Amphiphilic Peptides Research Articles

A Photo‐Switchable Peptide Fibril Esterase

AbstractRecent attempts to mimic enzyme catalysis using simple, short peptides have been successful in enhancing various reactions, but the on‐demand, temporal or spatial regulation of such processes by external triggers remains a great challenge. Light irradiation is an ideal trigger for regulating molecular functionality, since it can be precisely manipulated in time and space, and because most reaction mediums do not react to light. We herein report the selection of a photo‐switchable amphiphilic peptide catalyst from a small library of isomeric peptides, each containing an azobenzene‐based light responsive group and a catalytic histidine residue. In its native fibrillar form, the selected peptide is efficiently and enantio‐selectively active for ester hydrolysis, but after irradiation by UV light inducing trans‐to‐cis azobenzene isomerization, the fibrils disassemble to amorphous aggregates that are much less catalytically active. Significantly, this esterase‐like activity can be manipulated multiple times, as the fibrillar peptide assembly is reversibly reduced and restored upon alternate irradiation by UV and visible light, respectively. We propose that this research may shine light on the origin of complex functions in early chemical evolution. Furthermore, it paves the way to regulate additional functions for peptide nanotechnology, such as replication, charge transfer, and delivery.

Tumor Receptor-Mediated Morphological Transformation and In Situ Polymerization of Diacetylene-Containing Lipidated Peptide Amphiphile on Cell Membranes for Tumor Suppression.

In situ polymerization on cell membranes can decrease cell mobility, which may inhibit tumor growth and invasion. However, the initiation of radical polymerization traditionally requires exogenous catalysts or free radical initiators, which might cause side effects in normal tissues. Herein, we synthesized a Y-type diacetylene-containing lipidated peptide amphiphile (TCDA-KFFFFK(GRGDS)-YIGSR, Y-DLPA) targeting integrins and laminin receptors on murine mammary carcinoma 4T1 cells, which underwent nanoparticle-to-nanofiber morphological transformation and in situ polymerization on cell membranes. Specifically, the polymerized Y-DLPA induced 4T1 cell apoptosis and disturbed the substance exchange and metabolism. In vitro assays demonstrated that the polymerized Y-DLPA nanofibers decreased the migration capacity of 4T1 cells, potentially suppressing tumor invasion and metastasis. When administered locally to 4T1 tumor-bearing mice, the Y-DLPA nanoparticles formed a biomimetic extracellular matrix that effectively suppressed tumor growth. This study provides an in situ polymerization strategy that can serve as an effective drug-free biomaterial with low side effects for antitumor therapy.

3D Cryo-Electron Microscopy Reveals the Structure of a 3-Fluorenylmethyloxycarbonyl Zipper Motif Ensuring the Self-Assembly of Tripeptide Nanofibers.

Short peptide-based supramolecular hydrogels appeared as highly interesting materials for applications in many fields. The optimization of their properties relies mainly on the design of a suitable hydrogelator through an empirical trial-and-error strategy based on the synthesis of various types of peptides. This approach is in part due to the lack of prior structural knowledge of the molecular architecture of the various families of nanofibers. The 3D structure of the nanofibers determines their ability to interact with entities present in their surrounding environment. Thus, it is important to resolve the internal structural organization of the material. Herein, using Fmoc-FFY tripeptide as a model amphiphilic hydrogelator and cryo-EM reconstruction approach, we succeeded to obtain a 3.8 Å resolution 3D structure of a self-assembled nanofiber with a diameter of approximately 4.1 nm and with apparently "infinite" length. The elucidation of the spatial organization of such nano-objects addresses fundamental questions about the way short amphiphilic N-Fmoc peptides lacking secondary structure can self-assemble and ensure the cohesion of such a lengthy nanostructure. This nanofiber is organized into a triple-stranded helix with an asymmetric unit composed of two Fmoc-FFY peptides per strand. The three identical amphiphilic strands are maintained together by strong lateral interactions coming from a 3-Fmoc zipper motif. This hydrophobic core of the nanofiber is surrounded by 12 phenyl groups from phenylalanine residues, nonplanar with the six Fmoc groups. Polar tyrosine residues at the C-term position constitute the hydrophilic shell and are exposed all around the external part of the assembly. This fiber has a highly hydrophobic central core with an internal diameter of only 2.4 Å. Molecular dynamics simulations highlight van der Waals and hydrogen bonds between peptides placed on top of each other. We demonstrate that the self-assembly of Fmoc-FFY, whether induced by annealing or by the action of a phosphatase on the phosphorylated precursor Fmoc-FFpY, results in two nanostructures with minor differences that we are unable to distinguish.

