Accepted Manuscript Title: Chitosan Stabilized Prussian Blue Nanoparticles For Photothermally Enhanced Gene Delivery Author: Xiao-Da Li Xiao-Long Liang Fang Ma Li-Jia Jing Li Lin Yong-Bo Yang Shan-Shan Feng Guang-Lei Fu Xiu-Li Yue Zhi-Fei Dai PII: DOI: Reference:
S0927-7765(14)00532-3 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.10.001 COLSUB 6662
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
6-5-2014 19-9-2014 1-10-2014
Please cite this article as: X.-D. Li, X.-L. Liang, F. Ma, L.-J. Jing, L. Lin, Y.-B. Yang, S.S. Feng, G.-L. Fu, X.-L. Yue, Z.-F. Dai, Chitosan Stabilized Prussian Blue Nanoparticles For Photothermally Enhanced Gene Delivery, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.10.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chitosan Stabilized Prussian Blue Nanoparticles For Photothermally Enhanced Gene Delivery Xiao-Da Lia, Xiao-Long Liangb, Fang Maa, Li-Jia Jing a, Li Lina, Yong-Bo Yang a, Shan-Shan Feng a, Guang-Lei Fua, Xiu-Li Yue a,*, Zhi-Fei Dai b,* State Key Laboratory of Urban Water Resources and Environment, School of Life
ip t
a
Science and Technology, Harbin Institute of Technology, Harbin 150080, P. R.
Department of Biomedical Engineering, College of Engineering, Peking University,
us
b
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China.
an
Beijing 100871, P. R. China.
*Corresponding author. Tel/Fax: 86-010-62615542
M
Email address: [email protected] ; [email protected]
pt
Abstract:
ed
Homepage: http://bme.pku.edu.cn/~daizhifei
The lack of biosafety and insufficient delivery efficiency of gene-carriers are still
Ac ce
obstacles to human gene therapy. This paper reported highly biocompatible chitosan(CS) functionalized Prussian blue (PB) nanoparticles (designated as CS/PB NPs) for photocontrollable gene delivery. The ultra-small size (～3 nm), positive charge and high physiological stability of CS/PB NPs make it suitable to be a nonviral vector. In addition, CS/PB NPs could effectively convert the near infrared (NIR) light into heat due to its strong absorption in the NIR region, assisting the uptake of NPs by cells. Upon NIR light irradiation, CS/PB NPs showed superior gene transfection efficiency, much higher than that of free polyethylenimine (PEI). Both in vitro and in vivo experiments demonstrated that CS/PB NPs had excellent biocompatiblity. This 1
Page 1 of 31
work also encourages further exploration of the CS/PB NPs as a photocontrollable nanovector for combined photothermal and gene therapy. Keywords: Chitosan, Prussian blue, Biocompatible, Photothermal, Gene delivery Introduction
ip t
During the past two decades, gene therapy has been the subject of preclinical and clinical research due to its great potential to provide a lot of therapeutic application to
cr
tumor patients [1,2]. The development of gene carriers for effectively delivering genes
us
into particular organizations or specific cell types such as tumor cells has attracted a great deal of attention in recent years. Compared with viral vectors, non-viral vectors
an
such as liposomes [3,4], polymers [5-7] and other types of nanomaterials [8-10] show advantages in terms of simplicity of use, ease of large-scale production and lack of
M
specific immune response. However, the clinical applications of tumor gene therapies
safety and efficiency [11].
ed
are still largely hampered by the lack of a smart controllable gene carrier with high
In recent years, many methods based on external physical stimuli such as light
pt
[12], heat, ultrasound [13-15], magnetic field [16,17] and electrical field [18], have
Ac ce
been developed, aiming to improve the gene transfection efficiency and specificity [19,20]. Among these methods, near infrared (NIR) light induced temperature elevation and enhanced gene transfection has aroused extensive attention because of easy operation, highly localized controlling, and deep tissue penetration [21]. Liu et al. demonstrated that the photothermally enhanced intracellular transportation of polyethylene
glycol (PEG)
and polyethylenimine (PEI) dual-functionalized
nanographene oxide for light induced gene delivery [12]. It was demonstrated that the heat generated by NIR photothermal agent nanographene oxide could increase the permeability of tumor cells membrane, and greatly enhance the gene transfection
2
Page 2 of 31
efficiency. But the safety and the biocompatibility of nanographene oxide need more evidence. Prussian blue (PB) is a drug approved by USA Food and Drug Administration (FDA) in clinic for the treatment of radioactive exposure, and the important features
ip t
of PB in biological environments such as the stability in human blood serum and significantly low cytotoxicity have been studied for a long time [22,23]. Furthermore,
cr
its biosafety in the human body has been absolutely approved based on sufficient
us
clinical trials. Our group have demonstrated that PB has strong absorption in the NIR region, showing a superior NIR photothermal effect, good photothermal efficiency
an
and high photothermal stability [24]. It has been reported as a new generation of photothermal ablation agents and photoacoustic imaging contrast agent for cancer
M
therapy [25].
Chitosan (CS) is a naturally occurring linear cationic polysaccharide, showing
ed
good biocompatibility, biodegradability, low immunogenicity and perfect antibacterial property. In recent years, CS has been widely used in drug and gene delivery, tissue
pt
engineering, and as a pharmaceutical ingredient in medical science [26-29]. Chitosan
Ac ce
could be used for preparation of nanocomposites, and it could make the nanoparticles more biocompatible, and was an ideal candidate for gene delivery with high transfection efficiency without notable cytotoxicity [30-33]. In this paper, chitosan functionalized PB nanoparticles (CS/PB NPs) were
fabricated for photothermally enhanced gene delivery. CS/PB NPs were very small (about 3 nm) and stable, it had strong NIR optical absorbance, and could remarkably enhance green fluorescent protein (EGFP) transfection efficiency under NIR laser irradiation. In addition, CS/PB/pDNA showed no significant cytotoxicity to tumor cells, normal cells and immunity cells in vitro and no notable cytotoxicity in vivo.
