Phase transition of spindle-associated protein regulate spindle apparatus assembly.

Spindle assembly required during mitosis depends on microtubule polymerization. We demonstrate that the evolutionarily conserved low-complexity protein, BuGZ, undergoes phase transition or coacervation to promote assembly of both spindles and their associated components. BuGZ forms temperature-dependent liquid droplets alone or on microtubules in physiological buffers. Coacervation in vitro or in spindle and spindle matrix depends on hydrophobic residues in BuGZ. BuGZ coacervation and its binding to microtubules and tubulin are required to promote assembly of spindle and spindle matrix in Xenopus egg extract and in mammalian cells. Since several previously identified spindle-associated components also contain low-complexity regions, we propose that coacervating proteins may be a hallmark of proteins that comprise a spindle matrix that functions to promote assembly of spindles by concentrating its building blocks.

Self-assembling paclitaxel-mediated stimulation of tumor-associated macrophages for postoperative treatment of glioblastoma.

The unique cancer-associated immunosuppression in brain, combined with a paucity of infiltrating T cells, contributes to the low response rate and poor treatment outcomes of T cell-based immunotherapy for patients diagnosed with glioblastoma multiforme (GBM). Here, we report on a self-assembling paclitaxel (PTX) filament (PF) hydrogel that stimulates macrophage-mediated immune response for local treatment of recurrent glioblastoma. Our results suggest that aqueous PF solutions containing aCD47 can be directly deposited into the tumor resection cavity, enabling seamless hydrogel filling of the cavity and long-term release of both therapeutics. The PTX PFs elicit an immune-stimulating tumor microenvironment (TME) and thus sensitizes tumor to the aCD47-mediated blockade of the antiphagocytic "don't eat me" signal, which subsequently promotes tumor cell phagocytosis by macrophages and also triggers an antitumor T cell response. As adjuvant therapy after surgery, this aCD47/PF supramolecular hydrogel effectively suppresses primary brain tumor recurrence and prolongs overall survivals with minimal off-target side effects.

Therapeutic Supramolecular Polymers: Designs and Applications.

The self-assembly of low-molecular-weight building motifs into supramolecular polymers has unlocked a new realm of materials with distinct properties and tremendous potential for advancing medical practices. Leveraging the reversible and dynamic nature of non-covalent interactions, these supramolecular polymers exhibit inherent responsiveness to their microenvironment, physiological cues, and biomolecular signals, making them uniquely suited for diverse biomedical applications. In this review, we intend to explore the principles of design, synthesis methodologies, and strategic developments that underlie the creation of supramolecular polymers as carriers for therapeutics, contributing to the treatment and prevention of a spectrum of human diseases. We delve into the principles underlying monomer design, emphasizing the pivotal role of non-covalent interactions, directionality, and reversibility. Moreover, we explore the intricate balance between thermodynamics and kinetics in supramolecular polymerization, illuminating strategies for achieving controlled sizes and distributions. Categorically, we examine their exciting biomedical applications: individual polymers as discrete carriers for therapeutics, delving into their interactions with cells, and in vivo dynamics; and supramolecular polymeric hydrogels as injectable depots, with a focus on their roles in cancer immunotherapy, sustained drug release, and regenerative medicine. As the field continues to burgeon, harnessing the unique attributes of therapeutic supramolecular polymers holds the promise of transformative impacts across the biomedical landscape.

Constructing Antiretroviral Supramolecular Polymers as Long-Acting Injectables through Rational Design of Drug Amphiphiles with Alternating Antiretroviral-Based and Hydrophobic Residues.

One of the main challenges in the development of long-acting injectables for HIV treatment is the limited duration of drug release, which results in the need for frequent dosing and reduced patient adherence. In this context, we leverage the intrinsic reversible features of supramolecular polymers and their unique ability to form a three-dimensional network under physiological conditions to design a class of self-assembling drug amphiphiles (DAs) based upon lamivudine, a water-soluble antiretroviral (ARV) agent and nucleoside reverse transcriptase inhibitor. The designed ARV DAs contain three pairs of alternating hydrophobic valine (V) and hydrophilic lamivudine-modified lysine (K3TC) residues with a varying number of glutamic acids (E) placed on the C-terminus. Upon dissolution in deionized water, all three ARV DAs were found to spontaneously associate into supramolecular filaments of several micrometers in length, with varying levels of lateral stacking. Addition of 1× PBS triggered immediate gelation of the two ARV DAs with 2 or 3 E residues, and upon dilution in an in vitro setting, the dissociation from the supramolecular state to the monomeric state enabled a long-acting linear release of the ARV DAs. In vivo studies further confirmed their injectability, rapid in situ hydrogel formation, enhanced local retention, and long-acting therapeutic release over a month. Importantly, our pharmacokinetic studies suggest that the injected ARV supramolecular polymeric hydrogel was able to maintain a plasma concentration of lamivudine above its IC50 value for more than 40 days in mice and showed minimal systemic immunogenicity. We believe that these results shed important light on the rational design of long-acting injectables using the drug-based molecular assembly strategy, and the reported ARV supramolecular hydrogels hold great promise for improving HIV treatment outcomes.

