Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.     

An Experimental Investigation of the Effect of Bacillus megaterium on Apatite Dissolution

Field observations suggest that some mineral dissolution rates can be enhanced by microbial activity indirectly, without direct contact with the mineral surface. A series of apatite dissolution experiments was performed to better understand this rate enhancement process. Far-from equilibrium abiotic apatite dissolution rates, measured in mixed-flow reactors at 25°C were enhanced by increasing concentration of aqueous organic acids and decreasing aqueous phosphate activity, demonstrating the existence of indirect pathways for microbial rate enhancement. Further apatite dissolution experiments were performed in closed-system reactors in the presence of Bacillus megaterium , a common heterotrophic aerobe. Experiments were designed to allow the bacteria to be either in direct contact or indirect contact with the apatite; in the latter case, the microbes were physically separated from the apatite using dialysis bags. Apatite dissolution in indirect contact with Bacillus megaterium was 50 to 900% faster than abiotic controls. Bacterial rate enhancement was, however, 3 to over 10 times lower when Bacillus megaterium was in direct contract versus indirect contact with the apatite surfaces. These results show that (1) bacteria can accelerate rates without being in physical contact with the dissolving mineral, and (2) microbially mediated dissolution may be less effective when bacteria are in direct contact with mineral surfaces. Supression of mineral dissolution is interpreted to stem from the preferential colonization of reactive sites on the mineral surface.     

Disrupting actin filaments promotes efficient transfection of a leukemia cell line using cell adhesive protein-embedded carbonate apatite particles

Tumor cells such as leukemia and lymphoma cells are obvious and attractive targets for gene therapy. Gene transfer and expression for cytokine and immunomodulatory molecules in various kinds of tumor cells have been shown to mediate tumor regression and antimetastatic effects. Moreover, genetically modified leukemia cells expressing costimulatory molecules or cytokines are likely to have significant therapeutic roles for patients with leukemia. One of the major hurdles to the successful implementation of these promising approaches is the lack of a suitable nanocarrier for transgene delivery and expression in a safe and effective manner. Recently, we reported on the development of a safe, efficient nanocarrier system of carbonate apatite that can assist both intracellular delivery and release of DNA, leading to very high level of transgene expression in cancer and primary cells. However, its efficiency in human lymphocytes is poor. We show here that nanocrystals of carbonate apatite, when electrostatically associated with fibronectin and/or E-cadherin-Fc, accelerated transgene delivery in a human T leukemia cell line (Jurkat). Moreover, transgene expression efficiency could be enhanced dramatically with the cell adhesive protein-embedded particles finally up to 150 times by selectively disrupting the actin filaments.     

Influences of electrolytes and glucose on formulation of carbonate apatite nanocrystals for efficient gene delivery to mammalian cells

Genetic manipulation of human cells through delivery of a functional gene or a gene-silencing element is an attractive approach to treat critical diseases very precisely and effectively. Extensive research on the genetic basis of human diseases with complete sequencing of human genome has revealed many vital genes as possible targets in gene therapy programs. On the other hand, to facilitate cell- or tissue-directed delivery of genes and gene-silencing nucleic acid sequences, both genetic and chemical engineering approaches have led to the generation of various viral and nonviral carriers. However, considering the issues of both safety and efficacy, none of the existing vectors is an ideal candidate for clinical use. We recently established pH-sensitive inorganic nanocrystals of carbonate apatite with capability of efficient intracellular delivery and release of associated DNA molecules for subsequent protein expression. Here we show a new synthetic approach for carbonate apatite crystals with stronger affinity toward DNA, leading to significant increment in both transgene delivery and expression. Moreover, CaCl₂ and NaCl, existing as the major electrolytes in the bicarbonate-buffered solution, dose-dependently govern particle size and eventually internalization and expression of particle-associated DNA.     

