RESUMO
Long noncoding RNA (lncRNA) differentiation antagonizing noncoding RNA (DANCR) is overexpressed in human triple-negative breast cancer (TNBC) and promotes cell migration and proliferation. TNBC is limited in treatment options relative to hormone-receptor-positive breast cancer and is commonly treated with chemotherapy, which is often compromised by acquired resistance. DANCR has been implicated in the development of chemoresistance across multiple cancer types. Here, we applied magnetic resonance molecular imaging (MRMI) with a targeted contrast agent, MT218, specific to extradomain-B fibronectin (EDB-FN), a marker for epithelial-to-mesenchymal transition, to assess the therapeutic efficacy of the combination of paclitaxel and ZD2-PEG-ECO/siDANCR nanoparticles (ZD2-siDANCR-ELNP) to treat TNBC. The treatment of orthotopic MDA-MB-231 TNBC in mice with paclitaxel significantly suppressed tumor growth but with a significant increase of EDB-FN in the tumor, as revealed by MRMI and immunohistochemistry. Combining ZD2-siDANCR-ELNP with paclitaxel further reduced tumor sizes, along with reduced EDB-FN expression. Interestingly, MT218-MRMI revealed a lower reduction of tumor signal enhancement with the combination treatment than that with the siDANCR treatment alone, which was supported by higher cell density in the tumors treated with the combination therapy, as shown by histochemical analysis. MT218-MRMI clearly revealed the changes of the tumor microenvironment in response to various therapies and is effective to noninvasively assess the response of TNBC tumors to the therapies. Regulating oncogenic lncRNA DANCR is an effective strategy for improving the outcomes of chemotherapy in TNBC.
Assuntos
RNA Longo não Codificante , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Neoplasias de Mama Triplo Negativas/diagnóstico por imagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , RNA Longo não Codificante/genética , Interferência de RNA , Linhagem Celular Tumoral , Paclitaxel/uso terapêutico , Espectroscopia de Ressonância Magnética , Imagem Molecular/métodos , Proliferação de Células , Microambiente TumoralRESUMO
OBJECTIVE: The aim of this study was to evaluate the pharmacokinetics and safety profile of MT218, a peptide-targeted gadolinium-based contrast agent, in healthy males. MATERIALS AND METHODS: This was a double-blind, randomized, placebo-controlled, single-ascending-dose study including 30 healthy male subjects. In each dose group (0.01, 0.02, 0.04, and 0.08 mmol/kg), 4 subjects received MT218 and 2 subjects received placebo (saline) in bolus injections. The highest dose group (0.08 mmol/kg) was assessed in 2 cohorts, 1 fasted and 1 nonfasted. Clinical laboratory tests, vital signs, and electrocardiograms were investigated. Gadolinium concentrations were measured in plasma samples collected before administration and over a 24-hour period postinjection, and in urine specimens collected until 22 days. A noncompartmental model was used for pharmacokinetic analysis. A clinical and biological safety follow-up was carried out for up to 6 months. RESULTS: No clinically significant modifications in biochemistry, hematology, urinalysis, electrocardiogram parameters, or vital signs were reported at any time point for any treatment group. No serious adverse events were observed in any dose group. Transient dizziness, hyperhidrosis, and injection site coldness were the main adverse events reported in both the MT218 and placebo groups. The mean total apparent clearance decreased slightly with increasing dose, and the median plasma t 1/2 ranged from 1.7 hours in the 0.01 mmol/kg group to 2.7 hours in the 0.08 mmol/kg nonfasted group. MT218 was rapidly excreted via renal filtration with 42.9% to 52.8% of the injected dose measured in urine within the first hour after administration, and 92.5% to 117.3% in urine within 24 hours. No Gd was detected by inductively coupled plasma mass spectrometry in urine after 21 days. CONCLUSION: Single intravenous administration of MT218 was safely tolerated in the healthy males. Its pharmacokinetic parameters and safety profile are well aligned with those of other gadolinium-based contrast agents.
