Gene Ther Mol Biol Vol 9, 301-316, 2005

Designing smart nano-apatite composites: the emerging era of non-viral gene delivery

Review Article

Ezharul Hoque Chowdhury, Koichi Kutsuzawa & 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 __________________________________________________________________________________

*Correspondence: Akaike, Toshihiro, Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan; telephone: + 81 45 924-5790; fax: + 81 45 924-5815; e-mail: takaike@bio.titech.ac.jp

Key words: non-viral vector, gene delivery, transfection, magnesium, carbonate, apatite, endocytosis, pH sensitivity, protein expression, cell targeting, asialofetuin, transferrin

Abbreviations: octacalcium phosphate, (OCP); poly ethylene glycol, (PEG)

Received: 1 September 2005; Accepted: 6 October 2005; electronically published: October 2005

Summary

Transfer of desirable genetic sequences into mammalian cells is an essential tool for analysis of gene structure, functions and regulation, and pivotal for gene therapy and DNA vaccination strategies. Considering some severe limitations of viral systems including immunogenicity, carcinogenicity and so on, synthetic non-viral systems are highly desirable in the above applications. However, existing non-viral techniques are extremely inefficient compared to the viral ones. We have recently developed a new class of non-viral technology based on biodegradable apatite materials having simplicity and flexibility of preparation, size regulation and surface functionalization for carrying genetic materials to selective or a wide variety of cell types in an effective manner. Moreover, while the materials have high affinity for DNA due to their cationic surface charge and confers high stability of condensed DNA towards serum, they could release the associated DNA rapidly during endosomal acidification, resulting in notable level of transgene expression. We will particularly focus here on the recent development of the highly efficient synthetic device for gene delivery and expression based on controllable growth of nano-apatite particles. Mg²⁺ or CO₃^2- incorporation into the apatite particles caused significant inhibition of particle-growth, resulting in retention of nano-sized particles which contributed remarkably to the cellular uptake of DNA and its subsequent expression, leading to 5 to 100-fold higher transgene expression than the existing ones. Moreover, for cell-specific and more efficient transgene delivery, we could successfully assemble a desirable cell-recognizable protein and a highly hydrophilic protein onto the DNA/crystal surfaces, thereby conferring dual surface properties; one facilitating cell-specific delivery and the other blocking non-specific interactions. Thus, considering the efficiency, cell-targetability, biodegradability and simplicity, this newly developed gene delivery technology is highly promising over other existing ones for both basic research laboratories and clinical settings.

I. Introduction

Significant efforts are now being made for the development of non-viral gene-delivery techniques as alternatives to the viral vectors for basic research and clinical medicine (Luo et al, 2000). Despite existence of a wide variety of non-viral techniques particularly relying on synthetic lipids (liposomes), peptides (poly-L-lysine), dendrimers (polyamidoamine) and other polymers, such as polyethylenimine, limited understanding of the molecular and cellular basis in gene transfer hinders the development of a smart technology. Co-precipitation of DNA with calcium phosphate which is based on hydroxyapatite, is one of the most commonly used non-viral vectors (Graham et al, 1973; Gorman et al, 1983; Brash et al, 1987; Chen et al, 1987; Kjer et al, 1991; O’mahoney et al, 1994; Song et al, 1995; Jordan et al, 1996; Fasbender et al, 1998; Lee et al, 1999; Toyoda et al, 2000; 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 (Loyter et al, 1982; Chen et al, 1987; Jordan et al, 1996; Fasbender et al, 1998; Orrantia et al, 1990; Toyoda et al, 2000; Batard et al, 2001), an effective way of manipulating particle growth kinetics at the molecular level and a precise cell targeting concept have not been focused so far, which could overcome the hurdle dramatically (Chowdhury et al 2005). A time-dependent 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 procedure followed in the old system, transfection activity of the former was not better than the later (Seelos, 1996). Here, we report on the generation of Mg²⁺ and CO₃^2- substituted nanoapatite precipitates, which like Ca phosphate precipitates, adsorbed DNA, but unlike the later, could prevent the growth of the particles to a significant extent, resulting in huge cellular uptake of DNA, followed by notably high transgene expression. Moreover, in order to target the nanoapatite particle for smart delivery of a genetic material to a specific cell type, we successfully coated carbonate apatite nanocrystal with a cell-recognizable protein, such as asialofetuin for hepatocytes or transferrin for many cancer cell types as well as BSA for creating a highly hydrophilic crystal surface that could prevent non-specific interactions.

