RESUMO
Tumor-infiltrating lymphocyte (TIL) hypofunction contributes to the progression of advanced cancers and is a frequent target of immunotherapy. Emerging evidence indicates that metabolic insufficiency drives T cell hypofunction during tonic stimulation, but the signals that initiate metabolic reprogramming in this context are largely unknown. Here, we found that Meteorin-like (METRNL), a metabolically active cytokine secreted by immune cells in the tumor microenvironment (TME), induced bioenergetic failure of CD8+ T cells. METRNL was secreted by CD8+ T cells during repeated stimulation and acted via both autocrine and paracrine signaling. Mechanistically, METRNL increased E2F-peroxisome proliferator-activated receptor delta (PPARδ) activity, causing mitochondrial depolarization and decreased oxidative phosphorylation, which triggered a compensatory bioenergetic shift to glycolysis. Metrnl ablation or downregulation improved the metabolic fitness of CD8+ T cells and enhanced tumor control in several tumor models, demonstrating the translational potential of targeting the METRNL-E2F-PPARδ pathway to support bioenergetic fitness of CD8+ TILs.
Assuntos
Linfócitos T CD8-Positivos , Linfócitos do Interstício Tumoral , Mitocôndrias , Microambiente Tumoral , Linfócitos T CD8-Positivos/imunologia , Animais , Mitocôndrias/metabolismo , Mitocôndrias/imunologia , Camundongos , Microambiente Tumoral/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , Transdução de Sinais , Metabolismo Energético , PPAR delta/metabolismo , Linhagem Celular Tumoral , Neoplasias/imunologia , Glicólise , Camundongos Knockout , Fosforilação OxidativaRESUMO
Nanoparticle (NP)-based mRNA cancer vaccines hold great promise to realize personalized cancer treatments. To advance this technology requires delivery formulations for efficient intracellular delivery to antigen-presenting cells. We developed a class of bioreducible lipophilic poly(beta-amino ester) nanocarriers with quadpolymer architecture. The platform is agnostic to the mRNA sequence, with one-step self-assembly allowing for delivery of multiple antigen-encoding mRNAs as well as codelivery of nucleic acid-based adjuvants. We examined structure-function relationships for NP-mediated mRNA delivery to dendritic cells (DCs) and identified that a lipid subunit of the polymer structure was critical. Following intravenous administration, the engineered NP design facilitated targeted delivery to the spleen and preferential transfection of DCs without the need for surface functionalization with targeting ligands. Treatment with engineered NPs codelivering antigen-encoding mRNA and toll-like receptor agonist adjuvants led to robust antigen-specific CD8+ T cell responses, resulting in efficient antitumor therapy in in vivo models of murine melanoma and colon adenocarcinoma.
Assuntos
Adenocarcinoma , Vacinas Anticâncer , Neoplasias do Colo , Nanopartículas , Animais , Camundongos , Humanos , Células Dendríticas , Baço , Ligantes , RNA Mensageiro/genética , Adenocarcinoma/patologia , Neoplasias do Colo/terapia , Neoplasias do Colo/patologia , Antígenos , Adjuvantes Imunológicos , Vacinação , Nanopartículas/química , Polímeros/químicaRESUMO
T cell therapy shows promise as an immunotherapy in both immunostimulatory and immunosuppressive applications. However, the forms of T cell-based therapy that are currently in the clinic, such as adoptive cell transfer and vaccines, are limited by cost, time-to-treatment, and patient variability. Nanoparticles offer a modular, universal platform to improve the efficacy of various T cell therapies as nanoparticle properties can be easily modified for enhanced cell targeting, organ targeting, and cell internalization. Nanoparticles can enhance or even replace endogenous cells during each step of generating an antigen-specific T cell response - from antigen presentation and T cell activation to T cell maintenance. In this review, we discuss the unique applications of nanoparticles for antigen-specific T cell therapy, focusing on nanoparticles as vaccines (to activate endogenous antigen presenting cells (APCs)), as artificial Antigen Presenting Cells (aAPCs, to directly activate T cells), and as drug delivery vehicles (to support activated T cells).