3
Page 3 of 31
2. Materials and methods 2.1 Materials Potassium ferricyanide, iron(II)
chloride
tetrahydrate (FeCl2•4H2O),
Chitosan(200K), branched polyethyleneimine (b-PEI,25k), dimethyl sulfoxide
ip t
(DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
cr
(MTT) were purchased from Sigma-Aldrich. Agarose (molecular biology
us
grade), EGFP-C1 plasmid, 6×loading buffer, ethidium bromide (EB), acetone and fetal bovine serum (FBS) were purchased from gene company Ltd.
an
RPMI-1640 medium was purchased from Thermal scientific.
M
2.2. Preparation of CS/PB NPs.
Chitosan (200 k) powder was firstly dissolved in hydrochloric acid (0.5 mol
ed
L-1) and stirred at room temperature for 1 h, then filtered through a 0.45 μm
pt
cellulose filter film to give a 3 mg mL-1 chitosan soulution. Aqueous K3Fe(CN)6 solution (1 mM, 20 mL) was added to 80 mL chitosan solution
Ac ce
under the magnetic stirring. 30 minutes later, FeCl2 solution (1 mM, 20 mL) was slowly dropped into the mixture, and the reaction solution turned dark blue slowly. 1 hour later, acetone (200 mL) was added and the mixed solution was centrifuged at 5000 g for 30 min (Avanti J-25, Beckman Coulter). Then, the precipitate was collected carefully. This process including adding acetone, mixing the solution, centrifuging and collecting the precipitate was repeated three times to purify the synthesized CS/PB NPs. Finally, after being desiccated for one night, CS/PB NPs powder sample was obtained [34]. 4
Page 4 of 31
2.3. Characterization of CS/PB NPs. The morphology and structure of the CS/PB NPs were observed by transmission electron microscopy (TEM) (Hitachi, H-7650). The particle size
ip t
distribution and zeta potential of the CS/PB NPs were measured with a Brookhaven Zeta PALS instrument (BI-Zeta Plus-90 Plus). X-ray powder
cr
diffraction (XRD) data were collected on an EMPYREAN (Panalytical) X-ray powder
us
diffractometer. Fourier Translation Infrared Spectroscopy (FT-IR) Spectrum was obtained by a FTIR spectrometer (TENSOR 27, Bruker). Thermogravimetric analysis
an
(TGA) was performed using a TG/DTA NETZSCH STA 449F3 instrument by
M
scanning from 25 to 600 °C under argon at a heating rate of 10 °C min-1. X-ray photoelectron spectroscopy (XPS) data were obtained using an ESCALab250 electron
ed
spectrometer from Thermo Scientific Corporation with nonmonochromatic 300 W AlKα radiation. Pass energy for the narrow scan is 30 eV. The base pressure was
pt
about 6.5×10-10 mbar. The binding energies were referenced to the C1s line at 284.8
Ac ce
eV from alkyl or adventious carbon. The software for processing is Avantage 4.15. In CS/PB, the Fe content was detected by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). For detecting stability, CS/PB NPs were dissolved in deionized water, phosphate buffer solution (PBS) and cell culture medium to obtain the solutions with final concentration of 100 mg L-1, and placed in the room temperature for more than a week. UV-vis-NIR absorbance spectra of the CS/PB NPs in a wavelength range of 400–900 nm was tested by Varian 4000 UV-Vis spectrophotometer. 5
Page 5 of 31
2.4. Preparation of CS/PB/pDNA Complexes for Electrophoresis Assay. Assigned amounts of CS/PB NPs in 10 μL deionized water were mixed with 1 μg pDNA in 10 μL deionized water at different m/m ratios of 0:1, 0.1:1, 0.2:1,
ip t
0.5:1, 1:1, 2:1, 4:1 and 8:1. After incubation at room temperature for 20 min, the as-prepared complexes were analyzed by 0.8% agarose gel electrophoresis
cr
running in Tris-EDTA(TE) buffer at 120 V for 30 min. The gel was imaged by
us
a gel imaging analysis system.
The zeta potential of CS/PB/pDNA samples with different m/m ratio: 0:1,
an
0.2:1, 0.5:1, 1:1, 2:1, 4: 1 and 8:1 was also measured. Briefly, 0.3 mg CS/PB
M
NPs in 1.5 mL deionized water was gently mixed with designated amount of pDNA in 1.5 mL deionized water at room temperature for 20 min before
ed
measurements. Error bars representing standard deviation were measured on
pt
quintuplicate measurement.
Ac ce
2.5. NIR light Induced Temperature Change. An optical fiber coupled NIR laser (808 nm, 2 W) was used. 3.0 mL
aqueous solutions containing different concentrations of CS/PB/pDNA was added to a cuvette, which was irradiated at power densities of 2 W cm-2 for 15 min. The temperature changes were recorded by digital thermometer probe. The temperature was recorded at 10 seconds interval. Deionized water was used as control. 2.6. Cytotoxicity Induced by Laser Irradiation. 6
Page 6 of 31
Photothermal effect of CS/PB/pDNA was evaluated on Human cervical cancer HeLa cell line as determined by the standard MTT assay. Briefly, HeLa cells were seeded in 96 well plates at a density of 1×104 cell per well. After 24
ip t
h incubation at 37 °C, the cell medium was replaced with fresh medium containing 20 mg L−1 CS/PB/pDNA or pDNA. After preheating the cells to
cr
37°C for five minutes, cells were irradiated by the 808 nm laser for 0, 1, 2, 3, 4,
us
5, 6, 7, 8, 9 and 10 min at the power density of 2 W cm− 2 in a 37 °C incubator. Then the cells were replaced with fresh medium and continued to incubate for
an
24 h. After that, 20 μL MTT (5 mg mL-1) was added and incubated with cells
M
for 4 h at 37 °C, followed by 150 μL DMSO dissolving violet precipitations. The absorbance of formazan (produced by the cleavage of MTT by
ed
dehydrogenases in living cells) at 560 nm was directly proportional to the number of live cells. Cell viability was measured using a microplate reader
pt
(Multidkan MK3, Thermo). Cells incubated with serum-supplemented medium
Ac ce
represented 100% cell survival. For each sample, the final absorbance was the average of those measured from five wells in parallel. Error bars representing standard deviation were measured on quintuplicate measurement. 2.7. In vitro pEGFP-C1 Plasmid Transfection. HeLa cells were seeded in 24-well plate at a density of about 1×105 cell per well for pEGFP-C1 plasmid transfection. Briefly, 1 μg pEGFP-C1 plasmid was diluted in 50 μL FBS-free RPMI-1640 medium, and 2 μg CS/PB NPs were dissolved in 50 μL FBS-free RPMI-1640 medium. After being gently mixed, 7
Page 7 of 31
