- Email: [email protected]
Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles
FEBS 35708
No. of Pages 6, Model 5G
20 December 2012 FEBS Letters xxx (2012) xxx–xxx 1
journal homepage: www.FEBSLetters.org 5 6
Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles
3 4 7 8 9 10 11 12 13 1 3 5 0 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Q1
Chuan He a, Quanrong Gu a, Min Huang a, James Xing b, Jie Chen a,c,d,⇑ a
Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta, Canada Department of Laboratory Medicine & Pathology, University of Alberta, Edmonton, Alberta, Canada c Department of Biomedical Engineering, University of Alberta, Edmonton, Alberta, Canada d National Institute of Nanotechnology, Edmonton, Alberta, Canada b
a r t i c l e
i n f o
Article history: Received 31 October 2012 Accepted 5 December 2012 Available online xxxx Edited by Julian Schroeder Keywords: Canola cell Protoplast Centrifugation Microbubble Cell wall Membrane
a b s t r a c t We have successfully delivered FITC and FITC-Dextran (70, 250 kDa) into canola protoplasts by centrifuging cells with different amounts of microbubbles at variable centrifuge speed. The efficiency is around 90%, while cell viability remains high. Confocal microscopy images show that both FITC and FITC-Dextran are scattered inside the cytoplasm and the cell nucleus. Pores are observed on canola protoplast cell membranes and cell walls when centrifuged with microbubbles, while the membrane of cells centrifuged alone remain intact and smooth. We hypothesize that the collision between the microbubbles and cells or the bursting of microbubbles are the main reasons for the formation of these pores. Biomaterials can diffuse into the cells once the pathway is created. Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
31 32 33 34 35 36 37 38 39
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
1. Introduction Although numerous studies have been performed on the delivery of biomolecules into mammalian cells, normal chemical-based methods usually do not achieve desired results on plant cells [1]. The main reason is, unlike mammalian cells, plant cells have a solid cell wall made up of cellulose and hemicellulose. This stiff structure is very hard to penetrate. In addition, the cell wall also restricts the plant cells’ endocytotic ability [2], and thus the probability for the cells automatically uptaking the biomolecules is very low when biomolecule carriers, like polymers or lipids, approach the cell surface. The biolistic or particle bombardment system has a long history of being used in plant transformation [3–5]. In this method, biomolecules along with gold bullets enter the target cells due to air pressure acceleration [6–7]. Other methods have also been used including electroporation and sonoporation. For instance, Blackhall has achieved the delivery of FITC-Dextran (150 kDa) into plant protoplasts by electroporation [8], while Harold investigated the effect of electroporation transmembrane potential, acoustic energy exposure, uptake molecule size and the presence of a cell wall on
⇑ Corresponding author. Address: W6-019, ECERF, University of Alberta, Edmonton, Alberta, Canada T6G 2V4. E-mail address: [email protected] (J. Chen).
intracellular uptake and cell viability [9]. These methods can achieve high delivery efficiency, but cell viability is usually low as a trade-off. In this article, we developed a new method of delivering biomolecules such as fluorescein isothiocyanate (FITC) and FITC-Dextran into canola protoplasts using the microbubble assisted centrifuge process. FITC had been widely used as a cell labeling tool for a long time [10–11], and FITC-Dextran is usually considered a membrane impermeable molecule [12–13]. In this experiment, both FITC and FITC-Dextran of molecule weight 70 and 250 kDa, respectively were used for a delivery study. As Fig. 1 shows, the canola protoplasts are first extracted from the original canola cell clusters. The protoplasts were then centrifuged with a mixture of microbubbles and biomolecules. The role of microbubbles is similar to their application in sonoporation: the bursting of microbubbles in a high pressure environment induces cavitation or liquid streaming resulting in pores on the cell membrane [14–20]. In this system, microbubbles were broken possibly due to the centrifuge field or collisions with the canola cells. Flow cytometry (FACS) histograms and confocal microscopy images proved that the biomolecules chosen in our experiment entered the protoplasts after the treatment. The delivery efficiency was around 90%, and the cell viability was around 100% based on cell counts after staining by fluorescein diacetate (FDA). Overall, FITC and FITC-Dextran with molecule weight around 70 and 250 kDa, respectively were
0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.12.005
Q1 Please cite this article in press as: He, C., et al. Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles. FEBS Lett. (2012), http://dx.doi.org/10.1016/j.febslet.2012.12.005
62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86
FEBS 35708
No. of Pages 6, Model 5G
20 December 2012 Q1
2
C. He et al. / FEBS Letters xxx (2012) xxx–xxx
Canola Cell
Canola Protoplast
Separated canola Cell
MB
FITC or FITC-Dextran
Centrifuge Rotator
Fig. 1. Illustration of the proposed delivery method. The thick black circle stands for the cell wall surrounding the canola protoplasts.
89
efficiently delivered into the protoplasts with no compromise in cell viability. The pores formed on the protoplast as shown in the SEM image confirm the delivery mechanism of this method.
90
2. Materials and methods
91
2.1. Microbubble preparation and measurement
92
109
Twenty milligram 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (850365P, Avanti Polar Lipids), 2 mg Tween-80 (P1754, Sigma–Aldrich) and 6 ml chloroform (288306, Sigma–Aldrich) were mixed together in a round-bottom flask (30 ml, KontesÒ) and then evaporated on a rotatory evaporator (LABOROTA 4000, Heidolph). The dried lipid film was stirred with 4 ml PBS solution (14190– 250, Gibco) to a final concentration of 5 mg/ml at 60 °C for 2.5 h. Finally, 4 ml Glycerol (G5516, Sigma) was added to the previously prepared solution, and the mixture was then stirred at 60 °C for another 1.5 h to form the microbubble solution. VialmixÒ, equipment which was used to activate the commercialized ultrasound contrast agent DefinityÒ, was applied to integrate air into the lipidcoated monolayer through a high-speed agitation process at 4530 oscillations per minute for 45 s. To accurately measure the size of the microbubbles, 100 ll activated microbubble solution was diluted into a 50 ml beaker, and the microbubble solution was passed through a 20 lm aperture to obtain size distribution information with a Multisizer™ 3 Coulter CounterÒ.
110
2.2. Cell culture and protoplast preparation
111
Canola cell suspension was maintained on a rotary shaker (160 RPM) at 20 °C in NLN media (pH 6.0, containing 6.5% sucrose, 30 mg/l glutathione, 800 mg/l glutamine, 100 mg/l L-serine, 0.5 mg/l a-naphthaleneacetic acid (NAA), 0.05 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l 2,4-D). To extract the protoplasts,
87 88
93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108
112 113 114 115
(a)
plant cells were first incubated with CPW13M solution, consisting of 27.2 mg/l KH2PO4 (P5655, Sigma–Aldrich), 101 mg/l KNO3 (P8291, Sigma–Aldrich), 1480 mg/l CaCl22H2O (C7902, Sigma–Aldrich), 246 mg/l MgSO47H2O (63138, Sigma–Aldrich), 0.16 mg/l KI (60399, Sigma–Aldrich), 0.025 mg/l CuSO45H2O (C3036, Sigma–Aldrich), and 130 g/L Mannitol (M1902), pH 5.8 for 1 h at room temperature. The solution was then replaced with a digestion solution, consisting of MS salts, 0.06% 2-(N-Morpholino) ethanesulfonic acid (MES), 13% mannitol, 0.025% Pectolyase Y23 (Kanematsu-Gosho, Tokyo, Japan) and 0.4% Cellulase Onozuka R10 (Yakult Honsha Co., Japan), pH 5.8. The solution was then incubated for 5 h at 25 °C in the dark. The digestion mixture was then filtered through a sterile nylon cell strainer (40 lm, BD Falcon, USA) to remove the large cell clusters, and centrifuged (40g) for 8 min afterwards. The resulting pellet was resuspended in CPW13M solution.
