A Multiresponsive Nanohybrid to Enhance the Lysosomal Delivery of Oxygen and Photosensitizers.

Photodynamic therapy (PDT) is a promising cancer ablation method, but its efficiency is easily affected by several factors, such as the insufficient delivery of photosensitizers, low oxygen levels as well as long distance between singlet oxygen and intended organelles. A multifunctional nanohybrid, named MGAB, consisting of gelatin-coated manganese dioxide and albumin-coated gold nanoclusters, was designed to overcome these issues by improving chlorin e6 (Ce6) delivery and stimulating oxygen production in lysosomes. MGAB were quickly degraded in a high hydrogen peroxide, high protease activity, and low pH microenvironment, which is closely associated with tumor growth. The Ce6-loaded MGAB were picked up by tumor cells through endocytosis, degraded within the lysosomes, and released oxygen and photosensitizers. Upon near-infrared light irradiation, the close proximity of oxygen with photosensitizer within lysosomes enabled the production of cytotoxic singlet oxygen, resulting in more effective PDT.

Real-Time Visualization of Lysosome Destruction Using a Photosensitive Toluidine Blue Nanogel.

Breaking the lysosome helps its sequestered payloads access their molecular targets in cells and thus enhances the intracellular drug delivery. Current strategies for lysosomal escape involve direct physical interactions with the lipid membrane. These interactions pose a systemic toxicity and uncontrolled membrane rupture risk. Here, we report a light-detonated lysosome disruption using a hyaluronan (HA) nanogel packed with toludine blue (TB). The HA/TB nanogel is concentrated within the lysosomes. The applied light assists TB in generating reactive oxygen species and destroying the lysosome in situ, both in cells and isolated lysosomes. Real time fluorescent tracking reveals that quenched TB fluorescence recovers along with lysosome explosion, relocates to the nucleus, and is presented as a fluorescent sparkling in cells. This HA/TB, composed of all clinically approved materials, represents a biocompatible and facile strategy to "bomb" lysosomes in a spatiotemporally controlled fashion.

Facile metabolic glycan labeling strategy for exosome tracking.

BACKGROUND: Exosomes are nano-sized vesicles derived from the fusion of multivesicular bodies with the surrounding plasma membrane. Exosomes have various diagnostic and therapeutic potentials in cancer and other diseases, thus tracking exosomes is an important issue. METHODS: Here, we report a facile exosome labeling strategy using a natural metabolic incorporation of an azido-sugar into the glycan, and a strain-promoted azide-alkyne click reaction. In culture, tetra-acetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz) was spontaneously incorporated into glycans within the cells and later redistributed onto their exosomes. These azido-containing exosomes were then labeled with azadibenzylcyclooctyne (ADIBO)-fluorescent dyes by a bioorthogonal click reaction. RESULTS: Cellular uptake and the in vivo tracking of fluorescent labeled exosomes were evaluated in various cells and tumor bearing mice. Highly metastatic cancer-derived exosomes showed an increased self-homing in vitro and selective organ distribution in vivo. CONCLUSION: Our metabolic exosome labeling strategy could be a promising tool in studying the biology and distribution of exosomes, and optimizing exosome based therapeutic approaches. GENERAL SIGNIFICANT: A facile and effective exosome labeling strategy was introduced by presenting azido moiety on the surface of exosome through metabolic glycan synthesis, and then conjugating a strain-promoted fluorescent dye.

A cell surface clicked navigation system to direct specific bone targeting.

Cell therapies are promising up-and-coming therapeutic strategies for many diseases. For maximal therapeutic benefits, injected cells have to navigate their way to a designated area, including organ and tissue; unfortunately, the majority of therapeutic cells are currently administered without a guide or homing device. To improve this serious shortcoming, a functionalization method was developed to equip cells with a homing signal. Its application was demonstrated by applying an Azadibenzocyclooctyne-bisphosphonate (ADIBO-BP) and azide paired bioorthogonal chemistry on cells for bone specific homing. Jurkat T cells and bone marrow derived stromal cells (BMSCs) were cultured with tetraacetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) to place unnatural azido groups onto the cell's surface; these azido groups were then reacted with ADIBO-BP. The tethered bisphosphonates were able to bring Jurkat cells to hydroxyapatite, the major component of bone, and mineralized SAOS-2 cells. The incorporated BP groups also enhanced the specific affinity of BMSCs to mouse femur bone fragments in vitro. The introduced navigation strategy is expected to have a broad application in cell therapy, because through the biocompatible ADIBO and azide reactive pair, various homing signals could be efficiently anchored onto therapeutic cells.

