Differentiated mesenchymal stem cells-derived exosomes immobilized in decellularized sciatic nerve hydrogels for peripheral nerve repair.

Peripheral nerve injury (PNI) and the limitations of current treatments often result in incomplete sensory and motor function recovery, which significantly impact the patient's quality of life. While exosomes (Exo) derived from stem cells and Schwann cells have shown promise on promoting PNI repair following systemic administration or intraneural injection, achieving effective local and sustained Exo delivery holds promise to treat local PNI and remains challenging. In this study, we developed Exo-loaded decellularized porcine nerve hydrogels (DNH) for PNI repair. We successfully isolated Exo from differentiated human adipose-derived mesenchymal stem cells (hADMSC) with a Schwann cell-like phenotype (denoted as dExo). These dExo were further combined with polyethylenimine (PEI), and DNH to create polyplex hydrogels (dExo-loaded pDNH). At a PEI content of 0.1%, pDNH showed cytocompatibility for hADMSCs and supported neurite outgrowth of dorsal root ganglions. The sustained release of dExos from dExo-loaded pDNH persisted for at least 21 days both in vitro and in vivo. When applied around injured nerves in a mouse sciatic nerve crush injury model, the dExo-loaded pDNH group significantly improved sensory and motor function recovery and enhanced remyelination compared to dExo and pDNH only groups, highlighting the synergistic regenerative effects. Interestingly, we observed a negative correlation between the number of colony-stimulating factor-1 receptor (CSF-1R) positive cells and the extent of PNI regeneration at the 21-day post-surgery stage. Subsequent in vitro experiments demonstrated the potential involvement of the CSF-1/CSF-1R axis in Schwann cells and macrophage interaction, with dExo effectively downregulating CSF-1/CSF-1R signaling.

Multiscale embedded printing of engineered human tissue and organ equivalents.

Creating tissue and organ equivalents with intricate architectures and multiscale functional feature sizes is the first step toward the reconstruction of transplantable human tissues and organs. Existing embedded ink writing approaches are limited by achievable feature sizes ranging from hundreds of microns to tens of millimeters, which hinders their ability to accurately duplicate structures found in various human tissues and organs. In this study, a multiscale embedded printing (MSEP) strategy is developed, in which a stimuli-responsive yield-stress fluid is applied to facilitate the printing process. A dynamic layer height control method is developed to print the cornea with a smooth surface on the order of microns, which can effectively overcome the layered morphology in conventional extrusion-based three-dimensional bioprinting methods. Since the support bath is sensitive to temperature change, it can be easily removed after printing by tuning the ambient temperature, which facilitates the fabrication of human eyeballs with optic nerves and aortic heart valves with overhanging leaflets on the order of a few millimeters. The thermosensitivity of the support bath also enables the reconstruction of the full-scale human heart on the order of tens of centimeters by on-demand adding support bath materials during printing. The proposed MSEP demonstrates broader printable functional feature sizes ranging from microns to centimeters, providing a viable and reliable technical solution for tissue and organ printing in the future.

Digital Light Processing 4D Printing of Poloxamer Micelles for Facile Fabrication of Multifunctional Biocompatible Hydrogels as Tailored Wearable Sensors.

The lack of both digital light processing (DLP) compatible and biocompatible photopolymers, along with inappropriate material properties required for wearable sensor applications, substantially hinders the employment of DLP 3D printing in the fabrication of multifunctional hydrogels. Herein, we discovered and implemented a photoreactive poloxamer derivative, Pluronic F-127 diacrylate, which overcomes these limitations and is optimized to achieve DLP 3D printed micelle-based hydrogels with high structural complexity, resolution, and precision. In addition, the dehydrated hydrogels exhibit a shape-memory effect and are conformally attached to the geometry of the detection point after rehydration, which implies the 4D printing characteristic of the fabrication process and is beneficial for the storage and application of the device. The excellent cytocompatibility and in vivo biocompatibility further strengthen the potential application of the poloxamer micelle-based hydrogels as a platform for multifunctional wearable systems. After processing them with a lithium chloride (LiCl) solution, multifunctional conductive ionic hydrogels with antifreezing and antiswelling properties along with good transparency and water retention are easily prepared. As capacitive flexible sensors, the DLP 3D printed micelle-based hydrogel devices exhibit excellent sensitivity, cycling stability, and durability in detecting multimodal deformations. Moreover, the DLP 3D printed conductive hydrogels are successfully applied as real-time human motion and tactile sensors with satisfactory sensing performances even in a -20 °C low-temperature environment.