Geochemical environments of continental shelf-upper slope sediments in the northern Gulf of Mexico

Geochemical environments were characterized for 14 sites along the northern Gulf of Mexico continental shelf and upper slope, in an effort to examine the relationship between sediment geochemistry and carbonate shell taphonomy in a long-term study—Shelf and Slope Experimental Taphonomy Initiative (SSETI). Three groups of environments of preservation (seep, near-seep, and shelf-and-slope) were identified based on their geochemical characteristics (i.e., oxygen uptake rate and penetration depth, pore-water saturation states, and carbonate dissolution fluxes). Diffusive oxygen uptake rate increased in the order of shelf-and-slope, near-seep, and seep, although carbonate dissolution flux did not show significant correlation with O₂ flux, presumably due to non-diffusive behavior at some sites. Using pore-water saturation indices with respect to aragonite and calcite and sedimentation rates, we defined a semi-quantitative parameter, carbonate dissolution index (CDI), to predict carbonate preservation potential during the taphonomic processes. Our limited database suggests that both the seep and the shelf-and-slope sediments may have higher carbonate preservation potential than the near-seep sediments.     

Fluoride and carbonate incorporation into hydroxyapatite under condition of cyclic pH variation

Fluoride and carbonate ions, which are present in the inorganic phase of bone, enamel, and dentine, are known to play an important and opposite role in the process of recrystallization. We have investigated the incorporation of fluoride and carbonate ions into hydroxyapatite structure under conditions of cyclic pH fluctuation and the effect of these incorporations on the conversion of hydroxyapatite into β-tricalcium phosphate after heat treatment. Fluoro-hydroxyapatite has been obtained as unique crystalline phase up to 20 fluoride at. %. The degree of substitution of fluoride for hydroxyl does not depend on the extent of carbonate incorporated into the apatite structure. On the other hand, the carbonate incorporation into the apatite structure seems to be hindered by the contemporary presence of fluoride. Both fluoride and carbonate exhibit a stabilizing effect on the apatite structure, as far as its conversion into β-tricalcium phosphate is concerned. The order of efficiency in stabilizing the apatite structure is F⁻ > F⁻ + CO₃²⁻ > C0₃²⁻.     

Calcite and dolomite dissolution rates in the context of microbe–mineral surface interactions

Although microbes have been shown to alter the dissolution rate of carbonate minerals, a mechanistic understanding of the consequences of microbial surface colonization on carbonate dissolution has yet to be achieved. Here we report the use of vertical scanning interferometry (VSI) to study the effect of Shewanella oneidensis MR‐1 surface colonization on the dissolution rates of calcite (CaCO₃) and dolomite (CaMg(CO₃)₂) through qualitative analysis of etch pit development and quantitative measurements of surface‐normal dissolution rates. By quantifying and comparing the significant processes occurring at the microbe–mineral interface, the dominant mechanism of mineral dissolution during surface colonization was determined. MR‐1 attachment under aerobic conditions was found to influence carbonate dissolution through two distinct mechanistic pathways: (1) inhibition of carbonate dissolution through interference with etch pit development and (2) excavation of carbonate material at the cell–mineral interface during irreversible attachment to the mineral surface. The relative importance of these two competing effects was found to vary with the solubility of the carbonate mineral studied. For the faster‐dissolving calcite substrates, inhibition of dissolution by attachment and subsequent extracellular polysaccharide (EPS) production was the dominant effect associated with MR‐1 surface colonization. This interference with etch pit development resulted in a 40–70% decrease in the surface normal dissolution rate relative to cell‐free controls, depending primarily on the concentration of cells in solution. However, in the case of the slower‐dissolving dolomite substrates, carbonate material displaced during the entrenchment of cells on the surface far outweighed the abiotic dissolution rate. Therefore, during the initial stages of surface colonization, dolomite dissolution rates were actually enhanced by MR‐1 attachment. This study demonstrates the dynamic and competitive relationship between microbial surface colonization and mineral dissolution that may be expected to occur in natural environments.     

Effect of Microorganisms and Microbial Metabolites on Apatite Dissolution

The dissolution rate of apatite was determined in batch reactors in organic acid solutions and in microbial cultures. Inoculum for the cultures was from biotite plus apatite crystals from a granite weathering profile in South Eastern Australia. In both the biotic and the abiotic experiments, etching of the apatite surface leads to the formation of elongated spires parallel to the c axis. Apatite dissolution rates in the inorganic, acetate, and oxalate solutions increase as pH decreases from approximately 10 -11 mol/m -2 · s -1 at initial pH 5.5 to 10 -7 mol/m -2 · s -1 at initial pH 2. Under mildly acidic to near neutral pH conditions, both oxalate and acetate increased apatite dissolution by up to an order of magnitude compared to the inorganic conditions. Acetate catalyzed the reaction by forming complexes with Ca, either in solution or at the mineral surfaces. Oxalate forms complexes with Ca as well, and can also affect reaction rates and stoichiometry by forming Ca-oxalate precipitates, thus affecting solution saturation states. In all abiotic experiments, net phosphate release to solution approaches zero even when solutions are apparently undersaturated by several orders of magnitude with respect to the solubility of an ideal fluoroapatite mineral. In the microbial experiments, two enrichment cultures increased both apatite and biotite dissolution by producing organic acids, primarily pyruvate, fermentation products, and oxalate, and by lowering bulk solution pH to between 3 and 5. However, the microorganisms were also able to increase phosphate release from apatite (by two orders of magnitude) without lowering bulk solution pH by producing pyruvate and other compounds.     