Bidentate iminodiacetate modified dendrimer for bone imaging.

A new dendrimer probe was designed for bone imaging. Bidentate iminodiacetate groups were introduced to the probe to obtain strong bind to bones. The assembled dendrimeric probe, with four iminodiacetate moieties and a fluorescent tag, displayed good selectivity to hydroxyapatite, calcium oxalate and calcium phosphate salts. In mice, the probe offered vivid skeletal details after intravenous delivery.

Sequence-Independent DNA Nanogel as a Potential Drug Carrier.

DNA nanostructures largely rely on pairing DNA bases; thus, sequence designing is required. Here, this study demonstrates a sequence-independent strategy to fabricate DNA nanogel (NG) inspired by cisplatin, a chemotherapeutic drug that acts as a DNA crosslinker. A simple heating and cooling of the genomic DNA extracts and cisplatin produces DNA NG with a size controlled by the heating time. Furthermore, the drug-loaded NG is formulated by spontaneously mixing DNA segments, cisplatin, and doxorubicin. The in vitro cell studies demonstrate that the doxorubicin-loaded NG alters the drug distribution in cells while its cytotoxic potential is well-maintained. This chemotherapeutic-inspired method provides a facile one-pot and cost-effective strategy to fabricate size-controllable DNA NG that potentially acts as drug carrier.

Development of a fluorescent cardiomyocyte specific binding probe.

Cardiomyocytes are the major component of the heart. Their dysfunction or damage could lead to serious cardiovascular diseases, which have claimed numerous lives around the world. A molecule able to recognize cardiomyocytes would have significant value in diagnosis and treatment. Recently a novel peptide termed myocyte targeting peptide (MTP), with three residues of a non-natural amino acid biphenylalanine (Bip), showed good affinity to cardiomyocytes. Its selectivity towards cardiac tissues was concluded to be due to the ability of Bip to bind cardiac troponin I. With the aim of optimizing the affinity and the specificity towards cardiac myocytes and to better understand structure-activity relationship, a library of MTP derivatives was designed. Exploiting a fluorescent tag, the selectivity of the MTP analogs to myocardium over skeletal and stomach muscle tissues was assayed by fluorescence imaging. Among the tested sequences, the peptide probe Bip2, H-Lys(FITC)-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Ser-Gly-Ser-Bip-Bip-NH2, displayed the best selectivity for cardiomyocytes.

Osteotropic cancer diagnosis by an osteocalcin inspired molecular imaging mimetic.

BACKGROUND: Although microcalcifications of hydroxyapatite can be found in both benign and malignant osteotropic tumors, they are mostly seen in proliferative lesions, including carcinoma. The aim of this present study is to develop a molecular imaging contrast agent for selective identification of hydroxyapatite calcification in human osteotropic tumor tissues ex vivo and in human osteosarcoma cells in vitro. METHODS: A bioinspired biomarker, hydroxyapatite binding peptide (HABP), was designed to mimic natural protein osteocalcin property in vivo. A fluorescein isothiocyanate dye conjugated HABP (HABP-19) was utilized to characterize hydroxyapatite on human osteotropic tumor tissue sections ex vivo and to selectively image hydroxyapatite calcifications in human osteosarcoma cells in vitro. RESULTS: Using a HABP-19 molecular imaging probe, we have shown that it is possible to selectively image hydroxyapatite calcifications in osteotropic cancers ex vivo and in human SaOS-2 osteosarcoma cells in vitro. CONCLUSION: Hydroxyapatite calcifications were selectively detected in osteotropic tissues ex vivo and in the early stage of the calcification process of SaOS-2 human osteosarcoma in vitro using our HABP-19 molecular imaging probe. This new target-selective molecular imaging probe makes it possible to study the earliest events associated with hydroxyapatite deposition in various osteotropic cancers at the cellular and molecular levels. GENERAL SIGNIFICANCE: It potentially could be used to diagnose and treat osteotropic cancer or to anchor therapeutic agents directing the local distribution of desired therapy at calcified sites.

