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Photothermal-enhanced phase-transition nanodroplets for ultrasound-mediated diagnosis and gene transfection Jinbiao Gao, Baiqing Yu, Chao Li, Wei Wang, Ming Xu, Zhong Cao, Xiaoyan Xie, and Jie Liu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01611 • Publication Date (Web): 21 Jan 2019 Downloaded from http://pubs.acs.org on January 23, 2019
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Photothermal-enhanced phase-transition nanodroplets for ultrasound-mediated diagnosis and gene transfection Jinbiao Gao,†,‡ Baiqing Yu,†,‡ Chao Li,†,‡ Wei Wang,§ Ming Xu,§ Zhong Cao,† Xiaoyan Xie,§ and Jie Liu*,†
†
Department of Biomedical Engineering, School of Engineering, Sun Yat-sen University,
Guangzhou, Guangdong 510006, China §
Department of Medical Ultrasonics, Institute of Diagnostic and Interventional Ultrasound,
The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510080, China
† These
authors contribute equally to this work.
* To whom correspondence may be addressed. E-mail:
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ABSTRACT Gene therapy is one of the promising solutions in cancer therapeutics. Ultrasound-mediated gene delivery showed great potential as non-invasive strategy for gene therapy. However, the efficiency of gene transfection and incorporation of multiple functions remain key challenges in the development of gene delivery systems. In this study, we developed a perfluoropentane (PFP) and gold nanorods (AuNRs) loading nanodroplets for photothermal-enhanced ultrasound-mediated imaging and gene transfection. The nanodroplet theranostic system was formulated with fluorinated cationic poly(aspartamide) based polymer that encapsulated PFP, AuNRs and plasmid DNA, and was stabilized with negatively charged poly(glutamic acid)-g-MeO-poly(ethylene glycol) (PGA-g-mPEG) coating. The nanodroplets presented good stability, biocompatibility and DNA binding stability. Upon treatment with both near infrared and ultrasound, the photothermal and ultrasound-responsive system exerted a synergistic effect, in which strong adsorption of light induced hyperthermia that promoted phase transition of PFP and following ultrasound irradiation generated strong acoustic cavitation and sonoporation, thus leading to enhanced ultrasound contrast imaging and gene transfection efficiency both in vitro and in vivo. KEYWORDS: Ultrasound-mediated gene delivery; Near infrared; Ultrasound imaging; Hyperthermia; Gold nanorods; Phase-change agent; Theranostics
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1. Introduction In the prolonged fight with cancer, the study of gene therapy rises from various conventional therapeutics, attracting researchers with its potential to achieve efficient ablation of tumors at the genetic level1. Practical gene therapy aims to transfer the genetic agents, such as plasmid DNA and siRNA, through complex extracellular and intracellular barricades, into the target cells to activate the desired anti-cancer mechanisms, and thus requires an engineered gene vector capable of effectively stabilizing and precisely delivering its payload2. Viral vectors, represented by adeno-associated virus vectors and lentiviral vectors, become a potent gene therapy solution with high transfection efficacy3. However, the application of viral vectors is limited by concerns on undesirable immune response or mutagenesis on host cells. Non-viral gene vectors, including polymer-based, lipid-based and inorganic nano-systems, emerged as a promising alternative that emphasizes safety and synergies of multifunctional properties4. Exemplar non-viral gene vectors, such as PEI and DOTMA, utilize a mechanism involving the electrostatic adsorption of anionic nucleic acids to cationic materials that facilitates the cellular uptake of the complex, and the “proton sponge effect” that enables the subsequent endosomal escape of the therapeutic gene5-6. Strategies of stimuli responsiveness applied in advanced transfection systems achieve spatio-temporal controlled gene transfer and also inspire the development of theranostics making the advantages of imaging stimuli such as US, light or magnetic field4. A growing trend in the research of gene therapy has shifted towards the incorporation of additional therapeutic modalities to enhance gene therapy or overall treatment efficacy5. Ultrasound-mediated gene delivery emerged as one of the most promising strategies to improve the performance of gene therapy, as ultrasound is generally regarded to be clinically safe, noninvasive and has deep tissue penetration7. Ultrasound shock wave results in acoustic cavitation and sonoporation that increase the permeability of the affected cell membrane and thus facilitate the transfer of DNA, as revealed by early studies on DNA transfection with exposure to ultrasound8-9. The efficacy of gene therapy is further improved with the recent development of ultrasound-mediated gene delivery systems, which involve the combination of therapeutic gene and ultrasound-responsive contrast