Self-Assembly and Wound Healing Activity of Biomimetic Cycloalkane-Based Lipopeptides.

The self-assembly of lipopeptide (peptide amphiphile) molecules bearing single linear lipid chains has been widely studied, as has their diverse range of bioactivities. Here, we introduce lipopeptides bearing one or two cycloalkane chains (cycloheptadecyl or cyclododecyl) conjugated to the collagen-stimulating pentapeptide KTTKS used in Matrixyl formulations. The self-assembly of all four molecules is probed using fluorescence probe measurements to detect the critical aggregation concentration (CAC), and cryogenic-TEM and small-angle X-ray scattering (SAXS) to image the nanostructure. The peptide conformation is studied using circular dichroism (CD) and FTIR spectroscopies. All the cycloalkane lipopeptides show excellent compatibility with dermal fibroblasts. The compounds bearing one or two cyclododecyl chains (denoted as DKT and DDKT, respectively) show wound healing in diabetic rats, the improvement being markedly enhanced for DDKT. Interestingly, the revival of hair follicles and blood vessels in the dermis were observed, which are the critical markers of effective wound repair. Analysis of H&E-stained tissue images (from a rat model) shows that the rat groups treated with DDKT and DKT displayed a significantly increased amount of regenerated hair follicles, indicating a faster healing process for DDKT compared to the control group. Collagen deposition was also enhanced, especially for DDKT, and by day 20, the DDKT-treated groups had developed a dense collagen network accompanied by a regenerated epidermis. At the same time, the number of blood vessels in DDKT-treated diabetic wounds was significantly higher than in control groups and neovascularization was substantially enhanced, as assayed using α-SMA (a marker for vascular smooth muscle cells) and CD31 (a marker specific to vascular endothelial cells). These results suggest that the lead lipopeptide DDKT exhibits a remarkable pro-vascularization capability and shows great promise for future application as a wound-healing biomaterial.

A Photo-Switchable Peptide Fibril Esterase.

Recent attempts to mimic enzyme catalysis using simple, short peptides have been successful in enhancing various reactions, but the on-demand, temporal or spatial regulation of such processes by external triggers remains a great challenge. Light irradiation is an ideal trigger for regulating molecular functionality, since it can be precisely manipulated in time and space, and because most reaction mediums do not react to light. We herein report the selection of a photo-switchable amphiphilic peptide catalyst from a small library of isomeric peptides, each containing an azobenzene-based light responsive group and a catalytic histidine residue. In its native fibrillar form, the selected peptide is efficiently and enantio-selectively active for ester hydrolysis, but after irradiation by UV light inducing trans-to-cis azobenzene isomerization, the fibrils disassemble to amorphous aggregates that are much less catalytically active. Significantly, this esterase-like activity can be manipulated multiple times, as the fibrillar peptide assembly is reversibly reduced and restored upon alternate irradiation by UV and visible light, respectively. We propose that this research may shine light on the origin of complex functions in early chemical evolution. Furthermore, it paves the way to regulate additional functions for peptide nanotechnology, such as replication, charge transfer, and delivery.

Hydrogels with Independently Controlled Adhesion Ligand Mobility and Viscoelasticity Increase Cell Adhesion and Spreading.

