Enhancing bone regeneration and immunomodulation via gelatin methacryloyl hydrogel-encapsulated exosomes from osteogenic pre-differentiated mesenchymal stem cells.

Mesenchymal stem cell-derived exosomes (MSC-Exos) have emerged as promising candidates for cell-free therapy in tissue regeneration. However, the native osteogenic and angiogenic capacities of MSC-Exos are often insufficient to repair critical-sized bone defects, and the underlying immune mechanisms remain elusive. Furthermore, achieving sustained delivery and stable activity of MSC-Exos at the defect site is essential for optimal therapeutic outcomes. Here, we extracted exosomes from osteogenically pre-differentiated human bone marrow mesenchymal stem cells (hBMSCs) by ultracentrifugation and encapsulated them in gelatin methacryloyl (GelMA) hydrogel to construct a composite scaffold. The resulting exosome-encapsulated hydrogel exhibited excellent mechanical properties and biocompatibility, facilitating sustained delivery of MSC-Exos. Osteogenic pre-differentiation significantly enhanced the osteogenic and angiogenic properties of MSC-Exos, promoting osteogenic differentiation of hBMSCs and angiogenesis of human umbilical vein endothelial cells (HUVECs). Furthermore, MSC-Exos induced polarization of Raw264.7 cells from a pro-inflammatory phenotype to an anti-inflammatory phenotype under simulated inflammatory conditions, thereby creating an immune microenvironment conducive to osteogenesis. RNA sequencing and bioinformatics analysis revealed that MSC-Exos activate the p53 pathway through targeted delivery of internal microRNAs and regulate macrophage polarization by reducing DNA oxidative damage. Our study highlights the potential of osteogenic exosome-encapsulated composite hydrogels for the development of cell-free scaffolds in bone tissue engineering.

Purification, Structural Characterization, and Antitumor Activity of a Polysaccharide from Perilla Seeds.

A previous study found that a crude Perilla seed polysaccharide (PFSP) fraction exhibited obviously antitumor activity; however, the structural characterization and antitumor properties of this polysaccharide remain unclear. In this study, the PFSP was extracted and purified via combined column chromatography, and the structure of a single polysaccharide fraction was characterized by methylation, IC, GC-MS, NMR, and AFM. The results demonstrated that the efficient antitumor polysaccharide fraction PFSP-2-1 was screened from PFSP with a relative molecular weight of 8.81 × 106 Da. The primary structure of the PFSP main chain was →1)-Araf-(5→, →1,3)-Galp-(6→, →1)-Galp-(6→, →1,3)-Araf-(5→ and →1)-Xylp-(4→, and that of the side chains was →1)-Arap, →1,3)-Galp-(6→, →1)-Araf and →1)-Glcp-(4→, →1)-Galp-(3→ and →1)-Glcp, leading to a three-dimensional helical structure. CCK-8 experiments revealed that PFSP-2-1 significantly inhibited the growth of human hepatocellular carcinoma cells in vitro (p < 0.05), and its inhibitory effect positively correlation with the concentration of PFSP-2-1, and when the concentration of PFSP-2-1 was 1600 µg/mL, it showed the highest inhabitation rate on three hepatocellular carcinoma cells (HepG-2, Hep3b, and SK-Hep-1), for which the survival rates of HepG-2, Hep3b, and SK-Hep-1 were 53.34%, 70.33%, and 71.06%. This study clearly elucidated the structure and antitumor activity of PFSP-2-1, which lays a theoretical foundation for revealing the molecular mechanism of antitumor activity of Perilla seed polysaccharides and provides an important theoretical basis for the development of high-value Perilla.

An efficient metal-organic framework-based drug delivery platform for synergistic antibacterial activity and osteogenesis.

