA-binding and reporter assays support this with LMWF5A inhibition of STAT1α DNA-binding activity as well as a reduction in overall NF-κB-driven luciferase expression. Experiments using antagonists specific for the immunomodulatory and NF-κB/STAT-repressing transcription factors, peroxisome proliferator-activated receptor (PPAR)γ and aryl hydrocarbon receptor (AhR), indicate these pathways are involved in the LMWF5A mechanisms of action by reducing LMWF5A drug potency as measured by TNFα and IL-1ß release. CONCLUSION: In this report, we provide evidence that LMWF5A reduces pro-inflammatory cytokine release by activating the immunoregulatory transcription factors PPARγ and AhR. In addition, our data indicate that LMWF5A suppresses NF-κB and STAT1α pro-inflammatory pathways. This suggests that LMWF5A acts through these mechanisms to decrease pro-inflammatory transcription factor activity and subsequent inflammatory cytokine production.