A primary objective in designing hydrogels for cell culture is recreating the cell-matrix interactions found within human tissues. Identifying the most important biomaterial features for these interactions is challenging because it is difficult to independently adjust variables such as matrix stiffness, stress relaxation, the mobility of adhesion ligands and the ability of these ligands to support cellular forces. In this work we designed a hydrogel platform consisting of interpenetrating polymer networks of covalently crosslinked poly(ethylene glycol) (PEG) and self-assembled peptide amphiphiles (PA). We can tailor the storage modulus of the hydrogel by altering the concentration and composition of each network, and we can tune the stress relaxation half-life through the non-covalent bonding in the PA network. Ligand mobility can be adjusted independently of the matrix mechanical properties by attaching the RGD cell adhesion ligand to either the covalent PEG network, the dynamic PA network, or both networks at once. Interestingly, our findings show that endothelial cell adhesion formation and spreading is maximized in soft, viscoelastic gels in which RGD adhesion ligands are present on both the covalent PEG and non-covalent PA networks. The dynamic nature of cell adhesion domains, coupled with their ability to exert substantial forces on the matrix, suggests that having different presentations of RGD ligands which are either mobile or are capable of withstanding significant forces are needed mimic different aspects of complex cell-matrix adhesions. By demonstrating how different presentations of RGD ligands affect cell behavior independently of viscoelastic properties, these results contribute to the rational design of hydrogels that facilitate desired cell-matrix interactions, with the potential of improving in vitro models and regenerative therapies.

Versatile Cell Penetrating Peptide for Multimodal CRISPR Gene Editing in Primary Stem Cells.

CRISPR gene editing offers unprecedented genomic and transcriptomic control for precise regulation of cell function and phenotype. However, delivering the necessary CRISPR components to therapeutically relevant cell types without cytotoxicity or unexpected side effects remains challenging. Viral vectors risk genomic integration and immunogenicity while non-viral delivery systems are challenging to adapt to different CRISPR cargos, and many are highly cytotoxic. The arginine-alanine-leucine-alanine (RALA) cell penetrating peptide is an amphiphilic peptide that self-assembles into nanoparticles through electrostatic interactions with negatively charged molecules before delivering them across the cell membrane. This system has been used to deliver DNAs, RNAs, and small anionic molecules to primary cells with lower cytotoxicity compared to alternative non-viral approaches. Given the low cytotoxicity, versatility, and competitive transfection rates of RALA, we aimed to establish this peptide as a new CRISPR delivery system in a wide range of molecular formats across different editing modalities. We report that RALA was able to effectively encapsulate and deliver CRISPR in DNA, RNA, and ribonucleic protein (RNP) formats to primary mesenchymal stem cells (MSCs). Comparisons between RALA and commercially available reagents revealed superior cell viability leading to higher numbers of transfected cells and the maintenance of cell proliferative capacity. We then used the RALA peptide for the knock-in and knock-out of reporter genes into the MSC genome as well as for the transcriptional activation of therapeutically relevant genes. In summary, we establish RALA as a powerful tool for safer and effective delivery of CRISPR machinery in multiple cargo formats for a wide range of gene editing strategies.

Mind the Particle Rigidity: Blooms the Bioavailability via Rapidly Crossing the Mucus Layer and Alters the Intracellular Fate of Curcumin.

Overcoming intestinal epithelial barriers to enhance bioavailability is a major challenge for oral delivery systems. Desirable nanocarriers should simultaneously exhibit rapid mucus penetration and efficient epithelial uptake; however, they two generally require contradictory structural properties. Herein, we proposed a strategy to construct multiperformance nanoparticles by modifying the rigidity of amphiphilic nanostructures originating from soy polypeptides (SPNPs), where its ability to overcome multibarriers was examined from both in vitro and in vivo, using curcumin (CUR) as a model cargo. Low-rigidity SPNPs showed higher affinity to mucin and were prone to getting stuck in the mucus layer. When they reached epithelial cells, they tended to be endocytosed through the clathrin and macropinocytosis pathways and further transferred to lysosomes, showing severe degradation and lower transport of CUR. Increased particle rigidity generally improved the absorption of CUR, with medium-rigidity SPNPs bloomed maximum plasma concentration of CUR by 80.62-fold and showed the highest oral bioavailability. Results from monocultured and cocultured cell models demonstrated that medium-rigidity SPNPs were least influenced by the mucus layer and changes in rigidity significantly influenced the endocytosis and intracellular fate of SPNPs. Those with higher rigidity preferred to be endocytosed via a caveolae-mediated pathway and trafficked to the ER and Golgi, facilitating their whole transcytosis, and avoiding intracellular metabolism. Moreover, rigidity modulation efficiently induces the reversible opening of intercellular tight junctions, which synergistically improves the transport of CUR into blood circulation. This study suggested that rigidity regulation on food originated amphiphilic peptides could overcome multiple physiological barriers, showing great potential as natural building block toward oral delivery.

