PEGylated polyethylenimine-entrapped gold nanoparticles modified with folic acid for targeted tumor CT imaging

Colloids and Surfaces B: Biointerfaces 140 (2016) 489–496

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PEGylated polyethylenimine-entrapped gold nanoparticles modified with folic acid for targeted tumor CT imaging Benqing Zhou a,1 , Jia Yang b,1 , Chen Peng c , Jianzhi Zhu a , Yueqin Tang d , Xiaoyue Zhu a , Mingwu Shen a , Guixiang Zhang b,∗∗ , Xiangyang Shi a,c,e,∗ a

College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, People’s Republic of China Department of Radiology, Shanghai General Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200080, People’s Republic of China c Department of Radiology, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, Shanghai 200072, People’s Republic of China d Experiment Center, Shanghai General Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200080, People’s Republic of China e CQM—Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, 9000-390 Funchal, Portugal b

a r t i c l e

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Article history: Received 1 October 2015 Received in revised form 13 December 2015 Accepted 12 January 2016 Available online 14 January 2016 Keywords: Polyethyleneimine Folic acid Gold nanoparticles Tumors CT imaging

a b s t r a c t Development of various cost-effective contrast agents for targeted tumor computed tomography (CT) imaging still remains a great challenge. Herein, we present a facile approach to forming folic acid (FA)targeted multifunctional gold nanoparticles (AuNPs) using cost-effective branched polyethylenimine (PEI) modified with polyethylene glycol (PEG) as a template for tumor CT imaging applications. In this work, PEI sequentially modified with PEG monomethyl ether, FA-linked PEG, and fluorescein isothiocyanate was used as a template to synthesize AuNPs, followed by transformation of the remaining PEI surface amines to acetamides. The formed FA-targeted PEI-entrapped AuNPs (FA-Au PENPs) were fully characterized. We show that the formed FA-Au PENPs with an Au core size of 2.1 nm are water soluble, colloidally stable, and non-cytotoxic in a given concentration range. Flow cytometry and confocal microscopy data reveal that the FA-Au PENPs are able to target cancer cells overexpressing FA receptors (FAR). Importantly, the developed FA-Au PENPs can be used as a nanoprobe for targeted CT imaging of FAR-expressing cancer cells in vitro and the xenografted tumor model in vivo. With the demonstrated biocompatibility by organ biodistribution and histological studies, the designed FA-Au PENPs may hold great promise to be used as a nanoprobe for CT imaging of different FAR-overexpressing tumors. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Computed tomography (CT) has been considered as one of the most commonly utilized imaging techniques for disease diagnostics owing to its deep tissue penetration, better spatial and density resolution than other imaging modalities, and cost effectiveness [1–4]. High-quality CT imaging usually requires the use of contrast agents, however, the conventionally used iodinated small molecular CT contrast agents (e.g., Omnipaque) can be rapidly cleared by kidney [5–7], resulting in short imaging time. In addition, the iodinated small molecular CT contrast agents also suffer problems of

∗ Corresponding author at: College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, People’s Republic of China. Fax: +86 21 67792306 804. ∗∗ Corresponding author. E-mail addresses: [email protected] (G. Zhang), [email protected] (X. Shi). 1 Authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2016.01.019 0927-7765/© 2016 Elsevier B.V. All rights reserved.

renal toxicity and nonspecificity, quite limiting their applications in tumor CT imaging [7,8]. Recent progresses in nanotechnology have shown that various nanoparticulate CT contrast agents have been developed. Most of the developed nanoparticles (NPs) have enhanced X-ray attenuation property, low toxicity, and prolonged blood circulation time, enormously overcoming the drawbacks of iodinated small molecular CT contrast agents [9–11]. Among the currently available nanoparticulate CT contrast agents such as Au-, Pt-, Ta-, Yb-, and Bi-based inorganic NPs [12–16], gold NPs (AuNPs) have received immense interest owing to their better X-ray attenuation property than that of iodinated CT contrast agents, tunable surface chemical modifications, easy control of the size, and biocompatibility after surface functionalization [17–20]. For example, AuNPs modified with polyethylene glycol (PEG) with an average size of 30 nm possess long blood circulation time and good biocompatibility, and are able to be accumulated in phagocytic cells of the liver and spleen for CT imaging of hepatocelluar carcinoma [17,20]. Biocompatible AuNPs stabilized with gum-arabic (GA) matrix can be prepared and used as a CT

