ABSTRACT
The concept of delivering nanoformulations to desired tissues by means of targeting membrane receptors of high local abundance by ligands anchored to the nanocarrier has gained a lot of attention over the last decade. Currently, there is no unanimous opinion on whether surface functionalization of nanocarriers by targeting ligands translates into any real benefit in terms of pharmacokinetics or treatment outcomes. Having examined the published nanocarriers designed to engage with somatostatin receptors, we realized that in the majority of cases targetability claims were not supported by solid evidence of targeting ligand-targeted receptor coupling, which is the very crux of a targetability concept. Here, we present an approach to characterize targetability of mesoporous silica-based nanocarriers functionalized with ligands of somatostatin receptors. The targetability proof in our case comes from a functional assay based on a genetically-encoded cAMP probe, which allows for real-time capture of receptor activation in living cells, triggered by targeting ligands on nanoparticles. We elaborate on the development and validation of the assay, highlighting the power of proper functional tests in the characterization pipeline of targeted nanoformulations.
ABSTRACT
Drug delivery into the brain is impeded by the blood-brain-barrier (BBB) that filters out the vast majority of drugs after systemic administration. In this work, we assessed the transport, uptake and cytotoxicity of promising drug nanocarriers, mesoporous silica nanoparticles (MSNs), in in vitro models of the BBB. RBE4 rat brain endothelial cells and Madin-Darby canine kidney epithelial cells, strain II, were used as BBB models. We studied spherical and rod-shaped MSNs with the following modifications: bare MSNs and MSNs coated with a poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) block copolymer. In transport studies, MSNs showed low permeability, whereas the results of the cellular uptake studies suggest robust uptake of PEG-PEI-coated MSNs. None of the MSNs showed significant toxic effects in the cell viability studies. While the shape effect was detectable but small, especially in the real-time surface plasmon resonance measurements, coating with PEG-PEI copolymers clearly facilitated the uptake of MSNs. Finally, we evaluated the in vivo detectability of one of the best candidates, i.e. the copolymer-coated rod-shaped MSNs, by two-photon in vivo imaging in the brain vasculature. The particles were clearly detectable after intravenous injection and caused no damage to the BBB. Thus, when properly designed, the uptake of MSNs could potentially be utilized for the delivery of drugs into the brain via transcellular transport.
Subject(s)
Blood-Brain Barrier/metabolism , Drug Carriers , Endothelial Cells/drug effects , Nanoparticles/chemistry , Polyethylene Glycols/chemistry , Polyethyleneimine/analogs & derivatives , Silicon Dioxide/chemistry , Animals , Biological Transport , Brain/blood supply , Brain/metabolism , Brain/ultrastructure , Cell Line , Cell Survival/drug effects , Dogs , Endothelial Cells/cytology , Endothelial Cells/metabolism , Injections, Intravenous , Madin Darby Canine Kidney Cells , Mice , Microscopy, Fluorescence, Multiphoton , Models, Biological , Molecular Imaging , Nanoparticles/metabolism , Nanoparticles/ultrastructure , Particle Size , Permeability , Polyethyleneimine/chemistry , Rats , Surface Plasmon ResonanceABSTRACT
Cancer stem cells (CSCs) are a challenge in cancer treatment due to their therapy resistance. We demonstrated that enhanced Notch signaling in breast cancer promotes self-renewal of CSCs that display high glycolytic activity and aggressive hormone-independent tumor growth in vivo. We took advantage of the glycolytic phenotype and the dependence on Notch activity of the CSCs and designed nanoparticles to target the CSCs. Mesoporous silica nanoparticles were functionalized with glucose moieties and loaded with a γ-secretase inhibitor, a potent interceptor of Notch signaling. Cancer cells and CSCs in vitro and in vivo efficiently internalized these particles, and particle uptake correlated with the glycolytic profile of the cells. Nanoparticle treatment of breast cancer transplants on chick embryo chorioallantoic membranes efficiently reduced the cancer stem cell population of the tumor. Our data reveal that specific CSC characteristics can be utilized in nanoparticle design to improve CSC-targeted drug delivery and therapy.
