Hepatocyte-Targeted Nuclear Imaging Using ^99mTc-Galactosylated Chitosan: Conjugation, Targeting, and Biodistribution

Materials

Water-soluble chitosan (molecular weight ≈ 5,000, 97% deacetylated) was kindly provided by Dr. Jae-Woon Nah (Sunchon National University, Sunchon, Korea). Fluorescein isothiocyanate (FITC) and stannous chloride dihydrate were purchased from Sigma-Aldrich Co. ^99mTc-Pertechnetate was eluted from a technetium generator (Amersham Health) prepared in our hospital. Instant thin-layer chromatography (ITLC)-SG chromatographic strips were purchased from Gelman Sciences. Female BALB/c mice, 5–6 wk old and weighing 16–18 g (Harlan-Asia, Daehanbiolink, Co. Ltd.), were kept in cages (2 mice per cage) and fed standard laboratory chow and water. All animal experiments were approved by the Wonkwang University School of Medicine Committee and were performed in accordance with their guidelines.

Synthesis of GC and Galactosyl-Methylated Chitosan (GMC)

Chitosan was coupled with lactobionic acid (LA) (Tokyo Chemistry Industry) via an active ester intermediate using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Dojindo) and N-hydroxysuccinimide (NHS) (Pierce Chemicals) (15). Chitosan and equivalent moles of LA were dissolved in 10 mmol/L N,N,N′,N′-tetramethylethylenediamine/HCl buffer solution (pH 4.7). EDC and NHS were added to this solution and stirred for 72 h at room temperature. The resulting GC was dialyzed for 4 d using a Spectra/Por7 membrane (molecular weight cutoff = 3,500; Spectrum) against distilled water. After dialysis, GC was freeze-dried.

After 0.1 g of GC and 0.24 g of sodium iodide were dissolved in 4 mL of N-methyl-2-pyrrolidone (NMP) at 60°C, 0.55 mL of a 15% aqueous sodium hydroxide solution and 0.6 mL of methyl iodide were added and then reacted for 1 h with stirring (16,17). The product was isolated by precipitation with ethanol and subsequent centrifugation. To exchange the counterion iodide with chloride, the product was dissolved in 5 mL of 10% NaCl aqueous solution and precipitated with ethanol, isolated by centrifugation, and thoroughly washed with ethanol and ether. The final product was dried in a vacuum and measured for the degree of methylation by ¹H nuclear magnetic resonance (NMR). ¹H NMR spectra were measured in deuterated water, using a 600-MHz spectrometer (Bruker).

Conjugation of GMC with FITC

GMC was dissolved in sodium bicarbonate buffer (0.2 mol/L, pH 9.0). FITC (10 mg/mL in N,N-dimethylformamide) was slowly added to the buffer. The mixture was gently stirred at room temperature for 4 h. FITC-GMC was precipitated with ethanol and dried in a vacuum.

Radiolabeling with ^99mTc

GMC was dissolved in distilled water (1 μg/μL). Stannous chloride was dissolved in 0.02N HCl (0.2 μg/μL, w/v). One hundred microliters of GMC were mixed with 10 μL stannous chloride solution in a vial at room temperature. Labeling was performed by adding 111 MBq ^99mTc-sodium pertechnetate (0.3 mL saline) to this mixture. The final volume of the solution was brought to 1 mL with saline and then shaken gently. The pH of the final mixture was ∼7.0. The mixture was allowed to react for a further 30 min, with occasional shaking. Labeled GMC was then filtered through a 0.22-μm filter (Millipore) and the filter was washed with saline. Labeling efficiency was determined using ITLC-SG strips with saline and acetone as the mobile phase at 15 and 60 min after filtering.

Imaging and Biodistribution Studies

After injection of 18.5 MBq ^99mTc-GMC (200 μL containing 0.47 μmol GMC) and 18.5 MBq ^99mTc-methylated chitosan (MC) (200 μL containing 0.5 μmol MC) via the tail vein of mice, dynamic and static images were performed with a γ-camera (Vertex; ADAC Laboratories) with a 5-mm pinhole collimator, window setting of 140 keV, and 20% width. γ-Camera images were acquired at 10, 60, and 120 min after injection. The static images were stored in a 512 × 512 matrix size and acquisition times were 120 s. Regions of interest in the liver and heart were drawn and time-activity curves were acquired. Animals were killed and each organ was dissected at 10, 60, and 120 min after injection (n = 5). Dissected tissue samples were rinsed of excess blood, weighed, and counted in a γ-counter. The biodistribution of ^99mTc-GMC in each organ was calculated as a percentage of the injected dose per gram of tissue (%ID/g). The total counts injected per animal were calculated by the difference between the original syringe counts and the remaining syringe counts after injection. Correction was made for background radiation and physical decay during counting.

