Breaking Down the Barriers of Drug Resistance and Corneal Permeability with Chitosan-Poly(ethylene glycol)-LK13 Peptide Conjugate to Combat Fungal Keratitis.

Fungal keratitis (FK) is a leading cause of preventable blindness and eye loss. The poor antifungal activity, increased drug resistance, limited corneal permeability, and unsatisfactory biosafety of conventional antifungal eye drops are among the majority of the challenges that need to be addressed for currently available antifungal drugs. Herein, this study proposes an effective strategy that employs chitosan-poly(ethylene glycol)-LK13 peptide conjugate (CPL) in the treatment of FK. Nanoassembly CPL can permeate the lipophilic corneal epithelium in the transcellular route, and its hydrophilicity surface is a feature to drive its permeability through hydrophilic stroma. When encountering fungal cell membrane, CPL dissembles and exposes the antimicrobial peptide (LK13) to destroy fungal cell membranes, the minimum inhibitory concentration values of CPL against Fusarium solani (F. solani) are always not to exceed 8 µg peptide/mL before and after drug resistance induction. In a rat model of Fusarium keratitis, CPL demonstrates superior therapeutic efficacy than commercially available natamycin ophthalmic suspension. This study provides more theoretical and experimental supports for the application of CPL in the treatment of FK.

Histatin 5-Inspired Short-Chain Peptides Selectively Combating Pathogenic Fungi with Multifaceted Mechanisms.

Short-chain antifungal peptides (AFPs) inspired by histatin 5 have been designed to address the problem of antifungal drug resistance. These AFPs demonstrate remarkable antifungal activity, with a minimal inhibitory concentration as low as 2 µg mL-1. Notably, these AFPs display a strong preference for targeting fungi rather than bacteria and mammalian cells. This is achieved by binding the histidine-rich domains of the AFPs to the Ssa1/2 proteins in the fungal cell wall, as well as the reduced membrane-disrupting activity due to their low amphiphilicity. These peptides disrupt the nucleus and mitochondria once inside the cells, leading to reactive oxygen species production and cell damage. In a mouse model of vulvovaginal candidiasis, the AFPs demonstrate not only antifungal activity, but also promote the growth of beneficial Lactobacillus spp. This research provides valuable insights for the development of fungus-specific AFPs and offers a promising strategy for the treatment of fungal infectious diseases.

Guanidinylated/PEGylated chitosan in the bioink promotes the formation of multi-layered keratinocytes in a human skin equivalent.

The biological differences of skin between rodent and human beings and the strong appeal to replace the experimental animals have led to the development of alternative models with structures similar to the real human skin. Keratinocytes cultured in vitro on conventional dermal scaffolds tend to form monolayer rather than multi-layer epithelial tissue architectures. How to construct human skin or epidermal equivalents with multi-layered keratinocytes similar to real human epidermis remains one of the greatest challenges. Herein, a human skin equivalent with multi-layered keratinocytes was constructed by 3D bioprinting fibroblasts and subsequent culturing epidermal keratinocytes. Biocompatible guanidinylated/PEGylated chitosan (GPCS) was used as the main component of bioink to 3D bioprint tissue-engineered dermis. The function of GPCS to promote HaCat cell proliferation and connection was confirmed at the genetic, cellular, and histological levels. Compared with the skin tissues with mono-layered keratinocytes engineered with collagen and gelatin, adding GPCS in the bioink generated tissue-engineered human skin equivalents with multi-layered keratinocytes. Such human skin equivalents could be alternative models for biomedical, toxicological, and pharmaceutical research.

Surface decoration with leucine tetrapeptide: An antibacterial strategy against Gram-negative bacteria.

Surface-associated microbe contamination by Gram-negative bacteria poses a serious problem in medical care. Cationic peptides or polymers are the main materials used for antibacterial surface coating, but the positive charge may lead to blood coagulation. Therefore, exploiting surface coating which is free of positive charge and is effective for Gram-negative bacteria inactivation is in urgent need. In this study, inspired by the affinity between lipopolysaccharides of Gram-negative bacteria and Toll-like receptors of immune cells, we develop a leucine-based tetrapeptide coating strategy for combating Gram-negative bacteria. The obtained surface has excellent bactericidal activity against Gram-negative bacteria like Pseudomonas aeruginosa and Escherichia coli. A 1 mm2 coated glass surface could kill > 9.9 × 104 CFU bacteria in 1 h and has nearly no damage to mammal cells. Moreover, this surface coating strategy could be applied on various surfaces like glass slices, glass capillary cavity and thermoplastic polyurethane slices. And the coated surface could largely mitigate the microbe contamination in an in vivo subcutaneous implantation. This work paves a new way for antibacterial surface-coating which is behaving no positive charge and is of great importance for biomedical devices.