Evaluation of the Antibacterial Potential of Two Short Linear Peptides YI12 and FK13 against Multidrug-Resistant Bacteria.

The accelerating spread of antibiotic resistance has significantly weakened the clinical efficacy of existing antibiotics, posing a severe threat to public health. There is an urgent need to develop novel antimicrobial alternatives that can bypass the mechanisms of antibiotic resistance and effectively kill multidrug-resistant (MDR) pathogens. Antimicrobial peptides (AMPs) are one of the most promising candidates to treat MDR pathogenic infections since they display broad-spectrum antimicrobial activities and are less prone to achieve drug resistance. In this study, we investigated the antibacterial capability and mechanisms of two machine learning-driven linear peptide compounds termed YI12 and FK13. We reveal that YI12 and FK13 exhibit broad-spectrum antibacterial properties against clinically significant bacterial pathogens, inducing no or minimal hemolysis in mammalian red blood cells. We further ascertain that YI12 and FK13 are resilient to heat and acid-base conditions, and exhibit susceptibility to hydrolytic enzymes and divalent cations under physiological conditions. Initial mechanistic investigations reveal that YI12 and FK13 compromise bacterial membrane integrity, leading to membrane potential dissipation and excessive reactive oxygen species (ROS) generation. Collectively, our findings highlight the prospective utility of these two cationic amphiphilic peptides as broad-spectrum antibacterial agents.

3D-ink-extruded titanium scaffolds with porous struts and bioactive supramolecular polymers for orthopedic implants

Porous titanium addresses the longstanding orthopedic challenges of aseptic loosening and stress shielding. This work expands on the evolution of porous Ti with the manufacturing of hierarchically porous, low stiffness, ductile Ti scaffolds via direct-ink write (DIW) extrusion and sintering of inks containing Ti and NaCl particles. Scaffold macrochannels were filled with a subtherapeutic dose of recombinant bone morphogenetic protein-2 (rhBMP-2) alone or co-delivered within a bioactive supramolecular polymer slurry (SPS) composed of peptide amphiphile nanofibrils and collagen, creating four treatment conditions (Ti struts: microporous vs. fully dense; BMP-2 alone or with SPS). The BMP-2-loaded scaffolds were implanted bilaterally across the L4 and L5 transverse processes in a rat posterolateral lumbar fusion model. In-vivo bone growth in these scaffolds is evaluated with synchrotron X-ray computed microtomography (µCT) to study the effects of strut microporosity and added biological signaling agents on the bone formation response. Optical and scanning electron microscopy confirms the ∼100 µm space-holder micropore size, high-curvature morphology, and pore fenestrations within the struts. Uniaxial compression testing shows that the microporous strut scaffolds have low stiffness and high ductility. A significant promotion in bone formation was observed for groups utilizing the SPS, while no significant differences were found for the scaffolds with the incorporation of micropores. Statement of significanceBy 2050, the anticipated number of people aged 60 years and older worldwide is anticipated to double to 2.1 billion. This rapid increase in the geriatric population will require a corresponding increase in orthopedic surgeries and more effective materials for longer indwelling times. Titanium alloys have been the gold standard of bone fusion and fixation, but their use has longstanding limitations in bone-implant stiffness mismatch and insufficient osseointegration. We utilize 3D-printing of titanium with NaCl space holders for large- and small-scale porosity and incorporate bioactive supramolecular polymers into the scaffolds to increase bone growth. This work finds no significant change in bone ingrowth via space-holder-induced microporosity but significant increases in bone ingrowth via the bioactive supramolecular polymers in a rat posterolateral fusion model.

Maneuvering the mineralization of self-assembled peptide nanofibers for designing mechanically-stiffened self-healable composites toward bone-mimetic ECM.