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agents or microbubbles10. Microbubble gene carrier is commonly designed as a contrast gas bubble coated by shell materials with genetic payload, which achieves ultrasound imaging and ultrasound-enhanced uptake11-12. Nanobubbles appeared recently aiming to mitigate the size disadvantage of microbubbles and had shown increased tissue penetration by virtue of enhanced permeability and recruiting (EPR) effect and better stability13-14. Phase-change agents are a novel competitive alternative to gas bubbles that offers superior size advantage with the delivery system fabricated and stronger ultrasoundresponsiveness15-16. Phase-changing nanodroplets, stabilized in lipid or polymer shell, undergo acoustic droplet vaporization (ADV) with ultrasound that triggers the generation of microbubbles, producing strong imaging contrast and intensive acoustic cavitation17. Previous studies shown enhanced uptake and therapeutic effect with drug or gene loaded nanodroplets using phase-change agents such as perfluoropentane (PFP)18. Our group also developed a PFP-loaded nanodroplet with cationic polymer shell and targeting moiety for tumor imaging and gene transfection19. The nanodroplets generated strong cavitation effect following ADV and thereby enhanced imaging contrast and gene transfection efficacy. We noticed that the fabrication of the nanodroplets caused increased surface tension/Laplace pressure that raised the boiling point of PFP to as high as 37 ℃20, rendering decent stability in physiological environment. The potential of phase-change nanodroplet systems is dependent on the vaporization efficiency of the phase-change agent, and one possible way of reinforcement lies in heating, which reduces ADV pressure threshold15. Gold nanoparticle (AuNPs), a representative photothermal agent, has become attractive in cancer therapeutics due to the superior photothermal property21. Gold nanoparticles have large surface electric fields that are highly interactive with light radiation, leading to strong surface plasmon resonance absorption (PRA) in specific wavelengths, which is considerably stronger than the absorption of light-absorbing dyes22. Because of the penetration depth of NIR irradiation, specially tailored AuNPs are able to induce hyperthermia within tissue noninvasively23. AuNPs and its variations are playing an active role in photothermal therapy and multimodal therapeutics, which uses radical local hyperthermia over 50℃ to cause direct ablation of tumors23-24. AuNPs are also exploitable in another approach with mild hyperthermia of 42-45℃, which was proved to increase
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microvascular permeability and significantly boost the delivery of macromolecules22. Recent advancement of gene delivery features the corporation of photothermal agents and mechanisms25-27, and light-induced mild hyperthermia using gold nanorods (AuNRs), a easily tunable variation of AuNPs, also shown optimistic results28-29. In addition, AuNRs are also reported to improve phase-transition efficiency in PFP systems30-31. Therefore, AuNRs is a prospective augmenter with ultrasound-mediated phase-transition system, reinforcing gene transfection with high phase transition efficiency and synergistic effects.
Scheme 1. Fabrication of ultrasound-mediated phase-transition nanodroplets Au@TNDs and mechanisms of ultrasound-mediated imaging and gene transfection in vivo.
In this study, we developed a hybrid gene delivery system combining hyperthermiainducing AuNRs and ultrasound-mediated PFP-loaded nanodroplets (Scheme 1). This nanodroplet system presented high phase transition rate with NIR-induced hyperthermia preceding ultrasound irradiation, leading to strong cavitation and sonoporation, facilitating
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gene transfer and thereby further enhancing gene transfection efficiency. In our design, AuNRs were specially tailored with NIR adsorption, modified with fluorocarbon and dispersed in the phase-change agent PFP, which was then stabilized by a biocompatible, cationic and amphiphilic polymer C9F17-poly {N-[N’-(2-aminoethyl)]-aspartamide} [C9F17-PAsp(DET)]. The resulting nanodroplets were loaded with a reporter gene luciferase DNA (LucDNA) and augmented with PGA-g-mPEG coating for in vivo stability and cellular uptake. The optimized ternary nanodroplets, fluorinated silica-coated AuNRs/PFP/C9F17-PAsp(DET)/LucDNA/PGA-g-mPEG (Au@TNDs) was characterized with dynamic light scattering (DLS) and electron microscopy. Gene loading efficiency, stability and biocompatibility of the nanodroplets were tested. The photothermal properties of the nanodroplets and the effect of light-induced mild hyperthermia on phase transition of PFP were evaluated. Furthermore, gene transfection and ultrasound imaging experiments were performed in vitro and in vivo to evaluate the synergistic effect, imaging capability and overall gene transfection efficacy of the nanodroplet system.
2. Experimental 2.1. Materials For chemical synthesis, 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,9-Heptadecafluorononylamine (C9F17-NH2),
1H,1H,2H,2H-Perfluorodecyltriethoxysilane,
Tetraethyl
orthosilicate
(TEOS) and hexadecyltrimethylammonium bromide (CTAB) were obtained from SigmaAldrich (China). β-benzyl-L-aspartate N-carboxyanhydride (BLA-NCA) was obtained from
Beijing
HWRK
Chem
(China).