the mixtures were kept at room temperature for 20 min before being added into cells. After preheating the cells to 37°C for five minutes, the 808 nm laser light irradiated the cells for 0, 1, 2 and 3 min at the power density of 2 W cm− 2 in a
ip t
37°C incubator. After 4 h incubation under 37°C, cells were washed with PBS and incubated in 1 mL fresh RPMI-1640 medium for 48 h. Then the pEGFP-C1
cr
expression efficiency was detected by fluorescence microscope and flow
us
cytometer (FCM). For flow cytometry measurement, HeLa cells were collected, washed by phosphate buffered saline (PBS), re-suspended in 1 mL PBS containing 1
an
μg mL −1 PI and stained for 10 min. The cells were centrifuged at 500 g for 5 min,
M
rinsed by PBS, and analyzed by a BD Calibur flow cytometer. For FCM analysis, firstly, 1×106 control cells were used to set the position of the fluorescence strength
ed
as the boundary of the GFP-positive and negative cells. The cells with the fluorescence strength under the position were defined as negative cells and its number
pt
should be 99.9% of all the cells. Then the cells treated with PEI or CS/PB/pDNA with
Ac ce
different laser irradiation time were measured, and the cells with the fluorescence strength above the position were defined as GFP-positive cells.[35,36] Then the percent of the GFP-positive cells excluding the dead cells by PI staining was the gene transfection efficiency. [12, 37] For each sample, the final EGFP transfection efficiency was the average of those measured from five wells in parallel. The transfection rates of the pEGFP-C1 were also assessed by manual counting the percentage of GFP positive cells under fluorescence microscope. Every time, more than 300 cells were selected randomly to count the percentage of GFP
8
Page 8 of 31
positive cells. [38] Error bars representing standard deviation were measured on quintuplicate measurement. 2.8. Cytotoxicity Assay and Biocompatibility Evaluation.
ip t
Human cervical cancer HeLa cell line, HUVECs cell line, Bone marrow
cr
dendritic cells (BMDC) and T cells were selected to evaluate the cytotoxicity of
CS/PB NPs in vitro [39-45]. HeLa cells and HUVECs cells were seeded in 96
us
well plates at a density of 1×104 cell per well in a humidified atmosphere
an
containing 5% CO2 for 24 h. Then, different concentrations of CS/PB NPs and b-PEI were added in HeLa cells or HUVECs cells and incubated for another 24
ed
were assessed by MTT assay.
M
h or 48 h. The cytotoxicity of CS/PB NPs to HeLa cells and HUVECs cells
BMDC cells were generated by culturing bone marrow stem cells from SD
pt
rats in complete IMDM (Iscove’s Modified Dulbecco’s Medium) with 20
Ac ce
ng/mL recombinant granulocyte/ macrophage colony stimulating for 6 days at 37°C. Then, 5×104 cells per well were cocultured with different concentrations of CS/PB NPs in IMDM. T cells were obtained from SD rats and cultured in cIMDM at 37 °C. Then, 5 × 104 cells were cultured with different concentrations of CS/PB NPs in cIMDM. After incubation with nanoparticles for 24 h, cell viability was determined by double staining with both calcein acetoxymethyl ester (calcein-AM) and propidium iodide (PI). The cells were observed by fluorescence microscope. The cytotoxicities of CS/PB NPs to BMDC cells and T cells were also assessed using the standard MTT assay. Five 9
Page 9 of 31
replicate wells were run for each concentration and each experiment was repeated three times. For each sample, the final absorbance was the average of those measured from five wells in parallel. Error bars representing standard
ip t
deviation were measured on quintuplicate measurement. To further investigate the biocompatibility of CS/PB NPs, mice (n=5) were
cr
intravenously injected with CS/PB NPs (1000 mg L-1, 100 μL), PBS was used
us
as control. The body weight of control and treated mice were recorded at 2 days interval. Then, representative organs including heart, liver, spleen, lung, and
an
kidney were collected at day 1, 7 and 30, fixed in formaldehyde solution,
M
embedded in paraffin, followed by section, and stained with hematoxylin and eosin (H&E). The histological sections were observed under an optical
ed
microscope. For each point, the final weight was the average of those measured from five mice. Error bars representing standard deviation were measured on
pt
quintuplicate measurement.
Ac ce
3. Results and Discussion
3.1 Fabrication and characterization of CS/PB NPs The fabrication process of CS/PB NPs, was shown in Figure 1. Chitosan, a widely applied biological material in nanotechnology and medicine, can hold the nanoparticles stable in physiological environment and can be used as the gene delivery vector due to its strong positive charge [26-29]. When K3Fe(CN)6 solution was mixed with the chitosan solution, Fe(CN)63− ions would be bound to chitosan macromolecules and dispersed quite well in the biopolymer matrix. 10
Page 10 of 31
Upon addition of FeCl2 solution, PB nanoparticles were formed by reaction of Fe(CN)63− and FeCl2. In this process, chitosan acted as a template, stabilized the growth of the PB nanoparticles, prevented the nanoparticles aggregating and
ip t
offered the strong positive charge. Therefore, CS/PB NPs had the ability of binding pDNA to obtain CS/PB/pDNA complexes which could be used as the gene delivery
cr
nanovector. Due to the strong absorption in the NIR region and the capability to
us
convert light energy into heat, such complexes could be used as a
an
photocontrollable gene delivery nanovector.
Particle size and stability in physiological environment were very important
M
for the gene delivery vector. The TEM image of the CS/PB NPs revealed the presence of spherical nanoparticles homogeneously dispersed in water (Figure
ed
2A). The nanoparticles did not show any agglomeration, suggesting the CS/PB
pt
NPs were very stable in water. The average size of the CS/PB NPs was estimated to be about 3 nm, which was well consistent with the dynamic light
Ac ce
scattering (DLS) measurement (2.69±0.49 nm). The zeta potential was measured to be 32.04±8.05 mV, ensuring the CS/PB NPs could bind with pDNA.
