Javascript is currently disabled in your browser. When javascript is disabled, some functions of this website will not work.
Open access for scientific and medical research
From submission to the first editing decision.
From editor acceptance to publication.
The above percentage of manuscripts have been rejected in the past 12 months.
Open access to peer-reviewed scientific and medical journals.
Dove Medical Press is a member of OAI.
Batch reprints for the pharmaceutical industry.
We provide authors with real benefits, including fast processing of papers.
Register your specific details and specific drugs of interest, and we will match the information you provide with articles in our extensive database and send you a PDF copy via email in a timely manner.
Back to Journal »International Journal of Nanomedicine» Volume 16
Lactoferrin-containing gold nanocage used for in vitro gene delivery of prostate cancer cells
Author Almowalad J, Somani S, Laskar P, Meewan J, Tate RJ, Mullin M, Dufès C
Published on June 30, 2021, the 2021 volume: 16 pages 4391-4407
DOI https://doi.org/10.2147/IJN.S316830
Single anonymous peer review
Editor approved for publication: Dr. Yan Shen
Jamal Almowalad, 1 Sukrut Somani, 1 Partha Laskar, 1 Jitkasem Meewan, 1 Rothwelle J Tate, 1 Margaret Mullin, 2 Christine Dufès 1 1 Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, G40RE, UK; 2 School of Medicine, Veterinary Medicine and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK Mailing address: Christine Dufès Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Glasgow, G4 0RE, UK Phone 44 14154835254225 Faxclyde [Email protection] Background: Gold Nanocages have been widely used as multifunctional platforms for drug and gene delivery, as well as photothermal agents for cancer treatment. However, their potential as a gene delivery system for cancer treatment has been combined with chemotherapy and photothermal therapy, but it has not been isolated so far. The purpose of this work is to study whether the combination of gold nanocages and cancer-targeting ligands lactoferrin, polyethylene glycol and polyethyleneimine can improve the transfection efficiency of prostate cancer cells in vitro without the help of external stimuli. Method: A new type of lactoferrin-containing gold nanocage conjugated with polyethyleneimine and polyethylene glycol was synthesized and characterized. After complexing with plasmid DNA, their transfection efficacy and cytotoxicity were evaluated on the PC-3 prostate cancer cell line. Results: Lactoferrin-containing gold nanocages, alone or conjugated with polyethyleneimine and polyethylene glycol, can condense DNA with a conjugate:DNA weight ratio of 5:1 or higher. Among all gold conjugates, the highest gene expression was obtained after treatment with the gold complex conjugated with polyethyleneimine and lactoferrin, with a weight ratio of 40:1, which was 1.71 times higher than that of polyethyleneimine. This may be due to the increased cellular uptake of DNA observed with this conjugate, which is an 8.65-fold increase compared to naked DNA. Conclusion: Lactoferrin-containing gold nano clathrate conjugate is a very promising prostate cancer cell gene delivery system. Keywords: gold nanocage, cancer targeting, transfection effect, lactoferrin, polyethyleneimine, polyethylene glycol
Gold nanoparticles are widely used in biomedical applications to specifically target cancer cells due to their low toxicity, adjustable size, unique physicochemical properties, and easily surface-modified targeting moieties (such as proteins, peptides, and antibodies). 1-3 In addition to these properties, they are particularly attractive for gene delivery because they can condense nucleic acids through electrostatic interactions or through nucleic acid binding to their surface through thiol bonds. 4-7 Among these nanoparticles, gold nanocage is a new type of gold nanomaterial with cage shape, hollow interior and porous wall. In addition to its ability to convert light into heat in the near infrared region (700-900 nm) In addition, drugs can also be loaded, making them suitable candidates for biomedical applications and photothermal cancer treatments. 8-10 Due to the unique structure of gold nanocages, drugs can be loaded into the hollow interior of the nanocage and then be heated by thermosensitive polymers. The shell is wrapped, as previously reported. 11,12 The polymer shell prevents the release of the drug until there is an external thermal stimulus, such as near infrared light or high-intensity focused ultrasound.
However, recent advances in the use of gold nanocages in controlled drug delivery and photothermal cancer treatment have led to the need to explore the possibility of using gold nanocages for targeted gene delivery for cancer treatment without the help of external stimuli or the use of chemotherapy. combination. Therefore, we suggest combining the surface of gold nanocage with cancer targeting moieties such as lactoferrin, and functionalizing the surface of the nanocage with cationic polymers, and studying whether the combined gold nanocage can condense DNA on its surface and promote prostate cancer. Gene expression in cells in vitro. Recently, the receptor for the cancer-targeting ligand lactoferrin (Lf) is overexpressed in most cancers. Due to the significant increase in gene expression in tumors after intravenous administration, it has been proven to improve the therapeutic effect of gene cancer therapy Very hopeful. 13-15 Cationic polymers, such as polyethyleneimine (PEI), have been widely used to generate positively charged nanoparticles due to their ability to compound DNA or RNA and their high transfection efficiency. 7,16 In addition, low molecular weight PEI leads to a decrease in 17 Polyethylene glycol (PEG) binding to nanoparticles is another recent method that has helped improve transfection efficiency, while at the same time by shielding the charge of nanoparticles and preventing their interactions To reduce the cytotoxicity associated with nanoparticles. Plasma protein. 18–20
Therefore, the purpose of this study is to (1) synthesize and characterize gold nanocages with lactoferrin, PEG-conjugated gold nanocages with lactoferrin, and PEI-conjugated gold with lactoferrin. Nanocages, (2) evaluate their DNA condensation efficiency. Compound with plasmid DNA, and (3) evaluate the cell viability, transfection efficiency and cell uptake of these DNA-complexed conjugates on PC-3 prostate cancer cells.
Lactoferrin (Lf), branched polyethyleneimine (PEI, Mw 800 Da), diethylene glycol (DEG), silver trifluoroacetate (CF3COOAg), sodium hydrosulfide (NaSH), hydrogen tetrachloroaurate (III) ) Hydrate (HAuCl4), aqueous hydrochloric acid (HCl), 37%), polyvinylpyrrolidone (PVP, Mw 55,000 Da), sodium chloride (NaCl) and deionized (DI) water with a resistivity of 18.2 MΩ·cm From Sigma Aldrich (Poole, UK). Thiol PEG amine (HS-PEG3.5K-NH2) was from JenKem Technology (Plano, TX). N-[1-(2,3-Dioleoyloxy) propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP) liposome transfection reagent was purchased from Roche (Burgess Hill, UK). The expression plasmid encoding β-galactosidase (pCMVsport β-galactosidase) was obtained from Invitrogen (Paisley, UK). It was purified using endotoxin-free Giga Plasmid Kit (Qiagen, Hilden, Germany). Minimal modified Eagle medium (MEM) and fetal bovine serum (FBS), L-glutamine and penicillin-streptomycin were purchased from Life Technologies (Paisley, UK). Vectashield® mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) was purchased from Vector Laboratories (Peterborough, UK). Label IT® Fluorescein Nucleic Acid Labeling Kit was obtained from Cambridge Biosciences (Cambridge, UK). Passive lysis buffer was purchased from Promega (Southampton, UK). BioWare® androgen non-responsive PC-3M-luc-C6 human prostate adenocarcinoma expressing firefly luciferase was purchased from Caliper Life Sciences (Hopkinton, MA).
