Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: 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.

SUBMITTER: Bae Y

PROVIDER: S-EPMC8405440 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

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

Polyplexes of Functional PAMAM Dendrimer/Apoptin Gene Induce Apoptosis of Human Primary Glioma Cells In Vitro.

Project description:Highly efficient and safe gene delivery has become an important aspect of neuronal gene therapy. We evaluated the ability of polyamidoamine (PAMAM) dendrimer grafted with phenylalanine, histidine, and arginine (PAMAM-FHR), a nonviral gene delivery vector, to deliver a therapeutic, tumor cell-specific killer gene, apoptin, into the human primary glioma cell line GBL-14 and human dermal fibroblasts. We performed a transfection assay using plasmids of luciferase and enhanced green fluorescent protein (EGFP) and assessed cell viability. Both cell lines were treated with complexes of PAMAM-FHR and apoptin after which their intracellular uptake and localization were examined by fluorescence-activated cell sorting (FACS)analysis and confocal laser scanning microscopy. Confocal microscopy showed that the PAMAM-FHR escaped from the endo-lysosome into the cytosol. Cell cycle phase distribution analysis, annexin V staining, and a tetramethylrhodamine ethyl ester (TMRE) assay established that apoptin triggered apoptosis in the GBL-14 cell line but not in normal fibroblasts. These results indicated that the PAMAM-FHR/apoptin complex is an effective gene vehicle for cancer therapy in vitro.

| S-EPMC6419211 | biostudies-literature

An Effective Cationic Human Serum Albumin-Based Gene-Delivery Carrier Containing the Nuclear Localization Signal.

Project description:Considerable effort has been devoted to the development of gene carriers over the years. However, toxicity, immunogenicity, and low transfection efficiency are still major barriers. How to overcome these obstacles has become a burning question in gene delivery. In the present study, a simple cationic human serum albumin (CHSA)-based gene-delivery system containing nuclear localization signals (NLSs) was constructed to conquer the limitations. CHSA/NLS/plasmid DNA (pDNA) complexes were prepared and characterized by Hoechst 33258 intercalation, gel retardation assay, morphological analysis, circular dichroism (CD) spectroscopy, particle size, and zeta potential measurements. Results showed that CHSA/NLS/pDNA complexes were able to condense and protect pDNA with high encapsulation efficiency. The complexes displayed a nutritional effect on cells at a low concentration and there was no significant cytotoxicity or immunogenicity. In addition, CHSA/NLS/pDNA complexes exhibited excellent cellular uptake rates and the mechanism was mainly the clathrin or macropinocytosis-dependent endocytosis pathway. Furthermore, CHSA/NLS/pDNA significantly enhanced gene expression efficiency in vitro. More importantly, CHSA/NLS/pDNA complexes showed a desired antitumor effect in vivo, exhibiting the highest inhibition rate (57.3%) and significant upregulation in p53 protein. All these results confirm that CHSA/NLS/pDNA complexes have a bright future as a safe and effective delivery system for gene therapy.

| S-EPMC6920835 | biostudies-literature

A novel disulfide bond-mediated cleavable RGD-modified PAMAM nanocomplex containing nuclear localization signal HMGB1 for enhancing gene transfection efficiency.

Project description:Background:Polyamidoamine (PAMAM) dendrimers modified by polyethylene glycol (PEG) have frequently been investigated as a delivery carrier for gene therapy. However, modification of PAMAM with PEG using covalent linkage significantly reduces the cellular uptake rate and the transfection efficiency. How to conquer these barriers becomes a burning question in gene delivery. Materials and methods:The present study constructed an effective disulfide bond-mediated cleavable RGD modified gene delivery system to overcome the aforementioned limitations. The disulfide bond was introduced between PAMAM dendrimers and PEG chains to realize the cleavage of PEG from the carrier system, whereas the arginine-glycine-aspartate (RGD) peptide was expected to promote the cellular uptake rate. A high mobility group Box 1 (HMGB1) protein containing nuclear localization signal (NLS) was simultaneously introduced to further promote gene expression efficiency. A pDNA/HMGB1/PAMAM-SS-PEG-RGD (DHP) nanocomplex was prepared via electrostatic interaction and characterized. Results:The results showed that DHP generated small particles and was able to condense and protect pDNA against degradation. In addition, the RGD peptide could significantly promote the cellular uptake of a nanocomplex. Intracellular trafficking and in vitro expression study indicated that the DHP nanocomplex escaped from lysosomes and the disulfide bonds between PAMAM and PEG cleaved due to the high concentration of GSH in the cytoplasm, pDNA consequently became exclusively located in the nucleus under the guidance of HMGB1, thereby promoting the red fluorescence protein (RFP) expression. Importantly, an in vivo antitumor activity study demonstrated that the DHP nanocomplex had higher antitumor activity than any other reference preparation. Conclusion:All these results confirm that DHP could be a new strategy for improving the transfection and expression efficiency in gene delivery.

| S-EPMC6228086 | biostudies-literature

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

Project description: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.

| S-EPMC7313639 | 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

Nuclear localization signal peptides induce molecular delivery along microtubules.