XRD was used to characterize CS/PB NPs. As shown in Figure 2B, the XRD peak of CS/PB NPs at around 2θ = 24.3° was broader than that of PB NPs, and it was assigned to amorphous chitosan. The other peaks of CS/PB NPs at 2θ = 17.2°, 24.7°, 35.3°, 39.5° and 43.4° were well consistent to 200, 220, 400, 420 and 422 planes of the PB cubic space group Fm3m [34], indicating that PB 11
Page 11 of 31
was embedded within chitosan matrix. FTIR transmittance spectra of the CS/PB NPs are shown in Figure 2C, and chitosan and PB were used for comparison. Peaks of chitosan appeared at 3450 cm−1, 2865 and 2940 cm−1, 1550 and 1580
ip t
cm−1, 1375 and 1455 cm−1, 1020 cm−1 could be attributed to the –O–H, C–H, N–H, CH3 or CH2, and C–O–C groups, respectively. Peaks of PB at 2086 cm−1
cr
attributed to the CN stretching in the Fe2+–CN–Fe3+ of PB. The IR spectrum of
an
further confirming the formation of CS/PB NPs.
us
the CS/PB NPs showed both the corresponded peaks of chitosan and PB,
For quantitatively analyzing, the contents of PB moiety and chitosan in the
M
obtained nanoparticle-polymer complex CS/PB NPs was revealed by TGA technique. From Figure 2D, TGA data for the CS/PB NPs showed a 58.59%
ed
weight loss in a argon atmosphere at 600 °C, and CS and PB have weight losses
pt
of 61.59% and 54.61%, respectively. Therefore, it could be calculated that the composite CS/PB NPs contained about 57.0 wt.% CS and 43.0 wt.% PB. X-ray
Ac ce
photoelectron spectroscopy (XPS) data demonstrate the presence of Fe elements (Figure 2 E). From the XPS data of Fe 2p collected on CS/PB NPs (Figure 2 F), the molar ratio between Fe3+ and Fe2+ was evaluated to be 0.847. In CS/PB NPs, the Fe content was 18.90 ± 0.12 % in mass ratio as detected by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES), and we can determine the weight percentage of PB in the CS/PB NPs was about 41.43 wt.% and CS was 58.57 wt.%, which was very close to the results obtained from the TGA. 12
Page 12 of 31
Figure 2 G showed the CS/PB NPs could be dissolved quite well in water, PBS buffer and cell culture medium, and all three solutions were very stable at room temperature even more than one week. The UV-vis-NIR absorbance
ip t
spectra of CS/PB NPs was also detected. As shown in Figure 2H, aqueous CS/PB NPs exhibit strong absorption in the wavelength of 600-900 nm,
cr
providing a possibility to be exploited for tumor photothermal ablation and
an
3.2 DNA plasmid binding ability of CS/PB NPs
us
photothermally enhanced gene delivery.
The DNA plasmid binding ability of CS/PB NPs at various mass ratios
M
(DNA plasmid/CS/PB NPs) of 0:1, 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, 4: 1 and 8: 1
ed
was evaluated by the agarose gel electrophoresis assays (Figure 3 A), and the corresponding Zeta potential of CS/PB/pDNA complexes was tested (Figure 3
pt
B). The results showed that DNA plasmid could be firmly bound to CS/PB NPs
Ac ce
when the mass ratio was higher than 2:1, at this mass ratio the zeta potentials was 17.12±4.23 mV. Increasing the mass ratio led to higher zeta potentials but a plateau of 25.69±1.21 mV nearly reached at the mass ratio of 8:1. CS/PB/pDNA complexes with mass ratio of 2:1 were selected for the following experiments. According to the content of 57.0 wt.% CS in CS/PB NPs, the N/P ratios was evaluated to be 2.28. 3.3 Temperature elevation of CS/PB/pDNA upon NIR laser irradiation
13
Page 13 of 31
To investigate the temperature elevation of CS/PB/pDNA upon NIR laser irradiation, we measured the temperature changes of 3.0 mL aqueous solutions containing CS/PB/pDNA of different concentrations under the irradiation of an
ip t
808 nm laser (2 W cm-2, 15 min). As shown in Figure 3C, no significant temperature change was observed when pure water was exposed to NIR laser
cr
light. On the contrary, the temperature of CS/PB/pDNA solution increased
us
greatly. After irradiation for 15 min, the temperature elevation of 17.7 °C was achieved for the CS/PB/pDNA sample with concentration of 30 mg L−1, and it
an
was much higher than control (6.6 °C). These results showed CS/PB/pDNA
M
complexes could adsorb NIR light and may take its advantages to enhance gene delivery because of the photothermal effect. To optimize a safe irradiation time
ed
for further gene delivery experiments, 20 mg L−1 CS/PB/pDNA were added to HeLa cells and irradiated with an 808 nm laser (2 W cm-2) for different time.