N-methyl-2-pyrrolidone
(NMP)
and
Diethylenetriamine (DET) were purchased from the Aladdin Industrial (China). Silver nitrate was purchased from Alfa Aesar China. L-Ascorbic acid and Tetrachloroauric (Ⅲ) acid hydrate were purchased from Sinopharm Chemical Reagent (China). Sodium borohydride was purchased from Shanghai Macklin Biochemical (China). For nanodroplets preparation, perfluoro-n-pentane was obtained from Strem Chemicals (USA). LucDNA (pGL4.13 vector encoding luciferase reporter gene luc2), Assay Reagent
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and Reporter Lysis 5× Buffer Luciferase were obtained from Promega (USA). MTT formazan was obtained from Sigma-Aldrich (China). Regular agarose G-10 was purchased from Biowest (France). Micro-BCA protein assay, 6 × DNA loading dye and LipofectamineTM 2000 (LF2K) were obtained from Thermo Fisher Scientific (China). GelRedTM was purchased from Biotium (USA). in vivo-jetPEI® was purchased from Polyplue-transfection (France). Label IT® TrackerTM Intracellular Nucleic Acid Localization Kit was purchased from Mirus (USA). For cell culture, HepG2 were obtained from Cell Bank of Chinese Academy of Sciences and cultured in a 5% CO2-humidified atmosphere at 37 ℃. Dulbecco’s modified Eagle’s medium was supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillin− streptomycin antibiotic. 2.2. Synthesis of C9F17-poly {N-[N’-(2-aminoethyl)]-aspartamide} [C9F17PAsp(DET)] C9F17-PAsp(DET) was synthesized with a similar process in our previous study19 (process shown in Scheme S1). Briefly, BLA-NCA (0.5 g) and of C9F17-NH2 (37.5 mg) was first dissolved in dimethylformamide (DMF, 5 mL) followed by slow addition of dichloromethane (DCM, 5 mL). The mixture was kept at room temperature for 3 days protected by nitrogen. C9F17-PBLA was obtained after dialysis against DMF for 1 day and water for 2 days, followed by lyophilization. C9F17-PBLA (150 mg) was dissolved in NMP (5 mL) and slowly added with DET (1 mL dissolved with 4 mL of NMP). The reaction was kept at 25 ℃ for 6 h before addition of 1 M HCl (9.7 mL) in an ice bath. C9F17-PAsp(DET) was obtained after dialysis against 0.01 M HCl for 1 day and water for 2 days and lyophilization. All products were stored in -20 ℃. Products of each step were characterized by 1H-NMR with a 400 MHz Bruker AvanceIII spectrometer. The solvents for C9F17-PBLA and C9F17-PAsp(DET) were DMSO-d6 and D2O, respectively. The molecular weight and degree of polymerization were calculated from the spectra.
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2.3. Synthesis of fluorinated silica-coated AuNRs The AuNRs were synthesized according to a previously published method32 and tailored the NIR adsorption wavelength to around 808 nm. Briefly, CTAB (364 mg) was dissolved in deionized water (5 mL) and mixed with 0.5 mM HAuCl4 (5 mL), followed by fast addition of ice-cold solution of NaBH4 (0.6 mL) under vigorous stirring for 2 min. The gold seed solution was obtained after thorough hydrolysis for 2 h at 25 ℃. 3.64 g of CTAB (3.64g) was dissolved in deionized water (5 mL), followed by additions of 10 mM AgNO3 (1 mL), 1 M HAuCl4 (50 mL), HCl (0.2 mL) and 0.0788 M ascorbic acid (0.7 mL) solutions with an interval of 15 min and rapid stirring. The as prepared growth solution was added with seed solution (120 μL) for 12 h at 30 ℃. The generated AuNRs were washed with deionized water, centrifuged and preserved at 25 ℃. The silica-coated AuNRs were synthesized using the Stober method33. Fleshly prepared of AuNRs suspension (10 mL) was added with 0.1 M NaOH to adjust the pH to 10-10.4. TEOS solution (30 μL, diluted in anhydrous methanol, 20% v/v) was slowly added in the AuNRs solution under moderate agitation for three times and each time interval 30 min, followed by gentle stirring for 20 h at 25 ℃. Silica-coated AuNRs was obtained after washing with methanol and dispersed in methanol. Silica-coated AuNRs were fluorinated according
to
the
research
by
Gorelikov
et
al34.
