Molecularly imprinted polymers (MIPs) are promising artificial receptors for biomolecular recognition, yet protein imprinting remains challenging due to denaturation and mass-transfer limitations during polymerization. Here, we report a soft and straightforward strategy to create biomimetic imprints directly inside silica capillaries via sol-gel polymerization of silylated amino acids under biocompatible conditions. The approach relies on the polymerization of silylated amino acids around adsorbed protein templates, generating hybrid organic-inorganic cavities that combine shape complementarity with tailored chemical functionalities. The resulting open-tubular imprinted capillaries were evaluated by capillary electrochromatography using proteins of different size and isoelectric point. All imprinted materials exhibited strong and selective recognition of their target proteins, with selective retention ratios exceeding 5, whereas non-target proteins displayed values below 2. The coatings showed excellent repeatability and long-term stability, with variations below 5% in electrochromatographic performance. Selective separations were achieved for several protein templates, including lysozyme, ribonuclease A, cytochrome C, α-lactalbumin and bovine serum albumin. Importantly, the imprinted capillaries maintained their selectivity in human plasma samples. The strategy was further extended to a nanobody template, yielding selective antibody-like molecular recognition in a complex biological matrix. This work demonstrates a versatile and protein-compatible route to create biomimetic recognition sites directly within capillary stationary phases. The proposed platform offers new opportunities for selective bioseparation, biomolecular analysis, and affinity-based analytical technologies.

Radiometal-labeled peptide-based radiopharmaceuticals (RLPB-radiopharmaceuticals) are promising for cancer imaging and targeted radiotherapy; however, their effectiveness is often compromised by the high retention of nonspecific radioactivity in the kidneys due to renal excretion pathways. Current strategies to address this issue have limitations, highlighting the need for innovative approaches to improve targeting specificity and therapeutic efficacy. We aimed to evaluate the applicability of the Gly-Phe-Lys (GFK) tripeptide, a renal brush border (RBB) enzyme-cleavable linkage, to reduce renal radioactivity in RLPB-radiopharmaceuticals using the integrin-targeting radiopeptide [64Cu]Cu-cyclam-RAFT-c(-RGDfK-)4 ([64Cu]Cu-cyclam-RaftRGD). We designed and synthesized the model compound [64Cu]Cu-cyclam-GFK(benzoyl [Bz]), its predictive metabolites, and GFK-incorporated [64Cu]Cu-cyclam-RaftRGD derivatives [64Cu]Cu-cyclam-GFK-RaftRGD and [64Cu]Cu-cyclam-GFK(beta-alanine [βA])3-RaftRGD. In vitro studies showed that dual radiometabolites, namely, [64Cu]Cu-cyclam-G and [64Cu]Cu-cyclam-GF, were simultaneously released from [64Cu]Cu-cyclam-GFK(Bz) by different RBB enzymes, whereas both RaftRGD derivatives released only [64Cu]Cu-cyclam-GF. When injected into mice, [64Cu]Cu-cyclam-GFK(Bz) and the two RaftRGD derivatives led to the urinary excretion of [64Cu]Cu-cyclam-G and [64Cu]Cu-cyclam-GF, respectively. PET imaging and biodistribution studies showed the increased rates of reduction in renal radioactivity levels for the two RaftRGD derivatives compared to the parental [64Cu]Cu-cyclam-RaftRGD (e.g., PET: 1 to 24 h postinjection, 73.0 ± 2.3 and 75.6 ± 1.8 vs 43.0 ± 4.5%, p < 0.0001; biodistribution: 3 to 24 h, 61.1 and 74.4 vs 22.8%). Taken together, these results indicate that the designed renal cleavage occurred in vivo. We also noted the steric interference of the RaftRGD moiety on enzyme access, the spacer effect of the trimeric βA sequence (reduced steric hindrance), and the altered radiopharmacokinetics (e.g., initially increased renal accumulation) of the RaftRGD compounds upon linker incorporation. These findings provide important insights into the chemical design of RLPB-radiopharmaceuticals with reduced renal retention based on the RBB strategy.