Assuntos
Meios de Contraste , Neoplasias , Humanos , Masculino , Gadolínio , Área Sob a Curva , Imageamento por Ressonância Magnética , Método Duplo-Cego , Relação Dose-Resposta a DrogaRESUMO
Differentiation antagonizing noncoding RNA (DANCR) is recognized as an oncogenic long noncoding RNA (lncRNA) overexpressed in triple negative breast cancer (TNBC). We showed in a previous study that RNAi with targeted multifunctional ionizable lipid ECO/siRNA nanoparticles was effective to regulate this undruggable target for effective treatment of TNBC. In this study, we developed dual-targeted ECO/siDANCR nanoparticles by targeting a tumor extracellular matrix oncoprotein, extradomain B fibronectin (EDB-FN), and integrins overexpressed on cancer cells for enhanced delivery of siDANCR. The treatment of Hs578T TNBC cells and MCF-7 estrogen receptor-positive cells in vitro resulted in significant down-regulation of DANCR and EDB-FN and suppressed invasion and 3D spheroid formation of the cells. Magnetic resonance molecular imaging (MRMI) with an EDB-FN-targeted contrast agent, MT218, was used to noninvasively evaluate tumor response to treatment with the targeted ECO/siDANCR nanoparticles in female nude mice bearing orthotopic Hs578T and MCF-7 xenografts. MRMI with MT218 was effective to differentiate between aggressive TNBC with high DANCR and EDB-FN expression and ER+ MCF-7 tumors with low expression of the targets. MRMI showed that the dual-targeted ECO/siDANCR nanoparticles resulted in more significant inhibition of tumor growth in both models than the controls and significantly reduced EDB-FN expression in the TNBC tumors. The combination of MRMI and dual-targeted ECO/siDANCR nanoparticles is a promising approach for image-guided treatment of TNBC by regulating the onco-lncRNA.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Quinolinas , Humanos , Carcinoma Hepatocelular/diagnóstico por imagem , Neoplasias Hepáticas/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Radioisótopos de Gálio , Fluordesoxiglucose F18RESUMO
Alternatively spliced forms of fibronectin, called oncofetal fibronectin, are aberrantly expressed in cancer, with little to no expression in normal tissue, making them attractive biomarkers to exploit for tumor-targeted therapeutics and diagnostics. While prior studies have explored oncofetal fibronectin expression in limited cancer types and limited sample sizes, no studies have performed a large-scale pan-cancer analysis in the context of clinical diagnostics and prognostics to posit the utility of these biomarkers across multiple cancer types. In this study, RNA-Seq data sourced from the UCSC Toil Recompute project were extracted and analyzed to determine the correlation between the expression of oncofetal fibronectin, including extradomain A and extradomain B fibronectin, and patient diagnosis and prognosis. We determined that oncofetal fibronectin is significantly overexpressed in most cancer types relative to corresponding normal tissues. In addition, strong correlations exist between increasing oncofetal fibronectin expression levels and tumor stage, lymph node activity, and histological grade at the time of diagnosis. Furthermore, oncofetal fibronectin expression is shown to be significantly associated with overall patient survival within a 10-year window. Thus, the results presented in this study suggest oncofetal fibronectin as a commonly upregulated biomarker in cancer with the potential to be used for tumor-selective diagnosis and treatment applications.
Assuntos
Fibronectinas , Neoplasias , Humanos , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , RNA-SeqRESUMO
Purpose: We tested a recently developed short peptide radioligand for PET imaging of hepatocellular carcinoma (HCC) by targeting an oncoprotein, extra-domain B fibronectin (EDB-FN) in the tumor microenvironment. Methods: The radioligand consists of a small linear peptide ZD2 with 68Ga-NOTA chelator, and specifically binds to EDB-FN. PET images were acquired dynamically for 1 hour after intravenously (i.v.) injecting 37 MBq (1.0 mCi) of the radioligand into the woodchuck model of naturally occurring HCC. Woodchuck HCC originated from chronic viral hepatitis infection, which recapitulates the corresponding human primary liver cancer. The animals were euthanized post-imaging for tissue collection and validation. Results: For ZD2 avid liver tumors, the radioligand accumulation plateaued a few minutes after injection, while the liver background uptake stabilized 20 min post-injection. The status of EDB-FN in woodchuck HCC was confirmed by histology and validated by PCR and western blocking. Conclusion: We have showed the viability of using the ZD2 short peptide radioligand targeting EDB-FN in liver tumor tissue for PET imaging of HCC, which can potentially impact the clinical care for HCC patients.
RESUMO
Hereditary genetic diseases, cancer, and infectious diseases are affecting global health and become major health issues, but the treatment development remains challenging. Gene therapies using DNA plasmid, RNAi, miRNA, mRNA, and gene editing hold great promise. Lipid nanoparticle (LNP) delivery technology has been a revolutionary development, which has been granted for clinical applications, including mRNA vaccines against SARS-CoV-2 infections. Due to the success of LNP systems, understanding the structure, formulation, and function relationship of the lipid components in LNP systems is crucial for design more effective LNP. Here, we highlight the key considerations for developing an LNP system. The evolution of structure and function of lipids as well as their LNP formulation from the early-stage simple formulations to multi-components LNP and multifunctional ionizable lipids have been discussed. The flexibility and platform nature of LNP enable efficient intracellular delivery of a variety of therapeutic nucleic acids and provide many novel treatment options for the diseases that are previously untreatable.