Overcoming the Stromal Barrier of the Cornea with a Peptide Conjugate Nano-Assembly to Combat Fungal Keratitis.

Fungal hyphae deeply invade the cornea in fungal keratitis. The corneal stroma hinders the infiltration of antifungal drugs and reduces their bioavailability. Here, this work reports a peptide conjugate nano-assembly that permeates the stroma and kills the pathogen without irritating the ocular cornea. The hydrophilic surface of the nano-assembly ensures deep permeation into the stroma. When encountering a fungal hyphal cell, the nano-assembly disassembles and exposes the α-helical peptide to destroy the fungal membrane, thus inactivating the pathogen. In a rabbit model of fungal keratitis, the nano-assembly exhibits a better therapeutic effect than commercially available natamycin ophthalmic suspension. Peptide conjugates with a nano-assembled structure and assembly-disassembly behavior could serve as the foundation of a new therapy for fungal keratitis.

One-Dimensional Rod-like Tobacco Mosaic Virus Promotes Macrophage Polarization for a Tumor-Suppressive Microenvironment.

The phenotype of tumor-associated macrophages plays an important role in their function of regulating the tumor immune microenvironment. The M1-phenotype macrophages display tumor-killing and immune activating functions. Here we show that the tobacco mosaic virus (TMV), a rod-like plant virus, can polarize macrophages to an M1 phenotype and shape a tumor-suppressive microenvironment. RAW 264.7 cells and bone marrow derived-macrophages (BMDMs) can recognize TMV via Toll-like receptor-4, and then the MAPK and NF-κB signaling pathways are activated, leading to the production of pro-inflammatory factors. Furthermore, the in vivo assessments on a subcutaneous co-injection tumor model show that the TMV-polarized BMDMs shape a tumor-suppressive microenvironment, resulting in remarkable delay of 4T1 tumor growth. Another in vivo assessment on an established tumor model indicates the high tumor-metastasis-inhibiting capacity of TMV-polarized BMDMs. This work suggests a role for this plant virus in macrophage-mediated therapeutic approaches and provides a strategy for tumor immunotherapy.

[Effects and mechanism of water-soluble chitosan hydrogel on infected full-thickness skin defect wounds in diabetic mice].

