RESUMO
Background: While nanoplatform-based cancer theranostics have been researched and investigated for many years, enhancing antitumor efficacy and reducing toxic side effects is still an essential problem. Methods: We exploited nanoparticle coordination between ferric (Fe2+) ions and telomerase-targeting hairpin DNA structures to encapsulate doxorubicin (DOX) and fabricated Fe2+-DNA@DOX nanoparticles (BDDF NPs). This work studied the NIR fluorescence imaging and pharmacokinetic studies targeting the ability and biodistribution of BDDF NPs. In vitro and vivo studies investigated the nano formula's toxicity, imaging, and synergistic therapeutic effects. Results: The enhanced permeability and retention (EPR) effect and tumor targeting resulted in prolonged blood circulation times and high tumor accumulation. Significantly, BDDF NPs could reduce DOX-mediated cardiac toxicity by improving the antioxidation ability of cardiomyocytes based on the different telomerase activities and iron dependency in normal and tumor cells. The synergistic treatment efficacy is enhanced through Fe2+-mediated ferroptosis and the ß-catenin/p53 pathway and improved the tumor inhibition rate. Conclusion: Harpin DNA-based nanoplatforms demonstrated prolonged blood circulation, tumor drug accumulation via telomerase-targeting, and synergistic therapy to improve antitumor drug efficacy. Our work sheds new light on nanomaterials for future synergistic chemotherapy.
Assuntos
Doxorrubicina , Telomerase , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Doxorrubicina/administração & dosagem , Animais , Humanos , Telomerase/metabolismo , Linhagem Celular Tumoral , Camundongos , DNA/química , DNA/farmacocinética , DNA/administração & dosagem , Distribuição Tecidual , Nanopartículas/química , Neoplasias/tratamento farmacológico , Ferroptose/efeitos dos fármacos , Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/administração & dosagem , Camundongos Endogâmicos BALB C , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinéticaRESUMO
This study investigates the potential of iron oxide nanoparticles (Fe3O4) and quince seed mucilage as combined genetic carriers to deliver plasmid DNA (pDNA) through the gastrointestinal system. The samples are characterized by x-ray diffraction (XRD), zeta potential, dynamic light scattering, FT-IR spectroscopy, field emission scanning electron microscopy and vibrating sample magnetometry. The stability of pDNA loading on the nanocarriers and their release pattern are evaluated in simulated gastrointestinal environments by electrophoresis. The XRD patterns reveal that the nanocarriers could preserve their structure during various synthesis levels. The saturation magnetization (Ms) of the Fe3O4cores are 56.48 emu g-1without any magnetic hysteresis. Not only does the loaded pDNA contents experience a remarkable stability in the simulated gastric environment, but also, they could be released up to 99% when exposed to an alkaline environment similar to the intestinal fluid of fish. The results indicate that the synthesized nanoparticles could be employed as efficient low-cost pDNA carriers.
Assuntos
Técnicas de Transferência de Genes , Nanopartículas de Magnetita/química , Sistemas de Liberação de Fármacos por Nanopartículas/química , Mucilagem Vegetal/química , Plasmídeos , DNA/química , DNA/farmacocinética , Tamanho da Partícula , Plasmídeos/química , Plasmídeos/farmacocinética , Rosaceae/química , Sementes/químicaRESUMO
Cyclo-(D-Trp-Tyr) peptide nanotubes (PNTs) were reported to be potential carriers for oral gene delivery in our previous study; however, the effect of the aspect ratio (AR) of these PNTs on gene delivery in vivo could affect penetration or interception in biological environments. The aim of this study was to assess the feasibility of cyclo-(D-Trp-Tyr) PNTs with two ARs as carriers for oral pMBP-bcl-xL-hRluc delivery to the spinal cord to treat spinal cord injury (SCI). We evaluated the biodistribution of oligodendrocyte (OLG)-specific myelin basic protein gene promoter-driven antiapoptotic DNA (pMBP-bcl-xL) to the brain and spinal cord delivered with cyclo-(D-Trp-Tyr) PNTs with large (L) and small (S) PNTs with two ARs. After complex formation, the length, width, and AR of the L-PNTs/DNA were 77.86 ± 3.30, 6.51 ± 0.28, and 13.75 ± 7.29 µm, respectively, and the length and width of the S-PNTs/DNA were 1.17 ± 0.52 and 0.17 ± 0.05 µm, respectively, giving an AR of 7.12 ± 3.17 as detected by scanning electron microscopy. Each of these three parameters exhibited significant differences (p < 0.05) between L-PNTs/DNA and S-PNTs/DNA. However, there were no significant differences (p > 0.05) between the L-PNTs and S-PNTs for either their DNA encapsulation efficiency (29.72 ± 14.19 and 34.31 ± 