Browse
Submit Data
Databases
API
Help

Dataset Information

33 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Duplex of Polyamidoamine Dendrimer/Custom-Designed Nuclear-Localization Sequence Peptide for Enhanced Gene Delivery.

ABSTRACT: Background: Dendrimers are an attractive alternative to viral vectors due to the low cost of production, larger genetic insert-carrying capacity, and added control over immune- and genotoxic complications through versatile functionalization. However, their transfection rates pale in comparison to their viral counterparts, resulting in widespread research efforts in the attempt to improve transfection efficiency. Materials and Methods: In this work, we designed a synthetic diblock nuclear-localization sequence peptide (NLS) (DDDDDDVKRKKKP) and complexed it with polyamidoamine (PAMAM) dendrimer G4 to form a duplex for gene delivery. We conducted transmission electron microscopy, gel mobility shift assay, and intracellular trafficking studies. We also assessed its transfection efficiency for the delivery of a green fluorescent protein-encoding plasmid (pGFP) to NIH3T3 cells. Results: PAMAM dendrimer G4, NLS, and plasmid DNA can form a stable three-part polyplex and gain enhanced entry into the nucleus. We found transfection efficiency, in large part, depends on the ratio of G4:NLS:plasmid. The triplex prepared at the ratio of 1:60:1 for G4:NLS:pGFP has been shown to be more significantly efficient in transfecting cells than the control group (G4/pGFP, 0.5:1). Conclusions: This new diblock NLS peptide can facilely complex with dendrimers to improve dendrimer-based gene transfection. It can also complex with other polycationic polymers to produce more potent nonviral duplex gene delivery vehicles.

SUBMITTER: Cooper RC

PROVIDER: S-EPMC7313639 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Efficient siRNA delivery using a polyamidoamine dendrimer with a modified pentaerythritol core.

Project description:PurposeDelivery of siRNA into cells remains a critical challenge. Our lab has shown a novel polyamidoamine (PAMAM) dendrimer with modified pentaerythritol derivative core (PD dendrimer) to exhibit high plasmid DNA transfection efficiency and low cytotoxicity. Here, we evaluate PD dendrimer as a siRNA carrier.MethodsAgarose gel electrophoresis and AFM were used to confirm formation of generation 5 (G5)-PD dendrimer/siRNA nanoparticles (NPs). G5 PD dendrimer/anti-luciferase siRNA NPs were used to transfect SK Hep-1 cells with stable luciferase expression. Effects of various endocytic pathway inhibitors on uptake of G5 PD dendrimer/siRNA NPs in SK Hep-1 cells were also investigated.ResultsAgarose gel electrophoresis indicated that G5 PD dendrimer and siRNA formed NPs at weight ratios >0.5:1. G5 PD dendrimer showed effective luciferase gene silencing when weight ratio was 3.0:1 and above. Treatment with endocytosis inhibitors showed that clathrin-mediated endocytosis was the main endocytic pathway by which G5-PD dendrimer/siRNA NPs enter the cell.ConclusionsThese results show that the novel G5 PD dendrimer has high siRNA delivery activity and is promising as a delivery agent for its therapeutic application.

| S-EPMC4289905 | biostudies-literature

Apoptin gene delivery by a PAMAM dendrimer modified with a nuclear localization signal peptide as a gene carrier for brain cancer therapy.

Project description:In this study, we aimed to synthesize PAMAMG3 derivatives (PAMAMG3-KRRR and PAMAMG3-HKRRR), using KRRR peptides as a nuclear localization signal and introduced histidine residues into the KRRR-grafted PAMAMG3 for delivering a therapeutic, carcinoma cell-selective apoptosis gene, apoptin into human primary glioma (GBL-14) cells and human dermal fibroblasts. We examined their cytotoxicity and gene expression using luciferase activity and enhanced green fluorescent protein PAMAMG3 derivatives in both cell lines. We treated cells with PAMAMG3 derivative/apoptin complexes and investigated their intracellular distribution using confocal microscopy. The PAMAMG3-KRRR and PAMAMG3-HKRRR dendrimers were found to escape from endolysosomes into the cytosol. The JC-1 assay, glutathione levels, and Annexin V staining results showed that apoptin triggered cell death in GBL-14 cells. Overall, these findings indicated that the PAMAMG3-HKRRR/apoptin complex is a potential candidate for an effective nonviral gene delivery system for brain tumor therapy in vitro.

| S-EPMC8405440 | biostudies-literature

Polyamidoamine Dendrimer Grafted with an Acid-Responsive Charge-Reversal Layer for Improved Gene Delivery.

