Biocompatible nanogold carrier coated with hyaluronic acid for

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Biocompatible nanogold carrier coated with hyaluronic acid for efficient delivery of plasmid or siRNA to mesenchymal stem cells Shan-hui Hsu, Yang-Hao Yu, Chun-An Yeh, Wei-Shen Sun, Shinn-Zong Lin, Ru-Huei Fu, Hsien-Hsu Hsieh, Po-Yuan Wu, and Huey-Shan Hung ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00540 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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ACS Applied Bio Materials

CD68 expression (Anti-inflammatory)

SEM image FITC

Plasmid

CD68

MSCs (Uptake efficiency)

NP-FITC/F-actin

Sod-1 plasmid

(Gene delivery ability)

Nuclear/NP-FITC

siRNA

NP-FITC NP-FITC (uptake by MSCs)

Spleen

Nuclear/NP-FITC

ACS Paragon Plus Environment

Spleen

Nuclear/MSCs/NP-FITC

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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

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Biocompatible nanogold carrier coated with hyaluronic acid for efficient delivery of plasmid or siRNA to mesenchymal stem cells

Shan-hui Hsu1#, Yang-Hao Yu2,3#, Chun-An Yeh4, Wei-Shen Sun4, Shinn-Zong Lin5, Ru-Huei Fu4,6, Hsien-Hsu Hsieh7, Po-Yuan Wu8, Huey-Shan Hung4,6*

1Institute

of Polymer Science and Engineering, National Taiwan University, Taipei, Taiwan,

R.O.C. 2Department

of Acute and Critical Care, Chang-Hua Hospital, Ministry of Health and Welfare,

Changhua, Taiwan, R.O.C. 3School

of Medicine, China Medical University Hospital, Taichung, Taiwan, R.O.C.

4Graduate

Institute of Biomedical Science, China Medical University, Taichung, Taiwan,

R.O.C. 5Center

for Neuropsychiatry, China Medical University Hospital, Taichung, Taiwan, R.O.C.

6Translational

Medicine Research, China Medical University Hospital, Taichung, Taiwan,

R.O.C. 7Blood

Bank, Taichung Veterans General Hospital, Taichung, Taiwan, R.O.C.

8Department

#

of Dermatology, China Medical University Hospital, Taichung, Taiwan, R.O.C.

The authors have equal contribution to this article.

Address correspondence to: Huey-Shan Hung, Graduate Institute of Biomedical Science, China Medical University,

Taichung,

Taiwan.

Tel:

886-4-22052121

Ext

7827;

886-4-22333641; E-Mail: [email protected]


Fax:

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Abstract A fluorescein isothiocyanate (FITC)-labeled, hyaluronic acid (HA)-coated nanogld (NP-FITC) was developed to carry plasmid or siRNA into mesenchymal stem cells (MSCs). NP-FITC was characterized by scanning electron microscopy (SEM), ultraviolet–visible (UV/Vis) spectroscopy, and Fourier transform infrared (FTIR) spectrophotometry. Non-toxicity of NP-FITC in both normal cells and cancer cells was confirmed by the MTT assay. The cellular uptake of NP-FITC at different time points (30 min, 2 hr, and 24 hr) was verified using immunofluorescence assay. The delivery efficiency of plasmid was tested on the delivery of superoxide dismutase-1 (SOD-1) plasmid, where the protein expression of SOD-1 was analyzed by Western blots. In addition, the delivery efficiency of siRNA was tested using CXCR4 siRNA. Besides, the siRNA delivery by NP-FITC was employed to elucidate the molecular mechanism associated with the effect of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1). The biological function of MSCs delivered with chemokine (C-X-C motif) receptor 4 (CXCR4) siRNA was examined using ELISA, gelatin zymography, and migration assay. Finally, we evaluated the tissue distribution of NP-FITC after the direct injection in the retro orbital sinus of mice or after injection of NP-FITC internalized MSCs through the tail vein of mice. The data provided essential information for NP-FITC as a plasmid or siRNA carrier.

