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.

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.

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.

Nanoassembly of Oligopeptides and DNA Mimics the Sequential Disassembly of a Spherical Virus.

Protein subunits of a low aspect ratio (width over length) with stimuli-responsiveness are hallmark building blocks of spherical viruses. The interaction of these repeating subunits enables hierarchical assembly for genome packaging and sequential disassembly for optimal genome release. Here, we mimicked these features and constructed a functional spherical artificial virus. The rationally designed 22-amino acid peptide containing pH-sensitive histidines and aromatic residues self-assembled into homogenous nanodiscs of a low aspect ratio (≈7×7×4 nm). In the presence of DNA, the inter-nanodisc interactions drove the formation of a viral capsid-like structure enclosing DNA in the interior. This artificial virus roughly 50 nm in diameter underwent partial disassembly in response to acidic pH. The resulting intermediate with lowered DNA-binding affinity continued to protect DNA from nuclease digestion. Such nanostructures, which mimic the intricate morphology and the intracellular transformation of spherical viruses, can be useful for constructing synthetic gene delivery vehicles, as evidenced by their efficient transgene expression.

Investigating impacts of surface charge on intraocular distribution of intravitreal lipid nanoparticles.

In this paper, the effect of surface charge of intravitreal lipid nanoparticles (LNPs) on their intraocular accumulation and distribution was quantitatively and systematically studied. The retinal distribution of LNPs delivering model drug (siRNA) with optimal surface charge was visualized as well. LNPs and siRNA-loaded LNPs (siLNPs) were prepared with ethanol-injection method. C57BL/6 Mice with the age of 6-8 weeks were used for the animal experiments. Quantitative accumulation in the eye and respective intraocular distribution of the intravitreally injected LNPs with different surface charge were examined after predesignated time points. The fluorescence intensity of the fluorescent labeled siRNA was also quantified based on the confocal microscope images. The release of siRNA from siLNPs was studied in mimicked vitreous environment at 37 °C with the presence of 50% serum protein. We successfully prepared six types of LNPs had relatively uniform size around 70 nm and varied in zeta potential ranging from -30 mV to +50 mV. Negative, neutral and slightly positive charged LNPs were cleared much faster than strongly positive charged LNPs from the mice's eyes. After 6 h, LNPs with zeta potential of +35 mV had highest ratio of retinal to vitreous accumulation and penetrated through the entire layer of the retina. The siLNPs could successfully deliver the siRNA to the ganglion cells and inner plexiform layer of the retina. After 24 h, majority of LNPs and siLNPs were cleared, suggesting that the LNPs with pegylated surface in the absence of any targeting ligand were not internalized by retinal cells. The mechanism of how the surface charge of nanoparticles affects their intraocular retention and distribution was intensively discussed. The results suggested that intravitreally injected LNPs with optimal surface charge around +35 mV can distribute and penetrate the retina, which could be further modified as promising nanocarriers for retinal delivery.

Tuning the Inter-nanofibril Interaction To Regulate the Morphology and Function of Peptide/DNA Co-assembled Viral Mimics.

The ability to tune the inter-subunit interaction within the virus capsid may be critical to assembly and biological function. This process was extended here with peptide/DNA co-assembled viral mimics. The resulting co-assemblies, formed and stabilized by both peptide nanofibril-DNA and peptide nanofibril-nanofibril interactions, were tuned through hydrophobic packing interactions of the peptide sequences. By strengthening peptide side-chain complementarity and/or elongating the peptide chain (from 4 to 8 residues), we report strengthening the inter-nanofibril interaction to create stable nanococoons that give high gene-transfection efficacy.

Formulation of in situ chemically cross-linked hydrogel depots for protein release: from the blob model perspective.

