A novel gene delivery system in plants with calcium alginate micro-beads

JOURNALOF BIOSCIENCEAND BIOENGINEERMG Vol. 94, No. 1, 87-91.2002

A Novel Gene Delivery System in Plants with Calcium Alginate Micro-Beads TAKEFUMI SONE,1,2” EIJI NAGAMORI,‘,2 TOMOHIKO IKEUCHI,‘,2@ ATSUSHI MIZUKAMI,’ YUKIKO TAKAKURA,’ SHIN’ICHIRO KAJIYAMA,’ El-ICHIRO FUKUSAKI,’ SATOSHI HARASHIMA,’ AK10 KOBAYASHI,’ AND KIICHI FUKUI’* Department of Biotechnologv Graduate School of Engineering, Osaka UniversiQ, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan’ and Bio-oriented Technology Research Advancement Institution (BRAIN), I-40-2 Nisshin, Saitama, Saitama 331-8537, Japan2 Received 12 February 2002/Accepted 30 April 2002

We have produced micrometer-sized calcium alginate beads referred to as “bio-beads” that encapsulate plasmid DNA molecules carrying a reporter gene. In order to evaluate the efficiency of the bio-beads in mediating genetic transfection, protoplasts isolated from cultured tobacco cells (BY-2) were transfected with bio-beads containing a plasmid that carries the modified green fluorescent protein gene CaMV3.WsGFP With the bio-beads treatment, approximately ten-fold higher GFP expression was observed after 24 h incubation compared to that with the conventional method using a naked plasmid solution. Transfection was up to 0.22% efficient. These results indicate that bio-beads have a possibility for efftcient transformation in plants. [Key words: bio-beads, calcium alginate, transfection

efficiency, tobacco protoplast, GFP expression]

A highly efficient genetic transfer system for plant cells is needed by plant scientists. Demands for simultaneous transformation of a large number of genes are increasing for functional analysis of plant genes. The difficulty in introducing large DNA molecules and/or a large quantity of DNAs into a plant cell lies in the lack of appropriate and efficient methods for this process. Several methods have been developed for genetic transformation of plant cells. For examples, the Agrobacterium-mediated method (l), the electroporation method (2), and the particle bombardment method (3) have been utilized for the transformation of plants. There are several limitations, however, in these methods for transformation of plant cells. The Agrobacteriummediated and electroporation methods are limited in the range of plant species that can be transformed due to biological and technical restrictions. The bombardment method is limited in the molecular size of genetic material that can be delivered. Therefore, we have developed a kind of drag delivery system (DDS) in which highly concentrated DNA molecules are encapsulated into autonomously degradable small particles and transferred into a plant cell. In order to transfer chemicals, mRNAs, DNA molecules and proteins into fungi and animal cells, several kinds of DDS have already been developed. Most of these systems utilize liposome (4, 5) or organic polymer (6) beads that encapsulate the molecules of interest, such as DNA molecules

to genetically them. Oil/water and water/oil type emulsions been used produce small, particles. O/W emulsions are to produce for carrying molecules. On other hand, type emulsions used for production of for hydrophilic By carefully the reaction and concentration the solidified the mean and variation the sizes the particles be regulated. workers have a co-polymer 2-hydroxyethyl methacrylate acrylamide as core matrix ethyl cellulose the barrier (6). Boussif al. (7) Godbey al. reported polyethyleneimine as appropriate materials to obtain high transfection efficiency in cultured human cells. Chitosan, a cationic polymer of deacetylated chitin, is also useful for high efficiency transformation in mammalian cells (9). Alginate is a kind of hydrophilic polysaccharide that gelates in the presence of Ca2+ ions. Alginate is harmless to both animal and plant cells and has been used as a material to immobilize bacteria in bioreactors and to encapsulate plant somatic embryos as artificial seeds (10). When a fine W/O type emulsion of sodium alginate and some kind of organic solvent were mixed with a solution containing Ca2+ ions, the alginate solidified as uniformly small and spherical particles. When hydrophilic molecules such as DNA, chromosomes, or nuclei were added to the emulsion, they would be entrapped within the solidified calcium alginate beads. Here we report the successful production of small, uniformly shaped spherical calcium alginate beads with entrapped genetic material. Furthermore, we demonstrated that tobacco protoplasts were transfected using beads containing GFP genes. Therefore, the micrometer or submicrometersized beads with encapsulated genetic material that are ca-

* Corresponding author. e-mail: [email protected] phone: +8 l-(0)6-6879-7440 fax: +8 l-(0)6-6879-7441 Present address: Department of Biology, Research stitute for Diseases. Osaka 3-1 Yamadaoka. Suita, Osaka Japan and Research Institute Marine Cargo Kobe University Mercantile Marine, -1 Fukae, Kobe 658-0022, 87