II. Gene delivery based on Ca-Mg phosphate nanocrystals

A. 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 (Chowdhury et al, 2004). As shown in Figure 1, IR spectrum of Ca phosphate particles (generated in absence of Mg²⁺) suggests formation of hydroxyapatite as the peaks between 1000 – 1100 cm^-1 and 550 – 650 cm^-1 represents phosphate in the structure. X-ray diffraction patterns also shows typical apatitic features (Figure 2) (Okazaki et al, 2001).

Figure 1. Infrared spectra of Ca phosphate particles. Particles were prepared by addition of 125 mM Ca²⁺ to HBS, containing 0.75 mM Na₂HPO₄.2H2O, 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.

Figure 2. X-ray diffraction patterns of Ca phosphate particles. Particles were prepared by addition of 125 mM Ca²⁺ to HBS, containing 0.75 mM Na₂HPO₄.2H2O, 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.

To know the chemical composition of all types of the particles, elemental analysis was performed for sample 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). With 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 sample 1 to 3 (~12%) and sample 4 to 8 (~16%), indicating precipitation of 2 different types of apatite (Chowdhury et al, 2004). The calculated molar ratios of Mg, Ca and P predicted formation of hydroxyapatite with the formula Ca_10-Mg_x(PO4)₆(OH)₂ for sample 1 to 3 and octacalcium phosphate (OCP) with the formula Ca_4-xMg_x(PO4)₃ for sample 4 to 8, thereby suggesting that a high Mg²⁺ level drives the reaction to the formation of OCP (Salimi et al, 1985; Kibalczyc et al, 1990).

B. 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; Chowdhury et al, 2005). As shown in Figure 3A, at 1min 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 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 the sizes of the particles, we estimated the mean diameters of all types of particles during their growing stages. As shown in Figure 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 fast growing particles to more slowly growing ones having size distribution in the nano-meter 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⁺, that subsequently slows the growth of the particles (Blumenthal et al, 1989).

Figure 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 ml of 2xHBS (pH 7.05) and the other containing 250 mM Ca²⁺, in addition to 0.0 to 280 mM Mg²⁺ in 300 ml water, spectroscopic reading (320 nm) was taken at 1 to 30 min. B, Determination of particle diameter was performed in the same manner as described above using a DLS device.

C. High rate cellular uptake of DNA carried by nano-apatite

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 (Figure 3B) is thus a big hurdle which must be eliminated for efficient gene delivery and expression into the cells. Since 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 PI, a cell-impermeable DNA intercalating dye (Alvarez-Maya 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 Figure 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 (Figure 3). On the contrary, strong fluorescence

Figure 4. Nano-apatite-mediated DNA uptake in HeLa cells. Following mixing of the two solutions, one containing 1.5 mM inorganic phosphate in 300 ml of 2xHBS and the other containing 6 mg 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 300ml water, 100 ml 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 37⁰C for 4 hr, cells were rinsed with 5 mM EDTA in PBS to remove the extracellular particles and observed under a fluorescence microscope. (Scale bar, 50mm).

of PI -labelled DNA was observed inside the cells for Mg²⁺ -containing particles (Figure 4) which were sufficiently resistant to growth (Figure 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 (Figure 4) indicates formation of insufficient amount of nano-particles (Figure 3) since Mg²⁺ beyond a level, could abolish precipitation reaction (Salimi et al, 1985; Kibalczyc et al, 1990).

D. Notable level of transgene expression mediated by nano-precipitate

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 (Figures 5, 6). Surprisingly, depending on the level of Mg²⁺, particle generation time and cell type, at least 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 nano-sized particles (Figure 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 mm 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 nano-apatite preparation/ and enable to establish a super-efficient gene delivery system for mammalian cells.

