Prussian blue nanoparticle-loaded microbubbles for photothermally enhanced gene delivery through ultrasound-targeted microbubble destruction

Prussian blue nanoparticle-loaded microbubbles for photothermally enhanced gene delivery through ultrasound-targeted microbubble destruction

Sci. Bull. (2016) 61(2):148–156 DOI 10.1007/s11434-015-0988-4 Article Materials Science Prussian blue nanopar...

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Sci. Bull. (2016) 61(2):148–156 DOI 10.1007/s11434-015-0988-4


Materials Science

Prussian blue nanoparticle-loaded microbubbles for photothermally enhanced gene delivery through ultrasound-targeted microbubble destruction Xiaoda Li • Xiuli Yue • Jinrui Wang • Xiaolong Liang • Lijia Jing Li Lin • Yongbo Yang • Shanshan Feng • Yajun Qian • Zhifei Dai

Received: 9 August 2015 / Revised: 31 October 2015 / Accepted: 3 December 2015 / Published online: 14 January 2016 Ó Science China Press and Springer-Verlag Berlin Heidelberg 2016

Abstract By adsorbing chitosan (CS)-functionalized Prussian blue (PB) nanoparticles (CS/PB NPs) complexing DNA onto the surface of gas encapsulated microbubbles (MBs), a multifunctional gene delivery system of [email protected]/PB/DNA was fabricated for photothermally enhanced gene transfection through ultrasound-targeted microbubble destruction. CS/PB NPs of (2.69 ± 0.49) nm could complex DNA effectively when the mass ratio was 2:1. It was found that [email protected]/PB/DNA could enhance ultrasound imaging greatly both in vitro and in vivo. In addition, [email protected]/PB/DNA could be disrupted by applying a higher-intensity ultrasound irradiation to release CS/PB/DNA, which could effectively transform the nearinfrared (NIR) light into heat to assist the uptake of CS/PB/ DNA by cells. With the aid of ultrasound irradiation and NIR light irradiation, the gene transfection efficiency was significantly enhanced to (43.08 ± 1.13) %, much higher than polyethylenimine. Moreover, [email protected]/PB/DNA showed excellent biocompatibility, encouraging the further exploration of [email protected]/PB/DNA to be a platform for combined ultrasound image, photothermal therapy, drug delivery, and gene therapy.

Electronic supplementary material The online version of this article (doi:10.1007/s11434-015-0988-4) contains supplementary material, which is available to authorized users. X. Li  X. Yue (&)  L. Jing  L. Lin  Y. Yang  S. Feng School of Municipal and Environmental Engineering, School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China e-mail: [email protected] J. Wang  X. Liang  Y. Qian  Z. Dai College of Engineering, Peking University Third Hospital, Peking University, Beijing 100871, China


Keywords Ultrasound imaging  Microbubble  Gene delivery  Prussian blue nanoparticle

1 Introduction In recent years, gene therapy has progressed rapidly as a revolutionary therapeutic strategy of preclinical and clinical research to treat various types of diseases including tumor diseases [1, 2]. To satisfy the clinical applications of cancer gene therapies, the development of gene delivery systems with high efficiency, safety, and selective targeting ability is highly required for gene therapy because of easy hydrolysis in biological fluids and low cellular uptake efficiency of the nucleic acids with polyanionic nature. So far, a variety of gene delivery vectors have been developed and optimized. Particular research interests have been focused on developing gene delivery vectors which are responsive to external stimuli, such as light, temperature, radiofrequency, magnetic field, and ultrasound. Among of them, light takes more advantage to deliver genes at the desired site at a specific time with high efficiency and minimum adverse effects [3]. In particular, near-infrared (NIR) light has received much attention due to the ‘‘water window’’ (650–900 nm) with minimal absorption by skin and tissue, thus leading to deep tissue penetration with high spatial precision without damaging normal biological tissues [4]. It was reported that polyethylenimine dual-functionalized nanographene oxide could enhance intracellular transportation and the gene transfection efficiency greatly upon irradiation with NIR light [5]. Chitosan (CS), naturally occurring linear cationic polysaccharide with good biocompatibility, has been widely used to modify the nanoparticles for gene delivery, showing high transfection efficiency and little cytotoxicity

