Nectin cell adhesion molecule 4 (Nectin-4) is overexpressed in various malignant tumors and has emerged as a promising target for tumor imaging. Bicyclic peptides, known for their conformational rigidity, metabolic stability, and membrane permeability, are ideal tracers for positron emission tomography (PET) imaging. In this study, we evaluated the feasibility of visualizing Nectin-4-positive tumors using radiolabeled bicyclic peptide derivatives and optimized the pharmacokinetics of radiotracers by introducing PEG chains of different lengths. Five PEGylated radiotracers radiolabeled with 68Ga3+ exhibited high radiochemical purity and stability. As the chain length increased, the Log D values decreased from -2.32 ± 0.13 to -2.50 ± 0.16, indicating a gradual increase in the hydrophilicity of the radiotracers. In vitro cell-binding assay results showed that the PEGylated bicyclic peptide exhibits nanomolar affinity, and blocking experiments confirmed the specific binding of the tracers to the Nectin-4 receptor. In vivo PET imaging and biodistribution studies in SW780 and 5637 xenograft mice showed that [68Ga]Ga-NOTA-PEG12-BP demonstrated optimal pharmacokinetics, characterized by rapid and good tumor uptake, faster background clearance, and improved tumor-to-tissue contrast. Finally, compared with 18F-FDG, PET imaging, in vivo blocking assays of [68Ga]Ga-NOTA-PEG12-BP and histological staining confirmed that specific tumor uptake was mediated by Nectin-4 receptors. The results indicated that [68Ga]Ga-NOTA-PEG12-BP was a promising PET radiotracer for Nectin-4 targeting, with applications for clinical translation.

Although the discovery of insulin 100 years ago revolutionized the treatment of diabetes, its therapeutic potential is compromised by its short half-life and narrow therapeutic index. Current long-acting insulin analogs, such as insulin-polymer conjugates, are mainly used to improve pharmacokinetics by reducing renal clearance. However, these conjugates are synthesized without sacrificing the bioactivity of insulin, thus retaining the narrow therapeutic index of native insulin, and exceeding the efficacious dose still leads to hypoglycemia. Here, we report a kind of di-PEGylated insulin that can simultaneously reduce renal clearance and receptor-mediated clearance. By impairing the binding affinity to the receptor and the activation of the receptor, di-PEGylated insulin not only further prolongs the half-life of insulin compared to classical mono-PEGylated insulin but most importantly, increases its maximum tolerated dose 10-fold. The target of long-term glycemic management in vivo has been achieved through improved pharmacokinetics and a high dose. This work represents an essential step towards long-acting insulin medication with superior safety in reducing hypoglycemic events.

Overcoming the influence of interfering substances in the environment and achieving superior sensing performance are significant challenges in biomarker detection within complex matrices. Herein, an integrated electrochemical sensing platform for sensitive detection of biomarkers in complex biofluids was developed based on a newly designed PEGylated multifunctional peptide (PEG-MPEP). The designed PEG-MPEP contains a poly(serine) sequence (-ssssss-) as the antifouling part and recognition peptide sequence (-avwgrwh) specific for the target human immunoglobulin G (IgG). To improve the peptide stability to protease hydrolysis, d-amino acids were adopted to synthesize the whole peptide. Additionally, the PEGylation can further enhance the stability of the peptide, and the PEG itself was also antifouling, ensuring superstrong antifouling capability of the PEG-MPEP. The designed PEG-MPEP-based biosensor possessed a high sensitivity for the detection of IgG in the range of 1.0 pg mL-1 to 1.0 μg mL-1, with a low limit of detection (0.41 pg mL-1), and it was capable of assaying targets accurately in real serum samples. Compared with conventional peptide-modified biosensors, the PEG-MPEP-modified biosensor exhibited superior antifouling and antihydrolysis properties in complex biofluid, showcasing promising potential for practical assay applications.