III. Gene delivery based on carbonate apatite nanocrystals

A. Generation and molecular characterization of carbonate apatite particles

Addition of only 3 mM Ca²⁺ to the HCO₃^- - buffered cell culture medium (DMEM, pH 7.5) and incubation at 37^oC for 30 min, resulted in microscopically visible particles. SEM image showed most of the particles in the nanometer range (Figure 10). Generation of these particles only in HCO₃^- -, but not in Hepes-buffered media or solution (pH 7.5) containing the same amount of total Ca²⁺ (4.8 mM) and phosphate (0.9 mM), indicates the possible involvement of carbonate along with phosphate and Ca²⁺ in particle formation. Elemental analysis proved the existence of C (3%), P (17%) and Ca²⁺ (32%) and FT-IR spectra (Figure 7a) identified carbonate, as evident from the peaks between 1410 and 1540 cm^-1 and at approximately 880 cm^-1, along with phosphate in the particles, as shown by the peaks at 1000-1100 cm^-1 and 550-650 cm^-1. X-ray diffraction patterns (Figure 7b) indicated less crystalline nature, represented by broad diffraction peaks of the particles (Figure 7c) – an intrinsic property of carbonate apatite (Legeros et al, 1967).

Figure 5. Enhancement of luciferase expression in HeLa cells by nano-precipitates. Mixing of the two solutions, one containing 1.5 mM inorganic phosphate in 300 ml of 2xHBS and the other containing 6 mg 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 ml water, was immediately followed by incubation at room temperature according to a specified timetable (1 to 30 min) and 100 ml 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 hr, cells were rinsed with fresh medium and recultured for 1 day and luciferase expression was detected by a luminometer using luciferase detection kit.

Figure 6. Enhancement of luciferase expression in NIH 3T3 cells by nano-precipitates. Mixing of the two solutions, one containing 1.5 mM inorganic phosphate in 300 ml of 2xHBS and the other containing 6 mg 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 ml water, was immediately followed by incubation at room temperature according to a specified timetable (1 to 30 min) and 100 ml 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 hr, cells were rinsed with fresh medium and recultured for 1 day and luciferase expression was detected by a luminometer using luciferase detection kit.

Figure 7. (a) FT-IR spectrum, (b) X-ray diffraction patterns and (c) Scanning electron microscopy of generated carbonate apatite particles.

B. Efficient internalization of nano-crystal-associated plasmid DNA

We verified the size limiting effect of carbonate by observing cellular uptake of plasmid DNA with the help of Southern blotting of the total DNA isolated following 4 hr transfection by carbonate apatite, since large particles are phagocytosed less efficiently than small ones (Jordan et al, 1996; Urabe et al, 2000; Chowdhury et al, 2004). DNA was carried into HeLa cells by carbonate apatite nano-particles at least 10 times more efficiently than hydroxyapatite following initial 4 hr incubation of the cells with particle-bound 200 ng and 2 mg of pGL3-control vector (a luciferase gene-containing plasmid DNA), respectively (Figure 8a). It is worthy of mentioning that DNA could be bound to the particles with almost 100% efficiency (not shown).

Figure 8. (a) HeLa cell uptake of carbonate apatite-bound pGL3-control vector for 4 hrs as assessed by southern blotting of total isolated DNA. (b-d) Comparison of luciferase expression in HeLa cells, NIH 3T3 cells and primary hepatocytes, following transfection with the same plasmid vector for 4 hrs and incubation for 24 hrs. (e) MTT assay in HeLa cells after transfection and incubation in the same way as mentioned above.

C. Transfection efficiency achieved with nano-crystals of carbonate apatite

To evaluate the role of carbonate apatite as a carrier of genetic material, we compared transfection efficiency of different techniques including two frequently used ones- CaP co-precipitation method and lipofection. In HeLa cell, luciferase expression level for carbonate apatite-mediated transfection was over 25-fold higher than for lipofection and CaP co-precipitation (Figure 8b). Transfection efficiency was also tremendously high in NIH 3T3 cells conferring over 50-times higher efficiency compared to the existing methods (Figure 8c). Transgene expression was also significantly higher in mouse primary hepatocytes (Figure 8d). We performed MTT assay in HeLa cells (Figure 8e) to clarify that high transfection efficiency was accompanied by high viability of the cells.

IV. Bio-recognition device on crystal surface for targeted delivery

Viral systems are by far the most effective means of DNA delivery to mammalian cells as a result of their highly evolved and specialized structure basically composed of a protein coat surrounding a nucleic acid core. Such a highly organized structure can prevent viral particles from unwanted interactions with serum components while promoting subsequent internalization by cells, escape from endosomes and release of genetic material from the particle either before or after entering the nucleus. So development of a non-viral approach having the beneficial virus-like properties and lacking the disadvantageous ones, would emerge as the most attractive one for implementation in research laboratories and gene therapy.

