RESUMO
Identifying novel breast cancer biomarkers will improve patient stratification, enhance therapeutic outcomes, and help develop non-invasive diagnostics. We compared the proteomic profiles of whole-cell and exosomal samples of representative breast cancer cell subtypes to evaluate the potential of extracellular vesicles as non-invasive disease biomarkers in liquid biopsies. Overall, differentially-expressed proteins in whole-cell and exosome samples (which included markers for invasion, metastasis, angiogenesis, and drug resistance) effectively discriminated subtypes; furthermore, our results confirmed that the proteomic profile of exosomes reflects breast cancer cell-of-origin, which underscores their potential as disease biomarkers. Our study will contribute to identifying biomarkers that support breast cancer patient stratification and developing novel therapeutic strategies. We include an open, interactive web tool to explore the data as a molecular resource that can explain the role of these protein signatures in breast cancer classification.
Assuntos
Biomarcadores Tumorais , Neoplasias da Mama , Exossomos , Proteômica , Humanos , Exossomos/metabolismo , Feminino , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Biomarcadores Tumorais/metabolismo , Proteômica/métodos , Linhagem Celular Tumoral , Proteoma/metabolismoRESUMO
The importance of an adequate linking moiety design that allows controlled drug(s) release at the desired site of action is extensively studied for polymer-drug conjugates (PDCs). Redox-responsive self-immolative linkers bearing disulfide moieties (SS-SIL) represent a powerful strategy for intracellular drug delivery; however, the influence of drug structural features and linker-associated spacers on release kinetics remains relatively unexplored. The influence of drug/spacer chemical structure and the chemical group available for conjugation on drug release and the biological effect of resultant PDCs is evaluated. A "design of experiments" tool is implemented to develop a liquid chromatography-mass spectrometry method to perform the comprehensive characterization required for this systematic study. The obtained fit-for-purpose analytical protocol enables the quantification of low drug concentrations in drug release studies and the elucidation of metabolite presence. and provides the first data that clarifies how drug structural features influence the drug release from SS-SIL and demonstrates the non-universal nature of the SS-SIL. The importance of rigorous linker characterization in understanding structure-function correlations between linkers, drug chemical functionalities, and in vitro release kinetics from a rationally-designed polymer-drug nanoconjugate, a critical strategic crafting methodology that should remain under consideration when using a reductive environment as an endogenous drug release trigger.
Assuntos
Sistemas de Liberação de Medicamentos , Polímeros , Polímeros/química , Preparações Farmacêuticas , Liberação Controlada de Fármacos , Sistemas de Liberação de Medicamentos/métodos , NanoconjugadosRESUMO
Developing peptide-based materials with controlled morphology is a critical theme of soft matter research. Herein, we report the formation of a novel, patterned cross-ß structure formed by self-assembled C3 -symmetric peptide amphiphiles based on diphenylalanine and benzene-1,3,5-tricarboxamide (BTA). The cross-ß motif is an abundant structural element in amyloid fibrils and aggregates of fibril-forming peptides, including diphenylalanine. The incorporation of topological constraints on one edge of the diphenylalanine fragment limits the number of ß-strands in ß-sheets and leads to the creation of an unconventional offset-patterned cross-ß structure consisting of short 3×2 parallel ß-sheets stabilized by phenylalanine zippers. In the reported assembly, two patterned cross-ß structures bind parallel arrays of BTA stacks in a superstructure within a single-molecule-thick nanoribbon. In addition to a threefold network of hydrogen bonds in the BTA stack, each molecule becomes simultaneously bound by hydrogen bonds from three ß-sheets and four phenylalanine zippers. The diffuse layer of alkyl chains with terminal polar groups prevents the nanoribbons from merging and stabilizes cross-ß-structure in water. Our results provide a simple approach to the incorporation of novel patterned cross-ß motifs into supramolecular superstructures and shed light on the general mechanism of ß-sheet formation in C3 -symmetric peptide amphiphiles.