Assuntos
COVID-19 , Nanopartículas , Ácidos Nucleicos , Humanos , Vacinas contra COVID-19 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/química , SARS-CoV-2/genética , Lipídeos/química , Nanopartículas/químicaRESUMO
Stargardt disease (STGD) is the most common form of inherited retinal genetic disorders and is often caused by mutations in ABCA4. Gene therapy has the promise to effectively treat monogenic retinal disorders. However, clinically approved adeno-associated virus (AAV) vectors do not have a loading capacity for large genes, such as ABCA4. Self-assembly nanoparticles composed of (1-aminoethyl)iminobis[N-(oleoylcysteinyl-1-amino-ethyl)propionamide (ECO; a multifunctional pH-sensitive/ionizable amino lipid) and plasmid DNA produce gene transfection comparable with or better than the AAV2 capsid. Stable PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles produce specific and prolonged expression of ABCA4 in the photoreceptors of Abca4 -/- mice and significantly inhibit accumulation of toxic A2E in the eye. Multiple subretinal injections enhance gene expression and therapeutic efficacy with an approximately 69% reduction in A2E accumulation in Abca4 -/- mice after 3 doses. Very mild inflammation was observed after multiple injections of the nanoparticles. PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles are a promising non-viral mediated gene therapy modality for STGD type 1 (STGD1).
RESUMO
Magnetic resonance imaging (MRI) is a clinical imaging modality that provides high-resolution images of soft tissues, including cancerous lesions. Stable gadolinium(III) chelates have been used as contrast agents (CA) in MRI to enhance the contrast between the tissues of interest and surrounding tissues for accurate diagnostic imaging. Magnetic resonance molecular imaging (MRMI) of cancer requires targeted CA to specifically elucidate cancer-associated molecular processes and can provide high-resolution delineation and characterization of cancer for precision medicine. The main challenge for MRMI is the lack of sufficient sensitivity to detect the low concentration of the cellular oncogenic markers. In addition, targeted CA must satisfy regulatory safety requirements prior to clinical development. Up to now, there is no FDA-approved targeted CA for MRMI of cancer.In this Account, we discuss the latest developments in the design and development of clinically translatable targeted CA for MRMI of cancer, with an emphasis on our own research. The primary limitation of MRMI can be overcome by designing small molecular targeted CA to target abundant cancer-specific targets found in the tumor microenvironment (TME). For example, aggressive tumors have a unique extracellular matrix (ECM) composed of oncoproteins, which can be used as targetable markers for MRMI. We have designed and prepared small peptide conjugates of clinical contrast agents, including Gd-DTPA and Gd-DOTA, to target fibrin-fibronectin clots in tumors. These small molecular CA have been effective in enhancing MRMI detection of solid tumors and have demonstrated the ability to detect submillimeter cancer micrometastases in mouse tumor models, exceeding the detection limit of current clinical imaging modalities. We have also identified extradomain B fibronectin (EDB-FN), an oncofetal subtype of fibronectin, as a promising TME target to leverage in the design and development of small peptide targeted CA for clinical translation. The expression level of EDB-FN is correlated with invasiveness of cancer cells and poor patient survival of multiple cancer types. ZD2 peptide with a sequence of seven amino acids (TVRTSAD) was identified to specifically bind to the EDB protein fragment. Several ZD2 conjugates of macrocyclic GBCA, including Gd-DOTA and Gd(HP-DO3A), have been synthesized and tested in mouse tumor models. ZD2-N3-Gd(HP-DO3A) (MT218) with a high r1 relaxivity was selected as the lead agent for clinical translation. The physicochemical properties and preclinical assessments of MT218 are summarized in this Account. MRMI of EDB-FN with MT218 can effectively detect invasive tumors of multiple cancers with risk-stratification and monitor tumor response to anticancer therapies in mouse models. Currently, MT218 is in clinical trials for precision cancer MRMI. Herein, we will show that using targeted MRI contrast agents specific to abundant TME biomarkers is a pragmatic solution for effective precision cancer imaging in high spatial resolution. And thus, we illustrate a replicable approach for CA development that is vital for cancer MRMI.