Key words ： fluorescein isothiocyanate (FITC), hyaluronic acid (HA), nanogold particle, mesenchymal stem cell (MSC), gene delivery, superoxide dismutase-1 (SOD-1),

chemokine

(C-X-C

motif)

receptor

4

(CXCR4)

1


siRNA


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1. Introduction Nanotechnology has been widely explored for biomedical applications for drug carriers. Traditional drug administration faces the first pass metabolic effect, undergoes the reticulo-endothelial system degradation, risk from narrow therapeutic index and toxic adverse effects. Nanomaterials are designed to carry drugs overcome some of these problems. With the advantages of high surface to volume ratio, tunable modification, and good dispersion, nanomedicines are better effective in controlling drug target and release to achieve precision medicine. Liposome 1, micelle

2

and

nanogold particle (AuNP) 3 have been provided as effective nano-carriers. AuNP has unique chemical and physical properties as well as biocompatibility. AuNP is able to carry various molecules including DNA, peptide, antibody, protein, and drug 4, it is therefore an ideal vehicle for gene or drug treatment. Gene therapy directly targets at disease origin using specific genetic information to repair deranged proteins or cells to cure disease. Virus and non-viral mediated vehicle are two major delivery systems for gene therapy, both have the advantages and limitations5. Non-viral delivery system, merited from the physical and chemical characteristics, delivers constructed genetic materials into target cells through microinjection, gene gun, electroporation and transfection6 to achieve therapeutic effects. Inadequately delivery efficiency is a major concern. New biomaterials such as polylactic-co-glycolic acid (PLGA)7, polyethylene glycol (PEG)8, and hyaluronic acid (HA)9 are continued being created and improved, nanoparticles have emerged potentially prevailing tool for gene therapy. Hyaluronic acid, a natural polysaccharide polymer, are inborn products mainly present

in

human

connective

tissues.

For

their

favorable

characteristics,

bio-absorbable, bio-compatible, and bio-degradable, HA become more and more 2


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popular for biomedical applications7,

10-11.

CD44, a cell surface marker, has been

reported to over express in solid cancers12-13, and becomes an attractively therapeutic target. HA is the primary binding molecule for CD4414. It is preferred to be used to fabricate the surface of nanoparticles15. Mesenchymal stem cells (MSCs) are attractive source for cell therapy in tissue regeneration because of the essentially biocompatible with low immune reaction, and particularly the potential to be induced differentiating into various types of tissue cells16-17. MSCs are able to be obtained from plenty sources including umbilical cord blood, bone marrow, and adipose17. MSCs constitutively express CD44, which is beneficial for uptake of nanoparticles with HA conjugation. With these advantages, MSCs are used to target the lesion sites where to release the designed nanomedicines to achieve precision treatment18, and which may also be applied to be a carrier for cancer therapy. Oxidative stress may be toxic to tissue cells and induced various clinical diseases. In clinical, exposure of excessive reactive oxygen species (ROS) is associated with neurodegenerative disease19. Superoxide dismutase (SOD) is an highly efficient enzyme to catalyze toxic peroxide into demand oxygen20, thus is able to clear excessive superoxide in tissue cells, protecting body from oxidative damage. In a transgenic mice model for studying amyotrophic lateral sclerosis, the mutated SOD-1 gene led to accumulation of mutant proteins and eventually induced paralysis21. Angiogenesis is a pathophysiological process for tissue repairing, and is also employed by cancers tissues. For injured vessels, the main chemokine (stromal cell derived factor-1, SDF-1), secreted from local tissues, attracts stem cells migrating to the injured lesion22, where the damaged tissues secretes vascular endothelial growth factor (VEGF) to regulate stem cells differentiating into endothelial cells to repair 3



vessels23-24. Cancer cells, however, secrete VEGF, fibroblast growth factor (FGF), and many other bioactive substances to promote survival25. Focusing on the SDF-1/CXCR4 axis, Jeun et. al. constructed CXCR4 overexpressed MSCs showing the powerful migration capacity in vitro and in vivo toward glioma tissues26, suggesting overexpression of CXCR4 will be a potential strategy for stem cell-based cancer treatment. One our prior study showed that stem cells was able to uptake peptide conjugated nanogold-based cargo to impact differentiation27. Herein, we used FITC-labeled HA-coated AuNP (NP-FITC) to carry the SOD-1 plasmid and to observe whether the conjugated SOD-1 plasmid can be efficiently taken up by MSCs. We also explored the biological functions of MSCs after treating with CXCR4 siRNA conjucated NP-FITC. In vivo mice model was used to trace tissue distribution of these nanoparticles, and those carried by MSCs.

