3D bioprinting of high-performance hydrogel with in-situ birth of stem cell spheroids.

Digital light processing (DLP)-based bioprinting technology holds immense promise for the advancement of hydrogel constructs in biomedical applications. However, creating high-performance hydrogel constructs with this method is still a challenge, as it requires balancing the physicochemical properties of the matrix while also retaining the cellular activity of the encapsulated cells. Herein, we propose a facile and practical strategy for the 3D bioprinting of high-performance hydrogel constructs through the in-situ birth of stem cell spheroids. The strategy is achieved by loading the cell/dextran microdroplets within gelatin methacryloyl (GelMA) emulsion, where dextran functions as a decoy to capture and aggregate the cells for bioprinting while GelMA enables the mechanical support without losing the structural complexity and fidelity. Post-bioprinting, the leaching of dextran results in a smooth curved surface that promotes in-situ birth of spheroids within hydrogel constructs. This process significant enhances differentiation potential of encapsulated stem cells. As a proof-of-concept, we encapsulate dental pulp stem cells (DPSCs) within hydrogel constructs, showcasing their regenerative capabilities in dentin and neovascular-like structures in vivo. The strategy in our study enables high-performance hydrogel tissue construct fabrication with DLP-based bioprinting, which is anticipated to pave a promising way for diverse biomedical applications.

Gold Nano Frameworks with Mesopores for Synergistic Immune-Thermal Therapy in Hepatic Carcinoma: A Paradigm Shift in Immune Checkpoint Blockade.

Immune checkpoint blockade (ICB) therapy, while showing promise in various cancers, exhibits limited effectiveness in hepatic carcinoma due to the tumor's immunosuppressive microenvironment (TME) and challenges associated with immune cell infiltration. Efforts to transform the "cold" TME into an "inflamed" state, notably through chemo-immunotherapy, have sparked interest due to their potential to induce immunogenic cell death and augment the infiltration of cytotoxic T lymphocytes (CTLs). Nonetheless, the efficacy of chemo-immunotherapy is often compromised by suboptimal pharmacokinetics, poor tumor accumulation, and off-target toxicity. Herein, in response, we introduce an innovative, milder thermal therapeutic approach leveraging gold nano frameworks with mesopores for the targeted delivery of the immunostimulant imiquimod and NIR-II photothermal therapy. This strategy employs targeted molecule modifications to ensure precise tumor targeting, guided by photoacoustic imaging. Subsequent to mild thermal treatment, there is a release of immunogenic proteins (CRT and HSP90), enhancing tumor immunogenicity. Assisted by imiquimod, substantial CTL infiltration occurs, accompanied by pro-inflammatory factor release (TNF-α, IL-6), transforming M2 macrophages into the M1 phenotype. Ultimately, the proposed strategy combines PD-L1/PD-1 blockade, imiquimod and mild thermal treatment to synergistically enhance tumor immunogenicity, remodel the TME, and restrain hepatic carcinoma, making strides in ICB synergistic immune-thermal therapy.

NIR-II-Absorbing NDI Polymer with Superior Penetration Depth for Enhanced Photothermal Therapy Efficiency of Hepatocellular Carcinoma.

Introduction: Hepatocellular carcinomas (HCC) have a high morbidity and mortality rate, and is difficult to cure and prone to recurrence when it has already developed. Therefore, early detection and efficient treatment of HCC is necessary. Methods: In this study, we synthesized a novel NDI polymer with uniform size, long-term stability, and high near-infrared two-zone (NIR-II) absorption efficiency, which can greatly enhance the effect of photothermal therapy (PTT) after intravenous injection into Huh-7-tumor bearing mice. Results: The in vitro and in vivo studies showed that NDI polymer exhibited excellent NIR-guided PTT treatment, and the antitumor effect was approximately 88.5%, with obvious antimetastatic effects. Conclusion: This study developed an NDI polymer-mediated integrated diagnostic and therapeutic modality for NIR-II fluorescence imaging and photothermal therapy.

Dissolving microneedles for transdermal drug delivery in cancer immunotherapy.