Strong precorneal clearance mechanisms including reflex blink, constant tear drainage, and rapid mucus turnover constitute great challenges for eye drops for effective drug delivery to the ocular epithelium. In this study, cyclosporine A (CsA) for the treatment of dry eye disease (DED) was selected as the model drug. Two strategies, PEGylation for mucus penetration and cationization for potent cellular uptake, were combined to construct a novel CsA nanosuspension (NS@lipid-PEG/CKC) by coating nanoscale drug particles with a mixture of lipids, DSPE-PEG2000, and a cationic surfactant, cetalkonium chloride (CKC). NS@lipid-PEG/CKC with the mean size ∼173 nm and positive zeta potential ∼+40 mV showed promoted mucus penetration, good cytocompatibility, more cellular uptake, and prolonged precorneal retention without obvious ocular irritation. More importantly, NS@lipid-PEG/CKC recovered tear production and goblet cell density more efficiently than the commercial cationic nanoemulsion on a dry eye disease rat model. All results indicated that a combination of PEGylation and cationization might provide a promising strategy to coordinate mucus penetration and cellular uptake for enhanced drug delivery to the ocular epithelium for nanomedicine-based eye drops.

Polyethylene glycol modification (PEGylation) is a widely used strategy to improve the physicochemical properties of various macromolecules, especially protein drugs. However, its application in enhancing the performance of enzymes for molecular biology remains underexplored. This study explored the PEGylation of Bst DNA polymerase, determining optimal modification reaction conditions. In comparison to the unmodified wild-type counterpart, the modified Bst DNA polymerase exhibited significantly improved activity, thermal stability, and inhibitor tolerance during loop-mediated isothermal amplification. When applied for the detection of Salmonella in crude samples, the modified enzyme demonstrated a notably accelerated reaction rate. Therefore, PEGylation emerges as a viable strategy for refining DNA polymerases, helping in the development of novel molecular diagnostic reagents.

A series of water-soluble PEGylated 1,2,4-triazoles 5-8 were successfully synthesized from methyl 5-(chloromethyl)-1-aryl-1H-1,2,4-triazole-3-carboxylates 1. All of the water-soluble PEGylated 1,2,4-triazoles were characterized by FT-IR and 1H NMR spectroscopy. The solubility, in vitro plasma stability, and anti-inflammatory activity were also determined and compared to original methyl 5-(halomethyl)-1-aryl-1H-1,2,4-triazole-3-carboxylates. For SAR study, all PEGylated 1,2,4-triazoles 5-8 performed potential anti-inflammatory activity on LPS-induced RAW 264.7 cells (IC50 = 3.42-7.81 μM). Moreover, the western blot result showed PEGylated 1,2,4-triazole 7d performed 5.43 and 2.37 folds inhibitory activity over iNOS and COX-2 expressions. On the other hand, the cell viability study revealed PEGylated 1,2,4-triazoles 7 and 8 with PEG molecular weight more than 600 presented better cell safety (cell viability > 95 %). Through the solubility and in vitro plasma stability studies, PEGylated 1,2,4-triazoles 7a-d exhibited higher hydrophilicity and prolonged 2.01 folds of half-life in compound 7d. Furthermore, the in vivo anti-inflammatory and gastric safety results indicated PEGylated 1,2,4-triazole 7d more effectively decreased the inflammatory response in edema and COX-2 expression and exhibited higher gastric safety than Indomethacin. Following the in vitro and in vivo study results, PEGylated 1,2,4-triazole 7d possessed favorable solubility, plasma stability features, safety, and significant anti-inflammatory activity to become the potential water-soluble anti-inflammatory candidate.

BACKGROUND: Although its immunomodulatory properties make thymopentin (TP5) appealing, its rapid metabolism and inactivation in the digestive system pose significant challenges for global scientists. PEGylated niosomal nanocarriers are hypothesized to improve the physicochemical stability of TP5, and to enhance its intestinal permeability for oral administration.
METHODS: TP5-loaded PEGylated niosomes were fabricated using the thin film hydration method. Co-cultured Caco-2 and HT29 cells with different ratios were screened as in vitro intestinal models. The cytotoxicity of TP5 and its formulations were evaluated using an MTT assay. The cellular uptake and transport studies were investigated in the absence or presence of variable inhibitors or enhancers, and their mechanisms were explored.
CONCLUSIONS: All TP5 solutions and their niosomal formulations were nontoxic to Caco-2 and HT-29 cells. The uptake of TP5-PEG-niosomes by cells relied on active endocytosis, exhibiting dependence on time, energy, and concentration, which has the potential to significantly enhance its cellular uptake compared to TP5 in solution. Nevertheless, cellular transport rates were similar between TP5 in solution and its niosomal groups. The cellular transport of TP5 in solution was carried out mainly through MRP5 endocytosis and a passive pathway and effluxed by MRP5 transporters, while that of TP5-niosomes and TP5-PEG-niosomes was carried out through adsorptive- and clathrin-mediated endocytosis requiring energy. The permeability and transport rate was further enhanced when EDTA and sodium taurocholate were used as the penetration enhancers.
CONCLUSIONS: This research has illustrated that PEG-niosomes were able to enhance the cellular uptake and maintain the cellular transport of TP5. This study also shows this formulation\'s potential to serve as an effective carrier for improving the oral delivery of peptides.

