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T2-weighted MR Imaging of hepatic tumor guided by SPIOloaded nanostructured lipid carriers and ferritin reporter genes Chenying Lu, Jiansong Ji, Xiuliang Zhu, Peifeng Tang, Qian Zhang, Nannan Zhang, Zuhua Wang, Xiaojuan Wang, Weiqian Chen, Jingbo Hu, Yongzhong Du, and Risheng Yu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09879 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 27, 2017

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ACS Applied Materials & Interfaces

T2-weighted MR Imaging of hepatic tumor guided by SPIO-loaded nanostructured lipid carriers and ferritin reporter genes Chen-ying Luac1, Jian-song Jic1, Xiu-liang Zhua, Pei-feng Tangd, Qian Zhanga, Nan-nan Zhangc, Zu-hua Wange, Xiao-Juan Wangb, Wei-qian Chenc, Jing-bo Hub, Yong-Zhong Dub*, Ri-Sheng Yu a* a

Department of Radiology, Second Affiliated Hospital, School of Medicine,

Zhejiang University, Hangzhou 310009, China b

Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University,

Hangzhou 310058, China c

Department of Radiology, Lishui Hospital of Zhejiang University, Lishui 323000,

China d

Department of Paper and Bioprocesss Engineering, State University of New York,

College of Environmental Science and Forestry, New York 13210, United States e

College of Pharmaceutical Sciences, Guiyang College of Traditional Chinese

Medicine, Guiyang 550002, China （1）These authors contributed equally to this work （2）Correspondence should be addressed to the following: Corresponding author: Dr. Ri-sheng Yu, Department of Radiology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China. Tel: +8613757118387; Fax：+8657187784556. E-mail: [email protected]. Dr. Yong-zhong Du, Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Tel: +8657188208435; Fax:+8657188208435. E-mail: [email protected] 1

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ABSTRACT Nowadays, there is a high demand for supersensitive contrast agents for the early diagnostics of hepatocarcinoma. It has been recognized that accurate imaging information is able to be achieved by constructing hepatic tumor specific targeting probes, though it still faces challenges. Here, A AGKGTPSLETTP peptide (A54) functionalized superparamagnetic iron oxide (SPIO)-loaded nanostructured lipid carrier (A54-SNLC), which can be specifically uptaken by hepatoma carcinoma cell (Bel-7402) and exhibited ultralow imaging signal intensity with varied Fe concentration on T2-weighted imaging (T2WI), was first prepared as an effective gene carrier. Then an endogenous ferritin reporter gene for magnetic resonance imaging (MRI) with tumor-specific promoter (AFP-promoter) was designed, which can also exhibit a decrease in signal intensity on T2WI. At last, using protamine as a cationic mediator, novel ternary nanoparticle of A54-SNLC/protamine/DNA (A54-SNPD) as active dual-target T2-weighted MRI contrast agent for imaging hepatic tumor was achieved. Owing to the synergistic effect of A54-SNLC and AFP-promoted DNA targeting with Bel-7402 cells, T2 imaging intensity values of hepatic tumors were successfully decreased via the T2 contrast enhancement of ternary nanoparticles. It is emphasized that the novel A54-SNPD ternary nanoparticle as active dual-target T2-weighted MRI contrast agent were able to greatly increase the diagnostic sensitivity and specificity of hepatic cancer. Keywords: Magnetic resonance imaging (MRI), Superparamagnetic iron oxide, Nanostructured lipid carrier, Ferritin reporter gene, Hepatic tumor 2


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Introduction Hepatocarcinoma is of great seriousness and the most common causes of cancer death after lung cancer over the world.1 Early specific diagnosis is an effective strategy for patients treatment and prognosis and reducing the substantial portion of liver cancer death.1,2 MRI is an important and efficient non-invasive imaging tool for early cancer diagnosis. It exhibits the advantages of multi-parameter imaging, high spatial and temporal resolution, and avoiding of ionizing radiation.3,4 However, highly sensitive contrast agent is necessary for MRI. Recently, iron oxide based magnetic nanoparticles catch a great attention.5-7 In particular, superparamagnetic iron oxide-loaded nanostructured lipid carriers (SNLCs) have been widely studied as promising MRI contrast agents, regarding the non-toxicity, good biocompatibility as well as magnetic properties.8,9 Nanostructured lipid carrier (NLC) is a novel effective nanoparticle delivery system, which can improve drug bio-distribution at target sites and reduce non-specific diffusion in normal organs with reasonable modification.10-12 As for MR imaging applications, SNLCs can be well stabilized in biological systems to diagnose early stage cancer as T2-shortening contrast agents during to the high R2 relaxivity.8,9 However, commonly used SNLCs suffer another problem of nonspecificity in MR imaging

because

of

its

non-specific

uptake

by

phagocytic

cells

and

reticuloen-edothelial system (RES), or will be recognized as foreign intruders and easily opsonized.13-15 Therefore, development of various tumor-targeted SNLC systems for MR imaging is highly demanded.16 To generate SNLCs with tumor 3



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targeting specificity, various targeting ligands or antibody were used to surface modify SNLCs.17,18 A54, a hepatocarcinoma-specific binding peptide, can be specifically internalized by hepatocarcinoma cell Bel-7402 through the receptor on the cell surface.19-21 As the exogenous contrast agent, A54-SNLC demonstrate ascendancy in hepatocarcinoma early stage diagnosis, allowing them exhibited hypointense images by T2WI. Still, injecting exogenous contrast agents also exhibits potential side effects including renal toxicity and allergic reactions. The attempt to reduce side effects by decreasing the use of exogenous contrast agents may lead to poor outcomes of tumor imaging. Therefore, exploring an approach to satisfy the imaging outcomes at target sites with a safe dose of exogenous contrast agents is of clinical significance. Modern molecular imaging can supply early tumor diagnosis using novel reporter genes for MR imaging gene expression, which includes ferritin, transferrin receptor, β-galactosidase and tyrosinase.22-24 Ferritin is a wide-existing protein that stores iron, which is composed of 24 heavy and light subunits. Ferritin heavy chain (Fth) displays ferroxidase activity to promote iron oxidation and incorporation as the candidate major regulator of ferritin activity. It plays as an applicable MR imaging reporter gene because of its non-toxicity and non-allergy reaction.24-26 Cohen et.al, have demonstrated that the cells transfected with ferritin showed increased transverse relaxation rate and strong hypointensity on T2/T2*-weighted images.24 Thus enhancing intracellular ferritin as the endogenous MRI reporter by transfection could be a potential effective method to improve the MRI signal. 4