Assuntos
Amiloide , Peptídeos , Estrutura Secundária de Proteína , Peptídeos/química , Amiloide/química , Conformação Proteica em Folha beta , FenilalaninaRESUMO
Studying metabolism may assist in understanding the relationship between normal and dysfunctional mitochondrial activity and various diseases, such as neurodegenerative, cardiovascular, autoimmune, psychiatric, and cancer. Nuclear magnetic resonance-based metabolomics represents a powerful method to characterize the chemical content of complex samples and has been successfully applied to studying a range of conditions. However, an optimized methodology is lacking for analyzing isolated organelles, such as mitochondria. In this study, we report the development of a protocol to metabolically profile mitochondria from healthy, tumoral, and metastatic tissues. Encouragingly, this approach provided quantitative information about up to 45 metabolites in one comprehensive and robust analysis. Our results revealed significant differences between whole-cell and mitochondrial metabolites, which supports a more refined approach to metabolic analysis. We applied our optimized methodology to investigate aggressive and metastatic breast cancer in mouse tissues, discovering that lung mitochondria exhibit an altered metabolic fingerprint. Specific amino acids, organic acids, and lipids showed significant increases in levels when compared with mitochondria from healthy tissues. Our optimized methodology could promote a better understanding of the molecular mechanisms underlying breast cancer aggressiveness and mitochondrial-related diseases and support the optimization of new advanced therapies.
Assuntos
Mitocôndrias , Neoplasias , Camundongos , Animais , Mitocôndrias/metabolismo , Metabolômica/métodos , Neoplasias/metabolismo , Espectroscopia de Ressonância Magnética , Aminoácidos/metabolismoRESUMO
We currently lack effective treatments for the devastating loss of neural function associated with spinal cord injury (SCI). In this study, we evaluated a combination therapy comprising human neural stem cells derived from induced pluripotent stem cells (iPSC-NSC), human mesenchymal stem cells (MSC), and a pH-responsive polyacetal-curcumin nanoconjugate (PA-C) that allows the sustained release of curcumin. In vitro analysis demonstrated that PA-C treatment protected iPSC-NSC from oxidative damage in vitro, while MSC co-culture prevented lipopolysaccharide-induced activation of nuclear factor-κB (NF-κB) in iPSC-NSC. Then, we evaluated the combination of PA-C delivery into the intrathecal space in a rat model of contusive SCI with stem cell transplantation. While we failed to observe significant improvements in locomotor function (BBB scale) in treated animals, histological analysis revealed that PA-C-treated or PA-C and iPSC-NSC + MSC-treated animals displayed significantly smaller scars, while PA-C and iPSC-NSC + MSC treatment induced the preservation of ß-III Tubulin-positive axons. iPSC-NSC + MSC transplantation fostered the preservation of motoneurons and myelinated tracts, while PA-C treatment polarized microglia into an anti-inflammatory phenotype. Overall, the combination of stem cell transplantation and PA-C treatment confers higher neuroprotective effects compared to individual treatments.