Subject(s)
Antineoplastic Agents/administration & dosage , Breast Neoplasms/drug therapy , Enzyme Inhibitors/administration & dosage , Glucose/metabolism , Neoplastic Stem Cells/drug effects , Receptors, Notch/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Breast Neoplasms/enzymology , Breast Neoplasms/metabolism , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Female , Humans , MCF-7 Cells , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Signal Transduction/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Targeted delivery of drugs is required to efficiently treat intestinal diseases such as colon cancer and inflammation. Nanoparticles could overcome challenges in oral administration caused by drug degradation at low pH and poor permeability through mucus layers, and offer targeted delivery to diseased cells in order to avoid adverse effects. Here, we demonstrate that functionalization of mesoporous silica nanoparticles (MSNs) by polymeric surface grafts facilitates transport through the mucosal barrier and enhances cellular internalization. MSNs functionalized with poly(ethylene glycol) (PEG), poly(ethylene imine) (PEI), and the targeting ligand folic acid in different combinations are internalized by epithelial cells in vitro and in vivo after oral gavage. Functionalized MSNs loaded with γ-secretase inhibitors of the Notch pathway, a key regulator of intestinal progenitor cells, colon cancer, and inflammation, demonstrated enhanced intestinal goblet cell differentiation as compared to free drug. Drug-loaded MSNs thus remained intact in vivo, further confirmed by exposure to simulated gastric and intestinal fluids in vitro. Drug targeting and efficacy in different parts of the intestine could be tuned by MSN surface modifications, with PEI coating exhibiting higher affinity for the small intestine and PEI-PEG coating for the colon. The data highlight the potential of nanomedicines for targeted delivery to distinct regions of the tissue for strict therapeutic control.
Subject(s)
Cell Differentiation/drug effects , Drug Delivery Systems , Gastrointestinal Tract/cytology , Nanoparticles/administration & dosage , Polyethylene Glycols/chemistry , Polyethyleneimine/analogs & derivatives , Silicon Dioxide/chemistry , Administration, Oral , Animals , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Gastrointestinal Tract/drug effects , Male , Mice , Mice, Inbred C57BL , Nanomedicine , Nanoparticles/chemistry , Polyethyleneimine/chemistry , Polymers/chemistry , Tumor Cells, CulturedABSTRACT
Nanomedicine is gaining ground worldwide in therapy and diagnostics. Novel nanoscopic imaging probes serve as imaging tools for studying dynamic biological processes in vitro and in vivo. To allow detectability in the physiological environment, the nanostructure-based probes need to be either inherently detectable by biomedical imaging techniques, or serve as carriers for existing imaging agents. In this study, the potential of mesoporous silica nanoparticles carrying commercially available fluorochromes as self-regenerating cell labels for long-term cellular tracking is investigated. The particle surface is organically modified for enhanced cellular uptake, the fluorescence intensity of labeled cells is followed over time both in vitro and in vivo. The particles are not exocytosed and particles which escaped cells due to cell injury or death are degraded and no labeling of nontargeted cell populations are observed. The labeling efficiency is significantly improved as compared to that of quantum dots of similar emission wavelength. Labeled human breast cancer cells are xenotransplanted in nude mice, and the fluorescent cells can be detected in vivo for a period of 1 month. Moreover, ex vivo analysis reveals fluorescently labeled metastatic colonies in lymph node and rib, highlighting the capability of the developed probes for tracking of metastasis.
Subject(s)
Cell Tracking/methods , Fluorescent Dyes/chemistry , Optical Phenomena , Silicon Dioxide/chemistry , Animals , Cell Cycle , Cell Line, Tumor , Cell Movement , Cell Proliferation , Diagnostic Imaging , Exocytosis , Female , Flow Cytometry , Fluorescence , Humans , Mice, Nude , Nanoparticles/ultrastructure , Porosity , Quantum Dots/chemistry , Xenograft Model Antitumor AssaysABSTRACT
Intracellular 3'-5'-cyclic adenosine monophosphate (cAMP) is one of the principal second messengers downstream of a manifold of signal transduction pathways, including the ones triggered by G protein-coupled receptors. Not surprisingly, biochemical assays for cAMP have been instrumental for basic research and drug discovery for decades, providing insights into cellular physiology and guiding pharmaceutical industry. However, despite impressive track record, the majority of conventional biochemical tools for cAMP probing share the same fundamental shortcoming-all the measurements require sample disruption for cAMP liberation. This common bottleneck, together with inherently low spatial resolution of measurements (as cAMP is typically analyzed in lysates of thousands of cells), underpin the ensuing limitations of the conventional cAMP assays: (1) genuine kinetic measurements of cAMP levels over time in a single given sample are unfeasible; (2) inability to obtain precise information on cAMP spatial distribution and transfer at subcellular levels, let alone the attempts to pinpoint dynamic interactions of cAMP and its effectors. At the same time, tremendous progress in synthetic biology over the recent years culminated in drastic refinement of our toolbox, allowing us not only to bypass the limitations of conventional assays, but to put intracellular cAMP life-span under tight control-something, that seemed scarcely attainable before. In this review article we discuss the main classes of modern genetically-encoded tools tailored for cAMP probing and modulation in living systems. We examine the capabilities and weaknesses of these different tools in the context of their operational characteristics and applicability to various experimental set-ups involving living cells, providing the guidance for rational selection of the best tools for particular needs.
