Objective To design and synthesize a novel C-X-C motif chemokine receptor 4 (CXCR4)-targeted radiotracer using a positive charge linker modification strategy. Explore the effect of increasing positive charges on the target affinity and tumor retention performance of the targeted probe.

Methods A positively charged linker, D-Lys-D-Arg-D-Ala, was introduced between the core structure of the CXCR4-targeting ligand iodo cyclo (D-Tyr1-D-(NMe)Orn²(NH^*)Arg³-Nal⁴-Gly⁵) and the bifunctional chelator 1,4,7,10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid. The precursor compound was labeled with ⁶⁸Ga to synthesize the novel tracer ⁶⁸Ga-Pentikra, and its radiochemical purity was determined by radio-high performance liquid chromatography. A head-to-head comparative analysis was performed between Pentikra and the clinically commonly used tracer PentixaFor. Its physicochemical properties and biological characteristics were systematically evaluated through stability tests, lipid water partition coefficient determination tests, competitive binding tests, cell blockade tests, and cell uptake tests. The in vivo biological performance was validated via PET/CT imaging and biodistribution experiment in tumor-bearing mice following tail vein injection of the ⁶⁸Ga-labeled probe. For normally distributed data, comparisons between two groups were performed using independent sample t-test.

Results ⁶⁸Ga-Pentikra remained stable in phosphate buffered saline and mouse serum for 2 h. The lipophilic-hydrophilic partition coefficient LogD_7.4 of ⁶⁸Ga-Pentikra was −3.15±0.29, its hydrophilicity is similar to ⁶⁸Ga-PentixaFor. The half maximal inhibition concentration (IC₅₀) of Pentikra for CXCR4 was (24.76±1.01) nmol/L, significantly lower than that of PentixaFor ((306.40±0.87) nmol/L), Pentikra has a significantly higher affinity. The uptake and internalization of ⁶⁸Ga-Pentikra in stable transfected Chinese hamster ovarian (CHO)-human C-X-C motif chemokine receptor 4 (hCXCR4) cells showed a continuous increasing trend within 120 minutes. At 120 min, the total binding rate increased to (49.27±4.43)%, which was significantly higher than the corresponding levels of ⁶⁸Ga-PentixaFor at the same time points ((12.32±2.99)%) (t=9.78, P<0.01). PET/CT imaging of tumor-bearing mice revealed that ⁶⁸Ga-Pentikra and ⁶⁸Ga-PentixaFor exhibited significant tumor uptake within 1 h after injection, with mean standardized uptake values of 4.25±0.33 and 4.98±0.47. These values remained high until 4 h after injection. The biodistribution results showed that 1 h after injection of ⁶⁸Ga Pentikra and ⁶⁸Ga PentixaFor, the uptake of tumor tissue in mice were (27.93±0.84) %ID/g and (33.66±1.84) %ID/g, respectively.

Conclusions A novel CXCR4-targeted tracer ⁶⁸Ga-Pentikra based on a positive charge linker strategy was successfully prepared. The in vitro experimental results confirmed that modification with the positive charge linker significantly enhanced the in vitro affinity and tumor tissue uptake capacity of ⁶⁸Ga-Pentikra. However, the in vivo experimental results demonstrated that further increases in the positive charge of the probe led to a significant increase in non-specific uptake by normal tissues.