Assuntos
Curcumina/farmacologia , Transplante de Células-Tronco Mesenquimais , Nanoconjugados/uso terapêutico , Fármacos Neuroprotetores/farmacologia , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/terapia , Acetais/uso terapêutico , Animais , Células Cultivadas , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas , Células-Tronco Mesenquimais , Células-Tronco Neurais , Polímeros/uso terapêutico , Ratos , Ratos Sprague-DawleyRESUMO
M2-like tumor-associated macrophages (M2 TAMs) play important roles in the resistance of tumors to immunotherapies. Selective depletion or reprogramming of M2 TAMs may sensitize the nonresponsive tumors for immune-mediated eradication. However, precision delivery of payloads to M2 TAMs, while sparing healthy tissues, has remained an unresolved challenge. Here, we studied the application of a short linear peptide (CSPGAK, "mUNO") for the delivery of molecular and nanoscale cargoes in M2 TAMs in vitro and the relevance of the peptide for in vivo targeting of early-stage primary breast tumors and metastatic lung foci. First, we performed in silico modeling and found that mUNO interacts with mouse CD206 via a binding site between lectin domains CTLD1 and CTLD2, the same site previously demonstrated to be involved in mUNO binding to human CD206. Second, we showed that cultured M2 macrophages take up fluorescein-labeled (FAM) polymersomes conjugated with mUNO using the sulfhydryl group of its N-terminal cysteine. Pulse/chase studies of FAM-mUNO in M2 macrophages suggested that the peptide avoided lysosomal entrapment and escaped from early endosomes. Third, our in vivo studies with FAM-mUNO demonstrated that intraperitoneal administration results in better pharmacokinetics and higher blood bioavailability than can be achieved with intravenous administration. Intraperitoneal FAM-mUNO, but not FAM-control, showed a robust accumulation in M2-skewed macrophages in mouse models of early primary breast tumor and lung metastasis. This targeting was specific, as no uptake was observed in nonmalignant control organs, including the liver, or other cell types in the tumor, including M1 macrophages. Collectively, our studies support the application of the CD206-binding mUNO peptide for delivery of molecular and nanoscale cargoes to M2 macrophages and manifest the relevance of this mode of targeting primary and metastatic breast tumors.
Assuntos
Imunoterapia/métodos , Lectinas Tipo C/química , Neoplasias Pulmonares/diagnóstico , Metástase Linfática/diagnóstico , Lectinas de Ligação a Manose/química , Peptídeos/química , Receptores de Superfície Celular/química , Neoplasias de Mama Triplo Negativas/diagnóstico , Macrófagos Associados a Tumor/imunologia , Animais , Sítios de Ligação , Diferenciação Celular , Linhagem Celular Tumoral , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Portadores de Fármacos/farmacocinética , Feminino , Fluorescência , Humanos , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Metástase Linfática/diagnóstico por imagem , Metástase Linfática/imunologia , Lisossomos/metabolismo , Maleimidas/química , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Peptídeos/administração & dosagem , Peptídeos/metabolismo , Peptídeos/farmacocinética , Poliésteres/química , Polietilenoglicóis/química , Polímeros/administração & dosagem , Polímeros/química , Polímeros/farmacologia , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Neoplasias de Mama Triplo Negativas/diagnóstico por imagem , Neoplasias de Mama Triplo Negativas/patologia , Macrófagos Associados a Tumor/metabolismoRESUMO
Triple-negative breast cancer (TNBC), an aggressive, metastatic and recurrent breast cancer (BC) subtype, currently suffers from a lack of adequately described spontaneously metastatic preclinical models that faithfully reproduce the clinical scenario. We describe two preclinical spontaneously metastatic TNBC orthotopic murine models for the development of advanced therapeutics: an immunodeficient human MDA-MB-231-Luc model and an immunocompetent mouse 4T1 model. Furthermore, we provide a broad range of multifactorial analysis for both models that could provide relevant information for the development of new therapies and diagnostic tools. Our comparisons uncovered differential growth rates, stromal arrangements and metabolic profiles in primary tumors, and the presence of cancer-associated adipocyte infiltration in the MDA-MB-231-Luc model. Histopathological studies highlighted the more rapid metastatic spread to the lungs in the 4T1 model following a lymphatic route, while we observed both homogeneous (MDA-MB-231-Luc) and heterogeneous (4T1) metastatic spread to axillary lymph nodes. We encountered unique metabolomic signatures in each model, including crucial amino acids and cell membrane components. Hematological analysis demonstrated severe leukemoid and lymphoid reactions in the 4T1 model with the partial reestablishment of immune responses in the immunocompromised MDA-MB-231-Luc model. Additionally, we discovered ß-immunoglobulinemia and increased basal levels of G-CSF correlating with a metastatic switch, with G-CSF also promoting extramedullary hematopoiesis (both models) and causing hepatosplenomegaly (4T1 model). Overall, we believe that the characterization of these preclinical models will foster the development of advanced therapeutic strategies for TNBC treatment, especially for the treatment of patients presenting both, primary tumors and metastatic spread.