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contrast agent [21]. Recently, dendrimers have been shown to be used as templates or stabilizers to create dendrimer-entrapped or dendrimer-stabilized AuNPs for CT imaging applications, in particular tumor CT imaging [11,22–24]. For improved tumor CT imaging performance, it is desirable to modify AuNPs with targeting ligands to significantly improve the imaging specificity. AuNPs conjugated with peptides [25], antibody [9,18], or folic acid [11,26] can be used for targeted tumor CT imaging. In order to improve the cytocompatibility and colloidal stability of the NPs for applications in living organism, the surface of the NPs is often modified with polyethylene glycol (PEG) [17,27,28]. Meanwhile, PEGylation is able to effectively prevent the NPs from rapid uptake by scavenger cells, leading to prolonged circulation times, which is beneficial for tumor CT imaging. Unfortunately, the development of such NP-based CT contrast agents with targeting specificity, good biocompatibility, significant CT contrast performance and cost effectiveness still remain an enormous challenge. Branched polyethylenimine (PEI) with abundant surface primary amines has been used as a multifunctional nanocarrier for drug/gene delivery [29,30] and for NP stabilization [31–34]. In our previous work, we have shown that branched PEI modified with PEG can be used as a template for the synthesis of AuNPs, which can be subsequently used as a high-performance contrast agent for blood pool and tumor CT imaging [35]. The major advantages of the use of PEGylated PEI as a template is that (1) PEGylation modification of PEI surface amines enables enhanced entrapment of AuNPs within its interior, similar to the case of dendrimers [36]; (2) compared to highly expensive commercial high-generation dendrimers, the use of branched PEI is more cost-effective. Likewise, the molecular structure of dendrimers is significantly different from that of branched PEI. However, until now PEGylated PEIentrapped AuNPs (Au PENPs) modified with targeting ligand have not been reported for targeted tumor CT imaging applications. With the PEI amine-mediated conjugation chemistry similar to aminated dendrimers, it is expected that PEGylated Au PENPs with targeting specificity can be readily prepared and used for targeted tumor CT imaging. To test our hypothesis, in this present study, branched PEI sequentially modified with PEG monomethyl ether with one end of carboxyl group (mPEG-COOH), FA-linked PEG with the other end of carboxyl group (PEG-FA-COOH), and fluorescein isothiocyanate (FI) was used as a template to prepare AuNPs, followed by acetylation of the remaining PEI surface amines (Scheme 1). The formed multifunctional FA-targeted Au PENPs (FA-Au PENPs) were characterized using different techniques. The cytocompatibility of the NPs were evaluated by quantitative cell viability assay and qualitative cell morphology observation. The targeting specificity of the FA-Au PENPs to cancer cells overexpressing FA receptors (FAR) was investigated by flow cytometry and confocal microscopy. The potential to use the developed FA-Au PENPs as a nanoprobe for targeted CT imaging of FAR-overexpressing cancer cells in vitro and the xenografted tumor model in vivo was investigated in detail. Finally the FA-Au PENPs were subjected to in vivo biodistribution and organ hematoxylin and eosin (H&E) staining studies. To our knowledge, this is the first report related to the synthesis of PEGylated Au PENPs with targeting specificity for tumor CT imaging applications.

