pH-Responsive Carboxymethylcellulose Nanoparticles for 68Ga-WBC Labeling in PET Imaging

Abstract

Carboxymethylcellulose (CMC) is a well-known pharmaceutical polymer, recently gaining attention in the field of nanomedicine, especially as a polyelectrolyte agent for the formation of complexes with oppositely charged macromolecules. Here, we report on the application of pH-sensitive pharmaceutical grade CMC-based nanoparticles (NP) for white blood cells (WBC) PET imaging. In this context and as an alternative to ^99mTc-HMPAO SPECT labeling, the use of ⁶⁸Ga³⁺ as PET radionuclide was investigated since, at early time points, it could provide the greater spatial resolution and patient convenience of PET tomography over SPECT clinical practices. Two operator-friendly kit-type formulations were compared, with the intention of radiolabeling within a short time (10 min), under mild conditions (physiological pH, room temperature) and in agreement with the actual clinically applied guidelines. NP were labeled by directly using ⁶⁸Ga³⁺ eluted in HCL 0.05 N, from hospital suited ⁶⁸Ge/⁶⁸Ga generator and in absence of chelator. The first kit type approach involved the application of ⁶⁸Ga³⁺ as an ionotropic gelation agent for in-situ forming NP. The second kit type approach concerned the re-hydration of a proper freeze-dried injectable NP powder. pH-sensitive NP with 250 nm average diameter and 80% labeling efficacy were obtained. The NP dispersant medium, including a cryoprotective agent, was modulated in order to optimize the Zeta potential value (-18 mV), minimize the NP interaction with serum proteins and guarantee a physiological environment for WBC during NP incubation. Time-dependent WBC radiolabeling was correlated to NP uptake by using both confocal and FT-IR microscopies. The ready to use lyophilized NP formulation approach appears promising as a straightforward ⁶⁸Ga-WBC labeling tool for PET imaging applications.

Keywords: 68Ga; PET; WBC labeling; carboxymethylcellulose; nanoparticles; pH sensitive; radiolabeling.

Figure 1

ATR/FT-IR spectra of pristine CMC and purified NP from In-situNP sample.

Figure 2

Effect of concentration and incubation time on NPSusp radiolabeling: (a) RADst after 5 () and 15 min (), (b) LE% after 5 min (■) or 15 min (□).

Figure 3

Plot of DLS average diameter vs. solution pH. There is a clear trend of NP diameter reduction with pH acidification. Labels over the marks refer to the polydispersity index (PI) of the diameter distribution.

Figure 4

Effect of medium composition on NP zeta potential value (□) and time of clot formation in the presence of serum proteins (). All samples contained CMC-based NP (5 mg/mL) in 0.1 M Tris-buffered at pH 7.4 (Plain), then, salts and cryo-protector agent were added either alone or combined: CaCl₂ (0.4 mM), NaCl (0.9%), and Trehalose (5%).

Figure 5

Micrographs of FITC-NPLyo labeled WBC after different incubation periods: control (A,E), 15 min (B,F), 30 min (C,D), 45 min (D,H). Fluorescence images 20× magnification (A–D), and photo-micrograph of the sampled region, acquired using a brightfield camera with a 20× magnification (E–H). Scale bars correspond to 100 μm.

Figure 6

WBC NPLyo 30 min labeled sample. (A) Photo-micrograph at 15× magnification, at a pixel resolution of 5.5 μm. (B) The Amide I integrated peak area map, in false colors.

Figure 7

Area ratio of macromolecular main components. Reference band: Amide II (1533 cm⁻¹).