Assuntos
Modelos Animais de Doenças , Neoplasias Mamárias Experimentais/terapia , Neoplasias de Mama Triplo Negativas/terapia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Metástase Linfática , Neoplasias Mamárias Experimentais/diagnóstico , Neoplasias Mamárias Experimentais/metabolismo , Metabolômica/métodos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Reprodutibilidade dos Testes , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
The high incidence of prostate carcinogenesis has prompted the search for novel effective treatment approaches. We have employed curcumin (Curc) and diethylstilbestrol (DES) to synthesize a series of polyacetal (PA)-based combination conjugates for prostate cancer (PCa) treatment. Given their bihydroxyl functionalities, Curc and DES molecules were incorporated into a PA mainchain using a one-pot reaction between diols and divinyl ethers. The PA-conjugates released both drugs under acidic conditions, such as those found in the tumor microenvironment, endosomes, or lysosomes, while remaining stable at neutral pH 7.4. The drug ratio was optimized to achieve anticancer drug synergism with elevated cytotoxicity against LNCaP-hormone-dependent human PCa cells conferred via the induction of S phase cell cycle arrest by the upregulation of p53 and CDK inhibitors p21Waf/CIP1 and downregulation of cyclin D1. The application of rationally designed PA-Curc-DES combination conjugates represents a potentially exciting new treatment for prostate cancer.
Assuntos
Acetais/química , Antineoplásicos , Curcumina/química , Dietilestilbestrol/química , Polímeros/química , Neoplasias da Próstata/tratamento farmacológico , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Preparações de Ação Retardada/síntese química , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Humanos , Masculino , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficit causes the rare disorder Primary Hyperoxaluria Type I (PH1). We now describe the conjugation of poly(ethylene glycol)-co-poly(L-glutamic acid) (PEG-PGA) block-co-polymer to AGT via the formation of disulfide bonds between the polymer and solvent-exposed cysteine residues of the enzyme. PEG-PGA conjugation did not affect AGT structural/functional properties and allowed the enzyme to be internalized in a cellular model of PH1 and to restore glyoxylate-detoxification. The insertion of the C387S/K390S amino acid substitutions, known to favor interaction with the peroxisomal import machinery, reduced conjugation efficiency, but endowed conjugates with the ability to reach the peroxisomal compartment. These results, along with the finding that conjugates are hemocompatible, stable in plasma, and non-immunogenic, hold promise for the development of polypeptide-based AGT conjugates as a therapeutic option for PH1 patients and represent the base for applications to other diseases related to deficits in peroxisomal proteins.
Assuntos
Sistemas de Liberação de Medicamentos , Hiperoxalúria Primária/tratamento farmacológico , Peroxissomos/metabolismo , Polietilenoglicóis/química , Ácido Poliglutâmico/análogos & derivados , Transaminases/administração & dosagem , Transaminases/química , Substituição de Aminoácidos , Animais , Células CHO , Cricetulus , Terapia Enzimática , Glioxilatos/metabolismo , Humanos , Hiperoxalúria Primária/enzimologia , Hiperoxalúria Primária/metabolismo , Modelos Moleculares , Peroxissomos/efeitos dos fármacos , Transaminases/genética , Transaminases/farmacocinéticaRESUMO
In photodynamic therapy (PDT), the low absorptivity of photosensitizers in an aqueous environment reduces singlet oxygen generation efficiency and thereby decreases photosensitizing efficacy in biological conditions. To circumvent this problem, we designed a phthalocyanine-poly-L-glutamic acid conjugate (1-PG) made from a new phthalocyanine (Pc 1) monofunctionalized to allow adequate conjugation to PGA. The resulting 1-PG conjugate retained high absorptivity in the near-infrared (NIR) region at its λmax 675nm in an aqueous environment. The 1-PG conjugate demonstrated good singlet oxygen generation efficiency, increased uptake by 4 T1 breast cancer cells via clathrin-mediated endocytosis, and enhanced photocytotoxic efficacy. The conjugate also displayed a high light-dark toxicity ratio, approximately 1.5-fold greater than zinc phthalocyanine at higher concentration (10 µM), an important feature for the reduction of dark toxicity and unwanted side effects. These results suggest that the 1-PG conjugate could be a useful alternative for deep tissue treatment with enhanced anti-cancer (PDT) efficacy.