Self-Sorting in Diastereomeric Mixtures of Functionalized Dipeptides.

Self-sorting in functionalized dipeptide systems can be driven by the chirality of a single amino acid, both at a high pH in the micellar state and at a low pH in the gel state. The structures formed are affected to some degree by the relative concentrations of each component showing the complexity of such an approach. The structures underpinning the gel network are predefined by the micellar structures at a high pH. Here, we describe the systems prepared from two dipeptide-based gelators that differ only by the chirality of one of the amino acids. We provide firm evidence for self-sorting in the micellar and gel phases using small-angle neutron scattering and cryo-transmission electron microscopy (cryo-TEM), showing that complete self-sorting occurs across a range of relative concentrations.

The role of critical micellization concentration in efficacy and toxicity of supramolecular polymers.

The inception and development of supramolecular chemistry have provided a vast library of supramolecular structures and materials for improved practice of medicine. In the context of therapeutic delivery, while supramolecular nanostructures offer a wide variety of morphologies as drug carriers for optimized targeting and controlled release, concerns are often raised as to how their morphological stability and structural integrity impact their in vivo performance. After intravenous (i.v.) administration, the intrinsic reversible and dynamic feature of supramolecular assemblies may lead them to dissociate upon plasma dilution to a concentration below their critical micellization concentration (CMC). As such, CMC represents an important characteristic for supramolecular biomaterials design, but its pharmaceutical role remains elusive. Here, we report the design of a series of self-assembling prodrugs (SAPDs) that spontaneously associate in aqueous solution into supramolecular polymers (SPs) with varying CMCs. Two hydrophobic camptothecin (CPT) molecules were conjugated onto oligoethylene-glycol (OEG)-decorated segments with various OEG repeat numbers (2, 4, 6, 8). Our studies show that the lower the CMC, the lower the maximum tolerated dose (MTD) in rodents. When administrated at the same dosage of 10 mg/kg (CPT equivalent), SAPD 1, the one with the lowest CMC, shows the best efficacy in tumor suppression. These observations can be explained by the circulation and dissociation of SAPD SPs and the difference in molecular and supramolecular distribution between excretion and organ uptake. We believe these findings offer important insight into the role of supramolecular stability in determining their therapeutic index and in vivo efficacy.

Collagen-Binding Peptide-Enabled Supramolecular Hydrogel Design for Improved Organ Adhesion and Sprayable Therapeutic Delivery.

Spraying serves as an attractive, minimally invasive means of administering hydrogels for localized delivery, particularly due to high-throughput deposition of therapeutic depots over an entire target site of uneven surfaces. However, it remains a great challenge to design systems capable of rapid gelation after shear-thinning during spraying and adhering to coated tissues in wet, physiological environments. We report here on the use of a collagen-binding peptide to enable a supramolecular design of a biocompatible, bioadhesive, and sprayable hydrogel for sustained release of therapeutics. After spraying, the designed peptide amphiphile-based supramolecular filaments exhibit fast, physical cross-linking under physiological conditions. Our ex vivo studies suggest that the hydrogelator strongly adheres to the wet surfaces of multiple organs, and the extent of binding to collagen influences release kinetics from the gel. We envision that the sprayable organ-adhesive hydrogel can serve to enhance the efficacy of incorporated therapeutics for many biomedical applications.

Utilizing the Hofmeister Effect to Induce Hydrogelation of Nonionic Supramolecular Polymers into a Therapeutic Depot.