As previously reported, silver nanocubes were synthesized by the reduction of polyols in diethylene glycol (DEG). 21 In short, add 60 mL of DEG to a 250 mL round bottom flask equipped with a magnetic stir bar and heat it in an oil bath at 150 °C for 30 minutes. Then prepare reagents in DEG and add them to the reaction flask separately middle. Initially, pipet NaSH solution (0.72 mL, 3 mM) into the flask. After 4 minutes, add HCL solution (6 mL, 3 mM), and then add 15 mL PVP solution (20 mg/mL). After 2 minutes, CF3COOAg solution (4.8 mL, 282 mM) was then added. Throughout the process, the flask was closed loosely, except during the addition of reagents. After 90 minutes, the reaction was stopped by placing the flask in an ice water bath for 30 minutes. Then, 25 mL of the mixture was transferred to a 50 mL centrifuge tube and mixed with 20 mL of acetone, and then centrifuged at 9000 rpm (16,000 g) for 30 minutes. After removing the supernatant, add 20 mL of deionized (DI) water to the remaining product, and then sonicate for 10 minutes in an ultrasonic bath. Next, the product was centrifuged at 9000 rpm (16,000 g) for 10 minutes, then the supernatant was discarded and the remaining product was sonicated with 20 mL of deionized water. This process was repeated three more times. The final product was then dispersed in 20 mL of deionized water and stored at 4°C.
The gold nanocage (AuNC) was synthesized based on the previously published method, 22 with some modifications. In short, 600 mL of polyvinylpyrrolidone (PVP) solution (1 mg/mL in deionized water) was added to a 1 L round bottom flask containing a magnetic stir bar. Then, 60 mL of the prepared silver nanocube was added to the flask and heated at 100 °C while stirring for 30 minutes. An aqueous solution of 1 mM HAuCl4 (140 mL) was added to the reaction flask via a syringe pump at a rate of 45 mL/h. The reaction was stopped when the reaction sample was blue and showed a UV-Vis absorption spectrum at 740 nm. Heat the mixture at 100 °C for another 15 minutes until the reaction color becomes stable. After cooling the flask to 25 °C, mix 20 mL of NaCl saturated solution (0.36 g/mL) with every 25 mL aliquot of the mixture to remove AgCl. The product was then centrifuged at 9000 rpm (16,000 g) for 30 minutes, the supernatant was discarded and washed with 20 mL of deionized water. This process was repeated 3 times. Add the final product to 20 mL of deionized water and store at 4 °C for further use. The Au concentration of the gold nanocage was obtained by inductively coupled plasma mass spectrometry (ICP-MS). In short, 20 μL of AuNC in deionized water was digested in 180 μL of aqua regia at 20 °C for 1 hour. The mixture is further diluted with deionized water to a final aqua regia concentration of 2%. 197Au isotope and 175Lu isotope are used as internal standards for all measurements to measure the Au concentration (μg/L) in the sample. All ICP-MS measurements were performed in triplicate using an Agilent 7700X® instrument (Agilent Technologies, Santa Clara, USA).
The binding of lactoferrin (Lf) to AuNCs was accomplished by modifying the previously reported method. 23 In short, dissolve 8 mg of Lf in 2 mL of 50 mM sodium phosphate and 0.15 M sodium chloride buffer (pH 7.5), and mix with 5-2-iminothiolane (Traut reagent, distilled water 1 Mg/ml, 7.26 mm, 61.2 μl) molar excess at 20°C for 75 minutes. Use a Vivaspin-6 centrifuge tube with a molecular weight cut-off (MWCO) of 5000 Da to purify the modified Lf at 8000 rpm (14,000 g) for 20 minutes. Add thiolated Lf (100 μL, 8 mg/mL) to 1 mL AuNC, then vortex and incubate at 25 °C for 24 hours. The final product was purified twice with 1 mL of deionized water, a Vivaspin-6 centrifuge tube with a MWCO of 100,000 Da was used to remove any unreacted AuNC, and then freeze-dried.
Add thiol-PEG3.5K-amine aqueous solution (100 µL, 2 mM) to 1 mL of AuNCs (AuNCs-Lf) suspension containing lactoferrin and vortex for 10 seconds. The mixture was then incubated at 25°C for 24 hours. Before freeze-drying, the final product was purified twice with deionized water using a Vivaspin-6 centrifuge tube with a MWCO of 100,000 Da to remove any unreacted PEG.
The thiolation of polyethyleneimine (PEI) was carried out by modifying the previously reported method. 24 The formation of thiolated PEI is carried out by reacting PEI with a 2-fold molar excess of 2-iminosulfane (Traut's reagent). PEI is dissolved in PBS at a concentration of 10 mg/mL (12.5 mM). Add Traut reagent (3.44 mg/mL PBS, 25 mM, 4.28 mL) to the PEI solution (10 mg/mL PBS, 12.5 mM, 4.28 mL) and vortex for 5 seconds. The reaction mixture was then incubated at 25°C for 24 hours. The final compound was purified with 2 L of distilled water with 2000 Da MWCO at room temperature (20-22°C) using benzoylated dialysis tubing, and was replaced twice during the dialysis process. After 24 hours of dialysis, the solution was freeze-dried for 48 hours using a Christ Epsilon 2-4 LSC® freeze dryer (Osterode am Harz, Germany).
Add thiolated PEI (10 mg/mL in PBS, 400 μL) to each 1 mL of AuNCs-Lf prepared, vortex for 5 seconds, and then incubate at 25°C for 24 hours. Then use a Vivaspin-6 centrifuge tube with a MWCO of 5000 Da to purify AuNCs-Lf-PEI with 1 mL of deionized water at 8000 rpm (14,000 g) for 20 minutes. The purified AuNCs-Lf-PEI conjugate was freeze-dried for 48 hours.
A transmission electron microscope (TEM) was used to examine the morphology of AuNCs conjugates. Typically, a 20 μL sample of AuNCs conjugate is placed on a carbon-coated 200-mesh copper grid and dried at 20°C for 1 hour. Then observe the sample using a JEOL JEM-1200EX® transmission electron microscope (Jeol, Tokyo, Japan) operating at an accelerating voltage of 80 kV.
A circular dichroism (CD) spectrometer (Chirascan V100®, Applied Photophysics, Leatherhead, UK) was used to evaluate the conjugation of Lf, Lf-PEG and Lf-PEI with AuNC. All samples were prepared in deionized water at a concentration of 2 mg/mL. The CD spectrum was recorded using a quartz cuvette with a path length of 10 mm, a range of 190-900 nm, a scanning speed of 70 nm/min, and a bandwidth of 1 nm.
