ACS Biomaterials Science & Engineering最新文献

Biofabrication and three-dimensional (3D) bioprinting enable precise spatial arrangement of cells within biomaterial scaffolds. We developed an alginate-based and Förster resonance energy transfer (FRET)-responsive "turn-on" reporter ink platform to enable real-time monitoring of matrix metalloproteinase (MMP) activity. Three distinct MMP-cleavable turn-on peptide reporters were synthesized and characterized for their cell-specific cleavage profiles using recombinant MMPs, cell-derived media, and different cell cultures (NIH3T3, HEK293, and MelHo). All turn-on reporters were covalently and site-specifically incorporated into alginate dialdehyde (ADA) to yield an MMP reporter ink. The ADA reporter ink with an MMP 13 turn-on reporter was responsive to all tested cell types over time within the cast bulk constructs. The ADA reporter ink material blended with gelatin had comparable print resolution and structural fidelity as observed for ADA. The extrusion-based bioprinted MelHo cell grids, measuring 2 × 2 cm² and containing 1 × 10⁶ cells/mL, exhibited MMP activity responses comparable to those of the casted reporter ink system, with a 3-fold increase observed at 24 h. This study introduces a versatile, FRET-based alginate bioink platform for the real-time monitoring of MMP activities, expanding the toolkit to understand cellular performance in bioprinted 3D constructs.

Various immunotherapeutic strategies are being developed to fight cancer, which is one of the leading causes of mortality. Dendritic cells (DCs), being professional antigen-presenting cells, after efficient manipulation with tumor-associated antigens, can lead to effective T-cell recruitment and activation at the tumor site, resulting in cytotoxic T-cell-mediated cancer cell killing. To circumvent the inefficiencies of ex vivo DC modification and patient infusion, an alternative strategy involving in situ DC activation has been explored here. Here, the vaccine components are tumor lysates, as antigens, and polyinosinic:polycytidylic acid (poly(I:C)), a toll-like receptor-3 (TLR3) agonist, as an adjuvant. Our in vitro studies demonstrate that complexing poly(I:C) with a carrier molecule, chitosan, enhances its stability and accessibility to TLR3 in the DC endosomal membrane. Material-based localized delivery of immunomodulatory factors is known to improve their stability and reduce their off-target side effects. Here, PEGDA-PLL-based macroporous scaffolds allow easy recruitment of host cells, thereby enabling effective interaction between the vaccine components loaded on them and the infiltrating immune cells. The vaccine components present in the scaffold facilitate efficient DC activation and migration, leading to subsequent T-cell activation and antitumor response, as shown by our in vivo studies.

Nucleic acids, including DNA and RNA, have been used extensively as building blocks to construct sophisticated nanostructures through complementary base pairing with predetermined shapes and sizes. With remarkable biocompatibility, spatial addressability, and structural programmability, self-assembled nucleic acid biomaterials have found widespread applications in various biomedical researches, including drug delivery, bioimaging, or disease diagnosis. Notably, as one of the representative nanostructures, DNA origami has drawn much attention. In this review, we summarize the latest developments in DNA/RNA origami design based on single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and single-stranded RNA (ssRNA) scaffolds for a range of biomedical applications, including drug delivery, gene regulation, immunomodulation, and receptor recognition. Additionally, the challenges and future opportunities of DNA/RNA origami in biomedical applications will be discussed.

Hypoxia in solid tumors, including head and neck cancer (HNC), contributes to treatment resistance, aggressive tumor phenotypes, and poorer clinical outcomes. Perfluorocarbon nanodroplets have emerged as promising drugs to alleviate tumor hypoxia. These versatile nanocarriers can also encapsulate and deliver various therapeutic agents, offering a multifunctional approach to cancer treatment. However, a detailed characterization of hypoxia alleviation, particularly the duration of hypoxia treatment drug residence, has not been thoroughly investigated. In this study, we developed and characterized perfluoropentane nanodroplets (PFP NDs) for the codelivery of oxygen and the photoactivatable drug benzoporphyrin derivative (BPD) to hypoxic HNC spheroids. The PFP NDs exhibited excellent stability, efficient oxygen loading/release, and biocompatibility. Using 3D multicellular tumor spheroids of FaDu and SCC9 HNC cells, we investigated the spatiotemporal dynamics of hypoxia within these spheroids and the ability of oxygenated PFP NDs to alleviate hypoxia. Our results showed that oxygen-loaded PFP NDs effectively penetrated the core of tumor spheroids, significantly reducing hypoxia, as evidenced by the downregulation of hypoxia-inducible factors HIF-1α and HIF-2α. Importantly, we demonstrated sustained hypoxia alleviation for up to 3 h post-treatment with PFP NDs. BPD-loaded PFP NDs successfully delivered the photosensitizer into the spheroid core in a time-dependent manner. Furthermore, we evaluated the efficacy of oxygen-dependent treatment modality, namely, photodynamic therapy (PDT) with BPD and oxygen-loaded PFP NDs compared to free BPD. The NDs formulation exhibited superior PDT outcomes, which were attributed to improved oxygen availability during the treatment. This study provides comprehensive evidence for the potential of PFP NDs as a codelivery platform to overcome hypoxia-mediated treatment resistance and enhance PDT efficacy in HNC. Our findings pave the way for further investigation of this promising approach in more complex in vivo models, potentially leading to improved therapeutic strategies for hypoxic solid tumors.