pt
The relative cell viability was assessed by MTT (Figure 3 D). When the
Ac ce
irradiating time was less than 4 min, cell viability was still above 80%, relatively safe without causing significant cell death. The cell viability of HeLa cells irradiated for 3 min was 92.3±7.4%, and for 4 min was 84.7±15.0%. So irradiation time of 3 min was selected for further gene delivery experiments. 3.4 Photothermal effect of CS/PB NPs on gene transfection efficiency To investigate the photothermal effect of CS/PB NPs on gene transfection efficiency, the pEGFP-C1 was used as the model plasmid. pEGFP-C1 plasmid was first binding to CS/PB NPs in FBS-free RPMI-1640 medium, and then 14
Page 14 of 31
incubated with HeLa cells, followed by the NIR laser (808 nm, 2 W cm−2) irradiation for 0, 1, 2 and 3 min. After being incubated for 4 h and washed with PBS, cells were re-incubated in fresh cell medium for 48 h before fluorescence
ip t
microscope and flow cytometry analysis. Figure 4 A showed the fluorescent microscopic images of HeLa cells gene
cr
transfection efficiency of PEI and CS/PB/pDNA with different laser irradiation
us
time. In control experiments（Figure 4A a, b）, the gene transfection efficiency of PEI/pDNA was 18.31±3.36%, and it was higher than that of CS/PB/pDNA
an
without light irradiation (12.95±2.56%)（Figure 4A c）, but much lower than
M
that of CS/PB/pDNA with light irradiation for 3 min (32.39±3.54%)（Figure 4 A f）. These results were well consistent with the quantification measured by flow
ed
cytometry. As shown in Figure 4 B, when CS/PB/pDNA was used for transfection and the introduction of NIR laser irradiation could notably enhance
pt
the transfection efficiency from 8.59±4.16% (0 min) to 27.78±4.76% (3 min),
Ac ce
which was much higher than the PEI control group of 16.28±2.75%(p
S0927-7765(14)00532-3 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.10.001 COLSUB 6662
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
6-5-2014 19-9-2014 1-10-2014
Please cite this article as: X.-D. Li, X.-L. Liang, F. Ma, L.-J. Jing, L. Lin, Y.-B. Yang, S.S. Feng, G.-L. Fu, X.-L. Yue, Z.-F. Dai, Chitosan Stabilized Prussian Blue Nanoparticles For Photothermally Enhanced Gene Delivery, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.10.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chitosan Stabilized Prussian Blue Nanoparticles For Photothermally Enhanced Gene Delivery Xiao-Da Lia, Xiao-Long Liangb, Fang Maa, Li-Jia Jing a, Li Lina, Yong-Bo Yang a, Shan-Shan Feng a, Guang-Lei Fua, Xiu-Li Yue a,*, Zhi-Fei Dai b,* State Key Laboratory of Urban Water Resources and Environment, School of Life
ip t
a
Science and Technology, Harbin Institute of Technology, Harbin 150080, P. R.
Department of Biomedical Engineering, College of Engineering, Peking University,
us
b
cr
China.
an
Beijing 100871, P. R. China.
*Corresponding author. Tel/Fax: 86-010-62615542
M
Email address: [email protected] ; [email protected]
pt
Abstract:
ed
Homepage: http://bme.pku.edu.cn/~daizhifei
The lack of biosafety and insufficient delivery efficiency of gene-carriers are still
Ac ce
obstacles to human gene therapy. This paper reported highly biocompatible chitosan(CS) functionalized Prussian blue (PB) nanoparticles (designated as CS/PB NPs) for photocontrollable gene delivery. The ultra-small size (～3 nm), positive charge and high physiological stability of CS/PB NPs make it suitable to be a nonviral vector. In addition, CS/PB NPs could effectively convert the near infrared (NIR) light into heat due to its strong absorption in the NIR region, assisting the uptake of NPs by cells. Upon NIR light irradiation, CS/PB NPs showed superior gene transfection efficiency, much higher than that of free polyethylenimine (PEI). Both in vitro and in vivo experiments demonstrated that CS/PB NPs had excellent biocompatiblity. This 1
Page 1 of 31
work also encourages further exploration of the CS/PB NPs as a photocontrollable nanovector for combined photothermal and gene therapy. Keywords: Chitosan, Prussian blue, Biocompatible, Photothermal, Gene delivery Introduction
ip t
During the past two decades, gene therapy has been the subject of preclinical and clinical research due to its great potential to provide a lot of therapeutic application to
cr
tumor patients [1,2]. The development of gene carriers for effectively delivering genes
us
into particular organizations or specific cell types such as tumor cells has attracted a great deal of attention in recent years. Compared with viral vectors, non-viral vectors
an
such as liposomes [3,4], polymers [5-7] and other types of nanomaterials [8-10] show advantages in terms of simplicity of use, ease of large-scale production and lack of
M
specific immune response. However, the clinical applications of tumor gene therapies
safety and efficiency [11].
ed
are still largely hampered by the lack of a smart controllable gene carrier with high
In recent years, many methods based on external physical stimuli such as light
pt
[12], heat, ultrasound [13-15], magnetic field [16,17] and electrical field [18], have
Ac ce
been developed, aiming to improve the gene transfection efficiency and specificity [19,20]. Among these methods, near infrared (NIR) light induced temperature elevation and enhanced gene transfection has aroused extensive attention because of easy operation, highly localized controlling, and deep tissue penetration [21]. Liu et al. demonstrated that the photothermally enhanced intracellular transportation of polyethylene
glycol (PEG)
and polyethylenimine (PEI) dual-functionalized
nanographene oxide for light induced gene delivery [12]. It was demonstrated that the heat generated by NIR photothermal agent nanographene oxide could increase the permeability of tumor cells membrane, and greatly enhance the gene transfection
2
Page 2 of 31
efficiency. But the safety and the biocompatibility of nanographene oxide need more evidence. Prussian blue (PB) is a drug approved by USA Food and Drug Administration (FDA) in clinic for the treatment of radioactive exposure, and the important features
ip t
of PB in biological environments such as the stability in human blood serum and significantly low cytotoxicity have been studied for a long time [22,23]. Furthermore,
cr
its biosafety in the human body has been absolutely approved based on sufficient
us
clinical trials. Our group have demonstrated that PB has strong absorption in the NIR region, showing a superior NIR photothermal effect, good photothermal efficiency
an
and high photothermal stability [24]. It has been reported as a new generation of photothermal ablation agents and photoacoustic imaging contrast agent for cancer
M
therapy [25].
Chitosan (CS) is a naturally occurring linear cationic polysaccharide, showing
ed
good biocompatibility, biodegradability, low immunogenicity and perfect antibacterial property. In recent years, CS has been widely used in drug and gene delivery, tissue
pt
engineering, and as a pharmaceutical ingredient in medical science [26-29]. Chitosan
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could be used for preparation of nanocomposites, and it could make the nanoparticles more biocompatible, and was an ideal candidate for gene delivery with high transfection efficiency without notable cytotoxicity [30-33]. In this paper, chitosan functionalized PB nanoparticles (CS/PB NPs) were
fabricated for photothermally enhanced gene delivery. CS/PB NPs were very small (about 3 nm) and stable, it had strong NIR optical absorbance, and could remarkably enhance green fluorescent protein (EGFP) transfection efficiency under NIR laser irradiation. In addition, CS/PB/pDNA showed no significant cytotoxicity to tumor cells, normal cells and immunity cells in vitro and no notable cytotoxicity in vivo.