A Fourier transform infrared (FTIR) spectrophotometer equipped with an attenuated total reflection (ATR) probe (IRSpirit® QATR-S, Shimadzu, Kyoto, Japan) was used to perform the infrared spectroscopy of AuNC containing Lf. The transmission spectrum was collected in the range of 4000-400 cm-1 with a resolution of 4 cm-1, and then analyzed using LabSolutions IR® software (Shimadzu Corporation, Japan).
A Varian Cary® 50 Bio UV-Vis spectrophotometer was used to further evaluate the formation of AuNCs conjugates by UV-Vis spectrophotometer analysis in the wavelength range of 300-900 nm. Before measuring the sample, adjust the UV-Vis spectrum of deionized water to the baseline.
At 25°C, the size and zeta potential of AuNCs conjugates in deionized water were measured by photon correlation spectroscopy and laser Doppler electrophoresis using Malvern Zetasizer Nano-ZS® (Malvern Instruments, Malvern, UK). All dimensions and zeta potential measurements were performed using a He-Ne laser at 632.8 nm and a scattering angle of 173° for three repeated measurements.
The ability of AuNCs conjugates to condense DNA was evaluated by agarose gel blocking test. AuNCs conjugate complexes are prepared under various AuNCs conjugate:DNA weight ratios ranging from 0.5:1 to 40:1, and the DNA concentration is constant at 20 µg/mL. After mixing with the loading buffer, load the sample (15 µL) onto 1X Tris-Borate-EDTA (TBE) (89 mM Tris base, 89 mM boric acid, 2 mM Na2-EDTA, pH 8.3), the buffer is 0.8% (w/v) Agarose gel containing ethidium bromide (0.4 µg/mL) with 1x TBE as the running buffer. The DNA size marker is HyperLadder I. The gel was run at 50 V for 1 hour and then photographed under UV light.
For different AuNCs conjugates, the size of the complex and zeta potential: DNA weight ratio in 5% (w/v) glucose were measured (0.5:1, 1:1, 5:1, 10:1, 20:1 , 40: 1) Obtained by photon correlation spectroscopy and laser Doppler electrophoresis using Malvern Zetasizer Nano-ZS® (Malvern Instruments, Malvern, UK) at 37 °C. The DNA concentration (1 µg/mL) remained constant throughout the experiment.
PC-3-Luc prostate cancer cells are supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) L-glutamine and 0.5% (v/v) minimum essential medium (MEM) Medium as a monolayer growth) penicillin-streptomycin. Keep the cell culture flask in a humid atmosphere of 37°C and 5% carbon dioxide.
The β-galactosidase assay was used to evaluate the transfection efficiency of DNA complexed with AuNCs conjugates. PC-3 cells were seeded in a 96-well plate at a concentration of 10,000 cells/well and cultured at 37°C and 5% CO2 for 24 hours. Then they were treated with AuNCs conjugates complexed with plasmid DNA encoding β-galactosidase, with different AuNCs conjugate:DNA weight ratios (0.5:1, 1:1, 2:1, 5:1, 10:1 20:1) in five copies and 40:1). Naked DNA was used as a negative control, while DOTAP-DNA (weight ratio 5:1) was used as a positive control. Throughout the experiment, the DNA concentration (0.5 μg/well) remained constant. The cells were incubated with the treatment for 72 hours before analysis. Then they were lysed with 1x Passive Lysis Buffer (PLB) (50 μL/well) for 20 minutes, and then tested for β-galactosidase expression. 25 In short, 50 μL assay buffer (2 mM magnesium chloride, 100 mM mercaptoethanol, 1.33 mg/mL o-nitrophenyl-β-D-galactosidase, 200 mM sodium phosphate buffer, pH 7.3 ) Add the lysate to each well and incubate at 37°C for 2 hours. A Multiskan Ascent® plate reader (MTX Lab Systems, Bradenton, FL) was then used to read the absorbance of the sample at 405 nm.
The cellular uptake of DNA complexed with AuNCs conjugates was quantified by flow cytometry. PC-3 cells were seeded in a 6-well plate at a density of 3×105 cells per well and grown at 37°C for 24 hours, and then treated with fluorescein-labeled DNA (2.5 μg DNA per well) or paired with AuNCs The weight ratio of conjugate: DNA is 0.5:1 and 40:1. Untreated cells served as a negative control. After treatment and incubation for 24 hours, each well was washed twice with 2 mL of pH 7.4 PBS. Then prepare a single cell suspension (use 250 μL trypsin per well, and then use 500 μL medium per well after cell separation), and then analyze it using a FACSCanto® flow cytometer (BD, Franklin Lakes, NJ). Analyze their average fluorescence intensity using FACSDiva® software (BD, Franklin Lakes, NJ), and count 10,000 cells per sample (gated events).
A confocal microscope was used to qualitatively assess the cellular uptake of DNA complexed with AuNCs conjugates. Fluorescein labeling of plasmid DNA with a fluorescent probe is performed using the Label IT® Fluorescein Nucleic Acid Labeling Kit, as described by the manufacturer. PC-3 cells were seeded on the coverslip of a 6-well plate at a concentration of 3×105 cells per well, and grown at 37°C for 24 hours. Then the cells were compounded with AuNCs-Lf-PEI at a weight ratio of 40:1 with fluorescein-labeled DNA (2.5 μg/well) and treated at 37°C for 24 hours. Control wells were also prepared with naked DNA or not processed. Then the cells were washed twice with 3 mL PBS, and then fixed with 2 mL methanol at 20°C for 10 minutes. Then wash again with 3 mL PBS. After staining the nuclei with Vectashield® mounting medium containing DAPI, the cells were examined using a Leica TCS SP5® confocal microscope (Wetzlar, Germany). DAPI (stained cell nucleus) was excited with a 405 nm laser line (emission bandwidth: 415-491 nm), and fluorescein (labeled DNA) was excited with a 514 nm laser line (emission bandwidth: 550-620 nm).
A standard MTT assay was used to evaluate the viability of PC-3 prostate cancer cells treated with AuNCs conjugate. PC-3 cells were seeded in quintuples in 96-well plates at a density of 10,000 cells/well, and incubated at 37°C for 24 hours. The cells were then treated with different concentrations of AuNCs conjugates at 37 °C and 5% CO2, ranging from 2.5 to 200 μg/mL for 72 hours. After treatment and incubation, add 50 μL of MTT solution (5 mg/mL in PBS pH 7.4) to the medium, and incubate at 37 °C in the dark for 4 hours. Then replace the medium with 200 μL DMSO per well to dissolve the precipitated formazan. The absorbance was measured at 570 nm using a Multiskan Ascent® plate reader (Thermo Scientific, Waltham, MA).
The results are expressed as mean±standard error of mean (SEM). The statistical significance of the data was evaluated by one-way analysis of variance (ANOVA) and Tukey's post-multiple comparison test (Minitab® 17.1.0 software, State College, PE). For P values below 0.05, the difference is considered statistically significant.
The gold nanocage is synthesized by the electro displacement reaction between silver nanocube and chloroauric acid (HAuCl4). Their large batch (800 ml) has a gold content of 597.93 μg/L and was used in all experiments described in this manuscript.
