RESUMO
Currently, there is no effective treatment for glioblastoma multiforme (GBM), the most frequent and malignant type of brain tumor. The blood-brain (tumor) barrier (BB(T)B), which is composed of tightly connected endothelial cells and pericytes (with partial vasculature collapse), hampers nanomedicine accumulation in tumor tissues. We aimed to explore the effect of nanomedicine size on passive targeting of GBM. A series of size-tunable poly(ethylene glycol) (PEG)-grafted copolymers (gPEGs) were constructed with hydrodynamic diameters of 8-30 nm. Biodistribution studies using orthotopic brain tumor-bearing mice revealed that gPEG brain tumor accumulation was maximized at 10 nm with â¼14 doseâ¯%/g of tumor, which was 19 times higher than that in the normal brain region and 4.2 times higher than that of 30-nm gPEG. Notably, 10-nm gPEG exhibited substantially higher brain tumor accumulation than 11-nm linear PEG owing to the prolonged blood circulation property of gPEGs, which is derived from a densely PEG-packed structure. 10 nm gPEG exhibited deeper penetration into the brain tumor tissue than the larger gPEGs did (>10 nm). This study demonstrates, for the first time, the great potential of a nanomedicine downsizing strategy for passive GBM targeting.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Polietilenoglicóis , Glioblastoma/patologia , Glioblastoma/metabolismo , Animais , Polietilenoglicóis/química , Camundongos , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Humanos , Barreira Hematoencefálica/metabolismo , Distribuição Tecidual , Linhagem Celular Tumoral , Tamanho da Partícula , Nanopartículas/química , Polímeros/químicaRESUMO
Various cationic polymers are used to deliver polyplex-mediated antisense oligonucleotides (ASOs). However, few studies have investigated the structural determinants of polyplex functionalities in polymers. This study focused on the polymer hydrophobicity. A series of amphiphilic polyaspartamide derivatives possessing various hydrophobic (R) moieties together with cationic diethylenetriamine (DET) moieties in the side chain (PAsp(DET/R)s) were synthesized to optimize the R moieties (or hydrophobicity) for locked nucleic acid (LNA) gapmer ASO delivery. The gene knockdown efficiencies of PAsp(DET/R) polyplexes were plotted against a hydrophobicity parameter, logD7.3, of PAsp(DET/R), revealing that the gene knockdown efficiency was substantially improved by PAsp(DET/R) with logD7.3 higher than -2.4. This was explained by the increased polyplex stability and improved cellular uptake of ASO payloads. After intratracheal administration, the polyplex samples with a higher logD7.3 than -2.4 induced a significantly higher gene knockdown in the lung tissue compared with counterparts with lower hydrophobicity and naked ASO. These results demonstrate that the hydrophobicity of PAsp(DET/R) is crucial for efficient ASO delivery in vitro and in vivo.
Assuntos
Oligonucleotídeos Antissenso , Polímeros , Polímeros/químicaRESUMO
Muscle-targeted drug delivery is a major challenge in nanomedicine. The extravasation of nanomedicines (or nanoparticles) from the bloodstream into muscle tissues is hindered by the continuous endothelium, the so-called blood-muscle barrier. This study aimed to evaluate the optimal size of macromolecular drugs for extravasation (or passive targeting) into muscle tissues. We constructed a size-tunable polymeric delivery platform as a polymeric nanoruler by grafting poly(ethylene glycol)s (PEGs) onto the poly(aspartic acid) (PAsp) backbone. A series of PEG-grafted copolymers (gPEGs) with a narrow size distribution between 11 and 32 nm in hydrodynamic diameter (DH) were prepared by changing the molecular weight of the PEGs. Biodistribution analyses revealed that accumulation amounts of gPEGs in the muscle tissues of normal mice tended to decrease above their size of ~15 nm (or ~11 nm for the heart). The gPEGs accumulated in the skeletal muscles of Duchenne muscular dystrophy model mice (mdx mice) at a 2-3-fold higher level than in the skeletal muscles of normal mice. At the same time, there was a reduced accumulation of gPEGs in the spleen and liver. Intravital confocal laser scanning microscopy and immunohistochemical analysis showed extravasation and locally enhanced accumulation of gPEGs in the skeletal muscle of mdx mice. This study outlined the pivotal role of macromolecular drug size in muscle-targeted drug delivery and demonstrated the enhanced permeability of 11-32 nm-sized macromolecular drugs in mdx mice.