16.78%, respectively) or loading efficiency (5.15 ± 2.58 and 5.95 ± 2.91%). The results of the in vitro analysis showed that the S-PNT/DNA complexes had a significantly higher DNA release rate and DNA permeation in the duodenum than the L-PNT/DNA complexes. Using Cy5 and TM-rhodamine to individually and chemically conjugate the PNTs with plasmid DNA, we observed, using laser confocal microscopy, that the PNTs and DNA colocalized in complexes. We further confirmed the complexation between DNA and the PNTs using fluorescence resonance energy transfer (FRET). Data from an in vivo imaging system (IVIS) showed that there was no significant difference (p > 0.05) in PNT distribution between L-PNTs/DNA and S-PNTs/DNA within 4 h. However, the S-PNT/DNA group had a significantly higher DNA distribution (p < 0.05) in several organs, including the ilium, heart, lungs, spleen, kidneys, testes, brain, and spinal cord. Finally, we determined the bcl-xL protein expression levels in the brain and spinal cord regions for the L-PNT/DNA and S-PNT/DNA complex formulations. These results suggested that either L-PNTs or S-PNTs may be used as potential carriers for oral gene delivery to treat SCI.
Assuntos
Encéfalo/metabolismo , DNA/farmacocinética , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Nanotubos de Peptídeos/química , Peptídeos Cíclicos/química , Medula Espinal/metabolismo , Proteína bcl-X/metabolismo , Administração Oral , Animais , DNA/administração & dosagem , DNA/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas , Distribuição Tecidual , Proteína bcl-X/administração & dosagem , Proteína bcl-X/genéticaRESUMO
The utilization of the mRNA-based Pfizer-BioNTech and Moderna coronavirus disease 2019 (COVID-19) vaccines represents the culmination of many years of nonviral nucleic acid delivery, but more importantly, they signify a massive clinical scientific success. Scientists working in the area of nucleic acid delivery using lipid nanoparticles will undoubtedly be energized by the success of these vaccines and begin to collect much needed data in the realm of nonviral-based RNA and DNA delivery, specifically, the use of lipid nanoparticles, the immune response, safety, and efficacy. It is easily conceivable that in the future we can utilize these data to help streamline our approach for the delivery of DNA for gene therapy and regulatory RNAs for therapeutic and regenerative medicine (ie, wound repair) applications.
Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , DNA/farmacocinética , Técnicas de Transferência de Genes , RNA Mensageiro/farmacocinética , Vacina de mRNA-1273 contra 2019-nCoV , Vacina BNT162 , Biotecnologia/tendências , COVID-19/imunologia , COVID-19/virologia , Vacinas contra COVID-19/biossíntese , Vacinas contra COVID-19/química , Vacinas contra COVID-19/genética , DNA/química , Mineração de Dados , Dependovirus/genética , Dependovirus/imunologia , Humanos , Lipossomos/química , Lipossomos/farmacocinética , Nanopartículas/administração & dosagem , Nanopartículas/química , RNA Mensageiro/química , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidadeRESUMO
Characterizing in vivo cellular kinetics and biodistribution of chimeric antigen receptor T (CAR-T) cells is critical for toxicity assessment, nonclinical and clinical efficacy studies. To date, the standardized assay to characterize CAR-T cell distribution, expansion, contraction, and persistence profiles is not readily available. To overcome this limitation and increase comparability among studies, we have established a universal protocol for analysis. We established a duplexing ddPCR protocol for the CAR-T transgene and reference gene to normalize the genomic DNA input prepared from mouse blood and tissues. The high-throughput gDNA extraction method enabled highly reproducible gDNA extraction while eliminating labor-intensive steps. The investigational CAR-T cells were intravenously injected into immunodeficient mice bearing human colorectal cancer xenografts. The blood and tissue samples were collected to measure the cellular kinetics by ddPCR and flow cytometry. The standard curves were linear throughout the calibration range with acceptable intra- and inter-day precision and accuracy. The gDNA recovery study performed by spiking in the exo-gene plasmid DNA or CAR-T cells revealed that the recovery ranged from 60 to 100% in blood and tissue homogenates. The use of both units of copy/µg gDNA and copy/µL blood met the current regulatory requirement and allowed for a systematic understanding of CAR-T cell expansion and a direct comparison with the flow cytometry data. A standardized ddPCR assay, including automated gDNA extraction procedures, has been established for evaluating cellular kinetics and biodistribution in CAR-T cell therapies.