Bone implants for clinical application should be endowed with antibacterial activity, biocompatibility, and even osteogenesis-promoting properties. In this work, metal-organic framework (MOF) based drug delivery platform was used to modify titanium implants for improved clinical applicability. Methyl Vanillate@Zeolitic Imidazolate Framework-8 (MV@ZIF-8) was immobilized on the polydopamine (PDA) modified titanium. The sustainable release of the Zn2+ and MV causes substantial oxidative damage to Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The increased reactive oxygen species (ROS) significantly up-regulates the expression of oxidative stress and DNA damage response genes. Meanwhile, the structural disruption of lipid membranes caused by the ROS, the damage caused by Zinc active sites and the damage accelerated by the MV are both involved in inhibiting bacterial proliferation. The up-regulated expression of the osteogenic-related genes and proteins indicated that the MV@ZIF-8 could effectively promote the osteogenic differentiation of the human bone mesenchymal stem cells (hBMSCs). RNA sequencing and Western blotting analysis revealed that the MV@ZIF-8 coating activates the canonical Wnt/ß-catenin signaling pathway through the regulation of tumor necrosis factor (TNF) pathway, thereby promoting the osteogenic differentiation of the hBMSCs. This work demonstrates a promising application of the MOF-based drug delivery platform in bone tissue engineering.

LDHA-mediated metabolic reprogramming promoted cardiomyocyte proliferation by alleviating ROS and inducing M2 macrophage polarization.

AIMS: Metabolic switching during heart development contributes to postnatal cardiomyocyte (CM) cell cycle exit and loss of regenerative capacity in the mammalian heart. Metabolic control has potential for developing effective CM proliferation strategies. We sought to determine whether lactate dehydrogenase A (LDHA) regulated CM proliferation by inducing metabolic reprogramming. METHODS AND RESULTS: LDHA expression was high in P1 hearts and significantly decreased during postnatal heart development. CM-specific LDHA knockout mice were generated using CRISPR/Cas9 technology. CM-specific LDHA knockout inhibited CM proliferation, leading to worse cardiac function and a lower survival rate in the neonatal apical resection model. In contrast, CM-specific overexpression of LDHA promoted CM proliferation and cardiac repair post-MI. The α-MHC-H2B-mCh/CAG-eGFP-anillin system was used to confirm the proliferative effect triggered by LDHA on P7 CMs and adult hearts. Metabolomics, proteomics and Co-IP experiments indicated that LDHA-mediated succinyl coenzyme A reduction inhibited succinylation-dependent ubiquitination of thioredoxin reductase 1 (Txnrd1), which alleviated ROS and thereby promoted CM proliferation. In addition, flow cytometry and western blotting showed that LDHA-driven lactate production created a beneficial cardiac regenerative microenvironment by inducing M2 macrophage polarization. CONCLUSIONS: LDHA-mediated metabolic reprogramming promoted CM proliferation by alleviating ROS and inducing M2 macrophage polarization, indicating that LDHA might be an effective target for promoting cardiac repair post-MI.

Quantitative investigation of laser ablation based on real-time temperature variations and OCT images for laser treatment applications.

BACKGROUND AND OBJECTIVE: Lasers are widely employed in clinical applications. In vivo monitoring of real-time information about different-wavelength laser surgeries would provide important surgical feedback for surgeons or clinical therapy instruments. However, the quantitative effect of laser ablation or vaporization still needs to be further explored and investigated. Here, we investigate and quantitatively evaluate the ablation variations and morphological changes of two laser ablation models: point- and sweeping-based models. METHODS: An infrared thermal imager was used to monitor the temperature variations, and curve fitting was used to build the relationship between the laser radiation duration/sweeping speed and quantitative parameters of the ablated areas. Optical coherence tomography (OCT) images were used to visualize the inner structure and evaluate the depth of the ablated craters. Optical attenuation coefficients (OACs) were computed to characterize the normal and ablated tissues. RESULTS: The results demonstrated that there was a good linear relationship between radiation duration and temperature variation. Similarly, a linear relationship was observed between the sweeping speed and quantitative parameters of craters or scratches (width and depth). The mean OAC of normal tissues was significantly distinguished from the mean OACs of the ablated craters or scratches. CONCLUSION: Laser ablation was investigated based on a quantitative parameter analysis, thermal detection, and OCT imaging, and the results successfully demonstrated that there is a linear relationship between the laser parameters and quantitative parameters of the ablated tissues under the current settings. Such technology could be used to provide quantitative solutions for exploring the laser-tissue biological effect and improve the performance of medical image-guided laser ablation in the future.

LncRNA Snhg1-driven self-reinforcing regulatory network promoted cardiac regeneration and repair after myocardial infarction.