116
2.3. Microbubble-assisted centrifuge process
132
1 106 Canola protoplasts were transferred to a 1.5 ml centrifuge tube for the microbubble-assisted centrifuge experiments. A small amount of CPW13M solution was left to submerge the small canola protoplast pellets in the bottom of the centrifuge tube. Five microlitre FITC solution (0.01 mg/ml, dissolved in CPW13M) was added into the centrifuge tube and mixed with the microbubble solution. The solution was then centrifuged at different speeds (expressed in relative centrifugal force (RCF)/centrifuge speeds) (1g/100 RPM, 28g/500 RPM, 112g/1000 RPM, 252g/1500 RPM and 447g/2000 RPM) in an Allegra™ 25R centrifuge (Beckman Coulter) for 2 min. The process was repeated three times. The final amount of FITC solution added was 15 ll, and the final amount of microbubble solution tested was 5, 15, 25, 45 and 65 ll. Finally, the canola protoplasts were centrifuged at 252g (1500 RPM) for an additional 4 min. Before checking the
133
(b)
Diameter (µm) Fig. 2. Microbubble morphology, size distribution. (a) Microbubble size distribution measured by Multisizer™ 3; (b) schematic description of the microbubble structure.
Q1 Please cite this article in press as: He, C., et al. Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles. FEBS Lett. (2012), http://dx.doi.org/10.1016/j.febslet.2012.12.005
117 118 119 120 121 122 123 124 125 126 127 128 129 130 131
134 135 136 137 138 139 140 141 142 143 144 145 146 147
FEBS 35708
No. of Pages 6, Model 5G
20 December 2012 Q1
150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166
delivery results, 400 ll CPW13M solution was used to finally wash the sample at 252g (1500 RPM) for 4 min. To deliver FITC-Dextran, the experiment procedures were the same as the delivery of FITC. In short, each time, 10 ll 100uM FITC-Dextran (70 or 250 kDa, dissolved in CPW13M) was mixed with 5 ll microbubble solution and centrifuged at 252g (1500 RPM) for 2 min. Overall, the final amount of FITC-Dextran solution added was 30 ll, and the final amount of microbubble solution was 15 ll. After that, the canola protoplast solution was centrifuged at 252g (1500 RPM) for 4 min. FITC-Dextran is sticky. To wash down the FITC-Dextran remaining on protoplasts’ membranes, the canola protoplasts were washed in 400 ll CPW13M at 252g (1500 RPM) for 4 min. Comparison experiments were performed in the similar way as the delivery experiments. Except that, after 1 106 protoplasts were submerged in CPW13M solution which just covered the cell pellets’ surfaces, 15 ll FITC (0.01 mg/ml, dissolved in CPW13M) or 30 ll 100 lM FITC-Dextran (FD70 kDa or FD250 kDa, dissolved in CPW13M) were directly added in the protoplast solution and left
(a)
for 6 min. The solution was replaced with 300 ll CPW13M solution and washed at 252g (1500 RPM) for 4 min afterwards.
167
2.4. FACS analysis and cell viability detection
169
The protoplasts were resuspended in 300 ll CPW13M solution after the delivery and washing in order to evaluate the delivery efficiency using a FACSCalibur (Becton–Dickinson). For a cell viability assay, the protoplasts were stained with 3 ll FDA, and 10 ll protoplast solution was then transferred onto a hemocytometer. Images were taken under both the bright and fluorescent fields, and finally merged together by Photoshop CS4. Only the fluorescent cells will be considered as living. The number of fluorescent cells would be counted based on the merged image.
170
2.5. Confocal microscopy and SEM analysis
179
The canola protoplasts were firstly attached onto the cover slip by polylysine. The protoplasts were then dehydrated by 4% paraf-
180
(b) 120
120 Celll Onlyy
100 80
1 ((100R RPM)
60 40
10
5µL
80
65µL L
60 40
M1
M1
20
20 0
Cell Onlyy
100
FIT TC Onnly
Cell number
149
Cell number
148
3
C. He et al. / FEBS Letters xxx (2012) xxx–xxx
0
101
(c)
(e)
10 2 FL1-H
103
0
104
10
(d)
0
101
10 2 FL1-H
103 1
104
(f)
Fig. 3. FITC delivery into canola protoplasts. (a) Delivery efficiency of FITC into canola protoplasts with different centrifuge speeds; (b) delivery efficiency of FITC into canola protoplasts using different amounts of microbubble solution; (c) FACS histogram of canola protoplasts under various conditions. The black curve stands for canola protoplast centrifuged alone, while the blue and green curves stands for the result obtained by mixing FITC with canola protoplast only and FITC centrifuged with microbubles at 1g (100 RPM), respectively; (d) FACS histogram of delivery efficiency with 5 and 65 lL of microbubble solution. The black curve stands for canola protoplasts centrifuged alone, while the red and orange curves stand for result obtained by 5 and 65 lL microbubble solution, respectively; (e) confocal microscopy image of canola protoplast centrifuged alone; (f) confocal microscopy image of canola protoplasts centrifuged by the mixture of microbubbles and FITC.
Q1 Please cite this article in press as: He, C., et al. Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles. FEBS Lett. (2012), http://dx.doi.org/10.1016/j.febslet.2012.12.005
168
171 172 173 174 175 176 177 178
181
FEBS 35708
No. of Pages 6, Model 5G
20 December 2012 Q1
4
C. He et al. / FEBS Letters xxx (2012) xxx–xxx
190
ormadelne for 1.5 h. The cover slip was then turned over and transferred to a slide using Vectashield mounting medium containing DAPI. Images were taken under a Leica SP5 II confocal microscope. For SEM images, 40 104 canola protoplasts were centrifuged with 45 ll microbubble solution at 1006g (3000 RPM) as mentioned before. After that the protoplasts were fixed in pure methanol for 5–10 min, and then underwent critical point drying as mentioned in [21]. SEM images were taken by a JSM 6301 FX emission scanning electron microscope (JEOL) operated at 5 kV.
191
3. Results and discussion
192
3.1. Microbubble size and morphology
193
204
Size distribution of the microbubbles was analyzed by a Multisizer™ 3 and the results are shown Fig. 2a. The horizontal axis denotes the particle size, while the vertical axis conveys the particle number at a particular size. Since the measurement was performed under a 20 lm aperture, which had a proper measurement range from 0.4 to 12 lm. The microbubble size distribution curve started around 500 nm. The curve clearly shows that the microbubble size distribution peak was around 1 lm. DSPC and Tween80 are the main components of the microbubble monolayer, while air is trapped inside [22–23] (Fig. 2b). Glycerol is added in order to further stabilize the microbubble structure by slowing down the air diffusing rate to the surrounding environment.