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pable of plant cell transfection were named “bio-beads”. CaMV35S+GFP(S65T)-NOS3’, an improved synthetic green fluorescent protein gene, in which serine65 is substituted with threonine, sGFP(S65T), and to which a califlower mosaic virus (CaMV) 35s promoter and noparin synthase (NOS) terminator are attached, was kindly provided as an insertion in pUC18 by Dr. Yasuo Niwa, University of Shizuoka, Shizuoka, Japan (11, 12). SpUC 19, a variant of the pUC 19 vector in which the Amp’ gene was substituted with a spectinomycin resistance gene, Spc: was provided by Prof. Ikuo Nakamura, Chiba University, Chiba, Japan. The expression cassette of CaMV35S-sGFP(S65T)-NOS3’ was excised at the Hind111 and EcoRI sites and inserted into the multiple cloning site of the SpUC19 vector. The construct, SpUCGFP, was used in transfection experiment. Two methods for bio-beads production were compared. Sodium alginate salt (lOO-150mPas.s at l%, w/v) and other chemicals were purchased from Wako Pure Chemical Industries, Osaka. First, 100 ul of 0.5-2% sodium alginate solutions containing SpUCsGFP DNA were mixed with 900 ul of a water-insoluble but partially hydrophilic liquid such as the long chain alcohol, isoamyl alcohol, with a test tube mixer (CST-040; Asahi Technoglass, Tokyo) for 1 min to form W/O type emulsions. In these emulsions, the alginate solution of the aqueous phase form small micrometersize droplets. Then 500 ul of a 100 mM CaCl, solution was added to the emulsions and the alginate was solidified with Ca2’ ions. The bio-beads were collected by centrifugation at 4000 rpm for 5 min and resuspended in a 100 mM CaCl, solution. This washing step was repeated 4 times and the biobeads were stored in 100 mM CaCl,. In the second method, 100 ul of 0.5-2% sodium alginate

solutions were mixed with 900 ul of isoamyl alcohol using a sonicator (UR-20P; Tomy Seiko, Tokyo) to form W/O emulsions. In the emulsions, the alginate solution of the aqueous phase forms small submicrometer-size droplets. Then 500 ul of a 100 mM CaCl, solution containing the plasmid DNA was added to the emulsions and the alginate droplets were solidified with Ca2+ ions. The bio-beads were collected by centrimgation at 4000 rpm for 5 min and resuspended in a 100 mM CaCl, solution. This washing step was repeated 4 times. A schematic diagram of the two methods is shown in Fig. 1. The size distribution of bio-beads was determined by the measurement of their areas. Adequate amounts of bio-beads were resuspended to a fresh 100 mM CaCl, solution on a glass slide and digital images of bio-beads were captured through an inverted fluorescent microscope (1X-70; Olympus, Tokyo) equipped with a RGB color CCD video camera (ICD-740; Olympus). The original images of the bio-beads were introduced to a personal computer (VAIO; Sony, Tokyo) and areas of each bead in the images were measured with the image analysis software, Scion Image (Scion, MD, USA). The bio-beads were assumed to be spherical, and their diameters were determined from the projection area. The diameters of approximately 1000 beads were measured. The bio-beads were divided into 5 classes based on their diameters and the size distribution of the bio-beads was determined. Entrapment of plasmid DNA was examined by staining with the DNA-specific dye, YOYO-1 (Molecular Probe, OR, USA). The bio-beads produced by the tube mixer method were l-100 urn in diameter. Entrapment of plasmid DNA in the bio-beads was confirmed after washing 4 times with 100

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FIG. 2. Bio-beads encapsulating plasmid DNAs. Encapsulated DNAs are stained with YOYO-1. The YOYO-l-stained image contrast image were photographed under a fluorescent microscope (Axioplan2 Imaging; Carl Zeiss, Giittingen, Germany) by cooled (MicroMax 1401E; Roper Scientific, AZ, USA) and the images were merged using image analysis software (IPLab 3.2; Scanalytics, green and gray, respectively. Bars show 10 urn. (A) Bio-beads produced by the tube mixer method. (B) Bio-beads produced by method.