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2. Materials and methods 2.1 Materials Potassium ferricyanide, iron(II)
chloride
tetrahydrate (FeCl2•4H2O),
Chitosan(200K), branched polyethyleneimine (b-PEI,25k), dimethyl sulfoxide
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(DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
cr
(MTT) were purchased from Sigma-Aldrich. Agarose (molecular biology
us
grade), EGFP-C1 plasmid, 6×loading buffer, ethidium bromide (EB), acetone and fetal bovine serum (FBS) were purchased from gene company Ltd.
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RPMI-1640 medium was purchased from Thermal scientific.
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2.2. Preparation of CS/PB NPs.
Chitosan (200 k) powder was firstly dissolved in hydrochloric acid (0.5 mol
ed
L-1) and stirred at room temperature for 1 h, then filtered through a 0.45 μm
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cellulose filter film to give a 3 mg mL-1 chitosan soulution. Aqueous K3Fe(CN)6 solution (1 mM, 20 mL) was added to 80 mL chitosan solution
Ac ce
under the magnetic stirring. 30 minutes later, FeCl2 solution (1 mM, 20 mL) was slowly dropped into the mixture, and the reaction solution turned dark blue slowly. 1 hour later, acetone (200 mL) was added and the mixed solution was centrifuged at 5000 g for 30 min (Avanti J-25, Beckman Coulter). Then, the precipitate was collected carefully. This process including adding acetone, mixing the solution, centrifuging and collecting the precipitate was repeated three times to purify the synthesized CS/PB NPs. Finally, after being desiccated for one night, CS/PB NPs powder sample was obtained [34]. 4
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2.3. Characterization of CS/PB NPs. The morphology and structure of the CS/PB NPs were observed by transmission electron microscopy (TEM) (Hitachi, H-7650). The particle size
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distribution and zeta potential of the CS/PB NPs were measured with a Brookhaven Zeta PALS instrument (BI-Zeta Plus-90 Plus). X-ray powder
cr
diffraction (XRD) data were collected on an EMPYREAN (Panalytical) X-ray powder
us
diffractometer. Fourier Translation Infrared Spectroscopy (FT-IR) Spectrum was obtained by a FTIR spectrometer (TENSOR 27, Bruker). Thermogravimetric analysis
an
(TGA) was performed using a TG/DTA NETZSCH STA 449F3 instrument by
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scanning from 25 to 600 °C under argon at a heating rate of 10 °C min-1. X-ray photoelectron spectroscopy (XPS) data were obtained using an ESCALab250 electron
ed
spectrometer from Thermo Scientific Corporation with nonmonochromatic 300 W AlKα radiation. Pass energy for the narrow scan is 30 eV. The base pressure was
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about 6.5×10-10 mbar. The binding energies were referenced to the C1s line at 284.8
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eV from alkyl or adventious carbon. The software for processing is Avantage 4.15. In CS/PB, the Fe content was detected by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). For detecting stability, CS/PB NPs were dissolved in deionized water, phosphate buffer solution (PBS) and cell culture medium to obtain the solutions with final concentration of 100 mg L-1, and placed in the room temperature for more than a week. UV-vis-NIR absorbance spectra of the CS/PB NPs in a wavelength range of 400–900 nm was tested by Varian 4000 UV-Vis spectrophotometer. 5
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2.4. Preparation of CS/PB/pDNA Complexes for Electrophoresis Assay. Assigned amounts of CS/PB NPs in 10 μL deionized water were mixed with 1 μg pDNA in 10 μL deionized water at different m/m ratios of 0:1, 0.1:1, 0.2:1,
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0.5:1, 1:1, 2:1, 4:1 and 8:1. After incubation at room temperature for 20 min, the as-prepared complexes were analyzed by 0.8% agarose gel electrophoresis
cr
running in Tris-EDTA(TE) buffer at 120 V for 30 min. The gel was imaged by
us
a gel imaging analysis system.
The zeta potential of CS/PB/pDNA samples with different m/m ratio: 0:1,
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0.2:1, 0.5:1, 1:1, 2:1, 4: 1 and 8:1 was also measured. Briefly, 0.3 mg CS/PB
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NPs in 1.5 mL deionized water was gently mixed with designated amount of pDNA in 1.5 mL deionized water at room temperature for 20 min before
ed
measurements. Error bars representing standard deviation were measured on
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quintuplicate measurement.
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2.5. NIR light Induced Temperature Change. An optical fiber coupled NIR laser (808 nm, 2 W) was used. 3.0 mL
aqueous solutions containing different concentrations of CS/PB/pDNA was added to a cuvette, which was irradiated at power densities of 2 W cm-2 for 15 min. The temperature changes were recorded by digital thermometer probe. The temperature was recorded at 10 seconds interval. Deionized water was used as control. 2.6. Cytotoxicity Induced by Laser Irradiation. 6
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Photothermal effect of CS/PB/pDNA was evaluated on Human cervical cancer HeLa cell line as determined by the standard MTT assay. Briefly, HeLa cells were seeded in 96 well plates at a density of 1×104 cell per well. After 24
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h incubation at 37 °C, the cell medium was replaced with fresh medium containing 20 mg L−1 CS/PB/pDNA or pDNA. After preheating the cells to
cr
37°C for five minutes, cells were irradiated by the 808 nm laser for 0, 1, 2, 3, 4,
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5, 6, 7, 8, 9 and 10 min at the power density of 2 W cm− 2 in a 37 °C incubator. Then the cells were replaced with fresh medium and continued to incubate for
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24 h. After that, 20 μL MTT (5 mg mL-1) was added and incubated with cells
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for 4 h at 37 °C, followed by 150 μL DMSO dissolving violet precipitations. The absorbance of formazan (produced by the cleavage of MTT by
ed
dehydrogenases in living cells) at 560 nm was directly proportional to the number of live cells. Cell viability was measured using a microplate reader
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(Multidkan MK3, Thermo). Cells incubated with serum-supplemented medium
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represented 100% cell survival. For each sample, the final absorbance was the average of those measured from five wells in parallel. Error bars representing standard deviation were measured on quintuplicate measurement. 2.7. In vitro pEGFP-C1 Plasmid Transfection. HeLa cells were seeded in 24-well plate at a density of about 1×105 cell per well for pEGFP-C1 plasmid transfection. Briefly, 1 μg pEGFP-C1 plasmid was diluted in 50 μL FBS-free RPMI-1640 medium, and 2 μg CS/PB NPs were dissolved in 50 μL FBS-free RPMI-1640 medium. After being gently mixed, 7