205
3.2. FITC delivery into canola protoplasts
206
The effect of centrifuge speed and the amount of microbubbble solution on the delivery efficiency were separately identified and quantified by flow cytometry. Fig. 3a show the result of delivery efficiency at different centrifuge speeds: different centrifuge speeds
183 184 185 186 187 188 189
194 195 196 197 198 199 200 201 202 203
207 208 209
(a)
have no significant impact on delivery efficiency. The delivery efficiency could reach 90% even when the centrifuge speed is only 1g (100 RPM). Similarly, the delivery efficiencies were independent of the amount of microbubble solution, as shown in Fig. 3b. The efficiencies are around 85–90%. The FACS histograms of cell fluorescence are shown in Fig. 3c and d. The horizontal axis is the fluorescence strength, while the vertical axis stands for the cell number at certain fluorescence strength. On Fig. 3c protoplasts centrifuged alone are shown as a single population with low fluorescent background on the left. The peak of the fluorescence distribution of protoplasts mixed with FITC for 6 min (blue curve) shifted to the right. The fluorescence distribution of protoplasts centrifuged with microbubbles at 1g (100 RPM) (green curve) shows two peaks and shifted to the far right. This phenomenon shows that after the microbubble assisted centrifugation process, the protoplasts have a stronger fluorescence signal, which also means the microbubbles assisted centrifugation process did help improve the delivery efficiency. To clearly present the delivery difference between samples centrifuged with different amounts of microbubble solution, the FACS histograms were compared between samples centrifuged with 5 and 65 lL of microbubble solution as shown in Fig. 3d. The fluorescence distribution for canola protoplasts centrifuged with 65 lL of microubble solution was much sharper than that with 5 lL of microbubble solution. In other words, more canola protoplasts have up-taken a similar amount of FITC in the sample centrifuged with 65 lL of microbubble solution. The reasons might be that the extra amount of microbubbles opened extra pores on the cell membrane of one single protoplast, or a greater population of overall protoplasts increases diffusing FITC molecules into the cells. Fig. 3e and f show the confocal microscopy images of canola protoplasts centrifuged alone and canola protoplasts with FITC delivered by microbubbles assisted centrifugation. The blue color stands for the cell nucleus, while the green fluorescent color was emitted by FITC un-
(b)
(c)
120
Cell Only FD70KD Only FD250KD Only FD70KD with MB FD250KD with MB
100
Cell number
182
80 60 40
M1
20
(d)
0 10
0
101
10 2 FL1-H
103
104
Fig. 4. Confocal microscopy images of canola protoplasts with delivered FITC-Dextran (a) canola protoplasts centrifuged alone; (b) canola protoplasts centrifuged with microbubbles and FITC-Dextran 70 kDa; the image on the top-right corner of Fig. 4b shows the FITC-Dextran 70 kDa could be delivered even into canola cells with cell walls. The cell wall is marked by the red dashed line; (c) canola protoplasts centrifuged with microbubbles and FITC-Dextran 250 kDa; (d) FACS histogram of canola protoplasts under various conditions: the black curve stands for the canola protoplasts centrifuged alone, the pink and blue curve stand for the canola protoplasts mixed with FTICDextran (70 kDa) and FITC-Dextran (250 kDa) respectively, while the red curve and green curve stand for the canola protoplasts centrifuged with microbubbles and FITCDextran 70 kDa or FITC-Dextran 250 kDa, respectively.
Q1 Please cite this article in press as: He, C., et al. Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles. FEBS Lett. (2012), http://dx.doi.org/10.1016/j.febslet.2012.12.005
210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242
FEBS 35708
No. of Pages 6, Model 5G
20 December 2012 Q1
5
C. He et al. / FEBS Letters xxx (2012) xxx–xxx
(a)
(b)
Fig. 5. Cell viability of canola protoplasts detected by the FDA staining method. (a) Viable cell count with different amounts of microbubble solution; (b) viable cell count after centrifuged at 447g (2000 RPM) with microbubble solution.
(a)
1µm
10µm
(b)
10µm
(c)
1µm
(d) Fig. 6. SEM images of canola cells. (a) A canola protoplast with centrifugation alone; (b) canola protoplasts treated by microbubbles assisted centrifugation; the pores formed are pointed out by the red arrows; (c) canola cells with cell wall centrifuged alone; (d) canola cells with cell wall centrifuged with microbubbles. The red dashed circle marks the area with small pores.
245
der the proper laser stimulation. Fig. 3f clearly shows that most of the canola protoplasts were green confirming that FITCs have been delivered into the protoplasts.
246
3.3. FITC-Dextran delivery into canola protoplasts
247
Fig. 4b and c show the confocal microscopy images of samples centrifuged with the mixture of microbubbles and FITC-Dextran. Both of the images show an intense green fluorescent signal released by FITC-Dextran closely surrounding the cell nucleus (blue colored). On the top right corner of Fig. 4b, an image shows that the FITC-Dextran could even enter walled canola cells after treatment using microbubble assisted delivery. A small population of individual walled canola cells exist due to the fact that cell walls were not fully removed by the enzyme solution. Unlike canola protoplast cells, the
243 244
248 249 250 251 252 253 254 255
walled cells usually do not have a round morphology. The image on the top right of Fig. 4b shows that the bursting of microbubbles could even create pores on the cell wall when centrifuged with microbubbles. The FACS histogram is shown in Fig. 4d. Similar to the results obtained under FITC delivery experiment, when centrifuged with microbubbles, the fluorescence distribution clearly shifted to the right compared to the canola protoplasts centrifuged alone or canola protoplasts only mixed with FITC-Dextran (70 or 250 kDa).
256
3.4. Viability of canola protoplasts treated with microbubble assisted centrifuge process
264
The canola protoplast viability was checked using the FDA staining method. Only the fluorescent protoplasts are considered to be viable, and the number of fluorescent cells was counted using
266
Q1 Please cite this article in press as: He, C., et al. Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles. FEBS Lett. (2012), http://dx.doi.org/10.1016/j.febslet.2012.12.005
257 258 259 260 261 262 263
265
267 268
FEBS 35708
No. of Pages 6, Model 5G
20 December 2012 Q1 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289
290 291
6
C. He et al. / FEBS Letters xxx (2012) xxx–xxx
a hemocytometer. Although different amounts of microbubble solution were used, the number of live cells had no significant change compared to cell control as shown in Fig. 5a. The percentages of the number of viable protoplasts in various amount of microbubble solution compared to the protoplasts centrifuged alone are also shown in Fig. 5a. Except at the point where 25 ll of microbubble solution was used, the values of the percentages go down as the amount of microbubble solution increases. The cell viability of the sample treated with 447g (2000 RPM) was also measured using the same method shown in Fig. 5b. The number of viable canola protoplasts when centrifuged with microbubbles is very similar to the number of viable canola protoplasts when centrifuged alone. All of this data indicates that the damage caused by the microbubble assisted centrifuge process to the protoplasts is very limited. It was hypothesized that the bursting of microbubbles induces pores on the cell membrane, but protoplasts might recover from this temporary pore opening and most of the protoplasts survive after the treatment. This is an obvious advantage to the other physical-based methods (bombardment, sonoporation and electroporation), because the high delivery efficiency of the other methods usually leads to low cell viability.
3.5. SEM images of canola protoplasts treated with the microbubble assisted centrifuge process
312
Fig. 6a shows the canola protoplasts morphology when centrifuged alone. The cell surface is intact and smooth. Canola protoplasts’ surfaces change significantly when centrifuged with microbubbles (Fig. 6b). There are many small pores on the cell surface as pointed out by red arrows. The cell surface also becomes rougher. For canola cells with cell walls, the cells accumulate together and attach to each other (Fig. 6c and d). When centrifuged alone, the canola cells show an integrated cell wall (Fig. 6c). Contrary to the pores scattered on the canola protoplasts’ membranes, pores on cell walls are formed only in certain regions and are much smaller when centrifuged with microbubbles. The regions marked by the red dashed line are magnified and shown in the left part of Fig. 6d. The pores formed on the protoplasts’ membranes and cell walls might due to the bursting of microbubbles during the centrifuge process. Once the intracellular pathway had been created, the biomolecules such as FITC or FITC-Dextran could then be more easily diffused into the cells. The protoplasts’ membranes are mainly composed of a lipid double layer, while the cell walls are made of cellulose. As a result, the cell wall is much more rigid than the cell membrane. The pores created by the impact force are therefore much smaller on the cell wall than on the cell membrane.