ABSTRACT
INTRODUCTION: Decoding transcriptional effects of experimental tissue-tissue or cell-cell interactions is important; for example, to better understand tumor-stroma interactions after transplantation of human cells into mouse (xenografting). Transcriptome analysis of intermixed human and mouse cells has, however, frequently relied on the need to separate the two cell populations prior to transcriptome analysis, which introduces confounding effects on gene expression. METHODS: To circumvent this problem, we here describe a bioinformatics-based, genome-wide transcriptome analysis technique, which allows the human and mouse transcriptomes to be decoded from a mixed mouse and human cell population. The technique is based on a bioinformatic separation of the mouse and human transcriptomes from the initial mixed-species transcriptome resulting from sequencing an excised tumor/stroma specimen without prior cell sorting. RESULTS: Under stringent separation criteria, i.e., with a read misassignment frequency of 0.2 %, we show that 99 % of the genes can successfully be assigned to be of mouse or human origin, both in silico, in cultured cells and in vivo. We use a new species-specific sequencing technology-referred to as S(3) ("S-cube")-to provide new insights into the Notch downstream response following Notch ligand-stimulation and to explore transcriptional changes following transplantation of two different breast cancer cell lines (luminal MCF7 and basal-type MDA-MB-231) into mammary fat pad tissue in mice of different immunological status. We find that MCF7 and MDA-MB-231 respond differently to fat pad xenografting and the stromal response to transplantation of MCF7 and MDA-MB-231 cells was also distinct. CONCLUSIONS: In conclusion, the data show that the S(3) technology allows for faithful recording of transcriptomic changes when human and mouse cells are intermixed and that it can be applied to address a broad spectrum of research questions.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Communication , Stromal Cells/metabolism , Animals , Cell Line, Tumor , Disease Models, Animal , Female , Gene Expression Profiling , Heterografts , Humans , Ligands , Mice , Receptors, Notch/metabolism , Signal Transduction , Species Specificity , TranscriptomeABSTRACT
Recently reported photoluminescent nanographene oxides (nGOs), i.e. nanographene oxidised with a sulfuric/nitric acid mixture (SNOx method), have tuneable photoluminescence and are scalable, simple and fast to produce optical probes. This material belongs to the vast class of photoluminescent carbon nanostructures, including carbon dots, nanodiamonds (NDs), graphene quantum dots (GQDs), all of which demonstrate a variety of properties that are attractive for biomedical imaging such as low toxicity and stable photoluminescence. In this study, the nGOs were organically surface-modified with poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) copolymers tagged with folic acid as the affinity ligand for cancer cells expressing folate receptors. The functionalization enhanced both the cellular uptake and quantum efficiency of the photoluminescence as compared to non-modified nGOs. The nGOs exhibited an excitation dependent photoluminescence that facilitated their detection with a wide range of microscope configurations. The functionalized nGOs were non-toxic, they were retained in the stained cell population over a period of 8 days and they were distributed equally between daughter cells. We have evaluated their applicability in in vitro and in vivo (chicken embryo CAM) models to visualize and track migratory cancer cells. The good biocompatibility and easy detection of the functionalized nGOs suggest that they could address the limitations faced with quantum dots and organic fluorophores in long-term in vivo biomedical imaging.