Assuntos
Gadolínio , Neoplasias , Aminoácidos , Animais , Meios de Contraste/química , Fibrina , Fibronectinas/metabolismo , Gadolínio/química , Gadolínio DTPA , Compostos Heterocíclicos , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética , Camundongos , Imagem Molecular/métodos , Neoplasias/diagnóstico por imagem , Proteínas Oncogênicas , Compostos Organometálicos , Peptídeos , Microambiente TumoralRESUMO
AIMS/HYPOTHESIS: CD40 expressed in Müller cells is a central driver of diabetic retinopathy. CD40 causes phospholipase Cγ1 (PLCγ1)-dependent ATP release in Müller cells followed by purinergic receptor (P2X7)-dependent production of proinflammatory cytokines in myeloid cells. In the diabetic retina, CD40 and P2X7 upregulate a broad range of inflammatory molecules that promote development of diabetic retinopathy. The molecular event downstream of CD40 that activates the PLCγ1-ATP-P2X7-proinflammatory cytokine cascade and promotes development of diabetic retinopathy is unknown. We hypothesise that disruption of the CD40-driven molecular events that trigger this cascade prevents/treats diabetic retinopathy in mice. METHODS: B6 and transgenic mice with Müller cell-restricted expression of wild-type (WT) CD40 or CD40 with mutations in TNF receptor-associated factor (TRAF) binding sites were made diabetic using streptozotocin. Leucostasis was assessed using FITC-conjugated concanavalin A. Histopathology was examined in the retinal vasculature. Expression of inflammatory molecules and phospho-Tyr783 PLCγ1 (p-PLCγ1) were assessed using real-time PCR, immunoblot and/or immunohistochemistry. Release of ATP and cytokines were measured by ATP bioluminescence and ELISA, respectively. RESULTS: Human Müller cells with CD40 ΔT2,3 (lacks TRAF2,3 binding sites) were unable to phosphorylate PLCγ1 and release ATP in response to CD40 ligation, and could not induce TNF-α/IL-1ß secretion in bystander myeloid cells. CD40-TRAF signalling acted via Src to induce PLCγ1 phosphorylation. Diabetic mice in which WT CD40 in Müller cells was replaced by CD40 ΔT2,3 failed to exhibit phosphorylation of PLCγ1 in these cells and upregulate P2X7 and TNF-α in microglia/macrophages. P2x7 (also known as P2rx7), Tnf-α (also known as Tnf), Il-1ß (also known as Il1b), Nos2, Icam-1 (also known as Icam1) and Ccl2 mRNA were not increased in these mice and the mice did not develop retinal leucostasis and capillary degeneration. Diabetic B6 mice treated intravitreally with a cell-permeable peptide that disrupts CD40-TRAF2,3 signalling did not exhibit either upregulation of P2X7 and inflammatory molecules in the retina or leucostasis. CONCLUSIONS/INTERPRETATION: CD40-TRAF2,3 signalling activated the CD40-PLCγ1-ATP-P2X7-proinflammatory cytokine pathway. Src functioned as a link between CD40-TRAF2,3 and PLCγ1. Replacing WT CD40 with CD40 ΔT2,3 impaired activation of PLCγ1 in Müller cells, upregulation of P2X7 in microglia/macrophages, upregulation of a broad range of inflammatory molecules in the diabetic retina and the development of diabetic retinopathy. Administration of a peptide that disrupts CD40-TRAF2,3 signalling reduced retinal expression of inflammatory molecules and reduced leucostasis in diabetic mice, supporting the therapeutic potential of pharmacological inhibition of CD40-TRAF2,3 in diabetic retinopathy.
Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética , Camundongos , Humanos , Animais , Células Ependimogliais/metabolismo , Retinopatia Diabética/metabolismo , Diabetes Mellitus Experimental/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Antígenos CD40 , Retina/metabolismo , Inflamação/metabolismo , Citocinas/metabolismo , Peptídeos , Trifosfato de Adenosina/metabolismo , MutaçãoRESUMO
Long noncoding RNA (lncRNA) differentiation antagonizing noncoding RNA (DANCR) is a proven oncogenic lncRNA across multiple cancer types. Its effects on cancer cell migration and invasion position it as a potential target for therapy on multiple levels of gene regulation. DANCR is overexpressed in non-small cell lung cancer (NSCLC), the most common lung cancer subtype with poor patient survival. To effectively deliver small interfering RNA (siRNA) against DANCR for NSCLC therapy, we used arginine-glycine-aspartic acid (RGD)-poly(ethylene glycol) (PEG)-(1-aminoethyl)-iminobis[N-oleicylcysteinyl-1-aminoethyl)propionamide] (ECO)/small interfering RNA against DANCR (siDANCR) nanoparticles to transfect A549 and NCI-H1299 cells. Over 90% DANCR silencing was observed along with inhibition of cell migration, invasion, and spheroid formation relative to transfection with negative control siRNA in RGD-PEG-ECO nanoparticles. DANCR knockdown further showed efficacy in reducing migration and invasion of epidermal growth factor receptor (EGFR)-inhibitor resistant NSCLC along with resensitization to the inhibitor. RGD-PEG-ECO/siDANCR demonstrated silencing for up to 7 d following a single transfection. The results suggest nanoparticle-mediated RNA interference against DANCR as a potential approach for NSCLC treatment by regulating cell migration and invasion in addition to improving EGFR inhibitor response.