313
4. Conclusion
314
Not much research has been reported so far on biomolecule delivery into plant cells. In this study, we have successfully delivered biomolecules of up to 250 kDa into canola protoplasts using a microbubble assisted delivery method. The delivery efficiency was around 90%. Although the delivery efficiency is similar at a variety of centrifuge speeds and amounts of microbubble solution, the FACS histogram showed that a higher amount of microbubble solution could lead to stronger fluorescence. The advantage of this method is the cost is extremely low. In addition, the number of viable canola protoplast after microbubble assisted centrifugation is as high as canola protoplasts which are centrifuged alone with around 90% delivery efficiency. From confocal microscopy images, we observed that FITC-Dextran could be delivered even into canola cells with intact cell walls. This new delivery method holds a great potential. Studies of this method’s
292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311
315 316 317 318 319 320 321 322 323 324 325 326 327 328
applications in plant cell biology and transformation technology are the subjects for our future investigations.
329
Acknowledgments
331
We would like to acknowledge the funding support from Alberta Innovates - Bio Solutions. We would also like to thank Dr. Randall Weselake for providing canola samples and Mr. Scott MacKay for proofreading the manuscript.
332
References
336
[1] Rico, C.M., Majumdar, S., Duarte-Gardea, M., Peralta-Videa, J.R. and GardeaTorresdey, J.L. (2011) Interaction of nanoparticles with edible plants and their possible implications in the food chain. J. Agric. Food Chem. 59, 3485–3498. [2] Ortigosa, M. (2012) Parameters affecting the efficient delivery of mesoporous silica nanoparticle materials and gold nanorods into plant tissues by the biolistic method. Small 8 (3), 413–422. [3] Elghabi, Z., Ruf, S. and Bock, R. (2011) Biolistic co-transformation of the nuclear and plastid genomes. Plant J. 67 (5), 941–948. [4] Finer, J.J., Vain, P., Jones, M.W. and McMullen, M.D. (1992) Development of the particle inflow gun for DNA delivery to plant-cells. Plant Cell Rep. 11, 323–328. [5] Abumhadi, N., Trifonova, A., Takumi, S., Nakamura, C., Todorovska, E., Getov, L., Christov, N. and Atanassov, A. (2011) Development of the particle inflow gun and optimizing the particle bombardment method for efficient genetic transformation in mature embryos of cereals. Biotechnol. Biotech. Equip. 15, 87–96. [6] Cain, P., Keen, N. and Murillo, J. (1993) Development of the particle inflow gun. Plant Cell Tissue Organ Cult. 33 (3), 237–246. [7] Takeuchi, Y., Dotson, M. and Keen, N.T. (1992) Plant transformation – a simple particle bombardment device based on flowing helium. Plant Mol. Biol. 18 (4), 835–839. [8] Blackhall, N.W., Finch, R.P. and Power, J.B. (1995) Flow cytometric quantification of electroporation-mediated uptake of macromolecules into plant protoplasts. Protoplasma 186 (1–2), 50–56. [9] Azencott, R., Peter, F. and Prausnitz, R. (2007) Influence of the cell wall on intracellular delivery to algal cells by electroporation and sonication. Ultrasound Med. Biol. 33 (11), 1805–1817. [10] Vandam, G.J., Bogitsh, B.J. and Fransen Jam. Application of the fitc-anti-fitcgold system to ultrastructural-localization of antigens. J. Histochem. Cytochem. 39 (12), 1725–1728. [11] Kyoung, C.H., Salifullah, L. and Duk, K. (2009) Synthesis and characterization of fluorescein isothiocyanate (FITC)-labeled PEO–PCL–PEO triblock copolymers for topical delivery. Polymer 50 (11), 2357–2364. [12] Shmuel, B., Colin, B. and Jonathan, E. The role of seawater endocytosis in the biomineralization process in calcareous foraminifera. Proc. Natl. Acad. Sci. USA 106(51), 21500–21504. [13] Michaelis, S., Rommel, C., Endell, J., Goring, P., Wehrspohn, R., Steinem, C., Janshoff, A., Galla, H. and Wegener, J. (2012) Macroporous silicon chips for laterally resolved, multi-parametric analysis of epithelial barrier function. Lab Chip 12, 2329–2336. [14] Sakakima, Y., Hayashi, S., Yagi, Y., Hayakawa, A., Tachibana, K. and Nakao, A. (2005) Gene therapy for hepatocellular carcinoma using sonoporation enhanced by contrast agents. Cancer Gene Ther. 12, 884–889. [15] Fong, S.W., Klaseboer, E. and Khoo, B.C. (2008) Interaction of microbubbles with high intensity pulsed ultrasound. J. Acoust. Soc. Am. 123, 1784–1793. [16] Qiu, Y., Luo, Y., Zhang, Y., Cui, W., Zhang, D., Wu, J., Zhang, J. and Tu, J. (2010) The correlation between acoustic cavitation and sonoporation involved in ultrasound-mediated DNA transfection with polyethylenimine (PEI) in vitro. J. Control. Release 145, 40–48. [17] Doinikov, A.A. and Bouakaz, A. (2010) Theoretical investigation of shear stress generated by a contrast microbubble on the cell membrane as a mechanism for sonoporation. J. Acoust. Soc. Am. 128, 11–19. [18] Landini, L., Santarelli, M.F. and Positano, V. (2008) Ultrasound techniques for drug delivery in cardiovascular medicine. Curr. Drug Discov. Technol. 5 (4), 328–332. [19] Van Wamel, A., Kooiman, K., Harteveld, M., Emmer, M., ten Cate, F., Versluis, M. and de Jong, N. (2006) Vibrating microbubbles poking individual cells: drug transfer into cells via sonoporation RID F-3541-2011 RID F-1949-2011. J. Control. Release 112, 149–155. [20] Li, Y.S., Davidson, E., Reid, C.N. and McHale, A.P. (2009) Optimising ultrasoundmediated gene transfer (sonoporation) in vitro and prolonged expression of a transgene in vivo: potential applications for gene therapy of cancer. Cancer Lett. 273, 62–69. [21] Bomblies, K., Shukla, V. and Graham, C. Scanning Electron Microscopy (SEM) of Plant Tissues. Arabidopsis: A Laboratory Manual, Cold Spring harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2002 (Chapter 4). [22] Hettiarachchi, K., Talu, E., Longo, M.L., Dayton, P.A. and Leea, A.P. (2007) Onchip generation of microbubbles as a practical technology for manufacturing contrast agents for ultrasonic imaging. Lab Chip 7 (4), 463–468. [23] Borden, M.A. and Longo, M.L. (2004) Oxygen permeability of fully condensed lipid monolayers. J. Phys. Chem. B 108 (19), 6009–6016.