Immunotherapy is an important approach in cancer treatment. Transdermal administration is emerging as a promising method for delivering immunotherapeutics. Dissolving microneedles are made mainly of soluble or biodegradable polymers and have garnered widespread attention due to their painlessness, safety, convenience, excellent drug loading capacity, and easy availability of various materials, making them an ideal transdermal delivery system. This review comprehensively summarized the preparation methods, materials, and applications of dissolving microneedles in cancer vaccines, immune checkpoint inhibitors, and adoptive cell therapy. Additionally, the challenges and perspectives associated with their future clinical translation are discussed.

Non-viral Gene Therapy for Melanoma Using Lysenin from Eisenia Foetida.

Earthworms, long utilized in traditional medicine, serve as a source of inspiration for modern therapeutics. Lysenin, a defensive factor in the coelom fluid of the earthworm Eisenia fetida, has multiple bioactivities. However, the inherent toxicity of Lysenin as a pore-forming protein (PFP) restricts its application in therapy. Here, a gene therapy strategy based on Lysenin for cancer treatment is presented. The formulation consists of polymeric nanoparticles complexed with the plasmid encoding Lysenin. After transfection in vitro, melanoma cells can express Lysenin, resulting in necrosis, autophagy, and immunogenic cell death. The secretory signal peptide alters the intracellular distribution of the expressed product of Lysenin, thereby potentiating its anticancer efficacy. The intratumor injection of Lysenin gene formulation can efficiently kill the transfected melanoma cells and activate the antitumor immune response. Notably, no obvious systemic toxicity is observed during the treatment. Non-viral gene therapy based on Lysenin derived from Eisenia foetida exhibits potential in cancer therapy, which can inspire future cancer therapeutics.

Advanced Nanomaterials in Medical 3D Printing.

3D printing is now recognized as a significant tool for medical research and clinical practice, leading to the emergence of medical 3D printing technology. It is essential to improve the properties of 3D-printed products to meet the demand for medical use. The core of generating qualified 3D printing products is to develop advanced materials and processes. Taking advantage of nanomaterials with tunable and distinct physical, chemical, and biological properties, integrating nanotechnology into 3D printing creates new opportunities for advancing medical 3D printing field. Recently, some attempts are made to improve medical 3D printing through nanotechnology, providing new insights into developing advanced medical 3D printing technology. With high-resolution 3D printing technology, nano-structures can be directly fabricated for medical applications. Incorporating nanomaterials into the 3D printing material system can improve the properties of the 3D-printed medical products. At the same time, nanomaterials can be used to expand novel medical 3D printing technologies. This review introduced the strategies and progresses of improving medical 3D printing through nanotechnology and discussed challenges in clinical translation.

Antheraea pernyi silk fibroin bioinks for digital light processing 3D printing.

The application of three-dimensional (3D) bioprinting has increased in the biomedical field. The lack of bioinks with both biocompatibility and printability is still a problem to be solved. Silk fibroin materials have good biocompatibility and have a broad application prospect in the field of biomedical materials. At present, most research usually involves Bombyx mori silk fibroin (BSF). However, BSF has low cell adhesion. Compared with BSF, Antheraea pernyi silk fibroin (ASF) isolated from typical non-mulberry silk exhibits a unique arginine-glycine-aspartate (RGD) sequence with good cell adhesion enhancement. In this study, we developed a bioink based on ASF for digital light processing (DLP) 3D bioprinting. The ASF-based bioinks (ASF-MA) were produced by a methacryloylation process using methacrylic anhydride (MA) to achieve the properties of photopolymerization reaction. The ASF-MA hydrogel has mechanical properties, biocompatibility, and especially cell adhesion. Meanwhile, we found that the ASF-MA hydrogels promoted the adhesion, migration, and proliferation of S16 cells. Hence, the ASF-MA hydrogels had the potential applications in biomedical fields.

3D printed elastic hydrogel conduits with 7,8-dihydroxyflavone release for peripheral nerve repair.