Assuntos
Ácido Glutâmico/química , Indóis/química , Nanoconjugados/química , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Animais , Linhagem Celular Tumoral , Endocitose , Isoindóis , Luz , Camundongos , Estrutura Molecular , Oxigênio Singlete/químicaRESUMO
There is a need to develop new and innovative polymer carriers to be used as drug delivery systems and/or imaging agents owing to the fact that there is no universal polymeric system that can be used in the treatment of all diseases. Additionally, limitations with existing systems, such as a lack of biodegradability and biocompatibility, inevitably lead to side effects and poor patient compliance. New polymer therapeutics based on amino acids are excellent candidates for drug delivery, as they do not suffer from these limitations. This article reports on a simple yet powerful methodology for the synthesis of 3-arm star-shaped polyglutamic acid with well-defined structures, precise molecular weights (MW), and low polydispersity (D = <1.3). These were synthesized by ring-opening polymerization (ROP) of N-carboxyanhydrides (NCA) in a divergent method from novel multifunctional initiators. Herein, their exhaustive physicochemical characterization is presented. Furthermore, preliminary in vitro evaluation in selected cell models, and exhaustive in vivo biodistribution and pharmacokinetics, highlighted the advantages of these branched systems when compared with their linear counterparts in terms of cell uptake enhancement and prolonged plasma half-life.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Ácido Poliglutâmico/análogos & derivados , Linhagem Celular Tumoral/metabolismo , Dicroísmo Circular , Células Endoteliais/metabolismo , Citometria de Fluxo , Humanos , Técnicas In Vitro , Microscopia Confocal , Estrutura Molecular , Ácido Poliglutâmico/síntese química , Ácido Poliglutâmico/química , Ácido Poliglutâmico/farmacocinética , Polimerização , Veias Umbilicais/citologia , Veias Umbilicais/metabolismoRESUMO
New pH-responsive polymersomes for active anticancer oligonucleotide delivery were prepared from triblock copolymers. The delivery systems were formed by two terminal hydrophilic blocks, PEG and polyglycerolmethacrylate (poly-GMA), and a central weakly basic block, polyimidazole-hexyl methacrylate (poly-ImHeMA), which can complex with oligonucleotides and control vesicle formation/disassembly via pH variations. Targeted polymersomes were prepared by mixing folate-derivatized and underivatized copolymers. At pH 5, ds-DNA was found to complex with the pH-responsive copolymers at a N/P molar ratio above â¼2:1, which assisted the encapsulation of ds-DNA in the polymersomes, while low association was observed at pH 7.4. Cytotoxicity studies performed on folate receptor overexpressing KB and B16-F10 cells and low folate receptor expressing MCF-7 cells showed high tolerance of the polymersomes at up to 3 mg/mL concentration. Studies performed with red blood cells showed that at pH 5.0 the polymersomes have endosomolytic properties. Cytofluorimetric studies showed a 5.5-fold higher uptake of ds-DNA loaded folate-functional polymersomes in KB cells compared to nontargeted polymersomes. In addition, ds-DNA was found to be localized both in the nucleus and in the cytosol. The incubation of luciferase transfected B16-F10 cells with targeted polymersomes loaded with luciferase and Hsp90 expression silencing siRNAs yielded 31 and 23% knockdown in target protein expression, respectively.