337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406
330
333 334 335
Q2
407
Q1 Please cite this article in press as: He, C., et al. Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles. FEBS Lett. (2012), http://dx.doi.org/10.1016/j.febslet.2012.12.005
No. of Pages 6, Model 5G
20 December 2012 FEBS Letters xxx (2012) xxx–xxx 1
journal homepage: www.FEBSLetters.org 5 6
Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles
3 4 7 8 9 10 11 12 13 1 3 5 0 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Q1
Chuan He a, Quanrong Gu a, Min Huang a, James Xing b, Jie Chen a,c,d,⇑ a
Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta, Canada Department of Laboratory Medicine & Pathology, University of Alberta, Edmonton, Alberta, Canada c Department of Biomedical Engineering, University of Alberta, Edmonton, Alberta, Canada d National Institute of Nanotechnology, Edmonton, Alberta, Canada b
a r t i c l e
i n f o
Article history: Received 31 October 2012 Accepted 5 December 2012 Available online xxxx Edited by Julian Schroeder Keywords: Canola cell Protoplast Centrifugation Microbubble Cell wall Membrane
a b s t r a c t We have successfully delivered FITC and FITC-Dextran (70, 250 kDa) into canola protoplasts by centrifuging cells with different amounts of microbubbles at variable centrifuge speed. The efficiency is around 90%, while cell viability remains high. Confocal microscopy images show that both FITC and FITC-Dextran are scattered inside the cytoplasm and the cell nucleus. Pores are observed on canola protoplast cell membranes and cell walls when centrifuged with microbubbles, while the membrane of cells centrifuged alone remain intact and smooth. We hypothesize that the collision between the microbubbles and cells or the bursting of microbubbles are the main reasons for the formation of these pores. Biomaterials can diffuse into the cells once the pathway is created. Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
31 32 33 34 35 36 37 38 39
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
1. Introduction Although numerous studies have been performed on the delivery of biomolecules into mammalian cells, normal chemical-based methods usually do not achieve desired results on plant cells [1]. The main reason is, unlike mammalian cells, plant cells have a solid cell wall made up of cellulose and hemicellulose. This stiff structure is very hard to penetrate. In addition, the cell wall also restricts the plant cells’ endocytotic ability [2], and thus the probability for the cells automatically uptaking the biomolecules is very low when biomolecule carriers, like polymers or lipids, approach the cell surface. The biolistic or particle bombardment system has a long history of being used in plant transformation [3–5]. In this method, biomolecules along with gold bullets enter the target cells due to air pressure acceleration [6–7]. Other methods have also been used including electroporation and sonoporation. For instance, Blackhall has achieved the delivery of FITC-Dextran (150 kDa) into plant protoplasts by electroporation [8], while Harold investigated the effect of electroporation transmembrane potential, acoustic energy exposure, uptake molecule size and the presence of a cell wall on
⇑ Corresponding author. Address: W6-019, ECERF, University of Alberta, Edmonton, Alberta, Canada T6G 2V4. E-mail address: [email protected] (J. Chen).
intracellular uptake and cell viability [9]. These methods can achieve high delivery efficiency, but cell viability is usually low as a trade-off. In this article, we developed a new method of delivering biomolecules such as fluorescein isothiocyanate (FITC) and FITC-Dextran into canola protoplasts using the microbubble assisted centrifuge process. FITC had been widely used as a cell labeling tool for a long time [10–11], and FITC-Dextran is usually considered a membrane impermeable molecule [12–13]. In this experiment, both FITC and FITC-Dextran of molecule weight 70 and 250 kDa, respectively were used for a delivery study. As Fig. 1 shows, the canola protoplasts are first extracted from the original canola cell clusters. The protoplasts were then centrifuged with a mixture of microbubbles and biomolecules. The role of microbubbles is similar to their application in sonoporation: the bursting of microbubbles in a high pressure environment induces cavitation or liquid streaming resulting in pores on the cell membrane [14–20]. In this system, microbubbles were broken possibly due to the centrifuge field or collisions with the canola cells. Flow cytometry (FACS) histograms and confocal microscopy images proved that the biomolecules chosen in our experiment entered the protoplasts after the treatment. The delivery efficiency was around 90%, and the cell viability was around 100% based on cell counts after staining by fluorescein diacetate (FDA). Overall, FITC and FITC-Dextran with molecule weight around 70 and 250 kDa, respectively were
0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.12.005
Q1 Please cite this article in press as: He, C., et al. Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles. FEBS Lett. (2012), http://dx.doi.org/10.1016/j.febslet.2012.12.005
62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86
FEBS 35708
No. of Pages 6, Model 5G
20 December 2012 Q1
2
C. He et al. / FEBS Letters xxx (2012) xxx–xxx
Canola Cell
Canola Protoplast
Separated canola Cell
MB
FITC or FITC-Dextran
Centrifuge Rotator
Fig. 1. Illustration of the proposed delivery method. The thick black circle stands for the cell wall surrounding the canola protoplasts.
89
efficiently delivered into the protoplasts with no compromise in cell viability. The pores formed on the protoplast as shown in the SEM image confirm the delivery mechanism of this method.
90
2. Materials and methods
91
2.1. Microbubble preparation and measurement
92
109
Twenty milligram 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (850365P, Avanti Polar Lipids), 2 mg Tween-80 (P1754, Sigma–Aldrich) and 6 ml chloroform (288306, Sigma–Aldrich) were mixed together in a round-bottom flask (30 ml, KontesÒ) and then evaporated on a rotatory evaporator (LABOROTA 4000, Heidolph). The dried lipid film was stirred with 4 ml PBS solution (14190– 250, Gibco) to a final concentration of 5 mg/ml at 60 °C for 2.5 h. Finally, 4 ml Glycerol (G5516, Sigma) was added to the previously prepared solution, and the mixture was then stirred at 60 °C for another 1.5 h to form the microbubble solution. VialmixÒ, equipment which was used to activate the commercialized ultrasound contrast agent DefinityÒ, was applied to integrate air into the lipidcoated monolayer through a high-speed agitation process at 4530 oscillations per minute for 45 s. To accurately measure the size of the microbubbles, 100 ll activated microbubble solution was diluted into a 50 ml beaker, and the microbubble solution was passed through a 20 lm aperture to obtain size distribution information with a Multisizer™ 3 Coulter CounterÒ.
110
2.2. Cell culture and protoplast preparation
111
Canola cell suspension was maintained on a rotary shaker (160 RPM) at 20 °C in NLN media (pH 6.0, containing 6.5% sucrose, 30 mg/l glutathione, 800 mg/l glutamine, 100 mg/l L-serine, 0.5 mg/l a-naphthaleneacetic acid (NAA), 0.05 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l 2,4-D). To extract the protoplasts,
87 88
93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108
112 113 114 115
(a)
plant cells were first incubated with CPW13M solution, consisting of 27.2 mg/l KH2PO4 (P5655, Sigma–Aldrich), 101 mg/l KNO3 (P8291, Sigma–Aldrich), 1480 mg/l CaCl22H2O (C7902, Sigma–Aldrich), 246 mg/l MgSO47H2O (63138, Sigma–Aldrich), 0.16 mg/l KI (60399, Sigma–Aldrich), 0.025 mg/l CuSO45H2O (C3036, Sigma–Aldrich), and 130 g/L Mannitol (M1902), pH 5.8 for 1 h at room temperature. The solution was then replaced with a digestion solution, consisting of MS salts, 0.06% 2-(N-Morpholino) ethanesulfonic acid (MES), 13% mannitol, 0.025% Pectolyase Y23 (Kanematsu-Gosho, Tokyo, Japan) and 0.4% Cellulase Onozuka R10 (Yakult Honsha Co., Japan), pH 5.8. The solution was then incubated for 5 h at 25 °C in the dark. The digestion mixture was then filtered through a sterile nylon cell strainer (40 lm, BD Falcon, USA) to remove the large cell clusters, and centrifuged (40g) for 8 min afterwards. The resulting pellet was resuspended in CPW13M solution.