A. Analysis of adsorption of serum proteins or BSA with crystal surface

Nanoparticles of DNA/carbonate apatite were synthesized by the addition of an appropriate amount of Ca²⁺ and DNA to the HCO₃^- - buffered cell culture medium (DMEM, pH 7.5), followed by incubation at 37⁰C for 30 min (Chowdhury et al, 2005). When HepG2 cells were incubated with the DNA/nano-crystals in absence of serum, a larger pool of DNA was internalized than in presence of 10% FBS or 5 mg/ml BSA (bovine serum albumin) (Figure 9).

Figure 9. DNA uptake in HepG2 cells by carbonate apatite particles. Particles bearing DNA labeled with propidium iodide were generated by mixing 3 mM Ca²⁺, 2 mg DNA and 2 mg PI in 1 ml bicarbonate-buffered medium (DMEM), followed by incubation at 37⁰C for 30 min. Cells were incubated for 1 hr with the particles in absence of FBS or BSA (A), in presence of 10% FBS (B), or in presence of 5 mg/ml BSA (C). After treatment with EDTA in PBS, cells were observed by a fluorescence microscope to see uptake of DNA. Bar indicates 50mm.

Moreover, particles upon coming in contact with serum proteins or BSA showed microscopically visible brownian motion, thus enabling us to suggest that protein adsorption resulted in a hydrophilic surface on the crystal, necessary to avoid interactions with cell surface (Figure 9) and surrounding crystals - a prior need for cell- targeted delivery of genetic materials. And, indeed, we detected adsorption of the highly hydrophilic BSA (~1 mg/ ml reaction mixture) by Bio-Rad protein assay kit (not shown). The propensity of binding proteins to carbonate apatite could be explained by assuming the existence of anion (preferably) and cation binding sites onto the crystal provided by its constituents, calcium and phosphate or carbonate, respectively (Chowdhury et al, 2005).

B. Measurement of particle size and z potential

The three dimensional images of protein-free and protein-coated particles resemble each other with an average diameter of ~ 100 nm for a non-aggregated particle (Figure 10). The average z potential of the nano-particles was found – 17.8 and BSA (5 mg/ml) binding shifted the value to – 24.4, supporting the previous observation that acidic peptide like BSA binds to the anion-binding sites of hydroxyapatite causing the net surface charge more negative (Reynolds et al, 1982; Lopes et al, 1999).

C. Establishment of a cell-recognizable system on hydrophilic crystal surface

Our next goal was to develop a specific cell-recognizable system, on the hydrophilic surface of the crystals. To achieve this, we allowed a natural ligand, asialofetuin, which specifically binds hepatocytes or hepatocyte-derived cell lines, such as HepG2, via ASGPR (a C-type lectin only on hepatocyte cell membrane with specificity for galactose and N-acetyl galactosamine residues of desialylated glycoproteins), to interact with the crystals prior to inclusion of BSA for creation of a hydrophilic surface. The adsoption profile of this protein was analysed by polyacrylamide gel electrophoresis after dissolving the particles by 100 mM EDTA (Not shown).

As shown in Figure 11, asialofetuin having tri-antennary galactose terminated sugar chains (Hara et al, 1995), could still present galactose residues efficiently while being adsorbed onto the nano-crystals along with BSA, suggesting that this approach could be implemented for other cell-recognizable ligands, such as transferrin which has its specific receptor on certain cancer cells (Wagner et al, 1990; Kircheis et al, 1997; Joshee et al, 2002). To our knowledge, this is the first demonstration of physical adsorption of a cell-recognizable protein molecule onto a apatite-type nano-particle, restoring the functionality of the protein.

D. Receptor- mediated quick transfer of plasmid DNA into target cells

To investigate whether coating of DNA-carrying nano-crystal with cell-recognizable ligands, results in improved cellular uptake of DNA via the corresponding receptor-mediated endocytosis, we allowed the crystals to interact with increasing concentrations of asialofetuin, followed by incubation with HepG2 cells having ASGPR. DNA uptake was enhanced noticeably for 10 mg/ml asialofetuin used for coating the crystals and diminished gradually with increasing the concentrations of asialofetuin (Figure 12) suggesting that increasingly high level of free asialofetuin could saturate ASGPR – an evidence supporting that uptake was mediated by ASGPR.