Assuntos
Antineoplásicos/farmacologia , Núcleo Celular/genética , Polietilenoglicóis/química , RNA Interferente Pequeno/farmacologia , Citosol/metabolismo , Portadores de Fármacos/química , Receptores de Folato com Âncoras de GPI/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Células KB , Células MCF-7 , Metacrilatos/síntese química , Metacrilatos/química , Nanopartículas/químicaRESUMO
PURPOSE: miRNAs are noncoding RNAs that negatively regulate target mRNA gene expression. Aberrant miRNA expression is associated with prostate cancer pathogenesis. We identified miRNAs as potential biomarkers for prostate cancer diagnosis and prognosis. MATERIALS AND METHODS: Total RNA was obtained from 10 normal prostate and 50 prostate cancer samples, and analyzed using the GeneChip® miRNA 2.0 Array. At a median followup of 92 months (range 2 to 189) an independent cohort of 273 paraffin embedded prostate cancer samples was used for validation by quantitative reverse transcriptase-polymerase chain reaction. Another 92 urine samples from patients undergoing prostate biopsy were evaluated for these miRNAs. RESULTS: miR-182 and 187, the miRNAs most differentially expressed between normal and tumor tissue, were selected for further validation. miR-187 inversely correlated with cT (p = 0.125) and pT (p = 0.0002) stages, Gleason score (p = 0.003) and TMPRSS2-ERG status (p = 0.003). The log rank test showed associations of miR-182 with biochemical (p = 0.026) and clinical (p = 0.043) progression-free survival, as also noted on multivariate analysis. A significant independent improvement in the definition of risk of progression was achieved by combining miR-182 expression with Gleason score (p <0.0001). miR-187 detection in urine provided an independent predictive value for positive biopsy. A prediction model including serum prostate specific antigen, urine PCA3 and miR-187 provided 88.6% sensitivity and 50% specificity (AUC 0.711, p = 0.001). CONCLUSIONS: Results show that miR-182 and 187 are promising biomarkers for prostate cancer prognosis to identify patients at risk for progression and for diagnosis to improve the predictive capability of existing biomarkers.
Assuntos
Biomarcadores Tumorais , MicroRNAs , Prostatectomia , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/cirurgia , Idoso , Biomarcadores Tumorais/urina , Diagnóstico Precoce , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , MicroRNAs/urina , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/urina , Estudos RetrospectivosRESUMO
Protein therapeutics have become an important class of medicines for a large variety of diseases. However, they have disadvantages such as rapid elimination/metabolism leading to the need for repeated doses, immunogenicity/antigenicity, and aggregation/degradation during formulation and storage. The concept of polymer masked-unmasked protein therapy (PUMPT) makes use of polymer-protein multivalent conjugation with biodegradable carriers, which mask the protein activity during transport and increase its stability, but is capable of specifically triggering an unmasking effect at the disease site, allowing its therapeutic action. The aim of this study was to widen the PUMPT concept by designing reduction sensitive poly-l-glutamic acid (PGA)-based conjugates, in which the protein release and unmasking effect takes place in the reducing environments found intracellularly as well as in the tumor microenvironment. Lysozyme was used as the model protein to achieve proof of concept. Overall, the synthesized platform showed to be promising for the delivery of anticancer proteins as well as for enzyme replacement therapeutic approaches aiming to treat lysosomal storage disorders.
Assuntos
Portadores de Fármacos/química , Muramidase/química , Ácido Poliglutâmico/química , Polímeros/química , Portadores de Fármacos/administração & dosagem , Micrococcus , Muramidase/administração & dosagem , Ácido Poliglutâmico/administração & dosagem , Polímeros/administração & dosagem , Estrutura Secundária de ProteínaRESUMO
Enhanced targeting approaches will support the treatment of diseases associated with dysfunctional mitochondria, which play critical roles in energy generation and cell survival. Obstacles to mitochondria-specific targeting include the presence of distinct biological barriers and the need to pass through (or avoid) various cell internalization mechanisms. A range of studies have reported the design of mitochondrially-targeted nanomedicines that navigate the complex routes required to influence mitochondrial function; nonetheless, a significant journey lies ahead before mitochondrially-targeted nanomedicines become suitable for clinical use. Moving swiftly forward will require safety studies, in vivo assays confirming effectiveness, and methodologies to validate mitochondria-targeted nanomedicines' subcellular location/activity. From a nanomedicine standpoint, we describe the biological routes involved (from administration to arrival within the mitochondria), the features influencing rational design, and the techniques used to identify/validate successful targeting. Overall, rationally-designed mitochondria-targeted-based nanomedicines hold great promise for precise subcellular therapeutic delivery.