The TEM image confirmed the cage shape, hollow interior and thin walls of the gold nanocage, with an edge length of 50 nm (Figure 1). Figure 1 Transmission electron microscope images of gold nanocage (A) and lactoferrin-containing gold nanocage (B). Inset: magnification of the sample, showing the morphology of the nanocage.
Figure 1 Transmission electron microscope images of gold nanocage (A) and lactoferrin-containing gold nanocage (B). Inset: magnification of the sample, showing the morphology of the nanocage.
CD and FTIR spectra were used to prove the conjugation of Lf with the surface of AuNCs. The CD spectrum of Lf shows unique negative ellipticity peaks at 244 nm and 283 nm, and a positive ellipticity peak at 309 nm (Figure 2). These peaks are visible in the CD spectrum of AuNCs-Lf, which are different from unmodified AuNCs, indicating that Lf successfully binds to the surface of AuNCs. The highest molar ellipticity values of the Lf protein and the conjugate at the same wavelength (244 nm and 283 nm) indicate that there is no significant conformational change in protein folding after being grafted into the nanocage. Figure 2 Lactoferrin ("Lf"), thiolated lactoferrin ("Thiol Lf"), unmodified gold nanocages ("AuNCs") and gold nanocages containing lactoferrin ("AuNCs-Lf") CD spectrum.
Figure 2 Lactoferrin ("Lf"), thiolated lactoferrin ("Thiol Lf"), unmodified gold nanocages ("AuNCs") and gold nanocages containing lactoferrin ("AuNCs-Lf") CD spectrum.
The FTIR spectrum of AuNCs-Lf shows that there are characteristic peaks at 1645.01 cm-1 and 1534.86 cm-1 (corresponding to the amide-I and amide-II of bare Lf), but there are no characteristic peaks at 1049.94 cm-1 and 1398.97 cm-1. (Corresponding to the sulfoxide and sulfate groups of thiolated lactoferrin, respectively), compared with the starting material (Figure 3). Therefore, these findings confirm that Lf successfully binds to the surface of the gold nanocage through the Au-S bond, and prove that there is no significant change in the secondary structure of the protein, which is consistent with the data obtained from CD. Figure 3 (A) FTIR spectra of lactoferrin ("Lf"), (B) thiol lactoferrin ("Thiol Lf") and (C) lactoferrin-containing gold nanocages ("AuNCs-Lf") .
Figure 3 (A) FTIR spectra of lactoferrin ("Lf"), (B) thiol lactoferrin ("Thiol Lf") and (C) lactoferrin-containing gold nanocages ("AuNCs-Lf") .
The UV-visible spectroscopy analysis further confirmed the conjugation of Lf, PEG and PEI with the surface of AuNCs. AuNCs and AuNCs conjugates show UV-Vis spectral peaks in the near infrared region (AuNCs, AuNCs-Lf, AuNCs-Lf-PEG and AuNCs-Lf-PEI are 715 nm, 771 nm, 767 nm and 813 nm, respectively). Figure 4). Compared with AuNCs, the red shift of the UV-Vis spectra of AuNCs-Lf, AuNCs-Lf-PEG and AuNCs-Lf-PEI confirms the surface modification of AuNCs and Lf, and shows the effective co-existence of PEG and PEI with AuNCs-Lf. Yoke. Figure 4 UV-Vis spectra of AuNCs, AuNCs-Lf, AuNCs-Lf-PEG, AuNCs-Lf-PEI conjugates.
Figure 4 UV-Vis spectra of AuNCs, AuNCs-Lf, AuNCs-Lf-PEG, AuNCs-Lf-PEI conjugates.
The conjugation of Lf, Lf-PEG, and Lf-PEI with the surface of AuNCs results in AuNCs-Lf (105.40 ± 0.43 nm), AuNCs-Lf-PEG (103.30 ± 1.31 nm) and AuNCs-Lf-PEI (127 ± 1.62 nm) and Compared with AuNCs (88.69 ± 0.66 nm), determined by DLS measurement. It also increases the net surface charge of the gold nanocage, from the negative value of unmodified AuNCs (-20.50 ± 0.38 mV) to the positive value of AuNCs-Lf (3.05 ± 0.18 mV, 4.11 ± 0.38 mV and 14.10 ± 0.14 mV) , AuNCs-Lf-PEG and AuNCs-Lf-PEI, respectively.
Gel retardation test confirmed that AuNCs-Lf and AuNCs-Lf-PEG fully or partially aggregated DNA. When the AuNCs conjugate:DNA weight ratio is 40:1, DNA is completely agglomerated by AuNCs-Lf and AuNCs-Lf-PEG, thereby preventing ethidium bromide from intercalating into DNA. Therefore, there is no visible free DNA at this ratio (Figure 5A and B). In contrast, DNA is partially concentrated by AuNCs-Lf and AuNCs-Lf-PEG at a ratio of 20:1 and lower. Therefore, ethidium bromide can intercalate into DNA, and bands corresponding to free DNA can be seen. On the other hand, AuNCs-Lf-PEI can completely condense DNA at a weight ratio higher than 1:1, resulting in no bands corresponding to free DNA at these ratios (Figure 5C). Therefore, among the three AuNCs conjugates evaluated in this study, AuNCs-Lf-PEI is the most effective for condensing DNA when the conjugate:DNA weight ratio is higher than 1:1. Figure 5 AuNCs-Lf complex (A), AuNCs-Lf-PEG complex (B) and AuNCs-Lf-PEI complex (C) in various conjugates: DNA weight ratio (0.5:1, 1:1) , 5 :1, 10:1, 20:1, 40:1) (control: DNA solution).
Figure 5 AuNCs-Lf complex (A), AuNCs-Lf-PEG complex (B) and AuNCs-Lf-PEI complex (C) in various conjugates: DNA weight ratio (0.5:1, 1:1) , 5 :1, 10:1, 20:1, 40:1) (control: DNA solution).
AuNCs conjugates complexed with DNA showed less than conjugate: DNA weight ratio for AuNCs-Lf and AuNCs-Lf-PEG higher than 1:1 and for AuNCs-Lf-PEI higher than 5:1 The average hydrodynamic size of 250 nm (Figure 6A). The average size of the composite decreases as the weight ratio increases, from 0.5:1 to 5:1, and then reaches the plateau from the 10:1 weight ratio. Among the AuNCs conjugated complexes, AuNCs-Lf-PEI exhibited the largest complex size of 1.50 ± 0.02 μm at a weight ratio of 0.5:1, while the difference between AuNCs-Lf and AuNCs-Lf-PEG complexes The size is smaller, 464.00 ± 37.37 nm and 520.80 ± 28.48 nm respectively at the same ratio. In contrast, AuNCs-Lf-PEI showed the smallest complex size when the conjugate:DNA weight ratio was 40:1, with an average size of 144.30 ± 6.27 nm. These results prove that AuNCs conjugates can condense DNA into small complexes of a size suitable for gene delivery. Figure 6 The size (A) and zeta potential (B) of gold conjugates complexed with DNA in various AuNC conjugates: DNA weight ratios. The results are expressed as mean ± SEM (n=9).