Pharmacokinetics of i.p.-administered 64Cu-RaftRGD in ascites of IGR-OV1-OCPM mice

We explore a bioinspired approach to design tailored functionalized capillary electrophoresis (CE) surfaces based on covalent grafting for biomolecules analysis. First, the approach aims to overcome well-known common obstacles in CE protein analysis affecting considerably the CE performance (asymmetry, resolution, and repeatability) such as the unspecific adsorption on fused silica surface and the lack of control of electroosmotic flow (EOF). Then, our approach, which relies on new amino-amide mimic hybrid precursors synthesized by silylation of amino-amides (Si-AA) derivatives with 3-isocyanatopropyltriethoxysilane, aims to recapitulate the diversity of protein-protein interactions (π-π stacking, ionic, Van der Waals…) found in physiological condition (bioinspired approach) to improve the performance of CE protein analysis (electrochromatography). As a proof of concept, these silylated Si-AA (tyrosinamide silylation, serinamide silylation, argininamide silylation, leucinamide silylation, and isoglutamine silylation acid) have been covalently grafted in physiological conditions in different amount on bare fused silica capillary giving rise to a biomimetic coating and allowing both the modulation of EOF and protein-surface interactions. The analytical performances of amino-amide functionalized capillaries were assessed using lysozyme, cytochrome C and ribonuclease A and compared to traditional capillary coatings poly(ethylene oxide), poly(diallyldimethylammonium chloride), and sodium poly(styrenesulfonate). EOF, protein adsorption rate, protein retention factor k, and selectivity were determined for each coating. All results obtained showed this approach allowed to modulate the EOF, reduce unspecific adsorption, and generate specific interactions with proteins by varying the nature and the amount of Si-AA in the functionalization mixture.

Figure S1The treatment outcomes of radiotherapy with 37 MBq (1 nmol) of 64Cu-cyclam-RAFT-c(-RGDfK-)4; Figure S2Toxicity studies in normal mice that were injected with 74 MBq of 64Cu-cyclam-RAFT-c(-RGDfK-)4; Figure S3Preliminary study of U87MG tumors excised at day 13 p.i. of 37 MBq of 64Cu-cyclam-RAFT-c(-RGDfK-)4 for the changes in microvasculature; Figure S4Positive correlation between the tumor volume and the corresponding WBC in U87MG tumor-bearing mice; Figure S5Biodistribution of 64Cu-cyclam-RAFT-c(-RGDfK-)4 at various peptide doses (0.00510 nmol) in U87MG tumor-bearing mice.

Supplementary Files contain (1) Supplementary Materials and Methods for ï¡Vï¢3 expression analysis, autoradiography and histological analysis, and hematology and hepatorenal function; (2) Supplementary Tables S1-S5; and (3) Supplementary Figures S1-S5. Table S1Effects of 37 MBq (1 nmol) of 64Cu-cyclam-RAFT-c(-RGDfK-)4 {plus minus} GF/Lys on hepatorenal functions of U87MG tumor-bearing mice; Table S2Blood cell counts of U87MG tumor-bearing mice at various time points post-injection of 37 MBq (1 nmol) of 64Cu-cyclam-RAFT-c(-RGDfK-)4 {plus minus} GF/Lys; Table S3Blood cell counts of U87MG tumor-bearing mice at various time points after single dose administrations of 37 and 74 MBq of 64Cu-cyclam-RAFT-c(-RGDfK-)4; Table S4Effects of 74 MBq (2 nmol) of 64Cu-cyclam-RAFT-c(-RGDfK-)4 + GF/Lys on hepatorenal functions of normal mice; Table S5Human absorbed doses extrapolated from the biodistribution data of 64Cu-cyclam-RAFT-c(-RGDfK-)4 in U87MG tumor-bearing mice

Biodistribution and estimated ADs of i.p.-administered 64Cu-RaftRGD

Supplementary Materials and Methods for integrin expression analysis, autoradiography, ITD of Cy5.5-RaftRGD in relation to the tumor microenvironment (in s.c. tumors), PET/CT or PET/CECT imaging and quantification, Ki-67 immunostaining, TUNEL assay, and quantification, survival study, and toxicity evaluation.

