Bombesin/oligoarginine fusion peptides for gastrin releasing peptide receptor (GRPR) targeted gene delivery

The development of non-viral gene delivery systems, with the capacity to overcome most of the biological barriers facing gene delivery, is challenging. We have developed peptide-based, multicomponent, non-viral delivery systems, incorporating: a bombesin peptide ligand (BBN(6–14)), to selectively target the gastrin releasing peptide receptor (GRPR); oligoarginine peptides (hexa- (R₆) and nona-arginine (R₉)), for plasmid DNA (pDNA) condensation; and GALA, to facilitate endosome escape. The uptake and endosome escape efficiency of bombesin/oligoarginine and bombesin/oligoarginine/GALA fusion peptides for oligonucleotide delivery was evaluated in terms of their complex size, cellular uptake, endosome escape, and cellular toxicity. Complex size and cell uptake studies demonstrated that the nona-arginine/bombesin delivery system was more efficient at condensing and delivering pDNA into PC-3 prostate cancer cells compared to the hexa-arginine/bombesin delivery system. Further, competition with free bombesin peptide, and comparative uptake studies in Caco-2 cells, which express GRPR at a lower level, suggested that GRPR contributes to the targeted uptake of this system. The addition of GALA into the nona-arginine/bombesin-based system further increased the pDNA cellular uptake at all tested N/P ratios; facilitated endosomal pDNA release; and had limited effects on cell viability. In conclusion, the delivery system combining BBN(6–14) with nona-arginine and GALA had optimal characteristics for the delivery of pDNA into the GRPR overexpressing cell line PC-3.     

Epidermal growth factor-PEG functionalized PAMAM-pentaethylenehexamine dendron for targeted gene delivery produced by click chemistry

Aim of this study was the site-specific conjugation of an epidermal growth factor (EGF)-polyethylene glycol (PEG) chain by click chemistry onto a poly(amido amine) (PAMAM) dendron, as a key step toward defined multifunctional carriers for targeted gene delivery. For this purpose, at first propargyl amine cored PAMAM dendrons with ester ends were synthesized. The chain terminal ester groups were then modified by oligoamines with different secondary amino densities. The oligoamine-modified PAMAM dendrons were well biocompatible, as demonstrated in cytotoxicity assays. Among the different oligoamine-modified dendrons, PAMAM-pentaethylenehexamine (PEHA) dendron polyplexes displayed the best gene transfer ability. Conjugation of PAMAM-PEHA dendron with PEG spacer was conducted via click reaction, which was performed before amidation with PEHA. The resultant PEG-PAMAM-PEHA copolymer was then coupled with EGF ligand. pDNA transfections in HuH-7 hepatocellular carcinoma cells showed a 10-fold higher efficiency with the polyplexes containing conjugated EGF as compared to the ligand-free ones, demonstrating the concept of ligand targeting. Overall gene transfer efficiencies, however, were moderate, suggesting that additional measures for overcoming subsequent intracellular bottlenecks in delivery have to be taken.     

Synthesis and characterization of a head-tail type polycation block copolymer as a nonviral gene vector

A head-tail type polycation block copolymer, which is composed of the polyamidoamine (PAMAM) dendron and poly(L-lysine) (PLL) blocks, was newly designed as a nonviral gene vector in this study. This block copolymer (PAMAM dendron-PLL) was successfully synthesized in two steps: the synthesis of the PAMAM dendron block and the polymerization of the PLL block from the PAMAM dendron block. PAMAM dendron and PLL blocks in block copolymer showed independent deprotonation behavior, and their pK(a) were determined to be 6.8 and 9.0, respectively. The complexation with pDNA was evaluated by gel retardation assay and dye exclusion assay, and both assays indicated that pDNA was selectively complexed with PLL block of block copolymer. Also, the PAMAM dendron-PLL poplyplexes showed 10(2) fold higher transfection efficiency to HeLa cells as that for PLL polyplexes. This might be due to the buffering effect of the PAMAM dendron block. This block copolymer could produce a function share in each block, i.e., tail block complexed with pDNA and head block showed a buffering effect. This molecular design of the head-tail type block copolymer might provide a new approach for realizing in vivo gene therapy.     