2. Result and discussion 2.1. Synthesis and characterization of the FA-Au PENPs Different from the synthesis of PEGylated Au PENPs reported in our previous work [35], in this study branched PEI was sequentially modified with mPEG-COOH, PEG-FA-COOH, and FI. The formed multifunctional PEGylated PEI was used as a template for the syn-

thesis of AuNPs, followed by full acetylation of the remaining PEI surface amines (Scheme 1). The formed FA-Au PENPs were characterized via different techniques. The number of PEG, FA, and FI moieties conjugated onto each PEI can be estimated by comparing the difference among the 1 H NMR integration areas of FA-PEG-COOH, PEI-mPEG, and FI with PEI -CH2 - protons (Fig. S1, Supporting information). The average number of FA moieties conjugated onto each PEG was estimated to be 0.7 (Fig. S1a, Supporting information), and the numbers of PEG, FA and FI moieties linked to each PEI were estimated to be 24, 5.5, and 6.3, respectively (Fig. S1b and S1c, Supporting information). Mass spectrum of the formed FA-PEG-COOH conjugate shows that the Mw of the FA-PEG-COOH conjugate is 2322.7 (Fig. S2, Supporting information). By comparison the average Mw of the NH2 -PEG-COOH (Mw = 2000), the number of FA moieties linked to each PEG was calculated to be 0.73, which is consistent with the 1 H NMR data. Similarly, based on the Mw of PEI-mPEG (Mw = 76158.3) and PEI (Mw = 25000) (Fig. S3, Supporting information), the number of mPEG moieties linked to each PEI was calculated to be 25.6, quite similar to that caulated based on 1 H NMR inegration. 1 H NMR was also used to confirm the successful PEI amine acetylation (Fig. S1d, Supporting information). The FA-Au PENPs after acetylation display two new peaks, one is the acetyl protons linked to the secondary PEI amides (1.90 ppm), and the other is the acetyl protons linked to the PEI tertiary amides (2.05 ppm), in agreement with the literature [35,37]. UV–vis spectroscopy was used to follow the PEI surface modification and AuNPs synthesis (Fig. 1a). By comparison of the FA-PEG-COOH and the PEI-FI-(PEG-FA), the FA-Au PENPs display the peaks at 290 nm (which is attributed to the attached FA moieties) and 500 nm (which is associated to overlapped absorption peaks of FI and surface plasmon resonance (SPR) peak of AuNPs). This indicates the successful formation of FA-Au PENPs. In addition, the acetylation reaction does not seem to significantly affect the optical property of the FA-Au PENPs. TEM was used to characterize the size and morphology of the formed FA-Au PENPs (Fig. 2). It is clear that the Au core NPs possesses a spherical shape and are quite uniform with a mean diameter of 2.1 ± 0.4 nm (Fig. 2a and 2b). High resolution TEM shows the clear lattice structure, confirming the crystalline nature of the Au core NPs (Fig. 2c). The crystalline structure of the Au core NPs was further confirmed by selected area electron diffraction (SAED) pattern, where the (111), (200), (220), and (311) rings are typical for the face-centered-cubic (fcc) Au crystal structure (Fig. 3d). The hydrodynamic size of the FA-Au PENPs was measured to be 202.4 nm via DLS (Fig. S4, Supporting information). The larger size of the particles measured by DLS than that measured by TEM could be due to the fact that DLS measures the clustered Au PENPs that may consist of many single AuNPs in aqueous solution, while TEM only measures single Au core NPs. Zeta potential measurements were also employed to confirm the successful acetylation reaction (Table S1, Supporting information). We show that the surface potential of the {(Au0 )200 PEI.NH2 -FI-mPEG-(PEG-FA)} PENPs (14.4 ± 2.4 mV) decreases to 6.1 ± 0.1 mV after acetylation. Similarly, the surface potential of the {(Au0 )200 -PEI-mPEG} PENPs without FA and FI modification (29.77 ± 8.59 mV) decreases to 8.6 ± 1.8 mV after acetylation. Due to the nature of the incomplete acetylation reaction, the particles after acetylation still display a slight positive surface potential, in agreement with our previous work [38]. The stability of the FA-Au PENPs under different conditions was analyzed by UV–vis spectroscopy (Fig. S5, Supporting information). The results show that the FA-Au PENPs have good stability at different pHs and temperatures, similar to PEGylated Au PENPs [35]. Furthermore, the FA-Au PENPs dispersed in water, PBS, and cell culture medium are quite colloidally stable and no precipitation