Figure 10. Scanning electron microscopy of differentially formulated carbonate apatite particles. DNA/carbonate apatite particles were generated by incubation of 3 mM Ca²⁺ - supplemented bicarbonate-buffered DMEM (pH 7.5) for 30 min at 37⁰C. For creation of protein-coated particles, the resulting particle suspension was sequentially incubated with asialofetuin (10 mg/ml) for 30 min and BSA (5 mg/ml) for another 30 min at the same temperature. A drop of 10 timed diluted suspension of the particles either free (A) or coated with protein (B) was added to a carbon-coated SEM stage and dried before observation by SEM. Bar indicates 300 nm.

Figure 11. Galactose presentation on surface of asialofetuin-coated carbonate apatite particles. The particles were generated by incubation of Ca²⁺ (6 mM)-supplemented 1 ml bicarbonate-buffered medium at 37⁰C for 30 min. Generated particles were incubated with either no asialofetuin (A), or 10 (B) and 100 (C) mg/ml asialofetuin for 30 min and then with 10 mg/ml BSA for another 30 min at the same temperature. Protein-coated particles were further incubated with 1 mg/ml biotin-labeled PNA (peanut agglutinin, a galactose-specific lectin) for 1 hr and subsequently with 1mg/ml FITC-labeled streptavidin for 2 hr, before observation by a confocal laser scanning microscope. Bar indicates 50mm.

Figure 12. Enhanced uptake of DNA in HepG2 cells by asialofetuin-coated carbonate apatite particles. Particles carrying PI-labeled plasmid DNA were allowed to interact with increasing concentrations of asialofetuin (0 to 200 mg/ml) for 30 min and then with 10% FBS for additional 30 min at 37⁰C before incubation with the cells for 1 hr for DNA uptake (A-D). Extracellular particles were removed by EDTA prior to observation of the cells by a fluorescence microscope. Bar indicates 50mm.

In an attempt to replace serum by BSA for a more simplified strategy, we could fruitfully coat the nano-crystals sequencially with asialofetuin and BSA, for ASGPR-mediated effective delivery of DNA, while preventing non-specific cellular uptake (Figure 13). We studied nano-crystal-mediated DNA delivery to NIH 3T3 cells lacking ASGPR. As shown in Figure 14, uptake of DNA was significantly inhibited when the crystals were pre-coated with BSA, suggesting an universal phenomena of blocking particle endocytosis by adsorbed BSA. Furthermore, as expected, crystals pre-coated with asialofetuin and BSA, resulted in no additional increment in DNA uptake by NIH 3T3 cells (Figure 14), suggesting again that DNA uptake in HepG2 cells was mediated exclusively by ASGPR.

E. High efficiency expression following ligand-directed transgene delivery

To explore that effective delivery of DNA to the cells was followed by efficient transgene expression, we performed transfections in HepG2 cells in presence of 10% FBS using the DNA-carrying nano-crystals, coated with increasing concentrations of asialofetuin (Figure 15A) and transferrin (Figure 15B). As expected from the study of DNA uptake (Figure 12), transgene expression level was increased to a significant extent at a certain pre-coating concentration of asialofetuin and transferrin (10 mg/ml), but declined with increasing concentrations of ligand proteins (Figure 15A, B), suggesting again that uptake was mediated by the corresponding receptors. Although the optimal concentrations for asialofetuin are within a narrow range, those for transferrin lie in a wide range and this might be explained by the existence of relatively higher amount of transferrin receptors in HepG2 cells (Tros de IIarduya et al, 2002). As shown in Figure 15C, transferrin-coated particles also caused an increased transgene expression in NIH 3T3 cells as in HepG2 cells, suggesting the existence of transferrin receptors in both of the cell lines (Baker et al, 2000; Tros de IIarduya et al, 2002). To simplify the approach and improve the cell-specificity and efficiency of transfection, we transfected HepG2 cells with the crystals, coated sequentially with 10mg/ml cell-recognizable ligands and 5 mg/ml BSA. Surprisingly, transfection efficiency was dramatically increased to beyond 5- to 50-times in HepG2 cells, depending on the type of ligands (Figure 16A). In the same approach, when NIH 3T3 cells were transfected, although efficiency was increased significantly for transferrin probably due to the availability of the corresponding receptors (Baker et al, 2000), reduced extremely for asialofetuin (Figure 16B) probably due to the lack of the receptor and prevention of non-specific interaction by asialofetuin along with BSA.