Objective: To explore the effects and mechanism of water-soluble chitosan hydrogel on infected full-thickness skin defect wounds in diabetic mice. Methods: The experimental research method was adopted. The control hydrogel composed of polyvinyl alcohol and gelatin, and the water-soluble chitosan hydrogel composed of the aforementioned two materials and water-soluble chitosan were prepared by the cyclic freeze-thaw method. The fluidity of the two dressings in test tube before and after the first freeze-thawing was generally observed, and the difference in appearance of the final state of two dressings in 12-well plates were compared. According to random number table (the same grouping method below), the cell strains of L929 and HaCaT were both divided into water-soluble chitosan hydrogel group and control hydrogel group, respectively. After adding corresponding dressings and culturing for 24 h, the cell proliferation activity was measured using cell counting kit 8. Rabbit blood erythrocyte suspensions were divided into normal saline group, polyethylene glycol octyl phenyl ether (Triton X-100) group, water-soluble chitosan hydrogel group, and control hydrogel group, which were treated accordingly and incubated for 1 hour, and then the hemolysis degree of erythrocyte was detected by a microplate reader. Twenty-four female db/db mice aged 11-14 weeks were selected, and full-thickness skin defect wounds on their backs were inflicted and inoculated with the methicillin-resistant Staphylococcus aureus (MRSA), 72 h later, the mice were divided into blank control group, sulfadiazine silver hydrogel group, control hydrogel group, and water-soluble chitosan hydrogel group, which were treated accordingly. On post injury day (PID) 0 (immediately), 7, 14, and 21, the healing of the wound was observed. On PID 14 and 21, the wound healing rate was calculated. On PID 14, MRSA concentration in wounds was determined. On PID 21, the wounds were histologically analyzed by hematoxylin and eosin staining; the expression of CD31 in the wounds was detected by immunofluorescence method, and its positive percentage was calculated. Raw264.7 cells were taken and divided into interleukin-4 (IL-4) group, blank control group, control hydrogel group, and water-soluble chitosan hydrogel group, which were treated accordingly. At 48 h of culture, the percentages of CD206 positive cells were detected by flow cytometry. The number of samples was all 3. Data were statistically analyzed with independent sample t test, one-way analysis of variance, analysis of variance for repeated measurement, least significant difference test, and Dunnett T3 test. Results: Two dressings in test tube had certain fluidity before freeze-thawing and formed semi-solid gels after freeze-thawing for once. The final forms of two dressings in 12-well plates were basically stable and translucent sheets, with little difference in transparency. At 24 h of culture, the cell proliferation activities of L929 and HaCaT in water-soluble chitosan hydrogel group were significantly higher than those in control hydrogel group (with t values of 6.37 and 7.50, respectively, P<0.01). At 1 h of incubation, the hemolysis degree of erythrocyte in water-soluble chitosan hydrogel group was significantly lower than that in Triton X-100 group (P<0.01), but similar to that in normal saline group and control hydrogel group (P>0.05). On PID 0, the traumatic conditions of mice in the 4 groups were similar. On PID 7, more yellowish exudates were observed inside the wound in blank control group and control hydrogel group, while a small amount of exudates were observed in the wound in sulfadiazine silver hydrogel group and water-soluble chitosan hydrogel group. On PID 14, the wounds in blank control group and control hydrogel group were dry and crusted without obvious epithelial coverage; in sulfadiazine silver hydrogel group, the scabs fell off and purulent exudate was visible on the wound; in water-soluble chitosan hydrogel group, the base of wound was light red and obvious epithelial coverage could be observed on the wound. On PID 14, the wound healing rate in water-soluble chitosan hydrogel group was significantly higher than that in the other 3 groups (all P<0.01). On PID 21, the wound in water-soluble chitosan hydrogel group was completely closed, while the wounds in the other 3 groups were not completely healed; the wound healing rate in water-soluble chitosan hydrogel group was significantly higher than that in the other 3 groups (all P<0.01). On PID 14, the concentration of MRSA in the wound in water-soluble chitosan hydrogel group was significantly lower than that in blank control group (P<0.01), but similar to that in control hydrogel group and sulfadiazine silver hydrogel group (P>0.05). On PID 21, the new epidermis was severely damaged in blank control group; the epidermis on the wound in control hydrogel group also had a large area of defect; complete new epidermis had not yet being formed on the wound in sulfadiazine silver hydrogel group; the wound in water-soluble chitosan hydrogel group was not only completely covered by the new epidermis, the basal cells of the new epidermis were also regularly aligned. On PID 21, the percentage of CD31 positivity in the wound in water-soluble chitosan hydrogel group was (2.19±0.35)%, which was significantly higher than (0.18±0.05)% in blank control group, (0.23±0.06)% in control hydrogel group, and (0.62±0.25)% in sulfadiazine silver hydrogel group, all P<0.01. At 48 h of culture, the percentage of CD206 positive Raw264.7 cells in water-soluble chitosan hydrogel group was lower than that in IL-4 group (P>0.01) but significantly higher than that in blank control group and control hydrogel group (P<0.05 or P<0.01). Conclusions: The water-soluble chitosan hydrogel has good biosafety and can induce higher level of macrophage M2 polarization than control hydrogel without water-soluble chitosan, so it can enhance the repair effect of MRSA-infected full-thickness skin defect wounds in diabetic mice and promote rapid wound healing.

Amino Acid Dependent Performance of Modified Chitosan Against Bacteria and Red Blood Cell.

