High-efficiency gene delivery for expression in

Gene 341 (2004) 77 – 82 www.elsevier.com/locate/gene

High-efficiency gene delivery for expression in mammalian cells by nanoprecipitates of Ca–Mg phosphate E.H. Chowdhury, Megumi Kunou, Masato Nagaoka, A.K. Kundu, Takashi Hoshiba, Toshihiro Akaike* Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan Received 2 December 2003; received in revised form 30 June 2004; accepted 19 July 2004 Available online 12 September 2004 Received by F. Salvatore

Abstract Transfer of desirable genetic sequences into mammalian cells is an essential tool for analysis of gene structure, functions and regulation and industry-based production of therapeutically important proteins and pivotal for gene therapy and DNA vaccination strategies. Considering some severe limitations of viral systems including immunogenicity, carcinogenicity and so on, synthetic nonviral systems are highly desirable in the above applications. However, existing nonviral techniques are extremely inefficient compared to the viral ones. Therefore, we report here on the development of a highly efficient synthetic device for gene delivery and expression into mammalian cells, based on controllable growth of nanoapatite particles. Mg2+ incorporation into the apatite particles caused significant inhibition of particle growth, resulting in retention of nanosized particles which contributed remarkably to the cellular uptake of DNA and its subsequent expression (N10-fold) compared with classical calcium phosphate coprecipitation, one of the most widely used transfection methods. D 2004 Elsevier B.V. All rights reserved. Keywords: Transfection; DNA uptake; Crystal growth; Nanoparticles

1. Introduction Significant efforts are now being made for the development of nonviral gene delivery techniques as alternatives to the viral vectors for basic research and clinical medicine (Luo and Saltzman, 2000). Despite existence of a wide variety of nonviral techniques particularly relying on synthetic lipids (liposomes), peptides (poly-l-lysine), dendrimers (polyamidoamine) and other polymers, such as polyethylenimine, limited understanding of the molecular

Abbreviations: DLS, dynamic light scattering; HBS, HEPES-buffered solution; OCP, octaclacium phosphate; PI, propidium iodide; RLU, relative light unit. * Corresponding author. Tel.: +81 45 924 5790; fax: +81 45 924 5815. E-mail address: [email protected] (T. Akaike). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.07.015

and cellular basis in gene transfer hinders the development of a smart technology. Coprecipitation of DNA with calcium phosphate which is based on hydroxyapatite, is one of the most commonly used nonviral vectors (Fasbender et al., 1998; Toyoda et al., 2000; Graham and van der Eb, 1973; Gorman et al., 1983; Chen and Okayama, 1987; Brash et al., 1987; Kjer and Fallon, 1991; O’mahoney and Adams, 1994; Song and Lahiri, 1995; Jordan et al., 1996; Lee and Welsh, 1999; Urabe et al., 2000), having potential applications in gene therapy (Fasbender et al., 1998; Toyoda et al., 2000). Although inefficiency in particle-mediated uptake of DNA by the cells has been considered as a major barrier of low transgene expression in vitro and in vivo (Fasbender et al., 1998; Toyoda et al., 2000; Chen and Okayama, 1987; Jordan et al., 1996; Loyter et al., 1982a; Loyter et al., 1982b; Orrantia and Chang, 1990; Batard and Jordan, 2001), an effective way of manipulating particle growth

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E.H. Chowdhury et al. / Gene 341 (2004) 77–82

kinetics at the molecular level has not been focused so far, which could overcome the hurdle dramatically. A timedependent control in particle growth kinetics was shown to modulate transfection and short time incubation resulted in finer particles and thus better performance in transgene expression (Jordan et al., 1996). Although the method is fairly straightforward, relying on direct mixing of the components, instead of the laborious dropwise mixing followed in the old system, transfection activity of the former was not better than the latter (Seelos, 1996). Here, we report on the generation of Ca–Mg phosphate precipitates which, like Ca phosphate precipitates, adsorbed DNA, but unlike the latter, could prevent the growth of the particles to a significant extent, resulting in huge cellular uptake of DNA, followed by notably high transgene expression.

2. Materials and methods 2.1. Chemical analysis Particles were prepared by providing 125 mM final concentration of calcium chloride and 0.0 to 140 mM final concentrations of magnesium chloride to HEPES-buffered solution (HBS; 140 mM NaCl, 5 mM KCl, 25 mM HEPES; pH 7.05), containing 0.75 mM Na2HPO4d 2H2O, followed by incubation at room temperature. After generation and precipitation, all types of apatites were purified by centrifugation and repeated washing with distilled deionized water and then lyophilized. Fourier transform-infrared (FT/IR) spectrum and X-ray diffraction (XRD) spectrum of Ca phosphate particles were performed using FT/IR-230, JASCO and M18XHF-SRA diffractometer system (Mac Sci.), respectively. Calcium, magnesium and phosphorus contents were determined using a Seiko SPS 1500 VR Atomic Absorption Spectrophotometer.

