Tumor cell-targeted delivery of nanoconjugated oligonucleotides in composite spheroids.
Ontology highlight
ABSTRACT:
SUBMITTER: Carver K
PROVIDER: S-EPMC4250944 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Oligonucleotides have shown promise in selectively manipulating gene expression in vitro, but that success has not translated to the clinic for cancer therapy. A potential reason for this is that cells behave differently in monolayer than in the three-dimensional tumor, resulting in limited penetration and distribution of oligonucleotides in the tumor. This may be especially true when oligonucleotides are associated with nanocarriers such as lipoplexes and polyplexes, commonly used delivery vehicles for oligonucleotides. The multicellular tumor spheroid (MCTS), a three-dimensional model that closely resembles small avascular tumors and micrometastases, has been utilized as an intermediate between monolayer culture and in vivo studies for the screening of small-molecule drugs. However, spheroids have been little used for the study of various oligonucleotide delivery formulations. Here, we have evaluated the uptake and efficacy of splice-switching antisense oligonucleotides using various delivery modalities in two- and three-dimensional culture models. We find that the size of the delivery agent dramatically influences penetration into the spheroid and thus the biological effect of the oligonucleotides. We hypothesize that the MCTS model will prove to be a useful tool in the future development of oligonucleotide delivery formulations.Molecular Therapy-Nucleic Acids (2014) 3, e153; doi:10.1038/mtna.2014.5; published online 11 March 2014.
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Project description:BACKGROUND: Stromal fibroblasts are important determinants of tumor cell behavior. They act to condition the tumor microenvironment, influence tumor growth, support tumor angiogenesis and affect tumor metastasis. Heparan sulfate proteoglycans, present both on tumor and stromal cells, interact with a large number of ligands including growth factors, their receptors, and structural components of the extracellular matrix. Being ubiquitously expressed in the tumor microenvironment heparan sulfate proteoglycans are candidates for playing central roles in tumor-stroma interactions. The objective of this work was to investigate the role of heparan sulfate expressed by stromal fibroblasts in modulating the growth of tumor cells and in controlling the interstitial fluid pressure in a 3-D model. METHODOLOGY/PRINCIPAL FINDINGS: We generated spheroids composed of fibroblasts alone, or composite spheroids, composed of fibroblasts and tumor cells. Here we show that stromal fibroblasts with a mutation in the heparan sulfate elongating enzyme Ext1 and thus a low heparan sulfate content, formed composite fibroblast/tumor cell spheroids with a significant lower interstitial fluid pressure than corresponding wild-type fibroblast/tumor cell composite spheroids. Furthermore, immunohistochemistry of composite spheroids revealed that the cells segregated, so that after 6 days in culture, the wild-type fibroblasts formed an inner core and the tumor cells an outer layer of cells. For composite spheroids containing Ext1-mutated fibroblasts this segregation was less obvious, indicating impaired cell migration. Analysis of tumor cells expressing the firefly luciferase gene revealed that the changes in tumor cell migration in mutant fibroblast/tumor cell composite spheroids coincided with a lower proliferation rate. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration that stromal Ext1-levels modulate tumor cell proliferation and affect the interstitial fluid pressure in a 3-D spheroid model. Learning how structural changes in stromal heparan sulfate influence tumor cells is essential for our understanding how non-malignant cells of the tumor microenvironment influence tumor cell progression.
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Project description:Antisense oligonucleotide (ASO) silencing of the expression of disease-associated genes is an attractive novel therapeutic approach, but treatments are limited by the ability to deliver ASOs to cells and tissues. Following systemic administration, ASOs preferentially accumulate in liver and kidney. Among the cell types refractory to ASO uptake is the pancreatic insulin-secreting ?-cell. Here, we show that conjugation of ASOs to a ligand of the glucagon-like peptide-1 receptor (GLP1R) can productively deliver ASO cargo to pancreatic ?-cells both in vitro and in vivo. Ligand-conjugated ASOs silenced target genes in pancreatic islets at doses that did not affect target gene expression in liver or other tissues, indicating enhanced tissue and cell type specificity. This finding has potential to broaden the use of ASO technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of ASOs to additional cell types.