Rationale: Most current cardiac regeneration approaches result in very limited cell division and little new cardiomyocyte (CM) mass. Positive feedback loops are vital for cell division, but their role in CM regeneration remains unclear. We aimed to determine whether the lncRNA small nucleolar RNA host gene 1 Snhg1 (Snhg1) could form a positive feedback loop with c-Myc to induce cardiac regeneration. Methods: Quantitative PCR and in situ hybridization experiments were performed to determine the Snhg1 expression patterns in fetal and myocardial infarction (MI) hearts. Gain- and Loss-of-function assays were conducted to explore the effect of Snhg1 on cardiomyocyte (CM) proliferation and cardiac repair following MI. We further constructed CM-specific Snhg1 knockout mice to confirm the proliferative effect exerted by Snhg1 using CRISPR/Cas9 technology. RNA sequencing and RNA pulldown were performed to explore how Snhg1 mediated cardiac regeneration. Chromatin immunoprecipitation and luciferase reporter assays were used to demonstrate the positive feedback loop between Snhg1 and c-Myc. Results: Snhg1 expression was increased in human and mouse fetal and myocardial infarction (MI) hearts, particularly in CMs. Overexpression of Snhg1 promoted CM proliferation, angiogenesis, and inhibited CM apoptosis after myocardial infarction, which further improved post-MI cardiac function. Antagonism of Snhg1 in early postnatal mice inhibited CM proliferation and impaired cardiac repair after MI. Mechanistically, Snhg1 directly bound to phosphatase and tensin homolog (PTEN) and induced PTEN degradation, activating the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway to promote CM proliferation. The c-Myc protein, one of downstream targets of PI3K/AKT signaling, functioned as a transcription factor by binding to the promoter regions of Snhg1. Perturbation of the positive feedback between Snhg1 and c-Myc by mutation of the binding sequence significantly affected Snhg1-induced CM proliferation. Conclusions: Snhg1 effectively elicited CM proliferation and improved cardiac function post-MI by forming a positive feedback loop with c-Myc to sustain PI3K/Akt signaling activation, and thus may be a promising cardiac regeneration strategy in treating heart failure post-MI.

Inhibition of SENP2-mediated Akt deSUMOylation promotes cardiac regeneration via activating Akt pathway.

Post-translational modification (PTM) by small ubiquitin-like modifier (SUMO) is a key regulator of cell proliferation and can be readily reversed by a family of SUMO-specific proteases (SENPs), making SUMOylation an ideal regulatory mechanism for developing novel therapeutic strategies for promoting a cardiac regenerative response. However, the role of SUMOylation in cardiac regeneration remains unknown. In the present study, we assessed whether targeting protein kinase B (Akt) SUMOylation can promote cardiac regeneration. Quantitative PCR and Western blotting results showed that small ubiquitin-like modifier-specific protease 2 (SENP2) is up-regulated during postnatal heart development. SENP2 deficiency promoted P7 and adult cardiomyocyte (CM) dedifferentiation and proliferation both in vitro and in vivo. Mice with SENP2 deficiency exhibited improved cardiac function after MI due to CM proliferation and angiogenesis. Mechanistically, the loss of SENP2 up-regulated Akt SUMOylation levels and increased Akt kinase activity, leading to a decrease in GSK3ß levels and subsequently promoting CM proliferation and angiogenesis. In summary, inhibition of SENP2-mediated Akt deSUMOylation promotes CM differentiation and proliferation by activating the Akt pathway. Our results provide new insights into the role of SUMOylation in cardiac regeneration.

Hydrogen sulfide-loaded microbubbles combined with ultrasound mediate thrombolysis and simultaneously mitigate ischemia-reperfusion injury in a rat hindlimb model.

