Surface Modification of Decellularized Heart Valve by the POSS-PEG Hybrid Hydrogel to Prepare a Composite Scaffold Material with Anticalcification Potential.

Tissue-engineered heart valves (TEHVs) are the most promising replacement for heart valve transplantation. Decellularized heart valve (DHV) is one of the most common scaffold materials for TEHVs. In actual clinical applications, the most widely used method for treating DHV is cross-linking it with glutaraldehyde, but this method could cause serious problems such as calcification. In this study, we introduced polyhedral oligomeric silsesquioxane (POSS) nanoparticles into a poly(ethylene glycol) (PEG) hydrogel to prepare a POSS-PEG hybrid hydrogel, and then coated them on the surface of DHV to prepare the composite scaffold. The chemical structures, microscopic morphologies, cell compatibilities, blood compatibilities, and anticalcification properties were further investigated. Experimental results showed that the composite scaffold had good blood compatibility and excellent cell compatibility and could promote cell adhesion and proliferation. In vivo and in vitro anticalcification experiments showed that the introduction of POSS nanoparticles could reduce the degree of calcification significantly and the composite scaffold had obvious anticalcification ability. The DHV surface-coated with the POSS-PEG hybrid hydrogel is an alternative scaffold material with anticalcification potential for an artificial heart valve, which provides an idea for the preparation of TEHVs.

In Vitro Photodynamic Therapy of Mononuclear and Dinuclear Iridium(III) Bis(terpyridine) Complexes.

Three mononuclear or dinuclear bis(terpyridine) (tpy) iridium(III) complexes bearing pyren-1-yl (pyr) group(s) were synthesized. Their photophysical properties in water and in vitro photodynamic therapy (PDT) effects toward the human lung epithelial cancer cell line A549 and the human epidermal skin cancer cell line A431 were investigated to evaluate the effects of dinuclear versus mononuclear complexes and the impact of the oligoether substituent at the ligand. All complexes possessed pyr-tpy ligand-associated charge transfer (1CT)/1π,π* absorption bands at 350-550 nm, with the dinuclear complex Ir3 showing the much enhanced absorptivity of this band. These complexes exhibited dual emission upon excitation at >430 nm in most cases, with the emitting states being ascribed to 1ILCT (intraligand charge transfer) and 3π,π*/3CT states, respectively. All complexes exhibited relatively weak to moderate cytotoxicity in the dark but high photocytotoxicity upon broadband visible light irradiation. Among them, the dinuclear complex Ir3 showed the highest intracellular reactive oxygen species (ROS) generation and PDT efficiency compared to its mononuclear counterpart Ir1. Introducing an oligoether substituent on one of the tpy ligands in Ir2 also improved its intracellular ROS generation and PDT efficacy compared to those induced by Ir1. Ir3 induced both mitochondrial dysfunction and lysosomal damage upon light activation toward both cell lines, whereas Ir1 and Ir2 caused both mitochondrial dysfunction and lysosomal damage in A431 cells but only lysosomal damage in A549 cells. The dominant cell death pathway induced by Ir1-Ir3 PDT is apoptosis.

A near-infrared fluorescent probe for monitoring leucine aminopeptidase in living cells.

Leucine aminopeptidase (LAP), one of the important cancer-related biomarkers, is significantly over-expressed in many malignant tumor cells. Developing an effective fluorescent probe for high-specificity and in situ trapping of endogenous LAP in living samples is still challenging. In this project, we report a water-soluble near-infrared (NIR) fluorescent probe (CHMC-M-Leu) for specific monitoring of LAP in vitro and in vivo. The novel fluorescent probe (CHMC-M-Leu) contains a NIR-emitting fluorophore (CHMC-M) as the reporter and l-leucine as the enzyme-active trigger moiety which are linked together by a p-aminobenzyl alcohol (PABA) section. Upon exposure to LAP, the fluorescence at 625 nm gets impressively enhanced, which belongs to the near-infrared region and is beneficial for imaging in vivo. Furthermore, the novel fluorescent probe exhibits fast response and highly chemoselective detection of LAP in various bio-related species. In addition, CHMC-M-Leu shows favourable cellular uptake and was successfully used to monitor endogenous LAP in living cells.

A Leucine Aminopeptidase-Activated Theranostic Prodrug for Cancer Diagnosis and Chemotherapy.

Currently, chemotherapy is a widely used and important treatment for cancer. However, almost all of the treatments have shortcomings associated with poor specificity and high toxicity, which results in severe side effects to normal cells and tissue. This is a very important problem, and yet, it currently remains unanswered. Therefore, the development of the method for the more effective delivery of anticancer drugs to their targets and real-time monitoring of the localization of the drugs are very important. Herein, we designed a theranostic prodrug: CPT-p-Leu, which was constructed using fluorescent camptothecin (CPT), a self-immolative linker and leucine (Leu) residue. Upon exposure to LAP (leucine aminopeptidase: LAP), the amide bond in CPT-p-Leu will be cleaved, followed by an intramolecular 1,6-elimination, which triggers the active anticancer drug (CPT) release and recovers the fluorescence of CPT. With our design, the anticancer drug, CPT, can be used as both a drug and a fluorescence reporter, making our system suitable to accurately and effectively track the released CPT distribution. Based on this strategy, CPT-p-Leu could achieve the chemoselective detection of LAP and monitoring of the anticancer drug release. Furthermore, it also provides a very convenient way to accurately determine the location of the released drug in living samples. In addition, CPT-p-Leu shows a good cell membrane permeability and enhanced cytotoxicity toward LAP overexpressing cancer cells. We anticipate that our research will facilitate the development of improved theranostic systems for cancer therapy.