and the phase CCD camera VA, USA) as the sonication

mM CaCl, followed by staining with YOYO-1 (Fig. 2A). The sizes of the bio-beads are dispersed in a wide range and the bio-beads have teardrop shapes. Beads that are l-100 pm would be too large to transform small cells or protoplasts from carrot or Arubidopsis. Thus we developed the other method, which uses a handy sonicator to make a liner and more uniform emulsion. There was, however, one problem in the use of the sonication method to produce biobeads. DNA molecules can be sheared by sonication. In order to avoid fragmentation by sonication, the plasmid DNAs were added not into the alginate solution with isoamyl alcohol, which was to be sonicated, but into the CaClz solution. With these alterations to the procedure, a finer emulsion and thus smaller bio-beads (0. l-l 5 urn) with plasmid DNAs entrapped on the surface were obtained (Fig. 2B). The distribution in the size of the bio-beads produced by the two methods was calculated from the images of the bio-beads using an image analysis technique (Fig. 3). The results showed that 91% of the bio-beads produced by the sonication method were less than 10 l_trnin diameter. In comparison, 65% of the bio-beads produced by the tube mixer method were 10 urn in diameter. Although bio-beads were produced efficiently using 0.52% sodium alginate solutions, the concentration of sodium alginate affected the physical character of the bio-beads. Lower or higher concentrations of sodium alginate resulted in weak bio-beads or a viscous emulsion inadequate for producing fine bio-beads, respectively. The bio-beads were stable for more than a few weeks in the 100 mM CaClz solution. However, the beads gradually degraded if they were stored in a buffer containing a Ca2’ ion concentration lower than 10 mM. Tobacco cells of the BY-2 cell line were harvested 4 d after subculture and were treated simultaneously with 4% cellulase and 1% pectolyase simultaneously for 2 h at 25°C. Then the BY-2 protoplasts at the final concentration of 1 x 106/ml were suspended in modified Linsmaier and Skoog (LS) medium (13) containing 0.4 M mannitol and used for

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transfection experiments. Transfection with bio-beads were carried out using the PEG treatment method described by Negrutiu et al. (14). The 5 x 10’ protoplasts suspended in 500 ,ttl of a MaMg solution (15 mM MgCl,, 0.4 M mannitol, 0.1% MES, pH 5.8) were mixed with 50 ~1 of the bio-beads carrying entrapped SpUCsGFP plasmid DNA on a Petri dish, followed by the addition of 550 pl PEG solution [40% PEG 6000, 0.1 M Ca(NO,),, 0.4 M mannitol, pH 6-81. After incubation for 30 min, the solution was diluted with 4 ml of 0.2 M CaCl, solution (0.2 M CaCl,, 0.4 M mannitol, pH 5.8) and the protoplasts were gently collected by centrifttgation using a swinging bucket rotor (TS-38LB; Tomy Seiko) at 160 xg %

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FIG. 3. Size distribution of bio-beads generated with the tube mixer and sonication methods. In the case of bio-beads prepared with the tube mixer, about 65% of the beads were clarified into the fraction with a size greater than IO urn in diameter. The largest beads observed were approximately 70 urn. In the case of bio-beads prepared with the sonication method, about 91% of the beads were clarified into the fraction with a size of less than 10 urn in diameter, and no beads observed in the fraction were larger than 15 urn.

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for 5 min. The protoplasts were resuspended in 1 ml of a modified LS medium (13). The number of the protoplasts expressing GFP was counted under an inverted fluorescent microscope (1X-70) using a GFP fluorescent filter after a 24-h incubation in the dark. The efficiency of transfection was calculated as the number of protoplasts with GFP fluorescence divided by the total number of protoplasts (5 x 105) applied. The efficiency of transfection with the two kinds of biobeads was obviously different. The bio-beads produced by the sonication method resulted in two- or three-fold higher transfection efficiencies than the beads produced by the tube mixer method (data not shown). This may indicate that the physical uptake of the bio-beads depends on their size, at least in transfection of BY-2 protoplast. When 5 x lo5 protoplasts were mixed with the bio-beads produced by the method (which contained O-50 pg of SpUCGFP plasmid DNA) and used to transfect BY-2 protoplasts by the PEG method, the expression of GFP was observed 24 h after treatment. In order to quantify the transfection efficiency of the bio-beads, BY-2 protoplasts were mixed either with the improved bio-beads containing the SpUCGFP plasmid DNAs or with the naked plasmid DNAs and transfection rates with the PEG method were compared. The efficiency of transfection was 5- to lo-fold higher in the experiments using bio-beads than with naked plasmids (Fig. 4A). The duration of PEG treatment and the concentration of PEG are fixed to 30 min and 24%, respectively. The transfection efficiency increased as the amount of DNA harbored by the bio-beads increased. In contrast, such a dramatic increase in transfection efficiency was not observed upon addition of increasing amount of naked plasmid DNA. The conditions of the PEG treatment were then optimized. The effect of the duration of the PEG treatment was examined when the amount of DNA used and the concentration of PEG are fixed to 50 pg and 24%, respectively (Fig. 4B). The results indicate that the highest efficiency of transfection was attained after a 30 min treatment and longer treatments resulted in a decrease in efficiency. This means that it is important to balance the transfection promoting effect and the toxic effect of PEG. The efficiency of transfection increased with increasing PEG concentration when the amount of DNA used and the duration of PEG treatment are fixed to 50 pg and 30 min, respectively (Fig. 4C). It was, however, not possible to increase the final concentration of the PEG to more than 24% because of the high viscosity. As a result, the best conditions for BY-2 transfection with bio-beads are as follows. The bio-beads should be prepared with a solution containing as high of a concentration plasmid DNA as possible (50 pg was the limit in the plasmid DNA preparation used). The transfection mixture should contain 24% PEG and duration of the treatment should be 30 min. The efficiency of transfection reached 0.22% at the highest. The effects of the PEG treatment on the transfection efficiency were consistent with the result reported by Negrutiu et al. (14). Our results demonstrated usefulness of the bio-beads for transfection of BY-2 protoplasts. In transformation and/or transfection of animal cells by DDS, the mechanism is explained as active import of the delivery particles by endocy-

