Transfecting mammalian cells by DNA/calcium phosphate precipitates: Effect of temperature and pH on precipitation

Chowdhury, Ezharul Hoque; Sasagawa, Tadashi; Nagaoka, Masato; Kundu, Anup K.; Akaike, Toshihiro

doi:10.1016/s0003-2697(02)00648-6

Cited by 34 publications

(22 citation statements)

References 3 publications

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“…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: +supporting

confidence: 81%

“…In addition, by changing more than one parameter at a time, such as, an increase in Ca 2+ concentration and a decrease Pi level, it is possible to develop the same degree of supersaturation as required to produce effective co-precipitates [17]. Similarly, by increasing the pH of the HBS from 7.05 to 7.5 and incubation temperature from 25˚C to 37˚C and decreasing the Ca 2+ concentration from 125 mM to 14 mM, a similar level of supersaturation development was feasible, enabling to effectively co-precipitate DNA and carrying out transfection in serum-free condition [18]. Since the co-precipitation reaction and subsequent cell transfection were performed at the same temperature (37˚C), there was no further induction of particle formation or growth even in the absence of serum, whereas the existence of serum is essential during the transfection period for coating the particle surface and thus blocking further production and growth of the particles in the former case which involves lower temperature (25˚C) for the particle precipitation and higher temperature (37˚C) for the trans-fection [10].…”

Section: Approaches For Regulation Of Particle Growth: Influences On mentioning

confidence: 99%

“…Since the co-precipitation reaction and subsequent cell transfection were performed at the same temperature (37˚C), there was no further induction of particle formation or growth even in the absence of serum, whereas the existence of serum is essential during the transfection period for coating the particle surface and thus blocking further production and growth of the particles in the former case which involves lower temperature (25˚C) for the particle precipitation and higher temperature (37˚C) for the trans-fection [10]. However, the transfection in 10% serumsupplemented medium was significantly higher than in serum-free medium probably due to the particles of larger size formed in the latter case, since endocytosis is usually more effective for the particles of smaller size [18].…”

Section: Approaches For Regulation Of Particle Growth: Influences On 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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Section: +supporting

confidence: 81%

Section: Approaches For Regulation Of Particle Growth: Influences On mentioning

confidence: 99%

Section: Approaches For Regulation Of Particle Growth: Influences On 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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“…Binding of Radioligands-CHO cells were seeded on 150-mm plates and transfected with 50 g of the plasmid for eGFP-M 2 using calcium-phosphate precipitation (31)(32)(33). The efficiency of transfection was 12%.…”

Section: Methodsmentioning

confidence: 99%

Oligomeric Size of the M2 Muscarinic Receptor in Live Cells as Determined by Quantitative Fluorescence Resonance Energy Transfer

Pisterzi

Jansma

Georgiou

et al. 2010

Journal of Biological Chemistry

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Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence lifetime imaging, has been used to estimate the size of oligomers formed by the M 2 muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M 2 receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M 2 receptor to enhanced green fluorescent protein-M 2 receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values of E as follows: n ‫؍‬ 2, 0.495 ؎ 0.019; n ‫؍‬ 4, 0.202 ؎ 0.010; n ‫؍‬ 6, 0.128 ؎ 0.006; n ‫؍‬ 8, 0.093 ؎ 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20 -0.24, identifying the M 2 receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process.Much evidence now indicates that G protein-coupled receptors can exist as oligomers (1, 2), a development that has implications for all aspects of GPCR 4 -mediated signaling. Among the many questions prompted by the emergence of such structures is that of oligomeric size. Although commonly referred to as dimers, oligomers of GPCRs have been detected most often by means of coimmunoprecipitation or resonance energy transfer (3). As typically applied, neither technique can distinguish dimers from larger oligomers. The latter have been identified on the basis of their electrophoretic mobility (reviewed in Ref. 4), but the composition of the bands may be unclear, and the size under the conditions of electrophoresis may have little in common with that in the membrane. Larger oligomers also have been identified by approaches in which detection requires the colocalization of three or four proteins, each bearing a different tag (5-11), but such procedures place only a lower limit on the possible size of the array.There have been comparatively few attempts to examine the oligomeric status of a GPCR in a more quantitative and explicit manner. Measurements of BRET at different ratios of acceptor to donor have pointed to dimers of the melatonin receptor (12), the ␤ 1 -and ␤ 2 -adrenergic receptors (13), the M 1 , M 2 , and M 3 muscarinic receptors (14), and the neurotensin receptor (15)....

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“…To achieve effective gene transfection, the conditions for CaP-DNA co-precipitation method have been studied [242][243][244]. HAp nanoparticles can also be utilized as gene carriers because of their capability to absorb DNA molecules [245,246].…”

Section: Carriers For Drug Protein and Gene Deliverymentioning

confidence: 99%

Hydroxylapatite nanoparticles: fabrication methods and medical applications

Okada

Furuzono

2012

Science and Technology of Advanced Materials

126

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Hydroxylapatite (or hydroxyapatite, HAp) exhibits excellent biocompatibility with various kinds of cells and tissues, making it an ideal candidate for tissue engineering, orthopedic and dental applications. Nanosized materials offer improved performances compared with conventional materials due to their large surface-to-volume ratios. This review summarizes existing knowledge and recent progress in fabrication methods of nanosized (or nanostructured) HAp particles, as well as their recent applications in medical and dental fields. In section 1, we provide a brief overview of HAp and nanoparticles. In section 2, fabrication methods of HAp nanoparticles are described based on the particle formation mechanisms. Recent applications of HAp nanoparticles are summarized in section 3. The future perspectives in this active research area are given in section 4.

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Transfecting mammalian cells by DNA/calcium phosphate precipitates: Effect of temperature and pH on precipitation

Cited by 34 publications

References 3 publications

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

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

Oligomeric Size of the M2 Muscarinic Receptor in Live Cells as Determined by Quantitative Fluorescence Resonance Energy Transfer

Hydroxylapatite nanoparticles: fabrication methods and medical applications

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