Assuntos
Nanopartículas , Vacinas , Células Apresentadoras de Antígenos , Antígenos , Humanos , Fatores Imunológicos , Imunoterapia , Imunoterapia Adotiva , Nanopartículas/uso terapêutico , Linfócitos TRESUMO
BACKGROUND & AIMS: Acute diarrheal diseases are the second most common cause of infant mortality in developing countries. This is contributed to by lack of effective drug therapy that shortens the duration or lessens the volume of diarrhea. The epithelial brush border sodium (Na+)/hydrogen (H+) exchanger 3 (NHE3) accounts for a major component of intestinal Na+ absorption and is inhibited in most diarrheas. Because increased intestinal Na+ absorption can rehydrate patients with diarrhea, NHE3 has been suggested as a potential druggable target for drug therapy for diarrhea. METHODS: A peptide (sodium-hydrogen exchanger 3 stimulatory peptide [N3SP]) was synthesized to mimic the part of the NHE3 C-terminus that forms a multiprotein complex that inhibits NHE3 activity. The effect of N3SP on NHE3 activity was evaluated in NHE3-transfected fibroblasts null for other plasma membrane NHEs, a human colon cancer cell line that models intestinal absorptive enterocytes (Caco-2/BBe), human enteroids, and mouse intestine in vitro and in vivo. N3SP was delivered into cells via a hydrophobic fluorescent maleimide or nanoparticles. RESULTS: N3SP uptake stimulated NHE3 activity at nmol/L concentrations under basal conditions and partially reversed the reduced NHE3 activity caused by elevated adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, and Ca2+ in cell lines and in in vitro mouse intestine. N3SP also stimulated intestinal fluid absorption in the mouse small intestine in vivo and prevented cholera toxin-, Escherichia coli heat-stable enterotoxin-, and cluster of differentiation 3 inflammation-induced fluid secretion in a live mouse intestinal loop model. CONCLUSIONS: These findings suggest pharmacologic stimulation of NHE3 activity as an efficacious approach for the treatment of moderate/severe diarrheal diseases.
Assuntos
Enterotoxinas , Trocadores de Sódio-Hidrogênio , Camundongos , Animais , Humanos , Trocador 3 de Sódio-Hidrogênio/metabolismo , Enterotoxinas/farmacologia , Enterotoxinas/metabolismo , Células CACO-2 , Trocadores de Sódio-Hidrogênio/metabolismo , Enterócitos/metabolismo , Sódio/metabolismo , Diarreia/tratamento farmacológico , Diarreia/prevenção & controle , Diarreia/induzido quimicamente , Peptídeos/efeitos adversos , Microvilosidades/metabolismoRESUMO
Cancer immunotherapy has been the subject of extensive research, but highly effective and broadly applicable methods remain elusive. Moreover, a general approach to engender endogenous patient-specific cellular therapy, without the need for a priori knowledge of tumor antigen, ex vivo cellular manipulation, or cellular manufacture, could dramatically reduce costs and broaden accessibility. Here, we describe a biotechnology based on synthetic, biodegradable nanoparticles that can genetically reprogram cancer cells and their microenvironment in situ so that the cancer cells can act as tumor-associated antigen-presenting cells (tAPCs) by inducing coexpression of a costimulatory molecule (4-1BBL) and immunostimulatory cytokine (IL-12). In B16-F10 melanoma and MC38 colorectal carcinoma mouse models, reprogramming nanoparticles in combination with checkpoint blockade significantly reduced tumor growth over time and, in some cases, cleared the tumor, leading to long-term survivors that were then resistant to the formation of new tumors upon rechallenge at a distant site. In vitro and in vivo analyses confirmed that locally delivered tAPC-reprogramming nanoparticles led to a significant cell-mediated cytotoxic immune response with systemic effects. The systemic tumor-specific and cell-mediated immunotherapy response was achieved without requiring a priori knowledge of tumor-expressed antigens and reflects the translational potential of this nanomedicine.
Assuntos
Engenharia Genética/métodos , Fatores Imunológicos/uso terapêutico , Melanoma Experimental/genética , Melanoma Experimental/terapia , Animais , Antígenos de Neoplasias , Antineoplásicos/uso terapêutico , Feminino , Genes Reporter , Humanos , Imunoterapia/métodos , Células Matadoras Naturais , Camundongos , Camundongos Endogâmicos C57BL , Nanomedicina , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Linfócitos TRESUMO
The vast opportunities for biomaterials design and functionality enabled by mimicking nature continue to stretch the limits of imagination. As both biological understanding and engineering capabilities develop, more sophisticated biomedical materials can be synthesized that have multifaceted chemical, biological and physical characteristics designed to achieve specific therapeutic goals. Mimicry is being used in the design of polymers for biomedical applications that are required locally in tissues, systemically throughout the body, and at the interface with tissues.