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Project description:Nanoparticle-based delivery has become an important strategy to advance siRNA and antisense oligonucleotides into clinical reality. However, limited biodistribution of nanoparticles and the toxicity of some nanocarriers restrict the wider application of this strategy. To address these issues we aimed to construct oligonucleotide delivery systems which are non-cytotoxic and smaller than typical nanoparticles. Thus, a morpholino oligonucleotide was conjugated to a tumor-targeting RGD peptide, and then, multiple RGD-oligo conjugates were linked to a single molecule of human serum albumin via a reductively responsive linkage. The resultant nanoconjugates showed uniform and monodispersed size distribution with a diameter of 13 nm. A single nanoconjugate molecule contains 15 oligonucleotides as well as 15 targeting ligands on the surface of albumin. The nanoparticle demonstrated 61-fold enhancement in receptor-specific cellular delivery of oligonucleotides in integrin-expressing tumor cells compared to the non-targeted control nanoconjugates and were able to robustly enhance functional activity of the oligonucleotide at low nanomolar concentrations without causing cytotoxicity. Due to their small size, the targeted nanoconjugates could penetrate deeply and distribute throughout 3-D tumor spheroids, whereas the conventional nanoparticles with sizes over 300 nm could only deliver to the cells on the surface of the tumor spheroids. As a result of their greater cellular delivery, smaller size, and lack of cytotoxicity compared to conventional nanoparticles, the multivalent nanoconjugates may provide an effective tool for targeting oligonucleotides to tumors and other diseased tissues.
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Project description:Synthetic nucleic acids have shown great potential in the treatment of various diseases. Nevertheless, the selective delivery to a target tissue has proved challenging. The coupling of nucleic acids to targeting peptides, proteins, and antibodies has been explored as an approach for their selective tissue delivery. Nevertheless, the preparation of covalently coupled peptides and proteins that can also undergo intracellular release as well as deliver more than one copy of the nucleic acid has proved challenging. Recently, we have developed a novel method for the rapid noncovalent conjugation of nucleic acids to targeting single chain antibodies (scFv) using chemically self-assembled nanostructures (CSANs). CSANs have been prepared by the self-assembly of two dihydrofolate reductase molecules (DHFR(2)) and a targeting scFv in the presence of bis-methotrexate (bis-MTX). The valency of the nanorings can be tuned from one to eight subunits, depending on the length and composition of the linker between the dihydrofolate reductase molecules. To explore their potential for the therapeutic delivery of nucleic acids as well as the ability to expand the capabilities of CSANs by incorporating smaller cyclic targeting peptides, we prepared DHFR(2) proteins fused through a flexible peptide linker to cyclic-RGD, which targets ?v?3 integrins, and a bis-MTX chemical dimerizer linked to an antisense oligonucleotide (bis-MTX-ASO) that has been shown to silence expression of eukaryotic translation initiation factor 4E (eIF4E). Monomeric and multimeric cRGD-CSANs were then prepared with bis-MTX-ASO and shown to undergo endocytosis in the breast cancer cell line, MDA-MB-231, which overexpresses ?v?3. The bis-MTX-ASO was shown to undergo endosomal escape resulting in the knock down of eIF4E with at least the same efficiency as ASO delivered by oligofectamine. The modularity, flexibility, and common method of conjugation may prove to be a useful general approach for the targeted delivery of ASOs, as well as other nucleic acids to cells.
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Project description:Uncontrolled cell growth, division, and lack of enough blood supply causes low oxygen content or hypoxia in cancerous tumor microenvironments. 17β-Estradiol (E2), an estrogen receptor (ER) ligand, can be incorporated on the surface of nanocarriers for targeted drug delivery to breast cancer cells overexpressing ER. In the present study, we synthesized estradiol-conjugated hypoxia-responsive polymeric nanoparticles (polymersomes) encapsulating the anticancer drug doxorubicin (E2-Dox-HRPs) for targeted delivery into the hypoxic niches of estrogen-receptor-positive breast cancer microtumors. Estradiol-conjugated polymersomes released over 90% of their encapsulated Dox in a sustained manner within hypoxia (2% oxygen) after 12 h. However, they released about 30% of Dox in normal oxygen partial pressure (21% oxygen, normoxia) during this time. Fluorescence microscopic studies demonstrated higher cytosolic and nuclear internalization of E2-Dox-HRPs (targeted polymersomes) compared to those of Dox-HRPs (nontargeted polymersomes). Monolayer cell viability studies on ER-positive MCF7 cells showed higher cytotoxicity of targeted polymersomes in hypoxia compared to in normoxia. Cytotoxicity studies with hypoxic three-dimensional spheroid cultures of MCF7 cells treated with targeted polymersomes indicated significant differences compared to those of normoxic spheroids. The novel estradiol-conjugated hypoxia-responsive polymersomes described here have the potential for targeted drug delivery in estrogen-receptor-positive breast cancer therapy.