Assuntos
Bioensaio/métodos , DNA/farmacocinética , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Antígenos Quiméricos/metabolismo , Animais , Linhagem Celular Tumoral , DNA/isolamento & purificação , Feminino , Citometria de Fluxo , Dosagem de Genes , Humanos , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Reação em Cadeia da Polimerase , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/uso terapêutico , Linfócitos T/metabolismo , Distribuição Tecidual , Transgenes , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This study presents a controlled synthesis of NaYbF4@NaYF4 core-shell upconversion nanoparticles using the hot-injection technique. NaYF4 shells with tunable morphologies including long-rod, short-rod, and quasi-sphere are grown on identical NaYbF4 core nanoparticles by controlled injection of acetate or trifluoroacetate precursors. Mechanistic investigations reveal that anisotropic interfacial strain accounts for the preferential growth of shell layers along the c-axis. However, the strain effect can be offset by the fast injection of shell precursors, leading to nearly isotropic growth of NaYF4 shells over the NaYbF4 core nanoparticles. The core-shell nanoparticles are further modified with DNA molecules and incubated with adenocarcinomic human alveolar basal epithelial cells. Based on a combination of characterizations by flow cytometry and confocal microscopy, favorable cellular uptake and DNA delivery are observed for the quasi-sphere nanoparticles, owing to the high dispersibility and easy membrane wrapping. The method described here could be extended to synthesize other types of functional nanostructures for the study of morphology-dependent properties.
Assuntos
Portadores de Fármacos/química , Fluoretos/química , Nanopartículas/química , Itérbio/química , Ítrio/química , Células A549 , DNA/administração & dosagem , DNA/farmacocinética , Portadores de Fármacos/metabolismo , Fluoretos/metabolismo , Técnicas de Transferência de Genes , Humanos , Nanopartículas/metabolismo , Nanopartículas/ultraestrutura , Nanotecnologia , Itérbio/metabolismo , Ítrio/metabolismoRESUMO
BACKGROUND: AsiDNA, a first-in-class oligonucleotide-mimicking double-stranded DNA breaks, acts as a decoy agonist to DNA damage response in tumour cells. It also activates DNA-dependent protein kinase and poly (adenosine diphosphate [ADP]-ribose) polymerase enzymes that induce phosphorylation of H2AX and protein PARylation. METHODS: The aim of this Phase 1 study was to determine dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), safety and pharmacokinetics/pharmacodynamics of AsiDNA administered daily for 3 days in the first week then weekly thereafter. Twenty-two patients with advanced solid tumours were enrolled in 5 dose levels: 200, 400, 600, 900, and 1300 mg, using a 3 + 3 design. RESULTS: The MTD was not reached. IV AsiDNA was safe. Two DLTs (grade 4 and grade 3 hepatic enzymes increased at 900 and 1300 mg), and two related SAE at 900 mg (grade 3 hypotension and grade 4 hepatic enzymes increased) were reported. AsiDNA PK increased proportionally with dose. A robust activation of DNA-PK by a significant posttreatment increase of γH2AX was evidenced in tumour biopsies. CONCLUSION: The dose of 600 mg was identified as the optimal dose for further clinical development. CLINICAL TRIAL REGISTRATION: Clinical trial registration (NCT number): NCT03579628.