Assuntos
Materiais Biocompatíveis , Materiais Biomiméticos , Polímeros , Adesivos/química , Animais , Materiais Biocompatíveis/química , Materiais Biomiméticos/química , Humanos , Hidrogéis/química , Mimetismo Molecular , Nanoestruturas/administração & dosagem , Nanoestruturas/química , Polímeros/química , Alicerces Teciduais/química , Vírus/químicaRESUMO
Polyelectrolyte complex particles assembled from plasmid DNA (pDNA) and poly(ethylenimine) (PEI) have been widely used to produce lentiviral vectors (LVVs) for gene therapy. The current batch-mode preparation for pDNA/PEI particles presents limited reproducibility in large-scale LVV manufacturing processes, leading to challenges in tightly controlling particle stability, transfection outcomes, and LVV production yield. Here we identified the size of pDNA/PEI particles as a key determinant for a high transfection efficiency with an optimal size of 400-500 nm, due to a cellular-uptake-related mechanism. We developed a kinetics-based approach to assemble size-controlled and shelf-stable particles using preassembled nanoparticles as building blocks and demonstrated production scalability on a scale of at least 100 mL. The preservation of colloidal stability and transfection efficiency was benchmarked against particles generated using an industry standard protocol. This particle manufacturing method effectively streamlines the viral manufacturing process and improves the production quality and consistency.
Assuntos
DNA , Polietilenoimina , DNA/genética , Tamanho da Partícula , Plasmídeos/genética , Reprodutibilidade dos Testes , TransfecçãoRESUMO
Clinical translation of polymer-based nanocarriers for systemic delivery of RNA has been limited due to poor colloidal stability in the blood stream and intracellular delivery of the RNA to the cytosol. To address these limitations, this study reports a new strategy incorporating photocrosslinking of bioreducible nanoparticles for improved stability extracellularly and rapid release of RNA intracellularly. In this design, the polymeric nanocarriers contain ester bonds for hydrolytic degradation and disulfide bonds for environmentally triggered small interfering RNA (siRNA) release in the cytosol. These photocrosslinked bioreducible nanoparticles (XbNPs) have a shielded surface charge, reduced adsorption of serum proteins, and enable superior siRNA-mediated knockdown in both glioma and melanoma cells in high-serum conditions compared to non-crosslinked formulations. Mechanistically, XbNPs promote cellular uptake and the presence of secondary and tertiary amines enables efficient endosomal escape. Following systemic administration, XbNPs facilitate targeting of cancer cells and tissue-mediated siRNA delivery beyond the liver, unlike conventional nanoparticle-based delivery. These attributes of XbNPs facilitate robust siRNA-mediated knockdown in vivo in melanoma tumors colonized in the lungs following systemic administration. Thus, biodegradable polymeric nanoparticles, via photocrosslinking, demonstrate extended colloidal stability and efficient delivery of RNA therapeutics under physiological conditions, and thereby potentially advance systemic delivery technologies for nucleic acid-based therapeutics.
RESUMO
Together, medulloblastoma (MB) and atypical teratoid/rhabdoid tumors (AT/RT) represent two of the most prevalent pediatric brain malignancies. Current treatment involves radiation, which has high risks of developmental sequelae for patients under the age of three. New safer and more effective treatment modalities are needed. Cancer gene therapy is a promising alternative, but there are challenges with using viruses in pediatric patients. We developed a library of poly(beta-amino ester) (PBAE) nanoparticles and evaluated their efficacy for plasmid delivery of a suicide gene therapy to pediatric brain cancer models-specifically herpes simplex virus type I thymidine kinase (HSVtk), which results in controlled apoptosis of transfected cells. In vivo, PBAE-HSVtk treated groups had a greater median overall survival in mice implanted with AT/RT (Pâ¯=â¯0.0083 vs. control) and MB (Pâ¯<â¯0.0001 vs. control). Our data provide proof of principle for using biodegradable PBAE nanoparticles as a safe and effective nanomedicine for treating pediatric CNS malignancies.