In order to combat antibiotic resistance, the development of new antibacterial agents is essential. In this study, we prepared four types of amino acid modified chitosan (CS-AA). Compared with chitosan modified with hydrophobic amino acids, the chitosan modified with positively charged amino acids showed higher antibacterial efficiency against Escherichia coli (E. coli) under similar grafting rate. CS-AA achieves antibacterial properties mainly by destroying the integrity of bacterial cell membranes. All the four types of CS-AA show low toxicity towards red blood cells. This work indicates that positively charged groups are more important than hydrophobic groups in the design of chitosan-based antibacterial agents, and provides helpful information for the molecular design of effective antibacterial agents.

Conjugating Peptides onto 1D Rodlike Bionanoparticles for Enhanced Activity against Gram-Negative Bacteria.

Gram-negative bacteria, which possess an impermeable outer membrane, are responsible for many untreatable infections. The lack of development of new relevant antibiotics for over 50 years has increased threats. Peptides are regarded as the most promising alternatives to antibiotics. However, since the activities of existing peptides are not yet comparable to those of current antibiotics, there is an urgent need to improve their antibacterial efficiencies. Herein, we conjugate peptides onto one-dimensional rod-like tobacco mosaic virus (TMV). The peptides on the obtained nanoparticles (peptide-TMV) are hundreds of times superior to free peptides in combating Gram-negative bacteria. Through morphology and gene detection of Escherichia coli, it was revealed that following peptide-TMV application, the high osmotic pressure related to membrane damage and the generated reactive oxygen species cause Escherichia coli's death. In addition, peptide-TMV causes a downregulation of biofilm-related genes, inhibiting biofilm formation. This work paves the way to combat Gram-negative bacteria-related infection.

3D-printable supramolecular hydrogels with shear-thinning property: fabricating strength tunable bioink via dual crosslinking.

3-dimensional (3D) bioprinting technology provides promising strategy in the fabrication of artificial tissues and organs. As the fundamental element in bioprinting process, preparation of bioink with ideal mechanical properties without sacrifice of biocompatibility is a great challenge. In this study, a supramolecular hydrogel-based bioink is prepared by polyethylene glycol (PEG) grafted chitosan, α-cyclodextrin (α-CD) and gelatin. It has a primary crosslinking structure through the aggregation of the pseudo-polyrotaxane-like side chains, which are formed from the host-guest interactions between α-CD and PEG side chain. Apparent viscosity measurement shows the shear-shinning property of this bioink, which might be due to the reversibility of the physical crosslinking. Moreover, with ß-glycerophosphate at different concentrations as the secondary crosslinking agent, the printed constructs demonstrate different Young's modulus (p < 0.001). They could also maintain the Young's modulus in cell culture condition for at least 21 days (p < 0.05). By co-culturing each component with fibroblasts, CCK-8 assay demonstrate cellular viability is higher than 80%. After bioprinting and culturing, immunofluorescence staining with quantification indicate the expression of Ki-67, Paxillin, and N-cadherin is higher in day 14 than those in day 3 (p < 0.05). Oil red O and Nissl body specific staining reflect strength tunable bioink may have impact on the cell fate of mesenchymal stem cells (p < 0.05). This work might provide new idea for advanced bioink in the application of re-establishing complicated tissues and organs.

Combating Pseudomonas aeruginosa Biofilms by a Chitosan-PEG-Peptide Conjugate via Changes in Assembled Structure.

Pseudomonas aeruginosa (P. aeruginosa) biofilms are associated with a wide range of infections, from chronic tissue diseases to implanted medical devices. In a biofilm, the extracellular polymeric substance (EPS) causes an inhibited penetration of antibacterial agents, leading to a 100-1000 times tolerance of the bacteria. In view of the water-filled channels in biofilms and the highly negative charge of EPS, we design a chitosan-polyethylene glycol-peptide conjugate (CS-PEG-LK13) in this study. The CS-PEG-LK13 prefers a neutrally charged assembly at a size of ∼100 nm in aqueous environment, while undergoes disassembly to expose the α-helical peptide at the bacterial cell membrane. This behavior provides CS-PEG-LK13 superiorities in both penetrating the biofilms and inactivating the bacteria. At a concentration of 8 times the minimum inhibitory concentration, CS-PEG-LK13 has a much higher antibacterial efficiency (72.70%) than LK13 peptide (15.24%) and tobramycin (33.57%) in an in vitro P. aeruginosa biofilm. Moreover, CS-PEG-LK13 behaves comparable capability of combating an implanted P. aeruginosa biofilm to highly excess tobramycin. This work has implications for the design of new antibacterial agents in biofilm combating.

