Peptide dendrimer/lipid hybrid systems are efficient DNA transfection reagents: structure--activity relationships highlight the role of charge distribution across dendrimer generations.
ABSTRACT: Efficient DNA delivery into cells is the prerequisite of the genetic manipulation of organisms in molecular and cellular biology as well as, ultimately, in nonviral gene therapy. Current reagents, however, are relatively inefficient, and structure-activity relationships to guide their improvement are hard to come by. We now explore peptide dendrimers as a new type of transfection reagent and provide a quantitative framework for their evaluation. A collection of dendrimers with cationic and hydrophobic amino acid motifs (such as KK, KA, KH, KL, and LL) distributed across three dendrimer generations was synthesized by a solid-phase protocol that provides ready access to dendrimers in milligram quantities. In conjunction with a lipid component (DOTMA/DOPE), the best reagent, G1,2,3-KL ((LysLeu)8(LysLysLeu)4(LysLysLeu)2LysGlySerCys-NH2), improves transfection by 6-10-fold over commercial reagents under their respective optimal conditions. Emerging structure-activity relationships show that dendrimers with cationic and hydrophobic residues distributed in each generation are transfecting most efficiently. The trigenerational dendritic structure has an advantage over a linear analogue worth up to an order of magnitude. The success of placing the decisive cationic charge patterns in inner shells rather than previously on the surface of macromolecules suggests that this class of dendrimers significantly differs from existing transfection reagents. In the future, this platform may be tuned further and coupled to cell-targeting moieties to enhance transfection and cell specificity.
Project description:Cationic dendrimers are intriguing materials that can be used as antibacterial materials; however, they display significant cytotoxicity towards diverse cell lines at high generations or high doses, which limits their applications in biomedical fields. In order to decrease the cytotoxicity, a series of biocompatible hybrid hydrogels based on cationic dendrimers and carboxylated cellulose nanofibrils were easily synthesized by non-covalent self-assembly under physiological conditions without external stimuli. The cationic dendrimers from generation 2 (G2) to generation 4 (G4) based on trimethylolpronane (TMP) and 2,2-bis (methylol)propionic acid (bis-MPA) were synthesized through fluoride promoted esterification chemistry (FPE chemistry). FTIR was used to show the presence of the cationic dendrimers within the hybrid hydrogels, and the distribution of the cationic dendrimers was even verified using elemental analysis of nitrogen content. The hybrid hydrogels formed from G3 and G4 showed 100% killing efficiency towards <i>Escherichia coli</i> (<i>E. coli</i>), <i>Staphylococcus aureus</i> (<i>S. aureus</i>) and <i>Pseudomonas aeruginosa</i> (<i>P. aeruginosa</i>) with bacterial concentrations ranging from 10<sup>5</sup> CFU/mL to 10<sup>7</sup> CFU/mL. Remarkably, the hybrid hydrogels also showed good biocompatibility most probably due to the incorporation of the biocompatible CNFs that slowed down the release of the cationic dendrimers from the hybrid hydrogels, hence showing great promise as an antibacterial material for biomedical applications.
Project description:The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.
Project description:An ideal nucleic-acid transfection system should combine the physical and chemical characteristics of cationic lipids and linear polymers to decrease cytotoxicity and uptake limitations. Previous research described new types of carriers termed amphiphilic dendrimers (ADs), which are based on polyamidoamine dendrimers (PAMAM). These ADs display the cell membrane affinity advantage of lipids and preserve the high affinity for DNA possessed by cationic dendrimers. These lipid/dendrimer hybrids consist of a low-generation, hydrophilic dendron (G2, G1, or G0) bonded to a hydrophobic tail. The G2-18C AD was reported to be an efficient siRNA vector with significant gene silencing. However, shorter tail ADs (G2-15C and G2-13C) and lower generation (G0 and G1) dendrimers failed as transfection carriers. To date, the self-assembly phenomenon of this class of amphiphilic dendrimers has not been molecularly explored using molecular simulation methods. To gain insight into these systems, the present study used coarse-grained molecular dynamics simulations to describe how ADs are able to self-assemble into an aggregate, and, specifically, how tail length and generation play a key role in this event. Finally, explanations are given for the better efficiency of G2/18-C as gene carrier in terms of binding of siRNA. This knowledge could be relevant for the design of novel, safer ADs with well-optimized affinity for siRNA.