RESUMO
OBJECTIVES: Preclinical assessments were performed according to the US Food and Drug Administration guidelines to determine the physicochemical properties, pharmacokinetics, clearance, safety, and tumor-specific magnetic resonance (MR) imaging of MT218, a peptidic gadolinium-based MR imaging agent targeting to extradomain B fibronectin for MR molecular imaging of aggressive tumors. MATERIALS AND METHODS: Relaxivity, chelation stability, binding affinity, safety-related target profiling, and effects on CYP450 enzymes and transporters were evaluated in vitro. Magnetic resonance imaging was performed with rats bearing prostate cancer xenografts, immunocompetent mice bearing murine pancreatic cancer allografts, and mice bearing lung cancer xenografts at different doses of MT218. Pharmacological effects on cardiovascular, respiratory, and central nervous systems were determined in rats and conscious beagle dogs. Pharmacokinetics were tested in rats and dogs. Biodistribution and excretion were studied in rats. Single and repeated dosing toxicity was evaluated in rats and dogs. In vitro and in vivo genotoxicity, in vitro hemolysis, and anaphylactic reactivity were also performed. RESULTS: At 1.4 T, the r1 and r2 relaxivities of MT218 were 5.43 and 7.40 mM -1 s -1 in pure water, 6.58 and 8.87 mM -1 s -1 in phosphate-buffered saline, and 6.54 and 8.70 mM -1 s -1 in aqueous solution of human serum albumin, respectively. The binding affinity of MT218 to extradomain B fragment is 3.45 µM. MT218 exhibited no dissociation of the Gd(III) chelates under physiological conditions. The peptide degradation half-life ( t1/2 ) of MT218 was 1.63, 5.85, and 2.63 hours in rat, dog, and human plasma, respectively. It had little effect on CYP450 enzymes and transporters. MT218 produced up to 7-fold increase of contrast-to-noise ratios in the extradomain B fibronectin-rich tumors with a dose of 0.04 mmol/kg for at least 30 minutes. MT218 had little pharmacological effect on central nervous, cardiovascular, or respiratory systems. MT218 had a mean plasma elimination half-life ( t1/2 ) of 0.31 and 0.89 hours in rats and dogs at 0.1 mmol/kg, respectively. No detectable Gd deposition was observed in the brain at 6 hours postinjection of MT218 at 0.1 mmol/kg in rats. MT218 was not mutagenic and had no mortality or morbidity in the rats or dogs up to 1.39 and 0.70 mmol/kg/d, respectively. The no observed adverse effect level of MT218 in Sprague-Dawley rats was 1.39 mmol/kg for single dosing and 0.46 mmol/kg/d for repeated dosing. The no observed adverse effect level in dogs was 0.07 mmol/kg/d. MT218 exhibited no genotoxicity, hemolysis, and anaphylactic reactivity. CONCLUSION: The preclinical assessments showed that the targeted contrast agent MT218 has high r1 and r2 relaxivities, satisfactory physicochemical properties, pharmacokinetic, and safety profiles and produces effective tumor enhancement in multiple cancer types in rats and mice at reduced doses.
