Multiscale Computational Framework for the Liquid-Liquid Phase Separation of Intrinsically Disordered Proteins.

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 ß-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.

Self-adjuvanted L-arginine-modified dextran-based nanogels for sustained local antigenic protein delivery to antigen-presenting cells and enhanced cellular and humoral immune responses.

In the development of cancer vaccines, antigens are delivered to elicit potent and specific T-cell responses to eradicate tumour cells. Nonetheless, successful vaccines are often hampered by the poor immunogenicity of tumour antigens, rapid clearance by the innate immunity, and limited cross-presentation on MHC-I to activate CD8+ T-cells arm. To address these issues, we developed dextran-based nanogels to promote antigen uptake, storage, and cross-presentation on MHC-I, while directing immunogenic maturation of the antigen-presenting cells (APCs). To promote the nanocarriers interaction with cells, we modified DX with L-arginine (Arg), whose immunomodulatory activities have been well documented. The ArgDX nanogel performance was compared with the nanogel modified with L-histidine (His) and L-glutamate (Glut). Moreover, we introduced pH-sensitive hydrazone crosslinking during the nanogel formation for the conjugation and controlled release of antigen ovalbumin (OVA). The OVA-laden nanogels have an average size of 325 nm. We demonstrated that the nanogels could rapidly release cargoes upon a pH change from 7 to 5 within 8 days, indicating the controlled release of antigens in the acidic cellular compartments upon internalization. Our results revealed that the ArgDX nanogel could promote greater antigen uptake and storage in DCs in vitro and promoted a stronger immunogenic maturation of DCs and M1 polarization of the macrophages. The OVA signals were co-localized with lysosomal compartments up till 96 hours post-treatment and washing, suggesting the nanogels could facilitate prolonged antigen storage and supply from endo-lysosomal compartments. Furthermore, all the tested nanogel formulations retained antigens at the skin injection sites until day 21. Such delayed clearance could be due to the formation of micron-sized aggregates of OVA-laden nanogels, extending the interactions with the resident DCs. Amongst the amino acid modifications, ArgDX nanogels promoted the highest level of lymph node homing signal CCR7 on DCs. The nanogels also showed higher antigen presentation on both MHC-I and II than DX in vitro. In the in vivo immune studies, ArgDX nanogels were more superior in inducing cellular and humoral immunity than the other treatment groups on day 21 post-treatment. These results suggested that ArgDX nanogel is a promising self-adjuvanted nanocarrier for vaccine delivery.

Vaccine delivery by zwitterionic polysaccharide-based hydrogel microparticles showing enhanced immunogenicity and suppressed foreign body responses.

The controlled release of antigens from injectable depots has been actively pursued to achieve long-lasting immune responses in vaccine development. Nonetheless, subcutaneous depots are often susceptible to foreign body responses (FBRs) dominated by macrophage clearance and fibrotic encapsulation, resulting in limited antigen delivery to target dendritic cells (DCs) that bridge innate and adaptive immunity. Here, we aim to develop a long-term antigen depot that can bypass FBR and engage DCs to mature and migrate to lymph nodes to activate antigen-specific T-cells. Leveraging the immunomodulatory properties of exogenous polysaccharides and the anti-fouling characteristics of zwitterionic phosphorylcholine (PC) polymers, we developed a PC functionalized dextran (PCDX) hydrogel for long-term antigen delivery. We observed that PCDX in both injectable scaffold and microparticle (MP) forms could effectively evade FBR as the anionic carboxymethyl DX (CMDX) in vitro and in vivo. Meanwhile, PCDX provided slower and longer release of antigens than CMDX, resulting in local enrichment of CD11c+ DCs at the MP injection sites. DC cultured on PCDX exhibited stronger immunogenic activation with higher CD86, CD40, and MHC-I/peptide complex than CMDX. PCDX also generated DC with greater propensity in migration to lymph nodes, as well as antigen presentations to trigger both CD4+ and CD8+ arms of T-cell responses, as compared to other charge derivatives of DX. Besides cellular responses, PCDX could also induce more durable and potent humoral responses, with higher levels of antigen specific IgG1 and IgG2a by day 28, as compared to other treatment groups. In conclusion, PCDX can incorporate the benefits of both immunogenic DX and anti-fouling properties of zwitterionic PC and thus, shows great promise in providing long-term delivery of antigens for vaccine development.