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[6, 7]. Prussian blue (PB) has been a drug in clinic for many years for the treatment of radioactive exposure and approved by USA Food and Drug Administration (FDA). Its stability in biological environments and biosafety in the human body have been proved in clinic for several decades. Our group [8] has demonstrated that PB could be a new-generation photothermal ablation agent for cancer photothermal therapy because of its strong absorption in the NIR region and the superior photothermal efficiency. In addition, CS functionalized PB nanoparticles (CS/PB NPs) were fabricated as a photothermal ablation agent to enhance gene transfection efficiency under NIR laser irradiation [9]. Among all the diagnostic imaging techniques, ultrasound imaging has been the most widely used clinical diagnostic imaging modality due to its unique features including real time, low cost, high safety, and ready availability for portable devices [3]. With the use of ultrasound contrast agents (UCAs), such as gas-filled microbubbles (MBs), the resolution and sensitivity of clinical ultrasound imaging can be greatly improved. Moreover, MBs have been developed as novel controlled-release carrier vehicles for targeted gene or drug delivery through ultrasound-targeted microbubble destruction (UTMD) technique [10, 11]. The triggered microvessel ruptures can provide a focal delivery of colloidal particles in a given tissue. The main obstacles for successful gene therapy are the insufficient delivery of genes to target tissues and the difficulty to monitor gene delivery. The imaging strategies offer us an opportunity to optimize gene therapy by evaluating the effectiveness of gene delivery noninvasively and spatiotemporally. In this work, DNA was absorbed onto CS/PB NPs, and the resulting CS/PB/DNA complex was further loaded onto the surface of gas encapsulated microbubbles ST68 MBs, which were generated from Span 60 and Tween 80 (Fig. 1a). The resulting [email protected]/PB/DNA showed the outstanding ultrasound imaging capability, which may have the potential to monitor gene delivery. After reaching the targeting sites, the ST68 MBs could release CS/PB/DNA complex to penetrate into the tumor interstitium through UTMD by applying the higher-intensity ultrasound irradiation. Followed by the NIR light irradiation, CS/PB/DNA could enhance gene transfection efficiency greatly (Fig. 1b).

2 Materials and methods 2.1 Preparation of CS/PB/DNA complexes for electrophoresis assay CS/PB NPs was prepared according to the literature [12]. Assigned amounts of CS/PB NPs in 10 lL deionized water


were mixed with 1 lg DNA in 10 lL deionized water at different molar ratios of 0:1, 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, 4:1, and 8:1, and incubated at room temperature for 20 min. Then CS/PB/DNA complexes were analyzed by 0.8 % agarose gel electrophoresis running in the Tris– EDTA buffer at 120 V for 30 min. The gel was stained with EB and imaged by a gel imaging analysis system. 2.2 Formation of [email protected]/PB/DNA ST68 MBs encapsulating perfluoropropane gas were prepared from Span 60 and Tween 80 according to the literature [13]. A total of 0.2 mL [email protected]/PB/DNA aqueous solution (containing 0.5 mol/L NaCl) was added into 0.2 mL ST68 MBs suspension in the centrifuge tube. The mixture was slightly shaken for 10 min to allow the sufficient adsorption reaction and centrifuged at 500 r/min for 5 min. The obtained [email protected]/PB/DNA were resuspended and washed by 0.4 mL PBS for three times. A total of 1 9 107/mL [email protected]/PB/DNA contained 10 mg/L DNA, 20 mg/L CS/PB NPs, and 1 9 107/mL MBs. 2.3 UTMD and ultrasound imaging in vitro and in vivo An ultrasonic transfection instrument (SonoPore KTAC4000, NepaGene, Japan) was used to irradiate [email protected]/ PB/DNA solution by ultrasound for 30 s at the power density of 0.8 W/cm2 to simulate UTMD process in vitro. The sample was then filtered through 0.45-lm filters, and only CS/PB/DNA could pass through. Then, the concentration of Fe was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). A tube simulating as the blood vessel phantom was immersed in a water tank in which an ultrasound probe was pointing closely at the tube. [email protected]/PB/DNA was injected into the tube with an inner diameter of 5 mm for ultrasonography in vitro. Ultrasonograms were obtained using a broadband linear array L9-3 transducer (9–3 MHz extended) of the IU22 ultrasound system (Philips Medical Systems, Germany) from the longitudinal cross section of the tube. The pulse inversion harmonic imaging (PIHI) mode with a mechanical index (MI) of 0.06 was applied to acquire contrast-enhanced images. A New Zealand white rabbit was anesthetized with 2 % w/v pentobarbital sodium (2.0 mL/kg weight). In total, 200 lL [email protected]/PB/DNA was injected through ear vein and flushed with 1.0 mL saline. The right kidney was imaged using a broadband L9-3 transducer of the IU22 ultrasound system in PIHI mode with MI of 0.06. All applicable institutional and/or national guidelines for the care and use of animals were followed.