Assuntos
Meios de Contraste , Neoplasias da Próstata , Animais , Quelantes , Meios de Contraste/farmacocinética , Cães , Fibronectinas , Hemólise , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Neoplasias da Próstata/diagnóstico por imagem , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
Extradomain-B Fibronectin (EDB-FN) is an oncomarker that can be visualized with magnetic resonance molecular imaging (MRMI) to detect pancreatic ductal adenocarcinoma (PDAC) metastasis. In this study, we sought to assess the expression of EDB-FN in clinical samples of PDAC and to evaluate MRMI of PDAC metastasis with an EDB-FN-specific gadolinium-based contrast agent (MT218) in an orthotopic KPC-GFP-Luc mouse model. EDB-FN expression was evaluated in PDAC tissue samples through immunohistochemistry. RNA-Seq data obtained from the GEPIA2 project was evaluated to demonstrate EDB-FN expression in large patient cohorts. FLASH-3D MRI at 3 T of the KPC-GFP-Luc metastasis model was performed following injection of MT218. Tumor enhancement in MR images was correlated to postmortem distribution of KPC-GFP-Luc tumors using fluorescent and bright-field cryo-imaging and anatomical landmarks. EDB-FN immunohistochemical staining scores of human metastatic tumor stroma, (2.17 ± 0.271), metastatic tumor parenchyma (2.08 ± 0.229), primary tumor stroma (1.61 ± 0.26), and primary tumor parenchyma (1.61 ± 0.12) were significantly (p < 0.0001) higher than normal pancreas stroma (0.14 ± 0.10) and normal pancreas parenchyma (0.14 ± 0.14). EDB-FN mRNA expression in tumors is 4.98 log2(TPM + 1) and 0.18 log2(TPM + 1) in normal tissue (p < 0.01). A mouse model of EDB-FN rich PDAC metastasis exhibited T1-weighted contrast to noise (CNR) changes of 21.80 ± 4.34 in perimetastatic regions and 8.38 ± 0.79 in metastatic regions identified through cryo-imaging, significantly higher (p < 0.05) than CNR changes found in normal liver (-6.43 ± 0.92), mesentery (2.24 ± 0.92), spleen (-3.06 ± 2.38) and intestine (1.08 ± 2.15). We conclude that EDB-FN is overexpressed in metastatic and primary PDAC tumors and MRMI with MT218 enables the detection of metastatic and perimetastatic tissues.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Carcinoma Ductal Pancreático/diagnóstico por imagem , Linhagem Celular Tumoral , Fibronectinas/química , Fibronectinas/metabolismo , Humanos , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética , Camundongos , Imagem Molecular , Neoplasias Pancreáticas/diagnóstico por imagemRESUMO
Nucleic acids are promising for a variety of therapies, such as cancer therapy and the gene therapy of genetic disorders. The therapeutic efficacy of nucleic acids is reliant on the ability of their efficient delivery to the cytosol of the target cells. Amino lipids have been developed to aid in the cytosolic delivery of nucleic acids. This work reports a new and efficient synthetic pathway for the lipid carrier, (1-aminoethyl) iminobis [N-(oleicylcysteinyl-1-amino-ethyl)propionamide] (ECO). The previous synthesis of the ECO was inefficient and presented poor product quality control. A solution-phase synthesis of the ECO was explored, and each intermediate product was characterized with better quality control. The ECO was synthesized with a relatively high yield and high purity. The formulations of the ECO nanoparticles were made with siRNA, miRNA, or plasmid DNA, and characterized. The transfection efficiency of the nanoparticles was evaluated in vitro over a range of N/P ratios. The nanoparticles were consistent in size with previous formulations and had primarily a positive zeta potential. The ECO/siLuc nanoparticles resulted in potent luciferase silencing with minimal cytotoxicity. The ECO/miR-200c nanoparticles mediated the efficient delivery of miR-200c into the target cells. The ECO/pCMV-GFP nanoparticles resulted in substantial GFP expression upon transfection. These results demonstrate that the solution-phase synthetic pathway produced pure ECO for the efficient intracellular delivery of nucleic acids without size limitation.
RESUMO
Cryo-imaging sections and images a whole mouse and provides ~ 120-GBytes of microscopic 3D color anatomy and fluorescence images, making fully manual analysis of metastases an onerous task. A convolutional neural network (CNN)-based metastases segmentation algorithm included three steps: candidate segmentation, candidate classification, and semi-automatic correction of the classification result. The candidate segmentation generated > 5000 candidates in each of the breast cancer-bearing mice. Random forest classifier with multi-scale CNN features and hand-crafted intensity and morphology features achieved 0.8645 ± 0.0858, 0.9738 ± 0.0074, and 0.9709 ± 0.0182 sensitivity, specificity, and area under the curve (AUC) of the receiver operating characteristic (ROC), with fourfold cross validation. Classification results guided manual correction by an expert with our in-house MATLAB software. Finally, 225, 148, 165, and 344 metastases were identified in the four cancer mice. With CNN-based segmentation, the human intervention time was reduced from > 12 to ~ 2 h. We demonstrated that 4T1 breast cancer metastases spread to the lung, liver, bone, and brain. Assessing the size and distribution of metastases proves the usefulness and robustness of cryo-imaging and our software for evaluating new cancer imaging and therapeutics technologies. Application of the method with only minor modification to a pancreatic metastatic cancer model demonstrated generalizability to other tumor models.
