BACKGROUND: Microbial L-asparaginase (L-ASNase, EC 3.5.1.1) is a pivotal biopharmaceutical drug-protein that catalyzes the hydrolysis of the non-essential amino acid L-asparagine (L-Asn) into L-aspartic acid (L-Asp) and ammonia , resulting in deplenishing the cellular L-Asn pool, which leads to the ultimate death of the L-asparagine synthetase (L-ASNS) deficient cancerous cells.
OBJECTIVE: This study aimed to investigate the impact of conjugating low molecular weight polyethylene glycol to recombinant P. aeruginosa L-ASNase by examining the pharmacokinetic properties, affinity towards the substrate, and enzyme stability prior to and following the reaction.
METHODS: The recombinant P. aeruginosa L-ASNase was affinity purified and then PEGylated by attaching polyethylene glycol (MW= 330 Da) site-specifically to the protein\'s N-terminus end. After which, the PEGylated L-ASNase was examined by SDS-PAGE (15%), FTIR, and UV/Vis spectrophotometry and subsequently biochemically characterized.
RESULTS: The Km and Vmax values of free P. aeruginosa rL-ASNase were determined to be 0.318 ±1.76 mM and 2915 μmol min-1and following the PEGylation, they were found to be 0.396 ±1.736 mM and 3193 μmol min-1, respectively. Polyethylene glycol (330 Da) has markedly enhanced LASNase thermostability at 37, 45, 50, and 55 °C, as opposed to the free enzyme, which retained 19.5% after 1 h of incubation at 37 °C. The PEGylated L-ASNase was found to be stable upon incubation with human serum for 28 h, in contrast to the sharp decline in the residual bioactivity of the free rL-ASNase after 4 h incubation. Accordingly, an in vivo study was used for validation, and it demonstrated that PEGylated rL-ASNase exhibited longer bioactivity for 24 h, while the free form\'s activity vanished entirely from the rats\' blood sera after 8 h. Molecular dynamics simulation indicated that PEG (330 Da) has affected the hydrodynamic volume of L-ASNase and increased its structural stability. Docking analysis has explored the position of PEG with respect to binding sites and predicted a similar binding affinity to that of the free enzyme.
CONCLUSIONS: For the first time, recombinant L-ASNase was modified by covalently attaching PEG (330 Da). The resultant novel proposed PEGylated rL-ASNase with remarkably increased stability and prolonged in vivo half-life duration, which could be considered an alternative to mitigate the high molecular weight of PEGylation\'s drawbacks.

In drug development, it is not uncommon that an active substance exhibits efficacy in vitro but lacks the ability to specifically reach its target in vivo. As a result, targeted drug delivery has become a primary focus in the pharmaceutical sciences. Since the approval of Doxil® in 1995, liposomes have emerged as a leading nanoparticle in targeted drug delivery. Their low immunogenicity, high versatility, and well-documented efficacy have led to their clinical use against a wide variety of diseases. That being said, every disease is accompanied by a unique set of physiological conditions, and each liposomal product must be formulated with this consideration. There are a multitude of different targeting techniques for liposomes that can be employed depending on the application. Passive techniques such as PEGylation or the enhanced permeation and retention effect can improve general pharmacokinetics, while active techniques such as conjugating targeting molecules to the liposome surface may bring even further specificity. This review aims to summarize the current strategies for targeted liposomes in the treatment of diseases.

Covalent labeling of therapeutic drugs and proteins with polyethylene glycol (PEGylation) is an important modification for improving stability, solubility, and half-life. PEGylation alters protein solution behavior through its impact on thermodynamic nonideality by increasing the excluded volume, and on hydrodynamic nonideality by increasing the frictional drag. To understand PEGylation\'s impact, we investigated the thermodynamic and hydrodynamic properties of a model system consisting of PEGylated human serum albumin derivatives using analytical ultracentrifugation (AUC) and dynamic light scattering (DLS). We constructed PEGylated human serum albumin derivatives of single, linear 5K, 10K, 20K, and 40K PEG chains and a single branched-chain PEG of 40K (2 × 20K). Sedimentation velocity (SV) experiments were analyzed using SEDANAL direct boundary fitting to extract ideal sedimentation coefficients so, hydrodynamic nonideality ks, and thermodynamic nonideality 2BM1SV terms. These quantities allow the determination of the Stokes radius Rs, the frictional ratio f/fo, and the swollen or entrained volume Vs/v, which measure size, shape, and solvent interaction. We performed sedimentation equilibrium experiments to obtain independent measurements of thermodynamic nonideality 2BM1SE. From DLS measurements, we determined the interaction parameter, kD, the concentration dependence of the apparent diffusion coefficient, D, and from extrapolation of D to c = 0 a second estimate of Rs. Rs values derived from SV and DLS measurements and ensemble model calculations (see complementary study) are then used to show that ks + kD = theoretical 2B22M1. In contrast, experimental BM1 values from SV and sedimentation equilibrium data collectively allow for similar analysis for protein-PEG conjugates and show that ks + kD = 1.02-1.07∗BM1, rather than the widely used ks + kD = 2BM1 developed for hard spheres. The random coil behavior of PEG dominates the colloidal properties of PEG-protein conjugates and exceeds the sum of a random coil and hard-sphere volume due to excess entrained water.