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FIG. 4. Optimization of conditions of transfection mediated with bio-beads. (A) Comparison of the transfection efficiency of bio-beads mediated PEG treatment and that of conventional PEG treatment using naked plasmid DNA in variety of DNA amount used. The duration of PEG treatment and the concentration of PEG are fixed to 30 min and 24%, respectively. (B) Effect of the duration of PEG treatment when the amount of DNA used and the concentration of PEG are fixed to 50 pg and 24%, respectively. (C) Effect of PEG concentration when the amount of DNA used and the duration of PEG treatment are fixed to 50 pg and 30min, respectively. The efficiency of transfection reached 0.22% at the highest. Open diamonds show treatment using naked plasmid DNA. Closed squares show bio-beads mediated treatment.

tosis (4-6). It has been reported that plant protoplasts also actively import particles by endocytosis (15). On the other hand, Luo et al. (16) presented a hypothesis that transfection efficiency is due to the concentration of DNA molecules brought by nano-particles toward the surface of cell membrane. In both the models, there should be an optimal size of bio-beads for the efficient transfection. Further experiments will provide the higher transfection efficiency and the critical parameters. For another effect of bio-beads, it is anticipated that DNAs with high molecular weight can be transferred into the cells without damaging the DNA. The

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effect will be verified by using high molecular weight DNAs such as artificial chromosomes, chromosomes, or nuclei itself in the near future. This study was supported in part by the fund “Development of a novel transformation system for plant using laser manipulation techniques” Basic Research Activities for Innovative Biosciences, Japan. We greatly thank Dr. Yasuo Niwa and Dr. Ikuo Nakamura for permission to use their plasmids and technical advises. We also thank Satoru Fujimoto for his technical assistance.

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7. Boussif, O., Lezouak’h, F., Zanta, M. A., Mergny, M.D., Scherman, D., Demeneix, B., and Behr, J.-P.: A vesatile vector for gene and oligonucleotide transfer into cells in culture and in viva: polyethylenimine. Proc. Natl. Acad. Sci. USA, 92,7297-7301 (1995). 8. Godbey, W. T., Wu, K. K., and Mikos, A. G.: Tracking the intracellular path of poly (ethylenimine)/DNA complexes for gene delivery. Proc. Natl. Acad. Sci. USA, 96,5 177-5 18 1 (1999). 9. Borchard, G.: Chitosans for gene delivery. Adv. Drug Deliv. Rev., 52, 145-150 (2001). 10. Kersulec, A., Bazinet, C., Corbiueau, F., Come, D., Barbotin, J. N., Hervagault, J. F., and Thomas, D.: Physiological behaviour of encapsulated somatic embryos. Biomater. Artif. Cells Immobilization Biotechnol., 21, 375-381 (1993). II. Heim, R, Cubit& A. B., and Tsien, R Y.: Improved green fluorescence. Nature, 373,663-664 (1995). 12. Chiu, W., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H., and Sheen, J.: Engineered GFP as a vital reporter in plants. Curr. Biol., 6, 325-330 (1996). 13. Ishida, S., Takahashi, Y., and Nagata, T.: Isolation of cDNA of an auxin-regulated gene encoding a G protein beta subunit-like protein from tobacco BY-2 cells. Proc. Natl. Acad. Sci. USA, 90, 11152-l 1156 (1993). 14. Negrutiu, I., Shillito, R., Potrykus, I., Biasini, G., and Sala, F.: Hybrid genes in the analysis of transformation conditions. Plant Mol. Biol., 8, 363-373 (1987). 15. Fukunaga, Y., Nagata, T., Takebe, I., Kakei, T., and Matsui, C.: An ultrastructural study of the interaction of liposomes with plant protoplasts. Exper. Cell Res., 144, 18 l189 (1983). 16 Luo, D. and Saltzman, W. M.: Enhancement of transfection by physical concentration of DNA at the cell surface. Nat. Biotechnol., 18, 893-895 (2000).