Assuntos
Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Encefálicas/diagnóstico por imagem , Aprendizado Profundo , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Animais , Neoplasias Ósseas/secundário , Neoplasias Encefálicas/secundário , Feminino , Processamento de Imagem Assistida por Computador , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/patologia , Camundongos , Redes Neurais de ComputaçãoRESUMO
PURPOSE: To investigate the effectiveness of targeted ECO/miR-200c in modulating tumor microenvironment and treating triple negative breast cancer (TNBC) using non-invasive magnetic resonance molecular imaging (MRMI) of extradomain B fibronectin (EDB-FN) with a targeted MRI contrast agent. METHODS: MDA-MB-231 and Hs578T TNBC cells were transfected with RGD-PEG-ECO/miR-200c. Invasive and migratory potential was evaluated using transwell, scratch wound, and spheroid formation assays. Athymic nude mice bearing orthotopic MDA-MB-231 and Hs578T xenografts were treated with weekly i.v. injection of RGD-PEG-ECO/miR-200c nanoparticles at 1.0 mg/kg/week RNA for 6 weeks. MRMI of EDB-FN was performed using a targeted contrast agent MT218 [ZD2-N3-Gd(DO3A)] on a 3 T MRS 3000 scanner. T1-weighted images were acquired following intravenous injection of MT218 at dose of 0.1 mmol/kg using a fast spin echo axial sequence with respiratory gating. RESULTS: Systemic administration of RGD-PEG-ECO/miR-200c nanoparticles in mice bearing orthotopic TNBC xenografts significantly suppressed tumor progression without toxic side-effects. MRMI with MT218 revealed that the treatment significantly suppressed tumor proliferation as compared to the control. MRMI also showed that the miR-200c treatment altered tumor microenvironment by reducing EDB-FN expression, as evidenced by decreased contrast enhancement in both MDA-MB-231 and Hs578T tumors. The reduction of EDB-FN was confirmed by immunohistochemistry. CONCLUSIONS: Targeted delivery of miR-200c with RGD-PEG-ECO/miR-200c nanoparticles effectively modulates tumor microenvironment and suppresses TNBC proliferation in animal models. MRMI of tumor EDB-FN expression is effective to non-invasively monitor tumor response and therapeutic efficacy of RGD-PEG-ECO/miR-200c nanoparticles in TNBC.
Assuntos
MicroRNAs/administração & dosagem , Imagem Molecular/métodos , Nanopartículas/administração & dosagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Feminino , Fibronectinas/análise , Humanos , Imageamento por Ressonância Magnética/métodos , Camundongos , MicroRNAs/análise , Invasividade Neoplásica , Neoplasias de Mama Triplo Negativas/diagnóstico por imagem , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Safe and effective molecular therapeutics for prophylactic treatment of retinal degenerative diseases are greatly needed. Disruptions in the clearance of all-trans-retinal (atRAL) by the visual (retinoid) cycle of the retina can lead to the accumulation of atRAL and its condensation products known to initiate progressive retinal dystrophy. Retinylamine (Ret-NH2) and its analogues are known to be effective in lowering the concentration of atRAL within the eye and thus preventing retinal degeneration in mouse models of human retinopathies. Here, we chemically modified Ret-NH2 with amino acids and peptides to improve the stability and ocular bioavailability of the resulting derivatives and to minimize their side effects. Fourteen Ret-NH2 derivatives were synthesized and tested in vitro and in vivo. These derivatives exhibited structure-dependent therapeutic efficacy in preventing light-induced retinal degeneration in Abca4-/-Rdh8-/- double-knockout mice, with the compounds containing glycine and/or L-valine generally exhibiting greater protective effects than Ret-NH2 or other tested amino acid derivatives of Ret-NH2. Ret-NH2-L-valylglycine amide (RVG) exhibited good stability in storage; and effective uptake and prolonged retention in mouse eyes. RVG readily formed a Schiff base with atRAL and did not inhibit RPE65 enzymatic activity. Administered by oral gavage, this retinoid also provided effective protection against light-induced retinal degeneration in Abca4-/-Rdh8-/- mice. Notably, the treatment with RVG had minimal effects on the regeneration of 11-cis-retinal and recovery of retinal function. RVG holds promise as a lead therapy for effective and safe treatment of human retinal degenerative diseases.