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Fig. 1 (Color online) Schematic illustration of the preparation of [email protected]/PB/DNA (a), combined ultrasonic imaging and photothermally enhanced gene delivery with [email protected]/PB/DNA through UTMD (b)

2.4 NIR light-induced temperature change and cytotoxicity induced by UTMD and laser irradiation The solution of different concentrations of [email protected]/PB/ DNA in 3.0 mL aqueous solution was placed in a cuvette. The samples were irradiated for 15 min with an opticalfiber-coupled NIR laser (808 nm, 2 W) at the power densities of 2 W/cm2. The temperature was kept a recorded with a digital thermometer probe with 10-s interval. Cytotoxicity of [email protected]/PB/DNA induced by UTMD and laser irradiation was evaluated on human cervical cancer HeLa cell line as determined by the standard 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 2.5 The pEGFP-C1 plasmid transfection in vitro For pEGFP-C1 plasmid transfection in vitro, HeLa cells were plated in 24-well plate at a density of about 5 9 105 cell per well and incubated 24 h at 37 °C. For one well, 1 lg of pEGFP-C1 plasmid was diluted in 25 lL FBS-free RPMI-1640 medium, while 2 lg CS/PB NPs were dissolved in 25 lL FBS-free RPMI-1640 medium. After being gently mixed, the mixtures were kept at room temperature for 20 min. Then, 2 9 107/mL MBs in 50 lL FBS-free RPMI-1640 medium were added, gently mixed, and incubated for another 20 min before being added into cells. HeLa cells were irradiated by ultrasound for 30 s at the power density of 0.8 W/cm2 in 24-well culture plates to


simulate UTMD process in vitro. After preheating the cells to 37 °C for 5 min, the cells were irradiated by the 808-nm laser light for 0, 1, 2, and 3 min at the power density of 2 W/cm2 in an incubator at 37 °C. After incubation for another 4 h under 37 °C in the incubator, HeLa cells were washed twice with PBS solution and then incubated with 1 mL fresh RPMI-1640 medium for another 48 h. The pEGFP-C1 expression efficiency in HeLa cells was detected by both fluorescence microscope and flow cytometer (FCM). For FCM measurement, HeLa cells were collected and washed with PBS carefully. Then, the cells were stained with 1 lg/mL PI for 10 min, centrifuged for 5 min at 500 g, rinsed carefully with PBS, and analyzed with a BD Calibur flow cytometer. In this FCM analysis, 1 9 106 control cells were selected and evaluated to set as the single cells region. Then a negative control was used to set the position, which was the boundary of separate controls and positive cells, ensuring the number of positive cells was 0.1 % of negative cells. The percent of the positive cells without the dead cells was count as the gene transfection efficiency [5, 14, 15]. 2.6 Cytotoxicity assay and biocompatibility evaluation Four cell lines including human cervical cancer HeLa cell line, human umbilical vein endothelial cells (HUVECs cell line), bone marrow dendritic cells (BMDC), and T cells were selected to test the cytotoxicity of [email protected]/PB/ DNA in vitro [16–20], with the MTT reduction assays and double staining with both calcein acetoxymethyl ester

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(calcein-AM) and propidium iodide (PI). More details about experiments are shown in the Electronic Supplementary Material.