Nonionic hydrogels are of particular interest for long-term therapeutic implantation due to their minimal immunogenicity relative to their charged counterparts. However, in situ formation of nonionic supramolecular hydrogels under physiological conditions has been a challenging task. In this context, we report on our discovery of salt-triggered hydrogelation of nonionic supramolecular polymers (SPs) formed by self-assembling prodrug hydrogelators (SAPHs) through the Hofmeister effect. The designed SAPHs consist of two SN-38 units, which is an active metabolite of the anticancer drug irinotecan, and a short peptide grafted with two or four oligoethylene glycol (OEG) segments. Upon self-assembly in water, the resultant nonionic SPs can be triggered to gel upon addition of phosphate salts. Our 1 H NMR studies revealed that the added phosphates led to a change in the chemical shift of the methylene protons, suggestive of a disruption of the water-ether hydrogen bonds and consequent reorganization of the hydration shell surrounding the SPs. This deshielding effect, commensurate with the amount of salt added, likely promoted associative interactions among the SAPH filaments to percolate into a 3D network. The formed hydrogels exhibited a sustained release profile of SN-38 hydrogelator that acted potently against cancer cells.

Crystalline Nanoparticles of Water-Soluble Covalent Basket Cages (CBCs) for Encapsulation of Anticancer Drugs.

We herein describe the preparation, assembly, recognition characteristics, and biocompatibility of novel covalent basket cage CBC-11, composed of four molecular baskets linked to four trivalent aromatic amines through amide groups. The cage is tetrahedral in shape and similar in size to small proteins (Mw =8637 g/mol) with a spacious nonpolar interior for accommodating multiple guests. While 24 carboxylates at the outer surface of CBC-11 render it soluble in aqueous phosphate buffer (PBS) at pH=7.0, the amphiphilic nature prompts its assembly into nanoparticles (d=250 nm, DLS). Cryo-TEM examination of nanoparticles revealed their crystalline nature with wafer-like shapes and hexagonally arranged cages. Nanoparticulate CBC-11 traps anticancer drugs irinotecan and doxorubicin, with each cage binding up to four drug molecules in a non-cooperative manner. The inclusion complexation resulted in nanoparticles growing in size and precipitating. In media containing mammalian cells (HCT 116, human colon carcinoma), the IC50 value of CBC-11 was above 100 µM. While this work presents the first example of a large covalent organic cage operating in water at the physiological pH and forming crystalline nanoparticles, it also demonstrates its biocompatibility and potential to act as a polyvalent binder of drugs for their sequestration or delivery.

Targeting ACE2 for COVID-19 Therapy: Opportunities and Challenges.

The coronavirus disease (COVID-19) pandemic is sweeping the globe. Even with a number of effective vaccines being approved and available to the public, new cases and escalating mortality are climbing every day. ACE2 (angiotensin-converting enzyme 2) is the primary receptor for the COVID-19 causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and its complexation with spike proteins plays a crucial role in viral entry into host cells and the subsequent infection. Blocking this binding event or reducing the accessibility of the virus to the ACE2 receptor, represents an alternative strategy to prevent COVID-19. In addition, the biological significance of ACE2 in modulating the innate immune system and tissue repair cascades and anchors its therapeutic potential for treating the infected patients. In this viewpoint article, we review the current efforts of exploiting ACE2 as a therapeutic target to address this dire medical need. We also provide a holistic view of the pros and cons of each treatment strategy. We highlight the fundamental and translational challenges in moving these research endeavors to clinical applications.

Propagation-Instigated Self-Limiting Polymerization of Multiarmed Amphiphiles into Finite Supramolecular Polymers.

A fundamental goal in the noncovalent synthesis of ordered supramolecular polymers (SPs) is to achieve precise control over their size and size distribution; however, the reversible nature of noncovalent interactions often results in formation of living SPs with high dispersity in length. We report here on the self-limiting supramolecular polymerization (SPZ) of a series of multiarmed amphiphiles with propagation-attenuated reactivities that can automatically terminate the polymerization process, enabling effective control in both lengths and polydispersity. Through incorporating multiarmed oligoethylene-glycol (OEG) onto a quadratic aromatic segment, the lengths of the resultant SPs can be tuned from ∼1 µm to 130 and 50 nm with a polydispersity index of ∼1.2 for the last two SPs. We believe that the level of chain frustration of the multiarmed OEG segments, determined by both the number of arms and the degree of polymerization, poses physical and entropic constrains for supramolecular propagation to exceed a threshold length.

Valsartan nano-filaments alter mitochondrial energetics and promote faster healing in diabetic rat wounds.