Assuntos
Neoplasias Encefálicas , Terapia Genética , Herpesvirus Humano 1 , Nanopartículas , Timidina Quinase , Proteínas Virais , Animais , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Criança , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/genética , Humanos , Masculino , Camundongos , Camundongos Nus , Nanopartículas/química , Nanopartículas/uso terapêutico , Timidina Quinase/biossíntese , Timidina Quinase/genética , Proteínas Virais/biossíntese , Proteínas Virais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
DNA methylation has long been recognized as a tumor-promoting factor when aberrantly regulated in the promoter region of genes. However, the effect of intragenic DNA methylation remains poorly understood on the clinical aspects of cancer. Here, we first evaluated the significance of intragenic DNA methylation for survival outcomes of cancer patients in a genome-wide manner. Glioblastoma patients with hypermethylated intragenic regions exhibited better survival than hypomethylated patients. Enrichment analyses of intragenic DNA methylation profiles with epigenetic signatures prioritized the intragenic DNA methylation of ZMIZ1 as a possible glioblastoma prognostic marker that is independent of MGMT methylation in IDH1 wild-type patients. This intragenic region harbored molecular signatures of alternative transcription across many cell types. Furthermore, we found that the intragenic region of ZMIZ1 can serve as a molecular marker in multiple cancers including astrocytomas, bladder cancer and renal cell carcinoma according to DNA methylation status. Finally, in vitro and in vivo experiments uncovered the role of ZMIZ1 as a driver of tumor cell migration. Altogether, our results identify ZMIZ1 as a prognostic marker in cancer and highlight the clinical significance of intragenic methylation in cancer.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Metilação de DNA/genética , Glioblastoma/genética , Glioblastoma/patologia , Fatores de Transcrição/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Epigênese Genética/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Estudo de Associação Genômica Ampla/métodos , Camundongos Nus , Prognóstico , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genéticaRESUMO
Intracellular delivery of nucleic acids to mammalian cells using polyplex nanoparticles (NPs) remains a challenge both in vitro and in vivo, with transfections often suffering from variable efficacy. To improve reproducibility and efficacy of transfections in vitro using a next-generation polyplex transfection material poly(beta-amino ester)s (PBAEs), the influence of multiple variables in the preparation of these NPs on their transfection efficacy was explored. The results indicate that even though PBAE/pDNA polyplex NPs are formed by the self-assembly of polyelectrolytes, their transfection is not affected by the manner in which the components are mixed, facilitating self-assembly in a single step, but timing for self-assembly of 5-20 min is optimal. In addition, even though the biomaterials are biodegradable in water, their efficacy is not affected by up to eight freeze-thaw cycles of the polymer. It was found that there is a greater stability of nucleic acid-complexed polymer as a polyplex nanoparticle compared with free polymer. Finally, by exploring multiple buffer systems, it was identified that utilization of divalent cation magnesium or calcium acetate buffers at pH 5.0 is optimal for transfection using these polymeric materials, boosting transfection several folds compared with monovalent cations. Together, these results can improve the reproducibility and efficacy of PBAE and similar polyplex nanoparticle transfections and improve the robustness of using these biomaterials for bioengineering and biotechnology applications.
Assuntos
Materiais Biocompatíveis/química , DNA/química , Nanopartículas/química , Plasmídeos/química , Polímeros/química , Transfecção , Animais , Humanos , Concentração de Íons de HidrogênioRESUMO
Development of highly effective nonviral gene delivery vectors for transfection of diverse cell populations remains a challenge despite utilization of both rational and combinatorial driven approaches to nanoparticle engineering. In this work, multifunctional polyesters are synthesized with well-defined branching structures via A2 + B2/B3 + C1 Michael addition reactions from small molecule acrylate and amine monomers and then end-capped with amine-containing small molecules to assess the influence of polymer branching structure on transfection. These Branched poly(Ester Amine) Quadpolymers (BEAQs) are highly effective for delivery of plasmid DNA to retinal pigment epithelial cells and demonstrate multiple improvements over previously reported leading linear poly(beta-amino ester)s, particularly for volume-limited applications where improved efficiency is required. BEAQs with moderate degrees of branching are demonstrated to be optimal for delivery under high serum conditions and low nanoparticle doses further relevant for therapeutic gene delivery applications. Defined structural properties of each polymer in the series, including tertiary amine content, correlated with cellular transfection efficacy and viability. Trends that can be applied to the rational design of future generations of biodegradable polymers are elucidated.
