Glycol-Chitosan-Based Technetium-99m-Loaded Multifunctional Nanomicelles: Synthesis, Evaluation, and In Vivo Biodistribution

Abstract

We hereby propose the use of stable, biocompatible, and uniformly sized polymeric micelles as high-radiotracer-payload carriers at region-of-interest with negligible background activity due to no or low offsite radiolysis. We modified glycol chitosan (GC) polymer with varying levels of palmitoylation (P) and quaternization (Q). Quaternary ammonium palmitoyl glycol chitosan (GCPQ) with a Q:P ratio of 9:35 (Q9P35GC) offers >99% biocompatibility at 10 mg mL−1. Q9P35GC micelles exhibit >99% 99mTechnetium (99mTc) radiolabeling via the stannous chloride reduction method without heat. The 99mTc-Q9P35GC micelles (65 ± 3 nm) exhibit >98% 6 h serum stability at 37 °C and 7 day of radiochemical stability at 25 °C. HepG2 cells show a higher uptake of FITC-Q9P35GC than Q13P15GC and Q20P15GC. The in vivo 24 h organ cumulated activity (MBq h) order follows: liver (234.4) > kidneys (60.95) > GIT (0.73) > spleen (88.84). The liver to organ ratio remains higher than 2.4, rendering a better contrast in the liver. The radiotracer uptake decreases significantly in fibrotic vs. normal liver, whereas a blocking study with excess Q9P35GC significantly decreases the radiotracer uptake in a healthy vs. fibrotic liver. FITC-Q9P35GC shows in vivo hepato-specific uptake. Radiotracer liver uptake profile follows reversible binding kinetics with data fitting to two-tissue compartmental (2T), and graphical Ichise multilinear analysis (MA2) with lower AIC and higher R2 values, respectively. The study concludes that 99mTc-Q9P35GC can be a robust radiotracer for noninvasive hepatocyte function assessment and diagnosis of liver fibrosis. Furthermore, its multifunctional properties enable it to be a promising platform for nanotheranostic applications.

Keywords: biocompatible; glycol chitosan; kinetic modeling; noninvasive imaging; polymeric micelles; radiotracers; technetium-99m radiolabeling.

Figure 1

Schematic illustration of glycol chitosan modification, technetium (99mTc) labelling, and fluorescein isothiocyanate (FITC) conjugation. (a) Quaternary ammonium palmitoyl glycol chitosan (GCPQ) polymer synthesis scheme; (b) GCPQ-99mTc labelling using stannous-chloride-based reduction method; (c) GCPQ-FITC labeling; Yellow star represents the FITC molecule.

Figure 2

Alamar blue cell viability assay of GCPQ polymers against macrophage (RAW264.7). Q9P35GC showed cell viability equal to normal saline. Unpaired t-test. Abbreviations: ns, nonsignificant, NS, normal saline (0.9% NaCl), pH 7.4, ‘****’ shows statistically significant difference with p < 0.001.

Figure 3

Uptake of FITC-GCPQ micelles (a) Live-cell confocal microscopy images of HepG2 cells treated with FITC-Q9P35GC, Q13P15GC, and Q20P15GC micelles. Red fluorescence from FITC GCPQ micelles, the blue flourescence is from nuclei staining, and the last column is the merge. The scale is 20 µm; Characterization of 99mTc Q9P35GC micelles. (b) Representative TEM image of Q9P35GC micelles with an average size of 60 ± 9.4 nm, n = 6; (c) Representative TEM image of 99mTc labelled Q9P35GC micelles with an average size of 65 ± 8.3 nm, n = 6; (d,e) Particle size distribution of Q9P35GC micelles and 99mTc Q9P35GC micelles as determined by DLS, respectively; (f) Chromatogram of radiochemical analysis showing labelling efficiency of ^99mTc Q9P35GC micelles using double-strip method. Strip one, ITLC-SG Chromatogram of ^99mTc Q9P35GC micelles and hydrocolloids at RF = 0 and Free TcO₄⁻¹ at Rf = 1. Strip two, ITLC-SG Chromatogram of hydrocolloids at RF = 0 and Free TcO₄⁻¹ and ^99mTc Q9P35GC micelles at Rf = 0.672.

