RESUMO
Human transferrin protein (Tf) modified polyplexes have already displayed encouraging potential for receptor-mediated nucleic acid delivery into tumors. The use of a blood-derived targeting protein and polydisperse macromolecular cationic subunits however presents a practical challenge for pharmaceutical grade production. Here, Tf receptor (TfR) targeted small interfering RNA (siRNA) polyplexes are designed that are completely composed of synthetic, monodisperse, and sequence-defined subunits generated by solid-phase supported synthesis. An optimized cationizable lipo-oligoaminoamide (lipo-OAA) is used for siRNA core polyplex formation, and a retro-enantio peptide (reTfR) attached via a monodisperse polyethylene glycol (PEG) spacer via click chemistry is applied for targeting. Improved gene silencing is demonstrated in TfR-expressing KB and DU145 cells. Analogous plasmid DNA (pDNA) polyplexes are successfully used for receptor-mediated gene delivery in TfR-rich K562 cells and Neuro2a cells. Six lipo-OAAs differing in their lipidic domain and redox-sensitive attachment of lipid residues are tested in order to evaluate the impact of core polyplex stability on receptor-dependent gene transfer.
Assuntos
Técnicas de Transferência de Genes , Receptores da Transferrina , Inativação Gênica , Humanos , Polietilenoglicóis/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Receptores da Transferrina/genética , Transferrina/química , Transferrina/genéticaRESUMO
When optimizing nanocarriers, structural motifs that are beneficial for the respective type of cargo need to be identified. Here, succinoyl tetraethylene pentamine (Stp)-based lipo-oligoaminoamides (OAAs) were optimized for the delivery of plasmid DNA (pDNA). Structural variations comprised saturated fatty acids with chain lengths between C2 and C18 and terminal cysteines as units promoting nanoparticle stabilization, histidines for endosomal buffering, and disulfide building blocks for redox-sensitive release. Biophysical and tumor cell culture screening established clear-cut relationships between lipo-OAAs and characteristics of the formed pDNA complexes. Based on the optimized alternating Stp-histidine backbones, lipo-OAAs containing fatty acids with chain lengths around C6 to C10 displayed maximum gene transfer with around 500-fold higher gene expression than that of C18 lipo-OAA analogues. Promising lipo-OAAs, however, showed only moderate in vivo efficiency. In vitro testing in 90% full serum, revealing considerable inhibition of lytic and gene-transfer activity, was found as a new screening model predictive for intravenous applications in vivo.
Assuntos
Nanopartículas , Endossomos , Técnicas de Transferência de Genes , Histidina , Plasmídeos , TransfecçãoRESUMO
Carbon nanodots present resistance to photobleaching, bright photoluminescence, and superior biocompatibility, making them highly promising for bioimaging applications. Herein, nanoprobes were caged with four-armed oligomers and subsequently modified with a novel DBCO-PEG-modified retro-enantio peptide ligand reL57, enhancing cellular uptake into U87MG glioma cells highly expressing low-density lipoprotein receptor-related protein 1 (LRP1). A key point in the development of the oligomers was the incorporation of ε-amino-linked lysines instead of standard α-amino-linked lysines, which considerably extended the contour length per monomer. The four-armed oligomer 1696 was identified as the best performer, spanning a contour length of ~8.42 nm for each arm, and was based on an altering motive of two cationic ε-amidated lysine tripeptides and two tyrosine tripeptides for electrostatic and aromatic stabilization of the resulting formulations, cysteines for disulfide-based caging, and N-terminal azidolysines for click-modification. This work highlights that well-designed four-armed oligomers can be used for noncovalent coating and covalent caging of nanoprobes, and click modification using a novel LRP1-directed peptide ligand facilitates delivery into receptor-expressing target cells.