1H,1H,2H,2H-
Perfluorodecyltriethoxysilane (190 μL) was added in silica-coated AuNRs suspension (4 mL, containing 800 μg of AuNRs) with a 1:100 molar ratio of gold to fluorine, followed by stirring for 5 min. Ammonia-water solution (38 μL) was added to the mixture after stirring for 24 h. The fluorinated silica-coated AuNRs was precipitated and collected. The AuNRs, silica-coated AuNRs were observed with a transmission electron microscope (JEOLJEM 1400). The absorption profiles of AuNRs, silica-coated AuNRs and fluorinated silica-coated AuNRs were measured by UV-vis spectrophotometer (Beckman Coulter DU 370). 2.4. Fabrication of nanodroplets The fluorinated silica-coated AuNRs/PFP/C9F17-PAsp(DET) nanodroplets (Au@NDs) were fabricated by oil/water emulsification similar to our previous work. Briefly, C9F17-
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PAsp(DET) (10 mg) was dissolved in deionized water (1 mL) and the solution was precooled to 4 ℃ followed by injection of AuNRs dissolving PFP (200 μL dissolving 800 μg fluorinated AuNRs). The mixture was sonicated with a sonicator (Misonix S-4000) for 90 s (setting: amplitude 22%, power on 1 s, interval 1 s) in an ice-cold bath. The fluorinated silica-coated AuNRs/PFP/C9F17-PAsp(DET)/LucDNA binary nanodroplets (Au@BNDs) were prepared by electrostatic incorporation with LucDNA at an N/P ratio (molar ratio of primary amide groups in the polymer to phosphate groups in DNA) of 20 before incubation for 30 min at 4 ℃. Finally, the fluorinated silica-coated AuNRs/PFP/C9F17PAsp(DET)/LucDNA/PGA-g-mPEG ternary nanodroplets (Au@TNDs) were prepared by adding PGA-g-mPEG (prepared according to previous study35) at a C/N ratio (molar ratio of the carboxyl groups in PGA-g-mPEG to the primary amide groups in the polymer) of 0.4 followed by incubation for 30 min at 4 ℃. The Au@TNDs were preserved at 4 ℃ within 4 h before use. The particle size and surface potential of the nanodroplets were determined by DLS using Malvern Zetasizer Nano ZS90 and observed with TEM after staining with 1% uranyl acetate. 2.5. Stability and biocompatibility study To investigate the DNA binding capability of Au@TNDs after ultrasound irradiation, Au@TNDs were exposed to the ultrasound at various powers. 300 μL Au@TNDs (containing 9 μg DNA) solution (PBS/FBS, 0.01M, pH=7.4, 10% v/v FBS) were added to the 48-well plate and treated with ultrasound with the ultrasound transfection platform (1 MHz, duty cycle 30%, 60 s) at various powers. Following irradiation, Au@TNDs (10 μL) were mixed with 6×DNA loading buffer (2 μL, 0.1 μL/mL gel red) and added into the wells of a 1% (w/v) agarose gel in triacetate-ethylenediaminetetraacetic acid buffer (pH=7.4). Electrophoresis was conducted for 40 min at 100 V and visualized in ultraviolet. To investigate the integrity of DNA, Au@TNDs samples (75 μL) following ultrasound were added with heparin (10 μL) and incubated for 4 h to replace the DNA in the nanodroplets, followed by electrophoresis. Naked DNA were used as control.
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To evaluate the stability of the nanodroplets, Au@NDs, Au@BNDs and Au@TNDs were incubated in PBS (0.01M, pH=7.4, 10% v/v FBS) with constant shaking at 25 ℃ and 37 ℃. The particle size was measured with DLS at various time points. To evaluate the cytotoxicity of Au@TNDs, Au@TNDs with various concentrations of C9F17-PAsp(CET) was added to HepG2 cells seeded in 96-well plate (7×103 cells/well) and incubated for 48 h. The viability of the cells was measured with MTT assay. Briefly, the cells were washed by PBS twice and added with flesh medium containing 20 μL of MTT (5 mg/mL in PBS) per well and incubated for 4 h. The medium was removed and DMSO (200 μL) was added per well to dissolve the formazan crystals, followed by measurement of light absorbance at 570 nm using a BioTek Synergy 4 microplate reader. To evaluate the influence by NIR-induced hyperthermia, HepG2 cells treated with a fixed amount of Au@TNDs were irradiated with NIR laser (808 nm, 4 W/cm2) until the medium reached certain temperatures, which were monitored by an infrared thermometer (Fluke Ti27, USA). The cells were then incubated for another 24 h, followed by the MTT assay. Erythrocyte agglutination and hemolysis were performed to further evaluate the biocompatibility of Au@TNDs. Briefly, erythrocytes (extracted from human blood) were suspended in PBS (1×105 cells/mL) and mixed with Au@BNDs and Au@TNDs for 2 h at 37 ℃. Microscopic images were taken to observe erythrocyte agglutination in the samples, as well as control groups with erythrocytes treated with PBS or Triton X-100. For hemolysis test, erythrocytes were treated with various concentrations of Au@BNDs and Au@TNDs (10-160 μg/mL) and centrifuged after incubation. The absorbance at 413 nm of the supernatant was measured for quantification of hemolysis. 2.6. In vitro photothermal conversion of Au@TNDs Fleshly prepared Au@TNDs solutions (400 μL) with various AuNRs concentration were placed in 1.5 mL eppendorf tubes and were treated with the near-infrared laser at 808 nm (BWT Beijing LTD, China) for 10 min. The temperature of the samples was monitor and thermography were recorded by the infrared thermometer. Deionized water was used as the control. The power levels of near-infrared laser were 1, 2, 3, 4 W/cm2, respectively.