Damages to the supportive structure of the pelvic floor frequently result in pelvic organ prolapse (POP), which diminishes the quality of life. Surgical repair typically involves mesh implantation to reinforce the weakened tissues. However, the commonly used polypropylene (PP) mesh can lead to severe complications due to the mechanical mismatch of the mesh with the pelvic tissues. In this study, 3D-printed silk fibroin (SF) meshes are developed and optimized through cryogenic 3D printing followed by post-stretching treatment to enhance mechanical properties and biocompatibility for POP repair. Rheological analysis shows that the 30 wt % SF-based ink exhibited a zero shear viscosity of 1838 Pa·s and shear-thinning behavior, ensuring smooth extrusion during 3D printing. During the cryogenic incubation following 3D printing, self-assembly of SF occurs with the formation of β-sheet structures, leading to robust constructs with good shape fidelity. The post-stretching treatment further improves SF chain alignment and fibrilization, resulting in enhanced mechanical performance and a microstrip surface that promotes cell attachment, alignment, and differentiation. The SF mesh with a post-stretching ratio of 150% shows an ultimate tensile strength of 1.49 ± 0.14 MPa, an elongation at break of 104 ± 13%, and a Young's modulus of 5.0 ± 0.1 MPa at a hydrated condition, matching the properties of soft pelvic tissues. In vitro studies show that post-stretched SF meshes facilitated better cell alignment and myogenic differentiation than PP meshes. In vivo assessments demonstrate enhanced biocompatibility of the SF meshes, with better cellular infiltration and tissue integration than PP meshes in the long-term implantation, showing potential as a safe, effective alternative to traditional synthetic meshes for POP repair and other clinical applications.

Fibronectin (Fn) is an extracellular matrix glycoprotein with mechanosensitive structure-function. Extra domain A (EDA) Fn, a Fn isoform, is not present in adult tissue but is required for tissue repair. Curiously, EDA Fn is linked to both regenerative and fibrotic tissue repair. Given that Fn mechanoregulates cell behavior, EDA Fn organization during wound closure might play a role in mediating these differing responses. One mechanism by which cells sense and respond to their microenvironment is by activating a transcriptional coactivator, yes-associated protein (YAP). Interestingly, YAP activity is not only required for wound closure but similarly linked to both regenerative and fibrotic repair. Therefore, this study aims to evaluate how, during normal and fibrotic wound closure, EDA Fn organization might modulate YAP translocation by culturing human dermal fibroblasts on polydimethylsiloxane substrates mimicking normal (soft: 18 kPa) and fibrotic (stiff: 146 kPa) wounded skin. On stiffer substrates mimicking fibrotic wounds, fibroblasts assembled an aligned EDA Fn matrix comprising thinner fibers, suggesting increased microenvironmental tension. To evaluate if cell binding to the EDA domain of Fn was essential to overall matrix organization, fibroblasts were treated with Irigenin, which inhibits binding to the EDA domain within Fn. Blocking adhesion to EDA led to randomly organized EDA Fn matrices with thicker fibers, suggesting reduced microenvironmental tension even during fibrotic wound closure. To evaluate whether YAP signaling plays a role in EDA Fn organization, fibroblasts were treated with CA3, which suppresses YAP activity in a dose-dependent manner. Treatment with CA3 also led to randomly organized EDA Fn matrices with thicker fibers, suggesting a potential connected mechanism of reducing tension during fibrotic wound closure. Next, YAP activity was assessed to evaluate the impact of EDA Fn organization. Interestingly, fibroblasts migrating on softer substrates mimicking normal wounds increased YAP activity, but on stiffer substrates, they decreased YAP activity. When fibroblasts on stiffer substrates were treated with Irigenin or CA3, fibroblasts increased YAP activity. These results suggest that there may be disrupted signaling between EDA Fn organization and YAP translocation during fibrotic wound closure that could be restored when reestablishing normal EDA Fn matrix organization to instead drive regenerative wound repair.