3 Results and discussion Transmission electron micrograph showed that CS/PB NPs were spherical (Fig. S1a online), and the average size was measured to be (2.69 ± 0.49) nm with the dynamic light scattering (DLS) measurement (Fig. S1b online). X-ray powder diffraction (XRD) peaks of CS/PB NPs were all quite well consistent to PB (Fig. S1c online), indicating that PB was embedded within CS matrix. Fourier transform infrared spectroscopy (FTIR) spectrum of CS/PB NPs showed typical peaks of both CS and PB (Fig. S1d online), further confirming the formation of CS/PB NPs with CS and PB. Thermogravimetric analysis (TGA) showed the weight loss was 58.59 %, 61.59 %, and 54.61 % at 600 °C for the CS/PB NPs, CS, and PB, respectively. Therefore, the content of CS and PB was evaluated to be about


57.0 wt% and 43.0 wt% PB in the composite CS/PB NP, respectively. Optical microscope images revealed the good dispersity of ST68 MBs and [email protected]/PB/DNA, and their diameters were (1.28 ± 0.43) and (2.58 ± 1.09) lm using the static light scattering (SLS) measurements, respectively (Fig. 2a, b). The microbubbles with the diameter less than 7 lm met the requirement for clinical applications as ultrasonic contrast agent. The UV–Vis–NIR absorbance spectra showed that both CS/PB NPs and [email protected]/PB/DNA exhibited strong absorption at the wavelength of 700 nm (Fig. 2c). The dynamic absorption of [email protected]/PB/DNA at 700 nm was also measured (Fig. 2d). The reduction of absorbance vs. time indicated that [email protected]/PB/DNA floated up to the solution top in 60 min, which was confirmed by the picture of Fig. S1e (online). All these results demonstrated that most of the CS/PB/DNA was successfully adsorbed onto the surface of ST68 MBs, providing the possibility that [email protected]/PB/DNA could be exploited as a photocontrollable gene delivery system with NIR laser irradiation.

Fig. 2 (Color online) Optical microscope images of ST68 MBs (a), [email protected]/PB/DNA (b), insets show the size distributions. c UV–vis–NIR absorption spectra. d Absorbance of [email protected]/PB/DNA at 700 nm changes with time increasing



Agarose gel electrophoresis assay was chosen to evaluate the DNA plasmid binding ability of CS/PB NPs at various mass ratios. When the mass ratio of CS/PB and DNA was 2:1 (with the N/P ratio (the ratios for the amine groups moles of cationic polymers to those of the phosphate ones of DNAs) 2.28, 57.0 wt% CS in CS/PB NPs) or even higher, all the DNA plasmid could be firmly bound to CS/PB NPs without dissociative DNA detecting (Fig. 3a). As shown in Fig. 3b, the zeta potential of CS/PB was

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(32.04 ± 3.60) mV. In CS/PB NPs, CS could stabilize the PB nanoparticles and offer the positive charge to binding DNA because of electrostatic adsorption. After complexing with DNA, the zeta potential reduced to (17.12 ± 1.89) mV when the mass ratio of CS/PB and DNA was 2:1. After absorbing CS/PB/DNA onto the surface of MBs, the zeta potentials of [email protected]/PB/DNA decreased to (8.38 ± 1.17) mV. Therefore, the CS/PB/ DNA complexes with mass ratio of 2:1 were chosen for the

Fig. 3 (Color online) a Agarose gel electrophoresis assays of CS/PB/DNA complexes at various mass ratios. b Zeta potential of [email protected]/PB/ DNA complexes. c The cell survival rate of HeLa cells after treatment with different ultrasound intensity at different [email protected]/PB/DNA concentration. d The cell survival rate of HeLa cells after treatment with ultrasound intensity at 0.8 W/cm2 with the different times. e Temperature changes of [email protected]/PB/DNA under NIR laser irradiation. f Relative cell viability data of HeLa cells treated by [email protected]/PB/ DNA after laser irradiation by MTT assay