Nanoparticle-based drug delivery systems have emerged as promising platforms for enhancing therapeutic efficacy while minimizing off-target effects. Among various strategies employed to optimize these systems, polyethylene glycol (PEG) modification, known as PEGylation-the covalent attachment of PEG to nanoparticles, has gained considerable attention for its ability to impart stealth properties to nanoparticles while also extending circulation time and improving biocompatibility. PEGylation extends to different drug delivery systems, in specific, nanoparticles for targeting cancer cells, where the concentration of drug in the cancer cells is improved by virtue of PEGylation. The primary challenge linked to PEGylation lies in its confirmation. Numerous research findings provide comprehensive insights into selecting PEG for various PEGylation methods. In this review, we have endeavored to consolidate the outcomes concerning the choice of PEG and diverse PEGylation techniques.

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.

This note aims to inspire through providing a personal view of the development and potential Drug Delivery Nanocarriers functionalized with polythyleneglycol (PEG). This polymer has been used extensively in Pharmaceutical Technology in a variety of compositions, including polyethylene oxide (PEO)-based surfactants. However, the concept of PEGylation, which started in the 70\'s, differs from the functionality of a surfactant, already discloses in the 50\'s. Here, we strictly adhere to the biological functionality of PEGylated nanocarriers intended to have a reduced interaction with proteins and, therefore, modify their biodistribution as well as facilitate their diffusion across mucus and other biological barriers. We analyze how this concept has evolved over the years and the benefit obtained so far in terms of marketed nanomedicines and provide the readers with a prospect view of the topic.

Niosomes are arousing significant interest thanks to their low cost, high biocompatibility, and negligible toxicity. In this work, a supercritical CO2-assisted process was performed at 100 bar and 40 °C to produce niosomes at different Span 80/Tween 80 weight ratios. The formulation of cholesterol and 80:20 Span 80/Tween 80 was selected to encapsulate vancomycin, used as a model active compound, to perform a drug release rate comparison between PEGylated and non-PEGylated niosomes. In both cases, nanometric vesicles were obtained, i.e., 214 ± 59 nm and 254 ± 73 nm for non-PEGylated and PEGylated niosomes, respectively, that were characterized by a high drug encapsulation efficiency (95% for non-PEGylated and 98% for PEGylated niosomes). However, only PEGylated niosomes were able to prolong the vancomycin release time up to 20-fold with respect to untreated drug powder, resulting in a powerful strategy to control the drug release rate.

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.

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The search for novel anticancer agents to replace the current platinum-based treatments remains an ongoing process. Palladacycles have shown excellent promise as demonstrated by our previous work which yielded BTC2, a binuclear palladadycle with a non-ionisable polyethylene glycol (PEG) tether. Here, we explore the importance of the PEG-tether length on the anticancer activity of the binuclear palladacycles by comparing three analogous binuclear palladacycles, BTC2, BTC5 and BTC6, in the oestrogen receptor positive MCF7 and triple-negative MDA-MB-231 breast cancer cell lines. In addition, these are compared to another analogue with an ionisable morpholine tether, BTC7. Potent anticancer activity was revealed through cell viability studies (MTT assays) revealed that while BTC6 showed similar potent anticancer activity as BTC2, it was less toxic towards non-cancerous cell lines. Interestingly, BTC7 and BTCF were less potent than the PEGylated palladacycles but showed significantly improved selectivity towards the triple-negative breast cancer cells. Cell death analysis showed that BTC7 and BTCF significantly induced apoptosis in both the cancer cell lines while the PEGylated complexes induced both apoptosis and secondary necrosis. Furthermore, experimental and computational DNA binding studies indicated partial intercalation and groove binding as the modes of action for the PEGylated palladacycles. Similarly, experimental and computational BSA binding studies indicated and specific binding sites in BSA dependent on the nature of the tethers on the complexes.