Assuntos
Colesterol/análogos & derivados , DNA/administração & dosagem , DNA/efeitos adversos , DNA/farmacocinética , Neoplasias/tratamento farmacológico , Administração Intravenosa , Adulto , Idoso , Bélgica , Colesterol/administração & dosagem , Colesterol/efeitos adversos , Colesterol/farmacocinética , Reparo do DNA/efeitos dos fármacos , Progressão da Doença , Relação Dose-Resposta a Droga , Feminino , França , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
The immunostimulatory activity of unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) could be improved via delivery to immune cells expressing Toll-like receptor 9 (TLR9). Previously, we showed that the polypod-like structured nucleic acid (polypodna), a nanostructured DNA comprised of three or more ODNs, was an efficient system for the delivery of CpG ODNs to immune cells. Because some TLR9-positive immune cells express mannose receptors (MR), the uptake of polypodna by immune cells can be further increased by its modification with mannose. In this study, we selected the phosphodiester CpG ODN, ODN1668, which has a sequence identical to CpG1668, and a hexapodna, a polypodna with six pods, to design a hexapodna that harbored ODN1668 or the mannosylated CpG ODN (Man-ODN1668) synthesized via modification of the 5'-terminal of ODN1668 with a synthesized mannose motif. By mixing ODN1668 or Man-ODN1668 with the hexapodna, ODN1668/hexapodna and Man-ODN1668/hexapodna were successfully formed with high yields. However, Man-ODN1668/hexapodna was found to induce a greater tumor necrosis factor-α release from TLR9- and MR-positive mouse peritoneal macrophages and macrophage-like J774.1 cells than Man-ODN1668 or ODN1668/hexapodna. These results indicate that the combination of mannose modification and incorporation into nanostructured DNA is a useful approach for enhancing the immunostimulatory activity of CpG ODN.
Assuntos
Adjuvantes Imunológicos/síntese química , DNA/química , Nanoestruturas/química , Oligodesoxirribonucleotídeos/farmacologia , Adjuvantes Imunológicos/farmacologia , Animais , Células Cultivadas , DNA/farmacocinética , Feminino , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/química , Fator de Necrose Tumoral alfa/biossínteseRESUMO
BACKGROUND: Antisense oligonucleotides (ASOs) based technology is considered a potential strategy against antibiotic-resistant bacteria; however, a major obstacle to the application of ASOs is how to deliver them into bacteria effectively. DNA tetrahedra (Td) is an emerging carrier for delivering ASOs into eukaryotes, but there is limited information about Td used for bacteria. In this research, we investigated the uptake features of Td and the impact of linkage modes between ASOs and Td on gene-inhibition efficiency in bacteria. RESULTS: Td was more likely to adhere to bacterial membranes, with moderate ability to penetrate into the bacteria. Strikingly, Td could penetrate into bacteria more effectively with the help of Lipofectamine 2000 (LP2000) at a 0.125 µL/µg ratio to Td, but the same concentration of LP2000 had no apparent effect on linear DNA. Furthermore, linkage modes between ASOs and Td influenced gene-knockdown efficiency. Looped structure of ASOs linked to one side of the Td exhibited better gene-knockdown efficiency than the overhung structure. CONCLUSIONS: This study established an effective antisense delivery system based on loop-armed Td, which opens opportunities for developing antisense antibiotics.
Assuntos
Antibacterianos , DNA , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Oligonucleotídeos Antissenso , Antibacterianos/química , Antibacterianos/farmacocinética , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , DNA/química , DNA/farmacocinética , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lipídeos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacocinéticaRESUMO
Drug delivery depots boosting a local concentration of therapeutic agents have received great attention in clinical applications due to their low occurrence of side effects and high therapeutic efficacy. However, once the payload is exhausted, the local drug concentration will be lower than the therapeutic window. To address this issue, an injectable double-strand deoxyribonucleic acid (DNA)-architected nanoraspberry depot (DNR-depot) was developed that can refill doxorubicin (Dox, an anticancer drug) from the blood and remotely control drug release on demand. The large porous surface on a uniform nanoraspberry (NR) filled covalently with DNA serves as a Dox sponge-like refilling reservoir, and the NR serves as a magnetic electrical absorber. Via the strong affinity between Dox and DNA molecules, the refilling process of Dox can be achieved on DNR-depot both in vitro and in vivo. Upon high-frequency magnetic field (HFMF) treatment, the remotely triggered release of Dox is actuated by the dissociation of Dox and DNA molecules, facilitating an approximately 800% improvement in drug concentration at the tumor site compared to free Dox injection alone. Furthermore, the cycles of refilling and release can be carried out more than 3 times in vivo within 21 days. The combination of refilling and HFMF-programmable Dox release in tumors via DNR-depot can effectively inhibit tumor growth for 30 days.