Figure 13. Dual surface activities of carbonate apatite particles coated by asialofetuin and BSA. Particles carrying PI-labeled plasmid DNA were incubated at 37⁰C either with 5 mg/ml BSA for 1 hr (A), or sequentially with 10 mg/ml asialofetuin for 30 min and 5 mg/ml BSA for another 30 min (B). Cells were then incubated with the protein-coated particles for 1 hr, treated with EDTA and observed for DNA uptake by a fluorescence microscope. For more quantitative analysis (C), after EDTA treatment, cells were lysed by 200 ml of a lysis buffer (NP-40) followed by fluorescence imaging and counting by Typhoon 8600, a Variable Mode Imager (Amersham Biosciences). Bar indicates 50mm.

Figure 14. Uptake of DNA in NIH 3T3 cells by carbonate apatite particles. Particles bearing PI-labeled plasmid DNA were incubated at 37⁰C either without any BSA (A) or with 5 mg/ml BSA (B) for 1 hr, or sequentially with 10 mg/ml asialofetuin for 30 min and 5 mg/ml BSA for another 30 min (C). Cells were then incubated with the protein-coated particles for 1 hr, treated with EDTA and observed for DNA uptake by a fluorescence microscope. Bar indicates 50mm.

Figure 15. Luciferase expression profile for asialofetuin- and transferrin-coated carbonate apatite particles. Generation of DNA/carbonate apatite particles was followed by sequential incubation at 37⁰C with asialofetuin (0 to 200mg/ml) (A) or transferrin (0 to 1000mg/ml) (B, C) for 30 min and FBS (10%) for another 30 min. Cells were incubated with the protein-coated particles for 1 hr and extracellular particles were removed by 5 mM EDTA in PBS. Cells were then cultured for 1 day and luciferase expression was detected by a luminometer

Figure 16. Luciferase expression profile for carbonate apatite particles coated by asialofetuin or transferrin and BSA. Generation of DNA/carbonate apatite particles was followed by sequential incubation at 37⁰C with asialofetuin or transferrin (10 mg/ml) for 30 min and BSA (5 mg/ml) for another 30 min. Cells were incubated with the protein-coated particles for 1 hr and extracellular particles were removed by 5 mM EDTA in PBS. Cells were then cultured for 1 day and luciferase expression was detected by a luminometer

So, we have developed a completely new, the most simplified and an efficient technology for targeted delivery of a foreign DNA to the hepatocyte-originated cell lines and the tumor cells. Although an increasing number of reports could be found on targeted delivery using synthetic lipids, peptides and polymers bearing covalently bound specific ligand and occasionally with hydrophilic chain, such as poly ethylene glycol (PEG) (Wu et al, 1988; Fercol et al, 1993; Chen et al, 1994; Remy et al, 1995; Choi et al, 1998; Kawakami et al, 2000; Sudimack et al, 2000; Kircheis et al, 2002; Shi et al, 2002; Wolschek et al, 2002), to our knowledge not a single report has been demonstrated on targeted DNA delivery based on inorganic particles in such a simplified and versatile manner. Along with the controllable acid-mediated dissolution profile (Chowdhury et al, 2005), the smart targeting capacity of pH-sensitive nano-crystals of carbonate apatite would make it an attractive approach for both basic research and clinical medicine.

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 superior technology based on Mg²⁺ and CO₃^2--substituted nano-precipitates together with precise cell targetability and biocompatibility, would emerge as a tool of utmost importance in the above applications replacing the existing ones.

Acknowledgments

This work was partially supported by the grant from the Japan Society for Promotion of Science (JSPS).

References

Alvarez-Maya I, Navarro-Quiroga I, Meraz-Rios MA, Aceves J, Martinez-Fong D (2001) In vivo gene transfer to dopamine neurons of Rat substantia nigra via the high-affinity neurotensin receptor. Mol Med 7, 186-192.

Baker TL, Booden MA, Buss JE (2000) S-Nitrosocysteine increases palmitate turnover on Ha-ras in NIH3T3 cells. JBC 275, 22037-22047.