4


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2. Materials and Methods 2.1. Materials preparation The HA-coated Au nanoparticles were synthesized by following the previous report28. HAuCl4 (1000 l, 7.5 mM), HA (500 l, 6 mM), and deionized water (1500 l) were mixed in a sample flask for a final concentration of 2.5 mM HAuCl4 and 1 mM HA. The solution was then heated at 37℃ with a water bath for 60 min. FITC was dissolved in anhydrous DMSO at 0.1 mg/ml. For each 5 ml of HA-coated Au colloids, 50 ml of FITC solution was slowly added in 5 ml aliquots while the HA-coated Au colloids were gently and continuously stirred. After all the required amount of FITC solution was added, the reaction was continued in the dark for overnight at 4oC. The resulting FITC-labeled HA-coated AuNP (NP-FITC) was washed two times with deionized water and kept in a dark, oxygen-free environment.

2.2. Binding DNA or siRNA to NP-FITC The specific SOD-1 plasmid (0.5 g/ml) was incorporated with SNAP-Cell TMR-Star plasmid tag label SNAP-tag® fusion proteins inside (BioLabs, Inc, New England). SNAP-Cell® TMR-Star is a red fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells, on cell surfaces, or in vitro. The cell-permeable substrate (CP-6-TMR) is suitable for standard rhodamine filter sets. This package contains 30 nM of SNAP-Cell TMR-Star substrate, sufficient to make 10 ml of a 3 µM SNAP-tag fusion protein labeling solution. Following synthesis, NP-FITC was first concentrated up to 10-fold by membrane filtration. Then 150 mM 3-(3-(dimethylamino)propy)carbodiimide

hydrochloride

(EDC)

N-hydroxysuccinimide (NHS) (final concentrations) were added,

and

7.5

mM

with

pH value

controlled at 5.5, to react with the concentrated NP-FITC and SOD-1 plasmid or 5



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CXCR4 siRNA (25 nM) (Santa Cruz, USA) in a 1 ml total reaction volume in phosphate buffered saline (PBS) for 2 hr at room temperature and placed in 4oC until ready to use. The resulting conjugated nanoparticles were each abbreviated as NP-FITC-SOD-1 and NP-FITC-CXCR4 siRNA.

2.3. Nanoparticle characterization The

absorption

of

bare

AuNP,

NP-FITC,

NP-FITC-SOD-1,

and

NP-FITC-CXCR4 siRNA solutions was analyzed by a UV/Vis spectrophotometer (Helios Zeta) (Thermo Fisher Scientific Inc, USA). TEM images were acquired using a JEM 1010, JEOL electron microscope (JEOL Tokyo, Japan), setting acceleration voltage at 80 keV, to visualize particle size and microstructure. Samples for TEM imaging were prepared by adding 5 µL of the nanoparticle suspension onto a copper-coated TEM grid, then were dried out at room temperature. The hydrodynamic diameter and zeta potential were estimated using a DLS analyzer (ZETASIZER, Malvern ZS90, Taiwan) with the software provided by manufacturer. Practically, we prepared the tested sample as 1 ml in a disposable cuvette, and measured the parameters at operating temperature of 25°C. Data were analyzed in triplicate. Besides, each sample was mixed with KBr and put in a 60oC oven overnight and measured using a Fourier transform infrared spectrometer (FTIR) (Shimadzu FTIR Model IRPrestige-21, Japan), scanning in the spectral region of 600-2000 cm−1 to produce each spectrum.

2.4. Blood cell compatibility of NP-FITC Whole blood was obtained from healthy donors (IRB approval number CE12164) and monocytes were isolated by Percoll (Sigma, USA) density gradient centrifugation. 6


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Cells were resuspended and adjusted to the final concentration of 1×105 cells/ml in RPMI medium with 10% fetal bovine serum (FBS) and 1% (v/v) antibiotics (10,000 U/ml penicillin G and 10 mg/ml streptomycin).1 l of NP-FITC (stock concentration: 1 mg/ml) was added at final concentration (1 g/ml) to each well of 24 well culture plate and then 1 ml of monocytes was added per well. The plate was moved to a 37oC incubator with a humidified atmosphere and 5% CO2 for 96 hr. The cells were then trypsinized and the numbers of monocytes and macrophages were counted according to the cell morphology by an inverted phase contrast microscope and used for the calculation of the ratio of macrophages to monocytes (as the inflammatory index). The expression of CD68 on macrophages after 96 hr of incubation was examined by immunofluorescence staining with anti-CD68 antibody and FITC-conjugated secondary antibody under a fluorescence miroscope (Zeiss Axio Imager A1, USA). Cell nuclei were revealed by 4, 6-diamidino-2-phenylindole (DAPI) staining28. 1 ml of platelet-rich plasma (2106 platelets) (IRB, CE12164) was added in the 24-well culture plate and 1 l of NP-FITC (stocking concentration: 1 mg/ml) was added which containing NP-FITC at 1 g/ml (final concentration). The plasma was removed after incubation for 1 hr and the number of adherent platelets was counted by a cell counter (Metasizer, Coulter, USA). After the CPD process, samples were subjected to the standard procedures before SEM observation28.