Engineering Neurotoxin-Functionalized Exosomes for Targeted Delivery to the Peripheral Nervous System.

The administration of therapeutics to peripheral nerve tissue is challenging due to the complexities of peripheral neuroanatomy and the limitations imposed by the blood-nerve barrier (BNB). Therefore, there is a pressing need to enhance delivery effectiveness and implement targeted delivery methods. Recently, erythrocyte-derived exosomes (Exos) have gained widespread attention as biocompatible vehicles for therapeutics in clinical applications. However, engineering targeted Exos for the peripheral nervous system (PNS) is still challenging. This study aims to develop a targeted Exo delivery system specifically designed for presynaptic terminals of peripheral nerve tissue. The clostridium neurotoxin, tetanus toxin-C fragment (TTC), was tethered to the surface of red blood cell (RBC)-derived Exos via a facile and efficient bio-orthogonal click chemistry method without a catalyst. Additionally, Cyanine5 (Cy5), a reactive fluorescent tag, was also conjugated to track Exo movement in both in vitro and in vivo models. Subsequently, Neuro-2a, a mouse neuronal cell line, was treated with dye-labeled Exos with/without TTC in vitro, and the results indicated that TTC-Exos exhibited more efficient accumulation along the soma and axonal circumference, compared to their unmodified counterparts. Further investigation, using a mouse model, revealed that within 72 h of intramuscular administration, engineered TTC-Exos were successfully transported into the neuromuscular junction and sciatic nerve tissues. These results indicated that TTC played a crucial role in the Exo delivery system, improving the affinity to peripheral nerves. These promising results underscore the potential of using targeted Exo carriers to deliver therapeutics for treating peripheral neuropathies.

In Vitro and In Vivo Evaluation of Targeted Fluorescent Imaging Agents for Diagnosis and Resection of Cancer.

Local re-occurrence of cancer in patients with solid tumors is currently the most common reason for failure of treatment strategies. This fact indicates that prevailing approaches for tumor resection can cure only 50% of patients. A major cause of failure in tumor resection is off-target drug cytotoxicity and lack of sensitivity in tumor detection methods. These disadvantages are addressed with the development of targeted therapy and diagnostics, which significantly aid treatment strategies. Targeted diagnostics exploit properties of tumor cells that show significant up-regulation of tumor biomarkers. These biomarkers are targeted by a homing ligand attached to a fluorophore for visual inspection during surgery. However, these approaches suffer from disadvantages like high autofluorescence from background tissues, tissue absorption, and scattering, resulting in decreased image sensitivity and resolution. The use of near-infrared (NIR) fluorophores to overcome these drawbacks has generated unprecedented interest among researchers. The NIR window lies within the range of 650 to 1,700 nm, which results in reduced absorption and scattering by the tissues, thereby providing deeper tissue penetration and reduced autofluorescence. NIR fluorophores can be designed to target tumor biomarkers such as prostate specific membrane antigen (PSMA) or folate receptors found over-expressed on cancer tissues. These targeted fluorophores consist of small-molecule ligands conjugated with NIR dyes that bind with high specificity to PSMA and folic acid receptors. In this protocol, we have extensively described the methodology for the synthesis of targeted NIR agents for PSMA (DUPA-NIR bioconjugate) and folic acid (folate-NIR bioconjugate), along with detailed steps for preclinical evaluation. Procedures to calculate the binding affinity to cancer cells in vitro are described, along with uptake and biodistribution in different mice models in vivo. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis and purification of DUPA and folate-peptide linkers via a SPPS strategy Basic Protocol 2: Conjugation, purification, and characterization of targeted bioconjugates with NIR probe for deep-tissue imaging applications Basic Protocol 3: In vitro evaluation of binding affinity of targeted DUPA-NIR and folate-NIR bioconjugates using a spectrophotometer Basic Protocol 4: Induction of tumor in mice to develop CDX or metastatic tumor models Basic Protocol 5: Intravenous administration of targeted DUPA-NIR and folate-NIR bioconjugates in mouse CDX or metastatic tumor models for deep-tissue NIR imaging and tumor resection.