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following experiments. The cell survival rate of HeLa cells was investigated after treatment with UTMD with different [email protected]/PB/DNA concentration and ultrasound intensity and with fixed ultrasound intensity at different time, respectively. According to Fig. 3c, d, the UTMD treatment condition was optimized at 1 9 107/mL [email protected]/PB/ DNA, 0.8 W/cm2 ultrasound intensity for 30 s, and at this condition, the relative cell viability of HeLa cells was still above 90 %. To evaluate the targeted tumor effect of [email protected]/PB/DNA induced by UTMD, [email protected]/PB/ DNA in RPMI-1640 culture media was insonated and filtered through 0.45-lm filters to simulate leaky tumor vasculature, allowing only CS/PB/DNA to pass through. Then, the concentrations of PB NPs in [email protected]/PB/DNA and the PB NPs in CS/PB/DNA were measured by ICPOES [21]. The results revealed that more than 96 % of CS/ PB/DNA was successfully passed through the filters. As shown in Fig. 3e, after being exposed to 808-nm NIR laser light for 15 min, there were significant temperature changes (10.2 °C) of the [email protected]/PB/DNA solution with concentration of 30 mg/L (CS/PB), much higher than the pure water sample (5.0 °C). To optimize photocontrollable gene delivery, proper irradiation time was essential. As


shown in Fig. 3f, [email protected]/PB/DNA (20 mg/L CS/PB) were added to HeLa cells and treated with UTMD for 30 s and then followed by irradiation with an 808-nm laser for different time. After being irradiated for 3 min, the relative cell viability of HeLa cells was 83.9 % ± 4.2 %, which was still above 80 %, suggesting that the safe irradiation time should be less than 3 min. Within this time, NIR laser irradiation would not cause significant cell death of HeLa cells. To evaluate the acoustic enhancement effect of [email protected]/PB/DNA, ultrasound contrast imaging experiments were carried out in vitro by injecting [email protected]/PB/ DNA and CS/PB/DNA into a latex tube (Fig. 4a, b, and Fig. S2a, b online), and in the kidney of New Zealand white rabbits by injecting through ear vein (Fig. 4c, d, and Fig. S2c, d online) [22]. It could be found that [email protected]/ PB/DNA could significantly enhance the ultrasound imaging than CS/PB/DNA both in vitro and in vivo. [email protected]/PB/DNA had good blood stability in vivo, and the blood half-life of [email protected]/PB/DNA was evaluated to be (2.69 ± 0.4) h (Fig. S 1f online). Figure 5a shows the gene transfection efficiency of HeLa cells incubated for 4 h with enhanced green

Fig. 4 In vitro ultrasound contrast-enhanced images in a latex tube without (a) or with (b) [email protected]/PB/DNA. In vivo ultrasonograms in the rabbit right kidney before (c) and after (d) administration of [email protected]/PB/DNA (MI = 0.06)



fluorescent protein (EGFP) DNA alone, DNA mixed with PEI, and DNA with [email protected]/PB/DNA at different laser irradiation time after UTMD for 30 s. These data were well consistent with the quantification results measured by flow cytometry (Fig. 5b). The gene transfection efficiency of PEI/DNA (16.28 % ± 1.23 %) (Fig. 5a2) appeared to be higher than both control (Fig. 5a1) and [email protected]/PB/DNA without light irradiation after UTMD (11.50 % ± 1.86 %) (Fig. 5a3). Nevertheless, upon UTMD followed by laser irradiation, the gene transfection efficiency of [email protected]/ PB/DNA was improved apparently. When irradiating for 3 min, the gene transfection efficiency of [email protected]/PB/ DNA was increased to 43.08 % ± 1.13 % (Fig. 5a6), much higher than PEI/DNA (p \ 0.01). The data in Fig. 5b showed that the gene transfection efficiency would be significantly enhanced by assisting the delivery of DNA into the cells with the photothermal effect of [email protected]/PB/ DNA induced by NIR light after UTMD. Gene transfection enhancement was successfully obtained with laser irradiation that caused a temperature rise. The function of this carrier system was provided by a photothermal effect of the Prussian blue moiety and ultrasound-targeted microbubble destruction. To evaluate the biosafety of [email protected]/PB/DNA in vitro, four cell lines were selected to test the cytotoxicity, including HeLa cells, HUVECs cells, BMDC, and T cells. After incubated with [email protected]/PB/DNA for 24 or 48 h, cell viabilities were tested by MTT assay. As shown in Fig. 6a, the half maximal inhibitory concentration (IC50) of PEI with HeLa cells for 24 h incubation was about 10.0 mg/L. In contrast, the cell viability of HeLa cells