Uptake of FITC-GMC by Hepatocytes

Mice were sedated with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) administered intraperitoneally, and FITC-GMC was injected via the tail vein. To differentiate hepatocytes and Kupffer cells, India ink was injected via the tail vein in 1 mouse and FITC-GMC and India ink were injected systemically in the other mouse. Mice were sacrificed after 30 min, and the liver was isolated and embedded in 50% (v/v) optimal cutting compound (OCT; Sakura Finetek). Cryosections were cut at 6 μm using a microtome (Leica) and placed onto slides. The slides were dipped in acetone and then washed with phosphate-buffered saline. Fluorescence was visualized using a Zeiss Axioskop2 optical microscope. Green fluorescence was observed using the 450- to 490-nm excitation filter. The images were recorded by using Zeiss AxioVision software.

Synthesis of GMC

The synthetic scheme for GMC is outlined in Figure 1. Figure 2A shows NMR spectra of GC. The substitution value of galactose in GC was calculated by comparing the characteristic peak areas of the galactose group (4.1 ppm) with that of the 2.0-ppm peak attributed to the original acetamide group of chitosan. The substitution value of LA coupled with chitosan in GC was estimated to be 7.42 mol%. Figure 2B shows a peak at 2.2 ppm assigned to NH(CH₃), 3.1 ppm assigned to N(CH₃)₃⁺, and 3.4 ppm assigned to N(CH₃)₂. The composition of tri-, di-, and monomethylation of GC was 8.8, 46.0, and 35.2 mol%, respectively.

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FIGURE 1.

Schematic structures of GC and GMC.

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FIGURE 2.

¹H NMR spectra of GC (A) and GMC (B). Solid star, open square, and solid circle correspond to proton of acetamide group of chitosan (δ 2.0 ppm), proton attached at C2 in grafted galactose group (δ 4.5 ppm), and proton attached at C2 in opened pyranose ring linked between grafted galactose group and chitosan main chain (δ 4.2 ppm), respectively.

Radiolabeling with ^99mTc

The radiolabeling efficiency of ^99mTc-GC was increased >95% by incorporation of methyl groups and that lasted up to 1 h. As shown in Figure 3, the labeling stability is preserved by methylation. The labeling efficiency determined by ITLC-SG in acetone was at least 98% at 6 h in the case of ^99mTc-MC and was >95% at 6 h in the case of ^99mTc-GMC (data not shown). ^99mTc-GMC was incubated at 37°C with 1 mL of human serum. The result of human serum stability was >81% during 2 h.

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FIGURE 3.

Labeling efficiency of ^99mTc-GC and ^99mTc-GMC in saline (A) and acetone (B).

Imaging and Biodistribution Studies

The distributions of ^99mTc-MC and ^99mTc-GMC in mice are illustrated in Figures 4A and 4B. ^99mTc-GMC was transported to the liver within a few minutes of injection, whereas some faint liver uptake occurred in ^99mTc-MC-injected mice. The pattern of liver uptake was markedly different between the 2 materials because of the galactose residue positioned on chitosan’s exterior. The liver activity of ^99mTc-GMC gradually increased until 2 h (Fig. 4C). The calculations for the biodistribution of the ^99mTc-GMC are summarized in Table 1. The values of %ID/g of liver were as follows: 11.155 ± 2.332, 14.018 ± 6.081, and 14.082 ± 1.670 %ID/g (mean ± SD) at 10, 60, and 120 min after injection, respectively. ^99mTc-GMC also showed high activity in the kidney: 27.274 ± 3.061, 21.986 ± 5.108, and 19.615 ± 1.491 %ID/g (mean ± SD).

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FIGURE 4.

γ-Camera images of mice at 10, 60, and 120 min after injection of ^99mTc-MC (A) and ^99mTc-GMC (B). After ^99mTc-GMC injection into mice, ^99mTc-GMC was specifically localized to liver (B), but ^99mTc-MC uptake was faint (A). Solid arrows point to liver; open arrows indicate kidneys of each animal. (C) Time-activity curves of liver and heart of mouse injected with ^99mTc-GMC.

Uptake of FITC-GMC by Hepatocytes

As shown in Figure 5D, the fluorescence of the liver tissue of mice injected with FITC-GMC was visualized as scattered green-colored spots, in contrast with control liver tissue. No fluorescence was observed in the control liver tissue (Fig. 5B). When injected with India ink only, Kupffer cells appeared to be black, spindle-shaped cells under light and were shown as small fluorescent points under the 450- to 490-nm excitation filter (Figs. 5B and 5D). When injected with both FITC-GMC and India ink systemically, the fluorescent image showed larger spots or clusters in the space between Kupffer cells in addition to fluorescent points that were positioned in Kupffer cells under 10× magnification (Fig. 5D, arrows). These results suggest that accumulation of GMC in the liver was mediated by the ASGP-R.

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FIGURE 5.

(A and C) Light microscope images of liver tissue. (B and D) Fluorescence images of liver tissue. India ink trapped by Kupffer cells appears as black spots in A–D. Fluorescence of B was from India ink. Fluorescence from FITC-GMC was different from that of India ink trapped in Kupffer cells (D). Arrows indicate fluorescence of FITC-GMC combined with hepatocytes.