Glycopolymer modification on physicochemical and biological properties of poly(L-lysine) for gene delivery

Poly(L-lysine) (PLL) has excellent plasmid DNA (pDNA) condensation capacity. However, the relatively high cytotoxicity and low transfection efficiency limit its application as gene delivery vectors. Here, well-defined glycopolymers are synthesized by reversible addition fragmentation transfer polymerization and grafted onto PLL to improve the gene delivery performance. After glycopolymer modification, PLL shows reduced cytotoxicity. By regulating the glycopolymer length and amino group substitution degree, the glycopolymer modified PLL can condense pDNA with proper strength, protect the condensed pDNA from degradation and release them in time. Transfection with NIH3T3 and HepG2 cells shows that the glycopolymer modified PLL has improved transfection efficiencies. The low cytotoxicity, effective pDNA protection and enhanced transfection efficiencies indicate that glycopolymer modification would be an effective strategy to improve the polycation properties for gene delivery.     

Caesium uptake and toxicity in the cyanobacterium <Emphasis Type="Italic">Nostoc muscorum</Emphasis>

A NH₄⁺ transport-defective mutant and a K⁺ transport-defective mutant of the cyanobacterium Nostoc muscorum were analysed with regard to percentage survival as a function of CsCl toxicity and Cs⁺ uptake activity. Neither survival nor Cs⁺ uptake was affected in either of the two mutants when compared with the wild type. The results indicate that the toxicity of Cs⁺ is determined at more than one cellular site in this organism.     

Low‐generation asymmetric dendrimers exhibit minimal toxicity and effectively complex DNA

Conventional dendrimers are spherical symmetrically branched polymers ending with active surface functional groups. Polyamidoamine (PAMAM) dendrimers have been widely studied as gene delivery vectors and have proven effective at delivering DNA to cells in vitro. However, higher‐generation (G4‐G8) PAMAM dendrimers exhibit toxicity due to their high cationic charge density and this has limited their application in vitro and in vivo. Another limitation arises when attempts are made to functionalize spherical dendrimers as targeting moieties cannot be site‐specifically attached. Therefore, we propose that lower‐generation asymmetric dendrimers, which are likely devoid of toxicity and to which site‐specific attachment of targeting ligands can be achieved, would be a viable alternative to currently available dendrimers. We synthesized and characterized a series of peptide‐based asymmetric dendrimers and compared their toxicity profile and ability to condense DNA to spherical PAMAM G1 dendrimers. We show that asymmetric dendrimers are minimally toxic and condense DNA into stable toroids which have been reported necessary for efficient cell transfection. This paves the way for these systems to be conjugated with targeting ligands for gene delivery in vitro and in vivo. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and photoluminescence study of di‐dendron dendrimers derived from mono‐Boc‐protected ethylenediamine cores

This work is focused on the synthesis and optical properties of cone‐shaped structural feature di‐dendron polyamidoamine dendrimers up to the third generation with mono‐Boc‐protected ethylenediamine (EDA) as a core. Strong UV absorbance spectra and fluorescence spectra from di‐dendron dendrimers with different terminal groups (‐NH₂, ‐COOCH₃) were studied under different conditions by varying experimental parameters such as concentration and pH. The optical density and fluorescence intensities increased when di‐dendron dendrimers generation number increased from 0.5 to 3.0. It was confirmed that the concentration of di‐dendron dendrimers plays an important role in fluorescence intensity. The increase in fluorescence intensity was linear in low concentration regions, but the intensity increased slowly in high concentration regions. The results also showed a rapid increase in fluorescence intensity at low pH. The formation of a fluorescence‐emitting moiety had a close relationship to protonated tertiary amine groups in di‐dendron dendrimers derived from mono‐Boc‐protected EDA cores. Furthermore, the formation of fluorescent chemical species was irreversible. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis of ferrocenyl-bearing dendrimers with a resorcinarene core

A series of ferrocenyl ended dendrons containing π-conjugated systems were obtained using Wittig and Heck reactions. The dendrons were attached to eight functionalized resorcinarenes via Williamson reaction obtaining high molecular weight dendrimers. No electronic communication between metal centers was observed by cyclic voltammetry. All the dendrimers were characterized by ¹H, ¹³C NMR, FTIR, UV-Vis, MALDI-TOF, elemental analyses, and electrochemical studies.     

