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The reversible assembly of intrinsically disordered proteins (IDPs) to form membraneless organelles (MLOs) is a fundamental process involved in the spatiotemporal regulation in living cells. MLOs formed via liquid-liquid phase separation (LLPS) serve as molecule-enhancing hubs to regulate cell functions. Owing to the complexity and dynamic nature of the protein assembly via a network of weak inter- and intra-molecular interactions, it is challenging to describe and predict the LLPS behavior. We have developed a multiscale computational model for IDPs, using the fused in sarcoma (FUS) protein and its variants as illustrative examples. To simplify the description of protein, FUS is represented as a linear chain of stickers interspaced by spacers, as inspired by the associative polymer theory. Low-complexity aromatic-rich kinked segments (LARKS) available in FUS were identified using LARKSdb and represented as "stickers". The pairwise potential energies of each pair of stickers and their β-sheet-forming propensity were estimated via molecular docking and all atomistic molecular dynamics (AA-MD) simulations. Subsequently, FUS chains were randomly positioned in a cubic lattice as coarse-grained (CG) beads, with the bead assignment based on the Kuhn length estimation of stickers and spacers. Stochastic FUS movements were modeled by Monte Carlo (MC) simulations. In addition to the Metropolis algorithm, discretized pair potential distributions between stickers were considered in the move acceptance criteria. The chosen pair potential represents one of the possible binding energy states, with its probability determined by the frequency of the binding energy distribution histogram. The fluctuations of averaged radial distribution functions (RDFs) in successive MC trial move intervals of equilibrated lattice MC simulations were used to indicate the dynamic nature of assembly/disassembly of the protein chains. This multiscale computational framework provides an economical and efficient way of predicting and describing the LLPS behavior of IDPs.

A three-dimensional lattice-based model has been developed to describe the release of a macromolecular drug encapsulated in a degradable hydrogel. The degradation-induced network heterogeneity is considered by assigning varying diffusion coefficients to the lattice sites based on the fitted exponential node-diffusivity relationship. As time passes, due to the degradation of crosslink nodes, diffusivity values in lattice sites progress to higher values. To overcome the size limitation of the computational model and to compare it with experimental data, a scaling ratio based on the random walk equation is developed. The model was able to describe the experimental release data from chemically crosslinked dextran hydrogels. The results showed that the effect of the initial network and the chemistry of crosslink nodes (hydrolysis rate) on the drug release profile cannot be decoupled.

Using hydrogels to control the long-term release of protein remains challenging, especially for in-situ forming formulations. The uncontrollable burst release in the initial phase, the halted release in the subsequent phase, and the undesired drug dumping at the late stage are some obstacles hydrogel-based depots commonly encounter. In this study, we report hydrolyzable dextran-based hydrogels crosslinked by Michael addition to demonstrate a systematic solution to solve these problems. First, the polymer concentration was used as the critical parameter to control the proportion of releasable versus physically trapped protein molecules in the initial hydrogel meshwork. Subsequently, the dynamic change of the hydrogel meshwork was modulated by the crosslinking density and the cleavage rate of ester linkers. To this end, we designed and synthesized a series of ester linkers with hydrolytic half-life ranging from 4 h to 4 months and incorporate them into the hydrogel. Controlled release was demonstrated for model proteins varied in size, including lysozyme (14 kDa), bovine serum albumin (66 kDa), immunoglobulin G (150 kDa), and bevacizumab (149 kDa). In particular, sustained release of IgG ranging from 10 days to 8 months was achieved. Lastly, a tunable multi-phase release profile was made feasible by incorporating multiple ester linkers into one hydrogel formulation. The linker's half-life determined each phase's release duration, and the linkers' mixing ratio determined the corresponding release fraction. The reported hydrogel design engenders a versatile platform to address the needs for long-term and readily adjustable protein release for biomedical applications., (Copyright © 2021. Published by Elsevier B.V.)

A three-dimensional model has been developed to describe the multiphase release of macromolecular drugs encapsulated in a hydrogel. The heterogeneity of network mesh size was considered by assigning varying diffusion coefficients to the network lattices randomly. Using a stochastic approach, the random nature of diffusion of drug molecules was captured within the network. The simplest form of distribution containing two diffusion coefficients was tested. To generate the drug release profiles for experimental validation under the limitation of computational cost, a simple scaling relationship was employed. Unlike the single-diffusivity model, the dual-diffusivity model showed good agreement with the experimental data in describing the release profiles of macromolecular drugs of three different sizes from chemically cross-linked dextran hydrogels.

We have demonstrated the use of a simple 1 H NMR spectrometry-based method to directly measure the pseudo first-order hydrolytic cleavage rate constant (k obs ) of methacrylate-derived ester crosslinkers in hydrogels composed of PEG, dextran, carboxymethyl dextran (CM-dextran) and hyaluronic acid (HA). Using this technique, we systematically examined how the local environment in the hydrogel influenced the rate of ester hydrolysis. Within the formulations being studied, the esters in the crosslinked polymer network (gel state) degraded 1.8 times faster than esters of similar chemistry in soluble polymers (solution state). Furthermore, the value of k obs was independent of the polymer concentration or the hydrogel network structure, although these parameters affected the swelling profiles in response to the hydrolytic degradation. On the other hand, the presence of the negatively charged carboxylate groups in the polymer chains decreased k obs in gel state, while only minimally affecting k obs in solution state. Hydrogels composed of negatively charged polymers (HA and CM-dextran) had a k obs about 30% smaller than hydrogels composed of neutral polymers (dextran and PEG). The reported method provides a reliable tool to resolve conflicting views about hydrogel degradation, and to guide the rational design of degradable hydrogel. STATEMENT OF SIGNIFICANCE: Degradable hydrogels are widely used in biological applications. A common degradation mechanism of the crosslinked polymer is by hydrolytic cleavage. However, the hydrogel micro-milieu do affect the behavior of the hydrolysable bonds, for example esters. There have been several conflicting speculations on how hydrogel composition would affect the macroscopic degradation behavior. In this report, we simply, but innovatively applied ordinary 1 H NMR spectrometry-based method to probe the rate of ester cleavage in the native hydrogel milieu. We tried to answer whether these parameters will have direct influence on ester cleavage, or have indirect effect on the overall network disintegration behavior. This study provides quantitative evidences to assist theoretical modeling and to guide rational formulation design., (Copyright © 2019. Published by Elsevier Ltd.)

Antiangiogenic therapeutics, such as corticosteroids, VEGF targeting antibodies and aptamers have been demonstrated effective in controlling retinal and choroidal neovascularization related vision loss. However, to manage the chronic conditions, it requires long term and frequent intravitreal injections of these drugs, resulting in poor patient compliance and suboptimal treatment. In addition, emerging drugs such as tyrosine kinase inhibitors and siRNAs received much expectations, but the late stage clinical trials encountered various obstacles. Controlled release technology could improve the existing treatment regimen by extending therapeutic duration, reducing risks and burdens caused by frequent injections, and enabling new drugs to overcome the hurdles of translation. Here, we give qualitative and quantitative discussions about the principle mechanisms of polymeric reservoir, polymeric matrix and hydrogel systems. We also reveal the design rationales of the existing drug delivery and release systems in preclinical and clinical stages. Lastly, the animal models of ocular angiogenesis diseases are critically reviewed, which could help to facilitate the translation of controlled release technologies from bench to bedside., (Copyright © 2018 Elsevier B.V. All rights reserved.)