BACKGROUND: Thromboembolism and subsequent ischemia/reperfusion injury (IRI) remain major clinical challenges. OBJECTIVES: To investigate whether hydrogen sulfide (H2 S)-loaded microbubbles (hs-Mbs) combined with ultrasound (US) radiation (hs-Mbs+US) dissolve thrombi and simultaneously alleviate tissue IRI through local H2 S release. METHODS: hs-Mbs were manufactured and US-triggered H2 S release was recorded. White and red thromboembolisms were established ex vivo and in rats left iliac artery. All subjects randomly received control, US, Mbs+US, or hs-Mbs+US treatment for 30 minutes. RESULTS: H2 S was released from hs-Mbs+US both ex vivo and in vivo. Compared with control and US, hs-Mbs+US and Mbs+US showed comparable substantial decreases in thrombotic area, clot mass, and flow velocity increases for both ex vivo macrothrombi. In vivo, hs-Mbs+US and Mbs+US caused similarly increased recanalization rates, blood flow velocities, and hindlimb perfusion for both thrombi compared with the other treatments, with no obvious influence on hemodynamics, respiration, and macrophage vitality. More importantly, hs-Mbs+US substantially alleviated skeletal muscle IRI by reducing reactive oxygen species, cellular apoptosis, and proapoptotic Bax, caspase-3, and caspase-9 and increasing antiapoptotic Bcl-2 compared with other treatments. In vitro, hypoxia/reoxygenation-predisposed skeletal muscle cells and endothelial cells treated with normal saline solution exhibited similar trends, which were largely reversed by an H2 S scavenger or an inhibitor of Akt phosphorylation. CONCLUSION: hs-Mbs+US effectively dissolved both white and red macrothrombi and simultaneously alleviated skeletal muscle IRI through the US-triggered, organ-specific release of H2 S. This integrated therapeutic strategy holds promise for treating thromboembolic diseases and subsequent IRI.

Chip-Level Integration of Covalent Organic Frameworks for Trace Benzene Sensing.

State-of-the-art chemical sensors based on covalent organic frameworks (COFs) are restricted to the transduction mechanism relying on luminescence quenching and/or enhancement. Herein, we present an alternative methodology via a combination of in situ-grown COF films with interdigitated electrodes utilized for capacitive benzene detection. The resultant COF-based sensors exhibit highly sensitive and selective detection at room temperature toward benzene vapor over carbon dioxide, methane, and propane. Their benzene detection limit can reach 340 ppb, slightly inferior to those of the metal oxide semiconductor-based sensors, but with reduced power consumption and increased selectivity. Such a sensing behavior can be attributed to the large dielectric constant of the benzene molecule, distinctive adsorptivity of the chosen COF toward benzene, and structural distortion induced by the custom-made interaction pair, which is corroborated by sorption measurements and density functional theory (DFT) calculations. This study provides new perspectives for fabricating COF-based sensors with specific functionality targeted for selective gas detection.

Long noncoding RNA GAS5 induces abdominal aortic aneurysm formation by promoting smooth muscle apoptosis.

Objective: Long noncoding RNAs (lncRNAs) may serve as specific targets for the treatment of abdominal aortic aneurysms (AAAs). LncRNA GAS5, functionally associated with smooth muscle cell (SMC) apoptosis and proliferation, is likely involved in AAA formation, but the exact role of GAS5 in AAA is unknown. We thus explored the contribution of GAS5 to SMC-regulated AAA formation and its underlying mechanisms. Methods: Human specimens were used to verify the diverse expression of GAS5 in normal and AAA tissues. The angiotensin II (Ang II)-induced AAA model in ApoE-/- mice and the CaCl2-induced AAA model in wild-type C57BL/6 mice were used. RNA pull-down and luciferase reporter gene assays were performed in human aortic SMCs to detect the interaction between GAS5 and its downstream targets of protein or microRNA (miR). Results: GAS5 expression was significantly upregulated in human AAA specimens and two murine AAA models compared to human normal aortas and murine sham-operated controls. GAS5 overexpression induced SMC apoptosis and repressed its proliferation, thereby promoting AAA formation in two murine AAA models. Y-box-binding protein 1 (YBX1) was identified as a direct target of GAS5 while it also formed a positive feedback loop with GAS5 to regulate the downstream target p21. Furthermore, GAS5 acted as a miR-21 sponge to release phosphatase and tensin homolog from repression, which blocked the activation and phosphorylation of Akt to inhibit proliferation and promote apoptosis in SMCs. Conclusion: The LncRNA GAS5 contributes to SMC survival during AAA formation. Thus, GAS5 might serve as a novel target against AAA.

Inhibition of AZIN2-sv induces neovascularization and improves prognosis after myocardial infarction by blocking ubiquitin-dependent talin1 degradation and activating the Akt pathway.