New class of polymers for the delivery of macromolecular therapeutics

Cationic polymers show promise for the in vitro and in vivo delivery of macromolecular therapeutics. Known cationic polymers, e.g., poly(L)lysine (PLL) and polyethylenimine (PEI), have been employed in native and modified forms for the delivery of plasmid DNA (pDNA) and reveal varying levels of toxicity. Here, we report the preparation of a new class of cationic polymers that are specifically designed to deliver macromolecular therapeutics. Linear, cationic, beta-cyclodextrin (beta-CD)-containing polymers (CD-polymers) are synthesized by copolymerizing difunctionalized beta-CD monomers (AA) with other difunctionalized comonomers (BB) such that an AABBAABB product is formed. The beta-CD polymers are able to bind approximately 5 kbp pDNA above polymer to DNA (+/-) charge ratios of 1.5, compact the bound pDNA into particles of approximately 100-150 nm in size at charge ratios above 5+/-, and transfect cultured cells at charge ratios above 10+/-. In vitro transfections with the new beta-CD-polymers are comparable to the best results obtained in our hands with PEI and Lipofectamine. Some cell line-dependent toxicities are observed for serum-free transfections; however, no toxicity is revealed at charge ratios as high as 70+/- in transfections conducted in 10% serum. Single IV and IP doses as high as 200 mg/kg in mice showed no mortalities.     

Gene delivery into mesenchymal stem cells: a biomimetic approach using RGD nanoclusters based on poly(amidoamine) dendrimers

Poly(amidoamine) dendrimers (generations 5 and 6) with amine termini were conjugated with peptides containing the arginine-glycine-aspartic acid (RGD) sequence having in view their application as gene delivery vectors. The idea behind the work was to take advantage of the cationic nature of dendrimers and of the integrin targeting capabilities of the RGD motif to improve gene delivery. Dendrimers were used as scaffolds for RGD clustering and, by controlling the number of peptides (4, 8, and 16) linked to each dendrimer, it was possible to evaluate the effect of RGD density on the gene delivery process. The new vectors were characterized in respect to their ability to neutralize and compact plasmid DNA (pDNA). The complexes formed by the vectors and pDNA were studied concerning their size, zeta potential, capacity of being internalized by cells and ability of transferring genes. Transfection efficiency was analyzed, first, by using a pDNA encoding for Enhanced Green Fluorescent Protein and Firefly Luciferase and, second, by using a pDNA encoding for Bone Morphogenetic Protein-2. Gene expression in mesenchymal stem cells was enhanced using the new vectors in comparison to native dendrimers and was shown to be dependent on the electrostatic interaction established between the dendrimer moiety and the cell surface, as well as on the RGD density of nanoclusters. The use of dendrimer scaffolds for RGD cluster formation is a new approach that can be extended beyond gene delivery applications, whenever RGD clustering is important for modulating cellular responses.     

Microparticle‐mediated gene delivery for the enhanced expression of a 19‐kDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl‐terminal fragment of merozoite surface protein 1 (MSP1₁₉) is a major component of the invasion‐inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic‐co‐glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP1₁₉ is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h^?1. High levels of gene expression and moderate cytotoxicity in COS‐7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z‐average hydrodynamic diameters of 1.50–2.10 μm and zeta potentials of 17.8–23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA‐mediated microparticles can be employed as potential gene delivery systems to antigen‐presenting cells in the prevention of malaria. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Polyplex-mediated gene transfer and cell cycle: effect of carrier on cellular uptake and intracellular kinetics, and significance of glycosaminoglycans