Assuntos
Antineoplásicos , DNA , Doxorrubicina , Sistemas de Liberação de Medicamentos , Nanoestruturas , Neoplasias Experimentais/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , DNA/química , DNA/farmacocinética , DNA/farmacologia , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Feminino , Campos Magnéticos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanoestruturas/química , Nanoestruturas/uso terapêutico , Neoplasias Experimentais/metabolismo , RatosRESUMO
OBJECTIVES: To provide a new research direction for nerve regeneration and strategy for Alzheimer's disease treatment, tetrahedral DNA nanostructures (TDNs)-novel tetrahedral framework nucleic acid molecule nanoparticles (tFNA) that can inhibit the apoptosis of nerve cells are employed in the experiment. MATERIALS AND METHODS: To verify the successful preparation of TDNs, the morphology of TDNs was observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The expression of apoptosis-related genes and proteins was investigated by confocal microscope, flow cytometry, PCR and Western blot to detect the impact of TDNs on the Alzheimer's model. And finally, Morris water maze experiment was used to test behavioural changes and Nissl stain was detected to observe the morphology and quantity of neurons in the hippocampus. Immunofluorescence stain was used to observe the Aß stain, and TUNEL dyeing was utilized to observe neuronal apoptosis. RESULTS: In vitro and in vivo experiments confirm that TDNs, in a specific concentration range, have no toxic or side effects on nerve cells, can effectively inhibit apoptosis in an Alzheimer's disease cell model and effectively improve memory and learning ability in a rat model of Alzheimer's disease. CONCLUSIONS: These findings suggest that TDNs may be a promising drug for the treatment of Alzheimer's disease.
Assuntos
Doença de Alzheimer/tratamento farmacológico , DNA/uso terapêutico , Nanoestruturas/uso terapêutico , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/análise , Animais , Apoptose/efeitos dos fármacos , DNA/farmacocinética , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Aprendizagem/efeitos dos fármacos , Memória/efeitos dos fármacos , Modelos Moleculares , Nanoestruturas/ultraestrutura , Células PC12 , Ratos , Ratos Sprague-DawleyRESUMO
With the increasing number of identified intracellular drug targets, cytosolic drug delivery has gained much attention. Despite advances in synthetic drug carriers, however, construction of homogeneous and biocompatible nanostructures in a controllable manner still remains a challenge in a translational medicine. Herein, we present the modular design and assembly of functional DNA nanostructures through sequence-specific interactions between zinc-finger proteins (ZnFs) and DNA as a cytosolic drug delivery platform. Three kinds of DNA-binding ZnF domains were genetically fused to various proteins with different biological roles, including targeting moiety, molecular probe, and therapeutic cargo. The engineered ZnFs were employed as distinct functional modules, and incorporated into a designed ZnF-binding sequence of a Y-shaped DNA origami (Y-DNA). The resulting functional Y-DNA nanostructures (FYDN) showed self-assembled superstructures with homogeneous morphology, strong resistance to exonuclease activity and multi-modality. We demonstrated the general utility of our approach by showing efficient cytosolic delivery of PTEN tumour suppressor protein to rescue unregulated kinase signaling in cancer cells with negligible nonspecific cytotoxicity.