2.5. Culture of cells Human umbilical cord Wharton’s jelly tissue mesenchymal stem cells (MSCs) and non small cell lung cancer cell (A549) were grown in high glucose Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (FBS) serum and 1% (v/v) antibodies (10,000 U/ml penicillin G and 10 mg/ml streptomycin) in the 7



Page 10 of 54

37oC incubator. Bovine aortic endothelial cells (BAEC) and human skin fibroblasts (HSF) were grown in low glucose Dulbecco’s modified Eagle medium supplemented with 10% FBS and 1% (v/v) antibiotics (10,000 U/ml penicillin G and 10 mg/ml streptomycin) in the 37oC incubator. Breast cancer cells (Her-2) and colon cancer cells (Colo-205) were cultured using F12 medium supplemented with 10% FBS and 1% (v/v) antibiotics (10,000 U/ml penicillin G and 10 mg/ml streptomycin) in the 37oC incubator.

2.6. Cell viability and reactive oxygen species (ROS) assay To evaluate cell viability in the presence of NP-FITC, cells were seeded onto 96 well-plates at a density of 7103 cells per well. NP-FITC at 1 g/ml was added into the culture medium and incubated for 24 hr, 48 hr, and 72 hr. After incubation, culture medium were removed, then (3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) solution (0.5 mg/ml) were added with 100 l at 37°C for 2 hr. Thereafter, the formazan crystal was dissolved using dimethyl sulfoxide (DMSO), and the optical density was read by a UV-Vis Microplate reader (SpectraMax M2e, Molecular Devices, USA) at 570 nm wavelength. The generation of intracellular reactive oxygen species (ROS) was measured by the cell-permeable fluorogenic probe 2’,7’-dichlorofluorescin diacetate (DCFH-dA) (Sigma, USA). After cells were cultured with NP-FITC (1 g/ml) on different material, cells were trypsinized, washed twice with PBS, and incubated with 10 M of DCFH-dA at 37oC for 30 min. Cells were then subjected to cell sorting by a FACS Calibur flow cytometer (Becton Dickinson, USA) using the following setting: excitation/emission wavelength of 480/530 nm at 30-min intervals for 4 hr. The fluorescence intensity is increased with the generation of net intracellular ROS. The 8


Commented [u1]:

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fluorescein-positive cells were further analyzed by the FACS software (Becton Dickinson, USA) 28.

2.7. Cellular uptake of NP-FITC Cells were seeded onto 24 well-plates at a density of 1104 cells per well. After 24 hr of incubation, NP-FITC at 1 μg/ml was added into culture medium and incubated for 30 min, 2 hr and 24 hr. Cells were washed with PBS buffer, fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min, and permeabilized with 0.5% (v/v) Triton-X100 (Sigma, USA) in PBS for 10 min prior to staining. After then, the non-specific binding was blocked with 5% FBS. Next, cells were stained with rhodamine phalloidin for 30 min. Finally, the cell nuclei were then stained with DAPI (1 g/ml) for 10 min in the dark and observed by the fluorescence microscope. To quantify the cellular uptake, the fluorescence intensity of fluorescein positive cells was analyzed by Flow cytometer. The fluorescein-positive cells were further quantified by the FACS software (Becton Dickinson, USA) 28.

2.8. SOD-1 expression by Westem blotting The protein expression level of SOD-1 in MSCs was determined by Western blotting. After 48 hr of incubation with NP-FITC-SOD-1, cells were collected by trypsin (0.05%), centrifuged, and washed by PBS. Cell pellets were added with the lysis buffer, rotated for 1 hr, and centrifuged for 15 min at 13000 rpm. The protein supernatant was collected and quantified by a protein assay kit (Bio-Rad Labs, USA). Each sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the proteins and then transferred onto a PVDF membrane. After that, the membrane was blocked with 5% non-fat dry milk in 9



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wash buffer for 1 hr at room temperature before overnight incubation with the primary anti-SOD-1 antibody (ProSpect-Tany TechnoGene Ltd). The membrane was then incubated with HRP-conjugated secondary antibodies and detected with the ECL Western blotting detection system.