Nerve guide conduit is a promising treatment for long gap peripheral nerve injuries, yet its efficacy is limited. Drug-releasable scaffolds may provide reliable platforms to build a regenerative microenvironment for nerve recovery. In this study, an elastic hydrogel conduit encapsulating with prodrug nanoassemblies is fabricated by a continuous 3D printing technique for promoting nerve regeneration. The bioactive hydrogel is comprised of gelatin methacryloyl (GelMA) and silk fibroin glycidyl methacrylate (SF-MA), exhibiting positive effects on adhesion, proliferation, and migration of Schwann cells. Meanwhile, 7,8-dihydroxyflavone (7,8-DHF) prodrug nanoassemblies with high drug-loading capacities are developed through self-assembly of the lipophilic prodrug and loaded into the GelMA/SF-MA hydrogel. The drug loading conduit could sustainedly release 7,8-DHF to facilitate neurite elongation. A 12 â€‹mm nerve defect model is established for therapeutic efficiency evaluation by implanting the conduit through surgical suturing with rat sciatic nerve. The electrophysiological, morphological, and histological assessments indicate that this conduit can promote axon regeneration, remyelination, and function recovery by providing a favorable microenvironment. These findings implicate that the GelMA/SF-MA conduit with 7,8-DHF release has potentials in the treatment of long-gap peripheral nerve injury.

3D printing of microneedle arrays for hair regeneration in a controllable region.

Hair loss is a common skin disease that causes intense emotional suffering. Hair regeneration in a personalized area is highly desirable for patients with different balding conditions. However, the existing pharmaceutical treatments have difficulty precisely regenerating hair in a desired area. Here, we show a method to precisely control the hair regeneration using customized microneedle arrays (MNAs). The MNA with a customized shape is fast fabricated by a static optical projection lithography process in seconds, which is a 3D printing technology developed by our group. In the mouse model, MNA treatment could induce hair regrowth in a defined area corresponding to the customized shape of MNA. And the regenerated hair promoted by MNAs had improved quality. Cellular and molecular analysis indicated that MNA treatment could recruit macrophages in situ and then initiate the proliferation of hair follicle stem cells, thereby improving hair regeneration. Meanwhile, the activation of the Wnt/ß-catenin signaling pathway was observed in hair follicles. The expressions of Hgf, Igf 1 and Tnf-α were also upregulated in the treated skin, which may also be beneficial for the MNA-induced hair regeneration. This study provides a strategy to precisely control hair regeneration using customized microneedle arrays by recruiting macrophages in situ, which holds the promise for the personalized treatment of hair loss.

Biomimetic Peridontium Patches for Functional Periodontal Regeneration.

The unique structure of the periodontium, including the alveolar bone, cementum, and periodontal ligament (PDL), presents difficulties for the regeneration of its intricate organization. Irreversible structural breakdown of the periodontium increases the risk of tooth loosening and loss. Although the current therapies can restore the periodontal hard tissues to a certain extent, the PDL with its high directionality of multiple groups with different orientations and functions cannot be reconstructed. Here, biomimetic peridontium patches (BPPs) for functional periodontal regeneration using a microscale continuous digital light projection bioprinting method is reported. Orthotopic transplantation in the mandibles shows effective periodontal reconstruction. The resulting bioengineered tissues closely resembles natural periodontium in terms of the "sandwich structures," especially the correctly oriented fibers, showing different and specific orientation in different regions of the tooth root, which has never been found in previous studies. Furthermore, after the assessment of clinically functional properties it is found that the regenerative periodontium can achieve stable tooth movement under orthodontic migration force with no adverse consequences. Overall, the BPPs promote reconstruction of the functional periodontium and the complex microstructure of the periodontal tissue, providing a proof of principle for the clinical functional treatment of periodontal defects.

Acquired semi-squamatization during chemotherapy suggests differentiation as a therapeutic strategy for bladder cancer.

Cisplatin-based chemotherapy remains the primary treatment for unresectable and metastatic muscle-invasive bladder cancers (MIBCs). However, tumors frequently develop chemoresistance. Here, we established a primary and orthotopic MIBC mouse model with gene-edited organoids to recapitulate the full course of chemotherapy in patients. We found that partial squamous differentiation, called semi-squamatization, is associated with acquired chemoresistance in both mice and human MIBCs. Multi-omics analyses showed that cathepsin H (CTSH) is correlated with chemoresistance and semi-squamatization. Cathepsin inhibition by E64 treatment induces full squamous differentiation and pyroptosis, and thus specifically restrains chemoresistant MIBCs. Mechanistically, E64 treatment activates the tumor necrosis factor pathway, which is required for the terminal differentiation and pyroptosis of chemoresistant MIBC cells. Our study revealed that semi-squamatization is a type of lineage plasticity associated with chemoresistance, suggesting that differentiation via targeting of CTSH is a potential therapeutic strategy for the treatment of chemoresistant MIBCs.