Assuntos
Diterpenos/farmacologia , Peptídeos/farmacologia , Degeneração Retiniana/prevenção & controle , Visão Ocular/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Oxirredutases do Álcool/genética , Animais , Diterpenos/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Retiniana/fisiopatologiaRESUMO
RNA interference (RNAi) is a promising technology to regulate oncogenes for treating cancer. The primary limitation of siRNA for clinical application is the safe and efficacious delivery of therapeutic siRNA into target cells. Lipid-based delivery systems are developed to protect siRNA during the delivery process and to facilitate intracellular uptake. There is a significant progress in lipid nanoparticle systems that utilize cationic and protonatable amino lipid systems to deliver siRNA to tumors. Among these lipids, environment-responsive lipids are a class of novel lipid delivery systems that are capable of responding to the environment changes during the delivery process and demonstrate great promise for clinical translation for siRNA therapeutics. Protonatable or ionizable amino lipids and switchable lipids as well as pH-sensitive multifunctional amino lipids are the presentative environment-responsive lipids for siRNA delivery. These lipids are able to respond to environmental changes during the delivery process to facilitate efficient cytosolic siRNA delivery. Environment-responsive lipid/siRNA nanoparticles (ERLNP) are developed with the lipids and are tested for efficient delivery of therapeutic siRNA into the cytoplasm of cancer cells to silence target genes for cancer treatment in preclinical development. This review summarizes the recent developments in environment-response lipids and nanoparticles for siRNA delivery in cancer therapy.
Assuntos
Nanopartículas , Neoplasias , Sistemas de Liberação de Medicamentos , Lipídeos , Neoplasias/terapia , Interferência de RNA , RNA Interferente PequenoRESUMO
It is still a challenge to develop gene replacement therapy for retinal disorders caused by mutations in large genes, such as Stargardt disease (STGD). STGD is caused by mutations in ABCA4 gene. Previously, we have developed an effective non-viral gene therapy using self-assembled nanoparticles of a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid containing rhodopsin promoter (pRHO-ABCA4). In this study, we modified the ABCA4 plasmid with simian virus 40 enhancer (SV40, pRHO-ABCA4-SV40) for enhanced gene expression. We also prepared and assessed the formulations of ECO/pDNA nanoparticles using sucrose or sorbitol as a stablilizer to develop consistent and stable formulations. Results demonstrated that ECO formed stable nanoparticles with pRHO-ABCA4-SV40 in the presence of sucrose, but not with sorbitol. The transfection efficiency in vitro increased significantly after introduction of SV40 enhancer for plasmid pCMV-ABCA4-SV40 with a CMV promoter. Sucrose didn't affect the transfection efficiency, while sorbitol resulted in a fluctuation of the in vitro transfection efficiency. Subretinal gene therapy in Abca4-/- mice using ECO/pRHO-ABCA4 and ECO/pRHO-ABCA4-SV40 nanoparticles induced 36% and 29% reduction in A2E accumulation respectively. Therefore, the ECO/pABCA4 based nanoparticles are promising for non-viral gene therapy for Stargardt disease and can be expended for applications in a variety of visual dystrophies with mutated large genes.
Assuntos
Nanopartículas , Vírus 40 dos Símios , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Terapia Genética , Camundongos , Mutação , Doença de StargardtRESUMO
Radiation therapy is a mainstay in the standard of care for glioblastoma (GBM), thus inhibiting the DNA damage response (DDR) is a major strategy to improve radiation response and therapeutic outcomes. Small interfering RNA (siRNA) therapy holds immeasurable potential for the treatment of GBM, however delivery of the siRNA payload remains the largest obstacle for clinical implementation. Here we demonstrate the effectiveness of the novel nanomaterial, ECO (1-aminoethylimino[bis(N-oleoylcysteinylaminoethyl) propionamide]), to deliver siRNA targeting DDR proteins ataxia telangiectasia mutated and DNA-dependent protein kinase (DNApk-cs) for the radiosensitzation of GBM in vitro and in vivo. ECO nanoparticles (NPs) were shown to efficiently deliver siRNA and silence target protein expression in glioma (U251) and glioma stem cell lines (NSC11, GBMJ1). Importantly, ECO NPs displayed no cytotoxicity and minimal silencing of genes in normal astrocytes. Treatment with ECO/siRNA NPs and radiation resulted in the prolonged presence of γH2AX foci, indicators of DNA damage, and increased radiosensitivity in all tumor cell lines. In vivo, intratumoral injection of ECO/siDNApk-cs NPs with radiation resulted in a significant increase in survival compared with injection of NPs alone. These data suggest the ECO nanomaterial can effectively deliver siRNA to more selectively target and radiosensitize tumor cells to improve therapeutic outcomes in GBM.