RESUMO
Using a recently developed nucleic acid delivery platform, we demonstrate the effective delivery of metallodrug [AuIIIBr2(SSC-Inp-OEt)] (AP228; Inp = isonipecotic moiety), a hydrophobic, low solubility gold complex cytotoxic to cancer cells. It is shown that AP228 is delivered more effectively into HeLa cells using micellular surfactant assemblies compared to that of a more polar derivative [AuIIIBr2(SSC-Inp-GlcN1)] (AP209; GlcN1 = (α,ß)-d-glucosamino moiety). When AP228 is codelivered with siRNA targeting Bcl-2, a key regulator of apoptosis, the overall cytotoxic therapeutic effects of the drug are maximized. The optimized delivery and distribution of the compound is monitored by both fluorescence microscopy and inductively coupled plasma mass spectrometry. We show that codelivery of the AP228 and Bcl-2 targeting siRNA results in a substantial increase in drug efficacy, wherein the cytotoxic therapeutic effects of the drug are maximized, reducing the IC50 from 760 nM to 11 nM. This hybrid small molecule drug and therapeutic nucleic acid delivery vehicle is shown to enable both the improved solubility and uptake of the gold(III) metallodrugs and the delivery of chemically unmodified siRNA, resulting in enhanced cytotoxic effects.
Assuntos
Antineoplásicos/química , DNA/química , Portadores de Fármacos/química , Ouro/química , Nanopartículas Metálicas/química , RNA Interferente Pequeno/química , Tensoativos/química , Antineoplásicos/farmacologia , Células HeLa , Humanos , RNA Interferente Pequeno/genéticaRESUMO
Herein, we describe the characterization of a novel self-assembling and intracellular disassembling nanomaterial for nucleic acid delivery and targeted gene knockdown. By using a recently developed nucleic acid nanocapsule (NAN) formed from surfactants and conjugated DNAzyme (DNz) ligands, it is shown that DNz-NAN can enable cellular uptake of the DNAzyme and result in 60 % knockdown of a target gene without the use of transfection agents. The DNAzyme also exhibits activity without chemical modification, which we attribute to the underlying nanocapsule design and release of hydrophobically modified nucleic acids as a result of enzymatically triggered disassembly of the NAN. Fluorescence-based experiments indicate that the surfactant-conjugated DNAzymes are better able to access a fluorescent mRNA target within a mock lipid bilayer system than the free DNAzyme, highlighting the advantage of the hydrophobic surfactant modification to the nucleic acid ligands. In vitro characterization of DNz-NAN's substrate-cleavage kinetics, stability in biological serum, and persistence of knockdown against a proinflammatory transcription factor, GATA-3, are presented.
RESUMO
Herein we describe a nucleic acid functionalized nanocapsule in which nucleic acid ligands are assembled and disassembled in the presence of enzymes. The particles are fully degradable in response to esterases due to an embedded ester cross-linker in the particle's core. During synthesis the nanocapsules can be loaded with hydrophobic small molecules and post self-assembly undergo covalent cross-linking using copper catalyzed click chemistry. They can then be functionalized with thiolated DNA through stepwise thiolyne chemistry using UV light irradiation. Additionally, the capsule is compatible with enzyme mediated functionalization of a therapeutic mRNA-cleaving DNAzyme at the particle's surface. The resulting particle is highly stable, monodisperse in size, and maximizes the therapeutic potential of both the particles interior and exterior.
Assuntos
DNA Catalítico/metabolismo , DNA/metabolismo , Liberação Controlada de Fármacos , Esterases/metabolismo , Nanocápsulas/química , Sobrevivência Celular/efeitos dos fármacos , DNA/química , DNA/farmacologia , DNA Catalítico/química , Liberação Controlada de Fármacos/efeitos dos fármacos , Esterases/química , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Aberrant glycosylation has been linked to many different cancer types. The blood-brain barrier (BBB) is a region of the brain that regulates the entrance of ions, diseases, toxins, and so on. However, in breast cancer metastasis, the BBB fails to prevent the crossing of the cancer cells into the brain. Here we present a study of identifying and quantifying the glycosylation of six breast and brain cancer cell lines using hydrophilic interaction liquid chromatography (HILIC) and electrostatic repulsion liquid chromatography (ERLIC) enrichments and LC-MS/MS analysis. Qualitative and quantitative analyses of N-linked glycosylation were performed by both enrichment techniques for individual and complementary comparison. Potential cancer glycopeptide biomarkers were identified and confirmed by chemometric and statistical evaluations. A total of 497 glycopeptides were characterized, of which 401 were common glycopeptides (80.6% overlap) identified from both enrichment techniques. HILIC enrichment yielded 320 statistically significant glycopeptides in 231BR relative to the other cell lines out of 494 unique glycopeptides, and sequential HILIC-ERLIC enrichment yielded 214 statistically significant glycopeptides in 231BR compared with the other cell lines out of 404 unique glycopeptides. The results provide the first comprehensive glycopeptide listing for these six cell lines.