Assuntos
Nanopartículas , Neoplasias , Humanos , Nanomedicina/métodos , Mitocôndrias , Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológicoRESUMO
Targeting cancer cell exosome release and biogenesis represents a potentially efficient means to treat tumors and prevent cancer recurrence/metastasis; however, the complexity and time-consuming nature of currently employed methods to purify and characterize exosomes represent obstacles to progression. Herein, we describe a rapid, convergent, and cost-efficient strategy to analyze candidate U.S. Food and Drug Administration (FDA)-approved drugs that inhibit exosome release and/or biogenesis using breast cancer cell line models in the hope of repurposing them for the clinical treatment of metastatic tumors. We combined the ExoScreen assay based on AlphaScreenTM technology with the antibody-mediated detection of an atypical lipid (lysobisphosphatidic acid - LBPA) present in the intra-luminal vesicle/exosomal fraction to achieve both extracellular and intracellular information on exosome modulation after treatment. As proof of concept for this strategy, we identified docetaxel, biscurcumin, primaquine, and doxorubicin as potential exosome release inhibitors in the Her-2 positive MDA-MB-453 and luminal A MCF7 cell lines. Dinaciclib also functioned as an exosome release inhibitor in MCF7 cells. Further, we explored the expression of proteins involved in exosome biogenesis (TSG101, CD9 tetraspanin, Alix, SMase2) and release (Rab11, Rab27) to decipher and validate the possible molecular mechanisms of action of the identified exosome inhibitors. We anticipate that our approach could help to create robust high-throughput screening methodologies to accelerate drug repurposing when using FDA-approved compound libraries and to develop rationally-designed single/combination therapies (including nanomedicines) that can target metastasis progression by modulating exosome biogenesis or release in various tumor types.
Assuntos
Exossomos , Neoplasias , Estados Unidos , Humanos , Exossomos/metabolismo , Linhagem Celular Tumoral , Células MCF-7 , Neoplasias/metabolismoRESUMO
Brain tumors represent the second most common cause of pediatric cancer death, with malignant gliomas accounting for â¼75% of pediatric deaths. Palbociclib, a selective cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, has shown promise in phase I clinical trials of pediatric patients with progressive/refractory brain tumors using the oral administration route; however, pharmacokinetic limitations and toxicity issues remain. We synthesized a family of well-defined linear and star-shaped polyglutamate (PGA)-palbociclib conjugates using redox-sensitive self-immolative linkers to overcome limitations associated with free palbociclib. Exhaustive characterization of this conjugate family provided evidence for a transition towards the formation of more organized conformational structures upon increased drug loading. We evaluated the activity of conjugates in patient-derived glioblastoma and diffuse intrinsic pontine glioma cells, which display differing reducing environments due to differential glutathione expression levels. We discovered that microenvironmental parameters and the identified conformational changes determined palbociclib release kinetics and therapeutic output; furthermore, we identified a star-shaped PGA-palbociclib conjugate with low drug loading as an optimal therapeutic approach in diffuse intrinsic pontine glioma cells.