Low-intensity low-frequency ultrasound mediates riboflavin delivery during corneal crosslinking.

We employed the mechanical effect from 40 kHz ultrasound (US) to improve the delivery of riboflavin into corneal stroma for collagen crosslinking, which can benefit the treatment of keratoconus and other corneal ectasias. Experiments were conducted, first with porcine corneas ex vivo and then with New Zealand white rabbits in vivo, at varying mechanical index (MI) and sonication time. Results showed that 15 min of US applied on the cornea at MI = 0.8 in the presence of 0.5% of riboflavin solution enabled its delivery to deeper corneal stroma. Excessive heat was removed by a cooling setup to negate the thermal effect. The corneal absorption amount and penetration of riboflavin through cornea as detected by fluorotron, as well as the enhancement of corneal stiffness as measured by Young's modulus, were comparable to the conventional approach that requires complete corneal epithelium debridement. Histological analysis revealed minor exfoliation of superficial cell layers of corneal epithelium and loss of ZO-1 tight junctions immediately after US. Full recovery of the corneal epithelium and restoration of tight junctions occurred in 3-4 days. The study shows that low-intensity low-frequency ultrasound (LILF US) is a less invasive alternative to the conventional epithelium-off method for delivering riboflavin into the corneal stroma.

Construction of multiphasic membraneless organelles towards spontaneous spatial segregation and directional flow of biochemical reactions.

Many intracellular membraneless organelles (MLOs) appear to adapt a hierarchical multicompartment organization for efficient coordination of highly complex reaction networks. Recapitulating such an internal architecture in biomimetic platforms is, therefore, an important step to facilitate the functional understanding of MLOs and to enable the design of advanced microreactors. Herein, we present a modular bottom-up approach for building synthetic multiphasic condensates using a set of engineered multivalent polymer-oligopeptide hybrids. These hybrid constructs exhibit dynamic phase separation behaviour generating membraneless droplets with a subdivided interior featuring distinct chemical and physical properties, whereby a range of functional biomolecules can be spontaneously enriched and spatially segregated. The platform also attains separated confinement of transcription and translation reactions in proximal compartments, while allowing inter-compartment communication via a directional flow of reactants. With advanced structural and functional features attained, this system can be of great value as a MLO model and as a cell-free system for multiplex chemical biosynthesis.

Correction: Fusogenic peptide modification to enhance gene delivery by peptide-DNA nano-coassemblies.

Correction for 'Fusogenic peptide modification to enhance gene delivery by peptide-DNA nano-coassemblies' by Ruilu Feng et al., Biomater. Sci., 2022, 10, 5116-5120, https://doi.org/10.1039/D2BM00705C.

Recent advances in surface modification of micro- and nano-scale biomaterials with biological membranes and biomolecules.

Surface modification of biomaterial can improve its biocompatibility and add new biofunctions, such as targeting specific tissues, communication with cells, and modulation of intracellular trafficking. Here, we summarize the use of various natural materials, namely, cell membrane, exosomes, proteins, peptides, lipids, fatty acids, and polysaccharides as coating materials on micron- and nano-sized particles and droplets with the functions imparted by coating with different materials. We discuss the applicability, operational parameters, and limitation of different coating techniques, from the more conventional approaches such as extrusion and sonication to the latest innovation seen on the microfluidics platform. Methods commonly used in the field to examine the coating, including its composition, physical dimension, stability, fluidity, permeability, and biological functions, are reviewed.

Stochastic Lattice-Based Modeling of Macromolecule Release from Degradable Hydrogel.

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.

Fusogenic peptide modification to enhance gene delivery by peptide-DNA nano-coassemblies.