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Specific promoters can promote targeted gene expression, which enhance specificity and safety of gene diagnosis and therapy. For example, tumor-specific promoter driven reporter gene expression plays a significant role in cancer imaging, promoting expression of reporter genes at target tumors and preventing side effects at normal organs.27-31 Alpha-fetoprotein (AFP) is normally expressed at high levels in the fetal liver and is silenced after birth. The expression will rise again once liver cancer appeared. So it has been employed as a serum marker for diagnosis and prognosis of hepatocellular carcinoma (HCC).28 Therefore, AFP promoter, as a HCC-specific promoter, can be used as the liver cancer marker and monitored by imaging of reporter gene expression.29,30 In this study, a novel active targeted T2-weighted MR imaging contrast of exogenous

SPIO

and

endogenous

ferritin

reporter

protein

(A54-SNLC/protamine/DNA) to hepatic tumor was designed. A54-SNLC was prepared as exogenous reporter systems and proposed as an effective gene delivery imaging system to target Bel-7402 cells. Human Fth with AFP promoter was proposed as endogenous ferritin reporter protein for MR imaging of AFP-positive HCC, by promoting intracellular iron transferring and changing the MRI signal. This work reported a lab preparation of active dual-target T2 imaging contrast agent (A54-SNPD) of hepatic tumor. The specific binding ability of A54-SNPD to Bel-7402 was investigated in vitro and in vivo.

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Results and discussion Synthesis and Characterization of A54-PEG-SA The synthesis scheme of A54-PEG-SA is shown in Scheme 1. NH2-PEG-SA was firstly synthesized and A54-PEG-SA was further synthesized. The chemical structures of A54, NH2-PEG-SA and synthesized A54-PEG-SA were determined by 1H NMR spectra (Figure 1). The double peaks at about 3.41-3.76 ppm were the proton of -CH2- for PEG of NH2-PEG-SA, the sharp peak at about 2.16-2.19 ppm was attributed to the -CH2- close to carboxyl terminal for SA of NH2-PEG-SA. In contrast to NH2-PEG-SA, the 1H NMR result of the A54-PEG-SA demonstrated a peak at about 1.41-1.55 ppm, which were the proton of -CH2- for lysine of peptide A54. These results verify the successful synthesis of A54-PEG-SA. Furthermore, the chemical structures of physico-compounds of A54+PEG-SA and A54-PEG-SA conjugate were confirmed by infrared (IR). A54 polypeptide molecule contained four hydroxyl groups, two carboxyl groups and eleven amide bonds, in addition to the amino-groups. Therefore, in the IR spectra of physico-compounds of A54+PEG-SA, a strong and sharp characteristic peak of the carboxide in carboxyl groups at about 1680-1700 cm-1 (VC=O, blue arrow) appeared, which led to the characteristic peak of amide bond at 1640~1550 cm-1 to be covered up as shown in Figure 1B(a). In contrast, in the IR spectra of A54-PEG-SA conjugate, the characteristic peak of the carboxide in carboxyl groups was weakened after chemical reaction, and the characteristic peak of the amide bond of A54-PEG-SA conjugate was exposed (Figure 1B(b), βN-H, red arrow,1636 cm-1). In addition, in the IR spectra of physico-compounds of 6


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A54+PEG-SA, a wider and strong characteristic peak was appeared at 2500~3600 cm-1, it was attributed to the overlap peaks of amino, amide bond and the hydroxy in the carboxyl group. However, in the IR spectra of A54-PEG-SA conjugate, the hydroxyl characteristic peak of carboxyl (2500~3600) was strongly weakened, a weak and sharp characteristic peak at 3316 cm-1 was appeared (VN-H, green arrow). It indicated a successful amide bond formation between the carboxyl of A54 and the amido of PEG-SA. The results further indicated that the A54 polypeptide was successfully conjugated to PEG-SA molecule.

Scheme 1. Synthetic scheme of A54-PEG-SA

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Figure 1. (A) 1H NMR spectra of A54, PEG-SA and A54-PEG-SA. The important 8


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peaks were pointed out. (B) IR spectra of physico-compounds of A54+PEG-SA (a) and A54-PEG-SA conjugate (b). The important peaks were pointed out.

Preparation and Physicochemical characteristics of SNLCs, binary and ternary nanoparticles Size and zeta potential of SNLCs can affect transfection efficiency by influencing cellular uptake, nanoparticle stability and DNA compact ability.32 As shown in Table 1, Dynamic light Scattering (DLS) showed a uniform distribution (PI < 0.3) and negative charge of both A54-SNLC and SNLC. It demonstrated that the particle sizes were within 39.80 ± 4.15 nm and 40.06 ± 4.20 nm, while zeta potential around -24.07 ± 0.64 mV and -22.07 ± 0.70 mV. Thus, almost no changes of size and zeta potential in charge with the addition of peptide A54. Gene vector and DNA should be small and compact so as to enhance the efficiency of cellular endocytosis and gene transfer. Because of the negative charge of the SNLCs, cationic protamine was used to enhance the SNLCs condense to the DNA, forming ternary nanoparticles. Protamine can bind to DNA in a non-specific way for its high charge and arginine richness. Besides, protamine is with apparent nuclear localization signals domains and can enhance gene expression through increase of DNA translocation. Many studies show that addition of protamine sulfate can enhance the complex stability and DNA transfection efficiency.33,34 The size and zeta potential of the ternary nanoparticles with various weight ratios were shown in Table 1. It was found that the sizes of different weight ratios of A54-SNPD ternary nanoparticles 9