Batard P, Jordan M, Wurm F (2001) Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection. Gene 270, 61-68.

Blumenthal NC (1989) Mechanisms of inhibition of calcification. Clin Orthop 247, 279-289.

Brash DE et al (1987) Stronium phosphate transfection of human cells in primary culture: stable expression of theSimian virus 40 large-T-antigen gene in primary human bronchial epithelial cells. Mol Cell Biol 7, 2031-2034.

Chen C, Okayama H (1987) High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7, 2745-2752.

Chen J, Gamou G, Takayanagi A, Shimizu N (1994) A novel gene delivery system using EGF receptor-mediated endocytosis. FEBS Lett 338, 167-169.

Choi YH, Liu F, Park JS, Kim SW (1998) Lactose-poly-(ethylene glycol)-grafted poly-L-lysine as hepatoma cell-targeted gene carrier. Bioconjug Chem 9, 708-718.

Chowdhury EH et al (2004) High-efficiency gene delivery for expression in mammalian cells by nanoprecipitates of Ca-Mg phosphate. Gene 341, 77-82.

Chowdhury EH, Akaike T (2005) Advances in fabrication of calcium phosphate nano-composites for smary delivery of DNA and RNA to mammalian cells. Cur Analyt Chem 1, 187-192,

Chowdhury EH, Akaike T (2005) A Bio-recognition device developed onto nano-crystals of carbonate apatite for cell-targeted gene delivery. Biotechnol Bioeng 90, 414-421.

Chowdhury EH et al (2005) Integrin-supported fast rate intracellular delivery of plasmid DNA by extracellular matrix protein-embedded calcium phosphate complexes. Biochemistry (In press).

Fasbender A, Lee JH, Walters RW, Moninger TO, Zabner J, Welsh MJ (1998) Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway Epithelia in vitro and in vivo. J Clin Invest 102, 184-193.

Fercol T, Kaetzel CS, Davis PB (1993) Gene transfer into respiratory epithelial cells by targeting the polymeric immunoglobulin receptor. J Clin Invest 92, 2394-2400.

Gorman C, Padmanabhan R, Howard BH (1983) High efficiency DNA-mediated transformation of primate cells. Science 222, 551-553.

Graham FL, van der Eb AJ (1973) Transformation of rat cells by DNA of human adenovirus 5. Virology 52, 456-467.

Hara T, Aramaki Y, Takada S, Koike K, Tsuchiya S (1995) Receptor-mediated transfer of pSV2CAT DNA to a human hepatoblastoma cell line HepG2 using asialofetuin-labeled cationic liposomes. Gene 159, 167-174.

Jordan M, Schallhorn A, Wurm FM (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res 24, 596-60I.

Joshee N, Bastola DR, Cheng PW (2002) Transferrin-facilitated lipofection gene delivery strategy: characterization of the transfection complexes and intracellular trafficking. Hum Gene Ther 13, 1991-2004.

Kawakami S, Sato A, Nishikawa F, Yamashita F, Hashida M (2000) Mannose-receptor-mediated gene transfer into macrophages using novel mannosylated cationic liposomes. Gene Ther 7, 292-299.

Kibalczyc W, Christoffersen J, Christoffersen MR, Zielenkiewicz A, Zielenkiewicz W (1990) The effect of magnesium-ions on the precipitation of calcium phosphates. J Cryst Growth 106, 355-366.

Kircheis R, Kichler A, Wallner E, Kursa M, Ogris M, Felzmann T, Buchberger M, Wagner E (1997) Coupling of cell-binding ligands to polyethyleneimine for targeted gene delivery. Gene Ther 4, 409-418.

Kircheis R, Ostermann E, Wolschek MF, Lichtenberger C, Magin-lachmann C, Wightman L, Kursa M, Wagner E (2002) Tumor-targeted gene delivery of tumor necrosis factor-a induces tumor necrosis and tumor regression without systemic toxicity. Cancer Gene Ther 9, 673-680.

Kjer KM, Fallon AM (1991) Efficient transformation of mosquito cells is influenced by the temperature at which DNA-calcium phosphate coprecipitates are prepared. Arch Insect Biochem Physiol16, 189-200.

Lee JH, Welsh MJ (1999) Enhancement of calcium phosphate-mediated transfection by inclusion of adenovirus in coprecipitates. Gene Ther 6, 676-682.