Project description:We report the characterization of amphiphilic aminoglycoside conjugates containing luminophores with aggregation-induced emission properties as transfection reagents. These inherently luminescent transfection vectors are capable of binding plasmid DNA through electrostatic interactions; this binding results in an emission "on" signal due to restriction of intramolecular motion of the luminophore core. The luminescent cationic amphiphiles effectively transferred plasmid DNA into mammalian cells (HeLa, HEK 293T), as proven by expression of a red fluorescent protein marker. The morphologies of the aggregates were investigated by microscopy as well as ζ-potential and dynamic light-scattering measurements. The transfection efficiencies using luminescent cationic amphiphiles were similar to that of the gold-standard transfection reagent Lipofectamine® 2000.
Project description:Polyamidoamine (PAMAM) dendrimers have been widely explored as carriers of therapeutics and imaging agents. However, amine-terminated PAMAM dendrimers are rarely utilized in systemic applications due to their cytotoxicity and risk of opsonization, caused by their cationic charges. Such undesirable effects may be mitigated by shielding the PAMAM dendrimer surface with polymers that reduce the charges. However, this shielding may also interfere with the PAMAM dendrimers' ability to interact with target cells, thus reducing the cellular uptake and overall efficacy of the delivery system. Therefore, we propose to use zwitterionic chitosan (ZWC), a new chitosan derivative, which has a unique pH-sensitive charge profile, as an alternative biomaterial to modify the cationic surface of PAMAM dendrimers. A stable electrostatic complex of ZWC and PAMAM dendrimers was formed at pH 7.4, where the PAMAM dendrimer surface was covered with ZWC, as demonstrated by fluorescence spectroscopy and transmission electron microscopy. The presence of ZWC coating protected red blood cells and fibroblast cells from hemolytic and cytotoxic activities of PAMAM dendrimers, respectively. Confocal microscopy showed that the protective effect of ZWC disappeared at low pH as the complex dissociated due to the charge conversion of ZWC, allowing PAMAM dendrimers to enter cells. These results demonstrate that ZWC is able to provide a surface coverage of PAMAM dendrimers in a pH-dependent manner and, thus, enhance the utility of PAMAM dendrimers as a drug carrier to solid tumors with acidifying microenvironments.
Project description:Non-viral transfection reagents are continuously being developed in attempt to replace viral vectors. Among those non-viral vectors, dendrimers have gained increasing interest due to their unique molecular structure and multivalency. However, more improvements are still needed to achieve higher efficacy and lower toxicity. In this study, we have examined 18 peptide dendrimers conjugated to lipophilic moieties, such as fatty acids or hydrophobic amino acids, that were previously explored for siRNA. Reporter cells were employed to investigate the transfection of single strand splice-switching oligonucleotides (ONs) using these peptide dendrimers. Luciferase level changes reflecting efficiency varied with amino acid composition, stereochemistry, and complexation media used. 3rd generation peptide dendrimers with D-amino acid configuration were superior to L-form. Lead formulations with 3rd generation, D-amino acid peptide dendrimers increased the correction level of the delivered ON up to 93-fold over untreated HeLa Luc/705 cells with minimal toxicity. To stabilize the formed complexes, Polyvinyl alcohol 18 (PVA18) polymer was added. Although PVA18 addition increased activity, toxicity when using our best candidates G 2,3KL-(Leu)<sub>4</sub> (D) and G 2,3KL-diPalmitamide (D) was observed. Our findings demonstrate the potential of lipid-conjugated, D-amino acid-containing peptide dendrimers to be utilized as an effective and safe delivery vector for splice-switching ONs.