Extracellular matrix (ECM) elasticity remains a crucial parameter to determine cell-material interactions (viz. adhesion, growth, and differentiation), cellular communication, and migration that are essential to tissue repair and regeneration. Supramolecular peptide hydrogels with their 3-dimensional porous network and tuneable mechanical properties have emerged as an excellent class of ECM-mimetic biomaterials with relevant dynamic attributes and bioactivity. Here, we demonstrate the design of minimalist amyloid-inspired peptide amphiphiles, CnPA (n = 6, 8, 10, 12) with tuneable peptide nanostructures that are efficiently biomineralized and cross-linked using bioactive silicates. Such hydrogel composites, CnBG exhibit excellent mechanical attributes and possess excellent self-healing abilities and collagen-like strain-stiffening ability as desired for bone ECM mimetic scaffold. The composites exhibited the formation of a hydroxyapatite mineral phase upon incubation in a simulated body fluid that rendered mechanical stiffness akin to the hydroxyapatite-bridged collagen fibers to match the bone tissue elasticity eventually. In a nutshell, peptide nanostructure-guided temporal effects and mechanical attributes demonstrate C8BG to be an optimal composite. Finally, such constructs feature the potential for adhesion, proliferation of U2OS cells, high alkaline phosphatase activity, and osteoconductivity.

Fabrication of a peptide–AuNP–TiO2 nanocomposite and its application as a VOC sensor

AbstractWe fabricated a volatile organic compound (VOC) sensor with a peptide–Au nanoparticle (AuNP)–TiO2 nanocomposite in which AuNPs were linked with TiO2-coated conductive peptide nanowires. The conductive peptide nanowires were formed between the AuNPs via self-assembly through the complexation of amphiphilic peptides, LESEHEKLKSKHKSKLKEHESEL, and Co(II). Furthermore, TiO2 mineralization on the surface of the peptide nanowires yielded mixed crystals of rutile and anatase, which exhibited highly effective photolytic activity. In particular, the obtained TiO2 exhibited three times greater photodecomposition activity in the unsintered state toward organic matter than did commercially available TiO2. Next, we constructed a VOC sensor by immobilizing peptide–AuNP–TiO2 nanocomposites on a comb electrode. The electrochemical properties of the nanocomposite changed drastically under light irradiation in the presence of VOCs, indicating transport of the VOC-decomposition-generated photoexcited electrons of TiO2 to AuNPs through conductive peptide nanowires, which prevented electron–hole recombination. The obtained sensor exhibited a sensing range of 2–100 ppm for dichloromethane, which was used as a representative VOC. Therefore, nanocomposites made of AuNPs linked with conductive TiO2 nanotubes may be highly effective for TiO2-driven VOC decomposition. Moreover, we believe that this nanocomposite has high sensitivity for sensing VOCs.

Fluoroamphiphilic polymers exterminate multidrug-resistant Gram-negative ESKAPE pathogens while attenuating drug resistance.

ESKAPE pathogens are a panel of most recalcitrant bacteria that could "escape" the treatment of antibiotics and exhibit high incidence of drug resistance. The emergence of multidrug-resistant (MDR) ESKAPE pathogens (particularly Gram-negative bacteria) accounts for high risk of mortality and increased resource utilization in health care. Worse still, there has been no new class of antibiotics approved for exterminating the Gram-negative bacteria for more than 50 years. Therefore, it is urgent to develop novel antibacterial agents with low resistance and potent killing efficacy against Gram-negative ESKAPE pathogens. Herein, we present a class of fluoropolymers by mimicking the amphiphilicity of cationic antimicrobial peptides. Our optimal fluoroamphiphilic polymer (PD45HF5) displayed selective antimicrobial ability for all MDR Gram-negative ESAKPE pathogens, low resistance, high in vitro cell selectivity, and in vivo curative efficacy. These findings implied great potential of fluoroamphiphilic cationic polymers as promising antibacterial agents against MDR Gram-negative ESKAPE bacteria and alleviating antibiotic resistance.

Helicity-directed recognition of bacterial phospholipid via radially amphiphilic antimicrobial peptides.