Integrin expression in OVCAR-3 and IGR-OV1 s.c. xenografts

The files contain (1) Supplementary Materials and Methods for αVβ3 expression analysis, autoradiography and histological analysis, and hematology and hepatorenal function; (2) Supplementary Tables S1-S5; and (3) Supplementary Figures S1-S5. Table S1â^'Effects of 37 MBq (1 nmole) of 64Cu-cyclam-RAFT-c(-RGDfK-)4 {plus minus} GF/Lys on hepatorenal functions of U87MG tumor-bearing mice; Table S2â^'Blood cell counts of U87MG tumor-bearing mice at various time points post-injection of 37 MBq (1 nmole) of 64Cu-cyclam-RAFT-c(-RGDfK-)4 {plus minus} GF/Lys; Table S3â^'Blood cell counts of U87MG tumor-bearing mice at various time points after single dose administrations of 37 and 74 MBq of 64Cu-cyclam-RAFT-c(-RGDfK-)4; Table S4â^'Effects of 74 MBq (2 nmole) of 64Cu-cyclam-RAFT-c(-RGDfK-)4 + GF/Lys on hepatorenal functions of normal mice; Table S5â^'Human absorbed doses extrapolated from the biodistribution data of 64Cu-cyclam-RAFT-c(-RGDfK-)4 in U87MG tumor-bearing mice; Figure S1â^'The treatment outcomes of radiotherapy with 37 MBq (1 nmole) of 64Cu-cyclam-RAFT-c(-RGDfK-)4; Figure S2â^'Toxicity studies in normal mice that were injected with 74 MBq of 64Cu-cyclam-RAFT-c(-RGDfK-)4; Figure S3â^'Preliminary study of U87MG tumors excised at day 13 p.i. of 37 MBq of 64Cu-cyclam-RAFT-c(-RGDfK-)4 for the changes in microvasculature; Figure S4â^'Positive correlation between the tumor volume and the corresponding WBC in U87MG tumor-bearing mice; Figure S5â^'Biodistribution of 64Cu-cyclam-RAFT-c(-RGDfK-)4 at various peptide doses (0.005â^'10 nmole) in U87MG tumor-bearing mice.

64Cu-RaftRGD-based TRT (survival study)

Reducing effect of Gelofusine and L-lysine on the renal accumulation of 64Cu-RaftRGD

The development of artificial receptors able to selectively recognize a target protein is of particular in -terest in separation, diagnostics, and therapeutics fields. Herein, we disclose a method to prepare bio-mimetic and functionalized protein imprints in biocompatible conditions avoiding any protein denaturation. For that purpose, a set of different hybrid silylated amino acid derivatives were synthesized and used without tetraethyl orthosilicate to prepare our molecularly imprinted polymers, allowing to reduce to a minimum of the silicon amount, in order to obtain imprints made almost entirely of amino acids to mimic paratope surfaces of antibodies. Such functional building blocks were polymerized on the surface of magnetic silica nanoparticles at pH 8.5 in ultrapure water in the presence of two globular proteins: cytochrome C or lysozyme. The resulting imprinted hybrid materials were evaluated for their adsorption capacity, specificity, and selectivity by quartz-crystal microbalance with dissipation and magnetic enzyme-linked immunosorbent assay (ELISA) assays. High imprinting factors of 8.7 were measured for these biomimetic hybrid materials (corresponding to approximately 4000 and 450 ng of protein per cm2 immobilized on molecularly imprinted polymers and non-imprinted polymer nano-particles, respectively), representing a significant breakthrough in sol-gel-based molecular imprinting materials. Moreover, competition experiments performed by magnetic ELISA (mELISA) show very good specificity of our imprints at the usual concentrations of ELISA measurements.(c) 2022 Elsevier Ltd. All rights reserved.

Correlation between tumor length (mm) and tumor weight (g)

Blood clearance of i.p.-administered 64Cu-RaftRGD in normal and tumor-bearing mice

Spatial relationship of intratumoral Cy5.5-RaftRGD to the tumor microenvironment (detailed images for s.c. xenografts)

Proliferation and morphology of small intestine and kidney at 3 days after i.p. administration of 148 MBq/0.357 nmol 64Cu-RaftRGD

Table S1-Post-PET biodistribution data of i.v.- and i.p.-administered 64Cu-RaftRGD in IGR-OV1-OCPM mice; Table S2-Biodistribution data of i.p.-administered 64Cu-RaftRGD; Table S3-Inverse regression equations; and Table S4-Estimation of human absorbed doses.

64Cu-RaftRGD-based TRT (toxicity evaluation: hepatorenal functions and gastrointestinal state)

Intertumoral heterogeneity of 64Cu-RaftRGD uptake