A non-toxic fluorogenic dye for mitochondria labeling.

BACKGROUND: Mitochondria, powerhouses of cells, are responsible for many critical cellular functions, such as cell energy metabolism, reactive oxygen species production, and apoptosis regulation. Monitoring mitochondria morphology in live cells temporally and spatially could help with the understanding of the mechanisms of mitochondrial functional regulation and the pathogenesis of mitochondria-related diseases. METHODS: A novel non-cytotoxic fluorogenic compound, AcQCy7, was developed as a mitochondria-specific dye. RESULTS: AcQCy7 emitted no fluorescent signal outside of cells, but it became fluorescent after intracellular hydrolysis of the acetyl group. The hydrolyzed fluorescent product was well retained in mitochondria, enabling long-lasting fluorescence imaging of mitochondria without cell washing. A 2-day culture study using AcQCy7 showed no sign of cytotoxicity, whereas a commonly used mitochondria-staining probe, Mitochondria Tracker Green, caused significant cell death even at a much lower concentration. Apoptosis-causing mitochondria fission was monitored clearly in real time by AcQCy7. CONCLUSIONS: A simple add-and-read mitochondria specific dye AcQCy7 has been validated in various cell models. Bright mitochondria specific fluorescent signal in treated cells lasted several days without noticeable toxicity. GENERAL SIGNIFICANCE: The probe AcQCy7 has been proofed to be a non-toxic agent for long-term mitochondria imaging.

Effect of lyso-phosphatidylcholine and Schnurri-3 on osteogenic transdifferentiation of vascular smooth muscle cells to calcifying vascular cells in 3D culture.

BACKGROUND: In vitro cell culture is a widely used technique for investigating a range of processes such as stem cell behavior, regenerative medicine, tissue engineering, and drug discovery. Conventional cell culture is performed in Petri dishes or flasks where cells typically attach to a flat glass or plastic surface as a cell monolayer. However, 2D cell monolayers do not provide a satisfactory representation of in vivo conditions. A 3D culture could be a much better system for representing the conditions that prevail in vivo. METHODS AND RESULTS: To simulate 3D conditions, vascular smooth muscle cells (VSMCs) were loaded with gold-polyvmer-iron oxide hydrogel, enabling levitation of the cells by using spatially varying magnetic fields. These magnetically levitated 3D cultures appeared as freely suspended, clustered cells which proliferated 3-4 times faster than cells in conventional 2D cultures. When the levitated cells were treated with 10nM lysophosphatidylcholine (LPC), for 3days, cell clusters exhibited translucent extensions/rods 60-80µm wide and 200-250µm long. When 0.5µg/µl Schnurri-3 was added to the culture containing LPC, these extensions were smaller or absent. When excited with 590-650nm light, these extensions emitted intrinsic fluorescence at >667nm. When the 3D cultures were treated with a fluorescent probe specific for calcium hydroxyapatite (FITC-HABP-19), the cell extensions/rods emitted intensely at 518nm, the λmax for FITC emission. Pellets of cells treated with LPC were more enriched in calcium, phosphate, and glycosaminoglycans than cells treated with LPC and Schnurri-3. CONCLUSIONS: In 3D cultures, VSMCs grow more rapidly and form larger calcification clusters than cells in 2D cultures. Transdifferentiation of VSMC into calcifying vascular cells is enhanced by LPC and attenuated by Schnurri-3. GENERAL SIGNIFICANCE: The formation of calcified structures in 3D VSMC cultures suggests that similar structures may be formed in vivo.

Design and synthesis of a mitochondria-targeting carrier for small molecule drugs.

