Although numerous studies have been

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 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 FITCDextran 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  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
⇑ Corresponding author. Address: W6-019, ECERF, University of Alberta, Edmonton, Alberta, Canada T6G 2V4.
E-mail address: [e-mail] (J. Chen).
FEBS Letters 587 (2013) 285–290
journal homepage: www.FEBSLetters.org
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.

ผลลัพธ์ (ไทย) 1:

Although numerous studies have been performed on the delivery of biomolecules into mammalian cells, normal chemical-basedmethods usually do not achieve desired results on plant cells [1].The main reason is, unlike mammalian cells, plant cells have a solidcell 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 theprobability for the cells automatically uptaking the biomoleculesis very low when biomolecule carriers, like polymers or lipids, approach the cell surface.The biolistic or particle bombardment system has a long historyof being used in plant transformation [3–5]. In this method, biomolecules along with gold bullets enter the target cells due to airpressure acceleration [6–7]. Other methods have also been usedincluding electroporation and sonoporation. For instance, Blackhallhas achieved the delivery of FITC-Dextran (150 kDa) into plant protoplasts by electroporation [8], while Harold investigated the effectof electroporation transmembrane potential, acoustic energy exposure, uptake molecule size and the presence of a cell wall on intracellular uptake and cell viability [9]. These methods can achievehigh delivery efficiency, but cell viability is usually low as atrade-off.In this article, we developed a new method of delivering biomolecules such as fluorescein isothiocyanate (FITC) and FITCDextran into canola protoplasts using the microbubble assistedcentrifuge process. FITC had been widely used as a cell labeling toolfor a long time [10–11], and FITC-Dextran is usually considered amembrane 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 cellclusters. The protoplasts were then centrifuged with a mixture ofmicrobubbles 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 liquidstreaming resulting in pores on the cell membrane [14–20]. In thissystem, microbubbles were broken possibly due to the centrifugefield 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 thetreatment. The delivery efficiency was around 90%, and the cellviability was around 100% based on cell counts after staining byfluorescein diacetate (FDA). Overall, FITC and FITC-Dextran withmolecule weight around 70 and 250 kDa, respectively were0014-5793/$36.00  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⇑ Corresponding author. Address: W6-019, ECERF, University of Alberta, Edmonton, Alberta, Canada T6G 2V4.ที่อยู่อีเมล: (J. เฉิน) ใน [e-mail] อักษร 587 (2013) 285-290หน้าแรกของสมุดรายวัน: www.FEBSLetters.orgจัดส่งได้อย่างมีประสิทธิภาพเป็นโปด้วยในเซลล์นี้ รูขุมขนเกิดขึ้น protoplast มากรูป SEM ยืนยันกลไกส่งของวิธีการนี้