Project description:We report on a heterogeneous dendrimer (G3-acetal-NH2) derivative possessing an acid-responsive charge-reversal layer. The synthesis of G3-acetal-NH2 starts with a polyamidoamine (PAMAM) dendrimer G3 core and follows the aza-Michael addition with N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide synthesized by us and the thiol-ene click chemistry with cysteamine hydrochloride in sequence. In a weakly acidic environment, the surface of this newly formed dendrimer can turn from amine-terminated to hydroxyl-terminated due to the cleavage of the acetal groups. This charge conversion from 34.3 ± 2.7 to 18.0 ± 0.3 mV in 24 h at pH 5.3 enables its capacity as a gene delivery vehicle. G3-acetal-NH2 with a positively charged surface can condense pMAX GFP plasmid at similar weight ratios as native G4-NH2 (above 2:1), allowing for its protected uptake into cells and endosomal escape. Meanwhile, in the endosome, the drop in vesicle pH cleaves the acetal bond, releasing the genetic payload and limiting its recondensation by the reduction in the dendrimer surface charge. When the vector/plasmid weight ratio was 2:1, G3-acetal-NH2 improved transfection of pMAX GFP plasmid by 5-fold over native G4-NH2 in NIH3T3 cells in terms of GFP protein expression. Taken together, we show that this surface charge conversion performance makes the synthesized heterogeneous dendrimer an improved vehicle for gene transfection.

| S-EPMC7583228 | biostudies-literature

Second-Generation Polyamidoamine Dendrimer Conjugated with Oligopeptides Can Enhance Plasmid DNA Delivery In Vitro.

Project description:Poly(amidoamine) (PAMAM) dendrimers have attracted considerable attention in the field of gene therapy due to their flexibility in introducing different functional moieties and reduced toxicity at low generations. However, their transfection efficiency remains a limitation. Therefore, an essential approach for improving their transfection efficiency as gene carriers involves modifying the structure of PAMAM by conjugating functional groups around their surface. In this study, we successfully conjugated an RRHRH oligopeptide to the surface of PAMAM generation 2 (PAMAM G2) to create RRHRH-PAMAM G2. This construction aims to condense plasmid DNA (pDNA) and facilitate its penetration into cell membranes, leading to its promising potential for gene therapy. RRHRH-PAMAM G2/pDNA complexes were smaller than 100 nm and positively charged. Nano-polyplexes can enter the cell and show a high transfection efficiency after 24 h of transfection. The RRHRH-PAMAM G2 was non-toxic to HeLa, NIH3T3, A549, and MDA-MB-231 cell lines. These results strongly suggest that RRHRH-PAMAM G2 holds promise as a gene carrier for gene therapy owing to its biocompatibility and ability to deliver genes to the cell.

| S-EPMC10674462 | biostudies-literature

A Mixed-Surface Polyamidoamine Dendrimer for In Vitro and In Vivo Delivery of Large Plasmids.

Project description:Drug delivery to the brain is highly hindered by the presence of the blood-brain barrier (BBB), which prevents the entry of many potential drugs/biomolecules into the brain. One of the current strategies to achieve gene therapy for neurodegenerative diseases involves direct injection of a viral vector into the brain. There are various disadvantages of viral vectors, including limitations of cargo size and safety concerns. Nanomolecules, such as dendrimers, serve as an excellent alternative to viral delivery. In this study, as proof-of-concept, we used a surface-modified dendrimer complex and delivered large plasmids to cells in vitro and in vivo in healthy rats via intracranial injection. The dendrimers were biodegradable by chemicals found within cells and toxicity assays revealed that the modified dendrimers were much less toxic than unmodified amine-surface dendrimers. As mentioned in our previous publication, these dendrimers with appropriately modified surfaces are safe, can deliver large plasmids to the brain, and can overcome the cargo size limitations associated with viral vectors. The biocompatibility of this dendritic nanomolecule and the ability to finely tune its surface chemistry provides a gene delivery system that could facilitate future in vivo cellular reprograming and other gene therapies.

| S-EPMC7407876 | biostudies-literature

Click synthesis of a polyamidoamine dendrimer-based camptothecin prodrug.