Membrane intercalation-enhanced photodynamic inactivation of bacteria by a metallacycle and TAT-decorated virus coat protein.

Antibiotic resistance has become one of the major threats to global health. Photodynamic inactivation (PDI) develops little antibiotic resistance; thus, it becomes a promising strategy in the control of bacterial infection. During a PDI process, light-induced reactive oxygen species (ROS) damage the membrane components, leading to the membrane rupture and bacteria death. Due to the short half-life and reaction radius of ROS, achieving the cell-membrane intercalation of photosensitizers is a key challenge for PDI of bacteria. In this work, a tetraphenylethylene-based discrete organoplatinum(II) metallacycle (1) acts as a photosensitizer with aggregation-induced emission. It self-assembles with a transacting activator of transduction (TAT) peptide-decorated virus coat protein (2) through electrostatic interactions. This assembly (3) exhibits both ROS generation and strong membrane-intercalating ability, resulting in significantly enhanced PDI efficiency against bacteria. By intercalating in the bacterial cell membrane or entering the bacteria, assembly 3 decreases the survival rate of gram-negative Escherichia coli to nearly zero and that of gram-positive Staphylococcus aureus to ∼30% upon light irradiation. This study has wide implications from the generation of multifunctional nanomaterials to the control of bacterial infection, especially for gram-negative bacteria.

Phenotype Regulation of Smooth Muscle Cells Through Facial Crystallization of Poly(ɛ-Caprolactone).

Phenotype conversion of smooth muscle cells (SMCs) plays a key role in the formation of atherosclerosis. Understanding how SMCs respond to a micro/nano-topology and elucidating the cellular mechanism of phenotype conversion is critical to the atherosclerosis treatment. Herein, we prepared poly(ɛ-caprolactone) (PCL) spherulites with a radius more than 350 µm for the studying of radial microstructure influence on SMCs behaviors. We found that on the PCL spherulitic films, SMCs grew aligning the radial direction of PCL spherulites, overexpressed α-SMA gene than OPN gene, and preferred contractile phenotype. FAK signaling pathway and ROCK1 signaling pathway both contributed to the contractile phenotype maintenance of SMCs. This work illustrated the feasibility of spherulites in regulating SMCs behaviors, and elucidated the mechanism how SMCs respond to a radial micro/nano-topology. This research may provide theoretical basis for the atherosclerosis formation and treatment.

Integration of Cell-Penetrating Peptides with Rod-like Bionanoparticles: Virus-Inspired Gene-Silencing Technology.

Inspired by the high gene transfer efficiency of viral vectors and to avoid side effects, we present here a 1D rod-like gene-silencing vector based on a plant virus. By decorating the transacting activator of transduction (TAT) peptide on the exterior surface, the TAT-modified tobacco mosaic virus (TMV) achieves a tunable isoelectric point (from ∼3.5 to ∼9.6) depending on the TAT dose. In addition to enhanced cell internalization, this plant virus-based vector (TMV-TAT) acquired endo/lysosomal escape capacity without inducing lysosomal damage, resulting in both high efficiency and low cytotoxicity. By loading silencer green fluorescent protein (GFP) siRNA onto the TMV-TAT vector (siRNA@TMV-TAT) and interfering with GFP-expressing mouse epidermal stem cells (ESCs/GFP) in vitro, the proportion of GFP-positive cells could be knocked down to levels even lower than 15% at a concentration of ∼100% cell viability. Moreover, by interfering with GFP-expressing highly metastatic hepatocellular carcinoma (MHCC97-H/GFP) tumors in vivo, treatment with siRNA@TMV-TAT complexes for 10 days achieved a GFP-negative rate as high as 80.8%. This work combines the high efficiency of viral vectors and the safety of nonviral vectors and may provide a promising strategy for gene-silencing technology.

Fluorous interaction induced self-assembly of tobacco mosaic virus coat protein for cisplatin delivery.

Tobacco mosaic virus coat protein was modified with a small molecular fluorous ponytail at specific sites, and self-assembled into spherical nanoparticles through fluorous interaction induced self-assembly. By loading the anti-cancer drug cisplatin through metal-ligand coordination, this spherical assembly with high stability has potential as a drug carrier.