A novel mitochondria-targeting carrier QCy7HA was developed. QCy7HA transported the covalently attached doxorubicin (DOX) to mitochondria specifically. The conjugate limited the effects of P-glycoprotein (Pgp) efflux pumps of multidrug-resistant cells on DOX, indicating that diverting drugs to mitochondria is a potential promising method for treatment of drug-resistant cancers.

Distribution of calcification in carotid endarterectomy tissues: comparison of micro-computed tomography imaging with histology.

BACKGROUND: Calcification in atherosclerotic plaques has been viewed as a marker of plaque stability, but whether calcification accumulates in specific anatomic sites in the carotid artery is unknown. We determined the burden and distribution of calcified plaque in carotid endarterectomy (CEA) tissues. METHODS: A total of 22 CEA tissues were imaged with high-resolution micro-computed tomography (micro-CT). Total plaque burden and total calcium score using the Agatston method were quantified. The Agatston score (AS) was also normalized for tissue size. Plaque and calcium distribution were analyzed separately for three CEA regions: common segment (CS), bulb segment (BS), and internal/external segments (IES). RESULTS: The average CEA tissue length was 40.83 (interquartile range [IQR] 33.31-42.41) mm with total plaque burden of 103.45 (IQR: 78.84-156.81) mm(3) and total AS of 38.58 (IQR 11.59-89.97). Total plaque volume was 21.02 (IQR: 14.47-25.42) mm(3) in the CS, 37.89 (22.59-48.32) mm(3) in the BS, and 54.05 (36.87-74.52) mm(3) in the IES. Of the 22 tissues, 15 had no calcium in the CS compared with three in the bulb and two in the IES. Normalized calcified plaque was most prevalent in the BS, the IES and was least prevalent in the CS. The overall correlation of calcification between histology sections and matched micro-CT images was 0.86 (p<0.001). CONCLUSIONS: Calcified plaque is heterogeneously distributed in CEA tissues with most in the bulb and IES regions. The amount of calcification in micro-CT slices shows a high correlation with matched histology sections.

In vivo senescence imaging nanoprobe targets the associated reactive oxygen species.

Cellular senescence, a cell-cycle arrest state upon stress or damage, can adversely impact aging and cancers. We have designed a novel near infrared fluorogenic nanoprobe, named D3, which can only be turned on by highly elevated levels of reactive oxygen species (ROS), critical players for the induction and maintenance of senescence, for real-time senescence sensing and imaging. In contrast to glowing senescent cells, non-senescent cells whose ROS levels are too low to activate the D3 signal remain optically silent. Upon systemic injection into senescent tumor-bearing mice, the D3 nanoprobe quickly accumulates in tumors, and its fluorescence signal is turned on specifically by senescence-associated ROS in the senescent tumors. The fluorescence signal at senescent tumors was 3-fold higher than that of non-senescent tumors. This groundbreaking design introduces a novel activation mechanism and a powerful imaging nanoprobe to identify and assess cellular senescence in living organisms.

A fabricated siRNA nanoparticle for ultra-long gene silencing in vivo.

Persistent gene silencing is crucially required for the successful therapeutics of short interfering RNA (siRNA). Here, we describe a nanoparticle based delivery system which assembled by layering siRNAs between protease degradable polypeptides to extend the therapeutic window. These tightly packed nanoparticles are efficiently taken up by cells by endocytosis, and the fabricated siRNAs are gradually released following intracellular degradation of the polypeptide layers. During cell division, the particles are distributed to the daughter cells. Due to the slow degradation through the multiple layers, the particles continuously release siRNA in all cells. Using this controlled release construct, the in vivo gene silencing effect of siRNA is consistent for an ultra-long period of time (>3 weeks) with only a single treatment.

Exploring the structural requirements of collagen-binding peptides.