Assuntos
Nanopartículas/química , Plasmídeos/genética , Polímeros/química , Linhagem Celular , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Microscopia Confocal , Plasmídeos/administração & dosagem , Poliésteres/química , Transfecção/métodosRESUMO
Glioblastoma (GBMs) is the most common and aggressive type of primary brain tumor in adults with dismal prognosis despite radical surgical resection coupled with chemo- and radiotherapy. Recent studies have proposed the use of small-molecule inhibitors, including verteporfin (VP), to target oncogenic networks in cancers. Here we report efficient encapsulation of water-insoluble VP in poly(lactic- co-glycolic acid) microparticles (PLGA MP) of â¼1.5 µm in diameter that allows tunable, sustained release. Treatment with naked VP and released VP from PLGA MP decreased cell viability of patient-derived primary GBM cells in vitro by â¼70%. Moreover, naked VP treatment significantly increased radiosensitivity of GBM cells, thereby enhancing overall tumor cell killing ability by nearly 85%. Our in vivo study demonstrated that two intratumoral administrations of sustained slow-releasing VP-loaded PLGA MPs separated by two weeks significantly attenuated tumor growth by â¼67% in tumor volume in a subcutaneous patient-derived GBM xenograft model over 26 d. Additionally, our in vitro data indicate broader utility of VP for treatment for other solid cancers, including chordoma, malignant meningioma, and various noncentral nervous system-derived carcinomas. Collectively, our work suggests that the use of VP-loaded PLGA MP may be an effective local therapeutic strategy for a variety of solid cancers, including unresectable and orphan tumors, which may decrease tumor burden and ultimately improve patient prognosis.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Microesferas , Fármacos Fotossensibilizantes/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Polímeros/química , Verteporfina/farmacologia , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Ácido Láctico/química , Masculino , Camundongos , Camundongos Nus , Poliésteres/química , Ácido Poliglicólico/química , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite our growing molecular-level understanding of glioblastoma (GBM), treatment modalities remain limited. Recent developments in the mechanisms of cell fate regulation and nanomedicine provide new avenues by which to treat and manage brain tumors via the delivery of molecular therapeutics. Here, we have developed bioreducible poly(ß-amino ester) nanoparticles that demonstrate high intracellular delivery efficacy, low cytotoxicity, escape from endosomes, and promotion of cytosol-targeted environmentally triggered cargo release for miRNA delivery to tumor-propagating human cancer stem cells. In this report, we combined this nanobiotechnology with newly discovered cancer stem cell inhibiting miRNAs to develop self-assembled miRNA-containing polymeric nanoparticles (nano-miRs) to treat gliomas. We show that these nano-miRs effectively intracellularly deliver single and combination miRNA mimics that inhibit the stem cell phenotype of human GBM cells in vitro. Following direct intratumoral infusion, these nano-miRs were found to distribute through the tumors, inhibit the growth of established orthotopic human GBM xenografts, and cooperatively enhance the response to standard-of-care γ radiation. Co-delivery of two miRNAs, miR-148a and miR-296-5p, within the bioreducible nano-miR particles enabled long-term survival from GBM in mice.