Figure 6 The size (A) and zeta potential (B) of gold conjugates complexed with DNA in various AuNC conjugates: DNA weight ratios. The results are expressed as mean ± SEM (n=9).
The Zeta potential experiment showed that the AuNCs complex had a slight negative surface charge when the conjugate:DNA weight ratio was 0.5:1, indicating that the negatively charged DNA was not completely complexed with the AuNCs conjugate at this ratio. As the weight ratio of the complex increases, the zeta potential increases, and finally reaches the maximum positive charges of 19.90 ± 0.45, 26.70 ± 0.37 and 28.70 ± 0.38 mV for AuNCs-Lf, AuNCs-Lf-PEG and AuNCs-Lf-PEI, respectively 40:1 conjugate:DNA weight ratio (Figure 6B). These results are very consistent with the findings of gel blocking.
Compared with the PEI and DOTAP complexes on PC-3 cells, it was found that AuNCs-Lf, AuNCs-Lf-PEG and AuNCs-Lf-PEI complexed with DNA can increase genes at the best AuNCs conjugate:DNA weight ratio Expression (Figure 7). Figure 7 Transfection efficiency of AuNCs-Lf, AuNCs-Lf-PEG and AuNCs-Lf-PEI complexes in various conjugates: the weight ratio of DNA in PC-3 cells. The results are expressed as the mean ± SEM of three replicates (n=15). * P <0.05 compared with the highest transfection rate.
Figure 7 Transfection efficiency of AuNCs-Lf, AuNCs-Lf-PEG and AuNCs-Lf-PEI complexes in various conjugates: the weight ratio of DNA in PC-3 cells. The results are expressed as the mean ± SEM of three replicates (n=15). * P <0.05 compared with the highest transfection rate.
After treatment with the AuNCs-Lf-PEG complex at a conjugate:DNA ratio of 0.5:1, 1:1, 10:1, 20:1, higher gene expression was observed compared with the PEI complex. The results of the AuNCs-Lf-PEI complex treatment are 0.5:1, 5:1, 10:1, and 40:1, and the ratios are 0.5:1, 1:1, 20:1, and 40:1.
The three complexes also resulted in higher gene expression than the DOTAP complex at a ratio of 0.5:1 and the AuNCs-Lf-PEI complex at a ratio of 40:1.
The highest level of transfection was observed after treatment with the AuNCs-Lf-PEI complex at a ratio of 40:1 (2.49 ± 0.10 mU/mL). It is 2.1 times higher (1.16 ± 0.01 mU/mL) than when treated with DNA, 1.7 times higher than with PEI complex (1.46 ± 0.05 mU/mL), and 1.4 times (1.68 ± 0.09 mm) higher than after treatment with DOTAP complex. Unit/ml).
The AuNCs-Lf-PEG complex resulted in the highest gene expression level when the conjugate:DNA weight ratio was 0.5:1 (2.43 ± 0.21 mU/mL). This is 2.1, 1.6, and 1.4 times higher than the results obtained with DNA, PEI, and DOTAP, respectively.
Similar to the AuNCs-Lf-PEG complex, the AuNCs-Lf complex resulted in the highest gene expression when the conjugate:DNA weight ratio was 0.5:1 (2.22 ± 0.24 mU/mL).
Compared with unmodified AuNCs-Lf, the best conjugate:DNA ratio of PEI and AuNCs-Lf is 40:1, resulting in an increase in gene transfection (1.5 times), which is after conjugated with PEG and AuNCs-Lf The observed differences result in similar levels of transfection. Based on these findings and taking into account the results of DNA concentration, 40:1 and 0.5:1 conjugate:DNA ratios were selected for the cell uptake experiments.
The composite treatment of PC-3 cells with AuNCs-Lf-PEI and fluorescein-labeled DNA at a weight ratio of 40:1 resulted in the highest cell fluorescence (1726.17 ± 49.71 arbitrary units (au)), which was 2.3 times and the ratio at the same ratio The results observed with AuNCs-Lf and AuNCs-Lf-PEG complexes (725.17 ± 33.93 and 247.50 ± 3.53 au, respectively) are 6.9 times higher (Figure 8). It is similar to that observed with the PEI complex (1803.5 ± 25.42 au) and 8.6 times higher than that observed after treatment with the DNA solution (199.50 ± 1.31 au). After treatment with AuNCs-Lf, AuNCs-Lf-PEG and AuNCs-Lf-PEI complexes at a ratio of 0.5:1, there was no significant difference in DNA cell uptake. Figure 8 After 24 hours of incubation with PC-3 cells, flow cytometry was used (n = 6). *Compared with PEI-DNA, P <0.05.
Figure 8 After 24 hours of incubation with PC-3 cells, flow cytometry was used (n = 6). *Compared with PEI-DNA, P <0.05.
In addition, treatment of cells with naked DNA resulted in weaker DNA uptake, indicating that prostate cancer cells could not absorb DNA without the help of a carrier.
The cellular uptake of fluorescein-labeled DNA complexed with AuNCs-Lf-PEI in PC-3 cells was qualitatively confirmed using a confocal microscope (Figure 9). After treatment with the AuNCs-Lf-PEI complex, fluorescein-labeled DNA spreads in the cytoplasm. After 24 hours of incubation, no co-localization of DNA was observed in the nucleus. In contrast, the cells treated with the DNA solution did not show any fluorescein-derived fluorescence. Figure 9 Confocal microscope image taken by cells with fluorescein-labeled DNA (2.5 μg per well), compounded with AuNCs-Lf-PEI at a weight ratio of 40:1 or in a solution after 24 hours incubation with PC-3 cells (control : Untreated cells). Blue: cell nucleus stained with DAPI (excitation: 405 nm laser line; bandwidth: 415–491 nm), green: fluorescein-labeled DNA (excitation: 453 nm laser line; bandwidth: 550–620 nm) (bar: 20 µm).
Figure 9 Confocal microscope image taken by cells with fluorescein-labeled DNA (2.5 μg per well), compounded with AuNCs-Lf-PEI at a weight ratio of 40:1 or in a solution after 24 hours incubation with PC-3 cells (control : Untreated cells). Blue: cell nucleus stained with DAPI (excitation: 405 nm laser line; bandwidth: 415–491 nm), green: fluorescein-labeled DNA (excitation: 453 nm laser line; bandwidth: 550–620 nm) (bar: 20 µm).
At all tested concentrations, treatment of PC-3 cells with AuNCs-Lf, AuNCs-Lf-PEG and AuNCs-Lf-PEI resulted in cell viability higher than 70% (Figure 10). At the maximum treatment concentration (200 μg/mL), the cell viability of AuNCs-Lf-PEG and AuNCs-Lf-PEI (83.39 ± 1.10% and 79.27 ± 1.63%, respectively) was higher than that of AuNCs-Lf (73.12). ± 1.80%). This increase is attributed to the binding of PEG and PEI to AuNCs-Lf. Therefore, these results indicate that the AuNCs conjugate is safe against PC-3 cancer cells at the tested concentration. Figure 10 Cell viability of PC-3 prostate cancer cells treated with different concentrations of AuNCs-Lf, AuNCs-Lf-PEG and AuNCs-Lf-PEI (n=15).