Imaging of prostate cancer: optimizing affinity to prostate specific membrane antigen by spacer modifications in a tumor spheroid model.

Early diagnosis of prostate cancer (PCa) is crucial for staging, treatment and management of patients. Prostate specific membrane antigen (PSMA), highly over-expressed on PCa cells, is an excellent target for selective imaging of PCa. In recent years, various scaffolds have been explored as potential carriers to target diagnostic and therapeutic agents to PSMA+ tumour cells. Numerous fluorescent or radioisotope probes linked via a peptide linker have been developed that selectively binds to PCa cells. However, there are very few reports that examine the effects of chemical modifications in the peptide linker of an imaging probe on its affinity to PSMA protein. This report systematically investigates the impact of hydrophobic aromatic moieties in the peptide linker on PSMA affinity and in vitro performance. For this, a series of fluorescent bioconjugates 12-17 with different aromatic spacers were designed, synthesized, and their interactions within the PSMA pocket were first analysed in silico. Cell uptake studies were then performed for 12-17 in PSMA+ cell lines and 3D tumour models in vitro. Binding affinity values of 12-17 were found to be in the range of 36 to 157.9 nM, and 12 with three aromatic groups in the spacer exhibit highest affinity (KD = 36 nM) compared to 17 which is devoid of aromatic groups. These studies suggest that aromatic groups in the spacer region can significantly affect deep tissue imaging of fluorescent bioconjugates. Bioconjugate 12 can be a promising diagnostic tool, and conjugation to near-infrared agents would further its applications in deep-tissue imaging and surgery. Communicated by Ramaswamy H. Sarma.

Synthesis of 1-indolyl-3,5,8-substituted γ-carbolines: one-pot solvent-free protocol and biological evaluation.

1,5-Disubstituted indole-2-carboxaldehyde derivatives 1a-h and glycine alkyl esters 2a-c are shown to undergo a novel cascade imination-heterocylization in the presence of the organic base DIPEA to provide 1-indolyl-3,5,8-substituted γ-carbolines 3aa-ea in good yields. The γ-carbolines are fluorescent and exhibit anticancer activities against cervical, lung, breast, skin, and kidney cancer cells.

Agarose Micro-Well Platform for Rapid Generation of Homogenous 3D Tumor Spheroids.

In recent years, 3D culture of tumor spheroids has managed to revolutionize cancer research and drug discovery. 2D monolayer cells grown in cell culture flasks undergo radical changes in cell behavior, structure, and function owing to varying environmental cues and are unable to provide predictive data for preclinical evaluation. 3D tumor spheroids can better recapitulate tumor architecture, cell-cell and cell-matrix connectivity, and the tissue complexity of tumors grown in animal models. However, many of the existing techniques to culture 3D spheroids are time-consuming and ineffective and produce irregular-shaped spheroids that cannot be easily incorporated in biological assays. The set of protocols described herein makes use of a commercial hair brush as a template to create concave micro-well impressions in agarose. This technique is easy, inexpensive, and adaptable and also has the ability to produce uniform, homogenous cancer spheroids, with large diameter (∼1000 µm) and thickness (∼250 µm), within 24 to 48 hr after cell seeding. The 3D spheroids produced using the agarose micro-well platform function as an excellent 3D in vitro model for understanding the extent of penetration, uptake, and distribution of targeted cargos such as a diagnostic or therapeutic agents for identification and treatment of cancer. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Fabrication of agarose micro-well scaffold for growing tumor spheroids using a commercial hair brush Basic Protocol 2: Formation of homogenous tumor spheroids in agarose micro-well platform Basic Protocol 3: Assessing viability of 3D tumor spheroids grown in agarose micro-wells using confocal microscopy Basic Protocol 4: Analyzing uptake and penetration of targeted fluorescent bioconjugate in 3D tumor spheroids using two-photon imaging.

Tyrosine-based asymmetric urea ligand for prostate carcinoma: Tuning biological efficacy through in silico studies.

