Radiolabeling Technique

Radiolabeling is a sensitive and reliable tracking method widely used in biology, genetic engineering, pharmacy, and medicine. The analytes can be intrinsically labeled by replacing atoms in the molecular structure with its own radioisotope or chemically modified with an extra radionuclide contained residue. The commonly used radioactive isotopes are ³H, ¹⁴C, ³⁵S, ¹¹¹In, ¹²⁵I, and so on. As regards to PEG and PEGylated conjugates, radiolabeling is also a very effective tool to determine their pharmacokinetics in vivo.

Principles

Typically, a radioisotope is introduced to the PEG or PEGylated molecules by replacing one or several corresponding atoms with radioisotope, and is administrated in small amounts to minimize interference with the experimental system. The radionuclide atom continually emits radiation so that produces a copy of the conjugate with the same distribution path in the body, which can be detected and quantified by a γ-counter, liquid scintillation counter, Geiger counter or positron emission tomography computed tomography/computed tomography (PET/CT). Since the radioisotope labeling does not change the structure of the molecule and endows the analyte with fast identical physicochemical property, it can be used as a precise and sensitive tool to detect the pharmacokinetic behavior of the analytes. Up to date, radiolabeling has been successfully utilized in the studies of the in vivo absorption, distribution, metabolism, and excretion (ADME) processes of PEG, PEGylated small molecules, PEGylated peptides, PEGylated proteins, and PEGylated nanoparticles.

Radioisotopes Commonly Used

Methods for PEGs or PEG Derivatives Radiolabeling

Since methoxy-PEG (mPEG) is difficult to be directly labeled with radioisotope, it is necessary to use other functional end groups for radiolabeling.

• Amine-PEG

Fig. 1 Process demonstration of radiolabeling of amino-PEG. (Bioconjugate chemistry, 2012, 23(5): 881-899)

Amine-PEG can be directly labeled with 125I using Bolton Hunter reagent, as illustrated in the Fig. 1. Similarly, the PEG chain can react with 125I-labeled tyrosine (using the chloramine T method) through the terminal hydroxyl group, thereby being radioactive.

• Bi-functional PEG

Fig. 2 Process demonstration of radiolabeling through a chelating agent. (Bioconjugate chemistry, 2012, 23(5): 881-899)

Chelating agents can be directly introduced to one end of a bifunctional PEG molecule to allow chelation of an isotope and covalent attachment to the target protein or peptide. As shown in Fig. 2, an amine-PEG-succinimidyl ester first react with a cyclic peptide and then react with DOTA sulfosuccinimydyl ester to expose the terminal -NH2 group for labeling with 64Cu.

• PEGylated Peptides/Proteins

Fig. 3 Process demonstration of radiolabeling of PEGylated peptides/proteins through click reaction. (Bioconjugate chemistry, 2012, 23(5): 881-899)

Click reaction, for example, Cu(I)-catalyzed azide−alkyne cycloaddition, can be sufficiently utilized to label PEGylated peptides/proteins for pharmacokinetic studies in tumor imaging application.

• PEGylated Micelles/Liposomes

Fig. 4 Process illustration of surface radiolabeling of PEGylated liposomes. (Bioconjugate chemistry, 2012, 23(5): 881-899)

There are two major approaches to achieve the surface labeling of PEGylated liposomes or micelles: one is to use membrane-soluble chelating agents to incorporate radioisotopes into the conjugates, and the other choice is to covalently attach the chelating agents to the surface.

• PEGylated Nanoparticles

Inorganic nanoparticles, like Superparamagnetic iron oxide nanoparticles (SPION) and quantum dots (QDs), could also be reacted with the amine group at the PEG termini for binding of radioisotope, such as 64Cu. The biodistribution of these nanoparticles in mice could be followed by PET or magnetic resonance imaging (MRI).

Strengths & Weaknesses of Radiolabeling

References

1. Zhang Z, Zhang Y, et al. Recent advances in the bioanalytical methods of polyethylene glycols and PEGylated pharmaceuticals. Journal of Separation Science, 2020, 43(9-10): 1978-1997.
2. Cheng T L, Chuang K H, et al. Analytical measurement of PEGylated molecules. Bioconjugate chemistry, 2012, 23(5): 881-899.