A sulfhydryl blocking reagent BT-4 sensitizes cisplatin-based micelle prodrugs for efficient treatment of breast cancer.

Detoxification of glutathione (GSH) and insufficient cellular uptake of cisplatin (CDDP) severely compromised the therapeutic efficacy of CDDP. Here, a nano-delivery system (BT-4@PtPPNPs) for CDDP prodrug (C16-Pt(Ⅳ)-PEG) based on a novel sulfhydryl blocking reagent methyl 2-(methylsulfonyl) benzothiazole-6-carboxylate (BT-4) was developed. On the one hand, BT-4 can deplete GSH in tumor cells by directly interacting with reactive sulfhydryl group on GSH, thereby increasing the cytotoxicity of CDDP. On the other hand, the CDDP prodrug carrier C16-Pt(IV)-PEG can promote the distribution of CDDP in tumors, reduce the probability of unexpected inactivation of CDDP, and reduce the content of GSH in tumor cells during the conversion to CDDP, thereby making CDDP more effective for treatment. The results showed that the optimized BT-4@PtPPNPs with a small particle size (130 nm) exhibited notable cytotoxicity and apoptosis of 4T1 cells. BT-4@PtPPNPs not only significantly improved the uptake of drugs by tumor cells, but also rapidly targeted and accumulated in the tumors for a long time. Moreover, in vivo efficacy studies showed that BT-4@PtPPNPs could effectively inhibit tumor growth, inhibiting 60.85 % of tumors in a 4T1 breast cancer mice model, showing superior antitumor activity, which can be attributed to GSH-triggered CDDP tolerance reversal. Overall, this study provides an attractive and simple strategy to combine novel sulfhydryl blockers and CDDP prodrugs to potentiate the efficacy of CDDP in breast cancer.

Strategies for improving adipose-derived stem cells for tissue regeneration.

Adipose-derived stem cells (ADSCs) have promising applications in tissue regeneration. Currently, there are only a few ADSC products that have been approved for clinical use. The clinical application of ADSCs still faces many challenges. Here, we review emerging strategies to improve the therapeutic efficacy of ADSCs in tissue regeneration. First, a great quantity of cells is often needed for the stem cell therapies, which requires the advanced cell expansion technologies. In addition cell-derived products are also required for the development of 'cell-free' therapies to overcome the drawbacks of cell-based therapies. Second, it is necessary to strengthen the regenerative functions of ADSCs, including viability, differentiation and paracrine ability, for the tissue repair and regeneration required for different physiological and pathophysiological conditions. Third, poor delivery efficiency also restricts the therapeutic effect of ADSCs. Effective methods to improve cell delivery include alleviating harsh microenvironments, enhancing targeting ability and prolonging cell retention. Moreover, we also point out some critical issues about the sources, effectiveness and safety of ADSCs. With these advanced strategies to improve the therapeutic efficacy of ADSCs, ADSC-based treatment holds great promise for clinical applications in tissue regeneration.

3D-printed microneedle arrays for drug delivery.

Microneedle arrays provide an efficient tool for transdermal drug delivery in a minimally invasive and painless manner, showing great potential applications in medicine. However, it remains challenging to fabricate the desired microneedle arrays, because of their micron-scale size and fine structure. Novel manufacturing technologies are very wanted for the development of microneedle arrays, which would solidly advance the clinical translation of microneedle arrays. 3D printing technology is a powerful manufacturing technology with superiority in fabricating personalized and complex structures. Currently, 3D printing technology has been employed to fabricate microneedle arrays, which could push more microneedle arrays into clinic and inspire the development of future microneedle arrays. This work reviews the art of 3D printing microneedle arrays, the benefits of fabricating microneedle arrays with 3D printing, and the considerations for clinical translation of 3D-printed microneedle arrays. This work provides an overview of the current 3D-printed microneedle arrays in drug delivery.