Assuntos
Proteínas de Ligação a DNA , DNA , Sistemas de Liberação de Medicamentos , Nanoestruturas , Neoplasias , PTEN Fosfo-Hidrolase , Dedos de Zinco , DNA/química , DNA/farmacocinética , DNA/farmacologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/farmacocinética , Proteínas de Ligação a DNA/farmacologia , Humanos , Células MCF-7 , Nanoestruturas/química , Nanoestruturas/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/farmacocinética , PTEN Fosfo-Hidrolase/farmacologiaRESUMO
Spherical nucleic acids (SNAs) are a class of nanomaterials with a structure defined by a radial distribution of densely packed, short DNA or RNA sequences around a nanoparticle core. This structure allows SNAs to rapidly enter mammalian cells, protects the displayed oligonucleotides from nuclease degradation, and enables co-delivery of other drug cargoes. Here, we investigate the biodistribution of liposomal spherical nucleic acid (LSNA) conjugates, SNA architectures formed from liposome templates and DNA modified with hydrophobic end groups (tails). We compared linear DNA with two types of LSNAs that differ only by the affinity of the modified DNA sequence for the liposome template. We use single-stranded DNA (ssDNA) terminated with either a low-affinity cholesterol tail (CHOL-LSNA) or a high-affinity diacylglycerol lipid tail (DPPE-LSNA). Both LSNA formulations, independent of DNA conjugation, reduce the inflammatory cytokine response to intravenously administered DNA. The difference in the affinity for the liposome template significantly affects DNA biodistribution. DNA from CHOL-LSNAs accumulates in greater amounts in the lungs than DNA from DPPE-LSNAs. In contrast, DNA from DPPE-LSNAs exhibits greater accumulation in the kidneys. Flow cytometry and fluorescence microscopy of tissue sections indicate that different cell populations-immune and nonimmune-sequester the DNA depending upon the chemical makeup of the LSNA. Taken together, these data suggest that the chemical structure of the LSNAs represents an opportunity to direct the biodistribution of nucleic acids to major tissues outside of the liver.
Assuntos
Colesterol/farmacocinética , DNA/farmacocinética , Lipídeos/farmacocinética , Fígado/química , Animais , Colesterol/química , DNA/síntese química , DNA/química , Lipídeos/química , Lipossomos/química , Lipossomos/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Imagem Óptica , Tamanho da Partícula , Propriedades de Superfície , Distribuição TecidualRESUMO
In DNA nanotechnology, the aim in folding DNA origami is to find a good piece of rectangular DNA origami with desired sizes, which could be larger or smaller for different application purposes. In recent three years, the technique of folding smaller ones is paid heavily attentions. In this work, we design a programming rectangular DNA origami in size 20*30nm with M13p18, which is smallest and cheapest to the best acknowledge of the authors. Since it is not hard to prepare with 30 staple strands and short annealing time, the cost of folding our designed rectangular DNA origami is less than 100 dollars. Although the large origami give more space, the smaller ones are cheaper and has the potential applications in penetrating cancer cells. It is obtained by cell penetrating experiments that our designed rectangular DNA origami can penetrate ovarian cancer cells efficiently even loading doxorubicin, but the thermodynamic stability needs further improved. Our designed programming 20 30nm triangular DNA origami shows potential applications in precision control of nanoscale particles and anti-tumor drug delivery in vivo.
Assuntos
DNA/ultraestrutura , Doxorrubicina , Portadores de Fármacos , Nanotecnologia/métodos , Neoplasias Ovarianas/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacocinética , Simulação por Computador , DNA/química , DNA/metabolismo , DNA/farmacocinética , Doxorrubicina/química , Doxorrubicina/farmacocinética , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Portadores de Fármacos/farmacocinética , Feminino , Humanos , Tamanho da PartículaRESUMO
Non-viral gene delivery in its current form is largely dependent upon the ability of a delivery vehicle to protect its cargo in the extracellular environment and release it efficiently inside the target cell. Also a simple delivery system is required to simplify a GMP conform production if a marketing authorization is striven for. This work addresses these problems. We have developed a synthetic polycationic peptide-mimicking amphiphile, namely DiTT4, which shows efficient transfection rates and good biocompatibility without the use of a co-lipid in the formulation. The lipid-nucleic acid complex (lipoplex) was characterized at the structural (electron microscopy), physical (laser Doppler velocimetry and atomic force microscopy) and molecular levels (X-ray scattering). Stability studies of the lipoplexes in the presence of serum and heparin indicated a stable formation capable of protecting the cargo against the extracellular milieu. Hemocompatibility studies (hemolysis, complement activation and erythrocyte aggregation) demonstrated the biocompatibility of the formulation for systemic administration. The transfection efficiency was assessed in vitro using the GFP assay and confocal laser scanning microscopy studies. With the chorioallantoic membrane model, an animal replacement model according to the 3R strategy (replacement, refinement, and reduction), initial in vivo experiments were performed which demonstrate fast and efficient transfection in complex tissues and excellent biocompatibility.