Self-Assembly of Rod-Like Bionanoparticles at Interfaces and in Solution.

Rod-like nanoparticles show unique self-assembly behavior benefiting from their anisotropic properties. As a classic example of a one-dimensional (1D) rod-like plant virus, tobacco mosaic virus (TMV) can either assemble in a head-to-tail manner to form 1D long fibers, or align parallel to form crystal-like structures at interfaces or in solution. Here, the self-assembly behaviors of TMV at oil-water or air-liquid interfaces are summarized. In addition, the self-assembly of TMV with polymers in solution is also discussed in this chapter.

Balancing antimicrobial activity with biological safety: bifunctional chitosan derivative for the repair of wounds with Gram-positive bacterial infections.

Infection control and the promotion of healing are two key factors during the wound repair process. However, it is still a major challenge for one material to inhibit bacterial growth efficiently and promote wound healing at the same time. Here, a bifunctional chitosan derivative (CS-G/mPEG) was prepared by the successive modification of chitosan with carboxymethoxypolyethylene glycol (mPEG-COOH) and aminoiminomethanesulfonic acid (AIMSOA). We demonstrated that CS-G/mPEG can selectively disrupt bacterial membranes with high efficiency and thus kill Gram-positive bacteria without inducing hemolysis or cytotoxicity. In rats with full-thickness skin wounds infected with Staphylococcus aureus bacteria, CS-G/mPEG inhibited the growth of the bacteria and accelerated the healing of the wounds. Owing to the balance between the antimicrobial activity and biological safety of CS-G/mPEG, this bifunctional chitosan derivative is promising as an ideal anti-infective wound-repairing material for managing wounds with Gram-positive bacterial infections.

One-Pot Green Synthesis of Nitrogen-Doped Carbon Quantum Dots for Cell Nucleus Labeling and Copper(II) Detection.

The doping of nitrogen into carbon quantum dots is vitally important for improved fluorescence performance. However, the synthesis of nitrogen-doped carbon quantum dots (N-CQDs) is usually conducted under strong acid and high temperature, which results in environmental pollution and energy consumption. Herein, the N-CQDs were prepared by a mild one-pot hydrothermal process. The hydrothermal reaction temperature was adjusted to control the particle size, nitrogen/carbon atomic ratio, and quantum yield. The products were water soluble with a narrow particle size distribution and good dispersion stability over a wide pH range. The N-CQDs could penetrate into the HeLa cell nucleus without any further functionalization. Moreover, the fluorescence of N-CQDs could be selectively quenched by Cu2+ , which suggested applications for the detection of Cu2+ in human plasma.

Programming Self-Assembly of Tobacco Mosaic Virus Coat Proteins at Pickering Emulsion Interfaces for Nanorod-Constructed Capsules.

Pickering emulsion constructions on nanorods with high aspect ratio are a great challenge because of the geometry restrictions. On the basis of the theory that the stability of Pickering emulsion is highly dependent on the size and amphiphilicity of the nanoparticle at fluid interfaces, we report a novel strategy to fabricate long-time stable Pickering emulsion consisting of tobacco mosaic virus (TMV)-like nanorods through the programming self-assembly of TMV coat protein (TMVCP). The first step is the self-assembly of amphiphilic TMVCP at Pickering emulsion interfaces, and the second step is the in situ interfacial self-assembly of TMVCP into nanorods with increased particle size. The robust capsules can be further fabricated through cross-linking of the proteins. By taking advantage of both the amphiphilicity of TMVCP and the subsequent size growth induced by TMVCP self-assembly, this work provides a powerful strategy for constructing robust capsules consisting of nanorods with high aspect ratio, which may show potential applications for drug delivery and virus recognition.

Mussel-Inspired Polydopamine Coating on Tobacco Mosaic Virus: One-Dimensional Hybrid Nanofibers for Gold Nanoparticle Growth.

One-dimensional (1D) hybrid nanofibers with surface-deposited gold nanoparticles (AuNPs) have been fabricated by self-assembly of rod-like tobacco mosaic virus (TMV) with mussel-inspired polymerization of dopamine and in situ reduction of gold ion, providing a method for sensing the endocytic pathway of nanomaterial.