2.9. CXCR4 expression by immunofluorescence staining and flow cytometry MSCs were seeded onto 24 well-plates at a density of 1104 cells per well, and were co-treated with NP-FITC-CXCR4 siRNA (25 nM), as well as SDF-1 (50 ng/ml) or VEGF (50 ng/ml) for 48 hr. Cells were washed with PBS buffer, fixed with 4% paraformaldehyde (Sigma, USA) for 15 min, and permeabilized with 0.5% (v/v) Triton-X100 (Sigma,

USA) in PBS for 10 min prior to staining. After then, the

non-specific binding was blocked with 5% FBS. CXCR 4 siRNA which contained a pool of 3 different siRNA duplexes: A: sense CUAGCUUUCUUCCACUGUUTT anti-sense

AACAGUGGAAGAAAGCUAGTT,

B:

sense

CAGAGCGUGUAGUGAAUCATT anti-sense UGAUUCACUACACGCUCUGTT, C:

sense

GAUGGCACUUAUAACCAAATT

anti-sense

UUUGGUUAUAAGUGCCAUCTT. Next, cells were stained with CXCR4 antibody (Santa Cruz, USA) overnight and cell nuclei were stained with DAPI. In addition, the expression of CXCR4 was quantified using the flow cytometry. MSCs (2105/well) seeded in 6 well plates for 48 hr were harvested for analysis by the FACS Calibur flow cytometer. CXCR4-positive cells were calculated using the FCS software (Becton Dickinson, USA).

2.10. Analysis of SDF-1  expression, migration ability, and MMP expression The culture media of MSCs treated with NP-FITC-CXCR4 siRNA and SDF-1 10


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(or VEGF) for 48 hr were collected for quantitative analysis of SDF-1 expression using a human CXCL12/SDF-1 DuoSet® ELISA Development System kit (R&D system), where the optical density was measured at 450 nm by the microplate reader. Cell migration was assayed using the Oris Cell Migration Assay reagent kit (Platypus Technologies, Madison WI, USA) 28. Cells were stained with Calcein AM (2 M) (Sigma, USA) containing serum free medium. A cell seeding stopper (2 mm in diameter) was inserted in each well to prevent cell adhesion in the center region of the well. Cells were seeded (2105 cells /a well), treated with NP-FITC-CXCR4 siRNA and SDF-1 (or VEGF) as described, and incubated for 24 hr and 48 hr of incubation. The stopper was then removed from the well, which was defined as the pre-migration state. The plate was incubated again for 24 hr for observation of post-migration. The images were taken by the fluorescence microscope. The expression of MMP was measured by the gelatin zymography assay. The culture media of MSCs treated with NP-FITC-CXCR4 siRNA (25 nM) and SDF-1 (or VEGF) as described for 48 hr were collected for the assay. The resulting gel was then analyzed by Image Pro Plus 5.0 software.

2.11. In vivo tissue distribution of NP-FITC MSCs were seeded at a density of 2105 cells per well. NP-FITC (1 μg/ml) was added into each well and incubated for 2 hr. Cells were the collected by trypsinization and labeled with 1 μl of Qtracker 655 cell labeling kit in 200 μl medium by following the manufacturer’s instruction. 2105 cells in 50 l normal saline was used for each injection. NP-FITC was directly injected in the retro-orbital sinus of mice. In addition, the tail vein of other experiment, mice was injected with MSCs formerly treated with NP-FITC. After 24 hr or 48 hr, the mice were sacrificed. The heart, liver, spleen, lung, 11



kidney, and brain tissues were harvested and fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. The specimens were sectioned (in 4 m thickness) and stained with hematoxylin and eosin (H&E) (Sigma, USA) for histological examination. Samples were also observed by the fluorescence microscope to obtain the distribution of NP-FITC in heart, liver, spleen, lung, kidney, and brain tissue. The green color indicated the presence of NP-FITC previously taken up by MSCs and the red color indicated the quantum dot-labeled MSCs.

2.12. Statistical Analysis Experimental data were collected from different samples in each test (n=3-6). The results were given as mean ± standard deviation. We compared the difference between groups using method of single-factor analysis of variance (ANOVA). A p value less than 0.05 is considered significant.