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(5/4/1, 10/4/1, 20/4/1) were smaller than that of Protamine/DNA binary nanoparticle (0/4/1), while the size of A54-SNPD (30/4/1) ternary nanoparticle was bigger than that of Protamine/DNA binary nanoparticle (0/4/1). The reason maybe that the A54-SNPD ternary nanoparticle is electrostatically assembled by anionic A54-SNLC and cationic protamine/DNA complex, and the compress degree was dependent on the ratio of A54-SNLC to protamine/DNA. At the beginning, the protamine/DNA was condensed with the increase of the A54-SNLC, which led that the particle size of A54-SNPD ternary nanoparticle became smaller and smaller. However, the protamine/DNA can not be condensed without limitation, the particle size of protamine/DNA can not be smaller to a certain extent. So when the weight ratio of A54-SNPD reached to 30/4/1, the size of A54-SNPD (30/4/1) ternary nanoparticle was much bigger than that of binary nanoparticle (0/4/1). TEM was conducted to investigate the morphology and verify the successful formation of the SNLCs, binary and ternary nanoparticles (Figure 2A). TEM images indicated that anionic SNLC and A54-SNLC were mostly spherical or ellipsoidal shapes, with approximately 50 nm particle size. The successful entrapment of SPIO into A54-SNLC and SNLC was verified by the photographs. The result was similar to that from DLS. The protamine/DNA binary complexes also displayed spherical shapes. The ternary nanoparticles were prepared by adding anionic SNLCs to protamine/DNA binary complexes, which were appeared as round shapes with ‘bumpy’ surface, since the SNLCs were adsorbed onto the surface of the protamine/DNA complexes. This is for that the anionic SNLCs can recondense the 10


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protruding chains of components by electrostatic interaction. The stability of the A54-SNPD ternary nanoparticle was assessed by constant monitoring its hydrodynamic size and PDI using DLS. The time-dependent size and PDI curves of the nanoparticle (A54-SNPD) were showed in Figure 2B. The results showed that the A54-SNPD exhibited excellent stability after a 12-day incubation at 4 ℃, as reflected by the small changes in size and PDI. The results indicated that the A54-SNPD had good stability during used as MRI contrast agent.

Table 1．． Particle diameter and zeta potential of SNLC，A54-SNLC and different weight ratios of A54-SNPD ternary nanoparticles. Zeta potential Sample

Diameter (nm)

PI(-) (mV)

SNLC

40.06 ± 4.20

0.28 ± 0.02

-22.07 ± 0.70

A54-SNLC

39.80 ± 4.15

0.27 ± 0.03

-24.07 ± 0.64

A54-SNPD(0/4/1)

148.35 ± 5.86

0.21 ± 0.06

14.50± 0.87

A54-SNPD(5/4/1)

65.12 ± 4.95

0.29 ± 0.07

16.53 ± 1.79

A54-SNPD(10/4/1)

75.81 ± 5.61

0.22 ± 0.09

13.37 ± 0.25

A54-SNPD(20/4/1)

93.87 ± 6.52

0.28 ± 0.05

12.53 ± 0.67

A54-SNPD(30/4/1)

358.00 ± 6.94

0.24 ± 0.08

7.88 ± 0.10

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Figure 2. Characterization of A54-SNLC and A54-SNPD nanoparticles. (A) TEM images and size distribution of SNLC, A54-SNLC, Protamine/DNA (Weight Ratio: 4/1) 12


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and A54-SNPD (Weight Ratio: 20/4/1) nanoparticles. (B) The time-dependent size and PDI curves of the A54-SNPD. (C) Magnetic hysteresis loops of free SPIO, A54-SNLC and SNLC nanoparticles. (D) T2-weighted images of free SPIO, A54-SNLC and SNLC nanoparticles at various iron concentration. The diagram of T2 relaxation rates (1/T2 values, S-1) changing with Fe concentration for SPIO, A54-SNLC and SNLC.

Magnetic properties of SNLCs in vitro MR imaging and saturated magnetic intensity Magnetic property of a MRI contrast agent was a significant parameter. Figure

2C shows magnetization curves of free Fe3O4, A54-SNLC and SNLC nanoparticles. Saturated magnetization (Ms) values of the free Fe3O4, A54-SNLC and SNLC nanoparticles were 20.78 emu/g Fe，27.58 emu/g Fe and 30.08 emu/g Fe, respectively, demonstrating that both free Fe3O4 and SNLCs exhibited good superparamagnetic properties.

Interestingly,

both

A54-SNLC

and

SNLC

showed

better

superparamagnetic properties than free Fe3O4. As shown in Figure 2D, intensity (darkness) of T2-weighted MRI of free Fe3O4, A54-SNLC and SNLC nanoparticles in water solution was increased accompanied with Fe concentration increased from 0 to 100 µg mL-1. The MR signal intensity of 1/T2 was increased with increasing Fe concentration. The relaxation rate, R2 values (1/T2), was varied linearly with the Fe concentration, the slopes of which were defined as the transverse relaxivity. Furthermore, both A54-SNLC and SNLC showed 13



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more favorable contrast effect and significantly increased R2 relaxivity in comparison to free Fe3O4, which was in accordance to the results of the saturated magnetization values and earlier literatures.35 The great enhancement in the relaxivity of the SPIO nanoparticles within NLCs should be attributed to the reasons as follows. First, the highly loaded SPIO nanoparticles within NLCs may result in clustering effect of SPIO nanoparticles, which enhances the relaxivity of the SPIO nanoparticle. Second, the clustered SPIO nanoparticles jammed and crowded within NLCs may shortens the distance of the clustered SPIO, which gives rise to a synergistic increase in R2. Third, the diffusion of water close to the magnetic nanoparticles surface was impeded by NLCs and the interaction time between the water protons and the magnetic fields near the nanoparticle was extended, thus the relaxivity of the SPIO nanoparticles was enhanced. Therefore, the SNLC with unique structure can be used as a novel MR T2 negative contrast agent because of the high relaxivity coefficient.