Collagen, as the principal structural component of the cornea, has emerged as a promising biomaterial for artificial corneal owing to its excellent biocompatibility and degradability. However, the mechanical properties of current collagen membrane cannot match the requirements of artificial corneal materials. Inspired by the hierarchical lamellar organization of native corneal stromal collagen, a biomimetic collagen-based corneal repair material was designed via a "killing two birds with one stone" strategy. In this strategy, carboxymethyl-β-cyclodextrin (CM-β-CD) was incorporated into the collagen, serving dual functions: regulating the in vitro self-assembly process of collagen molecules and establishing multiple covalent cross-linking sites within the network. Concurrently, controlled external shear forces were applied to induce anisotropic alignment of collagen fibers, effectively replicating the highly organized structural hierarchy characteristic of native corneal stromal tissue. The resulting membrane exhibited a 67% enhancement in tensile strength (0.52 MPa) compared to pure collagen membranes. Notably, in vivo lamellar keratoplasty evaluations revealed accelerated tissue regeneration, achieving complete re-epithelialization within 14 days versus 28 days for controls. These findings establish the material's potential as an advanced artificial corneal for tissue engineering applications.

Single-domain antibodies (sdAbs) often exhibit superior thermal stability compared to traditional antibodies. Efforts are currently focused on enhancing their structural robustness and thermal refolding ability through protein engineering to achieve greater thermal properties and functionality in practical applications. Thermal aggregation is a key factor hindering the reversible thermal denaturation of sdAbs. While studies have explored the role of noncanonical disulfide bonds in camelid-derived VHH aggregation, research on thermal aggregation in shark-derived sdAbs (also known as VNARs) remains scarce, limiting their potential for further optimization. In this study, the role of noncanonical disulfide bonds in VNAR structural robustness, aggregation, and affinity has been simultaneously investigated. Enzyme-linked immunosorbent assay (ELISA), circular dichroism, and intrinsic fluorescence were carried out to compare thermal antigen-binding stability, refolding abilities, and melting temperatures of four wild VNARs B7, 1N9, 2E6, and 2E11 specific for different antigens. Meanwhile, nano differential scanning fluorimetry (nanoDSF) was applied, for the first time, to monitor the thermal aggregation of VNARs. Notably, 2E11, which lacked the noncanonical disulfide bond, demonstrated impressive performance in many aspects. When alanine mutation was engineered to remove the CDR1-CDR3 disulfide bond in 2E6, its refolding rate was increased, and thermal aggregation was prevented significantly. Furthermore, 2E6 exhibited enhanced thermal antigen-binding stability despite reduced structural robustness and affinity. This study provides deeper insights and theoretical support for improving VNAR biophysical properties, with potential applications in enhancing immunoassay performance.

The development of stable and efficient drug delivery systems is essential for advancing therapeutic applications. Here, we present an innovative approach using a mirror-image RNA (l-RNA) nanostructure to enhance the biostability and drug delivery efficiency. We engineered an l-RNA three-way junction structure conjugated with both small interfering RNA (siRNA) targeting MCL1 and the chemotherapeutic agent doxorubicin for targeted and synergistic drug delivery. This codelivery strategy leverages the combined effects of doxorubicin and MCL1 siRNA, achieving improved therapeutic outcomes. The l-RNA nanostructure demonstrates superior stability compared with natural d-RNA, resulting in reduced toxicity in healthy cells while maintaining therapeutic efficacy in cancer cells. This indicates that l-RNA nanostructures may offer enhanced biosafety when applied as therapeutic agents. The addition of folic acid (FA) to the nanostructure surface substantially increases both delivery specificity and endosomal escape efficiency, optimizing targeted delivery. Structural modeling also suggests a distinctive binding conformation of doxorubicin with l-DNA, setting it apart from native DNA interactions. This study highlights the potential of mirror-image nucleic acid nanostructures as robust and precise platforms for combinatorial drug delivery in cancer treatment.

Heterobivalent fusion aptamers that target a single protein show significant promise for studying protein-protein interactions. However, a major challenge is finding two distinct aptamers that can simultaneously recognize the same protein. In this study, we used a novel technique called Aptamer-Assisted DNA SELEX (AADS) to isolate two distinct aptamers capable of recognizing different sites on the programmed death-ligand 1 (PD-L1) protein. Initially, Aptamer 1 (P1C2) was identified by using conventional DNA SELEX targeting the PD-L1 protein. Subsequently, Aptamer 2 (P1CSC) was obtained via AADS, which was designed to bind to the PD-L1/P1C2 complex. After confirming that both aptamers could simultaneously recognize the PD-L1 protein, we engineered fusion aptamers by optimizing their orientation and linker sequences, resulting in the creation of the optimized fusion aptamer, P1CSC-T7-P1C1. Our fusion aptamer targeting PD-L1 demonstrated remarkable specificity and affinity, effectively inhibiting PD-1/PD-L1 interactions at both the protein and cellular levels. These findings highlight the potential of fusion aptamers via AADS as powerful tools for targeting the PD-L1 protein and cancer cells (A549 cells). This represents a significant advancement in aptamer-based molecular recognition and has the potential to drive innovation as a versatile method for targeting a wide range of proteins.