Fast Customization of Hollow Microneedle Patches for Insulin Delivery.

Hollow microneedle patches (HMNPs) have great promise for efficient and precise transdermal drug delivery in a painless manner. Currently, the clinical application of HMNPs is restricted by its complex manufacturing processes. Here, we use a new three-dimensional (3D) printing technology, static optical projection lithography (SOPL), for the fast fabrication of HMNPs. In this technology, a light beam is modulated into a customized pattern by a digital micromirror device (DMD) and projected to induce the spatial polymerization of monomer solutions which is controlled by the distribution of the light intensity in the monomer solutions. After an annulus picture is inputted into the DMD via the computer, the microneedles with hollow-cone structure can be precisely printed in seconds. By designing the printing pictures, the personalized HMNPs can be fast customized, which can afford the scale-up preparation of personalized HMNPs. Meanwhile, the obtained hollow microneedles (HMNs) have smooth surface without layer-by-layer structure in the commonly 3D-printed products. After being equipped with a micro-syringe, the HMNPs can efficiently deliver insulin into the skin by injection, resulting in effective control of the blood glucose level in diabetic mice. This work demonstrates a SOPL-based 3D printing technology for fast customization of HMNPs with promising medical applications.

3D Printing Mini-Capsule Device for Islet Delivery to Treat Type 1 Diabetes.

Transplantation of encapsulated islets has been shown to hold a promising potential treatment for type 1 diabetes (T1D). However, there are several obstacles to overcome, such as immune rejection by the host of the grafts, sustainability of islet function, and retrievability or replacement of the encapsulated system, hinder their clinical applications. In this study, mini-capsule devices containing islets were fabricated by using digital light processing (DLP) 3D printing. To ensure a high survival rate and low immunogenicity of the fabricated islets, 20s was selected as the most suitable printing condition. Meanwhile, the mini-capsule devices with a groove structure were fabricated to prevent islet cells leakage. Subcutaneous transplantations of encapsulated islets in immunocompetent C57BL/6 mice indicated significant improvement in the symptoms of streptozotocin-induced hyperglycemia without any immunosuppression treatment for at least 15 weeks. In vivo intraperitoneal glucose tolerance tests (IPGTT) performed at different time points demonstrated therapeutically relevant glycemic ameliorate of the device. The implants retrieved after 15 weeks still contained viable and adequate numbers of islet cells. The results of this study indicate that the proposed mini-capsule device can deliver sufficient islet cell mass, prevent islet cells leakage, and maintain long-term cell survival while allowing easy retrieval. Furthermore, the proposed encapsulated islets may help with T1D cellular treatment by overcoming the obstacles of islet transplantation.

Nerve transfer with 3D-printed branch nerve conduits.

Background: Nerve transfer is an important clinical surgical procedure for nerve repair by the coaptation of a healthy donor nerve to an injured nerve. Usually, nerve transfer is performed in an end-to-end manner, which will lead to functional loss of the donor nerve. In this study, we aimed to evaluate the efficacy of 3D-printed branch nerve conduits in nerve transfer. Methods: Customized branch conduits were constructed using gelatine-methacryloyl by 3D printing. The nerve conduits were characterized both in vitro and in vivo. The efficacy of 3D-printed branch nerve conduits in nerve transfer was evaluated in rats through electrophysiology testing and histological evaluation. Results: The results obtained showed that a single nerve stump could form a complex nerve network in the 3D-printed multibranch conduit. A two-branch conduit was 3D printed for transferring the tibial nerve to the peroneal nerve in rats. In this process, the two branches were connected to the distal tibial nerve and peroneal nerve. It was found that the two nerves were successfully repaired with functional recovery. Conclusions: It is implied that the two-branch conduit could not only repair the peroneal nerve but also preserve partial function of the donor tibial nerve. This work demonstrated that 3D-printed branch nerve conduits provide a potential method for nerve transfer.

Histones released by NETosis enhance the infectivity of SARS-CoV-2 by bridging the spike protein subunit 2 and sialic acid on host cells.