Gel retardation assay of binary and ternary nanoparticles In order to prepare protamine/DNA complexes, protamines were incubated with plasmid DNA at the ratio of 0.1:1, 0.3:1, 0.7:1, 1:1, 2:1 and 4:1 (w/w). The complete complexes retardation appeared when the ratio exceed 0.7 (Figure 3A), indicating that protamine/DNA became tight with redundant positive surface charge. Furthermore, anionic A54-SNLC absorbed on the surface of cationic protamine/DNA complexes formed A54-SNPD via electrostatic interactions. The weight ratio of A54-SNLC to protamine/DNA complexes was shown in Figure 3A. When the weight 14


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ratio of protamine/DNA complexes was 2, the A54-SNPD can be completely retarded when the weight ratio of A54-SNLC to protamine/DNA complexes was below 15. When the weight ratio of protamine/DNA complexes was 4, the A54-SNPD can be completely retarded when the weight ratio of A54-SNLC to protamine/DNA complexes was below 30. The ternary nanoparticles became stable when the weight ratio of A54-SNLC against protamine was under 7.5:1.

Figure 3. Assessment on cytotoxicity of A54-SNLC and A54-SNPD nanoparticles (A) Gel retardation analyses of different weight ratios of Protamine/DNA (WP/WD) and 15



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A54-SNPD (WA54-SN/WP/WD) complex nanoparticles. (B) Cytotoxicity of A54-SNLC and SNLC nanoparticles at various concentrations in Bel-7402 and LO2 cells. (n = 3). (C) Cytotoxicity of ternary nanoparticles with 2 ug/well DNA at various weight ratio of A54-SNPD ternary nanoparticles (WA54-SN/WP/WD) in Bel-7402 and LO2 cells. (n = 3).

The cytotoxixity of SNLCs and ternary nanoparticles The toxicity of the SNLCs and ternary nanoparticles was tested by MTT method against Bel-7402 and LO2 cells. The results in Figure 3B demonstrated that cell viabilities of Bel-7402 and LO2 were above 80% even when concentration of Fe3O4 reached up to 100 µg mL-1. The results in Figure 3C displayed that cell viability of binary and different ratios of ternary nanoparticles were still above 80%, which manifested the ternary nanoparticles also had a relatively high biocompatibility and low toxicity for both tumor cells and normal cells, even though there was a gradually decrease in viability when the cells were incubated with higher ratios of ternary nanoparticles. Furthermore, the cytotoxicity of the ternary nanoparticles was found to be no significance with the PEI/DNA (PEI25k:pDNA = 1.25:1, w/w) .

Identification of reconstructed pEGFP-AFP-Fth The digests and sequence analysis demonstrated that the DNA sequence of Fth was in accordance with that in the genbank, which testified that the pEGFP-AFP-Fth was constructed successfully.

Optimal ratio of ternary nanoparticles for transfection in vitro 16


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A green fluorescence protein assay was carried out to investigate the effect of different weight ratios of A54-SNLC to DNA on the effciency of gene transfection via protamine in vitro. The fluorescence images of naked DNA, PEI/DNA (PEI25k:pDNA = 1.25:1, w/w) and different weight ratios of A54-SNPD ternary nanoparticles in Bel-7402 cells were shown in Figure 4A, the related transfection efficiencies were shown in Figure 4B. PEI/DNA was employed as a positive control. Cell incubated with naked DNA (2 µg/well) was employed as a negative control. It indicated that the fluorescence of Bel-7402 cells treated with protamine/DNA binary complexes were too low to be detected by the flow cytometry assay. However, the A54-SNLC/Protamine/DNA with weight ratio of 20/4/1 owned the highest gene expression with the transfection efficiency of 24.7%, only slightly lower than that of PEI/DNA with the transfection efficiency of 29.4%. This indicates that A54-SNLC/Protamine/DNA with weight ratio of 20/4/1 on Bel-7402 cells is optimal and compatible for transfection in Bel-7402 cells. In contrast, the fluorescence images of naked DNA, PEI/DNA and different weight ratios of A54-SNPD ternary nanoparticles on LO2 cells were too low to be observed.

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Figure 4. Optimal ratio of A54-SNPD ternary nanoparticles for transfection in vitro (A) EGFP expression of transfected Bel-7402 cells were shown in inversed fluorescent images. Bel-7402 cells were incubated with different weight ratio of A54-SNPD nanoparticles for 48 h. Naked DNA and PEI were employed as the negative and positive control. Scale bars represent 50 µm in all images. (B) Percentages of transfected cells determined by FACS. (C) Western blots assay for AFP protein using anti-AFP antibody

(**p < 0.01, compared with LO2 cells). (D)

Western blot assay of Fth expression. Bel-7402 and LO2 cells were transfected with A54-SNPD nanoparticles at weight ratio of 20/4/1 for 48 h (**p < 0.01, *p < 0.05, compared with untransfected LO2 cells).

Expression of AFP proteins in Bel-7402 and LO2 cells We investigate AFP protein expression in Bel-7402 cells by performing western blot analysis. Whereas, little APF protein was detected in LO2 cells (Figure 4C). These results are consistent with previously published work.36,37 As expression of AFP protein is good indicator of AFP activity,29 Bel-7402 cells were considered as AFP-positive, while LO2 cells were considered as AFP-negative in this study.

AFP promoter drives Fth expression in AFP-positive cells Analysis by western blot showed that Fth protein expression level of the untransfected Bel-7402 cells exceed that of untransfected LO2 cells (p < 0.05, n = 3). Furthermore, Fth protein expression level in A54-SNPD (20/4/1) transfected AFP-positive Bel-7402 cells was prominently higher than that in untransfected Bel-7402 cells (p < 0.05, n = 3). In contrast, there was no remarkable difference 19



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between the A54-SNPD transfected LO2 cells and the untransfected LO2 cells (p > 0.05, n = 3) (Figure 4D). The results shown that the reporter gene plasmid pEGFP-AFP-Fth under the control of AFP promoter can be expressed in the AFP-positive Bel-7402 cells definitely and specifically. On the other hand, the reporter gene with AFP promoter had little effect in AFP-negative LO2 cells.