116
2.3. Microbubble-assisted centrifuge process
132
1 106 Canola protoplasts were transferred to a 1.5 ml centrifuge tube for the microbubble-assisted centrifuge experiments. A small amount of CPW13M solution was left to submerge the small canola protoplast pellets in the bottom of the centrifuge tube. Five microlitre FITC solution (0.01 mg/ml, dissolved in CPW13M) was added into the centrifuge tube and mixed with the microbubble solution. The solution was then centrifuged at different speeds (expressed in relative centrifugal force (RCF)/centrifuge speeds) (1g/100 RPM, 28g/500 RPM, 112g/1000 RPM, 252g/1500 RPM and 447g/2000 RPM) in an Allegra™ 25R centrifuge (Beckman Coulter) for 2 min. The process was repeated three times. The final amount of FITC solution added was 15 ll, and the final amount of microbubble solution tested was 5, 15, 25, 45 and 65 ll. Finally, the canola protoplasts were centrifuged at 252g (1500 RPM) for an additional 4 min. Before checking the
133
(b)
Diameter (µm) Fig. 2. Microbubble morphology, size distribution. (a) Microbubble size distribution measured by Multisizer™ 3; (b) schematic description of the microbubble structure.
Q1 Please cite this article in press as: He, C., et al. Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles. FEBS Lett. (2012), http://dx.doi.org/10.1016/j.febslet.2012.12.005
117 118 119 120 121 122 123 124 125 126 127 128 129 130 131
134 135 136 137 138 139 140 141 142 143 144 145 146 147
FEBS 35708
No. of Pages 6, Model 5G
20 December 2012 Q1
150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166
delivery results, 400 ll CPW13M solution was used to finally wash the sample at 252g (1500 RPM) for 4 min. To deliver FITC-Dextran, the experiment procedures were the same as the delivery of FITC. In short, each time, 10 ll 100uM FITC-Dextran (70 or 250 kDa, dissolved in CPW13M) was mixed with 5 ll microbubble solution and centrifuged at 252g (1500 RPM) for 2 min. Overall, the final amount of FITC-Dextran solution added was 30 ll, and the final amount of microbubble solution was 15 ll. After that, the canola protoplast solution was centrifuged at 252g (1500 RPM) for 4 min. FITC-Dextran is sticky. To wash down the FITC-Dextran remaining on protoplasts’ membranes, the canola protoplasts were washed in 400 ll CPW13M at 252g (1500 RPM) for 4 min. Comparison experiments were performed in the similar way as the delivery experiments. Except that, after 1 106 protoplasts were submerged in CPW13M solution which just covered the cell pellets’ surfaces, 15 ll FITC (0.01 mg/ml, dissolved in CPW13M) or 30 ll 100 lM FITC-Dextran (FD70 kDa or FD250 kDa, dissolved in CPW13M) were directly added in the protoplast solution and left
(a)
for 6 min. The solution was replaced with 300 ll CPW13M solution and washed at 252g (1500 RPM) for 4 min afterwards.
167
2.4. FACS analysis and cell viability detection
169
The protoplasts were resuspended in 300 ll CPW13M solution after the delivery and washing in order to evaluate the delivery efficiency using a FACSCalibur (Becton–Dickinson). For a cell viability assay, the protoplasts were stained with 3 ll FDA, and 10 ll protoplast solution was then transferred onto a hemocytometer. Images were taken under both the bright and fluorescent fields, and finally merged together by Photoshop CS4. Only the fluorescent cells will be considered as living. The number of fluorescent cells would be counted based on the merged image.
170
2.5. Confocal microscopy and SEM analysis
179
The canola protoplasts were firstly attached onto the cover slip by polylysine. The protoplasts were then dehydrated by 4% paraf-
180
(b) 120
120 Celll Onlyy
100 80
1 ((100R RPM)
60 40
10
5µL
80
65µL L
60 40
M1
M1
20
20 0
Cell Onlyy
100
FIT TC Onnly
Cell number
149
Cell number
148
3
C. He et al. / FEBS Letters xxx (2012) xxx–xxx
0
101
(c)
(e)
10 2 FL1-H
103
0
104
10
(d)
0
101
10 2 FL1-H
103 1
104
(f)
Fig. 3. FITC delivery into canola protoplasts. (a) Delivery efficiency of FITC into canola protoplasts with different centrifuge speeds; (b) delivery efficiency of FITC into canola protoplasts using different amounts of microbubble solution; (c) FACS histogram of canola protoplasts under various conditions. The black curve stands for canola protoplast centrifuged alone, while the blue and green curves stands for the result obtained by mixing FITC with canola protoplast only and FITC centrifuged with microbubles at 1g (100 RPM), respectively; (d) FACS histogram of delivery efficiency with 5 and 65 lL of microbubble solution. The black curve stands for canola protoplasts centrifuged alone, while the red and orange curves stand for result obtained by 5 and 65 lL microbubble solution, respectively; (e) confocal microscopy image of canola protoplast centrifuged alone; (f) confocal microscopy image of canola protoplasts centrifuged by the mixture of microbubbles and FITC.
Q1 Please cite this article in press as: He, C., et al. Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles. FEBS Lett. (2012), http://dx.doi.org/10.1016/j.febslet.2012.12.005
168
171 172 173 174 175 176 177 178
181
FEBS 35708
No. of Pages 6, Model 5G
20 December 2012 Q1
4
C. He et al. / FEBS Letters xxx (2012) xxx–xxx
190
ormadelne for 1.5 h. The cover slip was then turned over and transferred to a slide using Vectashield mounting medium containing DAPI. Images were taken under a Leica SP5 II confocal microscope. For SEM images, 40 104 canola protoplasts were centrifuged with 45 ll microbubble solution at 1006g (3000 RPM) as mentioned before. After that the protoplasts were fixed in pure methanol for 5–10 min, and then underwent critical point drying as mentioned in [21]. SEM images were taken by a JSM 6301 FX emission scanning electron microscope (JEOL) operated at 5 kV.
191
3. Results and discussion
192
3.1. Microbubble size and morphology
193
204
Size distribution of the microbubbles was analyzed by a Multisizer™ 3 and the results are shown Fig. 2a. The horizontal axis denotes the particle size, while the vertical axis conveys the particle number at a particular size. Since the measurement was performed under a 20 lm aperture, which had a proper measurement range from 0.4 to 12 lm. The microbubble size distribution curve started around 500 nm. The curve clearly shows that the microbubble size distribution peak was around 1 lm. DSPC and Tween80 are the main components of the microbubble monolayer, while air is trapped inside [22–23] (Fig. 2b). Glycerol is added in order to further stabilize the microbubble structure by slowing down the air diffusing rate to the surrounding environment.