The fundamental differences in phospholipids between bacterial and mammalian cell membranes present remarkable opportunities for antimicrobial design. However, it is challenging to distinguish bacterial anionic phospholipid phosphatidylglycerol (PG) from mammalian anionic phosphatidylserine (PS) with the same net charge. Here, we report a class of radially amphiphilic α helix antimicrobial peptides (RAPs) that can selectively discriminate PG from PS, relying on the helix structure. The representative RAP, L10-MMBen, can direct the rearrangement of PG vesicles into a lamellar structure with its helix axis parallel to the PG membrane surface. The helical structure imparts both the thermodynamic and kinetic advantages of L10-MMBen/PG assembly, and the hiding of hydrophobic regions in RAPs is crucial for PG recognition. L10-MMBen exhibits high selectivity against bacteria depending on PG recognition, showing low in vivo toxicity and significant treatment efficacy in mice infection models. Our study introduces a helicity-direct bacterial phospholipid recognition paradigm for designing highly selective antimicrobial peptides.

De Novo Design and Characterization of Amphiphilic Peptides with Basic Side Chains for Tailored Interfacial Chemistries.

Lysine-leucine (LK) peptides have been used as model systems and platforms for 2D material design for decades. LK peptides are amphiphilic sequences designed to bind and fold at hydrophobic surfaces through hydrophobic leucine side chains and hydrophilic lysine side chains extending into the aqueous subphase. The hydrophobic periodicity of the sequence dictates the secondary structure at the interface. This robust design makes them ideal candidates for controlling interfacial chemistry. This study presents the de novo design and characterization of two novel peptides: LRα14 and LHα14, which substitute lysine with arginine and histidine, respectively, in the helical LKα14 sequence. This modification is intended to expand the LK peptide platform to a new basic interfacial chemistry. We explore the stability of the new LRα14 and LHα14 designs with respect to changes in pH and salt concentration in bulk solution and at the interface using circular dichroism (UV-CD) and vibrational sum-frequency generation spectroscopy, respectively. Notably, the structural stability of the peptides remains unaffected across a wide range of pH and ionic strength values. At the same time, the variation of side-chain chemistry leads to a wide spectrum of interfacial water structures. By extension of the LK platform to include arginine and histidine, this study broadens the toolbox for designing tailored interfacial chemistries with applications in material and biomedical sciences.

Overcoming cisplatin resistance in ovarian cancer: A novel approach via mitochondrial targeting peptide Pal-pHK-pKV

Cisplatin (DDP) resistance in advanced stages of ovarian cancer significantly reduces survival rates. Mitochondria may serve as a potential therapeutic target for ovarian cancer. Pal-pHK-pKV is a mitochondrial targeting peptide synthesized by supramolecular assembly. Our study aims to investigate whether Pal-pHK-pKV serves as a useful strategy to reverse DDP resistance in ovarian cancer. Subcutaneous tumor implantation of the DDP-resistant ovarian cancer cell line A2780CP was conducted in nude mice, and drugs were administered intraperitoneally to compare the inhibitory effects of Pal-pHK-pKV and DDP on A2780CP cells in vivo. Combination index values were calculated for various concentrations of DDP and Pal-pHK-pKV to determine the optimal combination concentration. Mitochondrial membrane potential, cytochrome C distribution and immunofluorescence were also measured. Our studies demonstrated that Pal-pHK-pKV treatment reduced the proliferation, invasion and metastasis of ovarian cancer cells and impaired mitochondrial function. Furthermore, the combination of Pal-pHK-pKV and DDP exhibited a synergistic effect. Mechanistically, Pal-pHK-pKV can impair mitochondrial function, reduce mitochondrial membrane potential and release ROS. On the other hand, Pal-pHK-pKV can affect ERK pathway activation and inhibit tumor development. In conclusion, the mitochondria-specific amphiphilic peptide Pal-pHK-pKV provides a novel approach for treating ovarian cancer and may potentially overcome DDP drug resistance.