BACKGROUND: Here we report on studies that probe whether the intracellular kinetics of plasmid DNA (pDNA) and cell surface glycosaminoglycans (GAGs) are modified during the cell cycle in a way that can be correlated with changes in gene transfer efficiency with poly(ethyleneimine) (PEI) and poly-L-lysine (PLL) polyplexes. METHODS: Synchronized D407 retinal cells were transfected with PEI and PLL polyplexes using a luciferase reporter. The free and/or loosely complexed nuclear pDNA was determined by real-time PCR, and compared with transgene expression, the rate of pinocytosis by FITC-dextran uptake and the content of cell surface GAGs. RESULTS: The amount of free and/or loosely complexed nuclear pDNA between cell cycle phases varied approximately 4-20 times (G1 < S < G2/M). Both carriers delivered pDNA in a similar way into the nucleus (PLL vs. PEI < or = 3.5-fold), but PEI was approximately 10-100 times more efficient in gene expression than PLL (G1 < G2/M < S). The rate of pinocytosis increased up to 70-fold from G1 to middle S phase. Cell surface heparan and chondroitin sulfate increased 50-80%, and hyaluronan decreased 50% when the cells went from G1 through S to G2/M. CONCLUSIONS: The data obtained indicates that no single parameter (pinocytosis, cell surface GAGs, nuclear uptake) solely accounts for the differential pDNA uptake or expression during cell cycle, and that the main difference in PLL- and PEI-mediated transfections seems to be at the nuclear level.     

The impact of DNA topology on polyplex uptake and transfection efficiency in mammalian cells

The effect of DNA vector topology when complexed to poly-l-lysine (PLL) and its quantification in transfection efficiency has not been fully addressed even though it is thought to be of importance from both production and regulatory viewpoints. This study investigates and quantifies cell uptake followed by transfection efficiency of PLL:DNA complexes (polyplexes) in Chinese hamster ovary (CHO) cells and their dependence on DNA topology. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Characterization of PLL conjugated to a 6.9 kb plasmid was carried out. Dual labeling of both the plasmid DNA (pDNA) and PLL enabled quantitative tracking of the complexed as well as dissociated elements, within the cell, and their dependence on DNA topology. Polyplex uptake was quantified by confocal microscopy and image analysis. Supercoiled (SC) pDNA when complexed with PLL, forms a polyplex with a mean diameter of 139.06 nm (±0.84% relative standard error [RSE]), whereas open circular (OC) and linear-pDNA counterparts displayed mean diameters of 305.54 (±3.2% RSE) and 841.5 nm (±7.2% RSE) respectively. Complexes containing SC-pDNA were also more resistant to nuclease attack than its topological counterparts. Confocal microscope images reveal how the PLL and DNA remain bound post transfection. Quantification studies revealed that by 1 h post transfection 61% of SC-pDNA polyplexes were identified to be associated with the nucleus, in comparison to OC- (24.3%) and linear-pDNA polyplexes (3.5%) respectively. SC-pDNA polyplexes displayed the greatest transfection efficiency of 41% which dwarfed that of linear-pDNA polyplexes of 18.6%. Collectively these findings emphasize the importance of pDNA topology when complexed with PLL for gene delivery with the SC-form being a key pre-requisite.     

Enhanced Cellular Uptake of G-Rich Oligonucleotides

Abstract

Sequence-dependency of cellular uptake of oligonucleotides into Vero cells has been studied. Cellular uptake of 5′-[³⁵S]-labelled homopolymers decreased in the order (dG)₁₆ >> (dT)₁₆> (dA)₁₆ > (dC)₁₆. The change of two base-pairs (dG → dA) in a dG-rich antisense oligonucleotide with good antiviral activity dramatically decreased cellular uptake and abolished antiviral activity.     

Poly(glycoamidoamine)s for gene delivery: stability of polyplexes and efficacy with cardiomyoblast cells

