Protein stability remains one of the main factors limiting the realization of the full potential of protein therapeutics. Poly(ethylene glycol) (PEG) conjugation to proteins has evolved into an important tool to overcome instability issues associated with proteins. The observed increase in thermodynamic stability of several proteins upon PEGylation has been hypothesized to arise from reduced protein structural dynamics, although experimental evidence for this hypothesis is currently missing. To test this hypothesis, the model protein α-chymotrypsin (α-CT) was covalently modified with PEGs with molecular weights (MW) of 700, 2000 and 5000 and the degree of modification was systematically varied. The procedure did not cause significant tertiary structure changes. Thermodynamic unfolding experiments revealed that PEGylation increased the thermal transition temperature (Tm) of α-CT by up to 6°C and the free energy of unfolding (ΔGU (25°C)) by up to 5 kcal/mol. The increase in stability was found to be independent of the PEG MW and it leveled off after an average of four PEG molecules were bound to α-CT. Fourier-transformed infrared (FTIR) H/D exchange experiments were conducted to characterize the conformational dynamics of the PEG-conjugates. It was found that the magnitude of thermodynamic stabilization correlates with a reduction in protein structural dynamics and was independent of the PEG MW. Thus, the initial hypothesis proved positive. Similar to the thermodynamic stabilization of proteins by covalent modification with glycans, poly(ethylene glycol) thermodynamically stabilizes α-CT by reducing protein structural dynamics. These results provide guidance for the future development of stable protein formulations.

Cancer is the second largest cause of death worldwide with the number of new cancer cases predicted to grow significantly in the next decades. Biotechnology and medicine can and should work hand-in-hand to improve cancer diagnosis and treatment efficacy. However, success has been frequently limited, in particular when treating late-stage solid tumors. There still is the need to develop smart and synergistic therapeutic approaches to achieve the synthesis of strong and effective drugs and delivery systems. Much interest has been paid to the development of smart drug delivery systems (drug-loaded particles) that utilize passive targeting, active targeting, and/or stimulus responsiveness strategies. This review will summarize some main ideas about the effect of each strategy and how the combination of some or all of them has shown to be effective. After a brief introduction of current cancer therapies and their limitations, we describe the biological barriers that nanoparticles need to overcome, followed by presenting different types of drug delivery systems to improve drug accumulation in tumors. Then, we describe cancer cell membrane targets that increase cellular drug uptake through active targeting mechanisms. Stimulusresponsive targeting is also discussed by looking at the intra-and extracellular conditions for specific drug release. We include a significant amount of information summarized in tables and figures on nanoparticle-based therapeutics, PEGylated drugs, different ligands for the design of active-targeted systems, and targeting of different organs. We also discuss some still prevailing fundamental limitations of these approaches, eg, by occlusion of targeting ligands.

Enzymes are attractive catalysts for the production of optically active compounds in organic solvents. However, their often low catalytic activity in such applications hampers their practical use. To overcome this, we investigated the effectiveness of the covalent modification of alpha-chymotrypsin with methoxy poly(ethylene glycol) (PEG) with a Mw of 5,000 to enhance its activity. The model transesterification reaction between sec-phenethyl alcohol and vinyl butyrate in various neat dry organic solvents and at a controlled water activity of 0.008 in two solvents was employed to measure the effect of PEGylation on activity and enantioselectivity. Synthesis conditions were varied to obtain various conjugates with average molar ratios of PEG-to-chymotrypsin ranging from ca. 1 to 7. While the enantioselectivity increased only modestly from ca. 4.4 to 6.1 when averaging results in all solvents, PEG was very efficient in increasing the activity of alpha-chymotrypsin up to more than 400-fold compared to that of the powder lyophilized from buffer alone. The activity increase was more pronounced in apolar than in polar organic solvents and also depended on the amount of PEG bound to the enzyme. For example, the activity of the modified enzyme towards the most reactive "S" enantiomer in octane increased 440-fold but increasing the molar ratio of PEG-to-enzyme from 1.1 to 7.1 resulted in a more than twofold decrease in enzyme activity. Controlling the water activity did not prevent the drop in activity. To investigate the possible origin of the activity changes, Fourier transform infrared (FTIR) spectroscopy experiments were conducted. It was found that PEGylation reduced lyophilization-induced structural perturbations, but exposure to the organic solvents caused structural perturbations. These perturbations were more pronounced in polar than in apolar solvents. The pronounced activity drop in polar solvents at increasing PEG-modification levels correlated with an increasing level of solvent-induced structural perturbations. This correlation was less pronounced in apolar solvents where both, activity drop and structural perturbations, were less pronounced at increasing PEGylation levels. In summary, PEG-modified alpha-chymotrypsin might be an interesting system to catalyze reactions, particularly in apolar organic solvents.