Project description:<h4>Background</h4>siRNAs hold a great potential for cancer therapy, however, poor stability in body fluids and low cellular uptake limit their use in the clinic. To enhance the bioavailability of siRNAs in tumors, novel, safe, and effective carriers are needed.<h4>Results</h4>Here, we developed cationic solid lipid nanoparticles (cSLNs) to carry siRNAs targeting EphA2 receptor tyrosine kinase (siEphA2), which is overexpressed in many solid tumors including prostate cancer. Using DDAB cationic lipid instead of DOTMA reduced nanoparticle size and enhanced both cellular uptake and gene silencing in prostate cancer cells. DDAB-cSLN showed better cellular uptake efficiency with similar silencing compared to commercial transfection reagent (Dharmafect 2). After verifying the efficacy of siEphA2-loaded nanoparticles, we further evaluated a potential combination with a histone lysine demethylase inhibitor, JIB-04. Silencing EphA2 by siEphA2-loaded DDAB-cSLN did not affect the viability (2D or 3D culture), migration, nor clonogenicity of PC-3 cells alone. However, upon co-administration with JIB-04, there was a decrease in cellular responses. Furthermore, JIB-04 decreased EphA2 expression, and thus, silencing by siEphA2-loaded nanoparticles was further increased with co-treatment.<h4>Conclusions</h4>We have successfully developed a novel siRNA-loaded lipid nanoparticle for targeting EphA2. Moreover, preliminary results of the effects of JIB-04, alone and in combination with siEphA2, on prostate cancer cells and prostate cancer tumor spheroids were presented for the first time. Our delivery system provides high transfection efficiency and shows great promise for targeting other genes and cancer types in further in vitro and in vivo studies.
Project description:Water-soluble and cytocompatible polymers were investigated to enhance a transporting efficiency of biomolecules into cells in vitro. The polymers composed of a 2-methacryloyloxyethyl phosphorylcholine (MPC) unit, a hydrophobic monomer unit, and a cationic monomer unit bearing an amino group were synthesized for complexation with model biomolecules, siRNA. The cationic MPC polymer was shown to interact with both siRNA and the cell membrane and was successively transported siRNA into cells. When introducing 20-50 mol% hydrophobic units into the cationic MPC polymer, transport of siRNA into cells. The MPC units (10-20 mol%) in the cationic MPC polymer were able to impart cytocompatibility, while maintaining interaction with siRNA and the cell membrane. The level of gene suppression of the siRNA/MPC polymer complex was evaluated in vitro and it was as the same level as that of a conventional siRNA transfection reagent, whereas its cytotoxicity was significantly lower. We concluded that these cytocompatible MPC polymers may be promising complexation reagent for introducing biomolecules into cells, with the potential to contribute to future fields of biotechnology, such as in vitro evaluation of gene functionality, and the production of engineered cells with biological functions.
Project description:Despite advancements in the development of high generation cationic-dendrimer systems for delivery of nucleic acid-based therapeutics, commercially available chemical agents suffer from major drawbacks such as cytotoxicity while being laborious and costly to synthesize. To overcome the aforementioned limitations, low-generation cationic peptide asymmetric dendrimers with side arm lipid (cholic and decanoic acid) conjugation were designed, synthesized and systematically screened for their ability to self-assemble into micelles using dynamic light scattering. Cytotoxicity profiling revealed that our entire asymmetric peptide dendrimer library when trialled alone, or as asymmetric dendrimer micelle-nucleic acid complexes, were non-cytotoxic across a broad concentration range. Further, the delivery efficiency of asymmetric peptide dendrimers in H-4-II-E (rat hepatoma), H2K (mdx mouse myoblast), and DAOY (human medulloblastoma) cells demonstrated that cholic acid-conjugated asymmetric dendrimers possess far superior delivery efficiency when compared to the commercial standards, Lipofectamine 2000 or Lipofectin®.
Project description:A family of generation 1, 2, and 3 triazine dendrimers differing in their core flexibility was prepared and evaluated for their ability to accomplish gene transfection. Dendrimers and dendriplexes were analyzed by their physicochemical and biological properties such as condensation of DNA, size, surface charge, morphology of dendriplexes, toxic and hemolytic effects, and ultimately transfection efficiency in L929 and MeWo cells. Flexibility of the backbone was found to play an important role with generation 2 dendrimer displaying higher transfection efficiencies than 25 kDa poly(ethylene imine) or SuperFect at a lower cytotoxicity level. This result is surprising, as PAMAM dendrimers require generations 4 or 5 to become effective transfection reagents. The ability to delineate effects of molecular structure and generation of triazine dendrimers with biological properties provides valuable clues for further modifying this promising class of nonviral delivery system.