RESUMO
Sodium iodide symporter (NIS) gene transfer for active accumulation of iodide in tumor cells is a powerful theranostic strategy facilitating both diagnostic and therapeutic application of radioiodide. In glioblastoma (GBM), the blood-brain barrier (BBB) presents an additional delivery barrier for nucleic acid nanoparticles. In the present study, we designed dual-targeted NIS plasmid DNA complexes containing targeting ligands for the transferrin receptor (TfR) and the epidermal growth factor receptor (EGFR), thus providing the potential for active transport across the BBB followed by targeting of tumor cells. In vitro 125I transfection studies confirmed TfR- and EGFR-dependent transfection efficiency and NIS-specific iodide uptake of dual-targeted polyplexes. In vivo gene transfer in mice bearing orthotopic U87 GBM xenografts was assessed at 48 h after intravenous polyplex injection by positron emission tomography (PET) imaging using 18F-labeled tetrafluoroborate (TFB) as tracer. The tumoral 18F-TFB uptake of mice treated with dual-targeted polyplexes (0.56% ± 0.08% ID/mL) was significantly higher compared with mice treated with EGFR-mono-targeted (0.33% ± 0.03% ID/mL) or TfR-mono-targeted (0.27% ± 0.04% ID/mL) polyplexes. In therapy studies, application of 131I induced a superior therapeutic effect of the dual-targeted therapy, demonstrated by a significant delay in tumor growth and prolonged survival.
RESUMO
A cationizable sequence-defined lipo-oligoaminoamide (lipo-OAA) conferring stable assembly of siRNA into ~200 nm sized complexes contains an N-terminal azidolysine for covalent coating of formed nanoparticles with dibenzocyclooctyne-amine (DBCO)-modified hyaluronic acid (HA). Depending on the applied equivalents of DBCO-HA, stable nanoparticles with either cationic or anionic surface charge can be formed. The unmodified and two types of covalent HA-modified siRNA nanoparticles differ in their biological characteristics. Both types of HA coated siRNA complexes show an enhanced cellular uptake over uncoated complexes and facilitate efficient gene silencing, but differ in intracellular uptake pathways and distribution. Upon intravenous administration in mice, beyond our expectation and in contrast to the in vitro findings, only the cationic HA nanoparticles but neither the non-coated cationic nor the anionic HA complexes were able to target subcutaneous Huh 7 tumors and exert potent (78%) gene silencing. The favorable and very fast accumulation of cationic HA nanoparticles was confirmed in another subcutaneous tumor model. As evidenced by 3D nanoparticle distribution within Huh 7 tumors evaluated at early time points of 5 min and 45 min, only the cationic HA-based nanoparticles rapidly attach to the tumor endothelium and subsequently penetrate into tumor, in contrast to the analogous anionic HA coated or the cationic non-coated formulation.
Assuntos
Nanopartículas , Animais , Linhagem Celular Tumoral , Endotélio , Inativação Gênica , Ácido Hialurônico , Camundongos , RNA Interferente PequenoRESUMO
Lipo-oligomers, post-functionalized with ligands to enhance targeting, represent promising new vehicles for the tumor-specific delivery of therapeutic genes such as the sodium iodide symporter (NIS). Due to its iodide trapping activity, NIS is a powerful theranostic tool for diagnostic imaging and the application of therapeutic radionuclides. 124I PET imaging allows non-invasive monitoring of the in vivo biodistribution of functional NIS expression, and application of 131I enables cytoreduction. In our experimental design, we used epidermal growth factor receptor (EGFR)-targeted polyplexes (GE11) initially characterized in vitro using 125I uptake assays. Mice bearing an orthotopic glioblastoma were treated subsequently with mono-dibenzocyclooctyne (DBCO)-PEG24-GE11/NIS or bisDBCO-PEG24-GE11/NIS, and 24-48 h later, 124I uptake was assessed by positron emission tomography (PET) imaging. The best-performing polyplex in the imaging studies was then selected for 131I therapy studies. The in vitro studies showed EGFR-dependent and NIS-specific transfection efficiency of the polyplexes. The injection of monoDBCO-PEG24-GE11/NIS polyplexes 48 h before 124I application was characterized to be the optimal regime in the imaging studies and was therefore used for an 131I therapy study, showing a significant decrease in tumor growth and a significant extension of survival in the therapy group. These studies demonstrate the potential of EGFR-targeted polyplex-mediated NIS gene therapy as a new strategy for the therapy of glioblastoma.