BACKGROUND: We previously found that loss of lncRNA-AZIN2 splice variant (AZIN2-sv) increases cardiomyocyte (CM) proliferation and attenuates adverse ventricular remodelling post-myocardial infarction (MI). However, whether inhibition of AZIN2-sv can simultaneously induce angiogenesis and thus improve prognosis after MI is unclear. METHODS: We used in situ hybridization and quantitative PCR to determine AZIN2-sv expression in endothelial cells. Knockdown and overexpression were performed to detect the role of AZIN2-sv in endothelial cell function, angiogenesis and prognosis after MI. RNA pulldown, RNA immunoprecipitation and luciferase reporter assays were used to determine the interaction with talin1 (Tln1) protein and miRNA-214 (miR-214). DNA pulldown and chromatin immunoprecipitation (ChIP) assays were used to study AZIN2-sv binding to upstream transcription factors. FINDINGS: AZIN2-sv was enriched in cardiac endothelial cells. The loss of AZIN2-sv reduced endothelial cell apoptosis and promoted endothelial sprouting and capillary network formation in vitro. Moreover, in vivo, the loss of AZIN2-sv induced angiogenesis and improved cardiac function after MI. Mechanistically, AZIN2-sv reduced Tln1 and integrin ß1 (ITGB1) protein levels to inhibit neovascularization. AZIN2-sv activated the ubiquitination-dependent degradation of Tln1 mediated by proteasome 26S subunit ATPase 5 (PSMC5). In addition, AZIN2-sv could bind to miR-214 and suppress the phosphatase and tensin homologue (PTEN)/Akt pathway to inhibit angiogenesis. With regard to the upstream mechanism, Bach1, a negative regulator of angiogenesis, bound to the promoter of AZIN2-sv and increased its expression. INTERPRETATION: Bach1-activated AZIN2-sv could participate in angiogenesis by promoting the PSMC5-mediated ubiquitination-dependent degradation of Tln1 and blocking the miR-214/PTEN/Akt pathway. Inhibition of AZIN2-sv induced angiogenesis and myocardial regeneration simultaneously, thus, AZIN2-sv could be an ideal therapeutic target for improving myocardial repair after MI. FUND: National Natural Science Foundations of China.

SM22α (Smooth Muscle 22α) Prevents Aortic Aneurysm Formation by Inhibiting Smooth Muscle Cell Phenotypic Switching Through Suppressing Reactive Oxygen Species/NF-κB (Nuclear Factor-κB).

Objective- Vascular smooth muscle cell phenotypic transition plays a critical role in the formation of abdominal aortic aneurysms (AAAs). SM22α (smooth muscle 22α) has a vital role in maintaining the smooth muscle cell phenotype and is downregulated in AAA. However, whether manipulation of the SM22α gene influences the pathogenesis of AAA is unclear. Here, we investigated whether SM22α prevents AAA formation and explored the underlying mechanisms. Approach and Results- In both human and animal AAA tissues, a smooth muscle cell phenotypic switch was confirmed, as manifested by the downregulation of SM22α and α-SMA (α-smooth muscle actin) proteins. The methylation level of the SM22α gene promoter was dramatically higher in mouse AAA tissues than in control tissues. SM22α knockdown in ApoE-/- (apolipoprotein E-deficient) mice treated with Ang II (angiotensin II) accelerated the formation of AAAs, as evidenced by a larger maximal aortic diameter and more medial elastin degradation than those found in control mice, whereas SM22α overexpression exerted opposite effects. Similar results were obtained in a calcium chloride-induced mouse AAA model. Mechanistically, SM22α deficiency significantly increased reactive oxygen species production and NF-κB (nuclear factor-κB) activation in AAA tissues, whereas SM22α overexpression produced opposite effects. NF-κB antagonist SN50 or antioxidant N-acetyl-L-cysteine partially abrogated the exacerbating effects of SM22α silencing on AAA formation. Conclusions- SM22α reduction in AAAs because of the SM22α promoter hypermethylation accelerates AAA formation through the reactive oxygen species/NF-κB pathway, and therapeutic approaches to increase SM22α expression are potentially beneficial for preventing AAA formation.

Anti-tumor properties of anthocyanins from Lonicera caerulea 'Beilei' fruit on human hepatocellular carcinoma: In vitro and in vivo study.