Polymeric vectors have potential as nucleic acid delivery vehicles for novel gene therapy and oligonucleotide treatments for cardiovascular disease. In this report, poly(glycoamidoamine)s that contain four secondary amines and either two or four hydroxyl units in the repeat unit with D-glucarate (D4), meso-galactarate (G4), D-mannarate (M4), and l-tartarate (T4) stereochemistry have been investigated for their pDNA-binding affinity, DNase protection effect, and polyplex stability in the presence of salt and serum. Also, the luciferase gene delivery and cellular internalization of polyplexes formed with these polymers have been investigated with rat cardiomyoblast [H9c2(2-1)] cells. The results demonstrate that the number of hydroxyl groups and the stereochemistry affect the biological properties. Polymers T4 and G4 have higher pDNA binding affinity, protect pDNA from nuclease degradation, and do not release pDNA in the presence of serum. Polymers D4 and M4 bind pDNA with lower affinity, which allows for some pDNA degradation and release in the presence of serum. Although T4 forms the most stable polyplexes, vector G4 reveals the highest luciferase gene expression in serum-free media and the greatest cellular internalization of fluorescein-labeled pDNA both in serum-free and serum-supplemented media. The results of these studies indicate that the polymer-DNA binding affinity, nuclease protection capability, and polyplex stability are important parameters to facilitate effective pDNA delivery with poly(glycoamidoamine)s in cultured cardiomyoblast cells. The carbohydrate type also plays an important role to increase cellular uptake and gene expression where the polymer with the galactarate stereochemistry (in G4) is found to be the most effective vector for pDNA delivery to cardiomyoblast cells in vitro.     

Optimization of Lipoplex Formulations for Intravenous Gene Delivery

Abstract

A synthetic lipid-based gene delivery system, termed DLS, which meets some requirements to be suitable for systemic administration is under development. The DLS system was designed to account for the combinatory aspect of lipid composition and formulation. Optimized DLS preparation is highly reproducible and stable, exhibit great structural and low mean size homogeneity, and results in high efficacy following intravenous administration. Factors influencing pDNA biodistribution, transgene tissue specific activity, and toxicity are discussed.     

综述: 细胞穿透肽及其结构改造在siRNA传递中的应用

小RNA药物应用于临床的主要技术瓶颈在于如何高效、低毒地将小RNA分子传递到它发挥功能的场所．基于细胞穿透肽在小RNA透皮给药的临床应用中所取得的进展,本文系统评述了近年来细胞穿透肽在小RNA的体内、体外传递方面的研究动态,分析了细胞穿透肽的结构改造对肽/小RNA复合物转染进入细胞发挥功能的影响,展望了细胞穿透肽作为小RNA的体内药物传递载体的发展方向．     

Charge and acidity compensation during proton-sugar symport in Chlorella: The H+-ATPase does not fully compensate for the sugar-coupled proton influx

This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H⁺-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H⁺ and K⁺ fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H⁺ symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H⁺ by the hexoseproton symport and charge compensation by K⁺ loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H⁺-ATPase caused a slow-down of the K⁺ efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H⁺ uptake and K⁺ loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H⁺ fluxes. The quantitative participation of the several phases of H⁺ and K⁺ flow depended on the pH of the medium, the ambient Ca²⁺ concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H⁺-ATPase never fully compensated for H⁺ uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K⁺ efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.Abbreviations DMO dimethyloxazolidinedione - EDTA ethylcnediaminetetraacetic acid - p.c. packed cell volume     

The heterotrimeric G‐protein β subunit,AGB1, plays multiple roles in the Arabidopsis salinity response

Salinity stress includes both osmotic and ionic toxicity. Sodium homeostasis is influenced by Na⁺ uptake and extrusion, vacuolar Na⁺ compartmentation and root to shoot Na⁺ translocation via transpiration. The knockout mutant of the Arabidopsis heterotrimeric G‐protein Gβ subunit, agb1, is hypersensitive to salt, exhibiting a leaf bleaching phenotype. We show that AGB1 is mainly involved in the ionic toxicity component of salinity stress and plays roles in multiple processes of Na⁺ homeostasis. agb1 mutants accumulate more Na⁺ and less K⁺ in both shoots and roots of hydroponically grown plants, as measured by inductively coupled plasma atomic emission spectrometry. agb1 plants have higher root to shoot translocation rates of radiolabelled ²⁴Na⁺ under transpiring conditions, as a result of larger stomatal apertures and increased stomatal conductance. ²⁴Na⁺ tracer experiments also show that ²⁴Na⁺ uptake rates by excised roots of agb1 and wild type are initially equal, but that agb1 has higher net Na⁺ uptake at 90 min, implicating possible involvement of AGB1 in the regulation of Na⁺ efflux. Calcium alleviates the salt hypersensitivity of agb1 by reducing Na⁺ accumulation to below the toxicity threshold. Our results provide new insights into the regulatory pathways underlying plant responses to salinity stress, an important agricultural problem.