With the rapid advancement of genetic and protein engineering, proteins and peptides have emerged as promising drug molecules for therapeutic applications. Consequently, there has been a growing interest in the field of chemical modification technology to address challenges associated with their clinical use, including rapid clearance from circulation, immunogenicity, physical and chemical instabilities (such as aggregation, adsorption, deamination, clipping, oxidation, etc.), and enzymatic degradation. Polyethylene glycol (PEG) modification offers an effective solution to these issues due to its favorable properties. This review presents recent progress in the development and application of PEGylated therapeutic proteins and peptides (TPPs). For this purpose, firstly, the physical and chemical properties as well as classification of PEG and its derivatives are described. Subsequently, a detailed summary is provided on the main sites of PEGylated TPPs and the factors that influence their PEGylation. Furthermore, notable instances of PEG-modified TPPs (including antimicrobial peptides (AMPs), interferon, asparaginase and antibodies) are highlighted. Finally, we propose the chemical modification of TPPs with PEG, followed by an analysis of the current development status and future prospects of PEGylated TPPs. This work provides a comprehensive literature review in this promising field while facilitating researchers in utilizing PEG polymers to modify TPPs for disease treatment.

FGF21 plays an active role in the treatment of type 2 diabetes, obesity, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). However, the short half-life and poor stability of wild-type FGF21 limit its clinical application. Previous studies found that PEGylation can significantly increase the stability of FGF21. However, the uneven distribution of PEGylation sites in FGF21 makes it difficult to purify PEG-FGF21, thereby affecting its yield, purity, and activity. To obtain long-acting FGF21 with controlled site-specific modification, we mutated lysine residues in FGF21, resulting in PEGylation only at the N-terminus of FGF21 (mFGF21). In addition, we modified mFGF21 molecules with different PEG molecules and selected the PEG-mFGF21 moiety with the highest activity. The yield of PEG-mFGF21 in this study reached 1 g/L (purity >99 %), and the purification process was simple and efficient with strong quality controllability. The half-life of PEG-mFGF21 in rats reached 40.5-67.4 h. Pharmacodynamic evaluation in mice with high-fat, high-cholesterol- and methionine and choline deficiency-induced NASH illustrated that PEG-mFGF21 exhibited long-term efficacy in improving liver steatosis and reducing liver cell damage, inflammation, and fibrosis. Taken together, PEG-mFGF21 could represent a potential therapeutic drug for the treatment of NASH.

The growing prevalence of hyperuricemia necessitates the urgent development of more potent treatments. This study aimed to develop, optimize, and evaluate the safety and efficacy of porcine-human recombinant uricase (PHRU) both in vitro and in vivo. The study employed gene editing of PHRU through site-directed mutagenesis, with recombinant proteins expressed in vitro utilizing Escherichia coli. The polyethylene glycol (PEG) approach was employed to augment uricase stability and diminish immunogenicity. The pharmacokinetics and pharmacodynamics of PHRU were tested in vitro and in Sprague Dawley rats. Successful expression of the fusion protein in E. coli and the development of the PEGylated drug were achieved. In vitro experiments confirmed the efficacy of PEG-PHRU in degrading uric acid, with PEGylation not markedly affecting the biological activity of PHRU. Animal studies revealed that PEG-PHRU significantly lowered plasma uric acid levels and mitigated hyperuricemia-induced renal damage in rats. Both drug metabolism and pharmacokinetics exhibited favorable characteristics without observable adverse effects in experimental animals. This novel fusion protein shows the potential for ameliorating hyperuricemia and related renal complications, highlighting it as a promising drug candidate with substantial market applications.