Assuntos
Polietilenoglicóis , Polímeros , Animais , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Polietilenoglicóis/química , Polímeros/metabolismo , Distribuição TecidualRESUMO
Development of efficient delivery vehicles for in vitro transcribed mRNA (IVT mRNA) is currently a major challenge in nanomedicines. For systemic mRNA delivery, we developed a series of cationic amphiphilic polyaspartamide derivatives (PAsp(DET/R)s) carrying various alicyclic (R) moieties with diethylenetriamine (DET) in the side chains to form mRNA-loaded polyplexes bearing stability under physiological conditions and possessing endosomal escape functionality. While the size and ζ-potential of polyplexes were comparable among various PAsp(DET/R)s, the transfection efficiencies of polyplexes were considerably varied due to difference in the R moieties of PAsp(DET/R)s and were described by an octanol-water (or buffer at pH 7.3) distribution coefficient (logD7.3). The critical logD7.3 for the efficient in vitro transfection of mRNA was indicated at -2.7 to -1.8. The polyplexes with logD7.3 > -1.8 elicited the much higher in vitro transfection efficiencies. After systemic administration, the polyplexes with logD7.3 from -1.8 to -1.3 elicited the significant mRNA expression specifically in the lungs. The highest mRNA expression in the lungs was achieved by a polyaspartamide derivative having a cyclohexylethyl group (PAsp(DET/CHE)), which induced more than 10-fold increase in mRNA transfection efficiency compared to commercially available lipid nanoparticles. The higher mRNA expression by polyplexes in the lungs was explained well by the preferential lung accumulation of intact mRNA, as determined by quantitative real-time PCR. Our results demonstrate that PAsp(DET/R)s are a promising synthetic material for the enhanced systemic IVT mRNA delivery.
Assuntos
Lipossomos , Cátions , Nanopartículas , RNA Mensageiro/genética , TransfecçãoRESUMO
Current antisense oligonucleotide (ASO) therapies for the treatment of central nervous system (CNS) disorders are performed through invasive administration, thereby placing a major burden on patients. To alleviate this burden, we herein report systemic ASO delivery to the brain by crossing the blood-brain barrier using glycemic control as an external trigger. Glucose-coated polymeric nanocarriers, which can be bound by glucose transporter-1 expressed on the brain capillary endothelial cells, are designed for stable encapsulation of ASOs, with a particle size of about 45â nm and an adequate glucose-ligand density. The optimized nanocarrier efficiently accumulates in the brain tissue 1â h after intravenous administration and exhibits significant knockdown of a target long non-coding RNA in various brain regions, including the cerebral cortex and hippocampus. These results demonstrate that the glucose-modified polymeric nanocarriers enable noninvasive ASO administration to the brain for the treatment of CNS disorders.
Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Glucose/química , Nanoestruturas/química , Oligonucleotídeos Antissenso/química , Polímeros/química , Animais , Linhagem Celular Tumoral , Portadores de Fármacos/química , Corantes Fluorescentes/química , Humanos , Camundongos , Oligonucleotídeos Antissenso/metabolismo , Tamanho da Partícula , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Rapid and transient expression of in vitro transcribed mRNA (IVT mRNA) in target cells is a current major challenge in genome engineering therapy. To improve mRNA delivery efficiency, a series of amphiphilic polyaspartamide derivatives were synthesized to contain various hydrophobic moieties with cationic diethylenetriamine (DET) moieties in the side chain and systematically compared as mRNA delivery vehicles (or mRNA-loaded polyplexes). The obtained results demonstrated that the side chain structures of polyaspartamide derivatives were critical for the mRNA delivery efficiency of polyplexes. Interestingly, when the mRNA delivery efficiencies (or the luciferase expression levels in cultured cells) were plotted against an octanol-water partition coefficient (log P) as an indicator of hydrophobicity, a log P threshold was clearly observed to obtain high levels of mRNA expression. Indeed, 3.5 orders of magnitude difference in the expression level is observed between -2.45 and -2.31 in log P. This threshold of log P for the mRNA transfection efficiency apparently correlated with those for the polyplex stability and cellular uptake efficiency. Among the polyaspartamide derivatives with log P > -2.31, a polyaspartamide derivative with 11 residues of 2-cyclohexylethyl (CHE) moieties and 15 residues of DET moieties in the side chains elicited the highest mRNA expression in cultured cells. The optimized polyplex further accomplished highly efficient, rapid, and transient IVT mRNA expression in mouse brain after intracerebroventricular and intrathecal injection. Ultimately, the polyplex allowed for the highly efficient target gene deletion via the expression of Streptococcus pyogenes Cas9 nuclease-coding IVT mRNA in the ependymal layer of ventricles in a reporter mouse model. These results demonstrate the utility of log P driven polymer design for in vivo IVT mRNA delivery.