205
3.2. FITC delivery into canola protoplasts
206
The effect of centrifuge speed and the amount of microbubbble solution on the delivery efficiency were separately identified and quantified by flow cytometry. Fig. 3a show the result of delivery efficiency at different centrifuge speeds: different centrifuge speeds
183 184 185 186 187 188 189
194 195 196 197 198 199 200 201 202 203
207 208 209
(a)
have no significant impact on delivery efficiency. The delivery efficiency could reach 90% even when the centrifuge speed is only 1g (100 RPM). Similarly, the delivery efficiencies were independent of the amount of microbubble solution, as shown in Fig. 3b. The efficiencies are around 85–90%. The FACS histograms of cell fluorescence are shown in Fig. 3c and d. The horizontal axis is the fluorescence strength, while the vertical axis stands for the cell number at certain fluorescence strength. On Fig. 3c protoplasts centrifuged alone are shown as a single population with low fluorescent background on the left. The peak of the fluorescence distribution of protoplasts mixed with FITC for 6 min (blue curve) shifted to the right. The fluorescence distribution of protoplasts centrifuged with microbubbles at 1g (100 RPM) (green curve) shows two peaks and shifted to the far right. This phenomenon shows that after the microbubble assisted centrifugation process, the protoplasts have a stronger fluorescence signal, which also means the microbubbles assisted centrifugation process did help improve the delivery efficiency. To clearly present the delivery difference between samples centrifuged with different amounts of microbubble solution, the FACS histograms were compared between samples centrifuged with 5 and 65 lL of microbubble solution as shown in Fig. 3d. The fluorescence distribution for canola protoplasts centrifuged with 65 lL of microubble solution was much sharper than that with 5 lL of microbubble solution. In other words, more canola protoplasts have up-taken a similar amount of FITC in the sample centrifuged with 65 lL of microbubble solution. The reasons might be that the extra amount of microbubbles opened extra pores on the cell membrane of one single protoplast, or a greater population of overall protoplasts increases diffusing FITC molecules into the cells. Fig. 3e and f show the confocal microscopy images of canola protoplasts centrifuged alone and canola protoplasts with FITC delivered by microbubbles assisted centrifugation. The blue color stands for the cell nucleus, while the green fluorescent color was emitted by FITC un-
(b)
(c)
120
Cell Only FD70KD Only FD250KD Only FD70KD with MB FD250KD with MB
100
Cell number
182
80 60 40
M1
20
(d)
0 10
0
101
10 2 FL1-H
103
104
Fig. 4. Confocal microscopy images of canola protoplasts with delivered FITC-Dextran (a) canola protoplasts centrifuged alone; (b) canola protoplasts centrifuged with microbubbles and FITC-Dextran 70 kDa; the image on the top-right corner of Fig. 4b shows the FITC-Dextran 70 kDa could be delivered even into canola cells with cell walls. The cell wall is marked by the red dashed line; (c) canola protoplasts centrifuged with microbubbles and FITC-Dextran 250 kDa; (d) FACS histogram of canola protoplasts under various conditions: the black curve stands for the canola protoplasts centrifuged alone, the pink and blue curve stand for the canola protoplasts mixed with FTICDextran (70 kDa) and FITC-Dextran (250 kDa) respectively, while the red curve and green curve stand for the canola protoplasts centrifuged with microbubbles and FITCDextran 70 kDa or FITC-Dextran 250 kDa, respectively.
Q1 Please cite this article in press as: He, C., et al. Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles. FEBS Lett. (2012), http://dx.doi.org/10.1016/j.febslet.2012.12.005
210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242
FEBS 35708
No. of Pages 6, Model 5G
20 December 2012 Q1
5
C. He et al. / FEBS Letters xxx (2012) xxx–xxx
(a)
(b)
Fig. 5. Cell viability of canola protoplasts detected by the FDA staining method. (a) Viable cell count with different amounts of microbubble solution; (b) viable cell count after centrifuged at 447g (2000 RPM) with microbubble solution.
(a)
1µm
10µm
(b)
10µm
(c)
1µm
(d) Fig. 6. SEM images of canola cells. (a) A canola protoplast with centrifugation alone; (b) canola protoplasts treated by microbubbles assisted centrifugation; the pores formed are pointed out by the red arrows; (c) canola cells with cell wall centrifuged alone; (d) canola cells with cell wall centrifuged with microbubbles. The red dashed circle marks the area with small pores.
245
der the proper laser stimulation. Fig. 3f clearly shows that most of the canola protoplasts were green confirming that FITCs have been delivered into the protoplasts.
246
3.3. FITC-Dextran delivery into canola protoplasts
247
Fig. 4b and c show the confocal microscopy images of samples centrifuged with the mixture of microbubbles and FITC-Dextran. Both of the images show an intense green fluorescent signal released by FITC-Dextran closely surrounding the cell nucleus (blue colored). On the top right corner of Fig. 4b, an image shows that the FITC-Dextran could even enter walled canola cells after treatment using microbubble assisted delivery. A small population of individual walled canola cells exist due to the fact that cell walls were not fully removed by the enzyme solution. Unlike canola protoplast cells, the
243 244
248 249 250 251 252 253 254 255
walled cells usually do not have a round morphology. The image on the top right of Fig. 4b shows that the bursting of microbubbles could even create pores on the cell wall when centrifuged with microbubbles. The FACS histogram is shown in Fig. 4d. Similar to the results obtained under FITC delivery experiment, when centrifuged with microbubbles, the fluorescence distribution clearly shifted to the right compared to the canola protoplasts centrifuged alone or canola protoplasts only mixed with FITC-Dextran (70 or 250 kDa).
256
3.4. Viability of canola protoplasts treated with microbubble assisted centrifuge process
264
The canola protoplast viability was checked using the FDA staining method. Only the fluorescent protoplasts are considered to be viable, and the number of fluorescent cells was counted using
266
Q1 Please cite this article in press as: He, C., et al. Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles. FEBS Lett. (2012), http://dx.doi.org/10.1016/j.febslet.2012.12.005
257 258 259 260 261 262 263
265
267 268
FEBS 35708
No. of Pages 6, Model 5G
20 December 2012 Q1 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289
290 291
6
C. He et al. / FEBS Letters xxx (2012) xxx–xxx
a hemocytometer. Although different amounts of microbubble solution were used, the number of live cells had no significant change compared to cell control as shown in Fig. 5a. The percentages of the number of viable protoplasts in various amount of microbubble solution compared to the protoplasts centrifuged alone are also shown in Fig. 5a. Except at the point where 25 ll of microbubble solution was used, the values of the percentages go down as the amount of microbubble solution increases. The cell viability of the sample treated with 447g (2000 RPM) was also measured using the same method shown in Fig. 5b. The number of viable canola protoplasts when centrifuged with microbubbles is very similar to the number of viable canola protoplasts when centrifuged alone. All of this data indicates that the damage caused by the microbubble assisted centrifuge process to the protoplasts is very limited. It was hypothesized that the bursting of microbubbles induces pores on the cell membrane, but protoplasts might recover from this temporary pore opening and most of the protoplasts survive after the treatment. This is an obvious advantage to the other physical-based methods (bombardment, sonoporation and electroporation), because the high delivery efficiency of the other methods usually leads to low cell viability.
3.5. SEM images of canola protoplasts treated with the microbubble assisted centrifuge process
312
Fig. 6a shows the canola protoplasts morphology when centrifuged alone. The cell surface is intact and smooth. Canola protoplasts’ surfaces change significantly when centrifuged with microbubbles (Fig. 6b). There are many small pores on the cell surface as pointed out by red arrows. The cell surface also becomes rougher. For canola cells with cell walls, the cells accumulate together and attach to each other (Fig. 6c and d). When centrifuged alone, the canola cells show an integrated cell wall (Fig. 6c). Contrary to the pores scattered on the canola protoplasts’ membranes, pores on cell walls are formed only in certain regions and are much smaller when centrifuged with microbubbles. The regions marked by the red dashed line are magnified and shown in the left part of Fig. 6d. The pores formed on the protoplasts’ membranes and cell walls might due to the bursting of microbubbles during the centrifuge process. Once the intracellular pathway had been created, the biomolecules such as FITC or FITC-Dextran could then be more easily diffused into the cells. The protoplasts’ membranes are mainly composed of a lipid double layer, while the cell walls are made of cellulose. As a result, the cell wall is much more rigid than the cell membrane. The pores created by the impact force are therefore much smaller on the cell wall than on the cell membrane.