Highly Sensitive Ratiometric Self-Assembled Micellar Nanoprobe for Nitroxyl and Its Application In Vivo.

Nitroxyl (HNO) is a derivative of nitric oxide (NO) that plays an essential role in various biological and pharmacological events. Until now, the in situ trapping and specific detection of HNO in living samples is still challenging. In this project, we fabricated a novel BODIPY-based micellar nanoprobe for monitoring nitroxyl in vitro and in vivo in ratiometric mode in aqueous solution. The probe (P-BODIPY-N) contains an asymmetrical BODIPY dye for fluorescent signaling and a diphenylphosphinobenzoyl as the trigger moiety; then we encapsulated P-BODIPY-N into the hydrophobic interior of an amphiphilic copolymer (mPEG-DSPE) and prepared a novel BODIPY-based micellar nanoprobe: NP-BODIPY-N. As far as we know, this probe is the first reported ratiometric fluorescent nanoprobe for HNO, which exhibits ultrasensitivity, high selectivity, and good biocompatibility. Above all, this nanoprobe shows favorable cellular uptaken and was successfully used to detect intracellular HNO released by Angeli's salt in living cells and zebrafish larvae. These results indicate that our newly designed nanoprobe will provide a promising tool for the studies of HNO in living system.

A Ratiometric Fluorescent Probe for Monitoring Leucine Aminopeptidase in Living Cells and Zebrafish Model.

Leucine aminopeptidase (LAP) is an important cancer-related biomarker, which shows significant overexpression in malignant tumor cells like liver cancer. Developing an effective method to monitor LAP in tumor cells holds great potential for cancer diagnosis, treatment, and management. In this work, we report a novel BODIPY-based fluorescent probe (BODIPY-C-Leu) capable of monitoring LAP in vitro and in vivo in both ratiometric and turn-on model. BODIPY-C-Leu contains an asymmetrical BODIPY dye for fluorescent signaling and a dipeptide (Cys-Leu) as the triggered moiety. Activation occurs by cleavage of the amide bond in dipeptides and subsequently an intramolecular S → N conversion to convert sulfur-substituted BODIPY to amino-substituted BODIPY, resulting in a dramatic fluorescence variation to realize the detection of LAP. Furthermore, we have successfully employed BODIPY-C-Leu to monitor LAP activity in different cancer cells, indicating that HeLa cells have a higher level of LAP activity than A549 cells. Importantly, we demonstrated the capability of the probe for real-time monitoring the drug-induced LAP level changes in zebrafish.

Evaluation of a novel tetra-functional branched poly(ethylene glycol) crosslinker for manufacture of crosslinked, decellularized, porcine aortic valve leaflets.

To address concerns over limitations in the clinical use of glutaraldehyde (GA) fixation in bioprosthetic heart valves, we manufactured novel, branched poly(ethylene glycol) tetraacrylate (PEG-TA) crosslinked valve leaflets and evaluated cytotoxic, thrombogenic, hemolytic, and anticalcification effects, thermal stability, and mechanical properties, in comparison to decellularized valves (control) and GA crosslinked valves. Thermal denaturation temperatures were higher for PEG-TA valve leaflets compared to control and GA crosslinked valves (p < 0.001). Leaflet hydrolyzation rate was lower for the PEG-TA group than for GA and control groups (p < 0.05). Superior cytocompatibility was found for PEG-TA group leaflets (MTT, p < 0.01. apoptosis assay, p > 0.05). No thrombogenesis was found in platelet activation tests (p < 0.0001). Hemolysis assays showed that PEG-TA leaflets would not cause damage to blood cells (p > 0.05). Excellent anticalcification properties were confirmed by von Kossa staining, western blot, and atomic absorption spectroscopy (p < 0.0001) in a rat subcutaneous embedding model. Finally, the novel PEG-TA crosslinked material exhibits improved mechanical properties as compared to GA crosslinked materials (tensile strength, p < 0.001, Young's modulus, p < 0.001). On the basis of all results presented, it is clear that the performance characteristics of PEG-TA crosslinked valve leaflets make PEG-TA crosslinked leaflets a promising alternative for the next generation of bioprosthetic heart valve.

[PEG-mediated covalent binding of VEGF to decellularized aortic valves promotes adhesion and proliferation of endothelial progenitor cells].

OBJECTIVE: To improve the biological properties of decellularized aortic valves by polyethylene glycol (PEG)-mediated covalent incorporation of vascular endothelial growth factor (VEGF). METHODS: PEG crosslinking of decellularized aortic valves were completed via a Michael-type addition reaction, followed by covalent incorporation of VEGF through another Michael-type addition reaction between the unsaturated propylene acyl of PEG and the thiol groups on cysteine residues of VEGF. The effect of VEGF incorporation was evaluated by enzyme-linked immunosorbent assay (ELISA) and immune fluorescence assay. The endothelial progenitor cells (EPCs) were seeded on decellularized aortic valves with or without these modifications, and after 10 days of culture, the valves were examined for DNA content and by hematoxylin-eosin staining and scanning electron microscopy. RESULTS: Immune fluorescence and ELISA showed that the maximal VEGF incorporation on the decellularized aortic valve reached 908.94∓0.27 pg. Compared with the unmodified valves and the valves with PEG crosslinking, decellularized aortic valves with covalent incorporation of VEGF significantly promoted the adhesion and proliferation of EPCs, which formed a confluent cell monolayer on the valve surface. CONCLUSIONS: PEG-mediated covalent incorporation of VEGF in the decellularized aortic valves improves the adhesion and proliferation of the seeded EPCs to facilitate the construction of tissue-engineered heart valves.