Targeting ability of A54-SNLC and A54-SNPD to Bel-7402 cell in vitro T2-weighted images of Bel-7402 and LO2 cells incubated with A54-SNLC and SNLC nanoparticles for 3h and their intensity were shown in Figure 5A. Cellular internalization of the SNLCs induced a change in the T2-weighted MR images. The T2-weighted image intensity of Bel-7402 and LO2 cells incubated with A54-SNLC and SNLC were decreased obviously, and the decrease of the T2-weighted image intensity noted significantly in Bel-7402 cell incubated with A54-SNLC, which was significantly different in contrast to the others (P < 0.01 , n = 3). This attributed to the specific targeting ability of A54-SNLC toward Bel-7402 cell. Cellular uptake of FITC labeled A54-SNPD and SNPD on Bel-7402 and LO2 cells were observed by inversed fluorescent microscope to evaluate the targeting ability of A54-SNPD to Bel-7402 cell. The results were presented in Figure 5B and

Figure 5C. It was found that the uptake of the A54-SNPD and SNPD by two cells was time dependence. The uptake of A54-SNPD in Bel-7402 cells was faster than A54-blocking and SNPD, while there were no obvious difference among the A54-SNPD and SNPD in LO2 cell. Differences were significant at 3 h. The results confirmed specific targeting ability of the A54-SNPD to Bel-7402 cell. 20


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Figure 5. Assessment on targeting ability of A54-SNLC and A54-SNPD nanoparticles to Bel-7402 cell in vitro. (A) The T2-weighted image intensity of A54-SNLC and SNLC nanoparticles incubated with Bel-7402 and LO2 cells for 3 h. (**p < 0.01, *p < 0.05, compared with ddH2O group). (B) In vitro cellular uptake of ternary nanoparticles in different cells. Inversed fluorescent images of Bel-7402 and LO2 cells incubated with FITC-labeled A54-SNPD, A54-SNPD pre-treated with A54 blocking and SNPD ternary nanoparticles for 1, 3, 6 and 12 h, respectively. All pictures shown were merged images, which included the nuclei (blue) and nanoparticles (green). Scale bars represent 50 µm in all images. (C) Fluorescence intensity inside Bel-7402 and LO2 cells measured by flow cytometry when cells incubated with FITC-labeled A54-SNPD, A54-SNPD pre-treated with A54 blocking and SNPD ternary nanoparticles. The group without any treatment as negative control.

Targeting ability of A54-SNPD ternary nanoparticles in vivo In the study, an orthotropic hepatoma model was established in order to investigate the ability of A54-SNPD ternary nanoparticles to prolong drug drug circulation and specifically target Bel-7402 cells in vivo. As shown in Figure 6A, the fluorescence signals of major organs and tumors at 3, 6, 12, 24 and 48 h after injection of ICG-labeled A54-SNPD or SNPD nanoparticles were observed, and the fluorescence intensity of tumors at various time points was quantitated. The results shown that the nanoparticles were mainly accumulated in the liver, spleen, kidney, 22


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and tumor. The fluorescent signals of A54-SNPD in tumors were significantly stronger than those of SNPD in tumors at every time points (P < 0.01 , n = 3), which confirmed the targeting ability of A54-SNPD nanoparticles to Bel-7402 cells. Furthermore, the strongest fluorescent signal in tumors was observed at 6 h after injection, then gradually decreased along with time. Therefore, with modification of A54, the A54-SNPD nanoparticles could accumulate in tumor efficiently in vivo. Besides, liver was collected for histological analysis. As shown in Figure 6B, tumor cells became larger and pleomorphic with bigger nucleus, abundant cytoplasm, these results indicated the successful establishment of orthotopic hepatoma models. The Bel-7402 tumor tissues were observed with positive staining of AFP. In contrast, the liver tissues were detected with negative AFP. This result further to vertify that the Bel-7402 tumor was considered as AFP-positive tumor.

23



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Figure 6. Assessment on targeting ability of A54-SNPD ternary nanoparticles in vivo. (A) In vivo fluorescence images of major organs and tumors, and fluorescent semi-quantitative analysis of tumors at 3, 6, 12, 24 and 48 h after intravenous 24


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injection of ICG-labeled A54-SNPD or SNPD ternary nanoparticles. (**p < 0.01). (B) Orthotopic Bel-7402 tumor and histological analysis. (a) The red circle indicates the tumor model was successfully established in liver. (b) HE staining of the liver tissue with Bel-7402 tumor; “L” indicates the normal liver, “T” indicates the tumor (×200). (c) HE staining of the liver tissue with Bel-7402 tumor (×200); (d) Positive staining with AFP in tumor tissues (×200). (e) Negative staining with AFP in liver tissues (×200).

In vivo MR imaging of ternary nanoparticles T2 enhanced images of the tumors (Figure 7A) and the SNR of T2-weighted signal intensity of the tumors (Figure 7B) were obtained after the injection of A54-SNPD, SNPD, A54-SNP or SNP. The results indicated that the intensity of tumor images of post-contrast in four groups become significantly darkened at 1 h and recovered at 3 h in contrast with that of pre-contrast. Correspondingly, the SNR of MR signal intensity of tumors were obviously decreased at 1 h and then increased at 3 h, there is no significant difference between the four groups at 1 h and 3 h (P > 0.05), indicating that A54-SNPD showed nearly no targeting ability in 3 h ， and the decreased SNR of MR signal intensity of tumors should be associated with the known passive EPR effect.38 However, the image intensity of tumors in A54-SNPD and A54-SNP groups were darkened and the corresponding SNR of tumors were significantly decreased again at 6 h，while there was no significant change in SNPD and SNP groups. Furthermore, significant difference was observed between A54 25