Project description:Many essential processes in eukaryotic cells depend on regulated molecular exchange between its two major compartments, the cytoplasm and the nucleus. In general, nuclear import of macromolecular complexes is dependent on specific peptide signals and their recognition by receptors that mediate translocation through the nuclear pores. Here we address the question of how protein products bearing such nuclear localization signals arrive at the nuclear membrane before import, i.e., by simple diffusion or perhaps with assistance of cytoskeletal elements or cytoskeleton-associated motor proteins. Using direct single-particle tracking and detailed statistical analysis, we show that the presence of nuclear localization signals invokes active transport along microtubules in a cell-free Xenopus egg extract. Chemical and antibody inhibition of minus-end directed cytoplasmic dynein blocks this active movement. In the intact cell, where microtubules project radially from the centrosome, such an interaction would effectively deliver nuclear-targeted cargo to the nuclear envelope in preparation for import.

| S-EPMC1366715 | biostudies-literature

Oral absorption enhancement of probucol by PEGylated G5 PAMAM dendrimer modified nanoliposomes.

Project description:Probucol (PB), an antioxidant drug, is commonly used as a lipid concentration lowering drug to reduce blood plasma cholesterol levels in the clinic. However, the therapeutic effects of this drug are negatively impacted by its poor water solubility and low oral absorption efficiency. In this study, a PEGylated G5 PAMAM dendrimer (G5-PEG) modified nanoliposome was employed to increase water solubility, transepithelial transport, and oral absorption of PB. The uptake mechanism was explored in vitro in Caco-2 cells with the results suggesting that the absorption improvement of G5-PEG modified PB-liposome (PB-liposome/G5-PEG) was related to P-glycoprotein (P-gp) efflux pump but was independent of caveolae endocytosis pathways. Additionally, plasma lipid concentration lowering effects of PB-liposome/G5-PEG were evaluated in vivo in a LDLR-/- hyperlipidemia mouse model. Compared with saline treated group, treatment with PB-liposome/G5-PEG significantly inhibited the increase of plasma total cholesterol (TC) and triglyceride (TG) of mice induced by a high fat diet. Moreover, its lipid concentration lowering effects and plasma drug concentration were greater than PB alone or commercial PB tablets. Our results demonstrated that PB-liposome/G5-PEG significantly increased the oral absorption of PB and therefore significantly improved its pharmacodynamic effects.

| S-EPMC4770526 | biostudies-literature

Generation 3 PAMAM dendrimer TAMRA conjugates containing precise dye/dendrimer ratios.

Project description:The synthesis, isolation, and characterization of generation 3 poly(amidoamine) (G3 PAMAM) dendrimer containing precise ratios of 5-carboxytetramethylrhodamine succinimidyl ester (TAMRA) dye (n = 1-3) per polymer particle are reported. Stochastic conjugation of TAMRA dye to the dendrimer was followed by separation into precise dye-polymer ratios using rp-HPLC. The isolated materials were characterized by rp-UPLC, MALDI-TOF-MS, and 1H NMR spectroscopy, UV-vis, and fluorescence spectroscopies.

| S-EPMC4631223 | biostudies-literature

Spermine-modified Antheraea pernyi silk fibroin as a gene delivery carrier.

Project description:The development of a novel cationized polymer used as a gene delivery carrier that can conveniently and effectively transfect cells resulting in a stably expressed target gene remains a challenge. Antheraea pernyi silk fibroin (ASF) is a cytocompatible and biodegradable natural polymer, and it possesses Arg-Gly-Asp sequences but a negative charge. In order to render ASF amenable to packaging plasmid DNA (pDNA), spermine was used to modify ASF to synthesize cationized ASF (CASF), which was used as a gene delivery carrier. CASF was characterized using trinitrobenzene sulfonic acid assay, the zeta potential determination, and a Fourier transform infrared analysis, and the results of these characterizations indicated that the -NH2 in spermine effectively reacts with the -COOH in the side chains of ASF. Spermine grafted to the side chains of ASF resulted in the conversion of the negative charge of ASF to a positive charge. CASF packaged pDNA and formed CASF/pDNA complexes, which exhibited spherical morphology with average particle sizes of 215-281 nm and zeta potential of approximately +3.0 mV to +3.2 mV. The results of the MTT assay, confocal laser scanning microscopy, and flow cytometry analysis in a human endothelial cell line revealed that CASF/pDNA complexes exhibited lower cytotoxicity and higher transfection efficiency compared to the pDNA complexes of polyethyleneimine. These results indicate that our synthesized CASF, a cationized polymer, is a potential gene delivery carrier with the advantages of biodegradability and low cytotoxicity.

| S-EPMC4798211 | 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