Project description:In the present work we report on the click synthesis of a new camptothecin (CPT) prodrug based on anionic polyamidoamine (PAMAM) dendrimer intended for cancer therapy. We applied 'click' chemistry to improve polymer-drug coupling reaction efficiency. Specifically, CPT was functionalized with a spacer, 1-azido-3,6,9,12,15-pentaoxaoctadecan-18-oic acid (APO), via EDC/DMAP coupling reaction. In parallel, propargylamine (PPA) and methoxypoly(ethylene glycol) amine were conjugated to PAMAM dendrimer G4.5 in sequence using an effective coupling agent 4-(4,6-dimethoxy-(1,3,5)triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM). CPT-APO was then coupled to PEGylated PAMAM dendrimer G4.5-PPA via a click reaction using copper bromide/2,2'-bipyridine/ dimethyl sulfoxide (catalyst/ligand/solvent). Human glioma cells were exposed to the CPT-conjugate to determine toxicity and cell cycle effects using WST-1 assay and flow cytometry. The CPT-conjugate displayed a dose-dependent toxicity with an IC50 of 5 ?M, a 185-fold increase relative to free CPT, presumably as a result of slow release. As expected, conjugated CPT resulted in G2/M arrest and cell death while the dendrimer itself had little to no toxicity. Altogether, highly efficient click chemistry allows for the synthesis of multifunctional dendrimers for sustained drug delivery.

| S-EPMC4669072 | biostudies-literature

Apoptin Gene Delivery by the Functionalized Polyamidoamine (PAMAM) Dendrimer Modified with Ornithine Induces Cell Death of HepG2 Cells.

Project description:The use of tumor-specific therapeutic agents is a promising option for efficient and safe nonviral gene transfer in gene therapy. In this study, we describe the efficacy of polyamidoamine (PAMAM)-based nonviral gene delivery carriers, namely, an ornithine conjugated PAMAM (PAMAM-O) dendrimer in delivering apoptin, a tumor-specific killer gene, into human hepatocellular carcinoma (HepG2 cells) and dermal fibroblasts. We analyzed the transfection efficiency by the luciferase assay and assessed cell viability in both cell types. The transfection efficiency of the PAMAM-O dendrimer was found to be higher than that of the PAMAM dendrimer. Moreover, the cytotoxicity of the PAMAM-O dendrimer was very low. We treated both cell types with a polyplex of PAMAM-O dendrimer with apoptin, and analyzed its cellular uptake and localization by confocal microscopy. Cell cycle distribution, tetramethylrhodamine, ethyl ester (TMRE) analysis, and transmission electron microscopy imaging showed that apoptin induced cell death in HepG2 cells. We therefore demonstrated that a PAMAM-O/apoptin polyplex can be used as an effective therapeutic strategy in cancer owing to its effectiveness as a suitable nonviral gene vector for gene therapy.

| S-EPMC6432117 | biostudies-literature

Dendrimer probes for enhanced photostability and localization in fluorescence imaging.

Project description:Recent advances in fluorescence microscopy have enabled high-resolution imaging and tracking of single proteins and biomolecules in cells. To achieve high spatial resolutions in the nanometer range, bright and photostable fluorescent probes are critically required. From this view, there is a strong need for development of advanced fluorescent probes with molecular-scale dimensions for fluorescence imaging. Polymer-based dendrimer nanoconjugates hold strong potential to serve as versatile fluorescent probes due to an intrinsic capacity for tailored spectral properties such as brightness and emission wavelength. In this work, we report a new, to our knowledge, class of molecular probes based on dye-conjugated dendrimers for fluorescence imaging and single-molecule fluorescence microscopy. We engineered fluorescent dendritic nanoprobes (FDNs) to contain multiple organic dyes and reactive groups for target-specific biomolecule labeling. The photophysical properties of dye-conjugated FDNs (Cy5-FDNs and Cy3-FDNs) were characterized using single-molecule fluorescence microscopy, which revealed greatly enhanced photostability, increased probe brightness, and improved localization precision in high-resolution fluorescence imaging compared to single organic dyes. As proof-of-principle demonstration, Cy5-FDNs were used to assay single-molecule nucleic acid hybridization and for immunofluorescence imaging of microtubules in cytoskeletal networks. In addition, Cy5-FDNs were used as reporter probes in a single-molecule protein pull-down assay to characterize antibody binding and target protein capture. In all cases, the photophysical properties of FDNs resulted in enhanced fluorescence imaging via improved brightness and/or photostability.