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2.7. In vitro NIR-induced phase transition and US imaging with Au@TNDs To study the photothermal-induced phase transition of PFP, fleshly prepared Au@TNDs (60 μg/mL of AuNRs in water) were put in quartz colorimetric utensil and treated with NIR laser (808 nm, 2 W/cm2) until it reached certain temperatures. Specially, the laser probe was kept away from the utensil at a certain distance so that the area irradiated was 1 cm2. The generation of microbubbles from Au@TNDs at different temperatures were observed by an optical microscope (DM2500p, Leica) on a glass slide. Ultrasound imaging test in vitro with NIR irradiation was performed in plastic pipettes holding Au@TNDs solution (4 mL, the concentration of AuNRs was 40 μg/mL). The pipettes were irradiated by NIR laser and the temperature was detected by the thermometer. When the targeted temperature was reached, the pipettes were dipped into a tank filled with 25 ℃ degassed water and treated with focused US, generated by a diagnostic ultrasound platform (Toshiba Medical Systems Aplio500, Japan) equipping a transducer head (3 cm × 1 cm). All samples were recorded in contrast-enhanced ultrasonography (CEUS) and Bmode imaging at the frequency of 5.0 MHz and 0.7 of mechanical index (MI) value. Ultrasound contrast was quantified with ImageJ software. 2.8. In vitro gene transfection and cellular uptake with Au@TNDs In vitro gene transfection experiment was first performed with varying degree of NIR irradiation for optimization. Briefly, the Au@TNDs (the concentration of AuNRs was 20 μg/mL, containing 1 μg DNA) were added per well in a 48-well plate seeded with HepG2 cells (5×104 cells/well) and incubated for 15 min before irradiation of NIR laser until reaching certain temperatures, followed by 48 h incubation. Gene transfection level of samples was measured by luciferase/fluorescence assay. Briefly, the cells were washed twice with PBS, and added with 1× reporter lysis buffer (100 μL). The cell lysate was centrifuged (12000 rpm, 10 min) and the supernatant (20 μL) was blended with luciferase assay buffer (100 μL), followed by fluorescence detection (in relative light units or RLU) by a luminometer. Furthermore, the protein level was measured using a BCA protein assay kit and the gene transfection efficiency was shown as RLU/mg of protein. LF2K loaded with same amount of DNA was used as positive control.
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Gene transfection experiment was then performed with NIR and ultrasound. For the ultrasound groups, each well was treated with ultrasound twice with the ultrasound transfection platform. For the NIR groups, the NIR laser irradiated each well until reaching the targeted temperature. Specially, the distance between the laser probe and well was kept at 2.8 cm to insure the irradiation coverage. For the combined groups, the cells were treated with NIR laser and ultrasound. Finally, the cells were incubated for 48 h and the transfection levels were measured as described above. To evaluate the cellular uptake of DNA with NIR and ultrasound, the plasmid DNA was marked with fluorescence label according to the kit protocol from the manufacturer. The Au@TNDs (same formulation as above) were loaded with labeled DNA and added to a 48-well plate seeded with HepG2 cells (7×104 cells/well) for 15 min, followed by ultrasound or NIR treatments as described above. After incubation for 4 h, the cells were washed by PBS for three times, digested with trypsin and collected by centrifugation (1500 rpm, 5 min). The samples were re-suspended in PBS (0.4 mL) and analyzed with flow cytometry using NoveCyte (ACEC, USA) at the excitation wavelength of 488 nm. 2.9. Animal experiment and tumor inoculation Female balb/c mice, aged 3-4 weeks and weighed 18-20 g, were supplied by animal experimental center, Sun Yat-sen University (China) and maintained under standard specific pathogen-free conditions. Murine-derived CT-26 tumor bearing mice were established by subcutaneous injection of 1×106 cells suspended in 100 μL PBS into the back of mice. Mice with tumors grown to about 200-300 mm3 were separated into empty treatment groups for stand-by. All animal experiments were performed following guidelines supervised by the ethics committee of Sun Yat-sen University. 2.10. In vivo photothermal conversion of Au@TNDs 4 groups of mice (n=3 per group) were selected and the Au@TNDs (containing various concentration of AuNRs) were injected via intravenously. The saline was injected for control group. 30 min following administration, the mice was anesthetized by intraperitoneal injection of 2% pentobarbital and fixed on a platform. Tumor area was