2.2. Turbidity and particle size measurements After mixing two solutions, one containing 1.5 mM inorganic phosphate in 300 Al of 2 HEPES-buffered solution (HBS; pH 7.05) and the other containing 250 mM Ca2+, in addition to 0.0 to 280 mM Mg2+ in 300 Al water, spectroscopic reading at 320 nm was taken at 1 to 30 min for turbidity measurement (in accordance with previous report by Jordan et al., 1996) by SmartSpeck 3000 (BioRad). Average diameters of the particles at their growing stage (1 to 30 min) were estimated by a super-dynamic light scattering (DLS) spectrophotometer (Photal, Otsuka Electronics) at 75 mW Ar laser. 2.3. Transfection of cells HeLa and NIH 3T3 cells were cultured in 75-cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum (FBS), 50 Ag penicillin ml 1, 50 Ag streptomycin ml 1 and 100 Ag neomycin ml 1 at 37 8C in a humidified 5% CO2-containing atmosphere. Cells from the exponentially growth phase were seeded at 50,000 cells per well into 24-well plates the day before transfection to enable 50% cell confluency just prior to transfection. About 6 Ag of plasmid DNA containing a luciferase gene (pGL3; Promega) was added to 300 Al of a solution containing 250 mM CaCl2 and 0.0 to 280 mM MgCl2. In order to label DNA for cellular uptake study, 6 Ag of propidium iodide (PI) was also added to the solution. This solution was added to 300Al of a 2 HBS (50 mM HEPES, 140 mM NaCl, 1.5 mM Na2HPO4d 2H2O, pH 7.05) and mixed rapidly by gentle pipetting twice. The DNA/Ca phosphate or DNA/Ca–Mg phosphate mixture was incubated at room temperature for the period of time indicated. After addition of 100 Al of the incubated mixture dropwise to 1 ml of 10% serum-supplemented media of each well, cells were incubated for 4 h, and after replacement with fresh serum

Fig. 1. Infrared spectra of Ca phosphate particles. Particles were prepared by addition of 125 mM Ca2+ to HBS, containing 0.75 mM Na2HPO4d 2H2O, followed by incubation at room temperature. After generation and precipitation, all types of apatites were purified by centrifugation and repeated washing with distilled deionized water and were then lyophilized.


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Table 2 Calculation of molar ratios of Mg, Ca and P in nanoprecipitates

Fig. 2. X-ray diffraction patterns of Ca phosphate particles. Particles were prepared by addition of 125 mM Ca2+ to HBS, containing 0.75 mM Na2HPO4d 2H2O, followed by incubation at room temperature. After generation and precipitation, all types of apatites were purified by centrifugation and repeated washing with distilled deionized water and were then lyophilized.

Sample

Mg

Ca

P

1 2 3 4 5 6 7 8

0.0 0.36 0.64 0.84 1.13 1.16 1.3 1.43

10.1 9.83 9.39 7.76 7.67 7.37 7.21 7.04

6 6 6 6 6 6 6 6

diffraction patterns also shows typical apatitic features (see Fig. 2; Okazaki et al., 2001). To know the chemical composition of all types of the particles, elemental analysis was performed (Tables 1 and 2) for samples 1, 2, 3,4, 5, 6, 7 and 8, representing, respectively, 0, 20, 40, 60, 80, 100, 120 and 140 mM Mg2+ added for particle generation (described above). As shown in Table 1, with an increase in Mg2+ concentrations in solution, particle-associated Mg2+ level increased up to ~3%, with concomitant decrease in Ca2+ level, whereas phosphorus (P) level remains almost fixed for samples 1 to 3 (~12%) and samples 4 to 8 (~16%),

media, were grown for 1 day. Luciferase gene expression was monitored by using a commercial kit (Promega) and photon counting (TD-20/20 Luminometer, USA) according to the instructions provided by Promega. Each transfection experiment was done in triplicate and transfection efficiency was expressed as mean light units per milligram of cell protein.