Figure 10 Cell viability of PC-3 prostate cancer cells treated with different concentrations of AuNCs-Lf, AuNCs-Lf-PEG and AuNCs-Lf-PEI (n=15).
The possibility of using gold nanocages as a gene delivery system for cancer treatment has been previously reported in combination with photothermal therapy and chemotherapy, but so far has never been evaluated separately without external stimulation. In order to explore this possibility, we hypothesized that the combination of cancer-targeted lactoferrin, PEG or PEI and gold nanocage complexed with plasmid DNA will lead to an increase in the efficiency of gene expression in PC-3 prostate cancer cells.
In this study, gold nanocages have been successfully produced through an electro-replacement reaction between silver nanocubes and an aqueous solution of chloroauric acid. 22 The production of large batches of gold nanocages eliminates batch-to-batch differences that may have adverse effects on the surface conjugation of gold nanocages, their characterization and in vitro experimental results. 26
The binding of Lf, PEG, and PEI was confirmed by using CD and FTIR spectroscopy (in the case of Lf binding) and UV-Vis spectroscopy (used to prove the grafting of Lf, PEG, and PEI). After the combination of PEI and AuNCs-Lf, the surface plasmon resonance of AuNCs-Lf-PEI is 813 nm. Compared with unmodified AuNCs, this red-shifted resonance is attributed to the change of the surface refractive index of AuNCs, which proves the conjugation of PEI and AuNCs surface. As reported by other groups, this absorption point is particularly suitable for using near-infrared radiation to further promote gene delivery. 27 All the gold conjugates synthesized in this study are in the near-infrared region (700-900 nm), making it a suitable platform for imaging and photothermal applications.
Crucially, AuNCs-Lf, AuNCs-Lf-PEG and AuNCs-Lf-PEI conjugates all have a positive charge, which is different from unmodified AuNCs, which are essential for allowing DNA complexation. AuNCs conjugates can compound negatively charged DNA through electrostatic interactions. The weight ratio of conjugate:DNA is 5:1 and above to ensure effective DNA aggregation. These weight ratios of AuNCs conjugate complexes show a suitable size diameter, allowing them to penetrate into cancer cells because most tumors have an extravasation cutoff size of 400 nm. 28
In addition, the conjugation of AuNCs-Lf with PEG and PEI improves DNA complexation efficiency, because AuNCs-Lf-PEG and AuNCs-Lf-PEI conjugates show higher positive charges compared with unmodified AuNCs-Lf. The positive charge is mainly due to the presence of positively charged amino acids in Lf. Therefore, the AuNCs-Lf conjugate has the physicochemical properties required to become an efficient gene delivery system. It is worth noting that the fluorescence quenching properties of gold nanoparticles and the blue color of AuNCs samples interfere with the fluorescence intensity measurement, which may lead to false positive results. 29
Compared with the positive controls PEI and DOTAP, treatment of PC-3 cells with AuNCs-Lf resulted in enhanced transfection of certain conjugate:DNA ratios. This result is consistent with the previous publication, which reported that the combination of Lf and the delivery system significantly improved gene expression in various cancer cell lines. For example, Lim et al. demonstrated that on A431 and B16F10 cells, the combination of Lf and 3-diaminobutyrate-producing polypropyleneimine dendrimer (DAB) resulted in gene expression that was 1.4 times higher than that of unbound DAB. 13 Similarly, Altwaijry et al. reported that PC-3 cells with DAB-Lf dendritic complexes had 2 times higher gene expression compared with unmodified DAB. 14 In this study, the gene expression ratios after treatment with the AuNCs-Lf complex were PEI and DOTAP, respectively. In addition, compared with unmodified AuNCs-Lf, the combination of AuNCs-Lf with PEI and PEG resulted in enhanced gene transfection. Gene expression after treatment with AuNCs-Lf-PEI and AuNCs-Lf-PEG complex, the DNA ratio of AuNCs-Lf-PEI is 40:1, and the DNA ratio of AuNCs-Lf-PEG is 5:1, 1.58 times respectively And 1.33-fold higher than the results obtained from the AuNCs-Lf complex. Compared with DOTAP and PEI, the transfection efficiency of transplanting PEG onto AuNCs-Lf is increased by 1.45 times and 1.66 times. It has previously been shown to improve gene transfection in various delivery systems. For example, Somani et al. reported that in B16F10-Luc, A431, T98G, DU145 and PC-3-Luc cancer cell lines, the transfection level of PEGylated dendrimers was significantly higher than that of non-PEGylated dendrimers.状polymer. 20 In addition, Luan et al. demonstrated that PEGylation of gold nanoparticles resulted in a significant increase in gene silencing in PC-3 cells compared with Lipofectamine® 2000, a lipofection reagent used as a positive control. 30 Recently, it has been reported that PEGylation of gold nanoparticles leads to a 45% increase in transfection efficiency and cellular uptake compared with non-PEGylated nanoparticles, and low cytotoxicity. Therefore, PEGylated gold nanoparticles Become a suitable carrier for DNA delivery. 31 The transfection efficiency after treatment with the AuNCs-Lf-PEI complex is 1.71 times higher than that of PEI and 1.48 times higher than that of DOTAP. This result is consistent with previous studies that the combination of gold nanoparticles and PEI improves transfection efficiency. 32,33 Compared with unmodified, AuNCs-Lf-PEG and AuNCs-Lf-PEI conjugates induce increased β-galactosidase expression. AuNCs-Lf is most likely due to the higher zeta of its DNA complex Potential, which is due to the strong correlation between cellular uptake and the positive charge density of the complex. 34 In addition, the highest transfection efficiency of AuNCs-Lf-PEI is also due to the proton sponge effect of PEI, which greatly promotes endosomal escape. 24,35
Compared with unmodified AuNCs-Lf, the cellular uptake of DNA compounded with AuNCs-Lf-PEI is significantly enhanced. This is because the presence of PEI mediates the proton sponge effect, thereby improving the endosomal escape of DNA. On the other hand, compared to AuNCs-Lf, AuNCs-Lf-PEG resulted in a lower level of DNA uptake after 24 hours of incubation, leading to the hypothesis that DNA uptake after this treatment occurred at a later time because PEG reduces cellular uptake The effect of this is previously reported. 36
Future work should evaluate the interaction of AuNCs-Lf conjugates with protein corona for potential in vivo applications. When exposed to biological fluids, nanoparticles tend to interact with biomolecules, resulting in the formation of a complex protein layer called protein corona. It has previously been demonstrated that the presence of this protein corona on the surface of the nanoparticle can affect the internalization of the delivery system and the release of encapsulated drugs, thereby changing the biological response in the body. 37,38 AuNC with Lf, PEG, and PEI can minimize protein adsorption and change the corona composition, as a study by Assali et al. 39 previously reported, functionalized gold nanometers compared to their unmodified counterparts There are fewer interactions between the platform and proteins, and fewer opsonins are adsorbed.