Chronic wounds are a common and debilitating condition associated with aging populations that impact more than 6.5 million patients in the United States. We have previously demonstrated the efficacy of daily topical 1% valsartan in treating wounds in diabetic mouse and pig models. Despite these promising results, there remains a need to develop an extended-release formulation that would reduce patient burden by decreasing the frequency of daily applications. Here, we used nanotechnology to self-assemble valsartan amphiphiles into a filamentous structure (val-filaments) that would serve as a scaffold in wound beds and allow for steady, localised and tunable release of valsartan amphiphiles over 24 days. Two topical treatments of this peptide-based hydrogel on full-thickness wounds in Zucker Diabetic Fatty rats resulted in faster rates of wound closure. By day 23, all val-filament treated wounds were completely closed, as compared to one wound closed in the placebo group. Mechanistically, we observed enrichment of proteins involved in cell adhesion and energetics pathways, downregulation of Tgf-ß signalling pathway mediators (pSmad2, pSmad3 and Smad4) and increased mitochondrial metabolic pathway intermediates. This study demonstrates the successful synthesis of a sustained-release valsartan filament hydrogel, its impact on mitochondrial energetics and efficacy in treating diabetic wounds.

Selective Capture and Recovery of Monoclonal Antibodies by Self-Assembling Supramolecular Polymers of High Affinity for Protein Binding.

The separation and purification of therapeutic proteins from their biological resources pose a great limitation for industrial manufacturing of biologics in an efficient and cost-effective manner. We report here a supramolecular polymeric system that can undergo multiple reversible processes for efficient capture, precipitation, and recovery of monoclonal antibodies (mAbs). These supramolecular polymers, namely immunofibers (IFs), are formed by coassembly of a mAb-binding peptide amphiphile with a rationally designed filler molecule of varying stoichiometric ratios. Under the optimized conditions, IFs can specifically capture mAbs with a precipitation yield greater than 99%, leading to an overall mAb recovery yield of 94%. We also demonstrated the feasibility of capturing and recovering two mAbs from clarified cell culture harvest. These results showcase the promising potential of peptide-based supramolecular polymers as reversible affinity precipitants for mAb purification.

A Two-Pronged Pulmonary Gene Delivery Strategy: A Surface-Modified Fullerene Nanoparticle and a Hypotonic Vehicle.

Inhaled gene therapy poses a unique potential of curing chronic lung diseases, which are currently managed primarily by symptomatic treatments. However, it has been challenging to achieve therapeutically relevant gene transfer efficacy in the lung due to the presence of numerous biological delivery barriers. Here, we introduce a simple approach that overcomes both extracellular and cellular barriers to enhance gene transfer efficacy in the lung in vivo. We endowed tetra(piperazino)fullerene epoxide (TPFE)-based nanoparticles with non-adhesive surface polyethylene glycol (PEG) coatings, thereby enabling the nanoparticles to cross the airway mucus gel layer and avoid phagocytic uptake by alveolar macrophages. In parallel, we utilized a hypotonic vehicle to facilitate endocytic uptake of the PEGylated nanoparticles by lung parenchymal cells via the osmotically driven regulatory volume decrease (RVD) mechanism. We demonstrate that this two-pronged delivery strategy provides safe, wide-spread and high-level transgene expression in the lungs of both healthy mice and mice with chronic lung diseases characterized by reinforced delivery barriers.

Isotopic Control over Self-Assembly in Supramolecular Gels.

It is common to switch between H2O and D2O when examining peptide-based systems, with the assumption being that there are no effects from this change. Here, we describe the effect of changing from H2O to D2O in a number of low-molecular-weight dipeptide-based gels. Gels are formed by decreasing the pH. In most cases, there is little difference in the structures formed at high pH, but this is not universally true. On lowering the pH, the kinetics of gelation are affected and, in some cases, the structures underpinning the gel network are different. Where there are differences in the self-assembled structures, the resulting gel properties are different. We, therefore, show that isotopic control over gel properties is possible.

Supramolecular Design of Unsymmetric Reverse Bolaamphiphiles for Cell-Sensitive Hydrogel Degradation and Drug Release.

Self-assembly of peptide-based building units into supramolecular nanostructures creates an important class of biomaterials with robust mechanical properties and improved resistance to premature degradation. Yet, upon aggregation, substrate-enzyme interactions are often compromised because of the limited access of macromolecular proteins to the peptide substrate, leading to either a reduction or loss of responsiveness to biomolecular cues. Reported here is the supramolecular design of unsymmetric reverse bolaamphiphiles (RBA) capable of exposing a matrix metalloproteinase (MMP) substrate on the surface of their filamentous assemblies. Upon addition of MMP-2, these filaments rapidly break into fragments prior to reassembling into spherical micelles. Using 3D cell culture, it is shown that drug release is commensurate with cell density, revealing more effective cell killing when more cancer cells are present. This design platform could serve as a cell-responsive therapeutic depot for local chemotherapy.