Cooperative β-sheet coassembly controls intermolecular orientation of amphiphilic peptide-polydiacetylene conjugates

In this work, we elucidated the structural organization of stimuli-responsive peptide-polydiacetylene (PDA) conjugates that can self-assemble as 1D nanostructures under neutral aqueous conditions. The amino acid sequences bear positively or negatively charged domains at the periphery of the peptide segments to promote solubility in water while also driving assembly of the individual and combined components into β-sheets. The photopolymerization of PDA, as well as the sensitivity of the resulting optical properties of the polymeric material to external stimuli, highly depends on the structural organization of the assembly of amphiphilic peptide-diacetylene units into 1D-nanostructures. Solid-state NMR measurements on 13C-labeled and 15N-labeled samples show that positively charged and negatively charged peptide amphiphiles are each capable of self-assembly, but self-assembly favors antiparallel β-sheet structure. When positively and negatively charged peptide amphiphiles interact in stoichiometric solutions, cooperative coassembly dominates over self-assembly, resulting in the desired parallel β-sheet structure with a concomitant increase in structural order. These results reveal that rational placement of oppositely charged residues can control β-strand organization in a peptide amphiphile coassembly, which would have implications on the adaptive properties of stimuli-responsive biomaterials such as the peptide-PDAs studied here.

Chimeric peptide-engineered immunostimulant for endoplasmic reticulum targeted photodynamic immunotherapy against metastatic tumor

The combination of therapy-induced immunogenic cell death (ICD) and immune checkpoint blockade can provide a mutually reinforced strategy to reverse the poor immunogenicity and immune escape behavior of tumors. In this work, a chimeric peptide-engineered immunostimulant (ER-PPB) is fabricated for endoplasmic reticulum (ER)-targeted photodynamic immunotherapy against metastatic tumors. Among which, the amphiphilic chimeric peptide (ER-PP) is composed of ER-targeting peptide FFKDEL, hydrophilic PEG8 linker and photosensitizer protoporphyrin IX (PpIX), which could be assembled with a PD-1/PD-L1 blocker (BMS-1) to prepare ER-PPB. After passively targeting at tumor tissues, ER-PPB will selectively accumulate in the ER. Next, the localized PDT of ER-PPB will produce a lot of ROS to destroy the primary tumor cells, while increasing the ER stress to initiate a robust ICD cascade. Moreover, the concomitant delivery of BMS-1 can impede the immune escape of tumor cells through PD-1/PD-L1 blockade, thus synergistically activating the immune system to combat metastatic tumors. In vitro and in vivo results demonstrate the robust immune activation and metastatic tumor inhibition characteristics of ER-PPB, which may offer a promising strategy for spatiotemporally controlled metastatic tumor therapy.

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells.

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in, and base editing. PERC involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating the formulation with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) bearing homology-directed repair template DNA may be included. In contrast to electroporation, PERC is appealing as it requires no dedicated hardware and has less impact on cell phenotype and viability. Due to the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Here we report methods for improved PERC-mediated editing of T cells as well as novel methods for PERC-mediated editing of HSPCs, including knockout and precise knock-in. Editing efficiencies can surpass 90% using either Cas9 or Cas12a in primary T cells or HSPCs. Because PERC calls for only three readily available reagents - protein, RNA, and peptide - and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes this approach appealing for rapid deployment in research and clinical settings.

A Matter of Charge: Electrostatically Tuned Coassembly of Amphiphilic Peptides.

Coassembly of peptide biomaterials offers a compelling avenue to broaden the spectrum of hierarchically ordered supramolecular nanoscale structures that may be relevant for biomedical and biotechnological applications. In this work coassemblies of amphiphilic and oppositely charged, anionic and cationic, β-sheet peptides are studied, which may give rise to a diverse range of coassembled forms. Mixtures of the peptides show significantly lower critical coassembly concentration (CCC) values compared to those of the individual pure peptides. Intriguingly, the highest formation of coassembled fibrils is found to require excess of the cationic peptide whereas equimolar mixtures of the peptides exhibited the maximum folding into β-sheet structures. Mixtures of the peptides coassembled sequentially from solutions at concentrations surpassing each peptide's intrinsic critical assembly concentration (CAC), are also found to require a higher portion of the cationic peptide to stabilize hydrogels. This study illuminates a systematic investigation of oppositely charged β-sheet peptides over a range of concentrations, in solutions and in hydrogels. The results may be relevant to the fundamental understanding of such intricate charge-driven assembly systems and to the formulation of peptide-based nanostructures with diverse functionalities.