313
4. Conclusion
314
Not much research has been reported so far on biomolecule delivery into plant cells. In this study, we have successfully delivered biomolecules of up to 250 kDa into canola protoplasts using a microbubble assisted delivery method. The delivery efficiency was around 90%. Although the delivery efficiency is similar at a variety of centrifuge speeds and amounts of microbubble solution, the FACS histogram showed that a higher amount of microbubble solution could lead to stronger fluorescence. The advantage of this method is the cost is extremely low. In addition, the number of viable canola protoplast after microbubble assisted centrifugation is as high as canola protoplasts which are centrifuged alone with around 90% delivery efficiency. From confocal microscopy images, we observed that FITC-Dextran could be delivered even into canola cells with intact cell walls. This new delivery method holds a great potential. Studies of this method’s
292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311
315 316 317 318 319 320 321 322 323 324 325 326 327 328
applications in plant cell biology and transformation technology are the subjects for our future investigations.
329
Acknowledgments
331
We would like to acknowledge the funding support from Alberta Innovates - Bio Solutions. We would also like to thank Dr. Randall Weselake for providing canola samples and Mr. Scott MacKay for proofreading the manuscript.
332
References
336
[1] Rico, C.M., Majumdar, S., Duarte-Gardea, M., Peralta-Videa, J.R. and GardeaTorresdey, J.L. (2011) Interaction of nanoparticles with edible plants and their possible implications in the food chain. J. Agric. Food Chem. 59, 3485–3498. [2] Ortigosa, M. (2012) Parameters affecting the efficient delivery of mesoporous silica nanoparticle materials and gold nanorods into plant tissues by the biolistic method. Small 8 (3), 413–422. [3] Elghabi, Z., Ruf, S. and Bock, R. (2011) Biolistic co-transformation of the nuclear and plastid genomes. Plant J. 67 (5), 941–948. [4] Finer, J.J., Vain, P., Jones, M.W. and McMullen, M.D. (1992) Development of the particle inflow gun for DNA delivery to plant-cells. Plant Cell Rep. 11, 323–328. [5] Abumhadi, N., Trifonova, A., Takumi, S., Nakamura, C., Todorovska, E., Getov, L., Christov, N. and Atanassov, A. (2011) Development of the particle inflow gun and optimizing the particle bombardment method for efficient genetic transformation in mature embryos of cereals. Biotechnol. Biotech. Equip. 15, 87–96. [6] Cain, P., Keen, N. and Murillo, J. (1993) Development of the particle inflow gun. Plant Cell Tissue Organ Cult. 33 (3), 237–246. [7] Takeuchi, Y., Dotson, M. and Keen, N.T. (1992) Plant transformation – a simple particle bombardment device based on flowing helium. Plant Mol. Biol. 18 (4), 835–839. [8] Blackhall, N.W., Finch, R.P. and Power, J.B. (1995) Flow cytometric quantification of electroporation-mediated uptake of macromolecules into plant protoplasts. Protoplasma 186 (1–2), 50–56. [9] Azencott, R., Peter, F. and Prausnitz, R. (2007) Influence of the cell wall on intracellular delivery to algal cells by electroporation and sonication. Ultrasound Med. Biol. 33 (11), 1805–1817. [10] Vandam, G.J., Bogitsh, B.J. and Fransen Jam. Application of the fitc-anti-fitcgold system to ultrastructural-localization of antigens. J. Histochem. Cytochem. 39 (12), 1725–1728. [11] Kyoung, C.H., Salifullah, L. and Duk, K. (2009) Synthesis and characterization of fluorescein isothiocyanate (FITC)-labeled PEO–PCL–PEO triblock copolymers for topical delivery. Polymer 50 (11), 2357–2364. [12] Shmuel, B., Colin, B. and Jonathan, E. The role of seawater endocytosis in the biomineralization process in calcareous foraminifera. Proc. Natl. Acad. Sci. USA 106(51), 21500–21504. [13] Michaelis, S., Rommel, C., Endell, J., Goring, P., Wehrspohn, R., Steinem, C., Janshoff, A., Galla, H. and Wegener, J. (2012) Macroporous silicon chips for laterally resolved, multi-parametric analysis of epithelial barrier function. Lab Chip 12, 2329–2336. [14] Sakakima, Y., Hayashi, S., Yagi, Y., Hayakawa, A., Tachibana, K. and Nakao, A. (2005) Gene therapy for hepatocellular carcinoma using sonoporation enhanced by contrast agents. Cancer Gene Ther. 12, 884–889. [15] Fong, S.W., Klaseboer, E. and Khoo, B.C. (2008) Interaction of microbubbles with high intensity pulsed ultrasound. J. Acoust. Soc. Am. 123, 1784–1793. [16] Qiu, Y., Luo, Y., Zhang, Y., Cui, W., Zhang, D., Wu, J., Zhang, J. and Tu, J. (2010) The correlation between acoustic cavitation and sonoporation involved in ultrasound-mediated DNA transfection with polyethylenimine (PEI) in vitro. J. Control. Release 145, 40–48. [17] Doinikov, A.A. and Bouakaz, A. (2010) Theoretical investigation of shear stress generated by a contrast microbubble on the cell membrane as a mechanism for sonoporation. J. Acoust. Soc. Am. 128, 11–19. [18] Landini, L., Santarelli, M.F. and Positano, V. (2008) Ultrasound techniques for drug delivery in cardiovascular medicine. Curr. Drug Discov. Technol. 5 (4), 328–332. [19] Van Wamel, A., Kooiman, K., Harteveld, M., Emmer, M., ten Cate, F., Versluis, M. and de Jong, N. (2006) Vibrating microbubbles poking individual cells: drug transfer into cells via sonoporation RID F-3541-2011 RID F-1949-2011. J. Control. Release 112, 149–155. [20] Li, Y.S., Davidson, E., Reid, C.N. and McHale, A.P. (2009) Optimising ultrasoundmediated gene transfer (sonoporation) in vitro and prolonged expression of a transgene in vivo: potential applications for gene therapy of cancer. Cancer Lett. 273, 62–69. [21] Bomblies, K., Shukla, V. and Graham, C. Scanning Electron Microscopy (SEM) of Plant Tissues. Arabidopsis: A Laboratory Manual, Cold Spring harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2002 (Chapter 4). [22] Hettiarachchi, K., Talu, E., Longo, M.L., Dayton, P.A. and Leea, A.P. (2007) Onchip generation of microbubbles as a practical technology for manufacturing contrast agents for ultrasonic imaging. Lab Chip 7 (4), 463–468. [23] Borden, M.A. and Longo, M.L. (2004) Oxygen permeability of fully condensed lipid monolayers. J. Phys. Chem. B 108 (19), 6009–6016.
337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406
330
333 334 335
Q2
407
Q1 Please cite this article in press as: He, C., et al. Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles. FEBS Lett. (2012), http://dx.doi.org/10.1016/j.febslet.2012.12.005