Assuntos
Glioblastoma/tratamento farmacológico , MicroRNAs/genética , Nanopartículas/administração & dosagem , Células-Tronco Neoplásicas/química , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , MicroRNAs/administração & dosagem , MicroRNAs/química , Nanomedicina/tendências , Nanopartículas/química , Polímeros/administração & dosagem , Polímeros/químicaRESUMO
Amphiphilic polymers can be used to form micelles to deliver water-insoluble drugs. A biodegradable poly(ethylene glycol) (PEG)-poly(beta-amino ester) (PBAE)-PEG triblock copolymer was developed that is useful for drug delivery. It was shown to successfully encapsulate and pH-dependently release a water-insoluble, small molecule anticancer drug, verteporfin. PEG-PBAE-PEG micelle morphology was also controlled through variations to the hydrophobicity of the central PBAE block of the copolymer in order to evade macrophage uptake. Spherical micelles were 50 nm in diameter, while filamentous micelles were 31 nm in width with an average aspect ratio of 20. When delivered to RAW 264.7 mouse macrophages, filamentous micelles exhibited a 89% drop in cellular uptake percentage and a 5.6-fold drop in normalized geometric mean cellular uptake compared to spherical micelles. This demonstrates the potential of high-aspect-ratio, anisotropically shaped PEG-PBAE-PEG micelles to evade macrophage-mediated clearance. Both spherical and filamentous micelles also showed therapeutic efficacy in human triple-negative breast cancer and small cell lung cancer cells without requiring photodynamic therapy to achieve an anticancer effect. Both spherical and filamentous micelles were more effective in killing lung cancer cells than breast cancer cells at equivalent verteporfin concentrations, while spherical micelles were shown to be more effective than filamentous micelles against both cancer cells. Spherical and filamentous micelles at 5 and 10 µM respective verteporfin concentration resulted in 100% cell killing of lung cancer cells, but both micelles required a higher verteporfin concentration of 20 µM to kill breast cancer cells at the levels of 80% and 50% respectively. This work demonstrates the potential of PEG-PBAE-PEG as a biodegradable, anisotropic drug delivery system as well as the in vitro use of verteporfin-loaded micelles for cancer therapy.
Assuntos
Antineoplásicos/administração & dosagem , Micelas , Polietilenoglicóis/química , Polímeros/química , Verteporfina/administração & dosagem , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Células RAW 264.7RESUMO
There is a need for new tools to better quantify intracellular delivery barriers in high-throughput and high-content ways. Here, we synthesized a triple-fluorophore-labeled nucleic acid pH nanosensor for measuring intracellular pH of exogenous DNA at specific time points in a high-throughput manner by flow cytometry following non-viral transfection. By including two pH-sensitive fluorophores and one pH-insensitive fluorophore in the nanosensor, detection of pH was possible over the full physiological range. We further assessed possible correlation between intracellular pH of delivered DNA, cellular uptake of DNA, and DNA reporter gene expression at 24 hr post-transfection for poly-L-lysine and branched polyethylenimine polyplex nanoparticles. While successful transfection was shown to clearly depend on median cellular pH of delivered DNA at the cell population level, surprisingly, on an individual cell basis, there was no significant correlation between intracellular pH and transfection efficacy. To our knowledge, this is the first reported instance of high-throughput single-cell analysis between cellular uptake of DNA, intracellular pH of delivered DNA, and gene expression of the delivered DNA. Using the nanosensor, we demonstrate that the ability of polymeric nanoparticles to avoid an acidic environment is necessary, but not sufficient, for successful transfection.
Assuntos
Técnicas Biossensoriais/métodos , DNA/química , Corantes Fluorescentes/química , Técnicas de Transferência de Genes , Nanopartículas/química , Coloração e Rotulagem/métodos , Animais , Carbocianinas/química , Ácidos Carboxílicos/química , DNA/genética , DNA/metabolismo , Citometria de Fluxo/métodos , Fluoresceína/química , Expressão Gênica , Genes Reporter , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Tamanho da Partícula , Polietilenoimina/química , Polilisina/química , Análise de Célula Única/métodosRESUMO
Therapeutic cancer vaccines require adjuvants leading to robust type I interferon and proinflammatory cytokine responses in the tumor microenvironment to induce an anti-tumor response. Cyclic dinucleotides (CDNs), a potent Stimulator of Interferon Receptor (STING) agonist, are currently in phase I trials. However, their efficacy may be limited to micromolar concentrations due to the cytosolic residence of STING in the ER membrane. Here we utilized biodegradable, poly(beta-amino ester) (PBAE) nanoparticles to deliver CDNs to the cytosol leading to robust immune response at >100-fold lower extracellular CDN concentrations in vitro. The leading CDN PBAE nanoparticle formulation induced a log-fold improvement in potency in treating established B16 melanoma tumors in vivo when combined with PD-1 blocking antibody in comparison to free CDN without nanoparticles. This nanoparticle-mediated cytosolic delivery method for STING agonists synergizes with checkpoint inhibitors and has strong potential for enhanced cancer immunotherapy.