Collagen synthesis and tissue remodeling are involved in many diseases; therefore, collagen-specific binding agents have been developed to study collagen changes in various tissues. Based on a recently reported collagen binding peptide, which contains unnatural biphenylalanine (Bip) amino acid residue, constructs with various structure variations were synthesized to explore the contributions of unnatural Bip residue, conformational restrain, and amino acid sequence in collagen recognition. Their binding efficiency to collagens was evaluated in vitro using pure collagens. The results indicate that the C-terminal unnatural Bip residue, rather than the peptide sequence or conformational restrain, dominated the collagen I binding. Subsequent tissue binding study showed that the selected peptide did not offer preferential selectivity over collagen I in tissue, suggesting that a simple in vitro binding assay cannot adequately model the complex biological environment.

Ultra Pseudo-Stokes Shift Near Infrared Dyes Based on Energy Transfer.

Novel fluorescent dyes with ultra pseudo-Stokes Shift were prepared based on intramolecular energy transfer between a fluorescent donor and a Cyanine-7 acceptor. The prepared dyes could be excited at ~ 320 nm and emit fluorescence at ~ 780 nm. The energy transfer efficiencies of the system are found to be > 94 %.

Detection of pancreatic cancer tumours and precursor lesions by cathepsin E activity in mouse models.

BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA. Surgical resection is the only effective treatment; however, only 20% of patients are candidates for surgery. The ability to detect early PDAC would increase the availability of surgery and improve patient survival. This study assessed the feasibility of using the enzymatic activity of cathepsin E (Cath E), a protease highly and specifically expressed in PDAC, as a novel biomarker for the detection of pancreas-bearing pancreatic intraepithelial neoplasia (PanIN) lesions and PDAC. METHODS: Pancreas from normal, chronic pancreatitis and PDAC patients was assessed for Cath E expression by quantitative real-time PCR and immunohistochemistry. Human PDAC xenografts and genetically engineered mouse models (GEMM) of PDAC were injected with a Cath E activity selective fluorescent probe and imaged using an optical imaging system. RESULTS: The specificity of Cath E expression in PDAC patients and GEMM of pancreatic cancer was confirmed by quantitative real-time PCR and immunohistochemistry. The novel probe for Cath E activity specifically detected PDAC in both human xenografts and GEMM in vivo. The Cath E sensitive probe was also able to detect pancreas with PanIN lesions in GEMM before tumour formation. CONCLUSIONS: The elevated Cath E expression in PanIN and pancreatic tumours allowed in-vivo detection of human PDAC xenografts and imaging of pancreas with PanIN and PDAC tumours in GEMM. Our results support the usefulness of Cath E activity as a potential molecular target for PDAC and early detection imaging.

Enhancing the Cellular Delivery of Nanoparticles using Lipo-Oligoarginine Peptides.

Nanoparticles have great potential as nanotherapeutics, delivery vectors, and molecular imaging agents due to their flexible properties. Although intracellular and nuclear delivery of nanoparticles is desirable for therapeutic applications, it remains a challenge. Cell penetrating peptides (CPPs) are a powerful tool for the intracellular delivery of various cargoes. Here we report that functionalization of nanoparticles with a myristoylated oligoarginine CPP promoted cellular uptake without increased toxicity. It was evidenced that the myristoylated CPP is much more effective in transporting nanoparticles than the unmodified CPPs.

Layered nanoprobe for long-lasting fluorescent cell label.

A long-lasting particle-based fluorescent label is designed for extended cell imaging studies. This onion-like nanoprobe is constructed through layer-by-layer fabrication technology. The nanoprobes are assembled with multiple layers of optically quenched polyelectrolytes, the fluorescence signal of which can be released later by intracellular proteolysis. Upon incubation with cells, the assembled nanoprobes are taken up efficiently. The tight packing and layered assembly of the quenched polyelectrolytes slow subsequent intracellular degradation, and then result in a prolonged intracellular fluorescence signal for up to 3 weeks with no noticeable toxicity.

A benzothiazole alkyne fluorescent sensor for Cu detection in living cell.

A new type of alkyne dye, 6-dimethylaminobenzothiazole alkyne (1), was developed for Cu sensing in biological system. Dye (1) offered excellent selective over a panel of ions, only Cu(I) could change the fluorescence of dye (I) by forming copper acetylide between the terminal alkyne and Cu(I). Its potential of detecting Cu in biological system was demonstrated in cell culture.