In this article, we have explored the chemical interactions of tyrosine-based asymmetric urea ligands in the binding pockets of prostate specific membrane antigen (PSMA) through in silico studies. The S1 pocket of the PSMA protein offers better scope for modifications in the urea ligands to improve the binding affinity. Accordingly, tyrosine-based (S)-2-(3-((S)-1-carboxy-2-(4-(carboxymethoxy)phenyl)ethyl)ureido)pentanedioic acid (CYUE, 3) ligand was designed, synthesized and predicted to possess inhibition constant (Ki) of 55 nM with PSMA protein. The CYUE (3) ligand was further elaborated into a fluorescent diagnostic probe for detection of PSMA+ cancers. In vitro studies on human malignant cell lines such as LNCaP and PC-3 were performed to show the efficacy and specificity of the newly synthesized bio-construct. The fluorescent bio-conjugate was found to be very specific to the PSMA protein with an overall binding affinity constant (KD) of 88 nM.

Novel solid-phase strategy for the synthesis of ligand-targeted fluorescent-labelled chelating peptide conjugates as a theranostic tool for cancer.

In this article, we have successfully designed and demonstrated a novel continuous process for assembling targeting ligands, peptidic spacers, fluorescent tags and a chelating core for the attachment of cytotoxic molecules, radiotracers, nanomaterials in a standard Fmoc solid-phase peptide synthesis in high yield and purity. The differentially protected Fmoc-Lys-(Tfa)-OH plays a vital role in attaching fluorescent tags while growing the peptide chain in an uninterrupted manner. The methodology is versatile for solid-phase resins that are sensitive to mild and strong acidic conditions when acid-sensitive side chain amino protecting groups such as Trt (chlorotrityl), Mtt (4-methyltrityl), Mmt (4-methoxytrityl) are employed to synthesise the ligand targeted fluorescent tagged bioconjugates. Using this methodology, DUPA rhodamine B conjugate (DUPA = 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid), targeting prostate specific membrane antigen (PSMA) expressed on prostate, breast, bladder and brain cancers and pteroate rhodamine B, targeting folate receptor positive cancers such as ovarian, lung, endometrium as well as inflammatory diseases have been synthesized. In vitro studies using LNCaP (PSMA +ve), PC-3 (PSMA -ve, FR -ve) and CHO-ß (FR +ve) cell lines and their respective competition experiments demonstrate the specificity of the newly synthesized bioconstructs for future application in fluorescent guided intra-operative imaging.

Preparation of Ligand-Targeted Drug Conjugates for Cancer Therapy and Their Evaluation In Vitro.

Present treatment strategies focus on minimizing unwanted toxicity to healthy cells during chemotherapeutic treatment. This is achieved by developing strategies to selectively deliver drugs to malignant cells over-expressing specific protein biomarkers. The drugs are attached via a self-immolative linker to a small molecule homing ligand having affinity for protein biomarkers over-expressed during disease states. Several such targeting-ligand drug conjugates have now reached preclinical and clinical trials, and this article aims to show a general methodology to prepare the same. Using solid-phase peptide synthesis (SPPS) methodology, the targeting ligand is covalently linked to a peptide spacer having appropriate hydrophobic and hydrophilic amino acids. The targeting ligand-attached peptide spacer is next conjugated with the required drug molecule through a cleavable disulfide bond in a solution-phase reaction. This protocol further elucidates the step-by-step procedures to be followed for complete evaluation of newly synthesized ligand-targeted drug conjugates in vitro. © 2018 by John Wiley & Sons, Inc.

In Vivo Evaluation of Ligand Targeted Drug Conjugates for Cancer Therapy.

The development of small molecule ligand-targeted therapeutics is currently of paramount importance for treatment of cancer due to their potential to reduce system toxicity and increase potency of a delivered chemotherapeutic drug. The main aim of a targeted drug-delivery technique is to release the drug cargo selectively into tumor tissues, avoiding off-site toxicity to healthy tissues and organs during chemotherapy. In this strategy, a chemotherapeutic drug is conjugated to a homing ligand, which has high affinity for proteins over-expressed on cancer cells, via a peptide linker and a self-immolative segment that facilitates intracellular release of drug cargo. During development of targeted drug conjugates, preclinical evaluation in tumor models of small animals like mice adds valuable data on the clinical performance of the drug. This article contains a set of protocols for implantation of tumor, determination of optimum dosage required for effective treatment, and estimation of maximum tolerated dose required for any visible side effects during treatment of cancer in tumor models of mice. © 2018 by John Wiley & Sons, Inc.