In this study, the anthocyanin from Lonicera caerulea 'Beilei' fruit (ABL) was extracted and purified. The purified component (ABL-2) was then evaluated for its anti-tumor properties on human hepatoma cells (SMMC-7721) in vitro and the murine hepatoma cells (H22) in vivo. In vitro, ABL-2 not only significantly inhibited the growth of SMMC-7721 cells, but also remarkably blocked the cells' cycle in G2/M phase, inducing DNA damage and eventually leading to apoptosis. In vivo, ABL also killed tumor cells, inhibited tumor growth, and improved the survival status of H22 tumor-bearing mice. These effects were associated with an increase in the activities of antioxidase and a decrease in the level of lipid peroxidation, as evidenced by changes in SOD, GSH-Px, GSH, and MDA levels. In addition, ABL-2 also regulated the levels of immune cytokines including IL-2, IFN-γ, and TNF-α. These results revealed that ABL-2 exerts an effective anti-tumor effect by dynamically adjusting the REDOX balance and improving the immunoregulatory activity of H22 tumor-bearing mice. High performance liquid chromatography (HPLC) analysis revealed that cyanidin-3,5-diglucoside (8.16 mg/g), cyanidin-3-glucoside (387.60 mg/g), cyanidin-3-rutinoside (23.62 mg/g), and peonidin-3-glucoside (22.20 mg/g) were the main components in ABL-2, which may contribute to its anti-tumor activity.

Loss of AZIN2 splice variant facilitates endogenous cardiac regeneration.

Aims: Long noncoding RNAs (lncRNAs) are critical regulators of cardiovascular lineage commitment and heart wall development, but their roles in regulating endogenous cardiac regeneration are unclear. The present study investigated the role of human-derived lncRNA in regulating endogenous cardiac regeneration as well as the underlying mechanisms. Methods and results: We compared RNA sequencing data from human foetal and adult hearts and identified a novel lncRNA that was upregulated in adult hearts (Genesymbol NONHSAG000971/NONHSAT002258 or ENST00000497710.5), which was a splice variant of the AZIN2 gene (AZIN2-sv). We used quantitative PCR to confirm the increased expression of AZIN2-sv in adult rat hearts. Coexpression network analysis indicated that AZIN2-sv could regulate proliferation. Loss- and gain-of-function approaches demonstrated that AZIN2-sv negatively regulated endogenous cardiomyocyte proliferation in vitro and in vivo. Knockdown of AZIN2-sv attenuated ventricular remodelling and improved cardiac function after myocardial infarction. Phosphatase and tensin homolog (PTEN) was identified as a target of AZIN2-sv, their direct binding increased PTEN stability. Furthermore, AZIN2-sv acted as a microRNA-214 sponge to release PTEN, which blocked activation of the PI3 kinase/Akt pathway to inhibit cardiomyocyte proliferation. Conclusions: The newly discovered AZIN2-sv suppressed endogenous cardiac regeneration by targeting the PTEN/Akt pathway. Thus, AZIN2-sv may be a novel therapeutic target for preventing and treating heart failure.

Complete regression of cutaneous metastases with systemic immune response in a patient with triple negative breast cancer receiving p53MVA vaccine with pembrolizumab.

A heavily pretreated patient with triple negative breast cancer distinguished by cutaneous metastases received p53MVA vaccine in combination with pembrolizumab. Her cutaneous metastases regressed and after 2 cycles of therapy, a skin biopsy showed a complete pathological response. Systemic response was confirmed with restaging CT and bone scans. Activation of p53-specific T cell responses and elevation of multiple immune response genes in peripheral blood correlated with the rapid clinical response which lasted for 6 months after the initiation of combined therapy.

[Progress on pharmacokinetic study of antibody-drug conjugates].

Antibody-drug conjugate (ADC) is a new class of therapeutics composed of a monoclonal antibody and small cytotoxin moieties conjugated through a chemical linker. ADC molecules bind to the target antigens expressed on the tumor cell surfaces guided by the monoclonal antibody component. The binding ADC molecules can be internalized and subsequently the toxin moieties can be released within the tumor cells via chemical and/or enzymatic reactions to kill the target cells. The conjugation combines the merits of both components, i.e., the high target specificity of the monoclonal antibody and the highly potent cell killing activity of the cytotoxin moieties. However, such complexities make the pharmacokinetic and metabolic studies of ADCs highly challenging. The major challenges should include characterization of absorption, distribution, metabolism and excretion, investigation of underlying mechanisms, assessment of pharmacokinetic- pharmacodynamic relationship, and analytical method development of ADC drugs. This review will discuss common pharmacokinetic issues and considerations, as well as tools and strategies that can be utilized to characterize the pharmacokinetic and metabolic properties of ADCs.