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Project description:Selective delivery of antisense or siRNA oligonucleotides to cells and tissues via receptor-mediated endocytosis is becoming an important approach for oligonucleotide-based pharmacology. In most cases receptor targeting has been attained using antibodies or peptide-type ligands. Thus, there are few examples of delivering oligonucleotides using the plethora of small-molecule receptor-specific ligands that currently exist. In this report we describe a facile approach to the generation of mono- and multivalent conjugates of oligonucleotides with small-molecule ligands. Using the sigma-receptor ligand anisamide as an example, we describe conversion of the ligand to a phosphoramidite and direct incorporation of this moiety into the oligonucleotide by solid-phase DNA synthesis. We generated mono- and trivalent conjugates of anisamide with a splice switching antisense oligonucleotide (SSO) and tested their ability to modify splicing of a reporter gene (luciferase) in tumor cells in culture. The trivalent anisamide-SSO conjugate displayed enhanced cellular uptake and was markedly more effective than an unconjugated SSO or the monovalent conjugate in modifying splicing of the reporter. Significant biological effects were attained in the sub-100 nM concentration range.
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Project description:Tropism of neural stem cells (NSCs) to hypoxic tumor areas provides an opportunity for the drug delivery. Here, we demonstrate that NSCs effectively transport antisense oligonucleotides (ASOs) targeting oncogenic and tolerogenic signal transducer and activator of transcription 3 (STAT3) protein into glioma microenvironment. To enable spontaneous, scavenger receptor-mediated endocytosis by NSCs, we used previously described CpG-STAT3ASO conjugates. Following uptake and endosomal escape, CpG-STAT3ASO colocalized with CD63+ vesicles and later with CD63+CD81+ exosomes. Over 3 days, NSCs secreted exosomes loaded up to 80% with CpG-STAT3ASO. Compared to native NSC exosomes, the CpG-STAT3ASO-loaded exosomes potently stimulated immune activity of human dendritic cells or mouse macrophages, inducing nuclear factor κB (NF-κB) signaling and interleukin-12 (IL-12) production. Using orthotopic GL261 tumors, we confirmed that NSC-mediated delivery improved oligonucleotide transfer from a distant injection site into the glioma microenvironment versus naked oligonucleotides. Correspondingly, the NSC-delivered CpG-STAT3ASO enhanced activation of glioma-associated microglia. Finally, we demonstrated that NSC-mediated CpG-STAT3ASO delivery resulted in enhanced antitumor effects against GL261 glioma in mice. Peritumoral injections of 5 × 105 NSCs loaded ex vivo with CpG-STAT3ASO inhibited subcutaneous tumor growth more effectively than the equivalent amount of oligonucleotide alone. Based on these results, we anticipate that NSCs and NSC-derived exosomes will provide a clinically relevant strategy to improve delivery and safety of oligonucleotide therapeutics for glioma treatment.
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Project description:Oligonucleotide-based drugs have received considerable attention for their capacity to modulate gene expression very specifically and as a consequence they have found applications in the treatment of many human acquired or genetic diseases. Clinical translation has been often hampered by poor biodistribution, however. Cell-penetrating peptides (CPPs) appear as a possibility to increase the cellular delivery of non-permeant biomolecules such as nucleic acids. This review focuses on CPP-delivery of several classes of oligonucleotides (ONs), namely antisense oligonucleotides, splice switching oligonucleotides (SSOs) and siRNAs. Two main strategies have been used to transport ONs with CPPs: covalent conjugation (which is more appropriate for charge-neutral ON analogues) and non-covalent complexation (which has been used for siRNA delivery essentially). Chemical synthesis, mechanisms of cellular internalization and various applications will be reviewed. A comprehensive coverage of the enormous amount of published data was not possible. Instead, emphasis has been put on strategies that have proven to be effective in animal models of important human diseases and on examples taken from the authors' own expertise.
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Project description:Under hypoxic conditions, tumor cells undergo a series of adaptations that promote evolution of a more aggressive tumor phenotype including the activation of DNA damage repair proteins, altered metabolism, and decreased proliferation. Together these changes mitigate the negative impact of oxygen deprivation and allow preservation of genomic integrity and proliferative capacity, thus contributing to tumor growth and metastasis. As a result the presence of a hypoxic microenvironment is considered a negative clinical feature of many solid tumors. Hypoxic niches in tumors also represent a therapeutically privileged environment in which chemo- and radiation therapy is less effective. Although the negative impact of tumor hypoxia has been well established, the precise effect of oxygen deprivation on tumor cell behavior, and the molecular signals that allow a tumor cell to survive in vivo are poorly understood. Multicellular tumor spheroids (MCTS) have been used as an in vitro model for the avascular tumor niche, capable of more accurately recreating tumor genomic profiles and predicting therapeutic response. However, relatively few studies have used MCTS to study the molecular mechanisms driving tumor cell adaptations within the hypoxic tumor environment. Here we will review what is known about cell proliferation, DNA damage repair, and metabolic pathways as modeled in MCTS in comparison to observations made in solid tumors. A more precise definition of the cell populations present within 3D tumor models in vitro could better inform our understanding of the heterogeneity within tumors as well as provide a more representative platform for the testing of therapeutic strategies.
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