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the mixtures were kept at room temperature for 20 min before being added into cells. After preheating the cells to 37°C for five minutes, the 808 nm laser light irradiated the cells for 0, 1, 2 and 3 min at the power density of 2 W cm− 2 in a
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37°C incubator. After 4 h incubation under 37°C, cells were washed with PBS and incubated in 1 mL fresh RPMI-1640 medium for 48 h. Then the pEGFP-C1
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expression efficiency was detected by fluorescence microscope and flow
us
cytometer (FCM). For flow cytometry measurement, HeLa cells were collected, washed by phosphate buffered saline (PBS), re-suspended in 1 mL PBS containing 1
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μg mL −1 PI and stained for 10 min. The cells were centrifuged at 500 g for 5 min,
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rinsed by PBS, and analyzed by a BD Calibur flow cytometer. For FCM analysis, firstly, 1×106 control cells were used to set the position of the fluorescence strength
ed
as the boundary of the GFP-positive and negative cells. The cells with the fluorescence strength under the position were defined as negative cells and its number
pt
should be 99.9% of all the cells. Then the cells treated with PEI or CS/PB/pDNA with
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different laser irradiation time were measured, and the cells with the fluorescence strength above the position were defined as GFP-positive cells.[35,36] Then the percent of the GFP-positive cells excluding the dead cells by PI staining was the gene transfection efficiency. [12, 37] For each sample, the final EGFP transfection efficiency was the average of those measured from five wells in parallel. The transfection rates of the pEGFP-C1 were also assessed by manual counting the percentage of GFP positive cells under fluorescence microscope. Every time, more than 300 cells were selected randomly to count the percentage of GFP
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positive cells. [38] Error bars representing standard deviation were measured on quintuplicate measurement. 2.8. Cytotoxicity Assay and Biocompatibility Evaluation.
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Human cervical cancer HeLa cell line, HUVECs cell line, Bone marrow
cr
dendritic cells (BMDC) and T cells were selected to evaluate the cytotoxicity of
CS/PB NPs in vitro [39-45]. HeLa cells and HUVECs cells were seeded in 96
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well plates at a density of 1×104 cell per well in a humidified atmosphere
an
containing 5% CO2 for 24 h. Then, different concentrations of CS/PB NPs and b-PEI were added in HeLa cells or HUVECs cells and incubated for another 24
ed
were assessed by MTT assay.
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h or 48 h. The cytotoxicity of CS/PB NPs to HeLa cells and HUVECs cells
BMDC cells were generated by culturing bone marrow stem cells from SD
pt
rats in complete IMDM (Iscove’s Modified Dulbecco’s Medium) with 20
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ng/mL recombinant granulocyte/ macrophage colony stimulating for 6 days at 37°C. Then, 5×104 cells per well were cocultured with different concentrations of CS/PB NPs in IMDM. T cells were obtained from SD rats and cultured in cIMDM at 37 °C. Then, 5 × 104 cells were cultured with different concentrations of CS/PB NPs in cIMDM. After incubation with nanoparticles for 24 h, cell viability was determined by double staining with both calcein acetoxymethyl ester (calcein-AM) and propidium iodide (PI). The cells were observed by fluorescence microscope. The cytotoxicities of CS/PB NPs to BMDC cells and T cells were also assessed using the standard MTT assay. Five 9
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replicate wells were run for each concentration and each experiment was repeated three times. For each sample, the final absorbance was the average of those measured from five wells in parallel. Error bars representing standard
ip t
deviation were measured on quintuplicate measurement. To further investigate the biocompatibility of CS/PB NPs, mice (n=5) were
cr
intravenously injected with CS/PB NPs (1000 mg L-1, 100 μL), PBS was used
us
as control. The body weight of control and treated mice were recorded at 2 days interval. Then, representative organs including heart, liver, spleen, lung, and
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kidney were collected at day 1, 7 and 30, fixed in formaldehyde solution,
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embedded in paraffin, followed by section, and stained with hematoxylin and eosin (H&E). The histological sections were observed under an optical
ed
microscope. For each point, the final weight was the average of those measured from five mice. Error bars representing standard deviation were measured on
pt
quintuplicate measurement.
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3. Results and Discussion
3.1 Fabrication and characterization of CS/PB NPs The fabrication process of CS/PB NPs, was shown in Figure 1. Chitosan, a widely applied biological material in nanotechnology and medicine, can hold the nanoparticles stable in physiological environment and can be used as the gene delivery vector due to its strong positive charge [26-29]. When K3Fe(CN)6 solution was mixed with the chitosan solution, Fe(CN)63− ions would be bound to chitosan macromolecules and dispersed quite well in the biopolymer matrix. 10
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Upon addition of FeCl2 solution, PB nanoparticles were formed by reaction of Fe(CN)63− and FeCl2. In this process, chitosan acted as a template, stabilized the growth of the PB nanoparticles, prevented the nanoparticles aggregating and
ip t
offered the strong positive charge. Therefore, CS/PB NPs had the ability of binding pDNA to obtain CS/PB/pDNA complexes which could be used as the gene delivery
cr
nanovector. Due to the strong absorption in the NIR region and the capability to
us
convert light energy into heat, such complexes could be used as a
an
photocontrollable gene delivery nanovector.
Particle size and stability in physiological environment were very important
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for the gene delivery vector. The TEM image of the CS/PB NPs revealed the presence of spherical nanoparticles homogeneously dispersed in water (Figure
ed
2A). The nanoparticles did not show any agglomeration, suggesting the CS/PB
pt
NPs were very stable in water. The average size of the CS/PB NPs was estimated to be about 3 nm, which was well consistent with the dynamic light
Ac ce
scattering (DLS) measurement (2.69±0.49 nm). The zeta potential was measured to be 32.04±8.05 mV, ensuring the CS/PB NPs could bind with pDNA.