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incubated with [email protected]/PB/DNA for 24 h was 94.2 % ± 2.0 % at the concentration of 200 mg/L (CS/ PB). After incubating normal cell line HUVECs cells with [email protected]/PB/DNA for 24 and 48 h, the cell viability were 97.7 % ± 3.6 % and 91.8 % ± 2.1 % at the concentration of 800 mg/L (CS/PB) (Fig. 6b). BMDC and T cells were incubated with [email protected]/PB/DNA for 24 h, and the cell viability were 100.0 % ± 3.9 % and 100.1 % ± 3.0 % at the concentration of 500 mg/L (CS/ PB) (Fig. S3a, b online). Cells survival of BMDC and T cells was also detected by staining with both calcein-AM and PI. Figure S3c, d (online) shows the fluorescence microscopy images of BMDC cells and T cells which were stained with calcein-AM and PI after incubation with or without [email protected]/PB/DNA (500 mg/L CS/PB) for 24 h. No dead cells (red fluorescence from PI) could be observed. All these data showed the excellent biosafety of [email protected]/PB/DNA as the gene delivery vector in vitro. The in vivo biocompatibility of [email protected]/PB/DNA was evaluated with Kunming mice. The mice were randomly assigned to two groups, and 100 lL [email protected]/PB/ DNA (1,000 mg/L of CS/PB) or 100 lL PBS was injected via the tail vein, respectively. As shown in Fig. 6c, no remarkable changes were found between the two groups in four weeks. No apparent organs histological changes (including heart, liver, spleen, lung, and kidney) were found in Fig. 6d, which were collected at day 0, 1, 7, 30, and observed by H&E staining. These results demonstrated good in vivo biocompatibility of [email protected]/PB/ DNA.

Fig. 5 (Color online) a Microscopic fluorescent images of HeLa cells; control (a1), after treatment with PEI (a2) and [email protected]/PB/DNA combined with laser irradiation for different time (a3–a6); scale bars were 50 lm. b Quantification of pEGFP-C1 transfected for 48 h at different laser irradiation times by flow cytometry


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Fig. 6 (Color online) Cytotoxicity of [email protected]/PB/DNA to HeLa cells (a), HUVECs cells (b) as determined by MTT assay. c Body weight curves of the mice for 28 d after a single-dose intravenous injection of [email protected]/PB/DNA. d Histological changes of the mice at 1, 7 and 30 d after an intravenous injection of [email protected]/PB/DNA. The organs were stained with H&E and observed under a light microscope. The scale bars were 50 lm

4 Conclusions A multifunctional ultrasound contrast agent of [email protected]/ PB/DNA has been successfully developed by depositing CS/ PB/DNA onto the microbubbles surface. [email protected]/PB/ DNA could enhance ultrasound imaging greatly. With the aid of ultrasound irradiation and NIR light irradiation, the gene transfection efficiency was significantly enhanced to 43.08 % ± 1.13 %, much higher than polyethylenimine. In

addition, [email protected]/PB/DNA showed high biocompatibility and no toxicity was observed, showing great application potential for combined ultrasound image, photothermal therapy, drug delivery, and gene therapy. Acknowledgments This work was supported by the National Natural Science Foundation of China (81371580 and 21273014), the National Natural Science Foundation for Distinguished Young Scholars (81225011) and the State Key Program of National Natural Science of China (81230036).


156 Conflict of interest of interest.

Sci. Bull. (2016) 61(2):148–156 The authors declare that they have no conflict 13.

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