3. Results 3.1. Characterization of NP-FITC, NP-FITC-SOD-1, and NP-FITC-CXCR4 siRNA Fabrication of gold NP-FITC and conjugation of SOD-1 or CXCR4 siRNA for in vitro and in vivo study is depicted in Figure 1. The absorption peak of UVVis spectroscopy for bare gold nanoparticle (AuNP) was 525 nm as shown in Figure 2A. Using DLS to analyze the binding of AuNP with HA, the average particle size of HA-AuNP was estimated at 28 nm as shown in Figure 2B(a), with the zeta potential of 36.32.3 mV as shown in Figure 2C. TEM imaging gave the feature of HA conjugated AuNP as shown in Figure 2B (b). NP-FITC and NP-FITC-SOD-1 were characterized by UV/Vis spectroscopy, FITR, and SEM. The UV absorption peak of NP-FITC-EDC (with HA crosslink) was shift from 260 nm to 270 nm after 12


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conjugation with SOD-1 as shown in Figure. 3A. FTIR analysis also confirmed the interaction between NP-FITC and SOD-1 plasmid in Figure 3B. The main peaks of HA were observed at 1610-1550 cm−1 (C=O and C-O bands) and 1100 cm−1 (C-O band). When the SOD-1 plasmid was conjugated onto the NP-FITC, the absorption peak area of C=O and C-N bands at 1650-1550 cm−1 increased. SEM imaging confirmed that NP-FITC had a homogenous size distribution with an average value at about 14 nm28. When the SOD-1 plasmid was conjugated onto NP-FITC by EDC, the nanoparticles were assembled into a network of stringed beads (Figure 3C).

3.2. Blood compatibility of NP-FITC The activation of monocytes into macrophages by NP-FITC was first assessed by staining the treated cells with the macrophage marker CD68. As shown in Figure 4A, the fluorescence intensity of CD68 on cells incubated with NP-FITC at 1 μg/ml was lower than that in the control group (untreated cells). The conversion ratio of monocytes into macrophages is shown in Figure 4B. It was evident that NP-FITC had an anti-inflammatory effect. We further examined the effect of NP-FITC on the ROS generation of MSCs. As shown in Figure 4C, ROS generation was lower for NP-FITC treated MSCs compared to the control. The effect of NP-FITC on the platelet morphology was examined by SEM and results are shown in Figure 4D. The platelets treated with NP-FITC exhibited less platelet adhesion and activation, as compared to the control group. These findings indicated that NP-FITC had prominent blood compatibility and may be a good candidate for gene carriers.

3.3. Cell viability and uptake of NP-FITC by different cells The effect of NP-FITC on cell proliferation was evaluated by MTT assay and 13



data are shown in Figure 5. Normal cell lines (HSF, BAEC, and MSCs) appeared growing more in the presence of 1 μg/ml NP-FITC (Figure 5A), but the phenomenon was not obvious in cancer cell lines (HER-2, Colo-205, and A549) (Figure 5B). “These data showed that NP-FITC had no toxic effects to these test cells at the concentration of 2 μg/ml. However, a slightly cell growth promoting effect may present at the concentration of 1 g/ml to normal cell lines, particularly for HSF.” The cellular uptake of NP-FITC is shown in Figure 6. The fluorescence images confirmed that NP-FITC was internalized in both normal cells and cancer cells (Figure 6A and Figure 6B). The amount of uptake was quantified by Image J software (Figure 6C and Figure 6D). The uptake of NP-FITC started early (30 min and 2 hr) in normal cells (Figure 6C). However, cancer cells had more NP-FITC internalized after 24 hr (Figure 6D).

3.4. SOD-1 plasmid delivered to MSCs MSCs are the most popular cells for cell therapy. The production of SOD-1 can remove ROS to protect cells against damage. Figure 7A shows that NP-FITC-SOD-1 with SNAP-tag® fusion protein was observed in the cytosol after 30 min of incubation and subsequently entered the nucleus after 2 hr of incubation. Interestingly, NP-FITC-SOD-1-SNAP-tag® fusion protein diffused from the nucleus to the cytosol after 24 hr of incubation. The observation suggested that NP-FITC-SOD-1 may be internalized by MSCs. Western blotting confirmed the delivery of NP-FITC-SOD-1 into MSCs and translation of SOD-1 protein expression. These MSCs significantly expressed more SOD-1 protein than the control group (TCPS), as shown in Figure 7B. Quantification of SOD-1 protein expression followed the same trend (p

Biocompatible nanogold carrier coated with hyaluronic acid for

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