We demonstrated for the first time that a new type of gold nanocage combined with lactoferrin, PEI and PEG and complexed with plasmid DNA leads to an increase in gene expression in PC-3 prostate cancer cells. The cell viability data demonstrated the low toxicity of these gold conjugates, with cell viability higher than 70% at all tested concentrations. Among the conjugates, the gold nanocage conjugated with lactoferrin and PEI resulted in the highest transfection level of PC-3 cells compared with DOTAP and PEI. This may be due to the significantly increased cellular uptake of DNA in the PC-3 prostate cancer cell line after treatment with this complex compared to what was observed in cells treated with the DNA solution. As far as we know, this is the first time that lactoferrin-containing gold nanocages have been used to target prostate cancer cells, thereby enhancing the DNA uptake and gene expression of PC-3 prostate cancer cells without external stimulation. Therefore, lactoferrin-containing gold nano-clad conjugates are promising gene nanocarriers for prostate cancer cells and will be further studied alone or in combination with other cancer therapies.
AgCl, silver chloride; ANOVA, one-way analysis of variance; ATR, attenuated total reflection; AuNCs, gold nanocages; AuNCs-Lf, AuNCs containing lactoferrin; AuNCs-Lf-PEG, PEG-conjugated, containing lactoferrin Gold nanocage for protein; AuNCs-Lf-PEI, PEI-conjugated, lactoferrin-containing gold nanocage; CD, circular dichroism; CF3COOAg, silver trifluoroacetate; DAPI, 4',6-diamidino -2-Phenylindole; DEG, diethylene glycol; DI, deionized water; DNA, deoxyribonucleic acid; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N, N,N-trimethylammonium methyl sulfate; FBS, fetal bovine serum; FTIR, Fourier transform infrared; HAuCl4, chloroauric acid; HCl, aqueous hydrochloric acid; HS-PEG3.5K-NH2, mercaptoPEG amine; ICP -MS, inductively coupled plasma mass spectrometry; Lf, lactoferrin; MEM, modified Eagle medium; MWCO, molecular weight cut-off; NaCl, sodium chloride; NaSH, sodium hydrosulfide hydrate; PEG, polyethylene glycol; PEI, Polyethyleneimine; PLB, passive lysis buffer; PVP, polyvinylpyrrolidone; RNA, ribonucleic acid; SEM, standard error of the mean; TBE, triborate-EDTA; TEM, transmission electron microscope.
This work was funded by a doctoral scholarship from the Kingdom of Saudi Arabia and Umm Al-Qura University (Kingdom of Saudi Arabia). JASS was funded by Dunhill Medical Trust Fund [Grant No. R463/0216]. PL was funded by Global Cancer Research. The center’s research grant [Grant No. 16–1303] was funded. The author would like to thank Mr. Alexander Clunie for his help with the ICP-MS experiment and CMAC national facility, which is located in the Center for Technology and Innovation of the University of Strathclyde and funded by the UK Research Partnership Institute Fund (UKRPIF) to obtain CD musical instrument.
The authors report no conflicts of interest in this work.
1. Chen Jie, Wang De, Xi Jie, etc. An immune gold nanocage with customized optical properties for targeted photothermal destruction of cancer cells. Nanolet. 2007; 7: 1318-1322. doi:10.1021/nl070345g
2. Sun Tao, Zhang Yongsheng, Pang B, etc. Engineered nanoparticles for drug delivery in cancer treatment. Angew Chem Int Ed Engl. 2014;53:12320-12364. doi:10.1002/anie.201403036
3. Beik J, Khateri M, Khosravi Z, etc. Combination of gold nanoparticles in cancer treatment strategies. Coord Chem Rev. 2019;387:299-324. doi:10.1016/j.ccr.2019.02.025
4. McIntosh CM, Esposito EA, Boal AK, etc. Use a mixed monolayer of cations to protect gold clusters to inhibit DNA transcription. J Am Chem Soc. 2001;123(31):7626-7629. doi:10.1021/ja015556g
5. Oishi M, Nakaogami J, Ishii T, etc. Smart PEGylated gold nanoparticles for siRNA cytoplasmic delivery to induce enhanced gene silencing. Chemical Wright. 2006;35(9):1046-1047. doi:10.1246/cl.2006.1046
6. Rosi NL, Giljohann DA, Thaxton CS, etc. Oligonucleotide-modified gold nanoparticles for intracellular gene regulation. science. 2006;312:1027-1030. doi:10.1126/science.1125559
7. Fitzgerald KA, Rahme K, Guo J, etc. Anisamide targeting gold nanoparticles for siRNA delivery in prostate cancer synthesis, physicochemical characterization and in vitro evaluation. J Mater Chem B. 2016;4(13):2242-2252. doi:10.1039/C6TB00082G
8. Skrabalak SE, Chen J, Sun Y, etc. Gold nanocage: synthesis, properties and applications. Speed up chemical research. 2008; 41: 1587-1595. doi:10.1021/ar800018v
9. Chen J, Glaus C, Laforest R, etc. The gold nanocage is used as a photothermal sensor for cancer treatment. small. 2010; 6: 811-817. doi:10.1002/smll.200902216
10. Xia X, Xia Y. Gold nanocages are used as multifunctional materials for nanomedicine. Former physics. 2014; 9: 378-384. doi:10.1007/s11467-013-0318-8
11. Li W, Cai X, Kim C, et al. Gold nanocages are covered with thermally responsive polymers for controlled release by high-intensity focused ultrasound. nanoscale. 2011; 3(4): 1724-1730. doi:10.1039/c0nr00932f
12. Yavuz MS, Cheng Y, Chen J, etc. The gold nanocage covered by smart polymer is used for controlled release of near-infrared light. Nat. 2009; 8: 935-939. doi:10.1038/nmat2564
13. Lim LY, Koh PY, Somani S, etc. The tumor resolved after intravenous administration of lactoferrin and lactoferrin-containing dendritic complex. Nanomedicine. 2015; 11: 1445-1454. doi:10.1016/j.nano.2015.04.006
14. Altwaijry N, Somani S, Parkinson JA, etc. Prostate tumor regression after intravenous administration of lactoferrin-containing polypropyleneimine dendrimers encoding TNF-α, TRAIL and interleukin 12. Drug delivery. 2018; 25: 679-689. doi:10.1080/10717544.2018.1440666
15. Altwaijry N, Somani S, Dufès C. Targeted non-viral gene therapy for prostate cancer. International J Nanomedicine. 2018; 13: 5753-5767. doi:10.2147/IJN.S139080
16. Sullivan MO, Green J, Przybycien T. Development of a novel gene delivery scaffold using colloidal gold-polyethyleneimine conjugates for DNA condensation. Gene therapy. 2003;10:1882-1890. doi:10.1038/sj.gt.3302083
17. Hu C, Peng Qi, Chen Fei, et al. Low-molecular-weight polyethyleneimine-conjugated gold nanoparticles are used as effective gene carriers. Bioconjug Chemistry 2010;21(5):836–843. doi:10.1021/bc900374d
18. Kong WH, Sung DK, Shim YH, etc. Through fast and simple two-step mixing, including non-covalent post-pegylation, an effective intracellular siRNA delivery strategy. J Control release. 2009;138:141-147. doi:10.1016/j.jconrel.2009.04.034