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Scheme 1. Schematic illustration of the preparation of the FA-Au PENPs. Ac2 O and TEA represents acetic anhydride and triethylamine, respectively.

occurred after the particles were stored at room temperature for a period of one month (Fig. S5c, Supporting information).

2.2. X-ray attenuation property of the FA-Au PENPs To examine the potential to use the developed FA-Au PENPs for CT imaging applications, the X-ray attenuation property of the particles was measured (Fig. 3). Nontargeted Au PENPs and Omnipaque were also tested for comparison. It can be seen that with the increase of Au or iodine concentration, all three materials display increased CT contrast enhancement. However, the brightness of both Au PENPs is more prominent than that of Omnipaque at the same concentration of radiodense element (Au or I), especially at the high concentrations (Fig. 3a). Quantitative CT value measurements (Fig. 3b) show that the CT value of both Au PENPs is almost

similar at the same Au concentrations, but higher than that of Omnipaque at the iodine concentrations similar to Au concentrations, in agreement with our previous work [35,36]. These results suggest that the formed FA-Au PENPs have a great potential to be used for CT imaging applications.

2.3. Cytotoxicity assay The cytotoxicity of the FA-Au PENPs was evaluated by MTT cell viability assay (Fig. 1b) and morphology observation of cells (Fig. S6, Supporting information). Clearly, the viability and morphology of KB cells are not significantly affected after treatment with the FAAu PENPs at the Au concentration range of 100–800 ␮M, similar to the control cells treated with PBS. It is interesting to note that the developed FA-Au PENPs have a better cytocompatibility than

Fig. 1. (a) UV–vis spectra of PEG-FA-COOH, PEI-FI-mPEG-(PEG-FA), FA-Au PENPs (before acetylation), and FA-Au PENPs (after acetylation), respectively; (b) MTT viability assay of KB cells after treatment with FA-Au PENPs NPs at different Au concentrations (0–800 ␮M) for 24 h.

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Fig. 2. TEM images (a), size distribution histogram (b), high-resolution TEM image (c), and SAED pattern (d) of the FA-Au PENPs, respectively.

PEGylated G5 dendrimer-entrapped Au NPs (Au DENPs) [39]. When the Au concentration is up to 400 ␮M, the cell viability is less than 80% for PEGylated Au DENPs; whereas at the Au concentration as high as 800 ␮M, the cell viability is still higher than 80% for the FA-Au PENPs.

2.4. Specific targeting of FA-Au PENPs to FAR—overexpressing cancer cells FA has been widely adopted as a ligand for targeting various FAR-overexpressing cancer cells [40–42]. The binding of the FA-Au PENPs to KB-HFAR cells and KB-LFAR cells was investigated by flow cytometry (Fig. 4a, and Fig. S7, Supporting information). It is clear that the KB-HFAR cells treated with the FA-Au PENPs display significantly higher FI fluorescence intensity than the KB-HFAR cells at the

Fig. 3. CT images (a) and X-ray attenuation intensity (b) of the FA-Au PENPs (1), nontargeted Au PENPs (2), and Omnipaque (3) as a function of the molar concentration of the radiodense element (Au or iodine).