Endosomal escape is a major obstacle for non-viral nucleic acids delivery. Here, we attached by click reaction a fusogenic peptide (L17E) onto peptide self-assembled disks (∼17 nm), which mimicked the functional subunits of the virus capsid. These peptide disks then spontaneously co-assembled with DNA to form patterned nanostructures (∼100 nm) as viral mimics. This modification did not affect the cellular uptake but enhanced endosomal escape and led to improved transfection in cell culture.

Molecular logic operations from complex coacervation with aggregation-induced emission characteristics.

Leveraging complex coacervation of a polycation and a bivalent anion with aggregation-induced emission characteristics, we accomplish eight basic logic operations with environmental stimuli as inputs, producing Boolean-like fluorescence intensity or turbidity 'outputs' with contrast higher than one order of magnitude. Storage of information of a fluorescent pattern and thermo-sensor applications are also demonstrated.

mRNA Delivery and Storage by Co-Assembling Nanostructures with Designer Oligopeptides.

Broadening the applicable tools for mRNA delivery provides more flexibility in research and those proven effective and safe can potentially be translated for clinical use. We report here a 27-amino acid peptide sequence mimicking the viral capsid protein, termed pepMAX, capable of co-assembling with mRNA into 100-150 nm nanostructures for efficient transfection of multiple cell lines. The mRNA loading and N/P ratio have been systematically optimized for each cell line. In HeLa, HEK293, and SKNMC, the transfection attained (>80%) is comparable with that of commercially available vectors Lipofectamine MessengerMAXTM (LipoMMAX). Confocal microscopy reveals that pepMAX efficiently delivers mRNA into the cytosol and induces efficient protein production. The pepMAX/mRNA co-assemblies retain their transfection efficiency after storage up to one week at room temperature in lyophilized form.

Nucleic Acids Modulate Liquidity and Dynamics of Artificial Membraneless Organelles.

Liquid-liquid phase separation (LLPS) emerges as a fundamental underlying mechanism for the biological organization, especially the formation of membraneless organelles (MLOs) hosting intrinsically disordered proteins (IDPs) as scaffolds. Nucleic acids are compositional biomacromolecules of MLOs with wide implications in normal cell functions as well as in pathophysiology caused by aberrant phase behavior. Exploiting a minimalist artificial membraneless organelles (AMLO) from LLPS of IDP-mimicking polymer-oligopeptide hybrid (IPH), we investigated the effect of nucleic acids with different lengths and sequence variations on AMLO. The behavior of this AMLO in the presence of DNAs and RNAs resembled natural MLOs in multiple aspects, namely, modulated propensity of formation, morphology, liquidity, and dynamics. Both DNA and RNA could enhance the LLPS of AMLO, while compared with RNA, DNA had a higher tendency to solidify and diminish dynamics thereof. These findings suggest its potential as a concise model system for the understanding of the interaction between nucleic acids and natural MLOs and for studying the molecular mechanism of diseases involving MLOs.

Multifaceted Cargo Recruitment and Release from Artificial Membraneless Organelles.

Liquid-liquid phase separation (LLPS) drives membraneless organelles (MLOs) formation for organizing biomolecules. Artificial MLOs (AMLOs) have been constructed mostly via the LLPS of engineered proteins capable of regulating limited types of biomolecules. Here, leveraging a minimalist AMLO, driven by LLPS of polymer-oligopeptide hybrids, enrichment, recruitment, and release of multifaceted cargoes are quantitatively shown, including small fluorescent molecules, fluorophore-containing macromolecules, proteins, DNAs, and RNAs. Cargoes show up to 105 -fold enrichment, whilst recruitment and release are triggered by variations of temperature, pH, and/or ionic strength. Also, the first efficacious, rapid, and reversible control of aggregation-induced emission with over 30 folds of modulation of overall fluorescence intensity is achieved, by intensifying the aggregation of luminogens in AMLO. The AMLO is a simple yet versatile platform for potential drug delivery and biosensor applications.