RESUMO
Stabilisation of fragile oligonucleotides, typically small interfering RNA (siRNA), is one of the most critical issues for oligonucleotide therapeutics. Many previous studies encapsulated oligonucleotides into ~100-nm nanoparticles. However, such nanoparticles inevitably accumulate in liver and spleen. Further, some intractable cancers, e.g., tumours in pancreas and brain, have inherent barrier characteristics preventing the penetration of such nanoparticles into tumour microenvironments. Herein, we report an alternative approach to cancer-targeted oligonucleotide delivery using a Y-shaped block catiomer (YBC) with precisely regulated chain length. Notably, the number of positive charges in YBC is adjusted to match that of negative charges in each oligonucleotide strand (i.e., 20). The YBC rendezvouses with a single oligonucleotide in the bloodstream to generate a dynamic ion-pair, termed unit polyion complex (uPIC). Owing to both significant longevity in the bloodstream and appreciably small size (~18 nm), the uPIC efficiently delivers oligonucleotides into pancreatic tumour and brain tumour models, exerting significant antitumour activity.
Assuntos
Antineoplásicos/metabolismo , Neoplasias Encefálicas/terapia , Regulação Neoplásica da Expressão Gênica , Nanoestruturas/química , Oligonucleotídeos/genética , Neoplasias Pancreáticas/terapia , RNA Interferente Pequeno/genética , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Carbocianinas/química , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Portadores de Fármacos/síntese química , Portadores de Fármacos/farmacocinética , Corantes Fluorescentes/química , Humanos , Injeções Intravenosas , Masculino , Camundongos , Nanoestruturas/administração & dosagem , Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismo , Oligonucleotídeos/farmacocinética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Polietilenoglicóis/química , Polilisina/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacocinética , Eletricidade Estática , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase 1 Polo-LikeRESUMO
Crystals of [Pd(tmdt)2] (tmdt = trimethylenetetrathiafulvalenedithiolate) were prepared in order to investigate their physical properties. The electrical resistivity of [Pd(tmdt)2] was measured on single crystals using two-probe methods and showed that the room-temperature conductivity was 100 S·cm(-1). The resistivity behaviors implied that [Pd(tmdt)2] was a semimetal at approximately room temperature and became narrow-gap semiconducting as the temperature was decreased to the lowest temperature. X-ray structural studies on small single crystals of [Pd(tmdt)2] at temperatures of 20-300 K performed using synchrotron radiation at SPring-8 showed no distinct structural change over this temperature region. However, small anomalies were observed at approximately 100 K. Electron spin resonance (ESR) spectra were measured over the temperature range of 2.7-301 K. The ESR intensity increased as the temperature decreased to 100 K and then decreased linearly as the temperature was further decreased to 50 K, where an abrupt decrease in the intensity was observed. To investigate the magnetic state, (1)H nuclear magnetic resonance (NMR) measurements were performed in the temperature range of 2.5-271 K, revealing broadening below 100 K. The NMR relaxation rate gradually increased below 100 K and formed a broad peak at approximately 50 K, followed by a gradual decrease down to the lowest temperature. These results suggest that most of the sample undergoes the antiferromagnetic transition at approximately 50 K with the magnetic ordering temperatures distributed over a wide range up to 100 K. These electric and magnetic properties of [Pd(tmdt)2] are quite different from those of the single-component molecular (semi)metals [Ni(tmdt)2] and [Pt(tmdt)2], which retain their stable metallic states down to extremely low temperatures. The experimental results and the band structure calculations at the density functional theory level showed that [Pd(tmdt)2] may be an antiferromagnetic Mott insulator with a strong electron correlation.