The fast release rate and the undesirable covalent binding are two major problems often encountered in formulating in situ chemically cross-linked hydrogel as protein release depot, particularly when prolonged release over months is desirable. In this study, we applied the De Gennes' blob theory to analyze and tackle these two problems using a vinylsulfone-thiol (VS-SH) reaction based in situ hydrogel system. We showed that the simple scaling relation ξb ≈ Rg(c/c*)(-v/(3v-1)) is applicable to the in situ hydrogel and the mesh size estimated from the precursor polymer parameters is a reasonable match to experimental results. On the other hand, as predicted by the theory and confirmed by experiments, the drug diffusion within hydrogel depends mainly on polymer concentration but not the degree of modification (DM). The covalent binding was found to be caused by the mismatch of location between the reactive groups and the entanglement points. The mismatch and, thus, the protein binding were minimized by increasing the DM and concentration of the SH polymer relative to the VS polymer, as predicted by theory. Using these principles, an in situ hydrogel system for the controlled release of an antiangiogenic antibody therapeutics bevacizumab for 3 months was developed.

In vitro evaluation of the inhibitory effect of canine serum, canine fresh frozen plasma, freeze-thaw-cycled plasma, and Solcoseryl™ on matrix metalloproteinases 2 and 9.

OBJECTIVE: Compare the efficacy of canine serum, fresh frozen plasma (FFP), freeze-thaw-cycled plasma (FTCP), and Solcoseryl(™) at inhibiting matrix metalloproteinases (MMP) 2 and 9 in vitro. PROCEDURE: Matrix metalloproteinases 2 and 9 activity in the presence of serum, FFP, FTCP, or Solcoseryl(™) was assayed using a commercially available fluorogenic gelatinase activity kit. RESULTS: Matrix metalloproteinases 2 activity in the presence of serum, FFP, FTCP, and Solcoseryl(™) was 20.84%, 5.76%, 8.10%, and 83.03%, respectively of uninhibited MMP 2 activity. MMP 9 activity in the presence of serum, FFP, FTCP, and Solcoseryl(™) was 57.36%, 58.35%, 49.35%, and -8.69%, respectively of uninhibited MMP 9 activity. CONCLUSION: Serum, FFP, and FTCP exhibit similar levels of MMP 2 and 9 inhibitions. Solcoseryl(™) causes minimal MMP 2 inhibition, but profound MMP 9 inhibition.

Structural mimics of viruses through peptide/DNA co-assembly.

A synthetic mimic of viral structure has been constructed by the synergistic co-assembly of a 16-amino acid peptide and plasmid DNA. The rational design of this short peptide, including segments for binding DNA and forming ß-sheet, is inspired by viral capsid protein. The resulting nanostructures, which we term nanococoons, appear as ellipsoids of virus-like dimension (65 × 47 nm) and display repeating stripes of ∼4 nm wide. We propose that the co-assembly process involves DNA as a template to assist the organization of peptide strands by electrostatic interaction, while the bilayer ß-sheets and their lateral association stabilize the peptide "capsid" and organize the DNA within. The hierarchy affords an extremely stable structure, protecting peptide and DNA against enzymatic digestion. It opens a new and facile avenue to fabricate viral alternatives with diverse functions.

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.

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.

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.

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.

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.

Factorial analysis of adaptable properties of self-assembling peptide matrix on cellular proliferation and neuronal differentiation of pluripotent embryonic carcinoma.

An integrative and quantitative approach for systematically studying the effects of changing the matrix environment on pluripotent cell viability and neuronal differentiation was demonstrated. This approach, based on factorial analysis and a self-assembling peptide (SAP) matrix, was exemplified using P19 as a pluripotent cell model. In a two-level, three-factor factorial design of experiments, three niche factors, namely, culture dimensionality, fixed biochemical signal and mechanical stiffness, were simultaneously investigated. We found that cell growth was slowed in matrices containing IKVAV epitopes on the SAP constructs, and neuronal differentiation was promoted synergistically by culturing in a three-dimensional matrix and in the presence of IKVAV. Variation of the storage modulus from around 262 Pa to 672 Pa had no significant effect on either viability or differentiation. This approach should be applicable to studying how niche properties that are tunable using SAPs affect the behavior of pluripotent cells in general, thus generating guidelines for constructing artificial matrices. FROM THE CLINICAL EDITOR: In this basic science study, an integrative and quantitative approach to study the effects of matrix environment on pluripotent cell viability and neuronal differentiation is demonstrated. Approaches, like the one described in this paper, are applicable to studying how self assembling peptides affect the behavior of pluripotent cells in general.

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.

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.

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.

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.

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.