Minimalist Design of an Intrinsically Disordered Protein-Mimicking Scaffold for an Artificial Membraneless Organelle.

Liquid-liquid phase separation (LLPS) is an emerging and universal mechanism for intracellular organization, particularly, by forming membraneless organelles (MLOs) hosting intrinsically disordered proteins (IDPs) as scaffolds. Genetic engineering is generally applied to reconstruct IDPs harboring over 100 amino acid residues. Here, we report the first design of synthetic hybrids consisting of short oligopeptides of fewer than 10 residues as "stickers" and dextran as a "spacer" to recapitulate the characteristics of IDPs, as exemplified by the multivalent FUS protein. Hybrids undergo LLPS into micron-sized liquid droplets resembling LLPS in vitro and in living cells. Moreover, the droplets formed are capable of recruiting proteins and RNAs and providing a favorable environment for a biochemical reaction with highly enriched components, thereby mimicking the function of natural MLOs. This simple yet versatile model system can help elucidate the molecular interactions implicated in MLOs and pave ways to a new type of biomimetic materials.

Altered Peptide Self-Assembly and Co-Assembly with DNA by Modification of Aromatic Residues.

Aromatic residues are widely used as building blocks for driving self-assemblies in natural and designer biomaterials. The noncovalent interactions involving aromatic rings determine proteins' structure and biofunction. Here, we studied the effects of changes in the proximity of the aromatic rings in a self-assembling peptide for modulating interactions involving the aromatic residues. By changing the distance between the aromatic ring and peptide backbone and replacing the side chain with a sulfur atom, we altered the nanostructures and gene transfection efficiency of peptide-DNA co-assemblies. This study demonstrates the significance of subtle alterations in aromatic interactions and facilitates deeper understanding of the aromatic-involving interactions.

A bioinspired synthetic soft hydrogel for the treatment of dry eye.

Natural soft hydrogels are unique elastic soft materials utilized by living organisms for protecting delicate tissues. Under a theoretical framework derived from the Blob model, we chemically crosslinked high molecular weight hyaluronic acid at a concentration close to its overlap concentration (c*), and created synthetic soft hydrogels that exhibited unique rheological properties similar to a natural soft hydrogel: being dominantly elastic under low shear stress while being viscous when the stress is above a small threshold. We explored a potential application of the hyaluronic acid-based soft hydrogel as a long-acting ocular surface lubricant and evaluated its therapeutic effects for dry eye. The soft hydrogel was found to be biocompatible after topical instillation on experimental animals' and companion dogs' eyes. In a canine clinical study, twice-a-day ocular instillation of the soft hydrogel in combination with cyclosporine for 1 month improved the clinical signs in more than 65% of dog patients previously unresponsive to cyclosporine treatment.

The effect of polysaccharide-based hydrogels on the response of antigen-presenting cell lines to immunomodulators.

Hydrogel presents as foreign material to the host and participates in immune responses, which skew the biofunctions of immunologic loads (antigen and adjuvants) during in situ DC priming. This study aims to investigate the effect of the hydrogel made from different polysaccharides on macrophage (RAW264.7) activation and DC (JAWSII) modulation. We adopted polysaccharides of different sugar chemistry to fabricate hydrogels. Hyaluronate (HA), glycol chitosan (GC) and dextran (DX) were functionalized with vinyl sulfone and chemically cross-linked with dithiothreitol via thiol-click chemistry. We found that HA reduced macrophage adhesion and activation on the hydrogel surface. GC and DX promoted M1 polarization in terms of higher CCR7 expression and TNF-α, IL-6 production. In terms of DC engagement, GC promoted antigen uptake by JAWSII and all hydrogels promoted antigen presentation on MHC-I molecules. GC and DX favoured the generation of immunogenic DC while accommodating immunostimulatory functions of IFN-γ and polyI:C or LPS during co-incubation. Particularly, the co-incubation of IP with GC promoted CCR7 expression on JAWSII. Conversely, HA was more appropriate for the construction of a tolerogenic DC priming platform. We observed that HA did not induce co-stimulatory markers expression on DC but suppressed the action of LPS in inducing TNF-α generation. Moreover, when immunosuppressive cytokines, IL-10 and TGF-ß were added, cytokines' immunosuppressive action was amplified by hydrogel bedding, HA, GC and to a less extent DX in suppressing LPS-induced IL-6 generation from JAWSII. We concluded that HA is preferable for tolerogenic DC development while minimizing the macrophage response in conferring foreign body response, whereas DX and GC are more appropriate for immunogenic DC development. This study demonstrates the potential of polysaccharides in conferring in situ DC priming together with antigen and adjuvant loads while addressing the tradeoff between the foreign body responses and DC engagement by selecting appropriate polysaccharides for the hydrogel platform construction.