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targeted groups and no A54 targeted groups from 6 h to 48 h, which indicated that the target effect of A54 polypeptide was begin to work at 6 h and can maintain 48 h. Once again, there was an obvious signal intensity decrease in the targeted A54-SNPD treated tumor after 6h post injection in contrast to the other three groups. On the other hand, the SNR value of tumors in A54-SNPD group became lower than that in A54-SNP group at 12 h, and a distinctly different was found between this two groups from 12 h to 48 h, while the difference appeared most obviously at 48 h. This indicated that Fth reporter gene with AFP promoter was begin to work at 12 h and can maintain 48 h. Compared to SPIO, overexpression of Fth can maintain a persistent reduction in MRI signal intensity with time expanding. The primary result is that the decrease of SNR value of tumors was noted significantly in A54-SNPD group, mainly due to the greater accumulation of SPIO in tumors and more Fth protein expression compared to the other three types of ternary nanoparticles. It was confirmed that A54-SNPD ternary nanoparticles has active dual targeted ability to hepatic tumors. Given inevitably uptaken by the RES, nanoparticles were distributed in the liver and led to the decreased MR signal intensity in liver tissue. To quantitate the different contrast abilities of the tumors and liver tissues, we analyzed the SNRpost/SNRpre value in A54-SNPD group, A54-SNP group and SNP group at 6 h and 24 h (Figure 7C). The results showed that the signal decrease of the tumors were more obvious than that of liver tissues in A54-SNPD group at both 6 h and 24 h (p < 0.01), while the signal decrease of the liver tissues were more obvious than that of tumors in SNP group at both 6 h and 24 h (p < 0.01). The results also indicated that A54-SNPD ternary 26


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nanoparticle showed active dual targeted ability to hepatic tumors. To further verify the target ability of A54-SNPD and the expression of Fth in AFP positive tumor, the Fth protein expression level was studied via the western blotting method. Figure 7D, 7E indicated that A54-SNPD group clearly induced the highest Fth protein expression level in the tumors among the four types of ternary nanoparticles, which should be mainly attributed to the active dual target ability of A54-SNPD nanoparticles in tumors compared to the other groups. In contrast, no remarkable difference among the four types of ternary nanoparticles in liver tissues was observed, which further indicated that the reporter gene plasmid pEGFP-AFP-Fth was rarely expressed in normal liver tissues. Furthermore, the Fth protein expression level in liver tissues were much lower than that in tumor tissues, which was in accordance with the previous western blotting results in cells.

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Figure 7. Assessment on enhanced ability of A54-SNPD ternary nanoparticles in MR imaging in vivo. (A) In vivo T2-weighted mages of Bel-7402 tumor-bearing livers before and after intravenous administration of A54-SNPD, A54-SNP, SNPD and SNP ternary nanoparticles. The yellow dashed circles indicate the tumor region. (B) SNR of T2-weighted signal intensity of the tumors (**p < 0.01, *p < 0.05). (C) Quantification of signal intensity changes (SNRpost /SNRpre) in tumors and liver tissues at 6 h and 24 h after intravenous administration of A54-SNPD, A54-SNP and SNP ternary nanoparticles (**p < 0.01, *p < 0.05). (D,E) Fth protein expression level of the tumors and the liver tissues 48 h after intravenous administration of A54-SNPD, A54-SNP, SNPD and SNP ternary nanoparticles (**p < 0.01, *p < 0.05, n = 5).

Conclusion In this study, a novel A54-SNPD ternary nanoparticles combined A54 polypeptide modified SNLC and AFP promoted ferritin reporter gene was fabricated. The obtained A54-SNPD ternary nanoparticles have active dual targeted ability to Bel-7402 cells in vitro and hepatic tumors in vivo. Furthermore, A54-SNPD ternary nanoparticles were able to successfully reduce the T2 imaging intensity values of hepatic tumors via the T2 contrast enhancement of ternary nanoparticles. The A54-SNPD ternary nanoparticles used as an effective and specific T2 negative contrast agent demonstrated potential application for early diagnosis of hepatic tumors.

Experiment section 29



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Materials Superparamagnetic iron oxide (II,III) (SPIO, 5 nm), octadecylamine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), hoechst 33342 and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Amino-terminated polyethylene glycol (NH2-PEG-NH2, MW = 2000) was purchased from Yare Biotech, Inc. (Shanghai, China). A54 was acquired from Guangzhou Sinoasis Pharmaceuticals Inc. (Guangzhou, China). Di-tert-butyl dicarbonate ((Boc)2O), 1-Ethyl-3-(3-dimethylaminopropyl) carbo-diimide (EDC) and N-Hydroxysuccinimide (NHS) were got from Shanghai Medpep Co., Ltd. (Shanghai, China). SA was purchased from Shanghai Chemical Reagent Co.,Ltd. (Shanghai, China). Poloxamer 188 was from Shenyang Jiqi Pharmaceutical Co, Ltd. (Shenyang, China). Oleic acid was obtained from Hangzhou Shuanglin Chemical Industry Co. Ltd. (Hangzhou, China). Indocyanine green (ICG) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Otcadecylamine (ODA, 95%) was purchased from Fluka, USA. Protamine sulfate was purchased from Shanghai Sangon Biological Engineering Technology and Services Co, Ltd. (Shanghai, China). Branched PEI (25 kD) was purchased from Sigma (St. Louis, MO, USA). The pEGFP-AFP-Fth plasmid, encoding an AFP promoter and enhanced green fluorescent protein (EGFP), was synthesized by Shanghai Genechem Co.,Ltd. (Shanghai, China) according to the Fth gene sequence in GenBank (NM_013233). Antibodies including anti-AFP antibody and anti-ferritin heavy chain antibody were purchased from Abcam (Abcam, Cambridge, UK, ab65080). All other chemicals were of analytical grade and 30


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used without further purification. Bel-7402 and LO2 cells were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (IBCB, Shanghai, China). The cells were maintained in DMEM medium containing 10% fetal bovine serum as well as 1% penicillin and streptomycin at 37 °C in a humidified atmosphere of 5% CO2. BALB/c nude mices (body weight: 18 ± 2 g) were purposed from the Zhejiang Medical Animal Centre. All the animal studies were supported by National Institutes of Health (NIH, USA) guidelines. The project was approved by the Committee for Animal Care of Zhejiang University.