XRD was used to characterize CS/PB NPs. As shown in Figure 2B, the XRD peak of CS/PB NPs at around 2θ = 24.3° was broader than that of PB NPs, and it was assigned to amorphous chitosan. The other peaks of CS/PB NPs at 2θ = 17.2°, 24.7°, 35.3°, 39.5° and 43.4° were well consistent to 200, 220, 400, 420 and 422 planes of the PB cubic space group Fm3m [34], indicating that PB 11
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was embedded within chitosan matrix. FTIR transmittance spectra of the CS/PB NPs are shown in Figure 2C, and chitosan and PB were used for comparison. Peaks of chitosan appeared at 3450 cm−1, 2865 and 2940 cm−1, 1550 and 1580
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cm−1, 1375 and 1455 cm−1, 1020 cm−1 could be attributed to the –O–H, C–H, N–H, CH3 or CH2, and C–O–C groups, respectively. Peaks of PB at 2086 cm−1
cr
attributed to the CN stretching in the Fe2+–CN–Fe3+ of PB. The IR spectrum of
an
further confirming the formation of CS/PB NPs.
us
the CS/PB NPs showed both the corresponded peaks of chitosan and PB,
For quantitatively analyzing, the contents of PB moiety and chitosan in the
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obtained nanoparticle-polymer complex CS/PB NPs was revealed by TGA technique. From Figure 2D, TGA data for the CS/PB NPs showed a 58.59%
ed
weight loss in a argon atmosphere at 600 °C, and CS and PB have weight losses
pt
of 61.59% and 54.61%, respectively. Therefore, it could be calculated that the composite CS/PB NPs contained about 57.0 wt.% CS and 43.0 wt.% PB. X-ray
Ac ce
photoelectron spectroscopy (XPS) data demonstrate the presence of Fe elements (Figure 2 E). From the XPS data of Fe 2p collected on CS/PB NPs (Figure 2 F), the molar ratio between Fe3+ and Fe2+ was evaluated to be 0.847. In CS/PB NPs, the Fe content was 18.90 ± 0.12 % in mass ratio as detected by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES), and we can determine the weight percentage of PB in the CS/PB NPs was about 41.43 wt.% and CS was 58.57 wt.%, which was very close to the results obtained from the TGA. 12
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Figure 2 G showed the CS/PB NPs could be dissolved quite well in water, PBS buffer and cell culture medium, and all three solutions were very stable at room temperature even more than one week. The UV-vis-NIR absorbance
ip t
spectra of CS/PB NPs was also detected. As shown in Figure 2H, aqueous CS/PB NPs exhibit strong absorption in the wavelength of 600-900 nm,
cr
providing a possibility to be exploited for tumor photothermal ablation and
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3.2 DNA plasmid binding ability of CS/PB NPs
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photothermally enhanced gene delivery.
The DNA plasmid binding ability of CS/PB NPs at various mass ratios
M
(DNA plasmid/CS/PB NPs) of 0:1, 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, 4: 1 and 8: 1
ed
was evaluated by the agarose gel electrophoresis assays (Figure 3 A), and the corresponding Zeta potential of CS/PB/pDNA complexes was tested (Figure 3
pt
B). The results showed that DNA plasmid could be firmly bound to CS/PB NPs
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when the mass ratio was higher than 2:1, at this mass ratio the zeta potentials was 17.12±4.23 mV. Increasing the mass ratio led to higher zeta potentials but a plateau of 25.69±1.21 mV nearly reached at the mass ratio of 8:1. CS/PB/pDNA complexes with mass ratio of 2:1 were selected for the following experiments. According to the content of 57.0 wt.% CS in CS/PB NPs, the N/P ratios was evaluated to be 2.28. 3.3 Temperature elevation of CS/PB/pDNA upon NIR laser irradiation
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To investigate the temperature elevation of CS/PB/pDNA upon NIR laser irradiation, we measured the temperature changes of 3.0 mL aqueous solutions containing CS/PB/pDNA of different concentrations under the irradiation of an
ip t
808 nm laser (2 W cm-2, 15 min). As shown in Figure 3C, no significant temperature change was observed when pure water was exposed to NIR laser
cr
light. On the contrary, the temperature of CS/PB/pDNA solution increased
us
greatly. After irradiation for 15 min, the temperature elevation of 17.7 °C was achieved for the CS/PB/pDNA sample with concentration of 30 mg L−1, and it
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was much higher than control (6.6 °C). These results showed CS/PB/pDNA
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complexes could adsorb NIR light and may take its advantages to enhance gene delivery because of the photothermal effect. To optimize a safe irradiation time
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for further gene delivery experiments, 20 mg L−1 CS/PB/pDNA were added to HeLa cells and irradiated with an 808 nm laser (2 W cm-2) for different time.
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The relative cell viability was assessed by MTT (Figure 3 D). When the
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irradiating time was less than 4 min, cell viability was still above 80%, relatively safe without causing significant cell death. The cell viability of HeLa cells irradiated for 3 min was 92.3±7.4%, and for 4 min was 84.7±15.0%. So irradiation time of 3 min was selected for further gene delivery experiments. 3.4 Photothermal effect of CS/PB NPs on gene transfection efficiency To investigate the photothermal effect of CS/PB NPs on gene transfection efficiency, the pEGFP-C1 was used as the model plasmid. pEGFP-C1 plasmid was first binding to CS/PB NPs in FBS-free RPMI-1640 medium, and then 14
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incubated with HeLa cells, followed by the NIR laser (808 nm, 2 W cm−2) irradiation for 0, 1, 2 and 3 min. After being incubated for 4 h and washed with PBS, cells were re-incubated in fresh cell medium for 48 h before fluorescence
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microscope and flow cytometry analysis. Figure 4 A showed the fluorescent microscopic images of HeLa cells gene
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transfection efficiency of PEI and CS/PB/pDNA with different laser irradiation
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time. In control experiments（Figure 4A a, b）, the gene transfection efficiency of PEI/pDNA was 18.31±3.36%, and it was higher than that of CS/PB/pDNA
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without light irradiation (12.95±2.56%)（Figure 4A c）, but much lower than
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that of CS/PB/pDNA with light irradiation for 3 min (32.39±3.54%)（Figure 4 A f）. These results were well consistent with the quantification measured by flow
ed
cytometry. As shown in Figure 4 B, when CS/PB/pDNA was used for transfection and the introduction of NIR laser irradiation could notably enhance
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the transfection efficiency from 8.59±4.16% (0 min) to 27.78±4.76% (3 min),
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which was much higher than the PEI control group of 16.28±2.75%(p
