Multicomponent, peptide-targeted glycol chitosan nanoparticles containing ferrimagnetic iron oxide nanocubes for bladder cancer multimodal imaging

Abstract

While current imaging modalities, such as magnetic resonance imaging (MRI), computed tomography, and positron emission tomography, play an important role in detecting tumors in the body, no single-modality imaging possesses all the functions needed for a complete diagnostic imaging, such as spatial resolution, signal sensitivity, and tissue penetration depth. For this reason, multimodal imaging strategies have become promising tools for advanced biomedical research and cancer diagnostics and therapeutics. In designing multimodal nanoparticles, the physicochemical properties of the nanoparticles should be engineered so that they successfully accumulate at the tumor site and minimize nonspecific uptake by other organs. Finely altering the nano-scale properties can dramatically change the biodistribution and tumor accumulation of nanoparticles in the body. In this study, we engineered multimodal nanoparticles for both MRI, by using ferrimagnetic nanocubes (NCs), and near infrared fluorescence imaging, by using cyanine 5.5 fluorescence molecules. We changed the physicochemical properties of glycol chitosan nanoparticles by conjugating bladder cancer-targeting peptides and loading many ferrimagnetic iron oxide NCs per glycol chitosan nanoparticle to improve MRI contrast. The 22 nm ferrimagnetic NCs were stabilized in physiological conditions by encapsulating them within modified chitosan nanoparticles. The multimodal nanoparticles were compared with in vivo MRI and near infrared fluorescent systems. We demonstrated significant and important changes in the biodistribution and tumor accumulation of nanoparticles with different physicochemical properties. Finally, we demonstrated that multimodal nanoparticles specifically visualize small tumors and show minimal accumulation in other organs. This work reveals the importance of finely modulating physicochemical properties in designing multimodal nanoparticles for bladder cancer imaging.

Keywords:

• MRI
• NIRF
• multimodal imaging
• chitosan
• iron oxide
• bladder cancer

Supplementary materials

We observed the in vivo biodistribution of peptide-conjugated chitosan nanoparticles (pCNP) and chitosan nanoparticle (CNP). Before injecting the nanoparticles into the athymic mice, the near infrared fluorescent (NIRF) intensity was measured by in vivo imaging system. We prepared 1 mL each of pCNP and CNP in water and observed the NIRF intensities. Figure S1 showed that the average NIRF intensities of the two nanoparticles were not significantly different (P-value =0.968).

The high positive-charged nanoparticles might cause higher toxic effects to cells by nonspecific binding. It is possible that the positive nanoparticles are accumulated at organs.¹ We also observed a high accumulation of CNP and pCNP in kidneys at 24 and 48 hours (Figure 7). However, interestingly, peptide-conjugated chitosan nanoparticles (pMCNP) showed a very low accumulation in kidneys as well as the other organs only except for tumor. Therefore, we measured the zeta-potential of pMCNP in water, serum, and plasma, respectively. The serum and plasma were obtained from the blood of a dog. Table S1 showed the change of zeta-potential of pMCNP. The zeta-potential of the particles in water was highly positive (26.7 mV). However, when we suspended the particles in serum and plasma, the zeta-potential was altered to negative charges (−5.37 and −2.5 mV, respectively). The results might indicate that numerous counter ions or proteins in the blood can rapidly cover the surface of the pMCNP changing the overall zeta-potential. The change might explain the highly specific accumulation of pMCNPs in tumor minimizing the nonspecific accumulation by the positive surface at major organs. This shows why the zeta-potential should be measured in the actual fluid environment (eg, in serum) rather than in a totally different microenvironment (eg, water).

Table S2 and Figure S2 show the vinblastine (Vin) loading efficiency and release profile of pCNP. Vin is a commercially available bladder cancer drug. We loaded Vin of 20 mg into the pCNP. Briefly, we added 1M NaOH in Vin solution and sonicated them for 10 minutes at 4°C. The mixture was centrifuged at 13,000 rpm for 5 minutes and the supernatant was removed. We repeated this process three times. The pellet was resuspended in distilled water and centrifuged again at 13,000 rpm for 5 minutes two times. We removed the supernatant and lyophilized the pellet. We got 12 mg of Vin without sulfate. pCNP of 10 mg was dissolved in 5 mL water and 5 mL Methanol was added. Two milligram of Vin was dissolved in water and Methanol (1:1). The CNP and Vin were stirred overnight. Methanol was evaporated at 50°C for 2–3 minutes. All the remaining solution was centrifuged again at 3,000 rpm for 10 minutes and supernatants were collected, which were lyophilized. Vin loading condition and drug release profile of the particles were evaluated by high-performance liquid chromatography analysis. We used Column (C18, 5 µm, 3.9×150 mm, Symmetry^®, Waters Corporation, Milford, MA, USA). Samples were dissolved in 0.8 M sodium salicylate solution for better flow of Vin without sulfate. Gradient combination of solvents was applied by four steps: Methanol (5%) and water (95%) for 0–5 minutes, Methanol (95%) and water (5%) for 5–35 minutes, Methanol (95%) and water (5%) for 35–40 minutes, and Methanol (5%) and water (95%) for 40–45 minutes. The signals of Vin were taken at 38.5–39 minutes at 215 nm. For release test, Vin-loaded pCNP (4 mg/mL) was prepared and 100 kDa dialysis bags were utilized.

The loading content of Vin was ~19 wt% and loading efficiency was 94%. After loading Vin, the size was measured by 421 nm. The high loading content and efficiency indicate the potential of CNP to deliver Vin. We also demonstrated superiority of CNPs to deliver anticancer drugs.^2–4 Figure S2 shows the sustained release of Vin from the pCNPs.

Figure S1 1 mL each of CNP and pCNP were prepared in water.

Notes: The particles were imaged in IVIS and had similar NIRF intensity (P-value =0.968). We utilized these particles to evaluate the biodistribution of each particle in athymic mice.

Abbreviations: CNP, chitosan nanoparticle; IVIS, in vivo imaging system; NIRF, near infrared fluorescent; pCNP, peptide-conjugated chitosan nanoparticles.

Figure S2 Release profile of vinblastine from pCNP at pH 7.4.

Note: Vinblastine was continuously released in a sustained rate for 50 h.

Abbreviation: pCNP, peptide-conjugated CNP.

Table S1 Zeta-potential of pMCNP at different mediums

Table S2 Loading efficiency of vinblastine by pCNP

Acknowledgments

This work was supported by the Intramural Research Program (Theragnosis) of KIST, the Christopher Columbus Foundation support to JFL, and a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No 2015R1C1A1A01052592). We would like to especially thank Patty I Bonney, Lindsey M Fourez, Jane C Stewart, and Carol Ann Dowell at Purdue University for their assistance with this work. We also express our appreciation to Dr Aaron Taylor of the Bindley Bioscience Imaging Facility, Dr Tom Talavage and Greg Tamer of the Purdue MRI facility at Purdue Research Park, Dr Debby Sherman and Mr Chia-Ping Huang of the Life Sciences Microscopy Facility at Purdue University for the TEM images, and Lisa Reece of the Bionano Facility in the Birck Nanotechnology Center who was supported in part by the Indiana Clinical and Translational Sciences Institute from the National Institutes of Health, National Center for Research Resources, Clinical and Translational Sciences Award.

Disclosure

The authors report no conflicts of interest in this work.