Figure 3

Uptake of FITC-GCPQ micelles (a) Live-cell confocal microscopy images of HepG2 cells treated with FITC-Q9P35GC, Q13P15GC, and Q20P15GC micelles. Red fluorescence from FITC GCPQ micelles, the blue flourescence is from nuclei staining, and the last column is the merge. The scale is 20 µm; Characterization of 99mTc Q9P35GC micelles. (b) Representative TEM image of Q9P35GC micelles with an average size of 60 ± 9.4 nm, n = 6; (c) Representative TEM image of 99mTc labelled Q9P35GC micelles with an average size of 65 ± 8.3 nm, n = 6; (d,e) Particle size distribution of Q9P35GC micelles and 99mTc Q9P35GC micelles as determined by DLS, respectively; (f) Chromatogram of radiochemical analysis showing labelling efficiency of ^99mTc Q9P35GC micelles using double-strip method. Strip one, ITLC-SG Chromatogram of ^99mTc Q9P35GC micelles and hydrocolloids at RF = 0 and Free TcO₄⁻¹ at Rf = 1. Strip two, ITLC-SG Chromatogram of hydrocolloids at RF = 0 and Free TcO₄⁻¹ and ^99mTc Q9P35GC micelles at Rf = 0.672.

Figure 4

Biodistribution and kinetics of 99mTc Q9P35GC micelles in rabbits. (a) Representative SPECT images were taken by Dual-Head Gamma Camera (Anterior: right and Posterior: left) after IV administration of 99mTc Q9P35GC micelles at 10, 30, and 60 min; (b) Average SUV TACs in liver, kidney, and bladder during initial 25 min after IV administration of 80 ± 2 MBq of 99mTc Q9P35GC micelles, represented as mean ± SD, n = 5; (c) Organ cumulated activity following intravenous bolus injection of 99mTc Q9P35GC micelles (80 ± 2 MBq) expressed as MBq h of tissue in different organs (n = 3); (d) In vivo biodistribution following an intravenous bolus injection of 99mTc Q9P35GC micelles expressed as percentage injected dose with SEM in different organs at different time points. Error bars show SD.

Figure 5

Kinetic modeling of the rabbit liver. Mean liver time–activity curves: Liver standardized value uptake (SUV), blue; red fits by one-compartment model (1T); green fits by two-compartment model (2T); and orange fits by Ichise multilinear regression analysis model (MA2).

Figure 6

Cellular distribution of ^99mTc Q9P35GC micelles. (I) Excised liver tissue images captured after 1 h after FITC Q9P35GC/India ink administration; (a,b) confocal images, green spots of FITC Q9P35GC in hepatocytes and black spot of India ink in Kupffer cells; (c,d) light microscope images, white spots of FITC Q9P35GC in hepatocytes and black spot of India ink in Kupffer cells, n = 3; (II) Biodistribution of India ink in the liver 1 h after the administration (as determined by light microscope image at 10×, (A) Kupffer cells with India ink, (B) sinus spaces, (C) hepatocytes arranged in chords, (D) portal vein.

Figure 7

Characterization of a CCl₄-induced rabbit model of liver fibrosis. Representative images of: (a) Normal liver; (b) Fibrotic liver: Masson’s stained liver tissue of; (c) control; (d) fibrotic liver after CCl₄ administration for 9 weeks, fibrotic liver tissue is showing increased collagen (purple); (e) Representative ^99mTc-Q9P35GC dynamic SPECT images at different time points for fibrotic and healthy liver.

Figure 8

Blocking studies of 99mTc-Q9P35GC in fibrotic mice. (a) Representative SPECT image of healthy and fibrotic-liver rabbit at 60 min pi 99mTc-Q9P35GC (80 MBq) with or without Q9P35GC for blocking. Images were adjusted using the same scale for all animals; (b) Hepatic uptake of 99mTc-Q9P35GC derived from SPECT imaging by drawing the ROI of the whole liver, uptake is normalized for each group, ‘***’ shows statistically significant difference with p < 0.001.