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Fig. 4. (a) Mean fluorescence of KB-LFAR and KB-HFAR cells treated with the FA-Au PENPs with different Au concentrations (0–500 ␮M) for 2 h; (b) Cellular Au uptake in KB-LFAR and KB-HFAR cells incubated with the FA-Au PENPs with different Au concentrations (0–300 ␮M) for 3 h.

same Au concentrations (p < 0.001). These results suggest that the binding specificity of the FA-Au PENPs to KB-HFAR cells is through FA-mediated targeting pathway. Due to the presence of FI moieties linked to PEI, the intracellular uptake of the FA-Au PENPs was able to be monitored by confocal microscopic imaging (Fig. 5). We show that after treatment with the FA-Au PENPs for 3 h, KB-HFAR cells display remarkable fluorescence signal both inside the cytosol and on the surface of the cells. In contrast, KB-LFAR cells just display little fluorescence signal, similar to the PBS control. The confocal imaging results corroborate the flow cytometry data.

2.5. Cellular uptake For targeted CT imaging of cells, it is essential to investigate the ability of the developed FA-Au PENPs to be specifically taken up by cancer cells overexpressing FAR. ICP-OES was applied to quantify the Au uptake in both KB-HFAR and KB-LFAR cells after treatment with the FA-Au PENPs at different Au concentrations for 3 h (Fig. 4b). It can be seen that at the given Au concentrations, the Au uptake in KB-HFAR cells is significantly higher than in KB-LFAR cells (p < 0.001). These results together with the flow cytometry and confocal microscopic imaging data suggest that the developed FA-Au PENPs can specifically target the FAR-overexpressing cancer

Fig. 5. Confocal microscopic images of KB-LFAR and KB-HFAR cells treated with the FA-Au PENPs ([Au] = 100 ␮M) for 3 h. The scale bar in each panel represents 20 ␮m.

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cells via receptor-mediated pathway, which is critical for them to be used as a nanoprobe for targeted CT imaging of FAR-overexpressing cancer cells. 2.6. Targeted CT imaging of cancer cells in vitro We next explored the feasibility to use the formed FA-Au PENPs for targeted CT imaging of cancer cells in vitro. Both KB-HFAR and KB-LFAR cells were incubated with FA-Au PENPs at different Au concentrations for 3 h before CT imaging (Fig. 6). For CT imaging, it is quite difficult to visualize the brightness difference of the cell CT images (Fig. 6a), similar to our previous study [24]. Quantitative analysis of the CT values of the images is prerequisite (Fig. 6b). It is clear that the CT values of both KB-HFAR and KB-LFAR cells treated with the FA-Au PENPs are much higher than that of the corresponding cells treated with PBS, and the CT values of both cells increase with the Au concentration. Under a given Au concentration, the CT value of the KB-HFAR cells is higher than that of the KB-LFAR cells, implying that the developed FA-Au PENPs are able to be used for targeted CT imaging of FAR-overexpressing cancer cells. 2.7. Targeted CT imaging of a xenografted tumor model in vivo We next checked the potential to use the FA-Au PENPs for targeted tumor CT imaging in vivo (Fig. 7). Compared to the tumors of mice before injection, the tumor areas have an obvious enhancement in CT contrast with the time postinjection for all 3 groups. Zoomed CT images of tumor site can be seen in Fig. S8 (Supporting information). This suggests that the intravenously injected Au PENPs are able to be delivered to the tumor area with time postinjection for effective tumor CT imaging. Importantly, the CT values of the tumor region injected with the FA-Au PENPs are much higher than those treated with the nontargeted Au PENPs at the same time points. Furthermore, the preinjection of free FA to the tumor mice leads to much lower tumor CT values after the injection of the FA-Au PENPs than direct injection of the FA-Au PENPs without free FA injection at the same time points. Meanwhile, there is no significant difference in CT values of tumors injected with the nontargeted Au PENPs and FA-Au PENPs (with free FA preinjection) at

Fig. 6. In vitro CT images (a) and values (b) of KB-LFAR and KB-HFAR cells treated with the FA-Au PENPs at different Au concentrations (0–300 ␮M) for 3 h, respectively.