Subject(s)
Cell Tracking/methods , Graphite/chemistry , Microscopy, Fluorescence/methods , Nanoparticles/chemistry , Neoplasms, Experimental/chemistry , Neoplasms, Experimental/pathology , Animals , Cell Movement , HeLa Cells , Humans , Image Enhancement/methods , Luminescent Measurements/methods , Molecular Probe Techniques , Molecular Probes , Oxides/chemistry , Subcellular Fractions/chemistry , Subcellular Fractions/pathologyABSTRACT
PURPOSE: Adenocarcinoma of the pancreas remains one of the most lethal human cancers. The high mortality rates associated with this form of cancer are subsequent to late-stage clinical presentation and diagnosis, when surgery is rarely possible and of modest chemotherapeutic impact. Survival rates following diagnosis with advanced pancreatic cancer are very low; typical mortality rates of 50% are expected within 3 months of diagnosis. However, adjuvant chemotherapy improves the prognosis of patients even after palliative surgery, and successful newer neoadjuvant chemotherapeutical modalities have recently been reported. For patients whose tumours appear unresectable, chemotherapy remains the only option. During the past two decades, the nucleoside analogue gemcitabine has become the first-line chemotherapy for pancreatic adenocarcinoma. In this study, we aim to increase the delivery of gemcitabine to pancreatic tumours by exploring the effect of sonoporation for localised drug delivery of gemcitabine in an orthotopic xenograft mouse model of pancreatic cancer. EXPERIMENTAL DESIGN: An orthotopic xenograft mouse model of luciferase expressing MIA PaCa-2 cells was developed, exhibiting disease development similar to human pancreatic adenocarcinoma. Subsequently, two groups of mice were treated with gemcitabine alone and gemcitabine combined with sonoporation; saline-treated mice were used as a control group. A custom-made focused ultrasound transducer using clinically safe acoustic conditions in combination with SonoVue® ultrasound contrast agent was used to induce sonoporation in the localised region of the primary tumour only. Whole-body disease development was measured using bioluminescence imaging, and primary tumour development was measured using 3D ultrasound. RESULTS: Following just two treatments combining sonoporation and gemcitabine, primary tumour volumes were significantly lower than control groups. Additional therapy dramatically inhibited primary tumour growth throughout the course of the disease, with median survival increases of up to 10% demonstrated in comparison to the control groups. CONCLUSION: Combined sonoporation and gemcitabine therapy significantly impedes primary tumour development in an orthotopic xenograft model of human pancreatic cancer, suggesting additional clinical benefits for patients treated with gemcitabine in combination with sonoporation.
Subject(s)
Deoxycytidine/analogs & derivatives , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Sonication/methods , Tumor Burden , Xenograft Model Antitumor Assays , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Deoxycytidine/pharmacology , Deoxycytidine/therapeutic use , Drug Delivery Systems , Humans , Mice , Mice, SCID , Microbubbles , Reproducibility of Results , Survival Analysis , Transducers , Tumor Burden/drug effects , Ultrasonics/instrumentation , GemcitabineABSTRACT
MSNs have attracted increasing interest as drug carriers due to promising in vivo results in small-animal disease models, especially related to cancer therapy. In most cases small hydrophobic drugs have been used, but recent in vitro studies demonstrate that MSNs are highly interesting for gene delivery applications. This review covers recent advances related to the therapeutic use of mesoporous silica nanoparticles (MSNs) administered intravenously, intraperitoneally or locally. We also cover the use of MSNs in alternative modes of therapy such as photodynamic therapy and multidrug therapy. We further discuss the current understanding about the biodistribution and safety of MSNs. Finally, we critically discuss burning questions especially related to experimental design of in vivo studies in order to enable a fast transition to clinical trials of this promising drug delivery platform.
Subject(s)
Drug Delivery Systems/methods , Nanoparticles/chemistry , Silicon Dioxide/chemistry , Animals , Humans , Nanoparticles/administration & dosage , Neoplasms/chemistry , Neoplasms/drug therapy , Neoplasms/metabolism , Particle Size , Phototherapy/methods , Porosity/drug effects , Silicon Dioxide/administration & dosage , Silicon Dioxide/metabolism , Tissue Distribution/drug effects , Tissue Distribution/physiologyABSTRACT
Nanotechnology may help overcome persisting limitations of current cancer treatment and thus contribute to the creation of more effective, safer and more affordable therapies. While some nanotechnology-based drug delivery systems are already being marketed and others are in clinical trial, most still remain in the preclinical development stage. Mesoporous silica nanoparticles have been highlighted as an interesting drug delivery platform, due to their flexibility and high drug load potential. Although numerous reports demonstrate sophisticated drug delivery mechanisms in vitro, the therapeutic benefit of these systems for in vivo applications have been under continuous debate. This has been due to nontranslatable conditions used in the in vitro studies, as well as contradictory conclusions drawn from preclinical (in vivo) studies. However, recent studies have indicated that the encouraging cellular studies could in fact be repeated also in vivo. Here, we report on these recent advances regarding therapeutic efficacy, targeting and safety issues related to the application of mesoporous silica nanoparticles in cancer therapy.