High performance DNA nano-carriers of carbonate apatite: multiple factors in regulation of particle synthesis and transfection efficiency

Increasing attention is being paid on synthetic DNA delivery systems considering some potential life-threatening effects of viral particles, for development of gene-based nanomedicine in the 21st century. In the current nonviral approaches, most of the efforts have been engaged with organic macromolecules like lipids, polymers, and peptides, but comparatively fewer attempts were made to evaluate the potential of inorganic materials for gene delivery. We recently reported that biodegradable nanoparticles of carbonate apatite are highly efficient in transfecting a wide variety of mammalian cells. Here we show that a number of parameters actively regulate synthesis of the nanoparticles and their subsequent transfection efficacy. Development of "supersaturation", which is the prerequisite for generation of such particles, could be easily modulated by reactant concentrations, pH of the buffered solution, and incubation temperatures, enabling us to establish a flexible particle generation process for highly productive trans-gene delivery. Carbonate incorporation into the particles have been proposed for generating nano-size particles resulting in cellular uptake of huge amount of plasmid DNA as well as endosome destabilization facilitating significant release of DNA from the endosomes.     

A bio-recognition device developed onto nano-crystals of carbonate apatite for cell-targeted gene delivery

The DNA delivery to mammalian cells is an essential tool for analyzing gene structure, regulation, and function. The approach holds great promise for the further development of gene therapy techniques and DNA vaccination strategies to treat and control diseases. Here, we report on the establishment of a cell-specific gene delivery and expression system by physical adsorption of a cell-recognition molecule on the nano-crystal surface of carbonate apatite. As a model, DNA/nano-particles were successfully coated with asialofetuin to facilitate uptake by hepatocyte-derived cell lines through the asialoglycoprotein receptor (ASGPr) and albumin to prevent non-specific interactions of the particles with cell-surface. The resulting composite particles with dual surface properties could accelerate DNA uptake and enhance expression to a notable extent. Nano-particles coated with transferrin in the same manner dramatically enhanced transgene expression in the corresponding receptor-bearing cells and thus our newly developed strategy represents a universal phenomenon for anchoring a bio-recognition macromolecule on the apatite crystal surface for targeted gene delivery, having immediate applications in basic research laboratories and great promise for gene therapy.     

Nuclear accumulation of plasmid DNA can be enhanced by non-selective gating of the nuclear pore

One of the major obstacles in non-viral gene transfer is the nuclear membrane. Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-β have achieved limited success. It has been proposed that the nuclear pore complexes (NPCs) through which nucleocytoplasmic transport occurs are filled with a hydrophobic phase through which hydrophobic importins can dissolve. Therefore, considering the hydrophobic nature of the NPC channel, we evaluated whether a non-selective gating of nuclear pores by trans-cyclohexane-1,2-diol (TCHD), an amphipathic alcohol that reversibly collapses the permeability barrier of the NPCs, could be obtained and used as an alternative method to facilitate nuclear entry of plasmid DNA. Our data demonstrate for the first time that TCHD makes the nucleus permeable for both high molecular weight dextrans and plasmid DNA (pDNA) at non-toxic concentrations. Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes. In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.     

Non-viral gene therapy

Non-viral gene therapies are currently under development that employ drug-delivery methods for targeting genes to selected cells in the body, where they express therapeutic gene products. Various methods have been described for non-viral gene therapy, ranging from the direct intramuscular injection of purified DNA to the systemic administration of formulations comprising DNA and lipids, proteins, peptides, or polymers. Products for non-viral gene therapies are designed both for direct administration to patients by conventional routes and for expression of a therapeutic product over a finite period of time in a manner similar to conventional medicines. Initial preclinical and clinical studies indicate that non-viral gene delivery methods exhibit safety profiles similar to conventional pharmaceutical or biological products. Clinical trials have been proposed, or are currently under way, to assess the applicability of non-viral gene therapy for a variety of disorders, including cystic fibrosis, cancer, and peripheral vascular disease. Non-viral techniques may soon allow gene therapy to be applied in clinical practice alongside conventional medicines for the treatment of common diseases.     