3. Results and discussion 3.1. Generation and chemical characterization of Ca–Mg phosphate particles Addition of 0 to 140 mM Mg2+ along with 125 mM Ca2+ to HBS (pH 7.05) containing 0.75 mM inorganic phosphate, followed by incubation at room temperature, resulted in microscopically visible particles (not shown here). As shown in Fig. 1, IR spectrum of Ca phosphate particles (generated in absence of Mg2+) suggests formation of hydroxyapatite as the peaks between 1000–1100 and 550– 650 cm 1 represents phosphate in the structure. X-ray Table 1 Estimation of Mg, Ca and P contents in nanoprecipitates Sample

Mg (%)

Ca (%)

P (%)

1 2 3 4 5 6 7 8

0.0 0.58 1.03 1.76 2.38 2.54 2.88 3.16

27.31 26.06 24.89 26.73 26.63 26.52 26.46 25.57

12.53 12.27 12.35 15.95 16.05 16.67 16.67 16.88

Fig. 3. Monitoring precipitation kinetics and estimation of particle sizes. (A) Turbidity measurement at 320 nm was used as an indicator of precipitation or particle growth. Just after mixing two solutions, one containing 1.5 mM inorganic phosphate in 300 Al of 2 HBS (pH 7.05) and the other containing 250 mM Ca2+, in addition to 0.0 to 280 mM Mg2+ in 300 Al water, spectroscopic reading (320 nm) was taken at 1 (x), 5 (n), 10 (E) and 30 (x) min. (B) Determination of particle diameter was performed in the same manner as described above using a DLS device.

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indicating precipitation of two different types of apatite. The molar ratio values (Table 2) indicate formation of hydroxyapatite with the formula Ca10 x Mgx (PO4)6(OH)2 for samples 1 to 3 and octacalcium phosphate (OCP), with the formula Ca4 x Mgx (PO4)3 for samples 4 to 8, thereby suggesting that a high Mg2+ level drives the reaction to the formation of OCP (Kibalczyc et al., 1990; Salimi et al., 1985). 3.2. Regulating growth kinetics and sizes of particles Turbidity determination of a particle suspension could be interpreted to analyse time-dependent particle growth,

following nucleation in a supersaturated solution (Jordan et al., 1996). As shown in Fig. 3A, at 1 min following mixing all of the components in HBS (described above), turbidity declined continuously with increasing Mg2+ concentrations in the solution, suggesting clearly that incorporated Mg2+ slows down the growth of the particles to a significant extent. With incubation for additional periods (5 to 30 min), turbidity plot showed an up and down profile which could be explained with the notion that an increasingly high concentrations of Mg2+ (20 to 60 mM) could further induce the precipitation reaction depending on the incubation time, thus causing an increment in turbidity for an increase in particle numbers, and that with a more

Fig. 4. Nano-apatite-mediated DNA uptake in HeLa cells. Following mixing of the two solutions, one containing 1.5 mM inorganic phosphate in 300 Al of 2 HBS and the other containing 6 Ag DNA labeled with PI at a DNA/PI weight ratio of 1, 250 mM Ca2+, in addition to 0.0 to 280 mM Mg2+ in 300 Al water; 100 Al of each particle suspension was collected at a specified period of time (1 to 30 min) and added onto the cells being cultured in a well of 24-well plate in presence of 10% FBS-supplemented DMEM. After incubation at 37 8C for 4 h, cells were rinsed with 5 mM EDTA in PBS to remove the extracellular particles and observed under a fluorescence microscope (scale bar, 50 Am).


Fig. 5. Enhancement of luciferase expression in HeLa cells by nanoprecipitates. Mixing of the two solutions, one containing 1.5 mM inorganic phosphate in 300 Al of 2 HBS and the other containing 6 Ag DNA labeled with PI at a DNA/PI weight ratio of 1, 250 mM Ca2+, in addition to 0.0 to 280 mM Mg2+ in 300 Al water, was immediately followed by incubation at room temperature according to a specified timetable 1 (x), 5 (n), 10 (E) and 30 (x) min) and 100 Al of the resulting particle suspensions was collected and added onto the cells being cultured in a well of 24-well plate in presence of 10% FBS-supplemented 1 ml DMEM. After incubation for 4 h, cells were rinsed with fresh medium and recultured for 1 day and luciferase expression was detected by a luminometer using luciferase detection kit.

significant amount of Mg2+ (80 to 140 mM), inhibition of particle growth played the major role for the sharp decrease in turbidity. In order to make a better understanding of how Mg2+ inclusion into the particles contributes immensely to the reduction of the growth and consequently, to the sizes of the particles, we estimated the mean diameters of all types of particles during their growing stages. As shown in Fig. 3B, at a period of time from 1 to 30 min following initiation of precipitation reaction, an increasing dose of Mg2+ dramatically reduced the particle diameters from micro to nano level. Moreover, the figure enables us to predict a clear and reliable growth kinetics, indicating that an increasingly high Mg2+ incorporation could transform a fast growing particles to more slowly growing ones having size distribution in the nanometer range. The strong inhibitory effect of Mg2+ on particle growth could be explained by creation of a distorted atomic structure in hydroxyapatite upon replacement of Ca2+ with Mg2+, which subsequently slows the growth of the particles (Blumenthal, 1989).