Assuntos
Antineoplásicos/administração & dosagem , Imunoterapia , Melanoma Experimental/terapia , Proteínas de Membrana/agonistas , Nanopartículas/administração & dosagem , Nucleotídeos Cíclicos/administração & dosagem , Animais , Antineoplásicos/química , Feminino , Fator Regulador 3 de Interferon/metabolismo , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Nucleotídeos Cíclicos/química , Polímeros/química , Células Tumorais CultivadasRESUMO
Targeted, noninvasive neuromodulation of the brain of an otherwise awake subject could revolutionize both basic and clinical neuroscience. Toward this goal, we have developed nanoparticles that allow noninvasive uncaging of a neuromodulatory drug, in this case the small molecule anesthetic propofol, upon the application of focused ultrasound. These nanoparticles are composed of biodegradable and biocompatible constituents and are activated using sonication parameters that are readily achievable by current clinical transcranial focused ultrasound systems. These particles are potent enough that their activation can silence seizures in an acute rat seizure model. Notably, there is no evidence of brain parenchymal damage or blood-brain barrier opening with their use. Further development of these particles promises noninvasive, focal, and image-guided clinical neuromodulation along a variety of pharmacological axes.
Assuntos
Encéfalo/efeitos dos fármacos , Emulsões/química , Nanopartículas/química , Neurotransmissores/administração & dosagem , Anestésicos/administração & dosagem , Anestésicos/química , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Portadores de Fármacos , Liberação Controlada de Fármacos , Fluorocarbonos/química , Imageamento por Ressonância Magnética , Neurotransmissores/química , Imagem Óptica , Propofol/administração & dosagem , Propofol/química , Ratos , Convulsões/tratamento farmacológico , Distribuição Tecidual , Ondas UltrassônicasRESUMO
Extracellular vesicles such as exosomes and microvesicles (MVs) are shed by cancer cells, are detected in the plasma of cancer patients, and promote cancer progression, but the molecular mechanisms regulating their production are not well understood. Intratumoral hypoxia is common in advanced breast cancers and is associated with an increased risk of metastasis and patient mortality that is mediated in part by the activation of hypoxia-inducible factors (HIFs). In this paper, we report that exposure of human breast cancer cells to hypoxia augments MV shedding that is mediated by the HIF-dependent expression of the small GTPase RAB22A, which colocalizes with budding MVs at the cell surface. Incubation of naïve breast cancer cells with MVs shed by hypoxic breast cancer cells promotes focal adhesion formation, invasion, and metastasis. In breast cancer patients, RAB22A mRNA overexpression in the primary tumor is associated with decreased overall and metastasis-free survival and, in an orthotopic mouse model, RAB22A knockdown impairs breast cancer metastasis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Exossomos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Neoplasias da Mama/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Feminino , Adesões Focais/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos SCID , Invasividade Neoplásica , Metástase Neoplásica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Tissue engineering using mesenchymal stem cells (MSCs) holds great promise for regenerating critically sized bone defects. While the bone marrow-derived MSC is the most widely studied stromal/stem cell type for this application, its rarity within bone marrow and painful isolation procedure have motivated investigation of alternative cell sources. Adipose-derived stromal/stem cells (ASCs) are more abundant and more easily procured; furthermore, they also possess robust osteogenic potency. While these two cell types are widely considered very similar, there is a growing appreciation of possible innate differences in their biology and response to growth factors. In particular, reports indicate that their osteogenic response to platelet-derived growth factor BB (PDGF-BB) is markedly different: MSCs responded negatively or not at all to PDGF-BB while ASCs exhibited enhanced mineralization in response to physiological concentrations of PDGF-BB. In this study, we directly tested whether a fundamental difference existed between the osteogenic responses of MSCs and ASCs to PDGF-BB. MSCs and ASCs cultured under identical osteogenic conditions responded disparately to 20 ng/ml of PDGF-BB: MSCs exhibited no difference in mineralization while ASCs produced more calcium per cell. siRNA-mediated knockdown of PDGFRß within ASCs abolished their ability to respond to PDGF-BB. Gene expression was also different; MSCs generally downregulated and ASCs generally upregulated osteogenic genes in response to PDGF-BB. ASCs transduced to produce PDGF-BB resulted in more regenerated bone within a critically sized murine calvarial defect compared to control ASCs, indicating PDGF-BB used specifically in conjunction with ASCs might enhance tissue engineering approaches for bone regeneration.