Synthesis of A54 Peptide functionalized PEGylated SA (A54-PEG-SA) NH2-PEG-SA was firstly synthesized through the reaction between amine group of NH2-PEG-NH2 and carboxyl group of stearic acid in the presence of EDC and NHS according to the previous study.39 A54-PEG-SA was then synthesized through the coupling reaction between amine group of NH2-PEG-SA and carboxyl group of A54 in the catalytic condition of EDC. Briefly, (Boc)2O (10.32 µL) was added into A54 (40 mg) dissolved anhydrous DMSO (20 mL) in ice bath,

followed by stirring

for 12 h shielded from light at room temperature. Then EDC (66.8 mg) was added to the reaction mixture to stir for 1.5 h. After NH2-PEG-SA (78 mg) was added, the reaction mixture was stirred for another 24 h to synthesize t-Boc protected A54-PEG-NH2. 2 M of hydrochloric acid was used to remove the t-Boc group to obtain A54-PEG-SA. The final product was further purified via dialysis through a dialysis bag (3.5 MWCO) for 2 days and followed by lyophilization. The structure 31



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confirmation of A54-PEG-SA was determined via 1H NMR spectra and IR spectra.

Preparation of SPIO loaded A54-SNLC, binary and ternary nanoparticles A54-SNLC was prepared by emulsion-diffusion method. Fe3O4 dispersion (100 µl) was added into Poloxamer 188 (4.7 mL, 0.1%) and dispersed in DI water by a probe sonicator (Sonicator JY92-II DN, China) for 30 minutes to obtain a water dispersed magnetic nanoparticles.100 µl of OA in ethanol solution (10 mg/mL) was further added and followed by ultrasound waterbath for 10 minutes to stabilize the nanoparticles. Subsequently, monostearin (32 mg) and A54-PEG-SA (3 mg) were dissolved in ethanol solution (2 mL) at 70 °C, the mixture (200 µl) was immediately injected into the Fe3O4 nanoparticles and followed by ultrasound waterbath for 10 minutes to obtain the nanoparticle of A54-SNLC. As a control, magnetic nanoparticle of SNLC was prepared as described above. The construct Plasmid pEGFP-AFP-Fth (DNA) was chosen as a model plasmid for physicochemical property assessment of ternary nanoparticles.

Binary

nanoparticles with various protamine/DNA weight ratios were obtained by vortically mixing DNA-protamine solution for 30 s, followed by incubation for 30 minutes, A54-SNPD ternary nanoparticles containing various weights ratios of A54-SNLC to protamine/DNA were further obtained by vortically mixing A54-SNLC with protamine/DNA nanoparticles for 30 s. As a control, SNLC/protamine/DNA (SNPD), A54-SNLC/protamine/blank DNA (A54-SNP) and NLC/protamine/blank DNA (SNP) were prepared as described above.

Physicochemical

characteristics

of

SNLCs,

32


binary

and

ternary

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nanoparticles The average diameter, polydispersity, and zeta potential of the SNLC, A54-SNLC,

protamine/DNA

binary

nanoparticles

and

A54-SNPD

ternary

nanoparticles were measured wih a Zetasizer (3000HS, Malvern Instruments Ltd, UK). The morphologies were further captured via transmission electron microscopy (TEM, JEM-1200EX, JEOL, Ltd., Akishima, Tokyo, Japan). One drop of each sample was deposited onto a copper grid without any staining for viewing.

Magnetic properties of SNLCs Saturated magnetic intensity was also performed to evaluate the magnetic properties of SNLCs The solutions of free SPIO, SNLC and A54-SNLC were lyophilized, then a vibrating sample magnetometer (PPMS, Quantum Design, MPMS-XL-5, CA, USA) was used to measure the magnetic properties of SNLCs at room temperature. Free SPIO, SNLC and A54-SNLC solutions were diluted at various Fe3O4 concentrations of 0, 1, 10, 15, 25, 50, 100 µg/mL, their magnetic properties were evaluated using a 3.0 T MRI scanner (GE, Discovery MR 750, USA). R2 relaxivities, defined as 1/T2 with units of s−1, of the free SPIO, SNLC and A54-SNLC were measured with the following parameters: Field of View, 180 × 180 mm; TR, 2000 ms; TE, 12 ms; slice thickness, 3.0 mm; number of slices, 8.

Agarose Gel Electrophoresis Experiments. The condensation ability of protamine to DNA and A54-SNLC to protamine/DNA with DNA (0.5 µg/well) was performed by gel retardation assay. 33



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Binary nanoparticles were prepared at various protamine/DNA weights ratios (ranging from 0.3 to 4.0) by incubating at 37 ff for 30 min, ternary nanoparticles were further prepared at various weights ratios of A54-SNLC （ from 5 to 30 ） to protamine/DNA（from 2 to 4）by incubating at 37 ℃for 30 min. Electrophoresis was performed with a voltage of 120 V for 40 minutes. Images were obtained by a digital imaging system (GL 200, Kodak, Windsor, CO, USA) and an ultraviolet transilluminator.

In vitro cytotoxicity The cytotoxicity of the SNLC, A54-SNLC on LO2 and Bel-7402 cells were measured by MTT assay. Briefly, cells were seeded in 96-well plates and cultured for 24 h. Then, the cells were incubated to a series of concentrations of SNLC and A54-SNLC for 48 h afterwards. Subsequently, MTT solution was added for an additional 4 h. After removal of the medium and addition of DMSO, the absorbance values were recorded at 570 nm using a microplate reader (Bio-Rad, model 680, USA). All experiments were performed thrice. The cytotoxicity of the A54-SNPD with different weight ratios of A54-SNLC to protamine/DNA (DNA, 2 µg/well) on LO2 and Bel-7402 cells were measured by MTT assay as described above.

In vitro gene transfection test of ternary nanoparticles Bel-7402 and LO2 cells were seeded in 24-well plates at a density of 5 × 104 cells per well and cultured for 24 h. Binary or ternary nanoparticles (DNA, 2 µg/well) were incubated with cells for 48 h at 37 °C. PEI/DNA nanoparticles with weight ratio of 1.25/1 was used as the positive control. Untransfected cells transfected with naked 34


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DNA were performed as the negative control. The cells were observed by an inversed fluorescence microscope (LSM-510 META, ZEISS, Heidelberg, Germany). Transfection efficiency was further assessed by a flow-cytometer (FC500MCL, Beckman Coulter, Fullerton, CA, USA) after cells was fixed with 4% formalin.

