High-efficiency gene delivery for expression in mammalian cells by nanoprecipitates of Ca–Mg phosphate

Chowdhury, Ezharul Hoque; Kunou, Megumi; Nagaoka, Masato; Kundu, Anup K.; Hoshiba, Takashi; Akaike, Toshihiro

doi:10.1016/j.gene.2004.07.015

Cited by 75 publications

(51 citation statements)

References 24 publications

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“…Magnesium has chemical properties very similar to those of calcium and can also partially replace this ion in many crystals [21]. Chowdhury et al successfully incorporated fibronectin and Mg 2+ into calcium phosphate particles in order to achieve a higher transfection efficiency than the classical precipitation method [7]. They reached circa ten times higher efficiency for the plasmid expression then that shown by classic calcium phosphate transfection method.…”

Section: Advanced Bioceramics In Nanomedicine and Tissue Engineeringmentioning

confidence: 99%

Functionalized Calcium Phosphate Nanoparticles for Biomedical Application

Epple

Kovtun

2010

KEM

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Calcium phosphate is a natural biomineral and therefore possesses an excellent biocompatibility due to its chemical similarity to human hard tissue (bone and teeth). Calcium phosphate nanoparticles can be precipitated under controlled conditions and used as carrier in biological systems, e.g. to transfer nucleic acids or drugs. Such nanoparticles can also be suitably functionalized with fluorescing dyes, polymeric agents, pro-drugs or activators. The small monodisperse nanoparticles only mildly influence the intracellular calcium level and therefore are not toxic for cells.

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Section: Advanced Bioceramics In Nanomedicine and Tissue Engineeringmentioning

confidence: 99%

Functionalized Calcium Phosphate Nanoparticles for Biomedical Application

Epple

Kovtun

2010

KEM

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“…The resulting particles of Ca/MgPi precipitates, like CaPi ones, adsorb DNA, but unlike the latter, could prevent the growth of the precipitates to a significant extent, leading to huge uptake of DNA and consequential efficient transgene expression, which is 10 to 100 times higher than the classical CaPi co-precipitation method in HeLa and NIH3T3 cells [13].…”

Section: Development Of Ca-mg Phosphate (Ca/mgpi) Nanoparticles For Ementioning

confidence: 98%

“…Despite being one of the oldest transfection methods, the procedure has remained almost unchanged, basically composed of either direct or drop-wise mixing of two solutions, one containing DNA and calcium chloride (CaCl 2 ) and the other possessing inorganic phosphate (Pi) (either NaH 2 PO 4 or Na 2 HPO 4 ) in order to induce "supersaturation" of the final solution with respect to CaCl 2 and Pi, resulting in precipitation of microscopically visible and invisible particles with the DNA being adsorbed through electrostatic interactions [12][13][14]. Although the particles have net negative charge, they are capable of binding anionic DNA presumably through the Ca…”

Section: Mechanism Of Dna Delivery By Classical Capi Particlesmentioning

confidence: 99%

“…X-ray diffraction and FT-IR analysis of the precipitated particles established them as "hydroxyapatite" with the molecular formula of Ca 10 (PO 4 ) 6 (OH) 2 [12][13][14]. The resulting DNA/CaPi co-precipitates are usually dispersed onto the cultured cells from several hours to overnight in the incubator allowing them to be taken up by the cells via endocytosis.…”

Section: +mentioning

confidence: 99%

“…The estimated molar ratios of Ca, Mg and P present in the precipitates of Ca/MgPi indicated formation hydroxyapatite with the molecular formula of Ca 10−X Mg X (PO 4 ) 6 (OH) 2 for 0.58 and 1.03 percentage of Mg and octacalcium phosphate (OCP) with the formula of Ca 4−X Mg X (PO 4 ) 3 for 1.76 to 3.16 percentage of Mg substituted in the particles [13]. The uptake of fluorescence-labeled plasmid in HeLa cells indicated size-dependent endocytosis of the Ca/MgPi particles with the particles of the smaller size resulting in higher uptake of the DNA compared to those of bigger size, implying in agreement with the notion that internalization of apatite particles is indeed size-dependent [18,19].…”

Section: +mentioning

confidence: 99%

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pH-responsive magnesium- and carbonate-substituted apatite nano-crystals for efficient and cell-targeted delivery of transgenes

Chowdhury¹

2013

OJGen

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The short half-lives due to the enzymatic degradation in blood, the lack of tissue targetability and the incapability to passively diffuse across the plasma membrane and smoothly traffic across the harsh intracelluar environment are the major shortcomings for nucleic acid-based potential therapeutics, such as recombinant plasmid and antisense oligonucleotides or small interferring RNA (siRNA). Plasmid DNA containing a gene of interest could have immense impact as a promising therapeutic drug for treating genetic as well as acquired human diseases at the molecular level with high level of efficacy and precision. Thus both viral and non-viral synthetic vectors have been developed in the past decades to address the aforementioned challenges of naked DNA. While in the viral particles plasmid DNA is integrated into the viral genome, in most non-viral cases the DNA being anionic in nature is electrostatically associated with a cationic lipid or polymer forming lipoplex or polyplex, respectively, or a cationized inorganic gold, silica or iron oxide particle. Due to the potential immunogenicity and carcinogenicity issues with the viral particles, non-viral vectors have drawn much more attention for the clinical evaluation. However, the main concern of using non-biodegradable particles, specially the inorganic ones, is the adverse effects owing to their long term interactions with body components. We have recently developed biodegradable pH-sensitive inorganic nanoparticles of Mg/CaPi and carbonate apatite for efficient transgene delivery to primary, cancer and embryonic stem cells, by virtue of their high affinity binding with the DNA, ability to contact the cell membrane by ionic or ligand-receptor interactions and fast dissolution kinectis in endosomal acidic pH facilitating release of the DNA from the dissolving particles and also from the endosomes.

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Transient Gene Expression in Mammalian Cells: Promises and Challenges for Medical Biotechnology

Hacker

Baldi

Adam

et al. 2009

Encyclopedia of Industrial Biotechnology

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Recombinant protein‐based biologics are increasingly relevant in the pharmaceutical industry. Currently, mammalian cell‐based bioprocesses are a routine manufacturing procedure for complex proteins such as recombinant antibodies, and the number of new biologics produced by mammalian cells is progressively increasing. In addition, a growing number of proteins need to be rapidly screened to identify new candidates for clinical trials. To help fill this need, rapid mammalian‐based bioprocesses have been established with transient gene expression, the production of recombinant protein following gene delivery into cells without the establishment of a stable cell line. With current transient gene expression methods, the recombinant protein can be made available in milligram amounts within 1–2 weeks from the time the appropriate expression vector has been generated. Thus, pre‐clinical material can quickly enter the drug screening process ( in vitro activity tests and animal studies), and successful candidates can be identified within a short time‐frame. But the potential of this innovative technology is still far from being fully exploited and appreciated. The aim of this article is to provide an overview of the past achievements and current status of the field of transient gene expression in mammalian cells as well as to discuss the future perspectives and challenges of this promising technology.

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High-efficiency gene delivery for expression in mammalian cells by nanoprecipitates of Ca–Mg phosphate

Cited by 75 publications

References 24 publications

Functionalized Calcium Phosphate Nanoparticles for Biomedical Application

Functionalized Calcium Phosphate Nanoparticles for Biomedical Application

pH-responsive magnesium- and carbonate-substituted apatite nano-crystals for efficient and cell-targeted delivery of transgenes

Transient Gene Expression in Mammalian Cells: Promises and Challenges for Medical Biotechnology

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