Controllable multi-phase protein release from in-situ hydrolyzable hydrogel.

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.

Droplet-Based Microfluidic Synthesis of Hydrogel Microparticles via Click Chemistry-Based Cross-Linking for the Controlled Release of Proteins.

Hydrogel microparticles (HMPs) have been widely applied in biological, pharmacologic, and biomedical industries due to their versatility. Particle size is a paramount factor for controlling drug release profiles from HMPs. Conventional fabrication methods such as bulk emulsion, coacervation, and spray drying do not offer a precise size control and high reproducibility, which may compromise the utility of HMPs for controlled release. Here, we report a droplet-based microfluidic synthesis method for the precise fabrication of HMPs. Functionalized polysaccharides/protein fluid mixtures were emulsified into monodisperse droplets in light mineral oil using a flow-focusing device and well mixed in precursor droplets through a serpentine mixing channel before the solidification of HMPs. The homogenized precursor polymers cross-link in the droplets by catalyst-free Michael addition. As a demonstration of the controlled release of a model drug from the HMPs, fluorescein-labeled immunoglobulin G (F-IgG) and bevacizumab were encapsulated in the HMPs of different diameters for measuring its release dynamics over time. The release kinetics of F-IgG from the HMPs was shown to be controllable by altering the particle size while keeping other parameters unchanged. Around 70% of bevacizumab released from DX HMPs was functional. Both HA and DX HMPs showed no cytotoxicity in the HEK293 cell line. We anticipate that this approach could be used as a general method to fabricate HMPs made of hydrophilic polymers for the controlled release of biotherapeutics.

Enhanced Delivery of siRNA to Retinal Ganglion Cells by Intravitreal Lipid Nanoparticles of Positive Charge.

RNAi therapy has been developed and explored for treating retinal conditions since last decades. The progression of retinal diseases including the age-related macular degeneration and glaucoma is associated with the malfunction of specific retinal cells. Therefore, to deliver therapeutic RNAi to selective retinal tissues with desired gene downregulation is crucial for the treatment of retinal diseases via RNAi therapy. Lipid-based nanoparticles are potent delivery vectors for RNAi therapeutics to achieve high gene silencing efficiency. The surface charge has been demonstrated to affect the intraocular behaviors and retinal distribution of intravitreally administered lipid nanoparticles (LNPs), which could subsequently affect the gene knockdown efficiency in specific retinal layers. Here, we evaluated three charged LNPs for their ability to deliver siRNA and facilitate gene downregulation both in vitro and in vivo. LNPs with different surface charges ranging from neutral to positive (5-34 mV) were successfully formulated. All types of charged LNPs managed gene knockdown in both mammalian cell line and primary neurons. At 48 h post intravitreal injection, neutral LNPs (6.2 mV) and mildly positive LNPs (15.9 mV) mediated limited retinal gene suppression (<10%) and the more positive LNPs (31.2 mV) led to ∼25% gene suppression in the retinal ganglion cell (RGC) layer. No gene silencing in the retinal pigmented epithelium layer was facilitated by any LNPs independent of the charges. In summary, this study has shown that positive LNPs with an optimized charge managed specific gene downregulation in the RGC layer. These RNAi carriers hold potential for the treatment of RGC-associated retinal diseases.