Design,preparation and application of nucleic acid delivery carriers

Gene delivery vectors must deliver their cargoes into the cytosol or the nucleus, where DNA or siRNA functions in vivo. Therefore it is crucial for the rational design of the nucleic acid delivery carriers. Compared with viral vectors, non-viral vectors have overcome some fatal defections in gene therapy. Whereas the most important issue for the non-viral vectors is the low transfection efficiency, which hinders the progress of non-viral carriers. Sparked by the structures of the virus and understanding of the process of virus infection, various biomimic structures of non-viral carriers were designed and prepared to improve the transfection issues in vitro and in vivo. However, less impressive results are achieved. In this review, we will investigate the evolution of the virus-mimicking carriers of nucleic acids for gene therapy, especially in cancer therapy; explore and discuss the relationship between the structures, materials and functions of the carriers, to provide guidance for establishing safe and highly efficient non-viral carriers for gene therapy.     

Apatite genesis: A biologically induced or biologically controlled mineral formation process?

Apatite formation from organic matter (ribonucleic acid) and calcium carbonate (cuttlebone) requires intervention of microorganisms. We have attempted to characterize this mineral formation process by locating the alkaline phosphatase and the crystals formed. Alkaline phosphatase, which is important for the liberation of the necessary components, was localized in the periplasmic space of Providencia rettgeri in the same manner as in Escherichia coli. Accordingly, the release of inorganic phosphate and the formation of apatite may occur at this site. However, electron microscope observations revealed the presence of extracellular apatite; moreover, apatite particles that were formed with or without bacteria (with alkaline phosphatase from hydrolyzed ribonucleic acid as phosphorus source) were closely similar in size and appearance. The formation of apatite can thus be qualified as biologically induced mineralization. Nevertheless, a bacterial cell can also act as a nucleator for apatite crystallization, but this would appear exceptional.     

Nuclear-targeted minicircle to enhance gene transfer with non-viral vectors in vitro and in vivo

BACKGROUND: To develop more efficient non-viral vectors, we have previously described a novel approach to attach a nuclear localisation signal (NLS) to plasmid DNA, by generating a fusion protein between the tetracycline repressor protein TetR and an SV40 NLS peptide (TetR-NLS). The high affinity of TetR for the DNA sequence tetO is used to bind the NLS to DNA. We have now investigated the ability of this system displaying the SV40 NLS or HIV-1 TAT peptide to enhance nuclear import of a minimised DNA construct more suitable for in vivo gene delivery: a minicircle. METHODS: We have produced a new LacZ minicircle compatible with the TetR system. After transfection of the minicircle in combination with TetR-NLS or TetR-TAT using different transfection agents, we first measured beta-galactosidase activity in vitro. We then used a special delivery technique, in which DOTAP/cholesterol liposomes and DNA/protein complexes are sequentially injected intravenously, to evaluate the activity of this system in vivo. RESULTS: In vitro results showed a 30-fold increase in transfection efficiency of the nuclear-targeted minicircle compared to normal plasmid lipofection. Results on cell cycle arrested cells seem to indicate a different mechanism between the TetR-NLS and TetR-TAT. Finally, we demonstrate a more than 6-fold increase in beta-galactosidase expression in the mouse lung using the minicircle and the TetR-TAT protein. This increase is specific for the peptide sequence and is not observed with the control protein TetR. CONCLUSIONS: Our results indicate that the combination of a minicircle DNA construct with a TetR nuclear-targeting system is able to potentiate gene expression of non-viral vectors.     

A high-resolution electron microscope study of synthetic and biological carbonated apatites

A series of synthetic carbonated apatites and human dental enamels characterized by chemical analysis, infrared spectroscopy, and X-ray diffraction was studied using high-resolution transmission electron microscopy. Steps on the surfaces of apatite crystals, often only a few unit cells high and occasionally one unit cell high, were observed by their Fresnel diffraction contrast. Highly substituted synthetic carbonated apatites appeared to have more irregular and finer-textured surface features than materials with less carbonate substitution. The surface features of enamel apatite crystal were also irregular, but surface steps were less frequently aligned in crystallographic directions. Complex strain fields due to radiation damage centers were observed in some crystals and the fine structure of dislocations and grain boundaries in synthetic apatites was resolved at high magnification. Experimental lattice-image contrasts, in favorable circumstances, could be matched to computer-simulated images and were found to contain detail at near atomic resolution, around 2.0-2.5 A.     