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treated with NIR laser on one side and the infrared thermometer was placed on the other side to monitor the change of temperature and record thermography. 2.11. In vivo ultrasound imaging with Au@TNDs To investigate the enhancement effect of Au@TNDs for contrast ultrasound imaging, the mice were randomly divided into three groups (n=3 per group) and treated with normal saline without NIR irradiation (group I), normal saline with NIR irradiation (group II), Au@TNDs without NIR irradiation (group III) and Au@TNDs with NIR irradiation (group IV). The NIR laser irradiated the tumor area until reaching the target temperature. The concentration of AuNRs in the sample of Au@TNDs was 80 μg/mL. Samples were injected into the tumor bearing mice intravenously followed by ultrasound irradiation with a diagnostic ultrasound platform (Aplio500). In addition, all the samples recorded in contrast-enhanced ultrasonography (CEUS) and B-mode imaging at the frequency of 5.0 MHz and 0.7 MI value. 2.12. In vivo gene transfection with Au@TNDs To investigate the enhancement effect of Au@TNDs on gene transfection with NIR and ultrasound, fifteen balb/c mice with tumors were divided into five groups: naked DNA (group I) and jetPEI (group II), Au@TNDs with NIR irradiation (group III), Au@TNDs with ultrasound irradiation (group IV), Au@TNDs with NIR and ultrasound irradiation (group V). The tumor area was treated with NIR laser until reaching the target temperature, or treated with ultrasound (5.0 MHz, MI=1.53, 60 s) twice. Each sample contained 25 μg DNA and was injected intravenously. 30 min following administration, the mice were anesthetized by intraperitoneal injection of 2% pentobarbital and fixed on platform. For luciferase assay, the mice were fed for 48 h before tumor tissue was collected. The tissue was treated with 1× reporter lysis buffer according to tumor mass (25%, w/v), smashed by a homogenizer and centrifuged (12000 rpm, 10 min). Supernatant was collected and blended with luciferase assay buffer (1:1), and the fluorescence intensity was detected with a luminometer. The efficiency of gene transfection was quantified as RLU/g of tissue.
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2.13. Statistical Analysis Statistical studies of the data were conducted using a two-sided Student’s t-test. All data are expressed as mean ± standard deviation concluded from more than three repeated experiments. The difference was considered statistically significant when the P value was less than 0.05.
3. Results and discussion 3.1. Synthesis and characterizations of polymers and AuNRs In this study, we utilized a cationic variation of poly aspartic acid C9F17-PAsp(DET), which has a fluorinated moiety that renders affinity to perfluorocarbons. The two-step synthesis process as shown in Scheme S1 was developed based on the previous studies of our work3536.
The fluorinated poly(β-benzyl-L-aspartate), C9F17-PBLA, was synthesized by ring-
opening polymerization of BLA-NCA initiated by C9F17-NH2, and the 1H-NMR spectrum in Fig. S1A (7.28 [COOCH2Ph]) indicated the successful synthesis of C9F17-PBLA. The molecular weight was calculated to be 8440 Da. C9F17-PAsp(DET) was synthesized by aminolysis of benzyl side groups with cationic DET groups, which was confirmed by the presence of DET peaks on the spectrum in Fig. S1B (2.90 [NHCOCH2CH], 3.05 [NHCH2CH2NH], 3.16 [NH2CH2CH2NH, NHCH2CH2NH], 3.39 [NH2CH2CH2]). The photothermal agent AuNRs was synthesized using the prevalent approach of seedmediated anisotropic growth37. The light adsorption and scattering of AuNRs were known to be dependent on the anisotropy of the particles, which can be controlled by the synthesis process. In this work, the aspect ratio of the AuNRs was tailored to around 3.9 (110 nm in length and 30 nm in width on average) with a specified formulation, as confirmed in the TEM image of AuNRs in Fig. 1A. From the adsorption spectrum of AuNRs in Fig. 1D, AuNRs showed two adsorption peaks at 1) 510 nm, which was due to transverse oscillation of gold particles, and 2) around 810 nm, which was due to longitudinal plasma resonance relative to the aspect ratio of the AuNRs32. The strong light adsorption of AuNRs at around 810 nm, which is in a NIR region with preferable energy and penetration depth23, indicated
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that the AuNRs were capable to exert strong photothermal property with enough tissue penetration and thus desirable for cancer therapeutics.
Fig. 1. TEM images of (A) AuNRs and (B) silica-coated AuNRs. (C) Picture and schematic of fluorinated silica-coated AuNRs dispersed in PFP. (D) UV-vis spectrums of AuNRs (water), silicacoated AuNRs (methanol) and flourinated silica-coated AuNRs (PFP).