Western blot analysis The AFP and Fth expression level in Bel-7402 cells and LO2 cells before and after gene transfection of ternary nanoparticles were analyzed by the western blotting analysis. Briefly, the cells were harvested by adding cell-lysis buffer, followed by centrifugation (12,000 rpm, 10 min). The cell lysis and protein concentrations were measured by Bradford protein assay kit (Bio-Rad, Hercules, CA). After sample-loading buffer was added, the protein samples were subjected to SDS-PAGE. The blots were incubated with specific primary antibody at 4 °C overnight, followed by incubation with secondary antibodies for 1 h at room temperature. Immunoreactive bands were quantified by Image J (National Institute of Health, MD). GADPH or β-actin was used as internal standards.

Cellular uptake MR imaging of SNLCs For in vitro cell uptake measurements using MRI, LO2 and Bel-7402 cells were incubated with A54-SNLC and SNLC nanoparticles for 3 h, then rinsed thrice with PBS, detached with trypsin-EDTA solution and collected to 2 mL eppendorf tubes by centrifugation. T2-weighted MR image was obtained to scan these tubes using MR scanner. The parameters were as the above description.

Cellular uptake studies of ternary nanoparticles 35



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Octadecylamine-fluorescein

isothiocyanate

(ODA-FITC)

Page 36 of 45

was

synthesized

according to our previous study and employed as the fluorescent marker for the SNLCs.40 The FITC labeled A54-SNLC and SNLC were prepared using 3 mg ODA-FITC instead of monostearin refer to the procedure described above. Bel-7402 and LO2 cells were seeded in 24-well plates at a density of 5 × 104 cells per well and cultured for 24 h, then incubated with FITC labeled A54-SNPD and SNPD ternary nanoparticles (50 µg/mL). For the blocking uptake test, free A54 (20 µg) were added to the Bel-7402 cells 1 h before adding the ternary nanoparticles. Afterwards, 20 µL Hoechst (0.1 g/L) was added to stain the cell nuclei for 30 min. After incubation, removed the medium, rinsed the cells thrice with PBS and fixed with 4% paraformaldehyde for 20 min. The cells were observed under an inverted fluorescence microscope. The cellular uptake fluorescence intensity was further obtained by a flow-cytometer.

In vivo animal hepatoma models BALB/c nude mices (4-5 weeks, 18 ± 2 g) (Zhejiang Medical Animal Centre, Hangzhou, China) were transplanted with Bel-7402 cells to establish orthotopic hepatoma models. The abdominal cavity was cutted longitudinally to expose normal liver tissues, then the abdominal wall and skin were sutured using biodegradable stitches after implanting 1 mm3 tumor tissue into the liver parenchyma.

In vivo distribution of ternary nanoparticles Two weeks after orthotopic hepatoma models of nude mices were transplanted, the realtime biodistribution of ternary nanoparticles was investigated by the Maestro 36


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system (Cambridge Research & Instrumentation, Inc., Woburn, MA, USA). The tumor-bearing mices were divided into five groups (n = 3 per group), then sacrificed and imaged at the predetermined time (3, 6, 12, 24, 48 h) after intravenous injection of 0.2 mL ICG labeled A54-SNPD and SNPD ternary nanoparticles solution (SPIO content was 100 µg/mL) at a dose of 5.0 mg/kg, respectively. At every predeterminded time points, representative organs including tumors were collected followed by observation using the imaging system. Fluorescence intensity associated with the amount of A54-SNPD and SNPD nanoparticles in various organs and tumors was semi-quantified using the imaging system. The distribution and accumulation of A54-SNPD and SNPD nanoparticles in various tissues were calculated as %ID/g.

Histological analysis The tumor tissues were submerged into 10% formalin for 48 h, then embedded in paraffin. The sections were stained using haematoxylin-eosin (H&E) and immunohistochemistry based on the standard procedures. The microscopic images were observed under Leica fluorescence microscope.

In vivo MR imaging For T2-weighted MR imaging, Bel-7402 tumor-bearing mices were divided into four groups (n = 5 per group). 0.2 mL A54-SNPD, SNPD, A54-SNP and SNP ternary nanoparticles solution (SPIO, 100 µg/mL) were injected through tail vein, respectively. Mices were imaged both before and after injection at the predetermined time (1, 3, 6, 12, 24, 48 h) by 3.0 T MRI. The parameters were as follows: FOV, 60 × 60 mm; TR, 3000 ms; TE, 78.6 ms; slice thickness, 2.0 mm; number of slice, 10. 37



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After examination, T2 axial hepatic images were obtained and the signal-to-noise ratio (SNR) was calculated for further image evaluation and quantitative image intensity of tumors and liver tissues. SNRpost/SNRpre was used to evaluate the quantitative changes of signal intensity in tumors and liver tissues. In order to measure the SNR value on an image, place the first region of interest (ROI) on tumors with the most homogenous area three times, then record the average value of the signal intensity in that region. Subsequently, position the second ROI (in the largest possible size to include maximum noise), outside of the tumors in the noisy image background three times, record the average values of signal intensity in that region. Calculate the image SNR using this formula: SNR = signal/noise. After 48 h MR imaging, the tumors and the normal liver tissues were selected for the total protein extraction using RIPA lysis and the level of Fth protein expression were analyzed by the western blotting method.

Statistical analysis. Results were calculated by Student’s t test and one-way analysis of variance (ANOVA). Significance level was controlled at p < 0.05 (*) and p < 0.01 (**). All data are reported as the mean ± S.E.M.

Acknowledgments Thanks to the National Natural Science Foundations of China (81571662, 81171334, 81573657), the Nature Science Foundation of Zhejiang province, China (Y14H180018) and the Major Social Development Program of Major Science and Technology Project of Zhejiang province, China (Grant No.2013C03010), for their 38


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great support on this project.

Competing Interests The authors declare no competing interest.

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Bisphosphonate-anchored

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