Preparation, characterization and transfection efficiency of cationic PEGylated PLA nanoparticles as gene delivery systems

The cationic polylactic acid (PLA) nanoparticle has emerged as a promising non-viral vector for gene delivery because of its biocompatibility and biodegradability. However, they are not capable of prolonging gene transfer and high transfection efficiency. In order to achieve prolonged delivery of cationic PLA/DNA complexes and higher transfection efficiency, in this study, we used copolymer methoxypolyethyleneglycol-PLA (MePEG-PLA), PLA and chitosan (CS) to prepare MePEG-PLA-CS NPs and PLA-CS NPs by a diafiltration method and prepared NPs/DNA complexes through the complex coacervation of nanoparticles with the pDNA. The object of our work is to evaluate the characterization and transfection efficiency of MePEG-PLA-CS versus PLA-CS NPs. The MePEG-PLA-CS NPs have a zeta potential of 15.7 mV at pH 7.4 and size under 100 nm, while the zeta potential of PLA-CS NPs was only 4.5 mV at pH 7.4. Electrophoretic analysis suggested that both MePEG-PLA-CS NPs and PLA-CS NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed MePEG-PLA-CS NPs exhibit a low cytotoxicity to normal human liver cells. The potential of PLA-CS NPs and MePEG-PLA-CS NPs as a non-viral gene delivery vector to transfer exogenous gene in vitro and in vivo were examined. The pDNA being carried by MePEG-PLA-CS NPs, PLA-CS NPs and lipofectamine could enter and express in COS7 cells. However, the transfection efficiency of MePEG-PLA-CS/DNA complexes was better than PLA-CS/DNA and lipofectamine/DNA complexes by inversion fluorescence microscope and flow cytometry. It was distinctively to find that the transfection activity of PEGylation of complexes was improved. The nanoparticles were also tested for their ability to transport across the gastrointestinal mucosa in vivo in mice. In vivo experiments showed obviously that MePEG-PLA-CS/DNA complexes mediated higher gene expression in stomach and intestine of BALB/C mice compared to PLA-CS/DNA and lipofectamine/DNA complexes. These results suggested that MePEG-PLA-CS NPs have favorable properties for non-viral gene delivery.     

Efficient removal of LoxP-flanked genes by electroporation of Cre-recombinase mRNA

Introduction of Cre-recombinase in target cells is currently achieved by transfection of plasmid DNA or by viral-mediated transduction. However, efficiency of non-viral DNA transfection is often low in many cell types, and the use of viral vectors for transduction implies a more complex and laborious manipulation associated with safety issues. We have developed a non-viral non-DNA technique for rapid and highly efficient excision of LoxP-flanked DNA sequences based on electroporation of in vitro transcribed mRNA encoding Cre-recombinase. A K562-DSRed[EGFP] cell line was developed in order to measure Cre-mediated recombination by flow cytometric analysis. These cells have a stable integrated DSRed reporter gene flanked by two LoxP sites, and an EGFP reporter gene, which could only be transcribed when the coding sequence for DSRed was removed. The presented data show recombination efficiencies, as measured by appearance of EGFP-fluorescence, of up to 85% in Cre-recombinase mRNA-electroporated K562-DSRed[EGFP] cells. In conclusion, mRNA electroporation of Cre-recombinase is a powerful, safe, and clinically applicable alternative to current technologies used for excision of stably integrated LoxP-flanked DNA sequences.     

Correlation of the abundance of betaproteobacteria on mineral surfaces with mineral weathering in forest soils

Pyrosequencing-based analysis of 16S rRNA gene sequences revealed a significant correlation between apatite dissolution and the abundance of betaproteobacteria on apatite surfaces, suggesting a role for the bacteria belonging to this phylum in mineral weathering. Notably, the cultivation-dependent approach demonstrated that the most efficient mineral-weathering bacteria belonged to the betaproteobacterial genus Burhkolderia.