Subject(s)
Drug Carriers/chemistry , Drug Delivery Systems , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Neoplasms/drug therapy , Silicon Dioxide/chemistry , Animals , Metabolic Clearance Rate , Mice , Nanotechnology/methods , Particle Size , Porosity , Safety , Surface Properties , Tissue Distribution , Treatment OutcomeABSTRACT
A switch from oxidative phosphorylation to glycolysis is frequently observed in cancer cells and is linked to tumor growth and invasion, but the underpinning molecular mechanisms controlling the switch are poorly understood. In this report we show that Notch signaling is a key regulator of cellular metabolism. Both hyper- and hypoactivated Notch induce a glycolytic phenotype in breast tumor cells, although by distinct mechanisms: hyperactivated Notch signaling leads to increased glycolysis through activation of the phosphatidylinositol 3-kinase/AKT serine/threonine kinase pathway, whereas hypoactivated Notch signaling attenuates mitochondrial activity and induces glycolysis in a p53-dependent manner. Despite the fact that cells with both hyper- and hypoactivated Notch signaling showed enhanced glycolysis, only cells with hyperactivated Notch promoted aggressive tumor growth in a xenograft mouse model. This phenomenon may be explained by that only Notch-hyperactivated, but not -hypoactivated, cells retained the capacity to switch back to oxidative phosphorylation. In conclusion, our data reveal a role for Notch in cellular energy homeostasis, and show that Notch signaling is required for metabolic flexibility.
Subject(s)
Gene Expression Regulation, Neoplastic , Receptors, Notch/metabolism , Animals , Glycolysis , Homeostasis , Humans , Mice , Mitochondria/metabolism , Models, Biological , Neoplasm Transplantation , Oxidative Phosphorylation , Oxygen/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Signal Transduction , Tumor Suppressor Protein p53/metabolismABSTRACT
Notch signaling, a key regulator of stem cells, is frequently overactivated in cancer. It is often linked to aggressive forms of cancer, evading standard treatment highlighting Notch as an exciting therapeutic target. Notch is in principle "druggable" by γ-secretase inhibitors (GSIs), inhibitory peptides and antibodies, but clinical use of Notch inhibitors is restricted by severe side effects and there is a demand for alternative cancer-targeted therapy. Here, we present a novel approach, using imagable mesoporous silica nanoparticles (MSNPs) as vehicles for targeted delivery of GSIs to block Notch signaling. Drug-loaded particles conjugated to targeting ligands induced cell-specific inhibition of Notch activity in vitro and exhibited enhanced tumor retainment with significantly improved Notch inhibition and therapeutic outcome in vivo. Oral administration of GSI-MSNPs controlled Notch activity in intestinal stem cells further supporting the in vivo applicability of MSNPs for GSI delivery. MSNPs showed tumor accumulation and targeting after systemic administration. MSNPs were biocompatible, and particles not retained within the tumors, were degraded and eliminated mainly by renal excretion. The data highlights MSNPs as an attractive platform for targeted drug delivery of anticancer drugs with otherwise restricted clinical application, and as interesting constituents in the quest for more refined Notch therapies.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Breast Neoplasms/metabolism , Drug Delivery Systems/methods , Nanoparticles/therapeutic use , Receptors, Notch/antagonists & inhibitors , Signal Transduction/drug effects , Amyloid Precursor Protein Secretases/metabolism , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Line , Female , Humans , Mice , Nanoparticles/administration & dosage , Porosity , Receptors, Notch/metabolism , Silicon Dioxide/administration & dosage , Silicon Dioxide/therapeutic useABSTRACT
The etiology of pituitary adenomas remains largely unknown, with the exception of involvement of estrogens in the formation of prolactinomas. We have examined the molecular pathogenesis of prolactin-producing pituitary adenomas in transgenic female mice expressing the human choriongonadotropin (hCG) beta-subunit. The LH/CG bioactivity is elevated in the mice, with consequent highly stimulated ovarian progesterone (P(4)) production, in the face of normal estrogen secretion. Curiously, despite normal estrogen levels, large prolactinomas developed in these mice, and we provide here several lines of evidence that the elevated P(4) levels are involved in the growth of these estrogen-dependent tumors. The antiprogestin mifepristone inhibited tumor growth, and combined postgonadectomy estradiol/P(4) treatment was more effective than estrogen alone in inducing tumor growth. Evidence for direct growth-promoting effect of P(4) was obtained from cultures of primary mouse pituitary cells and rat somatomammotroph GH3 cells. The mouse tumors and cultured cells revealed stimulation of the cyclin D1/cyclin-dependent kinase 4/retinoblastoma protein/transcription factor E2F1 pathway in the growth response to P(4). If extrapolated to humans, and given the importance of endogenous P(4) and synthetic progestins in female reproductive functions and their pharmacotherapy, it is relevant to revisit the potential role of these hormones in the origin and growth of prolactinomas.