Assuntos
DNA/administração & dosagem , Lipídeos/química , Transfecção/métodos , Células A549 , Animais , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , DNA/química , DNA/farmacocinética , Técnicas de Transferência de Genes , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Teste de Materiais , Microscopia Confocal , PolieletrólitosRESUMO
The delivery of plasmid DNA (pDNA) using polycations has been investigated for several decades; however, obstacles that limit efficient gene delivery still hinder the clinical application of gene therapy. One of the major limiting factors is controlling pDNA binding affinity with polymers to control the complexation and decomplexation of polyplexes. To address this challenge, polycations of α-poly(L-lysine) (APL) and ε-poly(L-lysine) (EPL) were used to prepare variable complexation/decomplexation polyplexes with binding affinities ranging from too tight to too loose and sizes ranging from small to large. APL-EPL/ATP-pDNA polyplexes were also prepared to compare the effects of endosomolytic ATP on complexation/decomplexation and the sizes of polyplexes. The results showed that smaller and tighter polyplexes delivered more pDNA into the cells and into the nucleus than the larger and looser polyplexes. Larger polyplexes exhibited slower cytosolic transport and consequently less nuclear delivery of pDNA than smaller polyplexes. Tighter polyplexes exhibited poor pDNA release in the nucleus, leading to no improvement in transfection efficiency. Thus, polyplexes should maintain a balance between complexation and decomplexation and should have optimal sizes for effective cellular uptake, cytosolic transport, nuclear import, and gene expression. Understanding the effects of complexation/decomplexation and size is important when designing effective polymer-based electrostatic gene carriers.
Assuntos
DNA/química , Plasmídeos/química , Polímeros/química , Transfecção/métodos , Transporte Ativo do Núcleo Celular , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , DNA/genética , DNA/farmacocinética , Liberação Controlada de Fármacos , Células HEK293 , Células Hep G2 , Humanos , Tamanho da Partícula , Plasmídeos/genética , Plasmídeos/farmacocinética , Polilisina/química , Eletricidade EstáticaRESUMO
Recently, many researchers have reported that DNA nanostructures, such as tetrahedral framework nucleic acids (tFNAs), have great potential to be useful tools in clinical and laboratory applications due to their programmable shapes, functional sites, and biological responses. However, finite endocytosis and stability in cells and body fluids compromise the functions of DNA nanostructures as a result of various adverse factors. In this study, we successfully synthesized PEGylated protamine, and tFNAs were adsorbed to it in a proper ratio of nitrogen in protamine to phosphorus in tFNAs (N/P ratio) as the functional complex. Furthermore, we demonstrated that PEGylated protamine-adsorbed tFNAs show a more prominent positive effect on cell viability and proliferation than naked tFNAs do. An increase in endocytosis can be observed in three different tissue-derived cells with the PEG-protamine-tFNA (PPT) complex. The increased endocytic ability is mediated by multiple pathways; moreover, the stimulatory effect of the PPT complex on the endocytic ability is dramatically blocked by the inhibition of the caveola-dependent pathway. Consistently, when tFNAs are stabilized by PEGylated protamine, they often tend to escape from lysosomes and survive for a longer period in biological fluids rather than being rapidly eliminated from the kidneys. The in vitro and in vivo results of our study demonstrate that the PPT complex method is a feasible, potent, and low-cost strategy that improves tFNA biocompatibility, stability, and internalization. This study provides evidence supporting the possibility of implementing PPTs for use in drug delivery, bioimaging, and gene transfection in the future.
Assuntos
DNA , Portadores de Fármacos , Nanopartículas/química , Polietilenoglicóis , Protaminas , Animais , Cavéolas/metabolismo , Linhagem Celular , DNA/química , DNA/farmacocinética , DNA/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Endocitose/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Polietilenoglicóis/farmacologia , Protaminas/química , Protaminas/farmacocinética , Protaminas/farmacologia , RatosRESUMO
It is highly desirable to realize real-time monitoring of the drug delivery/release process in cancer treatment. Herein, a monitorable mitochondria-specific DNAtrain (MitoDNAtrs) was developed for image-guided drug delivery and synergistic cancer therapy. In this system, mitochondria-targeting Cy5.5 dye served as the "locomotive" to guide the DNA "vehicle" selectively accumulating in the cancer cells in a detectable manner. More importantly, Cy5.5 showed reactive oxygen species (ROS) generation ability, which made it a promising adjuvant chemotherapy amplifier for cancer theranostics.