Legeros RZ, Trautz OR (1967) Apatite crystallites: effects of carbonate on morphology. Science 155, 1409-1411.

Lopes MA, Monteiro JD, Santos JD, Serro AP, Saramago B (1999) Hydrophobicity, surface tension and z potential measurements of glass-reinforcedbhydroxyapatite composites. J Biomed Mater Res 45, 370-375.

Loyter A, Scangos GA, Ruddle FH (1982) Mechanisms of DNA uptake by mammalian cells: Fate of exogenously added DNA monitored by the use of fluorescent dyes. Proc Natl Acad Sci USA 79, 422-426.

Loyter A, Scangos G, Juricek D, Keene D Ruddle FH (1982) Mechanisms of DNA entry into mammalian cells. Exp Cell Res 139, 223-234.

Luo D, Saltzman WM (2000) Synthetic DNA delivery systems. Nat Biotechnol 18, 33-37.

Okazaki M, Yoshida Y, Yamaguchi S, Kaneno M, Elliott JC (2001) Affinity binding phenomena of DNA onto apatite crystals. Biomaterials 22, 2459-2464.

O’mahoney JV, Adams TE (1994) Optimization of experimental variables influencing reporter gene expression in hepatoma cells following calcium phosphate transfection. DNA & Cell Biology 13, 1227-1232.

Orrantia E, Chang PL (1990) Intracellular distribution of DNA internalized through calcium phosphate precipitation. Exp Cell Res 190, 170-174.

Remy JS, Kichler A, Mordvinov V, Schuber F, Behr JP (1995) Targeted gene transfer into hepatoma cells with lipopolyamine-condensed DNA particles presenting galactose ligands: a stage toward artificial viruses. Proc Natl Acad Sci USA 92, 1744-1748.

Reynolds EC, Wong A (1983) Effect of adsorbed protein on hydroxyapatite z potential and Streptococcus mutans adherence. Infect Immun 39, 1285-1290.

Salimi MH, Heughebaert JC, Nancollas GH (1985) Crystal growth of calcium phosphates in the presence of magnesium ions. Langmuir 1, 119-122.

Seelos C (1996) A critical parameter determining the aging of DNA-calcium-phosphate precipitates. Anal Biochem 245, 109-111.

Shi G, Guo W, Stephenson SM, Lee RJ (2002) Efficient intracellular drug and gene delivery using folate receptor-targeted pH-sensitive liposomes composed of cationic/anionic lipid combinations. J Control Release 80, 309-319.

Song W, Lahiri DK (1995) Efficient transfection of DNA by mixing cells in suspension with calcium phosphate. Nucleic Acids Res 23, 3609-3611.

Sudimack J, Lee RJ (2000) Targeted drug delivery via the folate receptor. Adv Drug Deliv Rev. 41, 147-162.

Toyoda K, Andresen J, Zabner J, Faraci F, Heistad D (2000) Calcium phosphate precipitates augment adenovirus-mediated gene transfer to blood vessels in vitro and in vivo. Gene Ther 7, 1284-1291.

Tros de IIarduya C, Arangoa MA, Moreno-Aliaga MJ, Duzgunes N (2002) Enhanced gene delivery in vitro and in vivo by improved transferrin-lipoplexes. Biochim Biophys Acta 1561, 209-221.

Urabe M, Kume A, Tobita K. Ozawa K (2000) DNA/calcium phosphate mixed with media are stable and maintain high transfection efficiency. Anal Biochem 278, 91-92.

Wagner E, Zenke M, Cotton M, Beug H, Birnsteil ML (1990) Transferrin-polycation conjugates as carriers for DNA uptake into cells. Proc Natl Acad Sci USA 87, 3410-3414.

Wolschek MF, Thallinger C, Kursa M, Rossler V, Allen M, Lichtenberger C, Kircheis R, Lucas T, Willheim M, Reinisch W, Gangl A, Wagner E, Jansen B (2002) Specific systemic non-viral gene delivery to human hepatocellular carcinoma xenografts in SCID mice. Hepatology 36, 1106-1114.

Wu GY, Wu CH (1998) Receptor-mediated gene delivery and expression in vivo. JBC 263, 14621-14624.

From left to right: Koichi Kutsuzawa, Toshihiro Akaike, Ezharul Hoque Chowdhury