Neutrophil extracellular traps (NETs) can capture and kill viruses, such as influenza viruses, human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV), thus contributing to host defense. Contrary to our expectation, we show here that the histones released by NETosis enhance the infectivity of SARS-CoV-2, as found by using live SARS-CoV-2 and two pseudovirus systems as well as a mouse model. The histone H3 or H4 selectively binds to subunit 2 of the spike (S) protein, as shown by a biochemical binding assay, surface plasmon resonance and binding energy calculation as well as the construction of a mutant S protein by replacing four acidic amino acids. Sialic acid on the host cell surface is the key molecule to which histones bridge subunit 2 of the S protein. Moreover, histones enhance cell-cell fusion. Finally, treatment with an inhibitor of NETosis, histone H3 or H4, or sialic acid notably affected the levels of sgRNA copies and the number of apoptotic cells in a mouse model. These findings suggest that SARS-CoV-2 could hijack histones from neutrophil NETosis to promote its host cell attachment and entry process and may be important in exploring pathogenesis and possible strategies to develop new effective therapies for COVID-19.

Fast Customization of Microneedle Arrays by Static Optical Projection Lithography.

Customized microneedle arrays (CMNAs) hold great promise for precise transdermal delivery in a minimally invasive manner. Currently, the fast customization of microneedle arrays remains a great challenge. Here, we show a static optical projection lithography (SOPL) technology for fast 3D printing CMNAs. In this technology, the digital light is statically projected to induce the spatial polymerization of monomer solutions, and therefore microneedle formation can be precisely controlled by the intensity distribution of the projected light. The obtained CMNAs do not have the stair-like surface and layer-by-layer structure that are associated with the common 3D-printing technologies. This method enables fast fabrication of CMNAs with designed shape, size, and distribution in seconds without mechanical motion system. Up-conversion nanoparticles (UCNPs) were delivered into skin by the CMNAs, to form a personalized dot matrix for in vivo information storage. Under the irradiation of near-infrared (NIR) light, the UCNPs in skin displayed a visible dot matrix, presenting information encoded in the structure of CMNAs. This work demonstrates a SOPL technology for rapidly customizing high-quality microneedle arrays and a CMNA-mediated in vivo information storage strategy.

Targeted Nanotherapeutics Using LACTB Gene Therapy Against Melanoma.

INTRODUCTION: ß-lactamase (LACTB) is a tumor suppressor gene in various tumors including melanoma. However, it remains challenging to efficiently deliver the LACTB gene into melanoma. Recently, we designed a nonviral nanocarrier iRGD/DOTAP/MPEG-PDLLA (iDPP) that could deliver gene targetedly to melanoma efficiently without obvious adverse effects. METHODS: In this study, the tumor-targeted nanoparticle iDPP was prepared to deliver LACTB gene to treat melanoma in vitro and in vivo. First, the expression level of LACTB in 6 clinical specimens of melanoma patients was evaluated. Subsequently, the characteristics of iDPP/LACTB nanocomplexes were studied. Afterwards, the in vitro and in vivo anti-tumor efficacy of the iDPP/LACTB nanocomplexes were explored utilizing the B16-F10 mouse melanoma cell line and the B16-F10 subcutaneous melanoma model. RESULTS: Compared with the normal epithelium, the expression level of LACTB in melanoma tissues was significantly downregulated. In vitro B16-F10 cell tests showed iDPP/LACTB nanocomplexes could increase the mRNA levels of P21, Bid, Bax, Pidd1, and Sival genes and up-regulate the p53 signaling pathway of melanoma cells, thus promoting cell apoptosis and blocking the cell cycle. Injected intravenously, iDPP nanoparticles could deliver DNA to the subcutaneous melanoma targetedly. Based on in vivo mouse xenograft model, iDPP/LACTB nanocomplexes could effectively inhibit tumor proliferation and induce tumor apoptosis, thus significantly inhibiting melanoma growth (tumor inhibition rate is about 68%) in the subcutaneous B16-F10 melanoma model. CONCLUSION: The downregulated LACTB might be a potential target for melanoma therapy. The iDPP/LACTB nanocomplexes could inhibit the growth of the mouse melanoma without obvious side effects, which provide a new option for melanoma gene therapy research.