In order to encapsulate AuNRs in the PFP-loaded nanodroplets, we designed a two-step process to render the AuNRs affinity to perfluorocarbon. AuNRs were first modified with a silica-coating, as visualized in Fig. 1B and then grafted with perfluorocarbon on the silica surface, as confirmed by AuNRs stably dispersing in PFP (Fig. 1C). The decreased adsorption at 810 nm of silica-coated AuNRs compared to bare AuNRs was possibly caused by the blocking of silica surface (Fig. 1D), and the shifting of adsorption peak of fluorinated silica-coated AuNRs was possibly due to the fluorinated surface and the change of refractive index of the solvent38.
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3.2. Fabrication and characterizations of nanodroplets Ultrasound- and NIR- responsive phase-change nanodroplet gene delivery system was fabricated with a three-step process. The first precursor of the nanodroplet system, Au@NDs, was formulated by emulsification of AuNRs-containing PFP and the fluorinated C9F17-PAsp(DET) and obtained as a milk-like dispersion (Fig. 2A). According to DLS results, Au@NDs has a relatively uniform particle distribution in a size of 396 nm and strong positive surface potential of about 55.9 mV (Table 1). LucDNA was electrostatically bond to the cationic Au@NDs, forming Au@BNDs.
Fig. 2. (A) Picture of Au@NDs before and after emulsification and (B) TEM image of the Au@TNDs.
The optimized formulation (N/P=20) was concluded from previous work and the loading of DNA was confirmed by the slight increase in particle size and decrease of surface charge. The encapsulation of AuNRs was also confirmed in the TEM image of Au@BNDs (Fig. 2B). Au@BNDs were further modified with PGA-g-mPEG at C/N=0.4, a functional coating that promoted in vivo stability and biocompatibility of the nanodroplets36. The resulted Au@TNDs enlarged and the surface charge was neutralized (Table 1). The promoted stability of the resulted Au@TNDs was partly demonstrated in the TEM images: Au@TNDs were more uniform in size and shape (Fig. 2B). The optimized Au@TNDs had an average diameter of 483 nm and a slightly positive surface charge of 11.7 mV.
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Table 1 Characterization of Au@NDs and the optimized Au@BNDs and Au@TNDs.
Sample
Z-average (nm)
PDI
Zeta potential(mV)
Au@NDs
395.7±8
0.114
55.9±2.2
Au@BNDs (N/P=20)a
415.7±6
0.237
38.7±1.3
Au@TNDs (C/N=0.4)b
483.9±4
0.332
11.7±0.2
a. N/P represents the molar ratio of the primary amide groups in C9F17-PAsp(DET) to the phosphate groups in DNA. b. C/N represents the molar ratio of the carboxyl groups in PGA-g-mPEG to the primary amide groups in C9F17-PAsp(DET).
3.3. Stability and biocompatibility of the nanodroplets From the results of stability test on nanodroplets (Fig. 3A), Au@TNDs presented good stability at 25 ℃ in that the size of the nanodroplets maintained below 450 nm for at least 24 h, while the size of Au@NDs and Au@BNDs increased rapidly at the beginning of incubation and then displayed a sharp decrease after 24 h. Meanwhile, the count rate of these two samples decreased from 230 to less than 100. These results indicated that the Au@NDs and Au@BNDs with strong positive charge are not stable in physiological condition due to particle aggregation and adsorption of proteins at the beginning, the following size decrease was the collapse of the nanodroplets caused by vaporization. In contrast, the stability of Au@TNDs was largely attributed to the coating of PGA-g-mPEG, which reduced the surface charge and shielded the nanodroplets from aggregation and adsorption of macromolecules in physiological environment. At 37 ℃, Au@TNDs enlarged to about 600 nm within 4h and collapsed afterwards, likely due to slight vaporization of PFP. PGA-g-mPEG also enhanced the biocompatibility of nanodroplets as Au@TNDs showed low cytotoxicity at high polymer concentrations (Fig. 3B), and good serum compatibility compared with the non-modified Au@BNDs (Fig. 3C, D). The stability of DNA loading in Au@TNDs was also tested with conditions of ultrasound irradiation. Electrophoresis results showed that Au@TNDs maintained a stable binding of DNA with ultrasound power as high as 2.2 W/cm2 (Fig. S2A). DNA band appeared at the same distance as control for all samples following addition of heparin (Fig. S2B),
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suggesting that the DNA loaded in Au@TNDs was integrated despite irradiation of ultrasound in different power.
Fig. 3. (A) Particle size of the Au@NDs, Au@BNDs and Au@TNDs incubated in PBS/FBS (0.01M, pH=7.4, 10% v/v) at 25 ℃ or 37 ℃ within 24 h. (B) Viability of the HepG2 cells treated with the Au@TNDs at different polymer concentrations for 48 h. (C) Microscopic images of erythrocyte agglutination in samples incubated with Au@BNDs, Au@TNDs, PBS or Triton X-100 (Scale bar: 10 μm). (D) Hemolysis levels in samples incubated with various polymer concentrations of Au@BNDs and Au@TNDs for 2 h.