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PI, a cell-impermeable DNA-intercalating dye (AlvarezMaya et al., 2001), by adding PI in DNA/Ca2+ solution prior to mixing with HBS at 1:1 weight ration of DNA to PI. As shown in Fig. 4, internalization of DNA by Mg2+-free particles was inefficient and gradually decreased depending on passage of time to the lowest level due to the growth of the particles (see Fig. 3). On the contrary, strong fluorescence of PI-labelled DNA was observed inside the cells for Mg2+-containing particles (Fig. 4) which were sufficiently resistant to growth (see Fig. 3), indicating that DNA/Ca–Mg phosphate particles are efficiently endocytosed owing to their potential ability of blocking particle growth. The decline in uptake efficiency level for the particles generated with a high Mg2+ dose (Fig. 4) indicates formation of insufficient amount of nanoparticles (see Fig. 3) because Mg2+, beyond a level, could abolish precipitation reaction (Kibalczyc et al., 1990; Salimi et al., 1985). 3.4. Notable level of transgene expression mediated by nanoprecipitate To reach the final goal of our strategy, we checked expression profile of a luciferase gene based on DNA/Ca– Mg phosphate particles isolated according to a specified timetable (Figs. 5 and 6). Surprisingly, depending on the level of Mg2+, particle generation time and cell type, at least

3.3. High rate cellular uptake of DNA carried by nanoapatite Particle size is a crucial factor for successful gene transfer into mammalian cells; fine particles mediate an efficient gene transfer, whereas coarse ones do not (Jordan et al., 1996; Urabe et al., 2000). Rapid growth of the particles resulting in sharp increase in diameter (Fig. 3B) is thus a big hurdle which must be eliminated for efficient gene delivery and expression into the cells. Because Ca–Mg phosphate could block the growth and limit the sizes of the particles at a desirable level, we investigated DNA uptake in the cells, mediated by the particles. DNA was labelled with

Fig. 6. Enhancement of luciferase expression in NIH 3T3 cells by nanoprecipitates. Mixing of the two solutions, one containing 1.5 mM inorganic phosphate in 300 Al of 2 HBS and the other containing 6 Ag DNA labeled with PI at a DNA/PI weight ratio of 1, 250 mM Ca2+, in addition to 0.0 to 280 mM Mg2+ in 300 Al water, was immediately followed by incubation at room temperature according to a specified timetable (1 (x), 5 (n), 10 (E) and 30 (x) min) and 100 Al of the resulting particle suspensions was collected and added onto the cells being cultured in a well of 24-well plate in the presence of 10% FBS-supplemented 1 ml DMEM. After incubation for 4 h, cells were rinsed with fresh medium and recultured for 1 day and luciferase expression was detected by a luminometer using luciferase detection kit.

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10- to 100-fold higher luciferase expression could be detected compared with Mg2+-free particles. Such a high transfection efficiency could be solely attributed to the intrinsic property of Ca–Mg phosphate to significantly block the growing process and consequential generation of nanosized particles (Fig. 3) needed for efficient cellular uptake of DNA. The profound effect of particle sizes on DNA delivery and subsequent expression could be clearly seen when the particles are allowed to grow for 30 min; Mg2+ inclusion caused a remarkable transition of particle diameter from 2.5 Am to 500 nm and finally enhanced gene expression efficiency by at least 40 times. Thus, instead of providing tremendous efforts for limited transfection activity by collecting the precipitates just after initiation of precipitation (Jordan et al., 1996), Mg2+-regulated particle growth profiling could confer a highly flexible way of nanoapatite preparation and/or enable to establish a superefficient gene delivery system for mammalian cells. Considering the high impact of a traditionally and widely used transfecting agent like Ca phosphate precipitate in basic research laboratories, biotech companies for production of recombinant cell lines and recently, in gene therapy (Fasbender et al., 1998; Toyoda et al., 2000), our newly developed technology based on Ca–Mg phosphate nanoprecipitate would emerge as a tool of utmost importance in the above applications replacing the old one.

Acknowledgements We thank Nawa Nozomi for technical assistance. This work was partially supported by the grant from the Japan Society for Promotion of Science (JSPS).

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