Measurement of zeta potential of ce

Measurement of zeta potential of cells and binding affinity of Bmattacin2. For zeta potential measurement,
the concentration of bacteria (E. coli ATCC25922 or S. aureus ATCC25923) was adjusted to about 1.5 × 108/
ml according to the Mcfarland standard 0.5. The zeta potentials of four human cells were measured as well, 1 × 106 cells
were incubated in the presence of Bmattacin2 for 30 minutes with different concentrations. After washing with HEPES,
the bacteria or human cells were re-suspended again and treated with Bmattacin2 (dissolved in HEPES as well) in
different concentrations. The pH value was monitored and maintained at 7.4. The suspension was dispersed into disposable
zeta cells with gold electrodes and allowed to equilibrate for 30 min at 25 °C. The zeta potential was measured
using a zeta potential analyzer (Zeta Plus) from Brookhaven Instruments Corporation. The value was automatically
calculated from electrophoretic mobility based on the Smoluchowski equation43,44. The binding affinity of Bmattacin2
was determined by visualizing cells in the presence of FITC labeled Bmattacin2. Cells were seeded on cell chambers
until 70% confluent, 0.5 μM FITC-Bmattacin2 was then added for 24 hours, after thoroughly wash, cytoskeleton were
stained by TRITC-phalloidin. Cells and remaining FITC-Bmattacin2 were observed under fluorescence microscopy.
Preparation of composite membranes by electrospinning. A PLLA solution with a concentration of
1.0% was prepared by dissolving PLLA in a mixed organic solution of chloroform (90 wt%) together with DMF
(10 wt%). 1 mg prepared Bmattacin2 powder was dispersed in 5 g PLLA solution to form the PLLA/Bmattacin2
suspension. A syringe with a needle which was connected to high-voltage electricity was used to load and deliver
this suspension. Electrospinning was carried out at a voltage of 15 KV, a flow rate of 0.3 ml/min and a 10 cm
distance from the needle tip to the receptor, a grounded aluminum foil. Meanwhile, pure PLLA nanofibrous
membrane was also prepared as a control based on the same parameters. For observation of microstructure of
electrospun membranes, samples were compressed to films together with potassium bromide power respectively,
and then their microstructures were investigated by Fourier transform infrared spectrometry (FTIR, Nicolet
5700, Thermo Co.). All spectra were recorded in absorption mode at 2 cm−1 interval wavenumbers from 500 cm−1
to 2500 cm−1. The morphology of electrospun nanofibers was observed by a scanning electron microscope (SEM)
(Stereoscan 440, LEICA). The diameter of electrospun nanofibers were measured by Nano Measurer 1.2 by selecting
fibers randomly at more than 3 different locations. Totally more than 100 different fibers were measured for
each sample. All collected data were analyzed by OrginPro8 (OriginLab Corporation). The values were reported
as the mean ± SD for all of the results. For measurement of mechanical property, the electrospun membrane were
cut into 50 × 10 mm strips respectively and then mechanical performance of these membranes were characterized
by Instrons 5560 (Instron, Canton, MA) with a load cell of 10 N. Each tensile test was performed at room
temperature with a crosshead speed of 5 mm/min and a preload of 0.1 MPa. All reported tensile strength and
tensile stress values represent the average of 7–8 measurements. All collected data were analyzed by OrginPro8
(OriginLab Corporation). The values were reported as mean ± SD for all of the results.
Characterization of PLLA/Bmattacin2 membranes. For investigation of anticancer-cell activity, the lyophilized
Bmattacin2 and 5-FU (sigma) was blended into PLLA solution and the electrospun PLLA/Bmattacin2
(2 wt%), PLLA/5-FU (7.5 wt%), and PLLA/5-FU/Bmattacin2 (7.5 wt%, 2 wt%) membrane were prepared.
Fibrous membranes were placed in multi-well plates. After UV exposure, the cells were seeded at the density
of 6 × 104 cells per cm2. Cells in the wells were washed with PBS before evaluation. The MTS solution and
100 μl of fresh medium were added to each well at the ratio of 1:5, the plates were incubated at 37 °C for 4 hours.
The absorbance of medium at 490 nm was recorded by micro-plate reader. Data are mean with standard deviation
from triplicate determinations. ANOVA was applied for multi-group comparison followed by the Least
Significant Difference (LSD) test for means comparison between the groups. For live/dead staining, cell permeable
esterase-substrate fluorescein diacetate (FDA) together with cell nucleic acid stain propidium iodide (PI)
were used to assess the viability of cells grown on the nanofibrous membrane. Each sample was washed by PBS
for three times and then the cells were stained by rinsing in 1 ug/ml FDA and 1 ug/ml PI for 5 minutes at dark and
room temperature. Then samples were observed by fluorescent microscope. The assessment of intracellular structure
staining and surface morphology observation of PLLA/Bmattac

Kết quả (Việt) 1:

Đo lường của zeta tiềm năng của các tế bào và mối quan hệ ràng buộc của Bmattacin2. Đo lường tiềm năng zeta,nồng độ của các vi khuẩn (E. coli ATCC25922 hoặc S. aureus ATCC25923) được điều chỉnh để khoảng 1,5 × 108 /ml theo Mcfarland chuẩn 0,5. Tiềm năng zeta của bốn tế bào của con người được đo bằng tốt, 1 × 106 tế bàođã được ủ sự hiện diện của Bmattacin2 trong 30 phút với nồng độ khác nhau. Sau khi rửa với HEPES,vi khuẩn hoặc tế bào của con người đã được tái treo một lần nữa và đối xử với Bmattacin2 (hòa tan trong HEPES cũng) trongnồng độ khác nhau. Giá trị pH là giám sát và duy trì ở 7.4. Hệ thống treo được phân tán vào dùng một lầnZeta tế bào với điện cực vàng và phép equilibrate trong 30 phút ở 25 ° C. Zeta tiềm năng được đobằng cách sử dụng một zeta tiềm năng phân tích (Zeta cộng) từ Brookhaven cụ tổng công ty. Giá trị tự độngtính từ electrophoretic mobility dựa trên Smoluchowski equation43, 44. Mối quan hệ ràng buộc của Bmattacin2đã được xác định bằng cách visualizing các tế bào sự hiện diện của FITC có nhãn Bmattacin2. Các tế bào được hạt trên di động chamberscho đến khi chi 70%, cách 0.5 μM FITC-Bmattacin2 sau đó thêm 24 giờ, sau khi triệt để rửa, tópkính màu bởi TRITC-phalloidin. Các tế bào và FITC-Bmattacin2 còn lại đã được quan sát dưới kính hiển vi huỳnh quang.Chuẩn bị của màng tế bào tổng hợp bởi electrospinning. Một giải pháp PLLA với nồng độ của1.0% was prepared by dissolving PLLA in a mixed organic solution of chloroform (90 wt%) together with DMF(10 wt%). 1 mg prepared Bmattacin2 powder was dispersed in 5 g PLLA solution to form the PLLA/Bmattacin2suspension. A syringe with a needle which was connected to high-voltage electricity was used to load and deliverthis suspension. Electrospinning was carried out at a voltage of 15 KV, a flow rate of 0.3 ml/min and a 10 cmdistance from the needle tip to the receptor, a grounded aluminum foil. Meanwhile, pure PLLA nanofibrousmembrane was also prepared as a control based on the same parameters. For observation of microstructure ofelectrospun membranes, samples were compressed to films together with potassium bromide power respectively,and then their microstructures were investigated by Fourier transform infrared spectrometry (FTIR, Nicolet5700, Thermo Co.). All spectra were recorded in absorption mode at 2 cm−1 interval wavenumbers from 500 cm−1to 2500 cm−1. The morphology of electrospun nanofibers was observed by a scanning electron microscope (SEM)(Stereoscan 440, LEICA). The diameter of electrospun nanofibers were measured by Nano Measurer 1.2 by selectingfibers randomly at more than 3 different locations. Totally more than 100 different fibers were measured foreach sample. All collected data were analyzed by OrginPro8 (OriginLab Corporation). The values were reportedas the mean ± SD for all of the results. For measurement of mechanical property, the electrospun membrane were
cut into 50 × 10 mm strips respectively and then mechanical performance of these membranes were characterized
by Instrons 5560 (Instron, Canton, MA) with a load cell of 10 N. Each tensile test was performed at room
temperature with a crosshead speed of 5 mm/min and a preload of 0.1 MPa. All reported tensile strength and
tensile stress values represent the average of 7–8 measurements. All collected data were analyzed by OrginPro8
(OriginLab Corporation). The values were reported as mean ± SD for all of the results.
Characterization of PLLA/Bmattacin2 membranes. For investigation of anticancer-cell activity, the lyophilized
Bmattacin2 and 5-FU (sigma) was blended into PLLA solution and the electrospun PLLA/Bmattacin2
(2 wt%), PLLA/5-FU (7.5 wt%), and PLLA/5-FU/Bmattacin2 (7.5 wt%, 2 wt%) membrane were prepared.
Fibrous membranes were placed in multi-well plates. After UV exposure, the cells were seeded at the density
of 6 × 104 cells per cm2. Cells in the wells were washed with PBS before evaluation. The MTS solution and
100 μl of fresh medium were added to each well at the ratio of 1:5, the plates were incubated at 37 °C for 4 hours.
The absorbance of medium at 490 nm was recorded by micro-plate reader. Data are mean with standard deviation
from triplicate determinations. ANOVA was applied for multi-group comparison followed by the Least
Significant Difference (LSD) test for means comparison between the groups. For live/dead staining, cell permeable
esterase-substrate fluorescein diacetate (FDA) together with cell nucleic acid stain propidium iodide (PI)
were used to assess the viability of cells grown on the nanofibrous membrane. Each sample was washed by PBS
for three times and then the cells were stained by rinsing in 1 ug/ml FDA and 1 ug/ml PI for 5 minutes at dark and
room temperature. Then samples were observed by fluorescent microscope. The assessment of intracellular structure
staining and surface morphology observation of PLLA/Bmattac