Paclitaxel-Promoted Supramolecular Polymerization of Peptide Conjugates.

Spontaneous association above a threshold concentration is a hallmark of supramolecular polymerization, in which monomeric units self-assemble into polymeric aggregates through noncovalent interactions. This self-initiated supramolecular process differs from the conventional covalent chain-growth polymerization in that the latter often involves the use of a different chemical entity as an initiator to trigger/control the polymerization process. We report here the use of a small molecule hydrophobe, paclitaxel (PTX), as an effective promoter to induce the supramolecular polymerization of a peptide-paclitaxel conjugate, Spheropax (Spax). We found that Spax monomers alone in water self-assemble into spherical micelles of approximately 6.5 nm in diameter but, in the presence of free PTX, undergo a supramolecular polymerization process to form filamentous assemblies of several micrometers in length. Increasing the ratio of promoter to monomer (PTX/Spax) induces Spax's directional polymerization and expedites its kinetic process. We believe these findings provide important insight into the initiator-controlled supramolecular polymerization process.

Macrocyclization of a Class of Camptothecin Analogues into Tubular Supramolecular Polymers.

Nanostructured supramolecular polymers (SPs) are filamentous assemblies possessing a high degree of internal order and have important uses in regenerative medicine, drug delivery, and soft matter electronics. Despite recent advances in functional SPs, a challenging topic is the development of robust assembly protocols enabling the incorporation of various functional units without altering its supramolecular architecture. We report here the robust tubular assembly of camptothecin (CPT) analogues into functional SPs. Covalent linkage of two CPT moieties to various short hydrophilic segments (e.g., nonionic, cationic, anionic, and zwitterionic) leads to a class of CPT analogues that self-assemble in water into tubular SPs. Systemic administration of nonionic SPs effectively suppresses tumor growth. Furthermore, these tubular SPs act as universal dispersing agents in water for low-molecular-weight hydrophobes.

Light-Triggered Transformation of Molecular Baskets into Organic Nanoparticles.

Discovering novel and functional photoresponsive materials is of interest for improving controlled release of molecules and scavenging toxic compounds for cleaning our environment or designing chemosensors. In this study, we report on the photoinduced decarboxylation of basket 16- , containing three glutamic acids at its rim. This concave compound is, in an aqueous environment (30 mm phosphate buffer at pH 7.0), monomeric (1 H NMR DOSY, DLS) with glutamic acid residues randomly oriented about its rim (1 H NMR and MM-OPLS3). The irradiation (300 nm) of 16- leads to the exclusive removal of its α-carboxylates to give amphiphilic 23- possessing γ-carboxylates. The photochemical transformation is a consecutive reaction with mono- and bis-decarboxylated products observed with 1 H NMR spectroscopy and ESI mass spectrometry. Amphiphilic 23- is a preorganized molecule (MM-OPLS3) that, in water, aggregates into organic nanoparticles (ca. 50-200 nm in diameter; DLS, TEM and cryo-TEM) having a critical aggregation concentration of 12 µm (UV/Vis). As the transition of monomeric 16- into nanoparticulate 23- is triggered with light, we reasoned that stimuli-responsive formation of the soft material lends itself to nanotechnology applications such as controlled release or scavenging of targeted compounds.

Peptide-based nanoprobes for molecular imaging and disease diagnostics.

Pathological changes in a diseased site are often accompanied by abnormal activities of various biomolecules in and around the involved cells. Identifying the location and expression levels of these biomolecules could enable early-stage diagnosis of the related disease, the design of an appropriate treatment strategy, and the accurate assessment of the treatment outcomes. Over the past two decades, a great diversity of peptide-based nanoprobes (PBNs) have been developed, aiming to improve the in vitro and in vivo performances of water-soluble molecular probes through engineering of their primary chemical structures as well as the physicochemical properties of their resultant assemblies. In this review, we introduce strategies and approaches adopted for the identification of functional peptides in the context of molecular imaging and disease diagnostics, and then focus our discussion on the design and construction of PBNs capable of navigating through physiological barriers for targeted delivery and improved specificity and sensitivity in recognizing target biomolecules. We highlight the biological and structural roles that low-molecular-weight peptides play in PBN design and provide our perspectives on the future development of PBNs for clinical translation.