| S-EPMC3617423 | biostudies-literature

Peptide-Functionalized Dendrimer Nanocarriers for Targeted Microdystrophin Gene Delivery.

Project description:Gene therapy is a good alternative for determined congenital disorders; however, there are numerous limitations for gene delivery in vivo including targeted cellular uptake, intracellular trafficking, and transport through the nuclear membrane. Here, a modified G5 polyamidoamine (G5 PAMAM) dendrimer-DNA complex was developed, which will allow cell-specific targeting to skeletal muscle cells and transport the DNA through the intracellular machinery and the nuclear membrane. The G5 PAMAM nanocarrier was modified with a skeletal muscle-targeting peptide (SMTP), a DLC8-binding peptide (DBP) for intracellular transport, and a nuclear localization signaling peptide (NLS) for nuclear uptake, and polyplexed with plasmid DNA containing the GFP-tagged microdystrophin (µDys) gene. The delivery of µDys has been considered as a therapeutic modality for patients suffering from a debilitating Duchenne muscular dystrophy (DMD) disorder. The nanocarrier-peptide-DNA polyplexes were prepared with different charge ratios and characterized for stability, size, surface charge, and cytotoxicity. Using the optimized nanocarrier polyplexes, the transfection efficiency in vitro was determined by demonstrating the expression of the GFP and the µDys protein using fluorescence and Western blotting studies, respectively. Protein expression in vivo was determined by injecting an optimal nanocarrier polyplex formulation to Duchenne model mice, mdx4Cv. Ultimately, these nanocarrier polyplexes will allow targeted delivery of the microdystrophin gene to skeletal muscle cells and result in improved muscle function in Duchenne muscular dystrophy patients.

| S-EPMC8708248 | biostudies-literature

Enhanced Delivery of Plasmid DNA to Skeletal Muscle Cells using a DLC8-Binding Peptide and ASSLNIA-Modified PAMAM Dendrimer.

Project description:Skeletal muscle is ideally suited and highly desirable as a target for therapeutic gene delivery because of its abundance, high vascularization, and high levels of protein expression. However, efficient gene delivery to skeletal muscle remains a current challenge. Besides the major obstacle of cell-specific targeting, efficient intracellular trafficking, or the cytosolic transport of DNA to the nucleus, must be demonstrated. To overcome the challenge of cell-specific targeting, herein we develop a generation 5-polyamidoamine dendrimer (G5-PAMAM) functionalized with a skeletal muscle-targeted peptide, ASSLNIA (G5-SMTP). Specifically, to demonstrate the feasibility of our approach, we prepared a complex of our G5-SMTP dendrimer with a plasmid encoding firefly luciferase and investigated its delivery to skeletal muscle cells. Luciferase assays indicated a threefold increase in transfection efficiency of C2C12 murine skeletal muscle cells using G5-SMTP when compared with nontargeting nanocarriers using unmodified G5. To further improve the transfection yield, we employed a cationic dynein light chain 8 protein (DLC8)-binding peptide (DBP) containing an internal sequence known to bind to the DLC8 of the dynein motor protein complex. Complexation of DBP with our targeting nanocarrier, that is, G5-SMTP, and our luciferase plasmid cargo resulted in a functional nanocarrier that showed an additional sixfold increase in transfection efficiency compared with G5-SMTP transfection alone. To our knowledge, this is the first successful use of two different functional nanocarrier components that enable targeted skeletal muscle cell recognition and increased efficiency of intracellular trafficking to synergistically enhance gene delivery to skeletal muscle cells. This strategy of targeting and trafficking can also be universally applied to any cell/tissue type for which a recognition domain exists.

| S-EPMC8008946 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data