3.4. In vitro photo-thermal conversion of Au@TNDs The photo-thermal conversion efficiency of Au@TNDs was tested in various conditions in vitro. The temperature curves in Fig. 4A demonstrated that the photothermal heating rate corresponded well with the amount of AuNRs in low ultrasound energy (2 W/cm2), indicating that Au@TNDs possessed a superior photothermal property. On the other hand, the temperature curves of various ultrasound energy in Fig. 4B showed intense hyperthermia generated from low content of AuNRs in high ultrasound energy (3-4 W/cm2)
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with the implication of tissue damage. Other than applying the common photothermal therapy that causes heat-induced tumor ablation, we utilized photothermal hyperthermia to improve the performance of the nanodroplet gene delivery system without causing undesirable damage. Therefore, a moderate energy level of ultrasound of 2 W/cm2 was preferable for investigating the effect of hyperthermia in a safe and controlled manner and was used in the following experiments.
Fig. 4. The temperature changes of Au@TNDs solution (400 μL) over time at different concentration of AuNRs (A) and different NIR irradiation power (B).
3.5. In vitro phase transition and ultrasound imaging with Au@TNDs To study the effect of hyperthermia on the nanodroplets, the temperature-dependent phase transition of PFP was investigated. As shown in the optical images of Au@TNDs in different temperatures (Fig. 5A), minimal bubble generation was observed below 37 ℃ despite the boiling point of PFP being 29 ℃, which was explained by the theory that fabrication of nanodroplets increases the energy threshold for vaporization16. Microbubble generation was noticeable at 45 ℃ and became considerable at 50 ℃, suggesting the accelerating phase transition of PFP influenced by hyperthermia. The amount of microbubbles plummeted at 55 ℃, possibly due to rapid vaporization and escape of PFP from the microbubbles. The following ultrasound imaging test of Au@TNDs showed similar results. Both CEUS and B-mode images (Fig. 5B) as well as the quantification graph (Fig. 5C) displayed increasing contrast or greyscale with rising temperature, which peaked at 50 ℃ and depleted at 55 ℃. These results suggested that hyperthermia indeed promoted phase
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transition of PFP in Au@TNDs and thereby enhanced the imaging capability of Au@TNDs, and we believe that the ultrasound irradiation at 37 ℃ to 45 ℃ could generate enough contrast signal for ultrasound mediated image diagnosis.
Fig. 5. (A) Microscopic images of Au@TNDs (the concentration of AuNRs is 60 μg/mL) and deionized water treated with NIR irradiation (808 nm, 2 W/cm2) and reaching targeted temperature from 37 ℃ to 55 ℃ (bar size: 200 μm). (B) Contrast-enhanced ultrasound CEUS (left) and B-mode (right) images of Au@TNDs (the concentration of AuNRs is 40 μg/mL) when the temperature reached the various temperature under the NIR irradiation. (C) Contrast and grey scale of each group were quantified.
3.6. In vitro gene transfection with Au@TNDs The cytotoxicity relative to photothermal degree was first investigated. The result (Fig. 6A) showed that cytotoxicity remained negligible below 45 ℃, started to increase at 50 ℃ and became considerably high at 55 ℃. The increased cytotoxicity was likely due to cell ablation in high temperatures. On the other hand, the gene transfection level detected at 45 ℃ was higher than that at 37 ℃, and transfection level noticeably decreased at higher temperatures of 50 ℃ and 55 ℃ (Fig. 6B). These results, combined with conclusions from
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section 3.5, suggested that below 45 ℃, the rise of temperature enhanced gene transfection efficiency, which was attributed to promoted phase transition of PFP; at temperatures higher than 45 ℃, the increased cytotoxicity complicated the effect of hyperthermia on gene transfection. Considering that phase transition and imaging capability of Au@TNDs was sufficient (Fig. 5) and cytotoxicity was low at 45 ℃, we concluded that this level of photothermal hyperthermia was preferable to achieve an accurate evaluation on the ultrasound imaging and gene transfection performance of the Au@TNDs and used in the following experiments.
Fig. 6. (A) Cell viability at different temperatures heated with NIR irradiation and incubated for 24 h (the concentration of polymer and AuNRs were 75 μg/mL and 5 μg/mL, respectively). (B) Luciferase activities in HepG2 cells treated with Au@TNDs and NIR irradiation (808nm, 2 W/cm2) for various temperature, followed by ultrasound irradiation (1 MHz, 2.2 W/cm2, 60 s) twice. LF2K was used as positive control. (C) Luciferase activities in HepG2 cells transfected with LucDNA using Au@TNDs with various conditions. (D) Fluorescence histograms of the HepG2 cells incubated for 4 h with the Au@TNDs (under various conditions) bearing fluorescein-labeled Luc DNA. *p