the same time points. This means that the free FA injected is able to bind the overexpressed FAR in the tumor region, thereby blocking the further specific binding of the FA-Au PENPs to the tumors. Due to the shorter half-decay time (t1/2 ) of the PEGylated Au PENPs (11.2 h) [35] than that of the PEGylated Au DENPs (31.76 h) [36], the CT value of the tumor area reaches the highest at 2.5 h postin-

Fig. 7. CT images (a) and values (b) of KB tumors in nude mice before (0 h) and at different time points postinjection of the FA-Au PENPs (Group 1), nontargeted Au PENPs (Group 2), or FA-Au PENPs (Group 3) with the tumor region pre-intratumorally injected with free FA (10 mM, 10 ␮L in PBS) for 30 min. In all 3 groups, a similar dose ([Au] = 0.1 M, 150 ␮L in PBS) was injected. The circle in each panel refers to the tumors site.

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jection of the FA-Au PENPs, while the highest tumor CT value can be achieved at 6 h postinjection of the FA-targeted PEGylated Au DENPs [39]. Therefore, we think that the developed FA-Au PENPs have a faster tumor targeting behavior than the FA-targeted PEGylated Au DENPs. Our study suggests that besides the enhanced permeability and retention (EPR)-based passive tumor targeting for all NPs, the FA-mediated active targeting is able to significantly enhance the tumor particle accumulation, resulting in enhanced tumor CT imaging. The tumor CT value starts to decline at 5 h postinjection, implying that the particles accumulated in the tumor region undergo a further metabolism process and diffuse to other tissues or organs.

2.8. In vivo biodistribution To assess the fate of the injected FA-Au PENPs, we explored the in vivo Au biodistribution at different time points postinjection of the particles (Fig. S9, Supporting information). The results reveal that the Au uptake mainly occurs in the spleen, followed by the liver and lung. Because of the PEGylation modification of the NPs, the formed FA-Au PENPs are able to escape from the recognition of reticuloendothelial system (RES) in the liver and spleen, and be accumulated in other organs including heart, kidney, and the tumor region. After 72 h post-injection, the Au uptake in all the major organs starts to decrease, and Au uptake is just 39.4%, 37.5%, and 15.6% of the maximum in the liver, spleen, and lung, indicating that the NPs can be metabolized and cleared out of the body. This means that the injected FA-Au PENPs do not show potential in vivo toxicity after intravenous injection. The in vivo biocompatibility of the FA-Au PENPs was further demonstrated by H&E staining of the major organs of mice injected with the particles (Fig. S10, Supporting information). Clearly, no histological changes in the liver, lung, spleen, kidney, and heart of the mice at one month post intravenous injection of the FA-Au PENPs can be observed. This suggests that the developed FA-Au PENPs have no toxicity to the major organs.

3. Conclusion In conclusion, we developed a facile approach to forming FA-targeted multifunctional Au PENPs using cost-effective PEI modified with PEG as a template for targeted tumor CT imaging. The formed FA-Au PENPs with an Au core size of 2.1 nm are water soluble, stable, and non-cytotoxic in a given concentration range. Thanks to the FA-mediated targeting, the FA-Au PENPs can be used as a nanoprobe for efficient targeted CT imaging of FAR-expressing cancer cells in vitro and xenografted tumor model in vivo. The developed FA-Au PENPs may hold great promise to be used for targeted CT imaging of different FAR-overexpressing tumors.

Acknowledgments This research is financially supported by the National Natural Science Foundation of China (21273032, 81371623, 81501518 and 81101150), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning, and the Sino-German Center for Research Promotion (GZ899). X. Zhu gratefully acknowledges the support from Shanghai Pujiang Program (14PJ1400400). C. P. thanks the financial support from the Shanghai Natural Science Foundation (14ZR1432400). B. Z. thanks the support from the Chinese Universities Scientific Fund (CUSFDH-D-2014034).

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.01. 019.

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