19. Ping Y, Liu C, Zhang Z, et al. Chitosan-graft-(PEI-β-cyclodextrin) copolymer and its supramolecular PEGylation for DNA and siRNA delivery. biomaterials. 2011;32(32):8328–8341. doi:10.1016/j.biomaterials.2011.07.038
20. Somani S, Laskar P, Altwaijry N, etc. Pegylation of polypropyleneimine dendrimers: effects on cytotoxicity, DNA concentration, gene delivery and expression in cancer cells. Scientific Representative 2018; 8:1-13. doi:10.1038/s41598-018-27400-6
21. Wang Y, Zheng Y, Huang CZ, et al. Synthesis of silver nanocubes with 18-32 nm side length: The effect of polyols on reduction kinetics, size control and reproducibility. J Am Chem Soc. 2013; 135: 1941-1951. doi:10.1021/ja311503q
22. Skrabalak SE, Au L, Li X, etc. Easily synthesize Ag nanocubes and Au nanocages. National agreement. 2007;2(9):2182-2190. doi:10.1038/nprot.2007.326
23. Johnsen KB, Bak M, Kempen PJ, etc. Antibody affinity and valence affect the brain uptake of gold nanoparticles targeted by transferrin receptors. Therapeutics. 2018;8(12):3416. doi:10.7150/thno.25228
24. Kang HC, Kang HJ, Bae YH. A reducible polycationic gene carrier derived from 2-iminosulfane-linked thiolated low molecular weight branched polyethyleneimine. biomaterials. 2011;32(4):1193–1203. doi:10.1016/j.biomaterials.2010.08.079
25. Zinselmeyer BH, Mackay SP, Schätzlein AG, Uchegbu IF. Low-generation polypropylene imine dendrimers are effective gene transfer agents. Medical research. 2002;19:960-967. doi:10.1023/A:1016458104359
26. Avvakumova S, Pandolfi L, soprano E, etc. Is the conjugation strategy important? Cetuximab conjugated gold nanocage for targeting triple-negative breast cancer cells. Nano Advanced 2019; 1:3626-3638. doi:10.1039/C9NA00241C
27. Assali A, Akhavan O, Adeli M, etc. The multifunctional core-shell nanoplatform ([email protection] oxide) has mediated NIR hyperthermia to promote the delivery of miRNA. Nanomedicine. 2018;14:1891-1903. doi:10.1016/j.nano.2018.05.016
28. Yuan F, Dellian M, Fukumura D, etc. Vascular permeability in human tumor xenografts: molecular size dependence and cutoff size. Cancer research. 1995;55:3752-3756.
29. Xia X, Yang Ming, Oetjen LK, et al. An enzyme-sensitive probe for photoacoustic imaging and fluorescence detection of protease activity. nanoscale. 2011; 3:950-953. doi:10.1039/c0nr00874e
30. Luan X, Rahme K, Cong Z, et al. Anisamide targeting PEGylated gold nanoparticles for prostate cancer mediation: enhanced systemic exposure of siRNA, tumor growth inhibition and combined treatment with paclitaxel Synergistic treatment response in mice. Eur J Pharm Biopharm. 2019;137:56-67. doi:10.1016/j.ejpb.2019.02.013
31. Zamora-Justo JA, Africa-González P, Vázquez-Martínez GR, etc. Polyethylene glycol-coated gold nanoparticles are used as DNA and atorvastatin delivery system and cytotoxicity evaluation. J nano materials. 2019;2019:. doi:10.1155/2019/5982047
32. Thomas M, Klibanov AM. Combining with gold nanoparticles can enhance the transfer of plasmid DNA into mammalian cells by polyethyleneimine. Proc Natl Acad Sci US A. 2003;100:9138-9143. doi:10.1073/pnas.1233634100
33. Sharma A, Tandon A, Tovey JC, etc. Polyethyleneimine-conjugated gold nanoparticles: gene transfer potential and low toxicity in the cornea. Nanomedicine. 2011; 7: 505-513. doi:10.1016/j.nano.2011.01.006
34. Futaki S, Ohashi W, Suzuki T, etc. Arginine peptides rich in stearic acid: a new type of transfection system. Bioconjug Chemistry 2001; 12: 1005-1011. doi:10.1021/bc015508l
35. Creusat G, Rinaldi AS, Weiss E, etc. Proton sponge technique for pH-sensitive decomposition of polyethyleneimine-based siRNA delivery systems. Bioconjug Chemistry 2010; 21:994-1002. doi:10.1021/bc100010k
36. Mishra S, Webster P, Davis ME. Pegylation significantly affects the cellular uptake and intracellular transport of non-viral gene delivery particles. European Journal of Cell Biology. 2004;83:97-112. doi:10.1078/0171-9335-00363
37. Monopoli MP, Åberg C, Salvati A, Dawson KA. Biomolecular corona provides the biological properties of nanomaterials. Nat Nanotechnology. 2012; 7: 779-786. doi:10.1038/nnano.2012.207
38. Nguyen VH, Lee BJ. Protein corona: a new approach to nanomedicine design. International J Nanomedicine. 2017; 12: 3137-3151. doi:10.2147/IJN.S129300
39. Assali A, Razzazan S, Akhavan O, etc. Functionalized Au [email protection] The biological interface between GO nanoplatform and protein corona and its impact on delivery and release systems. Colloidal surfing B Biological interface. 2019;173:891-898. doi:10.1016/j.colsurfb.2018.10.042
This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and include the Creative Commons Attribution-Non-commercial (unported, v3.0) license. By accessing the work, you hereby accept the terms. The use of the work for non-commercial purposes is permitted without any further permission from Dove Medical Press Limited, provided that the work has an appropriate attribution. For permission to use this work for commercial purposes, please refer to paragraphs 4.2 and 5 of our terms.
Contact Us• Privacy Policy• Associations and Partners• Testimonials• Terms and Conditions• Recommend this site• Top
Contact Us• Privacy Policy
© Copyright 2021 • Dove Press Ltd • Software development of maffey.com • Web design of Adhesion
The views expressed in all articles published here are those of specific authors and do not necessarily reflect the views of Dove Medical Press Ltd or any of its employees.
Dove Medical Press is part of Taylor & Francis Group, the academic publishing department of Informa PLC. Copyright 2017 Informa PLC. all rights reserved. This website is owned and operated by Informa PLC ("Informa"), and its registered office address is 5 Howick Place, London SW1P 1WG. Registered in England and Wales. Number 3099067. UK VAT group: GB 365 4626 36
In order to provide our website visitors and registered users with services that suit their personal preferences, we use cookies to analyze visitor traffic and personalize content. You can understand our use of cookies by reading our privacy policy. We also retain data about visitors and registered users for internal purposes and to share information with our business partners. By reading our privacy policy, you can understand what data we retain, how we process it, who we share it with, and your right to delete data.
If you agree to our use of cookies and the content of our privacy policy, please click "Accept".