Assuntos
Neoplasias Encefálicas/química , Neoplasias da Mama/química , Glicopeptídeos/análise , Proteínas de Neoplasias/análise , Neoplasias Encefálicas/patologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Interpretação Estatística de Dados , Feminino , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Espectrometria de Massas em Tandem/métodosRESUMO
Allergic asthma is one of the leading chronic lung diseases of both children and adults worldwide, resulting in significant morbidity and mortality in affected individuals. Many patients have severe asthma, which is refractory to treatment, illustrating the need for the development of new therapeutics for this disease. Herein, we describe the use of a peptide cross-linked nucleic acid nanocapsule (NAN) for the delivery of a GATA3-specific DNAzyme to immune cells, with demonstration of modulated transcriptional activity and behavior of those cells. The NAN, built from peptide cross-linked surfactants, is chemically designed to degrade under inflammation conditions releasing individual DNAzyme-surfactant conjugates in response to proteolytic enzymes. Using the NAN, GATA3 DNAzymes were delivered efficiently to human peripheral blood mononuclear cells, with clear evidence of uptake by CD4+ helper T cells without the need for harsh transfection agents. Knockdown of GATA3 was achieved in vitro using human Jurkat T cells, which express GATA3 under homeostatic conditions. Additionally, mice treated with DNAzyme-NANs during house dust mite (HDM)-induced asthma developed less severe allergic lung inflammation than HDM-only control mice, as measured by pulmonary eosinophilia. This study suggests that peptide cross-linked GATA3 DNAzyme-NANs may have the potential to decrease the severity of asthma symptoms in human patients, and development of this technology for human use warrants further investigation.
Assuntos
Asma , DNA Catalítico , Nanocápsulas , Animais , Asma/genética , Modelos Animais de Doenças , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/uso terapêutico , Humanos , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pyroglyphidae , Células Th2/metabolismoRESUMO
Intracellular trafficking and delivery of nucleic acids is an area of growing interest, particularly as it relates to therapeutic applications. Spectroscopic methods have been used to observe and quantitatively measure the delivery of oligonucleotides both in vitro and in vivo. Herein we demonstrate the use of a new fluorophore labeled surfactant presenting a solvatochromatic chromophore for tracking the assembly and degradation of a hybrid biomaterial we refer to as a nucleic acid nanocapsule (NAN). We show that the surfactant enables critical micelle concentration determination, monitoring of NAN disassembly in vitro, and the ability to track the cellular movement and activity of surfactant-oligonucleotide conjugates in cells when coupled with quantitative PCR analysis.
RESUMO
This work highlights a multifunctional nanoscale material which can effectively compartmentalize small molecules and biomolecules into a single, micellar structure with programmable degradation properties resulting in highly controllable release properties. The nanomaterial consists of a ZIF-8 metal organic framework (MOF) encapsulated within a DNA surfactant micelle assembly, referred to as a nucleic acid nanocapsule (NAN). NANs have been demonstrated to enter cells through endocytosis and result in intracellular cargo release upon enzyme-triggered degradation. By combining the favorable properties of MOFs (large storage capacity) with those of NANs (triggerable release), we show diverse molecular cargo can be integrated into a single, highly programmable nanomaterial with controllable release profiles. The hybrid MOF-NANs exhibit double-gated regulation capabilities as evidenced by kinetic studies of encapsulated enzymes that indicate individual layers of the particle influence the overall enzymatic rate of turnover. The degradation of MOF-NANs can be controlled under multiple combined stimuli (i.e. varying pH, enzymes), enabling selective release profiles in solutions representative of more complex biological systems. Lastly, the enhanced control over the release of small molecules, proteins and plasmids, is evaluated through a combination of cell culture and in vitro fluorescence assays, indicating the potential of MOF-NANs for both therapeutic and diagnostic applications.