Assuntos
Neoplasias Encefálicas , Glioma Pontino Intrínseco Difuso , Glioma , Humanos , Criança , Ácido Glutâmico , Glioma/metabolismo , Neoplasias Encefálicas/metabolismoRESUMO
The standardization of clinical studies using extracellular vesicles (EVs) has mainly focused on the procedures employed for their isolation and characterization; however, preanalytical aspects of sample collection, handling and storage also significantly impact the reproducibility of results. We conducted an online survey based on SPREC (Standard PREanalytical Code) among members of GEIVEX (Grupo Español de Investigación en Vesiculas Extracelulares) to explore how different laboratories handled fluid biospecimens destined for EV analyses. We received 70 surveys from forty-three different laboratories: 44% focused on plasma, 9% on serum and 16% on urine. The survey indicated that variability in preanalytical approaches reaches 94%. Moreover, in some cases, researchers had no access to all relevant preanalytical details of samples, with some sample aspects with potential impact on EV isolation/characterisation not coded within the current version of SPREC. Our study highlights the importance of working with common standard operating procedures (SOP) to control preanalytical conditions. The application of SPREC represents a suitable approach to codify and register preanalytical conditions. Integrating SPREC into the SOPs of laboratories/biobanks will provide a valuable source of information and constitute an advance for EV research by improving reproducibility and credibility.
RESUMO
Modifying biological agents with polymers such as polyethylene glycol (PEG) has demonstrated clinical benefits; however, post-market surveillance of PEGylated derivatives has revealed PEG-associated toxicity issues, prompting the search for alternatives. We explore how conjugating a poly-l-glutamic acid (PGA) to an anti-insulin growth factor 1 receptor antibody (AVE1642) modulates the bio-nano interface and anti-tumor activity in preclinical prostate cancer models. Native and PGA-modified AVE1642 display similar anti-tumor activity in vitro; however, AVE1642 prompts IGF-1R internalization while PGA conjugation prompts higher affinity IGF-1R binding, thereby inhibiting IGF-1R internalization and altering cell trafficking. AVE1642 attenuates phosphoinositide 3-kinase signaling, while PGA-AVE1642 inhibits phosphoinositide 3-kinase and mitogen-activated protein kinase signaling. PGA conjugation also enhances AVE1642's anti-tumor activity in an orthotopic prostate cancer mouse model, while PGA-AVE1642 induces more significant suppression of cancer cell proliferation/angiogenesis than AVE1642. These findings demonstrate that PGA conjugation modulates an antibody's bio-nano interface, mechanism of action, and therapeutic activity.
Assuntos
Ácido Glutâmico , Neoplasias da Próstata , Animais , Camundongos , Masculino , Humanos , Fosfatidilinositol 3-Quinases , Neoplasias da Próstata/tratamento farmacológico , Proliferação de Células , Fosfatidilinositol 3-Quinase , PolietilenoglicóisRESUMO
Although many studies have explored the depletion of tumor-associated macrophages (TAM) as a therapeutic strategy for solid tumors, currently available compounds suffer from poor efficacy and dose-limiting side effects. Here, we developed a novel TAM-depleting agent ("OximUNO") that specifically targets CD206+ TAMs and demonstrated efficacy in a triple-negative breast cancer (TNBC) mouse model. OximUNO comprises a star-shaped polyglutamate (St-PGA) decorated with the CD206-targeting peptide mUNO that carries the chemotherapeutic drug doxorubicin (DOX). In the TNBC model, a fluorescently labeled mUNO-decorated St-PGA homed to CD206+ TAMs within primary lesions and metastases. OximUNO exhibited no acute liver or kidney toxicity in vivo. Treatment with OximUNO reduced the progression of primary tumor lesions and pulmonary metastases, significantly diminished the number of CD206+ TAMs and increased the CD8/FOXP3 expression ratio (indicating immunomodulation). Our findings suggest the potential benefit of OximUNO as a TAM-depleting agent for TNBC treatment. Importantly, our studies also represent a novel design of a peptide-targeted St-PGA as a targeted therapeutic nanoconjugate. Significance: A peptide-targeted nanoformulation of DOX exclusively eliminates mannose receptor+ TAMs in breast cancer models, generating response without off-target effects (a drawback of many TAM-depleting agents under clinical study).