Assuntos
Antineoplásicos/administração & dosagem , Carbocianinas/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Mitocôndrias/efeitos dos fármacos , Carbocianinas/química , DNA/química , DNA/farmacocinética , Doxorrubicina/administração & dosagem , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Sinergismo Farmacológico , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Humanos , Células MCF-7 , Mitocôndrias/metabolismo , Sulfobromoftaleína/farmacologia , Nanomedicina Teranóstica/métodosRESUMO
The microalgal cell wall is a natural barrier that limits the efficiency of gene delivery in algae genetic engineering. Here, we report the role of hard-uptake nanoparticles (huNPs) in microalgae cell electroporation to enhance the delivery of genes in Chlamydomonas reinhardtii. This role can be divided into two categories: (i) a 'transient state' for short-term behavior under confocal visualization and (ii) a 'steady state' for long-term behavior in cell culture. First, the 'transient' role of gene-huNP complexes was investigated after washing for clear confocal imaging to observe the location of huNPs after electroporation. Second, the 'steady-state' role of the gene-huNP complexes was examined after electroporation by transferring cells to a fresh, medium-rich culture environment without washing to obtain a stable cell culture. For selection of the huNPs, we used two types of nanoparticles (NPs, 250 nm and 530 nm) larger than the threshold size of electroporation uptake to avoid unwanted endocytic uptake of NPs. In the transient state, the visualization results indicate that gene-NP (250 nm) complexes were positioned on the cells and helped to deliver more genes than did the 530 nm NPs. In the steady state, the gene-NP (530 nm) complexes helped stably deliver more genes to the cells by precipitation of NPs due to gravity. We believe that these findings illustrate how gene-NP complexes function in microalgae cell electroporation and could help set up a protocol for enhanced microalgae applications associated with NPs such as environmental waste removal and biofuel production.
Assuntos
DNA/farmacocinética , Técnicas de Transferência de Genes , Nanopartículas/química , Sobrevivência Celular/efeitos dos fármacos , Chlamydomonas reinhardtii , DNA/genética , Eletroporação/métodos , Vetores Genéticos/genética , Vetores Genéticos/farmacocinética , Proteínas de Fluorescência Verde/genética , Microalgas , Nanopartículas/toxicidade , Oxazinas/química , Oxazinas/toxicidade , Tamanho da Partícula , Poliestirenos/química , Poliestirenos/toxicidade , RNA Guia de Cinetoplastídeos/genéticaRESUMO
DNA nanostructures hold great potential for drug delivery. However, their specific targeting is often compromised by recognition by scavenger receptors involved in clearance. In our previous study in cell culture, we showed targeting specificity of a 180â¯nm, 4-layer DNA-built nanocarrier called 3DNA coupled with antibodies against intercellular adhesion molecule-1 (ICAM-1), a glycoprotein overexpressed in the lungs in many diseases. Here, we examined the biodistribution of various 3DNA formulations in mice. A formulation consisted of 3DNA whose outer-layer arms were hybridized to secondary antibody-oligonucleotide conjugates. Anchoring IgG on this formulation reduced circulation and kidney accumulation vs. non-anchored IgG, while increasing liver and spleen clearance, as expected for a nanocarrier. Anchoring anti-ICAM changed the biodistribution of this antibody similarly, yet this formulation specifically accumulated in the lungs, the main ICAM-1 target. Since lung targeting was modest (2-fold specificity index over IgG formulation), we pursued a second preparation involving direct hybridization of primary antibody-oligonucleotide conjugates to 3DNA. This formulation had prolonged stability in serum and showed a dramatic increase in lung distribution: the specificity index was 424-fold above a matching IgG formulation, 144-fold more specific than observed for PLGA nanoparticles of similar size, polydispersity, ζ-potential and antibody valency, and its lung accumulation increased with the number of anti-ICAM molecules per particle. Immunohistochemistry showed that anti-ICAM and 3DNA components colocalized in the lungs, specifically associating with endothelial markers, without apparent histological changes. The degree of in vivo targeting for anti-ICAM/3DNA-nanocarriers is unprecedented, for which this platform technology holds great potential to develop future therapeutic applications.